Fresh milk is important worldwide for providing essential nutrients for human consumption (Anusha Siddiqui et al., 2024). However, milk is perishable, and its nutritious qualities can trigger microbial growth, causing spoilage that can cause food-borne diseases (Gopal et al., 2015).

Pasteurized milk is the easiest, most economical process for providing safe, nutritious milk worldwide, while eliminating most pathogenic and spoilage organisms. While pasteurization inactivates most spoilage microorganisms, some can survive in low numbers, limiting the shelf life of milk to 10–21 days. Shelf life can be extended by 30–90 days using ultra-high-temperature (UHT) pasteurization; however, this process may produce off-flavors and physicochemical instability, resulting in a ‘cooked taste’ compared with conventionally pasteurized milk (Machado et al., 2017). Moreover, ultra-high temperature (UHT) sterilization entails substantially higher thermal intensity compared with conventional pasteurization. Consequently, developing non-thermal or mild-thermal strategies to extend the refrigerated shelf life of pasteurized milk remains both scientifically relevant and industrially valuable.

One strategy to extend and maintain the shelf life of pasteurized milk is fortification with natural antibacterial compounds or synthetic food preservatives. Natural antibacterial compounds, such as essential oils (EOs), are extracted from different plant parts, such as leaves, peels, barks, flowers, buds, and seeds (Sousa et al., 2022). Within a specific EO source, individual constituents vary widely, demonstrating various wide-spectrum antimicrobial activities, creating interest as a potential food preservative alternative (Calo et al., 2015). However, the use of synthetic preservatives is facing escalating scrutiny from both consumers and regulatory agencies, and their incorporation into liquid milk products is increasingly constrained by clean-label expectations and consumer concerns regarding “chemical” or “artificial” additives. Consequently, demand for natural, clean-label preservation alternatives continues to rise across the dairy industry.

The Origanum genus contains a variety of Origanum spp. (Marrelli et al., 2016) and exhibits antimicrobial activity against many foodborne bacteria and fungi (Kolypetri et al., 2023). Origanum’s positive benefits are attributed to its chemical diversity and its main EO constituents of carvacrol (60 to 74%) and thymol (45%) (Kubiliene et al., 2023; Lagouri et al., 1993). Addition of oregano EO (OEO) to mutton, beef, fried-salted peanuts, salad dressings, and cheese has increased preservation (Karabagias et al., 2011; Ribes et al., 2019; Liu et al., 2019). Although oregano essential oil (OEO) holds considerable promise for dairy applications, its effective in situ concentration in milk-based systems remains poorly defined. This uncertainty arises partly from potential interactions between milk constituents—particularly casein and milk fat—and the hydrophobic bioactive compounds in OEO, which may compromise its antimicrobial efficacy. Consequently, establishing the minimum effective concentration of OEO required to achieve microbial inhibition in pasteurized milk is a prerequisite for translational implementation.

However, the addition of OEO to milk for stabilizing milk quality and extending shelf life has not been reported, to our knowledge. The study objectives were to investigate OEO addition at increasing inclusion rates to pasteurized milk, while assessing milk’s quality, stability, and shelf life impacts during storage. It was hypothesized that the addition of OEO would increase the preservation and, thereby, shelf life of pasteurized milk.

All data were checked for normality and outliers using the UNIVARIATE procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) before any statistical analyses were conducted. The box and whisker plots and the Shapiro–Wilk Test were then used to verify that the remaining data were normally distributed (p > 0.15). Milk components and quality were subjected to least squares analysis of variance (ANOVA) for a randomized complete block design (RCBD) (Steel and Torrie, 1980) using the PROC MIXED procedure of SAS with study day as a repeated measure ANOVA. The statistical model used was as follows: Y ijk = μ + R i + T j + D k + ( T j x D k ) + e ijk

Where Y_ijk = dependent variable, μ = overall mean, R_i = replication, T_j = treatment, D_k = study day, T_j x D_k = interaction of treatment-by-day, and e_ijk = residual random error. Treatment, day, and the interaction of treatment-by-day were considered fixed effects, and replication was considered a random effect. Study day was modeled as a repeated measure over time with a Toeplitz covariance structure. Polynomial contrasts were used to test for linear, quadratic, cubic, and quartic treatment effects (i.e., OEO inclusion rate). Differences among least square means were separated using the probability difference (PDIFF) statement of statistical analysis system (SAS), and significance was declared at p < 0.05 and is extremely significant at 0.05 ≤ p < 0.10.

It is widely accepted that microbial growth limits the shelf life of milk (Ziyaina et al., 2018), and our results indicate that OEO addition can inhibit bacterial growth and thereby improve the shelf life of pasteurized milk. Specifically, pasteurized milk shelf life is largely determined by the presence and growth of aerobic psychrotrophic microorganisms, including endospore formers (Machado et al., 2017). OEO has demonstrated broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria (Gomes et al., 2020), and multiple studies have reported its preservative potential in different food systems (Cattelan et al., 2018; García-Díez et al., 2017; Kosakowska et al., 2021; Veenstra and Johnson, 2019). Antimicrobial activities can be attributed to OEO’s diversity of chemical constituents, mainly carvacrol, thymol, and eugenol phenolic compounds (Afonso et al., 2017; Kachur and Suntres, 2020). In this present study, the major OEO constituents were carvacrol (51.78%), thymol (0.38%), o-cymene (7.56%), γ-terpinene (5.27%), and caryophyllene (7.24%). Mechanistically, carvacrol is known to insert into bacterial membranes and disrupt membrane integrity, which increases permeability to ions (e g., H⁺ and K⁺), collapses the proton motive force, and impairs ATP generation and essential transport processes (Veldhuizen et al., 2006). Such membrane damage induces the leakage of intracellular contents, ultimately resulting in the inhibition of bacterial growth and even cell death. In the refrigerated pasteurized milk system, psychrotrophic bacteria are the primary spoilage microorganisms; the aforementioned mechanism of action of carvacrol provides a plausible explanation for the reduction in the total plate count (TPC) of milk treated with oregano essential oil (OEO), with this effect being particularly pronounced at higher OEO addition levels. Notably, fats and proteins in the milk matrix can bind to the hydrophobic components of essential oils, which may reduce their free antimicrobial activity. Thus, the observation of a distinct dose-dependent antimicrobial effect of OEO in the liquid dairy system validates its practical antibacterial efficacy under such storage conditions. Our findings also align with prior literature demonstrating oregano-based interventions in other food matrices. For example, oregano EO incorporated into films has been shown to suppress microbial growth during beef storage (Oussalah et al., 2004). It was reported that a film containing 1.5% (w/w) oregano oil reduced total viable counts and particularly Pseudomonas during refrigerated beef storage (Zinoviadou et al., 2009). Although these studies used a solid food matrix and a delivery system (film) that differs from direct addition to milk, the direction of the effect is consistent with the present work—OEO inhibits microbial proliferation during cold storage. Compared with meat systems, milk presents additional challenges for EO dispersion and activity due to its emulsion nature and protein interactions; thus, demonstrating that inhibition in pasteurized milk strengthens the evidence that oregano-derived phenolics can function as preservatives beyond solid matrices.

Other indicators of the shelf life of milk include titratable acidity and pH, which are indirect indicators of microbial growth (Ibáñez et al., 2019; Sommella et al., 2018). Increased microbial activity ferments milk lactose, increasing titratable acidity (Krishna et al., 2021) and decreasing pH (Boor, 2001). In the present study, titratable acidity was greater in the control samples than in OEO-supplemented samples by day 6, consistent with the reduced microbial growth observed under OEO inclusion. It has been reported that when titratable acidity exceeds 18⁰T, it is considered to have reached the end of its shelf life. By day 8, titratable acidity exceeded 18⁰T in all treatments, suggesting that OEO delayed but did not fully prevent spoilage under the current storage conditions. Because storage temperature strongly influences acidification dynamics (Sitohy et al., 2011), maintaining a constant 4 °C throughout the study supports that differences among treatments primarily reflect antimicrobial effects rather than temperature variation. Although pH is not always used as a primary shelf life endpoint, it has been applied to monitor pasteurized milk spoilage during storage (Pan et al., 2023), given that pH decline reflects lactic acid production. As reported previously, a sharp increase in titratable acidity coupled with a decrease in pH is commonly observed as microbial counts approach the regulatory threshold (e g., 5.0 log10 CFU/mL) during refrigeration (Ziyaina et al., 2018). Collectively, these data support that OEO inclusion can hinder titratable acidity development and help maintain pH in pasteurized milk by suppressing microbial growth.

Milk fat and protein concentrations were improved with OEO inclusion compared with control (0.0 mg/L OEO). This may be due to OEO inclusion reducing TPC, including psychrotrophic gram-negative bacteria that produce lipases and proteases for breaking down fat and protein concentrations, respectively (Sepulveda et al., 2005). In addition, the small numerical difference in lactose (Table 2) is most plausibly explained by reduced microbial fermentation of lactose to organic acids when OEO was present, which aligns with lower titratable acidity development in OEO treatments. Because these variables are expressed as percentages, the observed differences are best interpreted as improved retention of milk solids (less spoilage-related hydrolysis and fermentation) rather than true increases in fat, protein, or lactose content during storage. High concentrations of psychotropic bacteria prior to pasteurization produce heat-stable proteases and lipases that remain active after pasteurization, potentially causing milk spoilage during extended storage (Fusco et al., 2020). In addition, high protease and lipase activities can produce off-flavors during pasteurized milk storage (Barbano et al., 2006). Although sensory evaluation was not conducted in this study, the observed enhancement in the oxidative stability of milk—characterized by elevated superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels—demonstrates that the quantification of SOD and MDA contents can be used to assess the oxidative potential and antioxidant capacity of fats and proteins in pasteurized milk. Thus, oregano essential oil (OEO) may help inhibit oxidation-induced quality deterioration in milk, a factor that is critical for extending the shelf life of milk products. Malondialdehyde (MDA) is an oxidation end product, while SOD inhibits oxidation. In this study, increasing the OEO inclusion rate increased SOD while reducing MDA concentrations, suggesting OEO effectiveness as an antioxidant. Specifically, a high antioxidant potential of 96.6–137.5 mmol for oregano has been reported (Orimaye et al., 2024). This antioxidant potential is stronger than the synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Henning et al., 2011), which are commonly added food preservatives. There is growing interest in the use of naturally occurring antioxidants, such as OEO, because of their perception of being healthier (safer) than synthetic antioxidants. Furthermore, EO effectiveness as an alternative to synthetic antioxidants in preserving food products has been well demonstrated. It was reported that EO incorporated into milk protein-based edible films wrapped around muscle meat increased antioxidative activity during 7-day storage (Oussalah et al., 2004). In agreement, OEO application decreased lipid and protein oxidation of foal steaks during storage (Lorenzo et al., 2014). Specifically, oregano EO has demonstrated a protective effect against lipid oxidation and fermentation in flavored cheeses prepared from a cream cheese base (Asensio et al., 2015; Olmedo et al., 2013). It has been concluded that OEO in high lipid food products (such as peanuts) can replace synthetic antioxidants, preventing lipid oxidation, chemical, and sensory changes during storage (Olmedo et al., 2009). These findings suggest that OEO could be explored as a natural preservative to enhance the refrigerated stability of pasteurized milk, potentially offering a “clean-label” alternative or complement to conventional preservatives. Because the effective dose identified in this study was low (optimal at 2.0 mg/L), the approach may be economically feasible at scale, provided that industrial mixing/dispersal ensures uniform distribution.

While this study provides valuable insights into oregano essential oil (OEO) as a preservative for pasteurized milk, it has several limitations. First, no sensory evaluation was conducted, despite its critical role in assessing OEO’s impact on milk flavor and odor—key factors for consumer acceptance of natural preservatives. Future research should incorporate sensory testing to evaluate OEO-treated milk acceptability. Second, the 8-day storage period is relatively short; a longer duration and varied storage conditions beyond refrigeration would yield more comprehensive data on OEO’s long-term preservation efficacy. Third, using only yak milk limits the generalizability of the results; testing OEO on other milk types (e g., cow, goat, and sheep) is needed to confirm consistent effects across dairy products. Finally, OEO’s strong herbal scent may alter milk’s organoleptic profile, which was unassessed here. Future work should investigate these effects and strategies to minimize negative sensory impacts, aligning with growing consumer preference for clean-label natural products.

Measurement of pH, TA, and TPC demonstrated the OEO’s potential effectiveness to inhibit bacterial growth. Specifically, OEO’s high carvacrol concentration may have inhibited common pasteurized milk spoilage microorganisms, thereby increasing milk’s shelf life. This study did not assess the smell/flavor and/or palatability of pasteurized milk for human consumption when incorporating OEO. However, fat and protein, as well as SOD and MDA concentrations, suggest that OEO is effective at preventing lipid and protein oxidation, which has the potential to prevent off-flavors during storage, while increasing milk’s shelf life. The optimal dose for inhibiting microbial growth and preventing oxidation was an OEO inclusion rate of 2.0 mg/L of pasteurized milk, the highest dose in this study. Future experiment(s) examining sensory properties of pasteurized milk with OEO incorporated at 2.0 mg/L or higher doses are warranted.