Combined Ultrasonic and Thermal Treatment: Effects on Microbial Reduction and Protein Stability in Fresh Milk
Abstract
Purpose of the study: This study aims to evaluate the effect of ultrasonic treatment combined with temperature variation on the inhibition of Escherichia coli and the preservation of protein content in fresh cow’s milk under controlled experimental conditions.
Methodology: Experimental design; ultrasonic generator (60 kHz, 70 W); water bath temperature control (30°C, 40°C, 50°C); incubator shaker; laminar air flow; Total Plate Count (TPC) method; Kjeldahl method; colony counter; Nutrient Agar (NA) and Nutrient Broth (NB); two-way ANOVA; descriptive statistics; statistical software (SPSS).
Main Findings: Ultrasonic treatment combined with temperature significantly reduced Escherichia coli counts, with the highest reduction (95.9%) achieved at 50°C for 30 minutes. Bacterial counts decreased progressively with increasing temperature and exposure time. Protein content remained relatively stable, ranging from 2.02% to 2.20%, indicating minimal degradation under treatment conditions.
Novelty/Originality of this study: This study presents an integrated and statistically validated approach to simultaneously analyze microbial inactivation and protein stability using combined ultrasonic and thermal treatments. It demonstrates a synergistic interaction between acoustic cavitation and moderate temperature, offering a novel framework linking physical wave principles with biological systems in food processing.
References
C. Lauteri, G. Ferri, A. Piccinini, L. Pennisi, and A. Vergara, “Ultrasound technology as inactivation method for foodborne pathogens: A review,” 2023. doi: 10.3390/foods12061212.
D. Grace, “Burden of foodborne disease in low-income and middle-income countries and opportunities for scaling food safety interventions,” Food Secur., vol. 15, no. 6, pp. 1475–1488, 2023, doi: 10.1007/s12571-023-01391-3.
C. C. Adley and M. P. Ryan, “Chapter 1 - The Nature and Extent of Foodborne Disease,” J. B. T.-A. F. P. (Second E. Barros-Velázquez, Ed., San Diego: Academic Press, 2025, pp. 3–14. doi: 10.1016/B978-0-323-90747-7.00002-8.
K. Rathnakumar et al., “Ultrasonic processing: effects on the physicochemical and microbiological aspects of dairy products,” Crit. Rev. Biotechnol., vol. 44, no. 8, pp. 1638–1652, Nov. 2024, doi: 10.1080/07388551.2024.2332941.
A. S. El-Sayed, H. Ibrahim, and M. A. Farag, “Detection of potential microbial contaminants and their toxins in fermented dairy products: A comprehensive review,” Food Anal. Methods, vol. 15, no. 7, pp. 1880–1898, 2022, doi: 10.1007/s12161-022-02253-y.
E. A. Alexa, A. Papadochristopoulos, T. O’Brien, and C. M. Burgess, “Chapter 1 - Microbial contamination of food,” A. K. Jaiswal and S. B. T.-F. P. and P. Shankar, Eds., Academic Press, 2024, pp. 3–19. doi: 10.1016/B978-0-323-90044-7.00001-X.
S. Karanth, S. Feng, D. Patra, and A. K. Pradhan, “Linking microbial contamination to food spoilage and food waste: the role of smart packaging, spoilage risk assessments, and date labeling,” Front. Microbiol., vol. Volume 14-2023, 2023, doi: 10.3389/fmicb.2023.1198124
O. Misiou and K. Koutsoumanis, “Climate change and its implications for food safety and spoilage,” Trends Food Sci. Technol., vol. 126, pp. 142–152, 2022, doi: 10.1016/j.tifs.2021.03.031.
G. K. Mahunu, M. Osei-Kwarteng, M. C. Ogwu, and N. A. Afoakwah, “Safe Food Handling Techniques to Prevent Microbial Contamination BT - Food Safety and Quality in the Global South,” M. C. Ogwu, S. C. Izah, and N. R. Ntuli, Eds., Singapore: Springer Nature Singapore, 2024, pp. 427–461. doi: 10.1007/978-981-97-2428-4_14.
B. Mengstu et al., “Evaluation of microbial hygiene indicators in raw milk, pasteurised milk and cottage cheese collected across the dairy value chain in Ethiopia,” Int. Dairy J., vol. 136, p. 105487, 2023, doi: 10.1016/j.idairyj.2022.105487.
A. Deddefo, G. Mamo, M. Asfaw, and K. Amenu, “Factors affecting the microbiological quality and contamination of farm bulk milk by Staphylococcus aureus in dairy farms in Asella, Ethiopia,” BMC Microbiol., vol. 23, no. 1, p. 65, 2023, doi: 10.1186/s12866-022-02746-0.
D. T. Robi, T. Mossie, and S. Temteme, “A comprehensive review of the common bacterial infections in dairy calves and advanced strategies for health management,” Vet. Med. Res. Reports, vol. 15, no. null, pp. 1–14, Dec. 2024, doi: 10.2147/VMRR.S452925.
M. A. Alhadlaq et al., “Overview of pathogenic Escherichia coli, with a focus on Shiga toxin-producing serotypes, global outbreaks (1982–2024) and food safety criteria,” Gut Pathog., vol. 16, no. 1, p. 57, 2024, doi: 10.1186/s13099-024-00641-9.
A. A. Akinsemolu and H. N. Onyeaka, “Microorganisms Associated with Food Spoilage and Foodborne Diseases BT - Food Safety and Quality in the Global South,” M. C. Ogwu, S. C. Izah, and N. R. Ntuli, Eds., Singapore: Springer Nature Singapore, 2024, pp. 489–531. doi: 10.1007/978-981-97-2428-4_16.
M. Mkangara, “Prevention and control of human salmonella enterica infections: An implication in food safety,” Int. J. Food Sci., vol. 2023, no. 1, p. 8899596, Jan. 2023, doi: 10.1155/2023/8899596.
A. Kaushik, N. K. Taneja, V. K. Juneja, J. K. Salazar, A. Joshi, and H. S. Oberoi, “Enhancing microbial safety and quality of milk with ultrasonication: Kinetics modeling of pathogenic Bacteria and milk characteristics,” LWT, vol. 202, p. 116287, 2024, doi: 10.1016/j.lwt.2024.116287.
E. J. Sarba, W. Wirtu, E. Z. Gebremedhin, B. M. Borena, and L. M. Marami, “Occurrence and antimicrobial susceptibility patterns of Escherichia coli and Escherichia coli O157 isolated from cow milk and milk products, Ethiopia,” Sci. Rep., vol. 13, no. 1, p. 16018, 2023, doi: 10.1038/s41598-023-43043-8.
B. Gume et al., “Bacterial hazard identification and exposure assessment of raw milk consumption in Jimma zone, South West Ethiopia,” BMC Microbiol., vol. 23, no. 1, p. 166, 2023, doi: 10.1186/s12866-023-02910-0.
D. Bermudez-Aguirre and B. A. Niemira, “Pasteurization of foods with ultrasound: The present and the future,” 2022. doi: 10.3390/app122010416.
M. Azizi-Lalabadi, N. R. Moghaddam, and S. M. Jafari, “9 - Pasteurization in the food industry,” S. M. B. T.-T. P. of F. P. by S. and H. W. Jafari, Ed., Woodhead Publishing, 2023, pp. 247–273. doi: 10.1016/B978-0-12-818616-9.00009-2.
K. K. Dash et al., “A comprehensive review on heat treatments and related impact on the quality and microbial safety of milk and milk-based products,” Food Chem. Adv., vol. 1, p. 100041, 2022, doi: 10.1016/j.focha.2022.100041.
N. Nikmaram and K. M. Keener, “The effects of cold plasma technology on physical, nutritional, and sensory properties of milk and milk products,” LWT, vol. 154, p. 112729, 2022, doi: 10.1016/j.lwt.2021.112729.
I. Neoκleous, J. Tarapata, and P. Papademas, “Non-thermal processing technologies for dairy products: Their effect on safety and quality characteristics,” Front. Sustain. Food Syst., vol. Volume 6-, 2022, doi: 10.3389/fsufs.2022.856199.
D. Verma et al., “Feasibility of pulse protein in the formulation of dairy alternatives: a review of nutri-functional attributes, applications, processing, and sustainability impact,” Nutrire, vol. 50, no. 1, p. 12, 2025, doi: 10.1186/s41110-025-00315-9.
A. Karim et al., “1 - Introduction to thermal food processes by steam and hot water,” S. M. B. T.-T. P. of F. P. by S. and H. W. Jafari, Ed., Woodhead Publishing, 2023, pp. 3–26. doi: 10.1016/B978-0-12-818616-9.00001-8.
Ž. Pandur, J. Zevnik, D. Podbevšek, B. Stojković, D. Stopar, and M. Dular, “Water treatment by cavitation: Understanding it at a single bubble - bacterial cell level,” Water Res., vol. 236, p. 119956, 2023, doi: 10.1016/j.watres.2023.119956.
A. T. Mustapha, H. Wahia, Q. Ji, O. A. Fakayode, L. Zhang, and C. Zhou, “Multiple-frequency ultrasound for the inactivation of microorganisms on food: A review,” J. Food Process Eng., vol. 47, no. 4, p. e14587, Apr. 2024, doi: 10.1111/jfpe.14587.
M. Siddique, R. Rashid, and A. Ali, “Chapter 1 - Fundamentals of acoustic cavitation, ultrasound-assisted processes, and sonochemistry,” K. B. T.-M. and S. of S.-P. Kerboua, Ed., Elsevier, 2025, pp. 3–17. doi: 10.1016/B978-0-443-23651-8.00001-2.
A. Gholamhosseinpour, S. M. B. Hashemi, and H. Khosravi Mazydi, “Ultrasound-assisted enhancement of microbial, physicochemical, and bioactive properties in Lactobacillus helveticus-fermented milk,” Food Chem. X, vol. 29, p. 102724, 2025, doi: 10.1016/j.fochx.2025.102724.
Y. Alves de Aguiar Bernardo, D. Kaic Alves do Rosario, and C. Adam Conte-Junior, “Ultrasound on milk decontamination: Potential and limitations against foodborne pathogens and spoilage bacteria,” Food Rev. Int., vol. 39, no. 1, pp. 320–333, Jan. 2023, doi: 10.1080/87559129.2021.1906696.
S. Condón-Abanto, C. Arroyo, I. Álvarez, S. Condón, and J. G. Lyng, “Application of ultrasound in combination with heat and pressure for the inactivation of spore forming bacteria isolated from edible crab (Cancer pagurus).,” Int. J. Food Microbiol., vol. 223, pp. 9–16, Apr. 2016, doi: 10.1016/j.ijfoodmicro.2016.02.001.
M. Singla and N. Sit, “Application of ultrasound in combination with other technologies in food processing: A review.,” Ultrason. Sonochem., vol. 73, p. 105506, May 2021, doi: 10.1016/j.ultsonch.2021.105506.
B. Khadhraoui, V. Ummat, B. K. Tiwari, A. S. Fabiano-Tixier, and F. Chemat, “Review of ultrasound combinations with hybrid and innovative techniques for extraction and processing of food and natural products,” Ultrason. Sonochem., vol. 76, p. 105625, 2021, doi: 10.1016/j.ultsonch.2021.105625.
X. Chen et al., “A comparative study of ultrasound and thermal processing: Effects on goat milk stability and protein structure,” J. Dairy Sci., vol. 108, no. 4, pp. 3273–3286, 2025, doi: 10.3168/jds.2024-25918.
A. T. Hong Bui, D. Cozzolino, B. Zisu, and J. Chandrapala, “Infrared analysis of ultrasound treated milk systems with different levels of caseins, whey proteins and fat,” Int. Dairy J., vol. 117, p. 104983, 2021, doi: 10.1016/j.idairyj.2021.104983.
I. Lambropoulos and I. G. Roussis, “Inhibition of the decrease of volatile esters and terpenes during storage of a white wine and a model wine medium by caffeic acid and gallic acid,” Food Res. Int., vol. 40, no. 1, pp. 176–181, 2007, doi: 10.1016/j.foodres.2006.09.003.
S. H. K., P. Adhikari, A. E. Yasmeen Shaikh, and S. S. Arya, “Hydrodynamic and ultrasonic cavitation physically modifies the milk protein concentrates with improved functionality,” Sustain. Food Technol., vol. 4, no. 1, pp. 1004–1020, 2026, doi: 10.1039/D5FB00393H.
Y. Sun et al., “Influence of milk fat globule membrane and milk protein concentrate treated by ultrasound on the structural and emulsifying stability of mimicking human fat emulsions.,” Ultrason. Sonochem., vol. 82, p. 105881, Jan. 2022, doi: 10.1016/j.ultsonch.2021.105881.
A. Prosekov, “The effect of ultrasound on the functionality and health-improving properties of dairy products (review),” Food Prod. Process. Nutr., vol. 7, no. 1, p. 36, 2025, doi: 10.1186/s43014-025-00311-6.
O. Krasulya, S. Shestakov, V. Bogush, I. Potoroko, P. Cherepanov, and B. Krasulya, “Applications of sonochemistry in Russian food processing industry,” Ultrason. Sonochem., vol. 21, no. 6, pp. 2112–2116, 2014, doi: 10.1016/j.ultsonch.2014.03.015.
S. Manickam et al., “Ultrasonics and sonochemistry: Editors’ perspective,” Ultrason. Sonochem., vol. 99, p. 106540, 2023, doi: 10.1016/j.ultsonch.2023.106540.
D. Fernandez Rivas, P. Cintas, J. Glassey, and D. C. Boffito, “Ultrasound and sonochemistry enhance education outcomes: From fundamentals and applied research to entrepreneurial potential.,” Ultrason. Sonochem., vol. 103, p. 106795, Feb. 2024, doi: 10.1016/j.ultsonch.2024.106795.
Copyright (c) 2026 Mei Chi Lee, Atul Handayani, Sing Huat Po
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and acknowledge that the Schrödinger: Journal of Physics Education is the first publisher licensed under a Creative Commons Attribution 4.0 International License.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges and earlier and greater citation of published work.
.png)
.png)
.png)
.png)
.png)
