Cold Plasma Technology: A Sustainable Approach to Milk Preservation by Reducing Pathogens and Enhancing Oxidative Stability

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) technology 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 deterioration. Furthermore, the oxidative stability of cold plasma-treated milk was enhanced, with an induction period extending from approximately five to seven hours, demonstrating its superior resistance to oxidation. In conclusion, CP has emerged as a promising eco-friendly technology for prolonging the shelf life of milk by mitigating microbial growth and lipid oxidation. This method not only aligns with sustainability goals by reducing the need for chemical preservatives but also enhances the overall quality of milk products. Future research should focus on large-scale applications and the impacts of CP on other essential milk components, particularly fat-soluble vitamins, to fully understand its sustainability benefits in the dairy sector.

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 change in taste for thermally processed food. Conventional methods, such as sterilization or pasteurization, can have detrimental effects on the taste and nutritional quality of food. Hence, in recent decades, many non-thermal sterilization technologies, e.g., high-pressure homogenization, high-pressure carbon dioxide, pulsed electric field, high-intensity ultrasound, and cold plasma have been developed to prevent the growth of microorganisms and preserve the nutritional quality of food [1,2].

Cold plasma (CP) technology represents a significant advancement in sustainable food processing, particularly in milk preservation. Unlike traditional thermal methods, 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 methods 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 physical 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 proteolysis, 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, 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 better 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. 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.

2.2. Methods

2.2.1. Chemical Analysis

The gross chemical analyses, acidity, and peroxide value were performed according to A.O.A.C. [19].

2.2.2. The Microbiological Examinations for Milk Samples

Ten-fold dilutions were prepared and inoculated onto plates of selective media. The aerobic colony count (ACC), yeasts, and molds were performed according to A.O.A.C. [19]. For ACC and using plate count agar (oxoid), plates were incubated at 30 ± 1 °C for 72 ± 2 h. Using acidified potato dextrose agar (oxoid) as the medium, yeasts and molds were grown on plates that were incubated at 25 °C for 5 d. The coliform group was identified using violet-red bile agar (VRBA) (oxoid), with plates incubated at 37 °C for 24 h. The identification of Enterobacteriaceae was conducted using violet red bile glucose (VRBG) agar (oxoid), with plates incubated at 37 °C for 24 h, following the methodology of Wai et al. [21].

In order to examine Listeria monocytogenes, 25 g of each sample was combined with 225 mL of Listeria selective enrichment medium in 500 mL flasks, and the mixture was then incubated at 30 °C for 7 d. Following the preparation of serial dilutions, they were plated onto Oxford agar bases supplemented with Listeria supplement and incubated at 35 °C for 48 h [22].

Salmonella typhimurium was examined by combining 25 g of sample and 225 mL of sterile buffer peptone water, then incubating at 35 °C for 24 h. In brief, 1 mL mixture was pipetted to 10 mL selenite cystine broth and incubated at 35 °C for 72 h. Subsequently, serial dilutions were prepared, and plates were placed on Salmonella and Shigella agar and incubated at 35 °C for 24 h [23].

Bacillus cereus was identified through surface plating onto the Bacillus cereus agar medium, which was enhanced with egg yolk and polymyxin-B. While, Staphylococcus aureus 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 plating method, followed by an incubation period of 24 h at 44 °C [23]. Figure 2 presents an overall flowchart of the experiment.

2.2.3. Pathogenic Bacteria Examination for Milk Samples

Five different types of bacteria pathogens, namely E. coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 7644), Bacillus cereus (ATCC 33018), and Staphylococcus aureus (ATCC 20231), were utilized in the challenge test after being activated in tryptone soy broth at 37 °C for 24 h. The various methods used on milk samples mentioned earlier were categorized into three groups; one group was inoculated with E. coli, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus individually and served as the control. Another group was inoculated with each 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 pathogen content in the milk samples varied between 4.08 and 4.86 log CFU/mL. Subsequent to the inoculation, all milk samples (15 samples) were refrigerated for observation for a period of 7 days.

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 instrument (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 of fatty chains to fatty acid methyl esters (FAMEs) through trans-methylation. An HP 6890 plus gas chromatography system fitted with a Supelco^TM SP-2380 capillary column was then used to separate the FAMEs. A flame ionization detector (FID) was used for 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 a flow rate of 1.2 mL/min. A split injector was used to introduce a 1 µL sample volume that had been dissolved in n-hexane at a 100:1 splitting ratio. To identify the FAMEs, their retention times were compared to those of established FAME standards. The relative proportion of the entire peak area was used to indicate the fatty acid content.

2.2.6. Calculated Oxidizability (COX) Value

The proportion of unsaturated C18 fatty acids was used to determine the estimated oxidizability (COX) value using the technique outlined by Fatemi and Hammond [26] as follows:

$$\mathrm{COX}={\displaystyle \frac{\left[1\right(18:1\%)+10.3(18:2\%)+21.6(18:3\%\left)\right]}{100}}$$

2.3. Statistical Analysis

Using the Statistical Analysis System (SAS-STAT, Ver. 9.2), Duncan’s test and analysis of variance (ANOVA) were used to perform statistical analysis on the collected data (three repetitions). The statistical significance was established using a probability of p < 0.05 [27].

3. Results and Discussion

3.1. Microbiological Examination of Milk Sample

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

Microbiological Examination	Storage Period (Days)	log/CFU per mL
		Raw Milk	Pasteurized Milk	Cold Plasma Milk
Total bacterial count	1	8.4 ± 0.01 C,a	6.1 ± 0.00 C,b	4.2 ± 0.02 C,c
	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
Mold and Yeast	1	6.2 ± 0.03 A,a	3.6 ± 0.01 A,c	3.7 ± 0.02 A,b
	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
Coliform bacterial Group	1	4.5 ± 0.01 C,a	1.3 ± 0.01 C,b	1.2 ± 0.02 C,c
	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
Enterobacteriaceae	1	4.7 ± 0.01 C,a	<1 ± 0.01 C,b	<1 ± 0.2 B,b
	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).

and Figure 3 exhibited the changes in viable counts log/CFU per mL of the total bacterial, mold and yeast, and coliform as well as Enterobacteriaceae count during milk storage at 5 °C ± 2 °C for 7 d. Generally, the total bacterial count, coliform bacterial count, Enterobacteriaceae, and mold and yeast counts were significantly higher (p < 0.05) in control raw milk when compared with the pasteurized and cold plasma-treated milk samples. Meanwhile, cold plasma-treated milk samples had the lowest values compared to other treatments, indicating a strong effect in eliminating microorganisms. Moreover, notable variations (p < 0.05) were observed in the total bacterial count, coliform count, Enterobacteriaceae, and mold and yeast count for each treatment during storage in the refrigerator for 1, 3, and 7 days. Where they increased by increasing the storage period (Table 2).

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

Pathogenic Bacteria	Storage Period (Days)	log/CFU per mL
		Raw Milk	Pasteurized Milk	Cold Plasma Milk
Listeria monocytogenes	1	4.0 ± 0.03 C,a	1.0 ± 0.03 C,b	<1 ± 0.05 C,c
	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
Salmonella typhimurium	1	+	-	-
	3	+	-	-
	7	+	-	-
Bacillus cereus	1	4.2 ± 0.02 C,a	<1 ± 0.2 B,b	<1 ± 0.04 B,b
	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
Staphylococcus aureus	1	4.5 ± 0.02 C,a	<1 ± 0.0 A,b	<1 ± 0.0 A,b
	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
E. coli	1	4.4 ± 0.01 C,a	<1 ± 0.0 A,b	<1 ± 0.0 A,b
	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”.

, it was noticed that there were remarkably significant differences (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 pasteurized 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, 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 (H₂O₂), hydroxyl (OH−), peroxyl (ROO−), and hydroperoxides (ROOH).

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 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 severity 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 cellular components, ultimately resulting in cell lysis [37,38,39]. 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 indicate 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. Acidity, peroxide, and pH values of raw, pasteurized, and cold plasma-treated milk samples during 7 days of storage in a refrigerator.

Parameters	Storage Period (Days)	Raw Milk	Pasteurized Milk	Cold Plasma Milk
Acidity (%)	1	0.31 ± 0.01 a,C	0.31± 0.01 a,C	0.27 ± 0.02 b,C
	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
pH	1	5.90 ± 0.2 a,A	6.00 ± 0.2 ab,A	6.10 ± 0.2 b,A
	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
Peroxide value (meq/kg)	1	0.42 ± 0.01 a,C	1.52 ± 0.07 b,C	0.47 ± 0.05 a,C
	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).

, 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 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].

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 (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 and free radicals that affect stability, other studies highlighted the benefits of CP on lipid stability [45].

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

Fatty Acids	RT (Min)	Concentration %
		Zero-Time			7 days Storage
		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 (C17:1)	12.35	ND	ND	0.32 ± 0.01	1.16 ± 0.05	ND	0.31 ± 0.02
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 (C18:3n6, CLA)	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
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.

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 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.

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 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 treatment, potentially involving alterations in the chemical structure of the fatty acids or the reactive species presence that expedite oxidation [2,20].

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 oxidative stability. The higher COX value in the treated sample could be indicative of accelerated oxidation processes, which could either be advantageous in specific applications or require mitigation strategies to maintain product quality and shelf life.

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.