Subscribe Now Subscribe Today
Research Article

Microbiological Quality of Heat-Treated Milk During Storage

Nagla Bourae Ahmed Hassan, Mohamed Osman Mohamed Abdalla and Abdel Azim Ahmed Mohamed Nour
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

This study was carried out to evaluate the microbial load of heat treated milk during storage. Collected raw milk samples were subjected to heat treatment at 98oC for 1.87 min (High Pasteurization, HP) and 85oC for 40 min (Low Pasteurization, LP) in addition to Ultra High Temperature Treated (UHT) milk samples which were purchased from local market. Milk samples were subjected to microbial examination, titratable acidity and pH at 1, 10, 20 and 30 day intervals. Results indicated that total bacteria and lactic acid bacteria count significantly increased with time during storage period, together with increase in titratable acidity of all samples tested, while pH gradually decreased towards the end. The following genera were isolated from milk samples: Bacillus, Micrococcus, Saphylococcus, Enterobacter, Pseudomonas, Streptococcus, Pediococcus and Lactobacillus.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

Nagla Bourae Ahmed Hassan, Mohamed Osman Mohamed Abdalla and Abdel Azim Ahmed Mohamed Nour, 2009. Microbiological Quality of Heat-Treated Milk During Storage. Pakistan Journal of Nutrition, 8: 1845-1848.

DOI: 10.3923/pjn.2009.1845.1848



From farm to fork, milk shelf life was a responsibility shared by producers, processors, retailers and consumers. Extension of shelf life from hours to months has been a prime objective of the dairy industry for many years to meet the demands for increasing distribution times and distances (Goff and Griffiths, 2006). Fluid milk processors would like to achieve 60-90 days of refrigerated shelf life for High Temperature Short Time (HTST) pasteurized milk to allow more efficient marketing and distribution of product. Ultra pasteurization is one approach to do this but consumers do not like heat induced off flavors associated with high heat treatment and would prefer high temp short time treated milk (Champan and Boor, 2001).

During cold storage after milk collection, psycrotrophic bacterial populations dominate the microflora and their extracellular enzymes, mainly proteases and lipases contribute to the spoilage of dairy products (Hantsis-Zacharov and Halpern, 2007). The numbers of psychrotrophs that develop after milk collection depends on the storage temperature and time. Under sanitary conditions <10% of the total microflora is psychrotrophs in contrast to >75% under unsanitary conditions (Cousin, 1982).

Pathogens involved in foodborne outbreaks associated with consumption of milk include Salmonella, Listeria monocytogens, Campylobacter, Staphylococcus aureus, Bacillus cereus and Clostridium botulinum, beside other most common contagious mastitis pathogens such as Staphylococcus aureus, Streptococcus agalactiae and Mycoplasma bovis (Ryser, 1998; Zadoks, 2003).

Extended shelf life products, with shelf life of 21-28 days at refrigerated temperature, through normal pasteurization and shelf stable milk products through Ultra High Temperature (UHT) continuous flow sterilization, are available (Goff and Griffiths, 2006). The conditions of heat treatment used for pasteurization depend on the final product, lower temperatures are used for refrigerated products and higher heat treatments are used for products stored at room temperature (USCFR, 2006).

The main objective of milk heat treatment is to eliminate inborn pathogenic organisms or reduce them to a level safe for human consumption during extended-shelf life. The effectiveness of heat treatment depends on type of microorganisms present and concentration of each (CFDRA, 1992).

Heat treatment systems are vat treatment (85oC for 10-40 min), high temperature short time (HTST; 98oC for 0.5-1.87 min) and ultra high temperature (UHT; 140oC for 2-8 sec) (Parnell-Cluniess et al., 1986).

This investigation was carried out to evaluate the microbiological quality of heat treated milk during storage and isolate some microorganisms.

Source of milk:
Raw milk samples were obtained from Khartoum University farm and two private farms, one in Omdurman and the second in Khartoum North. Sixty four cow milk samples were collected during February-March, kept in sterilized screw-capped bottles (100 ml) and transported to the laboratory in ice boxes at <6oC. Ultra High Temperature Treated (UHT) samples were purchased from Khartoum supermarkets.

Heat treatment of milk samples: Milk samples were heat treated in glass containers, using temperature-adjusted water bath. Heat treatment involved two time-temperature combinations: 85oC for 40 min (Low Pasteurization, LP) and 98oC for 1.87 min (High Pasteurization, HP). Samples were cooled immediately and stored at 4oC and microbiological examination was carried out at day 1, 10, 20 and 30.

Microbiological examination: Milk samples were microbiologically examined for total bacteria count and lactic acid bacteria count as follows:

Total bacteria count was determined using Nutrient agar medium (Houghtby et al., 1992). The liquefied medium (45-46oC) was poured into each Petri dish and mixed thoroughly and dishes were inverted and incubated at 32oC for 48 h.

Lactic acid bacteria count was determined using MRS agar medium. Plates were incubated at 37oC for 72 h under anaerobic conditions using anaerobic jars. Plates containing 30-300 colonies were then enumerated (Harrigan, 1998).

Identification of bacteria (Biochemical tests): Gram staining, Catalase test, Oxidase test, Motility test, Oxidation Fermentation (O/F) test and Endospore staining were used as primary tests to identify bacterial isolates to the genus level (Barrow and Feltham, 1993):

Titrable acidity test was determined according to AOAC (1990), while pH of milk was determined using pH meter (Hanna-instrument).

Statistical analysis: Data were analyzed using Statistical Analysis Systems (SAS). Significant differences between means were determined by Duncan ’s Multiple Range Test at p<0.05.


Heat treatment had significant (p<0.01) effect in reducing the bcaterial load, however, no significant differences were observed at the beginning of storage period, but later with progressing storage period a significant difference was noted. Total bacterial count increased from Log10 1.06 ±0.00, 2.35 ±1.17 and 2.04 ±1.52 at day one to Log10 2.9 ±0.14 and 6.41 ±0.35, 7.17 ±0.07 at day 30 and lactic acid bacteria increased from Log10 1.09 ±0.00, 1.53 ±1.26 and 1.77 ±1.07 at day one to Log10 3.26 ±0.38, 4.84 ± 1.94 and 7.00 ± 0.19 at day 30 for UHT, HP and LP respectively.

The above results are in agreement with the findings of Sahan et al. (1996) who found that heat treatment had significant (p<0.01) effect on the total bacterial count. Korhonen et al. (1998) reported that heat sterilization of milk is essential to ensure total microbial safety and stability of enzymic activity. Our data are also in accord with Holsinger et al. (1997) who concluded that standard pasteurization is effective for the destruction of pathogens in raw milk, however microbial population counted significant beyond day 20 indicating that injury and recovery time preceded growth.

This is asserted by Buchanan and Klawitter (1991) who reported that, the smaller the degree of difference between the old and new environment, the shorter the period of lag phase, during which microbial cells adjust to new environments prior to growth.

Our results are in full agreement with the findings of Ziarno et al. (2005) who concluded that, the changes in total bacterial count in milk were not observed before 7-14 days of storage at 4oC and then a significant change was observed on day 21 of storage at the same conditions.

Table 2 shows that titratable acidity was 0.15 ±0.00, 0.16 ±0.00 and 0.17 ±0.00 at day one and then increased to 0.19 ±0.00, 0.22 ±0.00 and 0.25 ±0.00 at day 30. The significant increase (p<0.01) in acidity corresponded by significant lowering in pH from 6.69 ±0.00, 6.69 ±0.00 and 6.68 ±0.02 at day one to 6.64 ±0.01, 6.21 ±0.13 and 5.83 ±0.14 at day 30 for UHT, HP and LP respectively. It was evident that, acidity was higher at the end of the storage and this was attributed to increased lactic acid producing bacteria, particularly streptococci and lactobacilli, which ferment lactose into lactic acid.

Table 1:

Effect of heat treatment and storage conditions on total bacteria count and lactic acid bacteria count (Log cfu/ml)

Means bearing same superscripts in row (lower case) and column (upper case) are not significantly different (p>0.05); UHT = Ultra High Temperature, HP = High Pasteurized (98oC for 1.87 min), LP = Low Pasteurized (85oC for 40 min), d = day, cuf/ml = colony forming unit per milliliter

Table 2:

Effect of heat treatment and storage conditions on titratable acidity and pH value of milk

Means bearing same superscripts in row (lower case) and column (upper case) are not significantly different (p>0.05); UHT = Ultra High Temperature, HP = High Pasteurized (98oC for 1.87 min), LP = Low Pasteurized (85oC for 40 min), d = day

Table 3:

Identification of bacterial isolates from High Pasteurized (HP) samples

Table 4:

Identification of bacterial isolates from Low Pasteurized (LP) samples

Table 5:

Identification of lactic acid bacteria isolates

+ = Postitive; - = Negative; O = Oxidative; F = Fermentative

The above results are in agreement with the study of Parky (1991) who showed that titrable acidity was similar when milk was subjected to different heat treatments. Kuippers et al. (2000) reported that, the antimicrobial effect of lactic acid bacteria was mainly due to their lactic acid production, causing the pH of the growth environment to decrease. Moreover, defects were detected when microbial concentration reached 5 x 105-107 cfu/ml (Vyletelova et al., 2000).

Bacteria were isolated post heat treatments, then identified (only detected colonies using primary biochemical tests. Bacillus, Staphylococcus, Micrococcus, Enterobacter, Pseudomonas, Streptococcus, Pediococcus and Lactobacillus were isolated from heat treated milk (Table 3, 4 and 5).

Our findings are in agreement with the findings of Ruegg and Reinemann (2002) who concluded that thermoduric bacteria such as Bacillus, Clostridium, Micrococcus, Microbacterium, Lactobacillus and occasionally streptococci, can retain their activity and affect the quality of post pasteurized products.

O'connor (1999) reported that, the species of bacteria found in milk as it comes from the udder are limited to few genera, the micrococci are generally present in the greatest proportion followed by streptococci and rods.

Cousin (1982) reported that psychrotrophic bacteria from numerous genera have been isolated from milk such as Pseudomonas, Bacillus, Mmicrococcus and Lactobacillus.

Our results are also in agreement with the findings of Anonymous (1994) who concluded that, pasteurization does not destroy all pathogenic microorganisms, but reduces the number to a level at which they don ’t constitute a significant health hazard.

The study concludes that heat treatment of milk did not significantly affect the number of total bacteria as well as lactic acid bacteria. However, during shelf life the count significantly increased. Different bacteria including lactic acid, spoilage as well as pathogenic were isolated following heat treatment of milk indicating unhygienic conditions during production, processing and storage.

1:  Anonymous, 1994. Recommendations for the hygienic manufacture of milk and milk based products. Int. Dairy Fed. Brussels Belgium IDF Bull., 292: 32-32.

2:  Barrow, G.I. and R.K. Feltham, 1993. Cowan and Steel Manual for the Identification of Medical Bacteria. 3rd Edn., Cambridge University Press, Cambridge, UK.

3:  Buchanan, R.L. and L.A. Klawitter, 1991. Effect of temperature history on the growth of L. monocytogenes Scott A at refrigeration temperatures. Int. J. Food Microbiol., 12: 235-245.
PubMed  |  Direct Link  |  

4:  CFDRA, 1992. Campden food and drink research association. Food Pasteurization. Technical Manual No. 27, UK.

5:  Champan, K.W. and K.J. Boor, 2001. Acceptance of 2% ultra pasteurized milk by consumers, 6-11 years old. J. Dairy Sci., 84: 951-954.

6:  Cousin, M.A., 1982. Presence and activity of psychrotrophic microorganisms in milk and dairy products. J. Food Protein, 45: 172-207.

7:  Goff, H.D. and M.W. Griffiths, 2006. Major advances in fresh milk and milk products. J. Dairy Sci., 89: 1163-1173.
Direct Link  |  

8:  Hantsis-Zacharov, E. and M. Halpern, 2007. Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits. Applied Environ. Microbiol., 73: 7162-7168.
CrossRef  |  Direct Link  |  

9:  Harrigan, W.F., 1998. Laboratory Methods in Food Microbiology. 3rd Edn., Academic Press, San Diego, Carlifornia, ISBN: 0-12-326043-4, pp: 36-84.

10:  Holsinger, V.H., K.T. Rajkowski and J.R. Stabel, 1997. Milk pasteurization and safety: A brief history and update. Rev. Sci. Technol., 16: 441-451.
PubMed  |  Direct Link  |  

11:  Houghtby, G.A., L.J. Maturin and E.K. Koenig, 1992. Microbiological Count Methods. In: Standard Methods for the Examination of Dairy Products, Marshall, R.T. (Ed.). 16th Edn., American Public Health Association, Washington, DC., USA., ISBN-13: 9780875532080, pp: 213-246.

12:  Korhonen, H., A. Pihtanto-Leppala, P. Rantamaki and T. Tupasella, 1998. Impact of processing on bioactive proteins and peptides. Trends Food Sci. Technol., 9: 307-319.
CrossRef  |  

13:  Kuippers, O.P., G. Buist and J. Kok, 2000. Current strategies for improving food bacteria. Res. Microbiol., 151: 815-822.
CrossRef  |  

14:  Oconnor, C.B., 1999. Rural Dairy Technology. International Live Stock Research Institute, Nairobi, Kenya, pp: 119.

15:  Parky, Y.H., 1991. Changes of indenatured whey protein and available lysine contents in heat treated market milks. J. Dairy Sci., 54: 61-132.

16:  Parnell-Cluniess, M.E., Y. Kakuda, K. Mullen, R.D. Arnott and J.Y.M. de Mann, 1986. Physical properties of yoghurt: A comparison of vat versus continuous heating system of milk. J. Dairy Sci., 69: 2593-2603.
Direct Link  |  

17:  Ruegg, P.L. and D.J. Reinemann, 2002. Milk quality and mastitis tests. Univ. Wisconsin Extension Services Bovine Practitioner, 36: 1-13.

18:  Ryser, E.T., 1998. Public Health Concerns. In: Applied Dairy Microbiology, Marth, E.H. and J.L. Steele (Eds.). Marcel Dekker, Inc., New York, pp: 263-403.

19:  Sahan, N., A. Konar and A. Kleebeger, 1996. Effects of H2O2 addition and heat treatment on some physical, chemical and microbial quality of milk. Turk. J. Agric. For., 20: 1-7.

20:  USCFR., 2006. United States Code of Federal Regulation. Part 63, Vol. 6, Office of the Federal Register, National Archives and Records Administration, Washington, DC.

21:  Vyletelova, M., O. Hanovs, E. Urbanova and P. Kopunecz, 2000. The occurance and identification of psychrotrophic bacteria with proteolytic and lipolytic activity in bulk milk samples at storage in primary production conditions. Czech. J. Anim. Sci., 45: 373-383.
Direct Link  |  

22:  Ziarno, M., I. Molska and M. Gronczynska, 2005. Quality and shelf life of milk pasteurized at 84oC/19-22 sec. and free from post pasteurization contamination. Medycyna Weterynaryina, 61: 1165-1168.

©  2020 Science Alert. All Rights Reserved