Application of Advanced Analytical Techniques for Chemical and Microbial Characterization of Cheese: Emerging Trends in Dairy Quality Assessment

Cheese are complex, diverse matrices of fermented milk with dynamic rheology. Microbial succession coupled with biochemistry impacts the flavor, texture, and quality attributes such as safety, nutrition, and authenticity. Routine evaluation of cheese relies on conventional methods are insufficient for monitoring the multitude of changes that occur due to ripening. The intersection of advanced omic technologies, molecular cell biology, and instrumental analytical chemistry has revolutionized research in dairy science and quality assurance system in modern dairy industry. The objective of this paper is to provide a comprehensive overview of innovative analytical methods of cheese evaluation and the chemical and microbial characterization of cheese. This method includes HPLC, GC-MS, HRMS, FTIR, NMR, PCR, biosensors, metagenomics, and NGS. With these technologies, the detection of dairy pathogens, as well as metabolomic profiling of peptides, detection of volatile dairy flavor compounds, and the authentication of dairy products is achieved with great sensitivity and reproducibility. The development of flavor and ripening is further analysed by the combined approaches of chemical characterization and microbial genomics. Other technologies that will likely serve to create the future boundaries of quality assurance in the dairy industry are the use of AI and analytics, blockchain for end-to-end traceability, and precision fermentation. The integration methods of analysis developed in the dairy industry stand to greatly improve food safety, proactive compliance with regulatory standards and the competitiveness of the dairy industry on a global scale.

Cheese, GC-MS, HPLC, metagenomics, biosensors, PCR, dairy, flavor profiling, food authenticity, quality control, microbiology.

Ahmed, M. E., Hammam, A. R., Ali, A. E. F., Alsaleem, K. A., Elfaruk, M. S., Kamel, D. G., & Moneeb, A. H. (2023). Measurement of carbohydrates and organic acids in varieties of cheese using high-performance liquid chromatography. Food Science & Nutrition, 11(5), 2081–2085.

Alessandria, V., Ferrocino, I., De Filippis, F., Fontana, M., Rantsiou, K., Ercolini, D., & Cocolin, L. (2016). Microbiota of an Italian Grana-like cheese during manufacture and ripening unraveled by 16S rRNA-based approaches. Applied and Environmental Microbiology, 82(13), 3988–3995.

Amamcharla, J. K., & Metzger, L. E. (2011). Development of a rapid method for the measurement of lactose in milk using a blood glucose biosensor. Journal of Dairy Science, 94(10), 4800–4809.

Ampuero, S., & Bosset, J. O. (2003). The electronic nose applied to dairy products: A review. Sensors and Actuators B: Chemical, 94(1), 1–12.

Ames, J. M., Guy, R. C., & Kipping, G. J. (2001). Effect of pH, temperature, and moisture on the formation of volatile compounds in glycine/glucose model systems. Journal of Agricultural and Food Chemistry, 49(9), 4315–4323.

Atasever, M., & Mazlum, H. (2024). Biochemical processes during cheese ripening. Veterinary Sciences and Practices, 19(3), 174–182.

Banicod, R. J. S., Tabassum, N., Jo, D. M., Javaid, A., Kim, Y. M., & Khan, F. (2025). Integration of artificial intelligence in biosensors for enhanced detection of foodborne pathogens. Biosensors, 15(10), 690.

Basilicata, M. G., Pepe, G., Sommella, E., Ostacolo, C., Manfra, M., & Campiglia, P. (2018). Peptidome profiles and bioactivity elucidation of buffalo-milk dairy products after gastrointestinal digestion. Food Research International, 105, 1003–1010.

Bertrand, E., Machado-Maturana, E., Chevarin, C., Portanguen, S., Mercier, F., Tournayre, R., & Berdagué, J. (2011). Heat-induced volatiles and odor-active compounds in a model cheese. International Dairy Journal, 21, 806–814.

Brautsch, A., Wang, Y., Graber, H. U., et al. (2016). Fast detection and quantification of four dairy propionic acid bacteria in milk samples using real-time quantitative polymerase chain reaction. International Dairy Journal, 61, 37–43.

Caligiani, A., Nocetti, M., Lolli, V., Marseglia, A., Palla, G., & Sforza, S. (2016). Development of a quantitative GC–MS method for the detection of cyclopropane fatty acids in cheese as new molecular markers for Parmigiano Reggiano authentication. Journal of Agricultural and Food Chemistry, 64(20), 4158–4164.

Cocolin, L., Dolci, P., & Rantsiou, K. (2011). Biodiversity and dynamics of meat fermentations: The contribution of molecular methods for a better comprehension of a complex ecosystem. Meat Science, 89(3), 296–302.

Collins, Y. F., McSweeney, P. L. H., & Wilkinson, M. G. (2003). Lipolysis and free fatty acid catabolism in cheese: A review of current knowledge. International Dairy Journal, 13(11), 841–866.

Curioni, P. M. G., & Bosset, J. O. (2002). Key odorants in various cheese types as determined by gas chromatography-olfactometry. International Dairy Journal, 12(12), 959–984.

De Filippis, F., La Storia, A., & Ercolini, D. (2016). Metagenomics insights into food fermentations. Microbial Biotechnology, 10(1), 91–102.

De Filippis, F., Parente, E., & Ercolini, D. (2014). Metagenomics insights into food fermentations. Microbial Biotechnology, 10(1), 91–102.

Ding, S., Chen, J., Ye, X., et al. (2017). A multiplex RT-PCR assay for S. aureus, L. monocytogenes, and Salmonella spp. detection in raw milk with pre-enrichment. Frontiers in Microbiology, 8, 989.

El-tahlawy, A. S., Alawam, A. S., Rudayn, H. A., Allam, A. A., Mahmoud, R., Abd El-Raheem, H., & Alahmad, W. (2025). Advanced analytical and digital approaches for proactive detection of food fraud as an emerging contaminant threat. Talanta Open, 12, 100499.

Eltemur, D., Morozova, K., Ceccon, A., Riccio, G. M., Robatscher, P., Oberhuber, M., & Scampicchio, M. (2025). 1H NMR detection of cyclopropane fatty acids in yoghurt as molecular markers for haymilk authentication. Journal of Food Composition and Analysis, 108623.

Ercolini, D. (2013). High-throughput sequencing and metagenomics: Moving forward in the culture-independent analysis of food microbial ecology. Applied and Environmental Microbiology, 79(10), 3148–3155.

Food and Agriculture Organization. (2024). FAOSTAT statistical database: Dairy production and products. FAOSTAT.

Fusco, V., Quero, G. M., Cho, G. S., Kabisch, J., Meske, D., Neve, H., Bockelmann, W., & Franz, C. M. A. P. (2015). Culture-dependent and culture-independent nucleic acid-based methods used in the microbial safety assessment of milk and dairy products. Comprehensive Reviews in Food Science and Food Safety, 14(4), 493–537.

Jangra, S. (2022). Development of biosensor-based technology for the detection of pathogenic microorganisms and biomolecules in dairy products. In Advances in Dairy Microbial Products (pp. 377–384). Woodhead Publishing.

Kim, S., Kim, J., Kim, M., et al. (2016). Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp., and Listeria monocytogenes in food. 3 Biotech, 6, 205.

Kim, T. G., Jeong, S. Y., & Cho, K. S. (2014). Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil. Applied Microbiology and Biotechnology, 98, 6105–6113.

Law, J. W. F., Ab Mutalib, N. S., Chan, K. G., & Lee, L. H. (2015). Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology, 5, 770.

Liu, C., Hao, J., Lu, H., Ma, P., He, Y., Xue, Y., ... & Wang, S. (2026). Metagenomic and metabolomic insights into microbial-metabolite interactions underlying flavor formation in Hebei-style soybean paste fermentation. Food Research International, 118665.

Luykx, D. M. A. M., & van Ruth, S. M. (2008). An overview of analytical methods for determining the geographical origin of food products. Food Chemistry, 107(2), 897–911.

Marseglia, A., Caligiani, A., Comino, L., Righi, F., Quarantelli, A., & Palla, G. (2013). Cyclopropyl and ω-cyclohexyl fatty acids as quality markers of cow milk and cheese. Food Chemistry, 140(4), 711–716.

McSweeney, P. L. (2004). Biochemistry of cheese ripening. International Journal of Dairy Technology, 57(2–3), 127–144.

Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335–350.

Nero, L. A., Andretta, M., Almeida, T. T., Ferreira, L. R., Camargo, A. C., Yamatogi, R. S., ... & Call, D. R. (2021). Lactic microbiota of the minas artisanal cheese produced in the Serro region, Minas Gerais, Brazil. LWT, 148, 111698.

O'Sullivan, D. J., Giblin, L., McSweeney, P. L., Sheehan, J. J., & Cotter, P. D. (2013). Nucleic acid-based approaches to investigate microbial-related cheese quality defects. Frontiers in Microbiology, 4, 1.

Paton, A. W., & Paton, J. C. (1998). Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, and hlyA. Journal of Clinical Microbiology, 36(2), 598–602.

Pillonel, L., Ampuero, S., Tabacchi, R., & Bosset, J. (2003). Analytical methods for the determination of the geographic origin of Emmental cheese: Volatile compounds by GC/MS-FID and electronic nose. European Food Research and Technology, 216(2), 179–183.

Porcellato, D., Narvhus, J., & Skeie, S. B. (2016). Detection and quantification of Bacillus cereus group in milk by droplet digital PCR. Journal of Microbiological Methods, 127, 1–6.

Poveda, J. M., Cabezas, L., & McSweeney, P. L. H. (2008). Preliminary observations on the use of headspace solid-phase microextraction for studying the aroma of Manchego cheese. International Dairy Journal, 18(5), 551–557.

Qian, M., & Reineccius, G. (2002). Identification of aroma compounds in Parmigiano Reggiano cheese by gas chromatography/olfactometry. Journal of Dairy Science, 85(6), 1362–1369.

Quigley, L., O’Sullivan, O., Beresford, T. P., Ross, R. P., Fitzgerald, G. F., & Cotter, P. D. (2012). High-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses. Applied and Environmental Microbiology, 78(16), 5717–5723.

Riccio, G. M., Eltemur, D., Fava, F., Martini-Lösch, D., Peratoner, G., Venir, E., ... & Ceccon, A. (2025). Differentiating cyclopropane fatty acids to support milk authenticity through GC–MS and NMR spectroscopy. Food Chemistry: X, 103033.

Rodriguez-Saona, L. E., & Allendorf, M. E. (2011). Use of FTIR for rapid authentication and detection of adulteration of food. Annual Review of Food Science and Technology, 2, 467–483.

Sablé, S., & Cottenceau, G. (1999). Current knowledge of soft cheeses flavor and related compounds. Journal of Agricultural and Food Chemistry, 47(12), 4825–4836.

Savas, S. (2025). For the detection and diagnosis of common pathogens. In Current Developments in Biosensors and Emerging Smart Technologies (Vol. 12, p. 267).

Smit, G., Smit, B. A., & Engels, W. J. M. (2005). Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese. FEMS Microbiology Reviews, 29(3), 591–610.

Tarapoulouzi, M., Kyriacou, S., Ioannidis, I., Pashalidis, I., & Theocharis, C. R. (2026). Fourier-transformed infrared (FTIR) spectroscopy for detecting milk powder addition in Cyprus goat milk: A preliminary study for future adulteration detection in halloumi cheese. Quality Assurance and Safety of Crops & Foods, 18(1), 74–81.

Tekin, A., & Hayaloglu, A. A. (2023). Understanding the mechanism of ripening biochemistry and flavour development in brine ripened cheeses. International Dairy Journal, 137, 105508.

Tilocca, B., Costanzo, N., Morittu, V. M., Spina, A. A., Soggiu, A., Britti, D., Roncada, P., & Piras, C. (2020). Milk microbiota: Characterization methods and role in cheese production. Journal of Proteomics, 210, 103534.

Tsouggou, N., Korozi, E., Pemaj, V., Drosinos, E. H., Kapolos, J., Papadelli, M., ... & Papadimitriou, K. (2026). Advances in shotgun metagenomics for cheese microbiology: From microbial dynamics to functional insights. Foods, 15(2), 259.

United States Department of Agriculture. (2024a). Dairy data: Per capita consumption of dairy products. Economic Research Service.

United States Department of Agriculture. (2024b). Dairy: World markets and trade. Foreign Agricultural Service.

Vera-Bravo, R., Hernández, A. V., Peña, S., Alarcón, C., Loaiza, A. E., & Celis, C. A. (2022). Cheese whey milk adulteration determination using casein glycomacropeptide as an indicator by HPLC. Foods, 11(20), 3201.

Wolfe, B. E., Button, J. E., Santarelli, M., & Dutton, R. J. (2014). Cheese rind communities provide tractable systems for in situ and in vitro studies of microbial diversity. Cell, 158(2), 422–433.

Xu, Y., Chai, Y., & Wang, Y. (2022). Artificial intelligence and machine learning applications in dairy food processing and quality assessment: A review. Trends in Food Science & Technology, 122, 256–273.

Zheng, X., Shi,X., & Wang, B. (2021). A review on the general cheese processing technology, flavor biochemical pathways and the influence of yeasts in cheese. Frontiers in Microbiology, 12, 703284.