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Effect of Storage and Heat Treatment of Milk Destined for Cheese Production on Its Oxidative Characteristics

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The oxidative stability of milk and dairy products is a very interesting topic for the dairyindustry due to the growing demand for foods containing bioactive compounds with positive healtheffects. The aim was to evaluate the oxidative stability of milk intended for cheese production. Theeffect of storage time, heat pre-treatment, and milk pasteurization temperature on the characteristicsof milk and cheese was investigated. The cheese samples were produced with pasteurized milk atboth 72 and 77◦C for a time of 15 s using three types of milk: raw fresh milk processed within 48 hof milking, raw stored milk processed within 96 h, and thermized milk that was heat-treated uponarrival at the dairy and processed within 96 h of milking. In total, three repetitions were carriedout for each type of milk and pasteurization. Samples of milk before and after pasteurization andcheese at 14 days of storage were analyzed. Antioxidant activity decreased from starting milk to milkafter pasteurization to final cheese. The longer storage time of the milk had significant effects on theantioxidant stability of the cheese (64.95 vs. 59.05% of antioxidant activity). Thermization of the milkfurther reduced the stability of the cheese (54.05% of antioxidant activity). The greater antioxidantstability of fresh milk and cheeses produced with fresh milk is the first result that encourages theproduction of cheese from a milk that best preserves its original characteristics.
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Article
Effect of Storage and Heat Treatment of Milk Destined for
Cheese Production on Its Oxidative Characteristics
Carmela Tripaldi 1, Simona Rinaldi 1, * , Giuliano Palocci 1, Sabrina Di Giovanni 1, Salvatore Claps 2
and Luca Buttazzoni 1


Citation: Tripaldi, C.; Rinaldi, S.;
Palocci, G.; Di Giovanni, S.; Claps, S.;
Buttazzoni, L. Effect of Storage and
Heat Treatment of Milk Destined for
Cheese Production on Its Oxidative
Characteristics. Dairy 2021,2, 585–601.
https://doi.org/10.3390/dairy2040046
Academic Editor: Giovanni Bittante
Received: 7 June 2021
Accepted: 24 September 2021
Published: 18 October 2021
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Attribution (CC BY) license (https://
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4.0/).
1
Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria (CREA), Centro di Ricerca Zootecnia
e Acquacoltura, Via Salaria, 31, 00015 Monterotondo, Italy; carmela.tripaldi@crea.gov.it (C.T.);
giuliano.palocci@crea.gov.it (G.P.); sabrina.digiovanni@crea.gov.it (S.D.G.); luca.buttazzoni@crea.gov.it (L.B.)
2
Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria (CREA), Centro di Ricerca Zootecnia
e Acquacoltura, S.S. 7 Via Appia, 85051 Bella Muro, Italy; salvatore.claps@crea.gov.it
*Correspondence: simona.rinaldi@crea.gov.it
Abstract:
The oxidative stability of milk and dairy products is a very interesting topic for the dairy
industry due to the growing demand for foods containing bioactive compounds with positive health
effects. The aim was to evaluate the oxidative stability of milk intended for cheese production. The
effect of storage time, heat pre-treatment, and milk pasteurization temperature on the characteristics
of milk and cheese was investigated. The cheese samples were produced with pasteurized milk at
both 72 and 77
C for a time of 15 s using three types of milk: raw fresh milk processed within 48 h
of milking, raw stored milk processed within 96 h, and thermized milk that was heat-treated upon
arrival at the dairy and processed within 96 h of milking. In total, three repetitions were carried
out for each type of milk and pasteurization. Samples of milk before and after pasteurization and
cheese at 14 days of storage were analyzed. Antioxidant activity decreased from starting milk to milk
after pasteurization to final cheese. The longer storage time of the milk had significant effects on the
antioxidant stability of the cheese (64.95 vs. 59.05% of antioxidant activity). Thermization of the milk
further reduced the stability of the cheese (54.05% of antioxidant activity). The greater antioxidant
stability of fresh milk and cheeses produced with fresh milk is the first result that encourages the
production of cheese from a milk that best preserves its original characteristics.
Keywords: fresh milk; antioxidant activity; pasteurized milk; milk oxidation; cheese production
1. Introduction
In high-income countries, especially European ones, some dairy products may have
characteristics designed to satisfy the demands of the consumer who is increasingly inclined
to choose products with certain sensory or nutritional characteristics or made with methods
that respect the environment and animal welfare. The European provisions on drinking
milk establish rules aimed at safeguarding the nutritional value of heat-treated milk (EC
Regulation 2597/1997) [
1
]. As part of its quality policy, European legislation has protected
the names of foods with unique characteristics linked to their geographical origin (starting
from EEC Regulation 2081/92) [
2
] through the Protected Designation of Origin (PDO) and
Protected Geographical Indication (PGI). Recently the obligation on the label of the origin
of the product has been extended to all dairy products (EU Regulation No. 1169/2011) [
3
].
Some of these measures have helped to increase the added value of some dairy products.
In Italy, 52% of national milk used for cheese production is destined to become PDO
cheeses [
4
]. For the other cheeses, except for the so-called “traditional” cheeses, for which
the process is defined, there are no specifications to be respected. The Italian Ministry
of Agriculture financed a project on the effects of the use of “fresh” milk on the quality
of cheese. By “fresh” milk, we mean raw milk processed within 48 h of milking, as it
is practiced in drinking milk called “fresh pasteurized milk” and “high-quality fresh
Dairy 2021,2, 585–601. https://doi.org/10.3390/dairy2040046 https://www.mdpi.com/journal/dairy
Dairy 2021,2586
pasteurized milk.” To evaluate the effects of the use of “fresh” milk on some specific
characteristics of final cheese, we focused on the oxidative stability of milk processed at
different times from milking. The effect of a thermal pre-treatment carried out in the case
of longer storage of the milk, in addition to that of temperature of the pasteurization, was
also evaluated.
The oxidative stability of milk and dairy products is a very interesting topic for the
dairy industry. In recent years there has been an increasing demand for foods containing
bioactive compounds that have positive effects on health. Milk also contains antioxidant
compounds that reduce or prevent the risk of various diseases. Antioxidant activity in milk
is due to many bioactive compounds such as proteins, peptides, vitamins E and C, retinol,
β-carotene, glutathione (GSH), and glutathione peroxidase [57].
The main antioxidants present in milk can be grouped into lipophilic and hydrophilic
antioxidants. Carotenoids, retinol, and
α
-tocopherol are lipid-soluble antioxidants present
in milk fat globule and they have properties such as preventative, chain-breaking an-
tioxidant, and quencher of singlet oxygen in milk, protecting milk fat against autoxida-
tion [
6
,
8
,
9
]. In addition to highly active and more abundant lipophilic antioxidants, milk is
also a source of hydrophilic antioxidants constituted by ascorbic acid and a large group
of nitrogen compounds, such as casein fractions, whey proteins (in particular lactoferrin
and
β
-lactoglobulin), bioactive peptides, low molecular weight nitrogen compounds and
uric acid [
10
]. Another important group of antioxidant compounds are enzymes that
counteract free radicals in milk, including superoxide dismutase, catalase, lactoperoxidase,
and glutathione peroxidase. Those enzymes form a synergistic system that increases the
antioxidant potential of milk. Antioxidant systems in milk can inhibit the free radical
mechanism by donating the proton and thus inhibit the onset of autoxidation [9].
In particular, the presence and concentration of antioxidants such as vitamin E,
polyphenols, and
β
-carotene, strongly depend on feeding management [
11
]. The intake of
vitamins and antioxidants within the diet is transferred to milk [
12
,
13
]. Higher levels of
antioxidants (tocopherol,
β
-carotene, and retinol) have been reported in milk from cows
that consume fresh grass compared with diets rich in concentrate or silage [
11
,
14
]. The
vitamin E content of milk is determined by tocopherol levels in feed while vitamin A and
β-carotene concentrations are determined by carotenoids supplied with bovine feed [9].
In milk, as in dairy products, oxidative reactions can develop off-flavor and loss of
nutritional quality [
15
]. Spontaneous oxidation in milk may progress as a result of external
factors during storage and processing such as photo-oxidation or heat treatments. However,
it depends on inherent factors in the milk itself which include fatty acid composition,
content of antioxidant molecules, antioxidative enzyme systems, and transition metal
ion content [
6
]. Moreover, external factors such as preservation, handling, agitation,
temperature, exposure to light, and contamination by metals and microorganisms can
induce additional oxidative reactions in milk [10,16,17].
The oxidative stability of milk and dairy products in function of time can be mon-
itored by measuring lipid oxidation, protein oxidation, content of vitamins (riboflavin,
tocopherols, carotenoids), or the use of a sensory panel. Since oxidation occurs in case
of an imbalance between the presence of reactive oxidants and the antioxidant defense
mechanism, sensitivity to oxidation can also be monitored by measuring the antioxida-
tive capacity of a product [
9
]. Considering the heterogeneity of antioxidant molecules in
milk, different methods have been developed to investigate total milk antioxidant capacity
without distinguishing the contributions from individual compounds [18].
The general objective of the project is to evaluate the qualitative characteristics of
cheese produced with fresh milk and to identify the most suitable markers to identify this
type of cheese. The project activity includes innovative determinations as sensory analyses
of cheese and the
in vitro
digestion tests of milk and cheese, near-infrared spectroscopy
(NIR) and electronic nose analyses of cheese.
In this work, the oxidative stability of milk destined to produce fresh milk cheese was
evaluated. The effects of milk storage time and heat treatments were investigated. The
Dairy 2021,2587
latter include the thermal pretreatment practiced in the case of prolonged milk storage and
pasteurization temperature.
2. Materials and Methods
2.1. Raw Milk
The cow’s milk used in the trial to produce cheese samples was collected between
October 2019 and March 2020 from local farms and arrived in the experimental dairy
plant of the Council for Agricultural Research and Economics (CREA), Research Centre
for Animal Production and Aquaculture of Bella Muro, Potenza, Italy, within 24 h of the
first milking. Samples of raw milk upon arrival at dairy were collected and transferred to
laboratory for routine analyses.
2.2. Milk Treatments
The treatments undergone by the milk before the production of the experimental
cheese samples were as follows:
- raw fresh milk: pasteurization within 48 h of the first milking (F);
- raw stored milk: pasteurization within 96 h of the first milking (S);
-
thermized and stored milk: thermization process (65
C for 15 s) immediately upon
arrival at the dairy and cooling, pasteurization within 96 h of the first milking (T).
The storage of milk was performed in tank at 4
C. Each batch of milk was heat-
treated at 72
C for 15 s, the minimum recommended for milk pasteurization and called
low temperature (L), and at 77 C for 15 s called high temperature (H). A plate exchanger
was used (Comat Dairy Equipment, Bellizzi, Salerno, Italy) equipped with software for the
management and control of the pasteurization temperature.
Samples of raw milk immediately before pasteurization and pasteurized milk were
collected and frozen to
80
C for further analysis. Second the experimental design, six
different treated milks were produced for three repetitions (3 treatments
×
2 pasteurizations
×3 repetitions).
2.3. Cheese Production
Six tests were carried out to produce cheese samples. In each test, the three exper-
imental milks (F, P, T) were submitted to the same type of pasteurization, for a total of
three repetitions for each type of milk and pasteurization. The cheeses were produced
artisanally in the small experimental dairy plant of CREA (Bella Muro, Potenza, Italy).
Cheese manufacture was performed using a protocol detailed in Figure 1. A starter con-
taining Streptococcus thermophilus and Lactobacillus delbrueckii was used. After coagulation,
two curd cuts were made, and the final size of the curd grain was similar to a hazelnut.
Once it reached pH 5.5–5.6, the cheese was transferred from the hot room to the cold room
(10
C). The cheeses were salted when the temperature of the cheese reached about 15
C.
Dry salting was practiced and about 15 g of salt/kg of cheese was used. The next day, the
cheese was transferred to the cold room at 4 C.
For each production, six shapes per group were collected and carried to the laboratory
of CREA localized in Monterotondo, Rome, Italy. The analyses were carried out on the
cheese after 14 days of storage at refrigeration temperature (4
C). The cheeses were
sampled by homogenizing the whole shape as they were fresh soft cheeses without rind.
2.4. Milk Analysis
On the batches of raw milk, sampled the day of being carried into dairy plant, the
following routine analyses were carried out. pH values were determined using Metrohm
mobile 826 pHmeter (Metrohm Ltd., Herisau, Switzerland); fat, protein, casein, and lactose
levels (expressed as % of fresh weight), were determined using a MilkoScan
7 RM (Foss
Electric A/S, Hillerød, Denmark) calibrated according to ISO-IDF references (ISO 9622:2013;
IDF 141:2013) [19]. These analyses were carried out on fresh milk.
Dairy 2021,2588
Moreover, to test the changes due to storage and heat treatments, each type of milk
(F, S, T) was analyzed both before and after pasteurization determining lactoperoxidase
activity, tryptophane, advanced Maillard’s products, and antioxidant activity according to
the methods specified below. These analyses were carried out on frozen at
80
C samples.
Dairy 2021, 2, x FOR PEER REVIEW 4 of 18
Figure 1. Flow chart of production of cheese samples.
For each production, six shapes per group were collected and carried to the labora-
tory of CREA localized in Monterotondo, Rome, Italy. The analyses were carried out on
the cheese after 14 days of storage at refrigeration temperature (4 °C). The cheeses were
sampled by homogenizing the whole shape as they were fresh soft cheeses without rind.
2.4. Milk Analysis
On the batches of raw milk, sampled the day of being carried into dairy plant, the
following routine analyses were carried out. pH values were determined using Metrohm
mobile 826 pHmeter (Metrohm Ltd., Herisau, Switzerland); fat, protein, casein, and lac-
tose levels (expressed as % of fresh weight), were determined using a MilkoScan™ 7 RM
(Foss Electric A/S, Hillerød, Denmark) calibrated according to ISO-IDF references (ISO
9622:2013; IDF 141:2013) [19]. These analyses were carried out on fresh milk.
Moreover, to test the changes due to storage and heat treatments, each type of milk
(F, S, T) was analyzed both before and after pasteurization determining lactoperoxidase
activity, tryptophane, advanced Maillard’s products, and antioxidant activity according
to the methods specified below. These analyses were carried out on frozen at −80 °C sam-
ples.
Figure 1. Flow chart of production of cheese samples.
2.5. Cheese Analysis
Cheese samples were submitted to the following analyses: pH, moisture (IDF, 1986) [
20
],
ashes (AOAC, 2000) [21], protein, and fat (ISO 21543:2006; IDF 201:2006) [22].
To value oxidation level in cheese after storage for 14 days, tryptophane, advanced
Maillard’s products and antioxidant activity were determined such as for milk using the
same methods reported below. Moreover, Malondialdehyde (MDA) analysis was carried
out as reported by Rinaldi et al. [
23
] to test fat oxidation in cheese samples. The analyses
were carried out on clopped samples frozen at
80
C. For each analysis, at least two
repetitions of each sample were performed.
2.6. Lactoperoxidase Activity Method
In milk, enzymatic activity of lactoperoxidase was measured using 2,2
0
-Azinobis-3-
ethylbenzothiazoline-sulfonic acid (ABTS) as substrate, following the spectrophotometric
method of Marin et al. [
24
]. In the spectrophotometer cuvette, 50
µ
L milk was added with
1 mL 0.65 mM ABTS substrate prepared in 0.1 M sodium phosphate buffer (pH 6.0) and
50
µ
L of the same sodium phosphate buffer. The reaction was initiated by adding 1 mL of
0.1 mM hydrogen peroxide as a reaction activator. Increasing absorbance at 412 nm was
Dairy 2021,2589
measured for 1 min at 20
C by a double beam UV-VIS spectrophotometer (Lambda 25,
PerkinElmer, Waltham, MA, USA).
The blank was prepared with 50
µ
L milk, 1 mL of substrate (0.65 mM ABTS), and
1050
µ
L of phosphate buffer, without hydrogen peroxide. The ratio between absorbance
and time was linear, at least in the 1st 20 s of the reaction. Enzymatic activity was calculated
as the slope of the curve in the linear part, considering the molar extinction coefficient of
32.4 mM
1
cm
1
, and it was expressed in units per mL, where 1 unit is the quantity of
enzyme which catalyzes the oxidation of 1 micromole of substrate per min at 20 C.
2.7. FAST Method
The FAST method (Fluorescence of Advanced Maillard products and Soluble Trypto-
phan) was used to evaluate the modifications induced in milk by heat-treatments analyzing
tryptophane and advanced Maillard’s products in samples [
25
,
26
]. This method has also
been applied to Mozzarella cheese in a previous work [23].
Samples of milk (500
µ
L) or cheese (0.5 g) were mixed to 4.5 mL sodium acetate buffer
(0.1M, pH 4.6) and shanked vigorously for 30 s. After centrifugation at 4000
×
gfor 10 min
at 20
C, the supernatant was diluted (1:10), filtered through a 0.45
µ
m filter, and analyzed
by Spectrofluorometer (FP-6300, Jasco, Tokyo, Japan).
Two fluorescence intensities were measured:
- fluorescence of tryptophan (Trp-F) at 290 nm excitation and 340 nm emission,
-
fluorescence of advanced Maillard’s products (AMP-F) at 350 nm excitation and
440 nm emission and expressed in arbitrary units (a.u.).
The FAST index was calculated as follows:
(AMP-F/Trp-F) ×100
Tryptophan measurement is an estimate of soluble proteins content in solution. In
the acidic conditions (pH 4.6) used in this analysis, caseins precipitate, so that Trp-F is
positively related to whey protein soluble at pH 4.6. Denaturation and precipitation of
proteins due to heat treatments may result in decrease of Trp-F.
AMP-F is related to the content of advanced Maillard’s products, and generally, Mail-
lard’s reaction occurs in products subjected to high heat treatments. Milk thermized and/or
exposed to high pasteurization could be characterized by higher values of FAST index.
2.8. Antioxidant Activity—DPPH Method
The antioxidant activity of the samples was analyzed by 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH) method, as reported by Unal [
27
] and Rinaldi et al. [
23
]. The DPPH radical is
reduced by the antioxidant compounds of the sample and the antioxidant activity expresses
the capacity of the sample to inhibit the DPPH radical.
Milk samples (2 mL) or cheese samples (2 g) were mixed with 8 mL of 0.11 mM DPPH
ethanolic solution. As control, 2 mL of ethanol, in place of the sample, were added to
8 mL of 0.11 mM DPPH solution. The mixtures were vortexed and incubated at room
temperature for 20 min in the dark and centrifuged for 10 min at 9000×gat 22 C.
Absorbance was recorded at 517 nm on the supernatant by a double beam UV-VIS
spectrophotometer (Lambda 25, PerkinElmer, Waltham, MA, USA). The antioxidant activity
was reported in percentage according to the following equation:
% Antioxidant activity = (A0As)/A0×100
where A
0
is the absorbance of the control (without sample) and As is the absorbance of
the tested sample. Using Trolox as standard, antioxidant activity of the samples can be
expressed in Trolox equivalents (mmol eq Trolox/100 mL of milk or 100 g of cheese).
Dairy 2021,2590
2.9. Statistical Analysis
Data of characteristics of the milk before and after pasteurization (lactoperoxidase
activity, FAST index, and antioxidant activity) were statistically analyzed by GLM proce-
dure in the statistical software package SAS/STAT (version 9.4 SAS Institute Inc.) [
28
]. For
both kinds of milk, the model included 3 levels of treatment of milk (fresh, stored, and
thermized milk) as fixed effect. For pasteurized milk, the model also included two kinds
of pasteurization (low and high) for each level, as fixed effect, for a total of 6 levels. The
same 6 level model was used for the cheese data (pH, moisture, ashes, protein, fat, lactoper-
oxidase activity, oxidative characteristics, and FAST Index). Differences were considered
significant at p< 0.05, in each model.
3. Results
3.1. Chemical Characteristics of Raw Milk
The chemical characteristics of raw milk used for cheese-making in this study are
reported in Table 1. These are the average data of analyses carried out on raw milk sampled
upon arrival at the dairy and then used in the six trials.
Table 1.
Chemical characteristics of raw milk upon arrival at the dairy. Data are expressed as the
mean of six repetitions. sd = standard deviation. For each parameter, maximum and minimum
are reported.
Parameter Average ±sd Minimum Maximum
Fat (%) 3.91 ±0.08 3.78 4.01
Protein (%) 3.49 ±0.04 3.43 3.54
Casein (%) 2.74 ±0.03 2.69 2.77
Lactose (%) 4.80 ±0.03 4.73 4.86
pH 6.68 ±0.02 6.65 6.71
The determination of the characteristics of raw milk was carried out to evaluate the
initial condition of used milk. The hygienic and healthy characteristics of the milk were
within the limits established by current legislation. The rate fat/protein was 1.12. The
results show that raw milk had the fat and protein content slightly higher than average data
provided by the National Association of Italian Friesian and Jersey Breed (ANAFIJ) [
29
],
respectively 3.79 and 3.35%, for the year 2020.
3.2. Characteristics of Pasteurized Milk
Characteristics of stored (S) and thermized (T) milk compared to fresh (F) milk sub-
jected to low (L) or high (H) pasteurization are reported in Table 2. The results show that
modifications occurred in some parameters of stored and thermized milk compared to
those of fresh one, as discussed below.
Table 2.
Lactoperoxidase activity, oxidative characteristics, and FAST Index of fresh (F), stored (S), and thermized (T) milk
subjected to low (L) or high (H) pasteurization.
Milk Lactoperoxidase U mL1Trp-F a.u. AMP-F a.u. FAST Index Antioxidant Activity %
Fresh F-L 2.53 a 647 a 25.4 b 3.94 c 81.3 a
F-H 0.64 b 650 a 26.5 ab 4.09 bc 78.9 b
Stored S-L 2.37 a 633 a 26.1 ab 4.13 bc 76.8 c
S-H 0.68 b 612 ab 27.5 a 4.38 ab 74.7 d
Thermized T-L 2.10 a 594 b 25.7 b 4.33 abc 76.2 cd
T-H 0.56 b 581 b 26.9 ab 4.65 a 74.9 cd
Data are expressed as mean of three repetitions. For each parameter, means followed by a different letter in the column are significantly
different (p< 0.05). Trp-F = tryptophan fluorescence, AMP-F = Maillard’s advanced products fluorescence; FAST = Fluorescence of advanced
Maillard products and soluble tryptophan.
Dairy 2021,2591
3.2.1. Lactoperoxidase Activity
The Lactoperoxidase (LPO) enzyme is recognized for its high heat stability and, due
to this property, LPO is used as an index of pasteurization efficiency in milk.
Lactoperoxidase activity decreased significantly (p< 0.001) in milk subjected to high
pasteurization compared to milk subjected to low pasteurization (on average from 2.33 to
0.63 U mL
1
) independently by preservation and thermization of the milk. The reduction
of Lactoperoxidase activity in fresh low pasteurized milk (F-L) was about 7.3% compared to
this activity in raw milk that was on average 2.73 U mL
1
in our samples. These data show
that the parameters used in low pasteurization (72
C for 15 s) were suitable to maintain
good levels of LPO activity while the temperature of 77
C for 15 s induced inactivation of
about 73% of LPO activity compared to activity in low pasteurized milk.
Data for LPO activity in the literature vary widely because of the various chromogens
used for its assay and the variability in the assay conditions (pH, temperature). The levels
of LPO change in different types of milk as reported by Seifu et al. [
30
]. In bovine milk,
LPO is the second most abundant enzyme after xanthine oxidase. A report indicated that
the mean LPO activity in cow raw milk throughout lactation ranged from 1.5 to 2.7 U mL
1
with an overall mean of 2.3 U mL
1
[
31
] as reported even by Ozer [
32
]. However, other
authors reported lower LPO activity, on average 1.42 U mL1, in bovine milk [30,33].
The LPO is active (able to catalyze the reaction between H
2
O
2
and thiocyanate at a
sufficient level) after heat treatment at 74
C for a short time. Complete LPO deactivation
occurred at 80
C for 15 s, whereas at 72
C for 15 s residual LPO activity was about
70% [
34
]. LPO activity is used together alkaline phosphatase, another original milk enzyme,
to investigate the retrospective determination of the heat treatment undergone by milk.
Alkaline phosphatase activity must be negative as assessment of correct pasteurization,
and it is used as an index of adequate pasteurization of milk.
3.2.2. Protein Denaturation and the FAST Index
The FAST index (fluorescence of advanced Maillard products and soluble tryptophan)
is based on the quantification of two complementary indicators, the fluorescence of trypto-
phan (Trp-F) and that of advanced Maillard products (AMP-F) in the milk protein fraction
soluble at pH 4.6. This method has been used in other works to evaluate the changes due
to heat treatments in milk [25,26].
Trp-F decreased significantly (p< 0.01) in thermized milk compared to fresh milk
(from 648 to 587 a.u.), but no significant differences were found between milk subjected
to high and low pasteurization (Table 2). There was also a tendency to reduction in Trp-F
from 648 to 622 a.u. in stored milk compared to the fresh one.
The Trp fluorescence appears to be a good indicator of the protein concentration,
as shown by the high correlation with colorimetric methods of protein determinations
and even by a good correlation with the acid-soluble
β
-lactoglobulin content observed
in milk in precedent works [
25
]. So, Trp fluorescence may be used to determine protein
denaturation due to heat treatments. However, when high heat treatments are applied,
the decrease of Trp fluorescence is due to both protein conformational changes and Trp
degradation [25,35].
AMP-F was significantly higher (p< 0.05) in stored milk (S-H) than in T-L and F-L
(ranging from 27.5 a.u. to 25.4 a.u.) while the other treated milk had middle levels of
AMP-F (Table 2). AMP level usually increased in samples during high heat treatments,
in fact, there was no effect on AMP-F due to thermization (65
C) while a tendency of
increasing AMP-F level was found due to high pasteurization (77
C) compared to low
pasteurization (72
C). The fluorescence of AMP generally increases with prolonged heat
treatment due to extensive conversion of the early Maillard product to AMP. Consequently,
AMP should be a good indicator of nutritional damage to evaluate severe treatments (such
as indirect UHT treatment and sterilization) where the advanced step of the Maillard
reaction is reached [25,26,36].
Dairy 2021,2592
The FAST index was significantly (p< 0.05) lower in F-L milk than in T-H and S-H
(3.94 vs. 4.65 and 4.38) while all other milk samples showed middle values (4.33–4.09). The
FAST index, depending on AMP-F/Trp-F ratio, generally increases with contemporaneous
decrease in Trp-F and production of AMP due to heat-treatments. The relation between
Trp-F and FAST index is reported in Figure 2, where each pasteurized milk was plotted.
Dairy 2021, 2, x FOR PEER REVIEW 8 of 18
Trp-F decreased significantly (p < 0.01) in thermized milk compared to fresh milk
(from 648 to 587 a.u), but no significant differences were found between milk subjected to
high and low pasteurization (Table 2). There was also a tendency to reduction in Trp-F
from 648 to 622 a.u. in stored milk compared to the fresh one.
The Trp fluorescence appears to be a good indicator of the protein concentration, as
shown by the high correlation with colorimetric methods of protein determinations and
even by a good correlation with the acid-soluble β-lactoglobulin content observed in milk
in precedent works [25]. So, Trp fluorescence may be used to determine protein
denaturation due to heat treatments. However, when high heat treatments are applied,
the decrease of Trp fluorescence is due to both protein conformational changes and Trp
degradation [25,35].
AMP-F was significantly higher (p < 0.05) in stored milk (S-H) than in T-L and F-L
(ranging from 27.5 a.u. to 25.4 a.u.) while the other treated milk had middle levels of AMP-
F (Table 2). AMP level usually increased in samples during high heat treatments, in fact,
there was no effect on AMP-F due to thermization (65 °C) while a tendency of increasing
AMP-F level was found due to high pasteurization (77 °C) compared to low pasteurization
(72 °C). The fluorescence of AMP generally increases with prolonged heat treatment due
to extensive conversion of the early Maillard product to AMP. Consequently, AMP should
be a good indicator of nutritional damage to evaluate severe treatments (such as indirect
UHT treatment and sterilization) where the advanced step of the Maillard reaction is
reached [25,26,36].
The FAST index was significantly (p < 0.05) lower in F-L milk than in T-H and S-H
(3.94 vs. 4.65 and 4.38) while all other milk samples showed middle values (4.33–4.09). The
FAST index, depending on AMP-F/Trp-F ratio, generally increases with contemporaneous
decrease in Trp-F and production of AMP due to heat-treatments. The relation between
Trp-F and FAST index is reported in Figure 2, where each pasteurized milk was plotted.
Figure 2. Relation between tryptophan fluorescence (Trp-F) and FAST index in fresh (F), stored (S),
and thermized (T) milk subjected to low (L) or high (H) pasteurization.
The Trp-F decreased with heat treatment and in particular with thermization, so that
samples of fresh milk could be discriminated easily by those of thermized milk both on
the Trp-F and FAST Index axis. The main decrease in Trp-F was induced by the double
heat-treatment, thermization and high pasteurization (T-H) and it was related to highest
FAST index value. The highest Trp-F levels characterized fresh milk (F-L and F-H) and F-
Figure 2.
Relation between tryptophan fluorescence (Trp-F) and FAST index in fresh (F), stored (S),
and thermized (T) milk subjected to low (L) or high (H) pasteurization.
The Trp-F decreased with heat treatment and in particular with thermization, so that
samples of fresh milk could be discriminated easily by those of thermized milk both on
the Trp-F and FAST Index axis. The main decrease in Trp-F was induced by the double
heat-treatment, thermization and high pasteurization (T-H) and it was related to highest
FAST index value. The highest Trp-F levels characterized fresh milk (F-L and F-H) and
F-L was even characterized by the lowest FAST index value, while T-L and S-H milk had
middle levels of Trp-F and FAST Index (Figure 2).
The values of the FAST index in our samples were lower than those reported in other
works [
26
,
37
], demonstrating the good nutritional quality of the milk and in particular of the
low-pasteurized fresh milk. However, the fast index rises more when higher temperatures
are used and/or for longer times than those used in this trial, as in the case of UHT and
sterilized milk due mostly to AMP production [36].
3.2.3. Antioxidant Activity
Antioxidant activity determination is a useful tool in estimating the total ability of
milk to counteract the oxidation that normally occurs during storage and processing in
milk [
38
,
39
]. The antioxidant activity of milk (Table 2), determined as DPPH free-radical
scavenging activity, was significantly higher (p< 0.01) in fresh milk than in thermized
and stored milk, ranging from 81.3% in F-L to 74.7% in S-H (corresponding to 17.2 and
15.8 mmol eq Trolox/100 mL, respectively). No significant differences in antioxidant
activity were found between thermized and stored milk (non-thermized). Moreover, there
were significant (p< 0.05) differences between high and low pasteurized milk both in fresh
milk and stored milk.
These values could depend on the antioxidant activity in raw milk that decreased
with the heat-treatment of thermization and mostly with high pasteurization, but even
with preservation time. Milk is a complex system of pro and antioxidant components [
6
]
and oxidation occurs in case of an imbalance between the presence of reactive oxidants and
the antioxidant defense mechanism [
7
,
40
]. Among spectrophotometric methods, the DPPH
Dairy 2021,2593
method is useful to measure the total antioxidant activity without giving any detailed
information about individual antioxidant components [
18
]. The antioxidant components
neutralized DPPH radical using both hydrogen atom transfer and single electron transfer
mechanisms [
39
]. Antioxidant content in raw milk, in particular the content of lipophilic
antioxidants, is largely determined by the composition of animal diets [9].
The storage time may affect the antioxidant activity of milk favoring better antioxidant
stability in fresh milk. The thermal pre-treatment was finalized to reduce the microbial
load, especially psychrotrophic bacteria, and to increase the keeping quality of raw milk
until the final process [
41
,
42
], which did not affect antioxidant activity of the two milks
stored for a longer time. During pasteurization, milk is exposed to heat treatment for a
time performant to kill the pathogens in milk and this time and temperature combination
may have an impact on the antioxidant characteristics of milk.
3.3. Chemical and Physical Characteristics of Cheese
Table 3shows the chemical and physical characteristics of the cheese produced using
fresh, stored, and thermized milk subjected to low and high pasteurization temperatures.
The highest pH value (p< 0.05) was found in the S-L cheese, the lowest in the T-H cheese.
The moisture content of cheese varied from 59.1% in S-H cheese to 56.9% content in
T-L cheese.
Table 3.
Chemical and physical characteristics of cheese produced using fresh (F), stored (S), and thermized (T) milk
subjected to low (L) or high (H) pasteurization.
pH Moisture % Ashes % Protein % Fat %
Fresh F-L 5.32 ab 58.5 2.79 19.3 a 22.2 a
F-H 5.17 abc 58.0 2.79 18.3 bc 20.9 ab
Stored S-L 5.41 a 57.6 2.78 19.0 ab 21.2 ab
S-H 5.12 abc 59.1 2.75 18.0 c 20.4 ab
Thermized T-L 5.34 ab 56.9 2.69 19.6 a 22.0 a
T-H 4.99 c 58.2 2.70 18.3 bc 19.4 b
Data are expressed as mean of three repetitions. For each parameter, means followed by a different letter in the column are significantly
different (p< 0.05).
In Figure 3we can observe that in cheese from stored and thermized milk the pH
values were lower when the pasteurization temperature was higher. Furthermore, the pH
trend was opposite to that of the moisture content of the cheese. Cheese made with high
pasteurized milk had a lower protein and fat content. In particular, the lowest percentage
of proteins was found in the S-H cheese (p< 0.01) and the lowest percentage of fat in the
T-H one (p< 0.05). The highest protein and fat content was found in F-L and T-L cheeses
(Table 3).
Similar results on the characteristics of cheese made with pasteurized milk at different
temperatures were obtained from previous studies [
43
,
44
]. As the pasteurization tempera-
ture increased, the moisture content increased, the fat and protein content decreased, and
the pH values also decreased. The lowering of the fat and protein content is due to the
reduction of the dry matter of the cheese following the increase in moisture content. The
reduction in pH is to be attributed to the higher moisture content (Figure 3) which leads to
a greater presence of lactose and, consequently, of lactic acid [45].
3.4. Characteristics of Cheese in Comparison with Milk
The characteristics of cheese after 14 days of storage were compared with those of milk
immediately before pasteurization and pasteurized milk with the different treatments. The
following figures show the results of some characteristics in fresh, stored, and thermized
milk immediately before pasteurization and after low and high pasteurization, and in the
corresponding cheese made from these types of milk.
Dairy 2021,2594
Figure 4shows the results of the Trp-F level. In milk immediately before pasteurization,
Trp content in F milk (810 a.u.) and S milk (787 a.u.) was significantly (p< 0.05) higher than
that of T sample (709 a.u.). In the comparison between the three modes of milk storage, the
Trp-F content was more sensitive to longer storage in presence of thermization treatment.
Dairy 2021, 2, x FOR PEER REVIEW 10 of 18
Table 3. Chemical and physical characteristics of cheese produced using fresh (F), stored (S), and thermized (T) milk
subjected to low (L) or high (H) pasteurization.
pH Moisture
%
Ashes
%
Protein
%
Fat
%
Fresh F-L 5.32 ab 58.5 2.79 19.3 a 22.2 a
F-H 5.17 abc 58.0 2.79 18.3 bc 20.9 ab
Stored S-L 5.41 a 57.6 2.78 19.0 ab 21.2 ab
S-H 5.12 abc 59.1 2.75 18.0 c 20.4 ab
Thermized T-L 5.34 ab 56.9 2.69 19.6 a 22.0 a
T-H 4.99 c 58.2 2.70 18.3 bc 19.4 b
Data are expressed as mean of three repetitions. For each parameter, means followed by a different letter in the column
are significantly different (p < 0.05).
In Figure 3 we can observe that in cheese from stored and thermized milk the pH
values were lower when the pasteurization temperature was higher. Furthermore, the pH
trend was opposite to that of the moisture content of the cheese. Cheese made with high
pasteurized milk had a lower protein and fat content. In particular, the lowest percentage
of proteins was found in the S-H cheese (p < 0.01) and the lowest percentage of fat in the
T-H one (p < 0.05). The highest protein and fat content was found in F-L and T-L cheeses
(Table 3).
Similar results on the characteristics of cheese made with pasteurized milk at
different temperatures were obtained from previous studies [43,44]. As the pasteurization
temperature increased, the moisture content increased, the fat and protein content
decreased, and the pH values also decreased. The lowering of the fat and protein content
is due to the reduction of the dry matter of the cheese following the increase in moisture
content. The reduction in pH is to be attributed to the higher moisture content (Figure 3)
which leads to a greater presence of lactose and, consequently, of lactic acid [45].
Figure 3. pH values and moisture content in cheese produced using fresh (F), stored (S), and
thermized (T) milk subjected to low (L) or high (H) pasteurization.
Figure 3.
pH values and moisture content in cheese produced using fresh (F), stored (S), and
thermized (T) milk subjected to low (L) or high (H) pasteurization.
Dairy 2021, 2, x FOR PEER REVIEW 11 of 18
3.4. Characteristics of Cheese in Comparison with Milk
The characteristics of cheese after 14 days of storage were compared with those of
milk immediately before pasteurization and pasteurized milk with the different
treatments. The following figures show the results of some characteristics in fresh, stored,
and thermized milk immediately before pasteurization and after low and high
pasteurization, and in the corresponding cheese made from these types of milk.
Figure 4 shows the results of the Trp-F level. In milk immediately before
pasteurization, Trp content in F milk (810 a.u.) and S milk (787 a.u.) was significantly (p <
0.05) higher than that of T sample (709 a.u.). In the comparison between the three modes
of milk storage, the Trp-F content was more sensitive to longer storage in presence of
thermization treatment.
Figure 4. Comparison of Trp-F (Tryptophan fluorescence) in milk before pasteurization and after
low and high pasteurization, and in the corresponding cheese made from each type of milk.
Different letters indicate significant differences (p < 0.05) within the group of milk or cheese. In the
milk group, capital letters were used for milk before pasteurization (F, S and T) while lowercase
letters for milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before pasteurization: F =
fresh milk, S = stored milk, T = thermized milk. Milk after pasteurization: L = low pasteurization (72
°C for 15 s), H = high pasteurization (77 °C for 15 s).
After pasteurization, T-L and T-H milk are confirmed to have the lowest Trp-F
content, the highest values were obtained from F-L and F-H milk (p < 0.05) as detailed
above (Table 2). Moreover, in all samples, Trp was not affected by the low and high
pasteurization. The decrease in the Trp content found in milk before and after
pasteurization (average value of low and high pasteurization) was about 17% for fresh
milk and about 21% for the other two types of milk.
In opposite to milk, significant differences were found between the two types of
pasteurization within F and T cheese groups (Figure 4). Among the groups, the value of
F-L cheese was higher (730 a.u.; p < 0.01) compared to those of S-H (611 a.u.) and T-H
cheeses (580 a.u.). Trp-F is an indicator of the whey protein denaturation [46] and therefore
of the thermal damage that occurs in heat-treated milk. In our cheese samples, it was
effective in discriminating cheese made with milk subjected to different treatments.
Figure 5 shows the results of the AMP content. The AMP-F values of the three milk
samples before pasteurization were not significant and were similar to those found after
pasteurization. The AMP level after pasteurization was highest in S-H milk (p < 0.05) and
Figure 4.
Comparison of Trp-F (Tryptophan fluorescence) in milk before pasteurization and after low
and high pasteurization, and in the corresponding cheese made from each type of milk. Different
letters indicate significant differences (p< 0.05) within the group of milk or cheese. In the milk
group, capital letters were used for milk before pasteurization (F, S and T) while lowercase letters for
milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before pasteurization: F = fresh milk,
S = stored milk
, T = thermized milk. Milk after pasteurization: L = low pasteurization (72
C for
15 s), H = high pasteurization (77 C for 15 s).
Dairy 2021,2595
After pasteurization, T-L and T-H milk are confirmed to have the lowest Trp-F content,
the highest values were obtained from F-L and F-H milk (p< 0.05) as detailed above
(
Table 2
). Moreover, in all samples, Trp was not affected by the low and high pasteurization.
The decrease in the Trp content found in milk before and after pasteurization (average
value of low and high pasteurization) was about 17% for fresh milk and about 21% for the
other two types of milk.
In opposite to milk, significant differences were found between the two types of
pasteurization within F and T cheese groups (Figure 4). Among the groups, the value of F-L
cheese was higher (730 a.u.; p< 0.01) compared to those of S-H (611 a.u.) and T-H cheeses
(580 a.u.). Trp-F is an indicator of the whey protein denaturation [
46
] and therefore of the
thermal damage that occurs in heat-treated milk. In our cheese samples, it was effective in
discriminating cheese made with milk subjected to different treatments.
Figure 5shows the results of the AMP content. The AMP-F values of the three milk
samples before pasteurization were not significant and were similar to those found after
pasteurization. The AMP level after pasteurization was highest in S-H milk (p< 0.05) and
lowest in F-L and T-L milk, as reported above (Table 2), therefore it appears to be influenced
mainly by the pasteurization temperature. In cheese the lowest values were obtained in
F-L and F-H cheeses, respectively 50.6 and 51.2 a.u., significantly different (p< 0.05) from
T-H cheese, 58.0 a.u. These results show that, by the AMP content of the cheese, one could
discriminate fresh milk cheese from cheese whose milk has received the most intense heat
treatment, i.e., thermization and high pasteurization.
Dairy 2021, 2, x FOR PEER REVIEW 12 of 18
lowest in F-L and T-L milk, as reported above (Table 2), therefore it appears to be
influenced mainly by the pasteurization temperature. In cheese the lowest values were
obtained in F-L and F-H cheeses, respectively 50.6 and 51.2 a.u, significantly different (p <
0.05) from T-H cheese, 58.0 a.u. These results show that, by the AMP content of the cheese,
one could discriminate fresh milk cheese from cheese whose milk has received the most
intense heat treatment, i.e., thermization and high pasteurization.
Figure 5. Comparison of AMP-F (advanced Maillard products fluorescence) in milk before
pasteurization and after low and high pasteurization, and in the corresponding cheese made from
each type of milk. Different letters indicate significant differences (p < 0.05) within the group of milk
or cheese. For the milk group, different capital letters were used in milk before pasteurization (F, S
and T) while lowercase letters in milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before
pasteurization: F = fresh milk, S = stored milk, T = thermized milk. Milk after pasteurization: L = low
pasteurization (72 °C for 15 s), H = high pasteurization (77 °C for 15 s).
The results about the antioxidant activity in milk and cheese are represented in
Figure 6. The milk before pasteurization is more sensitive to long storage and thermization
and the three values were significantly different from each other (F: 84.8% vs. S: 81.0% vs.
T: 78.7%; p < 0.05). After pasteurization, the antioxidant activity was higher in milk F than
in S and T milk samples and was affected by the pasteurization temperature, as detailed
above (Table 2). The decrease in antioxidant activity found in milk before and after
pasteurization (average value of low and high pasteurization) was about 5% for all three
types of milk.
Figure 5.
Comparison of AMP-F (advanced Maillard products fluorescence) in milk before pasteur-
ization and after low and high pasteurization, and in the corresponding cheese made from each type
of milk. Different letters indicate significant differences (p< 0.05) within the group of milk or cheese.
For the milk group, different capital letters were used in milk before pasteurization (F, S and T) while
lowercase letters in milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before pasteurization:
F = fresh milk, S = stored milk, T = thermized milk. Milk after pasteurization:
L = low pasteurization
(72 C for 15 s), H = high pasteurization (77 C for 15 s).
The results about the antioxidant activity in milk and cheese are represented in
Figure 6
. The milk before pasteurization is more sensitive to long storage and thermization
and the three values were significantly different from each other (F: 84.8% vs. S: 81.0%
vs. T: 78.7%; p< 0.05). After pasteurization, the antioxidant activity was higher in milk
F than in S and T milk samples and was affected by the pasteurization temperature, as
Dairy 2021,2596
detailed above (Table 2). The decrease in antioxidant activity found in milk before and after
pasteurization (average value of low and high pasteurization) was about 5% for all three
types of milk.
Dairy 2021, 2, x FOR PEER REVIEW 13 of 18
Figure 6. Comparison of antioxidant activity in milk before pasteurization and after low and high
pasteurization, and in the corresponding cheese made from each type of milk. Different letters
indicate significant differences (p < 0.05) within the group of milk or cheese. For the milk group,
different capital letters were used in milk before pasteurization (F, S, and T) while lowercase letters
in milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before pasteurization: F = fresh milk,
S = stored milk, T = thermized milk. Milk after pasteurization: L = low pasteurization (72 °C for 15
s), H = high pasteurization (77 °C for 15 s).
It can also be observed that the reduction in antioxidant activity due to the cheese-
making process is more marked than that due to the pasteurization treatment. No
differences were found between the two pasteurization treatments within all three groups
of cheese, while the differences between the three groups were significant (p < 0.05). Fresh
milk cheeses had the greatest antioxidant activity, thermized milk cheeses had the worst
(64.9 and 65.0 vs. 55.0 and 53.1%). From these results, it seems that it is possible to
discriminate between the three cheese groups according to their antioxidant activity.
According to Lucas et al. [47], total antioxidant capacity varied mainly with the
cheese-making process. During the cheese-making, a variable quantity of some
compounds with antioxidant activity, such as β-carotene, vitamin A and vitamin E, is lost.
This loss could be due to oxidative degradation by atmospheric oxygen and light and
could be accelerated by increasing temperature and acidity [48,49]. In addition, some
compounds are lost in the whey [47]. The DPPH values found in buffalo Ricotta cheese at
1 day (64.6%) and at 21 days (66.3%) of storage [50] were similar in comparison to those
found in fresh milk cheese.
Furthermore, the reduction in antioxidant activity in S and T cheeses does not
negatively affect the oxidation of fat. In fact, the MDA content is often used as a marker
of oxidative damage in cheese, varied from 0.22 to 0.25 nmol g
1
and are not significantly
different (p = 0.4957) in the three groups. These values were lower than average values
found in buffalo Mozzarella cheese (0.36 nmol g
1
) [23] and in buffalo Ricotta cheese (1.51
nmol g
1
) [50]. The MDA content of our cheese samples was also lower than the data for
other dairy products [51] and well below the threshold indicated by some authors for
other foods [52].
4. Discussion
Oxidative processes are important factors regarding freshness and suitability of the
milk for further processing and the oxidative stability of milk is the result of a delicate
Figure 6.
Comparison of antioxidant activity in milk before pasteurization and after low and high
pasteurization, and in the corresponding cheese made from each type of milk. Different letters
indicate significant differences (p< 0.05) within the group of milk or cheese. For the milk group,
different capital letters were used in milk before pasteurization (F, S, and T) while lowercase letters in
milk after pasteurization (F-L, F-H, S-L, S-H, T-L, T-H). Milk before pasteurization: F = fresh milk,
S = stored milk
, T = thermized milk. Milk after pasteurization: L = low pasteurization (72
C for
15 s), H = high pasteurization (77 C for 15 s).
It can also be observed that the reduction in antioxidant activity due to the cheese-
making process is more marked than that due to the pasteurization treatment. No differ-
ences were found between the two pasteurization treatments within all three groups of
cheese, while the differences between the three groups were significant (p< 0.05). Fresh
milk cheeses had the greatest antioxidant activity, thermized milk cheeses had the worst
(64.9 and 65.0 vs. 55.0 and 53.1%). From these results, it seems that it is possible to
discriminate between the three cheese groups according to their antioxidant activity.
According to Lucas et al. [
47
], total antioxidant capacity varied mainly with the cheese-
making process. During the cheese-making, a variable quantity of some compounds with
antioxidant activity, such as
β
-carotene, vitamin A and vitamin E, is lost. This loss could be
due to oxidative degradation by atmospheric oxygen and light and could be accelerated by
increasing temperature and acidity [
48
,
49
]. In addition, some compounds are lost in the
whey [
47
]. The DPPH values found in buffalo Ricotta cheese at 1 day (64.6%) and at 21 days
(66.3%) of storage [50] were similar in comparison to those found in fresh milk cheese.
Furthermore, the reduction in antioxidant activity in S and T cheeses does not neg-
atively affect the oxidation of fat. In fact, the MDA content is often used as a marker of
oxidative damage in cheese, varied from 0.22 to 0.25 nmol g
1
and are not significantly
different (p= 0.4957) in the three groups. These values were lower than average values
found in buffalo Mozzarella cheese (0.36 nmol g
1
) [
23
] and in buffalo Ricotta cheese
(1.51 nmol g
1
) [
50
]. The MDA content of our cheese samples was also lower than the data
for other dairy products [
51
] and well below the threshold indicated by some authors for
other foods [52].
Dairy 2021,2597
4. Discussion
Oxidative processes are important factors regarding freshness and suitability of the
milk for further processing and the oxidative stability of milk is the result of a delicate
balance between the overall anti- and pro-oxidative processes. The imbalance of this
equilibrium is an advantage of the prooxidative activity which induces a deterioration
process that can decrease the technological quality of the milk and cause a lower sensory
quality of the dairy products [10,15,53].
Our results showed that oxidation takes place throughout storage of milk with dif-
ferences mainly between fresh milk and the two kinds of stored milk with the higher
effect on thermized milk. There are a few investigations about the changes in antioxidant
content during storage of milk [
54
]. Kant et al. [
54
] reported that up to 3 days of storage
there are no significant changes in the antioxidant activity measured by DPPH assay in
both raw and pasteurized milk while at 6 days of storage there is an increase in the level
of oxidation. DPPH assay after 6 days of storage period indicated significant decline in
antioxidant activity of both raw and heat-treated milk. The decrease in antioxidant activity
is mostly depending on the degradation of antioxidant components, such as vitamins and
enzymes. A prolonged period of storage could affect a decrease of vitamin C, A, and E
content in milk.
Ascorbic acid (vitamin C) is the major water-soluble antioxidant in milk and can act
as a strong free radical scavenger, but it is present in low concentration in milk, and it is
very sensitive to treatment and storage period that considerably affect its content in milk.
Vitamin C decreased in raw milk at 3 days storage and moreover, its content decreased in
pasteurized milk compared to raw milk [
54
]. Loss of vitamin C in pasteurization of cow
milk was 40%. With more intense heat treatments loss of vitamin C become even greater,
at 82% [54].
Vitamin A and tocopherol up to 3 days of storage was maintained, while they de-
creased at 6 days. However, their concentration in pasteurized milk was not significantly
different from their content in raw milk while when the milk is boiled their content de-
creased compared to raw milk [54].
Studies on human milk reported that glutathione peroxidase activity decreased while
MDA increased in refrigerated milk even after 24 h confirm that storage of mother’s
milk results in a decrease of its antioxidant capacity associated with lipid peroxidation
reactions [
55
]. However, contrasting data showed that refrigerated storage at 4
C did not
affect the oxidative status of human milk with no evidence of lipid peroxidation [56,57].
So, storage period and conditions could affect the antioxidant properties of milk, which
could be related to the oxidative stability of pasteurized milk and dairy products. The
same authors reported that the addition of ascorbic acid and tocopherol in milk enhanced
the flavor and photooxidative stability during prolonged storage increasing its antioxidant
activity as compared to non-supplemented milk [58].
Our results also show that the pasteurization treatment induced an evident protein
denaturation which is more pronounced in thermized milk. In the same milk, no effect
of the pasteurization temperature was found. Both results were most likely caused by
the double heat treatment. The reduction of the antioxidant activity in the pasteurized
milk compared to the starting milk was moderate. The effect of pasteurization on the
antioxidant activity was less evident in F-milk which was influenced by the pasteurization
temperature as was S-milk. Additionally, in this case, the milk subjected to thermization
was not sensitive to the pasteurization temperature.
There are many studies on the effects of pasteurization and other heat treatments
on antioxidants content in milk and the results showed that loss of natural antioxidants,
protein unfolding, and formation of oxidation products or novel antioxidants greatly
depends on the processing conditions [
59
]. In particular, heat treatment of milk can induce
both the loss of natural antioxidants and the formation of novel oxidative molecules with
a consequent decrease in the antioxidant activity of milk. However, milk antioxidant
activity may increase due to protein unfolding and exposure of thiol groups, potentially
Dairy 2021,2598
acting as hydrogen donors. Most of the consequences of milk heating are related to
the development of the Maillard reaction but under the processing conditions usually
carried out at industrial level to produce pasteurized milk, only the early phase of the
Maillard reaction takes place [
8
]. These results showed a potential depletion in the overall
antioxidant properties of milk depending on the time and temperature combinations of
heat treatments and should be taken into consideration in the industrial processing of milk
and milk products [16].
Although the effect of heat treatment on antioxidant activity and content of vitamins
in milk was extensively studied, little information is available regarding the transition of
antioxidant capacity during cheese-making with respect to alteration in vitamin A, E, and
C [
7
,
40
]. Total antioxidant capacity was strongly correlated with vitamins content in milk,
but chemical composition of cheese is considerably different from parent milk and changes
in antioxidant components of cheese occurred during processes and ripening.
Lipophilic milk antioxidants are characterized by high levels of thermal stability,
and they remain active in all dairy products regardless of the applied thermal processing
method [
9
]. Results of an earlier investigation on migration of carotenoids from milk to
cheese have shown that the content of retinol in cheese is influenced by heat treatment
during manufacturing [
60
]. According to other authors [
47
,
48
], during the cheese-making
process, an important amount of the lipophilic vitamins originally present in milk fat was
lost: for vitamin A on average one-third, while the loss of vitamin E was around two-thirds.
Even the casein fractions are characterized by high levels of antioxidant activity [
9
].
Zulueta et al. [
18
] demonstrated that total antioxidant status of milk subjected to pasteur-
ization and other processes was determined mostly by caseins which are characterized by
greater thermal stability. Moreover, it is noted that production of bioactive peptides due to
heat treatments and ripening can increase the antioxidant capacity of cheese [61].
Milk quality influences the quality of processed dairy products and offers a perspective
on the merit of investing in quality. Dairy processors may incentive to use high-quality raw
milk to improve processed product quality and manufacturing efficiencies. Using high-
quality milk could increase cheese yields and improve texture and flavor characteristics in
cheese and other products [54].
5. Conclusions
Antioxidant activity decreases from the starting milk to the milk after pasteurization
to the final cheese. This reduction could be attributed to some factors (oxygen, light,
temperature, acidity, whey loss) that affect the main compounds responsible for this
activity, such as vitamins and enzymes. However, despite the losses that occurred during
the milk treatments and in the cheese-making process, the residual antioxidant activity of
the cheese was found to protect the fat from oxidation. Under our experimental conditions,
the longer storage time of the milk, especially if preceded by heat pre-treatment, had
a significant effect on the quality of milk and cheese. The greater antioxidant stability
of cheeses made from fresh milk, closely linked to the greater presence of antioxidant
compounds, is a valid reason to favors the production and consumption of these products.
Author Contributions:
Conceptualization, C.T.; methodology, C.T., S.R., G.P. and S.D.G.; formal
analysis, S.R., G.P. and S.D.G.; data curation, S.R., G.P. and S.D.G.; writing—original draft preparation,
C.T., S.R. and G.P.; writing—review and editing, C.T. and S.R.; project administration, C.T., S.C. and
L.B.; funding acquisition, C.T., S.C. and L.B. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the Italian Ministry of Agriculture (MiPAAF) within the
project CALAFRE “Caseificazione con latte fresco—Cheese production with fresh milk” (Ministry of
Agriculture D.M. 20591/7100/2017).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Dairy 2021,2599
Acknowledgments:
We would like to thank Associazione Regionale Allevatori della Basilicata
(Potenza) for their contribution to the chemical analysis of milk and cheese, the organization of dairy
cow breeders Platano Melandro Latte for the supply of cow’s milk for the production of the cheese
samples and Giuseppe Morone, Giovanni Masino and Antonio Capece for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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