Journal of Food and Nutrition Research
ISSN (Print): 2333-1119 ISSN (Online): 2333-1240 Website: Editor-in-chief: Prabhat Kumar Mandal
Open Access
Journal Browser
Journal of Food and Nutrition Research. 2023, 11(3), 185-192
DOI: 10.12691/jfnr-11-3-3
Open AccessArticle

Microbiological Assessment of Pasteurized Milk in Japan by Different Testing Methods

Hodaka Suzuki1, , Amalia Widya Rizky Sahoed1, Rikako Midoh1 and Yumiko Okada2

1College of Agriculture, Ibaraki University, Ami, Ibaraki, Japan

2Division of Biomedical Food Research, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan

Pub. Date: March 10, 2023

Cite this paper:
Hodaka Suzuki, Amalia Widya Rizky Sahoed, Rikako Midoh and Yumiko Okada. Microbiological Assessment of Pasteurized Milk in Japan by Different Testing Methods. Journal of Food and Nutrition Research. 2023; 11(3):185-192. doi: 10.12691/jfnr-11-3-3


A total of 73 samples of pasteurized milk retailed in Japan, which comprised 47 low-temperature long-time pasteurized samples (LTLT; 63–66°C, 30 min), 13 high-temperature short-time pasteurized samples (HTST; 72–79°C, 15 sec), and 13 high-temperature long-time pasteurized samples (HTLT; 75–85°C, 15–30 min), were analyzed for hygiene indicator microorganisms (standard plate counts [SPCs], coliforms, Enterobacteriaceae, Escherichia coli, and coagulase-positive Staphylococcus aureus) to assess microbiological quality using the Japanese Official Method (JOM), International Standard Organization Methods (ISO), and commercial dehydrated medium sheets. Of the 73 milk samples, one LTLT milk sample was positive for both coliforms and Enterobacteriaceae, and another LTLT milk sample exceeded the Japanese microbiological criterion for the SPC (≤50,000 colony forming units (CFU)/mL). All the samples tested were negative for E. coli and S. aureus. The SPC values obtained using ISO 4833-1:2013 (incubation at 30°C for 72 h) were significantly higher than those obtained using JOM (incubation at 32°C for 48 h); however, a high correlation was observed (R² = 0.7130). In contrast, the correlations between SPCs based on JOM and the two types of dehydrated medium sheets were relatively low (R² = 0.1023 and 0.4046, respectively) although these products obtained third-party certification for analyzing many food products. Our results indicate the importance of verifying alternative methods when new methods are introduced for testing new types or categories of food samples.

dehydrated medium sheet hygiene indicator method comparison pasteurized milk

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit


[1]  Asao, T., Kumeda, Y., Kawai, T., Shibata T, Oda, H., Haruki, K., Nakazawa, H. and Kozaki, S. “An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk,” Epidemiology and Infection, 130(1). 33-40. 2003.
[2]  De Buyser, M.L., Dufour, B., Maire, M. and Lafarge, V. “Implication of milk and milk products in food-borne diseases in France and in different industrialised countries,” International Journal of Food Microbiology, 67(1-2), 1-17. 2001.
[3]  Gruber, J.F., Morris, S., Warren, K. A., Kline, K. E., Schroeder, B., Dettinger, L., Husband, B., Pollard, K., Davis, C., Miller, J., Weltman, A., Mattioli, M., Ray, L., Tarr, C. and Longenberger, A.H, “Yersinia enterocolitica outbreak associated with pasteurized milk,” Foodborne Pathogens and Disease, 18(7), 448-454. 2021.
[4]  Heidinger, J.C., Winter, C.K. and Cullor, J.S. “Quantitative microbial risk assessment for Staphylococcus aureus and Staphylococcus enterotoxin A in raw milk,” Journal of Food Protection, 72(8), 1641-1653. 2009.
[5]  Masiello, S.N., Martin, N.H., Trmčić, A., Wiedmann, M. and Boor, K.J. “Identification and characterization of psychrotolerant coliform bacteria isolated from pasteurized fluid milk,” Journal of Dairy Science, 99(1), 130-140. 2016.
[6]  Ministry of Health and Welfare, Ministry of Health and Welfare Ordinance No. 52, “Ministerial Ordinance on Milk and Milk products Concerning Compositional Standards, etc.” 1951. (in Japanese)
[7]  Food and Drug Administration (FDA), Grade “A” Pasteurized Milk Ordinance 2019 Revision. 2020. [accessed on December 25, 2022].
[8]  European Union (EU), COMMISSION REGULATION (EU) No 365/2010 amending Regulation (EC) No. 2073/2005 on microbiological criteria for foodstuffs as regards Enterobacteriaceae in pasteurised milk and other pasteurised liquid dairy products and Listeria monocytogenes in food grade salt. 2010. [accessed on December 25, 2022].
[9]  Linton, R.H., Eisel, W.G. and Muriana, P.M. “Comparison of conventional plating methods and Petrifilm for the recovery of microorganisms in a ground beef processing facility,” Journal of Food Protection, 60(9), 1084-1088. 1997.
[10]  Takagi, K., Yoda, K., Miyazawa, K., Harata, G., He, F., Hiramatsu, M. “Physicochemical properties and sensory attributes of LTLT milk and UHT milk,” Japanese Journal of Sensory Evaluation, 20: 10-15. 2016.
[11]  International Organization for Standardization. ISO 4833-1:2013. Microbiology of the food chain-Horizontal method for the enumeration of microorganisms-Part 1: Colony count at 30°C by the pour plate technique. 2013.
[12]  International Organization for Standardization. ISO 21528-1:2017. Microbiology of the food chain-Horizontal method for the detection and enumeration of Enterobacteriaceae-Part 1: Detection of Enterobacteriaceae. 2017.
[13]  International Organization for Standardization. ISO 21528-2:2017. Microbiology of the food chain-Horizontal method for the detection and enumeration of Enterobacteriaceae-Part 2: Colony-count technique. 2017.
[14]  Ministry of Health and Welfare, Standards set by the Ministry of Health notice No. 370. 1959.
[15]  International Organization for Standardization. ISO 16649-2:2001. Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of b -glucuronidase-positive Escherichia coli-Part 2: Colony-count technique at 44 using 5-bromo-4-chloro-3-indolyo b-D-glucuronidase. 2001.
[16]  International Organization for Standardization. ISO 6888-1:1999. Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species)-Part 1: Technique using Baird-Parker agar medium. 1999.
[17]  Ludbrook, J. “Comparing methods of measurements,” Clinical and Experimental Pharmacology and Physiology, 24(2), 198-203. 1997.
[18]  R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2021. URL
[19]  Angelidis, A.S., Tsiota, S., Pexara, A. and Govaris, A. “The microbiological quality of pasteurized milk sold by automatic vending machines,” Letters in Applied Microbiology, 62(6), 472-479. 2016.
[20]  Valík, Ľ., Görner, F. and Laukova, D. “Growth dynamics of Bacillus cereus and shelf-life of pasteurised milk,” Czech Journal of Food Sciences, 21(6), 195-202. 2003.
[21]  Freitas, R., Nero, L. A. and Carvalho, A. F. “Enumeration of mesophilic aerobes in milk: Evaluation of standard official protocols and Petrifilm aerobic count plates,” Journal of Dairy Science, 92(7), 3069-3073. 2009
[22]  Thomas, P., Mujawar, M. M., Sekhar, A. C. and Upreti, R. “Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms,” Journal of Applied Microbiology, 116(4), 911-922. 2014.
[23]  Donnelly, C.B., Gilchrist, J.E., Peeler, J.T. and Campbell, J.E. “Spiral plate count method for the examination of raw and pasteurized milk,” Applied and Environmental Microbiology, 32(1), 21-27. 1976.
[24]  Alonso-Calleja, C., Carballo, J., Capita, R., Bernardo, A. and García-López, M.L. “Evaluation of the spiral plating method for the enumeration of microorganisms throughout the manufacturing and ripening of a raw goat's milk cheese,” Journal of Food Protection, 65(2), 339-344. 2002.
[25]  Ginn, R.E., Packard, V.S. and Fox, T.L. “Evaluation of the 3M dry medium culture plate (Petrifilm™ SM) method for determining numbers of bacteria in raw milk,” Journal of Food Protection, 47(10), 753-756. 1984.
[26]  Casillas-Buenrostro, R.M., Heredia, N.L., Benesh, D.L. and García, S. “Efficacy of 3M™ Petrifilm™ aerobic count plates for enumerating Bacillus sporothermodurans and Geobacillus stearothermophilus in UHT milk,” International Dairy Journal, 25(2), 147-149. 2012.
[27]  International Organization for Standardization. ISO 16140-3. Microbiology of the food chain-Method validation-Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory. 2021.