sustainability

Article
Cold Plasma Technology: A Sustainable Approach to Milk
Preservation by Reducing Pathogens and Enhancing
Oxidative Stability
Hayam M. Abbas 1 , Ebtehal A. Altamim 2 , Mohamed Salama 1,3 , Mohamed T. Fouad 1 and Hamdy A. Zahran 4, *

1 Dairy Department, Food Industries and Nutrition Research Institute, National Research Centre, Dokki,
Cairo 12622, Egypt; [email protected] (H.M.A.); [email protected] (M.S.);
[email protected] (M.T.F.)
2 Department of Physical Sport Sciences, College of Sport Sciences and Physical Activity, Princess Nourah Bint
Abdulrahman University, Airport Road, Riyadh 84428, Saudi Arabia; [email protected]
3 Hubei Hongshan Laboratory, National Research and Development Centre for Egg Processing, College of Food
Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
4 Fats and Oils Department, Food Industries and Nutrition Research Institute, National Research Centre, Dokki,
Cairo 12622, Egypt
* Correspondence: [email protected]; Tel.: +20-101-126-2510

Abstract: Pathogenic microorganisms and lipid oxidation are critical challenges in the dairy industry,
influencing both food safety and quality. This study explores the potential of cold plasma (CP) tech-
nology as a sustainable alternative for milk preservation compared to conventional pasteurization.
CP treatment utilizes ionized gas to generate reactive species, which effectively disrupt microbial
cell membranes and inactivate pathogens, thereby sterilizing the milk. We assessed raw, pasteurized,
and cold plasma-treated milk samples, focusing on microbial growth, lipid oxidation, and oxidative
stability. Our findings indicate that CP treatment significantly reduced microbial contamination,
effectively inhibiting the growth of pathogenic bacteria and delaying acidity development in milk.
In contrast, pasteurized milk exhibited a notable increase in peroxide values, indicating lipid de-
Citation: Abbas, H.M.; Altamim, E.A.; terioration. Furthermore, the oxidative stability of cold plasma-treated milk was enhanced, with
Salama, M.; Fouad, M.T.; Zahran, H.A. an induction period extending from approximately five to seven hours, demonstrating its superior
Cold Plasma Technology: A
resistance to oxidation. In conclusion, CP has emerged as a promising eco-friendly technology for
Sustainable Approach to Milk
prolonging the shelf life of milk by mitigating microbial growth and lipid oxidation. This method
Preservation by Reducing Pathogens
not only aligns with sustainability goals by reducing the need for chemical preservatives but also en-
and Enhancing Oxidative Stability.
hances the overall quality of milk products. Future research should focus on large-scale applications
Sustainability 2024, 16, 8754. https://
doi.org/10.3390/su16208754
and the impacts of CP on other essential milk components, particularly fat-soluble vitamins, to fully
understand its sustainability benefits in the dairy sector.
Academic Editors: Hugo Miguel
Lisboa Oliveira and Ana
Keywords: non-thermal treatment; cold plasma; milk preservation; pathogenic bacteria; oxidative
Paula Trindade
stability
Received: 22 August 2024
Revised: 4 October 2024
Accepted: 7 October 2024
Published: 10 October 2024 1. Introduction
The food industry faces the challenge of adapting to the shifting consumer preference
towards minimally processed alternatives, driven by a desire for healthier options and a
Copyright: © 2024 by the authors.
change in taste for thermally processed food. Conventional methods, such as sterilization
Licensee MDPI, Basel, Switzerland. or pasteurization, can have detrimental effects on the taste and nutritional quality of
This article is an open access article food. Hence, in recent decades, many non-thermal sterilization technologies, e.g., high-
distributed under the terms and pressure homogenization, high-pressure carbon dioxide, pulsed electric field, high-intensity
conditions of the Creative Commons ultrasound, and cold plasma have been developed to prevent the growth of microorganisms
Attribution (CC BY) license (https:// and preserve the nutritional quality of food [1,2].
creativecommons.org/licenses/by/ Cold plasma (CP) technology represents a significant advancement in sustainable
4.0/). food processing, particularly in milk preservation. Unlike traditional thermal methods,

Sustainability 2024, 16, 8754. https://doi.org/10.3390/su16208754 https://www.mdpi.com/journal/sustainability

Sustainability 2024, 16, 8754 2 of 17

which can degrade nutritional quality and alter sensory properties, cold plasma operates at
ambient temperatures, thus preserving the integrity of milk while effectively inactivating
pathogens such as Escherichia coli and Staphylococcus aureus without the use of chemicals [2].
This non-thermal approach not only enhances food safety but also aligns with sustainability
goals by minimizing energy consumption and waste production. For instance, studies
indicate that cold plasma technique can achieve a decrease in microbial load by 3–4 log
units within minutes, significantly outperforming conventional methods that often require
longer processing times and higher temperatures [3].
Moreover, CP technology contributes to economic sustainability by extending shelf life
and reducing spoilage, which is critical given the high perishability of dairy products. The
circular economy benefits from CP as it utilizes renewable resources primarily electricity
and produces non-toxic by-products, thereby supporting environmentally friendly practices
in food production [4,5]. Quantitatively, CP has been shown to reduce energy usage by up
to 50% compared to traditional pasteurization methods while maintaining product quality.
This positions CP not only as an innovative solution for enhancing food safety but also
as a pivotal technology in the pursuit of sustainable food systems that respect economic,
environmental, and socio-cultural dimensions. Yepez et al. [6] stated that CP is considered
a sustainable technique, including decontamination, non-thermal food processing, and non-
conventional technologies. Furthermore, CP is essential in the circular economy concept
because it can utilize renewable resources to create safe and sustainable food products,
using electricity and non-toxic gases as processing technology. It also plays a significant role
in the circular economy concept by utilizing renewable resources to create environmentally
friendly and sustainable food products [2].
In the dairy sector, many products are manufactured using heat as preserved meth-
ods to make the product safe, such as pasteurization and high-temperature-short-time
(HTST) pasteurization. These techniques affect the nutritional value of the product as
well as the sensory acceptability [7]. In this context, Segat et al. [8] studied the effect of
CP on the characteristics of treated whey protein isolate. Meanwhile, Ribeiro et al. [9]
evaluated the impact of CP on the physicochemical, functional, and sensorial properties
of whey beverages at different times (0, 5, 10, or 15 min). On the other hand, CP has been
shown to have minimal impacts on various attributes, including sensory characteristics
(such as taste and aroma), nutritional value (preserving vitamins and proteins), and phys-
ical properties (maintaining texture and appearance) of dairy products Nikmaram and
Keener [10]. Nikmaram and Keener [10] mentioned that thermal processes such as HTST,
which are utilized in the dairy industry, had negative effects on milk quality, which may
include vitamin loss, protein denaturation, non-enzymatic browning, and changes in flavor.
However, this recent technique can pasteurize milk while maintaining the milk quality.
Additionally, a study by Akarca et al. [11] demonstrated that CP significantly enhanced the
physicochemical characteristics of Kashar cheese, achieving a 3–4 log reduction in mold
counts of Aspergillus flavus and Penicillium chrysogenum. According to their statement, CP
offers numerous benefits in terms of reducing microorganisms and their enzymes in raw
milk and dairy products, while also causing minimal impact on the quality of the milk and
its products, including the sensory characteristics (such as taste and aroma), nutritional
value (preserving vitamins and proteins), and physical properties (maintaining texture and
appearance) of dairy products [2]. CP can alter the function of food ingredients in order
to attain specific properties in particular food items. In addition, plasma-induced mild
oxidation can enhance the functional properties of proteins by promoting favorable changes
in their secondary and tertiary structures. These modifications can increase the functional
properties of protein such as solubility, foaming, and emulsifying activity and stability,
which are crucial for various food applications. Furthermore, this process may lead to pro-
teolysis, generating new peptides that can improve flavor and nutritional profiles, thereby
expanding their technological capabilities in the food industry [12,13]. Furthermore, Wang
et al. [14] reported that sheep milk processed by the CP technique caused a reduction in pH,
Sustainability 2024, 16, 8754 3 of 17

which is associated with a rise in acidity caused by hydrogen ions, forming acids. Moreover,
the exposure to CP for 5 min was comparable to pasteurized sheep milk.
The changes in fatty acid profile reveal the intricate relationship between treatments
and milk fat composition. Notably, polyunsaturated fatty acid (PUFA) content rises in
the case of CP technique utilization, demanding a deeper understanding of causative
mechanisms. Oxidative processes, reactive species, and other treatment-related factors
likely influence PUFA formation or degradation, warranting thorough investigation [15,16].
Other studies reported that when the CP treatment was extended to 10–20 min, it led to a bet-
ter fatty acid profile with increased PUFA and monounsaturated fatty acids (MUFA) along
with a decrease in saturated fatty acids (SFAs) compared to pasteurized milk drinks [17,18].
Consequently, this work aimed to explore the application of cold plasma technology as a
sustainable preservation method for buffalo milk. Moreover, evaluate the effects of CP on
key quality parameters such as acidity, pH levels, and oxidation, while also quantifying
total bacterial counts, coliforms, molds, and yeasts. This study assessed the efficacy of CP
in inhibiting the growth of specific pathogenic bacteria, thereby improving food safety and
prolonging the shelf life without compromising the nutritional quality of the milk.

2. Materials and Methods

Raw Egyptian buffalo milk was obtained from a private farm in the Giza governorate.
The gross composition of milk samples was performed according to A.O.A.C. [19], the data
presented in Table 1.

Table 1. Gross chemical analysis of raw buffalo milk samples.

Gross Composition Content (%)

Total solids 17.33 ± 0.3
Fat 7.19 ± 0.5
Protein 3.84 ± 0.4
Lactose 5.50 ± 0.4
Ash 0.87 ± 0.1
All data represent the means of triplicates ± standard deviation (SD).

2.1. Sample Treatments

This study involved dividing the buffalo milk samples into three distinct portions: one
portion was flash pasteurized, another underwent cold plasma treatment, and the third
served as raw milk (control). Each treatment was replicated three times to ensure reliability,
meaning that three separate batches of raw milk were pasteurized, and three batches
remained untreated as raw milk. All samples were stored at refrigerated temperatures
(5 ◦ C ± 2 ◦ C) for a duration of 7 days, during which various parameters were monitored.

2.1.1. Pasteurization of Milk

Raw milk samples underwent pasteurization treatment, where they were flash heated
to 85 ◦ C for 1 min, cooled immediately, and then stored in the refrigerator (5 ◦ C ± 2 ◦ C) for
7 days.

2.1.2. Treatments of Milk by Cold Plasma Dose

For the cold plasma treatment, 50 mL portions of raw milk were placed in sterilized
Petri dishes (50 mm diameter) and treated using a cold plasma apparatus (Suzhou Opus
Plasma Technol. Co., Suzhou, China). The treatments were conducted at a power setting of
70 kV for a duration of 15 min, following the method reported by Sedik et al. [20]. After
treatment, the cold plasma-treated milk was stored in the refrigerator for 7 days until
subsequent analyses were performed. Each treatment condition had three replicates to
ensure consistency in results. The setup for cold plasma generation is illustrated in Figure 1.
 Plasma Technol. Co., Suzhou, China). The treatments were conducted at a power s
of 70 kV for a duration of 15 min, following the method reported by Sedik et al. [20].
treatment, the cold plasma-treated milk was stored in the refrigerator for 7 days unt
Sustainability 2024, 16, 8754 sequent analyses were performed. Each treatment condition had three 4replicates
of 17 to e
consistency in results. The setup for cold plasma generation is illustrated in Figure

Figure 1. Cold plasma

Figure generation layout.
1. Cold plasma generation layout.
2.2. Methods
2.2. Methods
2.2.1. Chemical Analysis
2.2.1. Chemical Analysis
The gross chemical analyses, acidity, and peroxide value were performed according to
A.O.A.C. [19]. The gross chemical analyses, acidity, and peroxide value were performed acco
to A.O.A.C. [19].
2.2.2. The Microbiological Examinations for Milk Samples
2.2.2. Thewere
Ten-fold dilutions Microbiological
prepared and Examinations for Milk
inoculated onto Samples
plates of selective media. The
aerobic colony count Ten-fold
(ACC), dilutions
yeasts, and were prepared
molds and inoculated
were performed ontotoplates
according of selective
A.O.A.C. [19]. medi
For ACC and aerobic
using plate
colonycount
count agar (oxoid),
(ACC), plates
yeasts, andwere
moldsincubated
were performed ◦
at 30 ± 1according
C for to A.O
72 ± 2 h. Using acidified
[19]. For ACC potato
and dextrose
using plate agar (oxoid)
count agaras the medium,
(oxoid), yeasts
plates were and molds
incubated at 30 ± 1 °C
were grown on ± 2plates thatacidified
h. Using were incubated at 25 ◦agar
potato dextrose C for(oxoid)
5 d. The coliform
as the medium, group
yeasts wasand molds
identified using violet-red bile that
agarwere
(VRBA) (oxoid), ◦ C for
grown on plates incubated at with
25 °Cplates incubated
for 5 d. at 37 group
The coliform was iden
24 h. The identification of Enterobacteriaceae
using violet-red bile agar (VRBA) was(oxoid),
conducted
withusing
platesviolet red bile
incubated glucose
at 37 °C for 24 hour
(VRBG) agar (oxoid), with plates
identification incubated at 37 ◦ C
of Enterobacteriaceae forconducted
was 24 h, following
usingtheviolet
methodology
red bile ofglucose (V
Wai et al. [21]. agar (oxoid), with plates incubated at 37 °C for 24 hours, following the methodolo
In order toWaiexamine Listeria monocytogenes, 25 g of each sample was combined with
et al. [21].
225 mL of Listeria selective
In orderenrichment
to examinemediumListeria in 500 mL flasks,25and
monocytogenes, g oftheeach
mixture
samplewaswasthencombined
incubated at 30225 ◦ C for 7 d. Following the preparation of serial dilutions, they were plated
mL of Listeria selective enrichment medium in 500 mL flasks, and the mixtur
onto Oxford agar thenbases supplemented
incubated at 30 °Cwith
for 7Listeria supplement
d. Following and incubated
the preparation at 35 ◦dilutions,
of serial C for they
48 h [22]. plated onto Oxford agar bases supplemented with Listeria supplement and incuba
Salmonella35typhimurium was examined by combining 25 g of sample and 225 mL of
°C for 48 h [22].
sterile buffer peptone water, then incubating at 35 ◦ C for 24 h. In brief, 1 mL mixture was
Salmonella typhimurium was examined by combining 25 g of sample and 225
pipetted to 10 sterile
mL selenite ◦ for 72 h. Subsequently,
buffer cystine
peptonebrothwater,and incubated
then at 35
incubating at 35C°C for 24 h. In brief, 1 mL mixtur
serial dilutionspipetted
were prepared, and plates were placed on Salmonella
to 10 mL selenite cystine broth and incubated andatShigella agar72and
35 °C for h. Subsequ
◦ C for 24 h [23].
incubated at 35serial dilutions were prepared, and plates were placed on Salmonella and Shigella aga
Bacillus cereus was identified
incubated at 35 °C forthrough
24 h [23].surface plating onto the Bacillus cereus agar
medium, which was enhanced with egg yolk and polymyxin-B. While, Staphylococcus
Bacillus cereus was identified through surface plating onto the Bacillus cereus aga
aureus was identified using Baird–Parker medium (oxoid) with egg yolk and potassium
dium, which was enhanced with egg yolk and polymyxin-B. While, Staphylococcus
tellurite following Polo et al. [24] method. Plates were left to incubate at 37 ◦ C for 48 h.
Additionally, E. coli was placed onto the oxoid tryptone bile–glucuronic medium using the
surface plating method, followed by an incubation period of 24 h at 44 ◦ C [23]. Figure 2
presents an overall flowchart of the experiment.
 was identified using Baird–Parker medium (oxoid) with egg yolk and potassium tellurite
following Polo et al. [24] method. Plates were left to incubate at 37 °C for 48 h. Additionally,
E. coli was placed onto the oxoid tryptone bile–glucuronic medium using the surface plat-
Sustainability 2024, 16, 8754 ing method, followed by an incubation period of 24 h at 44 °C [23]. Figure 2 presents 5 of an
17
overall flowchart of the experiment.

Figure2.2. Overall
Figure Overall flowchart
flowchart for
for the
theexperiment
experiment of
ofraw
rawmilk
milk(RM),
(RM),pasteurized
pasteurizedmilk
milk(PM),
(PM),and
andcold
cold
plasma-treatedmilk
plasma-treated milk(CPM)
(CPM)samples.
samples.

2.2.3.
2.2.3. Pathogenic
Pathogenic Bacteria
Bacteria Examination
Examinationfor forMilk
MilkSamples
Samples
Five different types of bacteria pathogens,
Five different types of bacteria pathogens, namely namely E. E.
colicoli
(ATCC
(ATCC 25922), Salmonella
25922), ty-
Salmonella
phimurium (ATCC 14028), Listeria monocytogenes (ATCC 7644), Bacillus
typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 7644), Bacillus cereus (ATCCcereus (ATCC 33018),
and Staphylococcus
33018), aureus (ATCC
and Staphylococcus aureus 20231), were utilized
(ATCC 20231), in the challenge
were utilized test after
in the challenge testbeing
after
activated in tryptone soy broth at 37 ◦ C for 24 h. The various methods used on milk
being activated in tryptone soy broth at 37 °C for 24 h. The various methods used on milk
samples
samplesmentioned
mentionedearlier
earlierwere
werecategorized
categorizedinto
intothree
threegroups;
groups;one onegroup
groupwaswasinoculated
inoculated
with E. coli, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus,
with E. coli, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and and Staphylococcus
Staphylococ-
aureus individually
cus aureus andand
individually served as the
served control.
as the Another
control. Another group
groupwas wasinoculated
inoculatedwith
witheach
each
pathogenic bacteria and then pasteurized at 85 ◦ C for 1 min. The third group was given
pathogenic bacteria and then pasteurized at 85 °C for 1 min. The third group was given
the pathogenic bacteria and then exposed to a dose of cold plasma. Post-inoculation, the
the pathogenic bacteria and then exposed to a dose of cold plasma. Post-inoculation, the
pathogen content in the milk samples varied between 4.08 and 4.86 log CFU/mL. Subse-
pathogen content in the milk samples varied between 4.08 and 4.86 log CFU/mL. Subse-
quent to the inoculation, all milk samples (15 samples) were refrigerated for observation
quent to the inoculation, all milk samples (15 samples) were refrigerated for observation
for a period of 7 days.
for a period of 7 days.
2.2.4. Induction Period and Oxidative Stability Index (OSI)
2.2.4. Induction Period and Oxidative Stability Index (OSI)
Fat from milk samples was extracted by an official method to determine the induction
period [19]. Sample 3 ± 0.1 g was placed in the reaction vessels. Using the Rancimat instru-
ment (Metrohm 892 Professional Rancimat, 9100 Herisau Switzerland), the temperature
and airflow rate were set at 120 ◦ C and 20 L/h.

2.2.5. Identification of Fatty Acids by Gas Chromatography (GC-FID)

The composition of fatty acids was examined using a modified approach based on
the method outlined by Zahran and Tawfeuk [25]. This procedure involved the alteration
Sustainability 2024, 16, 8754 6 of 17

of fatty chains to fatty acid methyl esters (FAMEs) through trans-methylation. An HP

6890 plus gas chromatography system fitted with a SupelcoTM SP-2380 capillary column
was
Sustainability 2024, 16, xthen used
FOR PEER to separate the FAMEs. A flame ionization detector (FID) was used7 for
REVIEW of 17

the detection process. The column temperature started at 140 ◦ C and rose gradually at a
pace of 4 ◦ C per minute to reach 240 ◦ C. At this point, it was maintained for ten min. The
carrier gas, helium, was employed at a7flow rate9.6 of±1.2
0.02mL/min.
A,a 6.4A± 0.02
splitA,binjector4.9was
± 0.0used
A,c

1 6.2 ± 0.03 A,a 3.6 ± 0.01 A,c 3.7

to introduce a 1 µL sample volume that had been dissolved in n-hexane at a 100:1 splitting ± 0.02 A,b

Mold and Yeast 3 5.0 ± 0.0 B,a 3.6 ± 0.0 A,b

ratio. To identify the FAMEs, their retention times were compared to those of established2.3 ± 0.01 B,c

7 4.6 ± 0.01 C,a 1.3 ± 0.03 B,b

FAME standards. The relative proportion of the entire peak area was used to indicate the <1 ± 0.04 C,c

fatty acid content. 1 4.5 ± 0.01 C,a 1.3 ± 0.01 C,b 1.2 ± 0.02 C,c
Coliform bacterial
3 4.8 ± 0.01 B,a 1.6 ± 0.0 B,b 1.4 ± 0.01 B,c
Group
7
2.2.6. Calculated Oxidizability (COX) Value 4.9 ± 0.05 A,a 1.7 ± 0.0 A,b 1.5 ± 0.01 A,c
1 4.7 ± 0.01 C,a <1 ± 0.01 C,b <1 ± 0.2 B,b
The proportion of unsaturated C18 fatty acids wasA,a used to determine the estimated
Enterobacteriaceae 3 5.1 ± 0.05 1.3 ± 0.02 A,b 1.2 ± 0.02 A,c
oxidizability (COX) value using the technique outlined B,aby Fatemi andB,cHammond [26]A,bas
7 4.9 ± 0.0 1.0 ± 0.0 1.1 ± 0.06
follows: Results are presented as mean ± standard deviation (SD). The distinct capital letters (A, B, C) within
the same column
[1(18 : 1%) + 10.3(18 : 2%) + 21.6(18 : 3%)]
COX = for each test indicate significant variations across the storage durations of 1, 3, and
100the same row indicate statistically significant differ-
7 days. Distinct lowercase letters (a, b, c) within
ences across the three treatments (p < 0.05).
2.3. Statistical Analysis
Furthermore, Abdelnasser Ahmed et al. [28] discovered that the present research as-
Using the Statistical Analysis System (SAS-STAT, Ver. 9.2), Duncan’s test and analysis
sessed the milk samplesʹ microbiological quality and safety. The TBC for raw milk sam-
of variance (ANOVA) were used to perform statistical analysis on the collected data (three
ples that were randomly collected varied from 1.2 × 104 to 5 × 108 cfu/ mL with a mean
repetitions). The statistical
value significance
of 5.46 × 10 7 ± 1.57 × 108 was established
cfu/ mL. usingexceeded
These findings a probability
those ofof p < 0.05and
El-Diasty [27].
El-
Kaseh [29], whereas El Zubeir and Ahmed [30] reported lower results, with mean levels
3. Results andofDiscussion
5.63 × 109 CFU/mL and 7.32 × 107 ± 1.12 × 107, respectively. We believe that inadequate
3.1. Microbiological Examination
sanitary of Milk
practices during Sample
milking, collection, and transportation are significant contribu-
tors to elevated bacterial loads in milk. Furthermore, inadequate cooling equipment
Table 2 and Figure 3 exhibited the changes in viable counts log/CFU per mL of the
throughout the phases of milk production, handling, and distribution, an inadequate un-
total bacterial,derstanding
mold andofyeast, and coliform as well as Enterobacteriaceae count during
sanitary milk processing and inappropriate handling practices in super-
5 ◦ C ±may
milk storage atmarkets 2 ◦C allfor 7 d. Generally,
contribute to the highthe totalcounts.
bacteria bacterialThesecount,
factorscoliform
collectivelybacterial
compro-
count, Enterobacteriaceae,
mise milk safety andand mold andleading
quality, yeast counts were
to potential significantly
health higher (p
risks for consumers. < 0.05)
Ledenbach
in control rawandmilk when [31]
Marshall compared with
support this theby
point pasteurized
highlighting and coldhygiene
that poor plasma-treated
during milk milk
han-
dling andcold
samples. Meanwhile, storage can lead to unacceptable
plasma-treated milk sampleslevelshadof microorganisms,
the lowest values including patho-
compared
genic bacteria
to other treatments, like Escherichia
indicating a strongcoli and Enterobacter
effect in eliminatingspecies, which pose health risks
microorganisms. and can
Moreover,
affect the sensory qualities of dairy products. Moreover, we observed that our results
notable variations (p < 0.05) were observed in the total bacterial count, coliform count,
closely aligned with those of Hasan et al. [32] who reported that the TCC (MPN/mL) of
Enterobacteriaceae,
market andmilkmold andtested
samples yeastin count for each
the Egyptian treatment
governorates during
of Cairo and storage
Giza varied in from
the
refrigerator for7.51,× 3,
102and
to 2.17×days.
107 withWhere theyofincreased
an average 1.8 × 106 ± 4by
× 10increasing
5 and from 2.1the storage
× 10 2 to 2.3 ×period
106 with
(Table 2). an average of 2 × 105 ± 7.7 × 104, in the same order.

Figure 3. Microbiological examination of raw (RM), pasteurized (PM), and cold plasma-treated milk
(CPM) samples; (A) total bacterial count; (B) mold and yeast.
Sustainability 2024, 16, 8754 7 of 17

Table 2. Microbiological examination of raw, pasteurized, and cold plasma-treated milk samples
during 7 days of storage in the refrigerator.

log/CFU per mL
Microbiological Examination Storage Period (Days) Pasteurized Cold Plasma
Raw Milk
Milk Milk
1 8.4 ± 0.01 C,a 6.1 ± 0.00 C,b 4.2 ± 0.02 C,c
Total bacterial count 3 8.8 ± 0.01 B,a 6.2 ± 0.0 B,b 4.6 ± 0.01 B,c
7 9.6 ± 0.02 A,a 6.4 ± 0.02 A,b 4.9 ± 0.0 A,c
1 6.2 ± 0.03 A,a 3.6 ± 0.01 A,c 3.7 ± 0.02 A,b
Mold and Yeast 3 5.0 ± 0.0 B,a 3.6 ± 0.0 A,b 2.3 ± 0.01 B,c
7 4.6 ± 0.01 C,a 1.3 ± 0.03 B,b <1 ± 0.04 C,c
1 4.5 ± 0.01 C,a 1.3 ± 0.01 C,b 1.2 ± 0.02 C,c
Coliform bacterial Group 3 4.8 ± 0.01 B,a 1.6 ± 0.0 B,b 1.4 ± 0.01 B,c
7 4.9 ± 0.05 A,a 1.7 ± 0.0 A,b 1.5 ± 0.01 A,c
1 4.7 ± 0.01 C,a <1 ± 0.01 C,b <1 ± 0.2 B,b
Enterobacteriaceae 3 5.1 ± 0.05 A,a 1.3 ± 0.02 A,b 1.2 ± 0.02 A,c
7 4.9 ± 0.0 B,a 1.0 ± 0.0 B,c 1.1 ± 0.06 A,b
Results are presented as mean ± standard deviation (SD). The distinct capital letters (A, B, C ) within the same
column for each test indicate significant variations across the storage durations of 1, 3, and 7 days. Distinct
lowercase letters (a, b, c ) within the same row indicate statistically significant differences across the three treatments
(p < 0.05).

Furthermore, Abdelnasser Ahmed et al. [28] discovered that the present research
assessed the milk samples’ microbiological quality and safety. The TBC for raw milk
samples that were randomly collected varied from 1.2 × 104 to 5 × 108 cfu/mL with a mean
value of 5.46 × 107 ± 1.57 × 108 cfu/mL. These findings exceeded those of El-Diasty and
El-Kaseh [29], whereas El Zubeir and Ahmed [30] reported lower results, with mean levels
of 5.63 × 109 CFU/mL and 7.32 × 107 ± 1.12 × 107 , respectively. We believe that inadequate
sanitary practices during milking, collection, and transportation are significant contributors
to elevated bacterial loads in milk. Furthermore, inadequate cooling equipment throughout
the phases of milk production, handling, and distribution, an inadequate understanding of
sanitary milk processing and inappropriate handling practices in supermarkets may all
contribute to the high bacteria counts. These factors collectively compromise milk safety
and quality, leading to potential health risks for consumers. Ledenbach and Marshall [31]
support this point by highlighting that poor hygiene during milk handling and storage
can lead to unacceptable levels of microorganisms, including pathogenic bacteria like
Escherichia coli and Enterobacter species, which pose health risks and can affect the sensory
qualities of dairy products. Moreover, we observed that our results closely aligned with
those of Hasan et al. [32] who reported that the TCC (MPN/mL) of market milk samples
tested in the Egyptian governorates of Cairo and Giza varied from 7.5 × 102 to 2.1 × 107
with an average of 1.8 × 106 ± 4 × 105 and from 2.1 × 102 to 2.3 × 106 with an average of
2 × 105 ± 7.7 × 104 , in the same order.
As illustrated in Table 3, it was noticed that there were remarkably significant differ-
ences (p < 0.05) in Salmonella typhimurium, Listeria monocytogenes, and Bacillus cereus among
the three treatments. The raw milk had the highest values, whereas cold plasma-treated
milk samples had the lowest compared to others. At the same time, there were insignificant
differences (p < 0.05) in Staphylococcus aureus and Escherichia coli counts between pasteur-
ized milk and cold plasma-treated milk. Additionally, all pathogenic bacteria in raw milk
showed notable variations (p < 0.05) after storage in the refrigerator for 1, 3, and 7 days. For
Staphylococcus aureus and Escherichia coli in pasteurized milk and cold plasma-treated milk,
Sustainability 2024, 16, 8754 8 of 17

no significant differences (p < 0.05) were found during storage for 7 days. However, other
pathogenic bacteria significantly increased during the storage period. Furthermore, the
populations of Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus,
and Staphylococcus aureus were decreased by three logarithmic cycles after treatment by
cold plasma for 15 min. In the same context, Baggio et al. [33] reported that cold plasma is
an emerging technology that could be used as an alternative method for food sanitization.
They explained that through reactive oxygen and nitrogen species (ROS and RNS), which
are produced by CP, its action can damage the DNA of microorganisms. In yeast and
bacteria, ROS and RNS generated by plasma may cause DNA–protein crosslinks (DPCs).
According to [34], a few instances of highly oxidized free radicals seen in the CP cloud
include O2 molecular oxygen (O2 ), superoxide anion (O2 −), ozone (O3 ), hydrogen peroxide
(H2 O2 ), hydroxyl (OH−), peroxyl (ROO−), and hydroperoxides (ROOH).

Table 3. Pathogenic bacteria examination of raw, pasteurized, and cold plasma-treated milk samples
during 7 days of storage in the refrigerator.

log/CFU per mL
Pathogenic Bacteria Storage Period (Days) Pasteurized Cold Plasma
Raw Milk
Milk Milk
1 4.0 ± 0.03 C,a 1.0 ± 0.03 C,b <1 ± 0.05 C,c
Listeria monocytogenes 3 4.5 ± 0.01 B,a 1.3 ± 0.02 B,b 1.1 ± 0.03 B,c
7 4.7 ± 0.01 A,a 1.7 ± 0.01 A,b 1.5 ± 0.01 A,c
1 + - -
Salmonella typhimurium 3 + - -
7 + - -
1 4.2 ± 0.02 C,a <1 ± 0.2 B,b <1 ± 0.04 B,b
Bacillus cereus 3 4.6 ± 0.01 B,a 1.3 ± 0.02 A,b 1.2 ± 0.02 A,c
7 4.8 ± 0.01 A,a 1.3 ± 0.02 A,b 1.2 ± 0.02 A,c
1 4.5 ± 0.02 C,a <1 ± 0.0 A,b <1 ± 0.0 A,b
Staphylococcus aureus 3 4.7 ± 0.02 B,a <1 ± 0.0 A,b <1 ± 0.0 A,b
7 4.9 ± 0.01 A,a <1 ± 0.0 A,b <1 ± 0.0 A,b
1 4.4 ± 0.01 C,a <1 ± 0.0 A,b <1 ± 0.0 A,b
E. coli 3 4.7 ± 0.01 B,a <1 ± 0.0 A,b <1 ± 0.0 A,b
7 4.8 ± 0.01 A,a <1 ± 0.0 A,b <1 ± 0.0 A,b
Results are presented as mean ± standard deviation (SD). The distinct capital letters (A, B, C ) within the same
column for each pathogenic test indicate substantial variations across the storage durations of 1, 3, and 7 days.
Distinct lowercase letters (a, b, c ) in the same row indicate significant differences across the three treatments
(p < 0.05). The findings for Salmonella typhimurium indicate its presence or absence in 25 grams of samples. The
symbol “+” denotes “Positive” while “-” signifies “Negative”.

The acquired findings were similar to those reported by Dezest et al. [35], who used
three different plasma or gas combinations (helium alone, with 1% oxygen, and with 1%
nitrogen) against E. coli. The results demonstrated that He-O2 plasma displayed the most
potent effects against E. coli, displaying rapid bactericidal activity. Oxidative stress induced
by plasma treatment significantly impairs bacteria, particularly for membrane permeability
and morphological changes. Biochemical assessments of E. Coli macromolecules indicated
significant oxidation of intracellular proteins. Reactive oxygen species and reactive nitrogen
species are not the exclusive contributors to the mortality of E. coli. Nonetheless, the electric
field and charged particles may be significant variables. Previous investigations assessed
the efficacy of surface barrier discharge (SBD) and dielectric barrier discharge (DBD) from
relyon plasma GmbH, 93055 Regensburg, Germany, against Listeria monocytogenes strains
Sustainability 2024, 16, 8754 9 of 17

and Salmonella typhimurium biofilms at varying plasma intensities (13.88, 17.88, 21.88 V
input voltage). Utilising the DBD electrode, with 0.0 (v/v) O2 % and an input voltage of
21.88 V, they accomplished a reduction of up to 3.5 log10 in both bacterial populations [36].
Numerous investigations have shown cell rupture after CP therapy, with the sever-
ity contingent upon the treatment length. Upon exposure to CP from various sources,
microorganisms exhibit structural modifications, a reduction in cell size, deformation,
and significant electroporation, leading to damage to the cell surface and leakage of cel-
lular components, ultimately resulting in cell lysis [37–39]. Additionally, other forms of
damage or modifications in cellular morphology were documented by Han, Patil, Boehm,
Milosavljević, Cullen, and Bourke [38]. The damage included cell membrane disruption
leading to cytoplasmic leakage, surface deformities in Staphylococcus aureus, shrinkage of
Listeria monocytogenes, compromised cell walls, the generation of cellular debris, and the
inactivation of Lactobacillus acidophilus and Streptococcus mutans. Moreover, it was found
that CP substantially altered the spores, potentially compromising preservation. Liang
et al. [40] noted that the surface and internal spore membranes had significantly damaging
dimensions. Bermudez-Aguirre [41] asserts that CP induces the fragmentation of genetic
material and obstructs several genetic pathways, eventually leading to cellular demise.
The phenomenon of CP, including charged particles like electrons, radiation, both excited
and non-excited molecules, as well as positive and negative ions, was elucidated by Ali,
Kim, Lee, Lee, Uhm, Cho, Park, and Choi [37]. The efficacy of these particles relies on the
substance and method used to produce the plasma, resulting in varying modes of action
against various microbes (Gram-positive and Gram-negative). Conversely, unique actions
are documented using CP obtained from diverse sources. Numerous investigations indi-
cate that cells underwent explosion when subjected to CP therapy, with intensity varying
based on treatment time. Upon exposure to CP from diverse sources, microorganisms
exhibited deformation, increased electroporation, reduction in cell size, damage to the
cellular membrane, and leaking of cellular constituents, culminating in cell lysis [38,39,41].
Additional forms of damage or alterations in cell morphology were also verified [38,42].
These included ruptures of the cell membrane permitting cytoplasmic leakage, surface
abnormalities in Staphylococcus aureus, shrinkage in Listeria monocytogenes, compromised
cell walls, development of cellular debris, and the inactivation of Lactobacillus acidophilus
and Streptococcus mutans.
Additionally, CP was shown to significantly change the spores, which could interfere
with preservation. There have been reports of spores with severely damaging interior
and exterior spore membranes [40,43]. According to Bermudez-Aguirre [41], CP cleaves
genetic material and inhibits a number of genetic processes that cause cells to die. It has
also been reported by Nwabor et al. [44] that bacteria may be killed or rendered inactive
by CP. Studies on the impact of CP on Gram-positive bacteria have been carried out.
The findings demonstrated that G was successfully destroyed by exposure to CP in the
vegetative cells of Stearothermophilus and B. cereus spores. Nevertheless, the inactivation
of G. stearothermophilus spores was not substantial. They also said that the development
of resistant spores by certain food-borne microbes and the generation of toxins restrict
the efficacy of most green technologies. On the other hand, CP has shown impressive
superiority in deactivating enzymes and toxins as well as inactivating spores. Moreover, it
was suggested by Nikmaram and Keener [10] that the CP approach might be improved
to maximize the decrease in germs without causing a substantial influence on the milk
and dairy products quality. According to these findings, CP may greatly increase the shelf
life of dairy products by lowering the microbial burden, which enhances food safety and
promotes sustainability.

3.2. Changes in Quality Parameters for Milk Samples

As shown in Table 4, there were significant differences between the treatments. Where
the cold plasma treatment decreased significantly (p < 0.05) in acidity and pH compared to
pasteurized and raw milk samples. While pasteurized milk samples showed a significant
Sustainability 2024, 16, 8754 10 of 17

increase (p < 0.05) in peroxide values compared to raw and cold plasma samples. This
increase might be due to the heat exposure. The acidity of raw samples was normally
increased through the days of storage until 7 days (0.31, 0.75, and 1.49%) in the refrigerator.
Their corresponding values for pasteurized samples were 0.31, 0.60, and 1.45%. In contrast,
the values for cold plasma-treated milk were 0.27, 0.47, and 0.92%. The action of CP
treatment in preserving milk and preventing the development of acidity could be detected.
All pH values took the opposite trend and confirmed the data of acidity percent. Although
the pH of the milk samples was slightly lower, it did not reach the isoelectric point of casein
(pH = 4.6), at which casein proteins precipitate. This increase in pH could be attributed to
higher levels of bacteria in milk samples obtained from the farm [32].

Table 4. Acidity, peroxide, and pH values of raw, pasteurized, and cold plasma-treated milk samples
during 7 days of storage in a refrigerator.

Pasteurized
Parameters Storage Period (Days) Raw Milk Cold Plasma Milk
Milk
1 0.31 ± 0.01 a,C 0.31± 0.01 a,C 0.27 ± 0.02 b,C
Acidity (%) 3 0.75 ± 0.05 a,B 0.60 ± 0.07 b,B 0.47 ± 0.02 c,B
7 1.49 ± 0.07 a,A 1.45 ± 0.03 a,A 0.92 ± 0.05 b,A
1 5.90 ± 0.2 a,A 6.00 ± 0.2 ab,A 6.10 ± 0.2 b,A
pH 3 5.97 ± 0.3 a,A 5.85 ± 0.4 a,A 5.55 ± 0.3 a,B
7 4.80 ± 0.3 a,B 4.80 ± 0.2 a,B 5.10 ± 0.3 b,B
1 0.42 ± 0.01 a,C 1.52 ± 0.07 b,C 0.47 ± 0.05 a,C
Peroxide value (meq/kg) 3 0.51 ± 0.03 a,B 1.86 ± 0.08 b,B 0.52 ± 0.06 a,B
7 0.78 ± 0.05 a,A 1.97 ± 0.07 b,A 0.79 ± 0.06 a,A
Results are presented as mean ± standard deviation (SD). The distinct capital letters (A, B, C ) inside the same
column for each parameter signify substantial changes across the storage durations of 1, 3, and 7 days. Distinct
lowercase letters (a, b, c ) in the same row indicate statistically significant differences across the three treatments
(p < 0.05).

For the values of the peroxide test, it was noted that their values increased as the effect
of cold plasma-treated samples increased through 7 days of storage, where their values
were 0.47, 0.52, and 0.79 meq/kg after 1, 3, and 7 days of storage, in comparison to 1.52,
1.86, and 1.97 meq/kg for pasteurized milk and 0.42, 0.51, and 0.78 meq/kg for raw milk.
This increase in peroxide value for pasteurized milk samples is attributed to the effect
of heat treatment, where exposure to heat increases the peroxide content. This could be
attributed to the fact that heat exposure accelerated the breakdown of unsaturated fatty
acids, leading to the free radicals formation and, subsequently, peroxide compounds [17].
Our results were consistent with the results of Nasiru, Boateng, Alnadari, Umair, Wang,
Senan, Yan, Zhuang, and Zhang [12] who reported that plasma-induced mild oxidation can
increase the peroxide values as a result of the generation of ROS, which react with lipids
to form peroxides. Additionally, Segat, Misra, Cullen, and Innocente [8] explained the
effects of CP on the pH value and the oxidation of lipids. They demonstrated how whey
protein isolate (WPI) model solutions interacted with CP under different times (from 1 to
60 min). The proteins and lipids showed moderate oxidation after 15 min of CP treatment.
Along with the decrease in free SH groups, this was shown by the rise in carbonyl groups
and the surface hydrophobicity. Ribeiro, Coutinho, Silveira, Rocha, Arruda, Pastore, Neto,
Tavares, Pimentel, Silva, Freitas, Esmerino, Silva, Duarte, and Cruz [9] assessed the impact
of CP at various intervals (0, 5, 10, or 15 min) on the physicochemical, functional, and
sensory properties of whey beverages supplemented with xylo oligosaccharide (XOS,
1.5% p/v). Both pasteurised and untreated whey beverages were evaluated. Reduced
colour intensity (L* = 87.4–87.9, a* = 0.24–0.60, b* = 2.41–5.19), diminished consistency
Sustainability 2024, 16, 8754 11 of 17

(K = 4.31–42.21 mPa.sn and N = 0.57–0.95), and similar apparent viscosity were seen in CP
and pasteurised goods. Furthermore, our results were inconsistent with those of Wang, Liu,
Zhang, Lü, Zhao, Song, Zhang, Jiang, Zhang, and Ge [14], which demonstrated that sheep
milk subjected to CP treatment exhibited a reduction in pH, correlating with an increase
in acidity. Moreover, there were significant changes (p < 0.05) between storage periods
(1, 3, and 7 days) in acidity, pH, and peroxide values for each treatment separately. The
same trend was observed for all three treatments during storage periods. By increasing
the storage period, the acidity and peroxide values increased, whereas pH increased
insignificantly for raw and pasteurized milk and then reduced significantly after 7 d of
storage. For cold plasma-treated milk, the pH significantly decreased after 3 d of storage
and then demonstrated a slight but no longer significant decrease (p < 0.05). These findings
were in accordance with those of Akarca, Atik, Atik, and Denizkara [11]. In conclusion, we
can say that cold plasma treatment, in particular, could offer a better and more sustainable
solution than pasteurization by slowing the rate of spoilage, thus contributing to the
sustainable management of the dairy sector.

3.3. Oxidative Stability of Raw, Pasteurized, and Cold Plasma-Treated Milk Samples
during Storage
Milk constitutes a blend of fat particles, or globules, dispersed within water. The
pricing of milk is contingent on its fat content; buffalo milk, richer in fat, has a higher price
than cow milk, which contains less fat [15,18]. Researchers employed cold plasma technique
(CP) to safeguard milk and improve the performance of its proteins. The Rancimat method
was employed to gauge the duration for fats in both treated and untreated milk samples to
undergo oxidation. The findings revealed that the induction period was extended through
the use of CP. Specifically, it increased significantly (p < 0.05) from around five hours for the
fat in raw milk to about seven hours for the fat in plasma-treated milk (Figure 4), at 120 ◦ C
and 20 L/h for temperature and airflow rate, respectively. This lengthened period might be
due to the impact of CP in diminishing free radicals and reactive species, which impede
the oxidation process in lipids. Although some prior research indicated that CP could
diminish the oxidative stability of lipids as a result of the generation of reactive species
Sustainability 2024, 16, x FOR PEER REVIEW 12 of 17
and free radicals that affect stability, other studies highlighted the benefits of CP on lipid
stability [45].

8 a a a
7
Induction period (h)

6 b b
b b
b b
5

4 RM

3 PM

2 CP

0
1 3 7
Storage time (d)

Figure4.4.Induction
Figure Inductionduration
durationofofraw,
raw,pasteurised,
pasteurised,and
andcold
coldplasma-treated
plasma-treated milk
milk samples
samples throughout
throughout a
a 7-day storage period. Distinct lowercase letters (a, b) at the top of each column indicate significant
7-day storage period. Distinct lowercase letters (a, b) at the top of each column indicate significant
differences across the three treatments (p < 0.05).
differences across the three treatments (p < 0.05).

Previous studies have shown that applying CP for 5 and 30 min increased the amount
of saturated fatty acids (SFAs) while decreasing the amounts of MUFA and PUFA. In con-
trast to pasteurized milk drinks, the fatty acid profile is improved when the CP processing
time is extended to 10–20 min, with higher levels of PUFA and MUFA and a decrease in
SFAs [18].
Sustainability 2024, 16, 8754 12 of 17

Subsequently, few studies have addressed the impact of CP on lipids derived from
complex matrices. Significant progress has been achieved in investigating the effects of
CP on lipids in actual food systems. To address the issue of lipid oxidation, potential
solutions include incorporating antioxidants into food prior to cold plasma (CP) treatment,
limiting food exposure to CP, employing lower voltage during treatment, decreasing the
oxygen concentration in the carrier gas, and refining the CP application process before its
implementation in food products [2].
Previous studies have shown that applying CP for 5 and 30 min increased the amount
of saturated fatty acids (SFAs) while decreasing the amounts of MUFA and PUFA. In con-
trast to pasteurized milk drinks, the fatty acid profile is improved when the CP processing
time is extended to 10–20 min, with higher levels of PUFA and MUFA and a decrease in
SFAs [18].

3.4. Changes in Fatty Acids Profile

Table 5 illustrates the examination of changes in the fatty acid composition of milk
fat across several treatment groups, namely the control, pasteurised, and cold plasma-
treated samples, offering valuable insights into the impact of these treatments on milk fat
stability. Upon the commencement of storage, the cumulative SFA content for the control,
pasteurised, and cold plasma-treated samples was 66.03, 64.02, and 66.19, respectively. This
little difference indicated very similar SFA levels among the groups. Nevertheless, after
a week in storage, notable changes occurred. The control sample exhibited a significant
rise (p < 0.05) in SFA content to 68.93, suggesting a tendency for elevated saturation during
storage. In contrast, the pasteurised sample exhibited a little reduction in SFA level to
66.02, indicating a reasonably consistent composition. The cold plasma-treated sample
demonstrated a significant alteration, with the SFA level reduced to 59.94. The alteration
in the cold plasma-treated sample indicates a possible influence of the therapy on the
degradation or transformation of SFAs [2].
The changes in PUFA within the studied samples reveal intriguing insights into
the influence of different treatments on the milk fat composition. The initial levels of
PUFA, recorded at 4.26, 4.86, and 5.81 for the control, pasteurized, and cold plasma-treated
samples, respectively, indicate slight variations in PUFA content among the groups at
the beginning of the experiment. After the designated observation period, the recorded
values for PUFA demonstrated notable fluctuations. In the control group, the PUFA
content increased to 4.77, suggesting a moderate increase in polyunsaturation. On the other
hand, the pasteurized sample exhibited a more substantial change, with the PUFA content
rising to 5.61. This marked increase could point to alters in the fatty acid composition
driven by the pasteurization process, potentially related to heat-induced transformations
or interactions [17].
The cold plasma-treated sample is particularly interesting, which displayed the most
dramatic shift in PUFA content, soaring to 8.29. This significant increase in polyunsaturation
levels implies a substantial modification in the composition of fatty acid altered by the
CP treatment. Moreover, CP treatment might have triggered reactions that led to the
generation of new PUFA or the breakdown of existing SFA or MUFA. The present study
highlights how pasteurization and cold plasma treatment affect milk fat stability and
composition. Pasteurization preserves the initial composition well, while cold plasma
treatment significantly transforms fatty acid composition, likely due to the treatment’s
transformative effects. On the contrary, the present results were not consistent with the
results of Korachi et al. [46] who found that the SFA concentrations were increased by
increasing the exposure time to CP.
These findings encourage further research into underlying mechanisms and their
impact on milk’s nutritional and functional qualities. Factors such as temperature, oxidative
stability, and reactive species should be considered to understand the observed shifts.
Coutinho, Silveira, Pimentel, Freitas, Moraes, Fernandes, Silva, Raices, Ranadheera, Borges,
Neto, Tavares, Fernandes, Nazzaro, Rodrigues, and Cruz [18] reported that when the CP
Sustainability 2024, 16, 8754 13 of 17

processing time was extended to 10–20 min, it resulted in an improved fatty acid profile
along with higher levels of PUFA and MUFA, as well as a decrease in SFA compared to
pasteurized milk drinks.

Table 5. Fatty acid profile of milk fat; raw, pasteurized, and cold plasma-treated samples after 7 days
of storage at 5 ◦ C ± 2 ◦ C.

Concentration %
RT
Fatty Acids Zero-Time 7 days Storage
(Min)
RMF PMF CPMF RMF PMF CPMF
Butyric acid (C4:0) 1.81 3.21 ± 0.15 1.91 ± 0.14 1.89 ± 0.23 3.23 ± 0.16 4.76 ± 0.37 3.15 ± 0.29
Caproic acid (C6:0) 2.38 3.03 ± 0.22 2.88 ± 0.26 1.67 ± 0.21 2.68 ± 0.29 2.53 ± 0.54 2.35 ± 0.36
Caprylic acid (C8:0) 3.63 2.41 ± 0.10 1.82 ± 0.09 1.35 ± 0.19 1.56 ± 0.55 1.43 ± 0.31 1.28 ± 0.18
Capric acid (C10:0) 5.35 2.97 ± 0.11 3.89 ± 0.25 3.21 ± 0.12 3.55 ± 0.43 2.35 ± 0.26 2.31 ± 0.11
Caproleic acid (C10:1) 5.87 0.53 ± 0.17 0.44 ± 0.06 0.23 ± 0.05 0.41 ± 0.09 0.38 ± 0.09 0.18 ± 0.05
Lauric acid (C12:0) 7.26 4.16 ± 0.15 4.13 ± 0.22 3.41 ± 0.65 3.98 ± 0.53 2.98 ± 0.14 3.17 ± 0.45
Tridecanoic acid (C13:0) 8.43 0.34 ± 0.05 0.52 ± 0.03 0.18 ± 0.02 0.52 ± 0.10 0.78 ± 0.11 0.85 ± 0.09
Tridecenoic acid (C13:1) 8.75 0.69 ± 0.04 0.61 ± 0.06 ND 0.47 ± 0.04 ND 0.22 ± 0.10
Myristic acid (C14:0) 9.11 9.65 ± 0.56 10.63 ± 0.62 9.59 ± 0.94 11.72 ± 0.72 9.56 ± 0.87 10.28 ± 0.83
Myristoleic acid (14.1) 9.39 1.49 ± 0.18 0.97 ± 0.16 0.76 ± 0.11 1.16 ± 0.49 1.24 ± 0.06 1.39 ± 0.18
Myristolinoleic acid (14.2) 9.75 1.18 ± 0.09 0.94 ± 0.11 0.66 ± 0.08 0.89 ± 0.23 1.06 ± 0.13 1.52 ± 0.25
Pentadecanoic acid (C15:0) 10.01 1.78 ± 0.05 1.35 ± 0.08 1.12 ± 0.11 0.75 ± 0.12 2.14 ± 0.47 2.13 ± 0.10
Cis-10-Pentadecenoic (C15:1) 10.56 0.56 ± 0.01 0.42 ± 0.02 0.53 ± 0.07 0.62 ± 0.08 0.93 ± 0.15 1.58 ± 0.11
Palmitic acid (C16:0) 11.04 31.27 ± 1.34 28.24 ± 1.04 34.21 ± 2.05 31.84 ± 1.35 27.73 ± 1.19 22.97 ± 1.12
Palmitoleic acid (C16:1 n9) 11.17 1.87 ± 0.22 1.77 ± 0.13 1.24 ± 0.63 1.98 ± 0.22 2.46 ± 0.33 3.40 ± 0.35
Palmitoleic acid (C16:1 n7) 11.21 0.46 ± 0.15 0.26 ± 0.05 0.77 ± 0.12 0.87 ± 0.16 1.02 ± 0.25 2.01 ± 0.08
Heptadecanoic acid (C17:0) 12.28 0.62 ± 0.11 0.22 ± 0.02 0.62 ± 0.08 1.09 ± 0.15 1.34 ± 0.22 1.65 ± 0.04
Cis-10-Heptadecanoic acid
12.35 ND ND 0.32 ± 0.01 1.16 ± 0.05 ND 0.31 ± 0.02
(C17:1)
Stearic acid (C18:0) 13.54 6.59 ± 0.37 8.27 ± 0.37 8.94 ± 0.87 7.64 ± 0.85 9.98 ± 1.16 9.51 ± 0.85
Oleic acid (C18:1n9c) 13.58 24.11 ± 0.95 26.65 ± 0.99 24.15 ± 1.22 19.63 ± 0.86 22.34 ± 1.12 22.68 ± 1.03
Linoleic acid (C18:2n6c) 14.18 2.33 ± 0.73 2.74 ± 0.35 4.27 ± 0.48 2.11 ± 0.07 2.33 ± 0.15 2.49 ± 0.63
γ- Linolenic acid
14.99 0.32 ± 0.03 0.82 ± 0.06 0.37 ± 0.03 0.65 ± 0.04 1.07 ± 0.11 2.83 ± 0.22
(C18:3n6, CLA)
α- Linolenic acid (C18:3n3) 15.45 0.43 ± 0.01 0.36 ± 0.02 0.51 ± 0.09 1.12 ± 0.10 1.15 ± 0.07 1.45 ± 0.16
Arachididc acid (C20:0) 16.62 ND 0.16 ± 0.03 ND 0.37 ± 0.05 0.44 ± 0.20 0.29 ± 0.03
∑ SFA --- 66.03 ± 1.94 64.02 ± 1.15 66.19 ± 1.02 68.93 ± 1.78 66.02 ± 1.34 59.94 ± 1.34
∑ UFA --- 33.97 ± 1.55 35.98 ± 2.17 33.81 ± 1.82 31.07 ± 1.11 33.98 ± 1.35 40.06 ± 1.15
∑ MUFA --- 29.71 ± 1.09 31.12 ± 1.83 28.00 ± 0.86 26.3 ± 0.88 28.37 ± 1.27 31.77 ± 1.52
∑ PUFA --- 4.26 ± 0.65 4.86 ± 0.78 5.81 ± 0.66 4.77 ± 0.35 5.61 ± 0.66 8.29 ± 0.55
Results are expressed as mean ± SD; RT, retention time, “min”; ND, not detectable; SFA, saturated fatty acids;
UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; RMF, raw
milk fat; PMF, pasteurized milk fat; CPMF, cold-plasma milk fat. The bold values were significantly (p < 0.05)
different from other values in the same row for each period of storage.

As depicted in Figure 5, the calculated oxidizability (COX) value is a significant

indicator of the oxidative stability of the raw milk (control) and cold plasma-treated samples.
The observed range, starting at approximately 0.55 for the control sample and extending
to around 0.75 for the cold plasma-treated sample, signifies (p < 0.05) distinct differences
in their susceptibility to oxidation. The control samples had a lower COX value of 0.55,
suggesting a lower oxidative risk vulnerability. This might be attributed to the inherent
composition or the initial condition of the sample. On the other hand, the treated sample
 suggesting a lower oxidative risk vulnerability. This might be attributed to the inherent
composition or the initial condition of the sample. On the other hand, the treated sample
subjected to CP displayed a higher COX value of 0.75, indicating increased susceptibility
to oxidation. The elevated COX value could stem from changes induced by the CP treat-
Sustainability 2024, 16, 8754 ment, potentially involving alterations in the chemical structure of the fatty acids 14 or the
of 17
reactive species presence that expedite oxidation [2,20]
The difference in COX values among the samples treated with cold plasma and the
subjected to CP displayed
control group a higher COX
makes it necessary value how
to analyze of 0.75,
theindicating
treatment increased
affects thesusceptibility to
milk fat’s overall
oxidation.
oxidativeThe elevated
stability. TheCOX value
higher COXcould stem
value in from changes
the treated induced
sample by be
could theindicative
CP treatment,
of ac-
potentially
celerated involving alterationswhich
oxidation processes, in the could
chemical structure
either of the fattyin
be advantageous acids or the
specific reactive
applications
species presence that expedite oxidation [2,20].
or require mitigation strategies to maintain product quality and shelf life.

Zero time 7 days of storage

a a
b
b c
c

Figure 5. The oxidisability (COX) values of both raw and cold plasma-treated samples. RMF refers
to raw milk fat; PMF denotes pasteurised milk fat; CPMF signifies cold plasma milk fat. Distinct
Figure 5. The oxidisability (COX) values of both raw and cold plasma-treated samples. RMF refers
lowercase letters
to raw milk (a,PMF
fat; b, and c) at thepasteurised
denotes top of each milk
column
fat;indicate significant
CPMF signifies differences
cold plasma across theDistinct
milk fat. three
treatments (p < 0.05).
lowercase letters (a, b, and c) at the top of each column indicate significant differences across the
three treatments (p < 0.05).
The difference in COX values among the samples treated with cold plasma and the
control group makes it necessary to analyze how the treatment affects the milk fat’s overall
4. Conclusions
oxidative stability. The higher COX value in the treated sample could be indicative of
This research sheds light on the largely untapped potential of cold plasma (CP) as a
accelerated oxidation processes, which could either be advantageous in specific applications
sustainable technique
or require mitigation for milk
strategies preservation
to maintain in the
product dairyand
quality sector.
shelfInlife.
line with food pro-
cessing sustainability objectives, CP is emerging as a viable substitute for conventional
4. Conclusions
This research sheds light on the largely untapped potential of cold plasma (CP) as a
sustainable technique for milk preservation in the dairy sector. In line with food processing
sustainability objectives, CP is emerging as a viable substitute for conventional preservation
techniques as interest in environmentally friendly solutions grows worldwide. According
to our research, CP treatment successfully suppresses the development of microorganisms,
including harmful bacteria, and lowers the acidity of milk when compared to control
and pasteurized samples. Remarkably, raw or cold plasma-treated milk did not exhibit
the notable rise in peroxide value that pasteurized milk did, which is a marker of lipid
breakdown. Additionally, the oxidative stability of milk was improved by CP treatment,
which resulted in an increase in the induction duration from around five hours to seven
hours in raw milk. By increasing shelf life, this enhancement not only improves product
quality but also lowers food waste. In summary, CP technique has important advantages
such as decreased microbial growth and improved oxidative stability, making it a practical
and environmentally responsible substitute for milk preservation. To improve treatment
settings and minimize lipid degradation while optimizing sustainability advantages, further
study is needed. This research contributes to the expanding body of information on CP
technology by highlighting how it might improve dairy product quality and safety and
encourage environmentally friendly practices in the food sector.
Sustainability 2024, 16, 8754 15 of 17

Author Contributions: H.M.A. and M.S. conceptualised, developed, supervised, amended, executed
this work, and authored the original draft. M.T.F.: conducted microbiological tests and composed
the draft. H.A.Z. and E.A.A.: conducted studies of estimated oxidative stability and fatty acids,
and authored the original draft. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported through research project no. 11040107, funded by the National
Research Centre (NRC), Cairo, Egypt.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare that there are no conflicts of interest.

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