Effect of Sonication Associated with Pasteurization on the Inactivation of Methicillin-Resistant Staphylococcus aureus in Milk Cream

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge to the dairy industry, necessitating robust strategies to ensure food safety. This study focuses on the efficacy of thermosonication, a novel technology combining ultrasound and heat, in reducing MRSA in milk cream. Comparative analysis is conducted with conventional pasteurization, the industry standard. Results indicate that thermosonication effectively reduces MRSA counts by up to 4.72 log CFU/mL, akin to pasteurization’s reduction of 4.82 log CFU/mL. This finding highlights the potential of thermosonication as a rapid, energy-efficient alternative to pasteurization in the dairy industry, significantly reducing processing time while maintaining microbial safety. Further exploration and optimization of these techniques promise enhanced food safety and quality control in dairy products, addressing the growing concern of antibiotic-resistant strains like MRSA. This research lays a foundation for innovative approaches and underscores the significance of quantitative data in food safety research.

1. Introduction

Milk cream holds a significant place within the realm of dairy products in the diets of Brazilians, primarily due to its versatility. It plays a pivotal role as an essential ingredient in a diverse spectrum of culinary preparations, serving as an integral component in various sweet and savory dishes [1]. It is worth noting that creams available on the Brazilian market exhibit discernible disparities, primarily stemming from variances in their heat treatment processes and fat content. Among the available options, UHT (Ultra-High Temperature) creams, characterized by an average fat content of 20%, offer a unique proposition [2]. These creams undergo processing that imparts an extended shelf life and allows them to be stored at ambient temperatures until opened, enhancing their convenience and accessibility. In contrast, pasteurized creams, which, on average, contain approximately 35% fat, represent a distinct category [2].

The pasteurization process entails subjecting the cream to a specific temperature for a predetermined duration, thereby ensuring the elimination of harmful microorganisms while preserving the cream’s organoleptic attributes. This differentiation in fat content and heat treatment provides consumers with a valuable array of choices, enabling them to select the cream that aligns with their culinary requirements and dietary preferences. However, pasteurized cream is inherently less microbiologically stable due to the lower temperature employed during its production process. The lower temperature range used in pasteurization, while sufficient to eliminate harmful microorganisms, does not provide the same level of microbiological stability as UHT processing. Consequently, pasteurized cream is more susceptible to microbiological variations during storage and possesses a relatively shorter shelf life. In this context, to safeguard the quality and safety of pasteurized cream, it is essential to adhere to stringent microbiological criteria. These evaluations encompass the quantification of total and thermotolerant coliforms, mesophilic aerobic microorganisms, and coagulase-positive Staphylococcus [2], which collectively provide valuable insights into the cream’s microbiological status and suitability for consumption.

Staphylococcus aureus, particularly its methicillin-resistant strains (MRSAs), raises significant concerns due to its capacity to induce foodborne illnesses and its growing resistance to conventional antibiotics [3]. Notably, recent times have witnessed a substantial upsurge in the exploration of innovative techniques aimed at ensuring the safety and quality of dairy products. This surge is partly attributed to the increasing challenges posed by antibiotic-resistant pathogens, prompting a heightened focus on the development of novel solutions [4,5,6]. This drive for innovation arises from the need to meet the demands of the global market, as well as consumers’ expectations for products that are minimally processed, palatable, healthy, and, above all, safe for consumption [7,8]. Thus, the microbiological safety of dairy products, including creams, stands as a paramount concern, representing a pivotal nexus between the food industry and public health. In response to these imperatives, the application of ultrasound, often referred to as sonication, emerges as a promising alternative technology with the potential to significantly enhance various facets of dairy production processes.

High-intensity ultrasound (HIUS) is a promising approach for enzymatic inactivation, nutritional and sensory quality improvement, and microbiological safety in dairy products [8]. According to Bernardo et al. [9], the integration of ultrasound in milk processing offers advantages like flavor preservation, better homogeneity, and energy efficiency compared with traditional methods. However, it can lead to mild physicochemical changes, like lipid oxidation in raw milk and volatile compound production in pasteurized milk. Some improvements include changes in the freezing point, reduced fat globule size, and dry milk reconstitution. Ultrasound treatment varies in effectiveness based on factors like frequency, amplitude, and duration, and its impact on dairy matrices is influenced by factors like microbiology and energy dissipation from cavitation bubbles [9]. Despite its potential, the implementation of ultrasound technology in the dairy industry requires considerations beyond technical aspects. Factors like cost, profit, consumer perceptions, and financial viability play essential roles in the adoption of this technology [9].

Moreover, thermosonication, an innovative methodology that integrates ultrasound with concurrent heat application, establishes a synergistic process, amplifying the effectiveness of conventional pasteurization techniques. This technique offers compelling advantages in terms of the inactivation of pathogenic microorganisms and the preservation of dairy product quality [10]. Indeed, the existing body of research pertaining to ultrasound treatment in dairy products has predominantly concentrated on milk and dairy beverages. However, it is imperative to extend this investigative framework to encompass a broader array of dairy commodities, including cream [11]. The lipophilic nature of fat renders it an effective sanctuary for microorganisms, shielding them from the rigors of conventional pasteurization procedures. Therefore, it becomes crucial to gain a comprehensive understanding of the effectiveness of ultrasound treatment, especially when combined with pasteurization, as it pertains to milk cream. Such exploration represents a viable strategy for enhancing the safety and quality of this dairy product. Within this context, this communication aims to assess the impact of the pasteurization time (slow and fast) reduction via the association of sonication-pasteurization on the inactivation of MRSA strains in milk cream.

2. Materials and Methods

2.1. Preparation of MRSA Suspensions

Five strains of mecA-positive β-lactam-resistant Staphylococcus aureus (MRSA) associated with livestock activities in Brazilian territory were used in this study. Specifically, strains 30PD.1 and 32AD.1 were isolated from cow milk, strain 3N originated from a milker’s nasal cavity, and strains 1T.1 and SFT.1 were isolated from cow teats [12].

Each isolate was stored in tryptic soy broth (TSB; HiMedia, Mumbai, India) supplemented with 20% glycerol and maintained at −80 °C. To initiate the experiment, the isolates were thawed at room temperature and individually cultured in 10 mL of Brain Heart Infusion (BHI) broth (Difco, Detroit, MI, USA) at 36 °C for 24 h. To ensure the purity of colonies, each isolate was streaked onto Baird-Parker agar (Merck, Darmstadt, Germany) and incubated at 36 °C for 48 h. Subsequently, a single colony from each plate was transferred to 10 mL of BHI broth, followed by two successive 24 h cultivations at 36 °C. After incubation, the contents of the test tubes were centrifuged at 3500 rpm for 10 min at 4 °C using a centrifuge (Spin Max 80-2B; Medmax, Baueri, Brazil). The resulting supernatants were discarded, and the pellets were washed with phosphate-buffered saline (PBS) at pH 7.2, repeating this washing process twice. The cells were then resuspended in PBS using a vortex mixer. These resultant suspensions were combined in equal proportions within a sterile Erlenmeyer flask, resulting in a single pooled suspension containing all five strains. To ensure uniformity, the concentration of the pooled suspension was standardized to an optical density of 0.5 (OD₆₀₀), corresponding to 10⁸ CFU/mL, as confirmed by colony counting on Baird-Parker agar plates following a 48 h incubation at 36 °C, and measured using a spectrophotometer (Specord 200plus, Analytik Jena, Jena, Germany).

2.2. Inoculation, Pasteurization, and Sonication Treatment

Raw cream was acquired from Taquipe Agropecuária (São Sebastião do Passé, Bahia, Brazil; coordinates: 12°30′45.5″ S, 38°29′27.2″ W), with a standardized fat content of 35%. Artificial contamination was performed by introducing a bacterial suspension at a concentration of 10⁶ CFU/mL into 200 mL of milk cream. Specifically, 2 mL of the inoculum was added, followed by a 5 min stand period before proceeding with the treatment. The treatments were divided into two categories, (1) pasteurization and (2) pasteurization and sonication (thermosonication groups), resulting in a total of ten treatment variations (

Table 1. Processing parameters used in the milk cream treatments.

Treatment	Processing Parameters
	Power (W)	Pulse (s)	Energy Density (J/cm3)	Time	Temperature (°C)
IUNT	-	-	-	-	-
NIP-72	-	-	-	15 s	72
NIP-65	-	-	-	40 min	65
IP-72	-	-	-	15 s	72
IP-65	-	-	-	40 min	65
US1	31	0	78	3 s	72
US2	31	0	140	5 s	72
US3	31	0	290	10 s	72
US4	35	30	4900	5 min	65
US5	33	30	9700	10 min	65
US6	35	30	23,800	25 min	65

IUNT, inoculated and untreated; NIP-72, not inoculated and rapid pasteurization; NIP-65, not inoculated and slow pasteurization; IP-72, inoculated and rapid pasteurization; IP-65, inoculated and slow pasteurization; US1, inoculated, rapid pasteurization, and 3 s sonication; US2, inoculated, rapid pasteurization, and 5 s sonication; US3, inoculated, rapid pasteurization, and 10 s sonication; US4, inoculated, slow pasteurization, and 5 min sonication; US5, inoculated, slow pasteurization, and 10 min sonication; US6, inoculated, slow pasteurization, and 25 min sonication.

). Each sample designated for thermosonication was heated to pasteurization temperature and meticulously placed into a 250 mL jacketed beaker to maintain pasteurization temperature.

HIUS technology was employed, utilizing a 13 mm ultrasound probe with a frequency of 20 kHz and a maximum input ultrasonic power of 500 W (VC-505 Ultrasonic Processor; Sonics Materials, Newtown, CT, USA). Within 250 mL glass tubes, 200 mL of each sample underwent the sonication process, with the probe consistently positioned 20 mm below the surface of the sample in all experiments. Detailed processing parameters, including power, duration, temperature, and energy density, can be found in Table 1. To maintain a stable and controlled temperature during the thermosonication process, a jacketed beaker with a continuous water circulation system was employed. This system was linked to a thermostatic water bath (Tecnal, Model: TE2000), ensuring precise temperature control throughout the process, monitored using a calibrated thermometer. Following pasteurization and thermosonication treatments, all samples were promptly cooled in an ice-water bath maintained at a temperature of 2 ± 1 °C and then stored in a refrigerator at 4 ± 0.5 °C until analysis.

2.3. Bacterial Enumeration

To assess the efficacy of the treatments, microbiological analyses of S. aureus counts were conducted immediately after processing (day 0), adhering to the guidelines outlined by the American Public Health Association [13]. Additionally, a positive control group, consisting of an inoculated and untreated sample, was included to facilitate comparisons of log reductions. For the enumeration process, three cream portions, each weighing 10 g, were homogenized and subsequently serially diluted in 90 mL of 0.1% peptone water (Merck, Darmstadt, Germany) utilizing a stomacher (Stomacher 80, Seward, London, UK). These dilutions were then spread-plated onto Baird-Parker agar and incubated at a controlled temperature of 36 ± 1 °C for 48 h. Finally, to ensure precise quantification, electronic counting was carried out using a Flash & Go electronic counter (IUL Instruments, Barcelona, Spain). The resulting data were expressed as log CFU/mL for comprehensive reporting.

2.4. Statistical Analysis

All analyses were conducted in analytical and experimental triplicate. The results were presented as the mean ± standard deviation (SD). This was carried out using a one-way analysis of variance (ANOVA) with subsequent Tukey post hoc testing, performed at a significance level of 0.05. This statistical evaluation was accomplished using XLSTAT version 2022.1 (Addinsoft, Paris, France). Furthermore, the graphic was plotted using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA, USA).

3. Results and Discussion

The survival levels of MRSA in milk cream are exhibited in Figure 1. The initial analysis revealed that MRSA counts in the treated samples presented a reduction of up to 3.41-fold following pasteurization (4.82 log CFU/mL reduction) and 3.25-fold after thermosonication (4.72 log CFU/mL reduction), in comparison with the untreated group (IUNT). These findings emphasize the significant impact of both pasteurization and thermosonication in reducing MRSA levels, thereby enhancing the safety and quality of the cream. In addition, the treatments subjected to thermosonication, denoted as US1 to US6, exhibited no significant differences (p > 0.05) when compared with the inoculated treatments subjected to conventional pasteurization (IP-72 and IP-65) or the thermosonicated treatments themselves. This intriguing observation indicates that thermosonication showcases potential antimicrobial effectiveness on par with conventional pasteurization methods for reducing MRSA levels. Importantly, it achieves this comparable efficacy with significantly reduced processing time during cream treatment, leading to improved productivity and resource utilization.

Further, it is essential to highlight that all treatments subjected to either pasteurization or thermosonication displayed significant differences (p < 0.05) when compared with the inoculated and untreated control group (IUNT), underscoring the efficacy of both technological approaches. Thus, the absence of significant differences among these treatments suggests their equal viability as options for enhancing product safety. Furthermore, the clear disparity between these treatments and the untreated control underscores the pivotal role of these processing methods in mitigating bacterial contamination in dairy products, especially when dealing with antibiotic-resistant strains like MRSA. In the dairy industry, the battle against MRSA emerges as a significant concern, and our research demonstrates that both pasteurization and thermosonication yield statistically substantial reductions in MRSA counts in milk cream. The deliberate creation of acoustic cavitation, facilitated by the application of low-amplitude frequencies, has emerged as a pivotal mechanism for cell destruction [9]. This controlled process results in the formation of microbubbles, whose subsequent collapse generates substantial rates of micro-shearing within the cream. It is worth noting that variables such as the frequency, intensity, and duration of ultrasound exposure have surfaced as critical determinants influencing the observed effects on reducing microbial load in milk [14].

Prior investigations have explored the inactivation of S. aureus strains in dairy products, shedding light on the pursuit of enhanced food safety. Notably, Li et al. [11] reported the effects of different hurdle techniques (single ultrasound for 5 min, single heat at 63 °C for 5 min, 5 min ultrasound followed by 5 min heat, 5 min heat followed by 5 min ultrasound, and simultaneous ultrasound and heat for 5 min) at 20 kHz frequency and with a total input power of 600 W, focused on S. aureus inactivation in milk. Comparing the diverse treatment techniques to the untreated control, the authors reported up to 1.24-fold cell inactivation when simultaneous ultrasound and heat were applied. This finding not only emphasizes the synergistic potential of thermosonication but also paves the way for the present study, which builds upon this foundation to achieve even higher levels of inactivation.

Moreover, additional investigations have illuminated the substantial potential of these techniques in combating a diverse array of harmful microorganisms, offering a valuable complement to traditional technological methodologies [4]. For instance, Bernardo et al. [15] reported an optimized thermosonication process lasting 1–20 min at temperatures between 62 and 74 °C. This approach resulted in a remarkable reduction of 6.6 log CFU/mL of Shiga toxin-producing Escherichia coli in goat milk. In a separate study, Machado et al. [16] noted a reduction of less than 1 log CFU/mL of heat-resistant E. coli isolated from milk. These findings not only underscore the significance and consistency of the techniques employed herein for mitigating microbiological risks in milk cream but also provide further affirmation of ultrasound’s efficacy as a valuable tool in combatting staphylococci within the realm of dairy products. The cumulative knowledge derived from these studies reinforces the versatility and potential of these innovative approaches, offering compelling opportunities for enhancing food safety in the dairy industry and beyond. The tailored optimization of factors such as time and temperature showcases the adaptability and nuanced nature of these techniques, encouraging further exploration and refinement for real-world application.

Additionally, it is important to highlight that our analysis did not reveal any statistically significant differences (p > 0.05) among the MRSA counts in the inoculated cream subjected to rapid pasteurization (IP-72) and the non-inoculated cream samples that underwent either rapid (NIP-72) or slow (NIP-65) pasteurization, as depicted in Figure 1. This finding is particularly intriguing, as both NIP-72 and NIP-65 demonstrated a notable absence of MRSA growth. Finally, the results conclusively indicate that the synergy of these two technologies significantly reduces the pasteurization time of milk cream by 15 min, all the while maintaining the process’s efficiency. Further research and exploration of these methods throughout the storage of cream may provide valuable insights into food safety and quality control.

4. Conclusions

This study highlights the significant impact of ultrasound and heat treatment, emphasizing its viability as a complementary technology to conventional pasteurization. Thermosonication effectively reduced MRSA strains by up to 4.72 log CFU/mL in milk cream, while pasteurization reduced cell counts by 4.82 log CFU/mL. This finding is noteworthy as it demonstrates bacterial inactivation similar to conventional treatment, resulting in a significant reduction in processing time for slow pasteurization—a highly advantageous consideration for the industry. Therefore, thermosonication can be deemed a promising approach for application in cream matrices. This work paves the way for continued exploration and refinement of these methods, ultimately benefiting both consumers and the food industry by delivering safer and more resilient products. In this context, further research is needed to optimize conditions and assess the impact on the physicochemical composition and stability.