Compositional and microbial quality of heat-treated milk brands marketed in Lusaka, Zambia

B Kunda, G S Pandey¹, C Mubita¹, J B Muma¹ and C Mumba¹

Zambia National Service Headquarters P.O. Box 32251, Lusaka, Zambia,
¹ University of Zambia, School of Veterinary Medicine, Department of Disease Control, PO Box 32379, Lusaka, Zambia
pandeygs@gmail.com

Abstract

The study was conducted to assess the compositional and microbial quality of pasteurized and Ultra High Temperature (UHT) treated milk on the market in Lusaka, Zambia. A total of 18 brands of milk (7 pasteurized and 11 UHT treated) were purchased from supermarkets and this formed the sample size of the study. The quality of milk was evaluated by assessing it for Total Bacteria Count (TBC), Total Coliform Count (TCC), isolation of Staphylococcus spp, Salmonella spp and Clostridium perfringes by culturing the samples of milk on standard plate count agar, violet red bile glucose agar and selective media. Milk components, freezing point and added water were determined using LactiCheck ultrasonic milk analyser while antibiotic residue using Copan Milk Test and Beta Star Combo Test kit.

The butter fat content of heat treated milk ranged from 2.91–3.74 %, protein from 2.05-3.47% and lactose 2.94-5.07%. Solid Not Fat (SNF) content ranged from 7.28- 9.23%, with 5 samples recording below the recommended standard of 8.3%. Freezing point ranged from -0.450 ⁰ C to -0.607 ⁰ C. About 16.6 % of the milk samples contained antibiotic residue. Total bacterial count of pasteurized milk ranged from 6,000–38,000 cfu/ml. There was no bacterial growth in UHT treated milk samples and thus within the acceptable standard. The coliform count in 4 of the 7 pasteurized milk samples ranged from 68-188 cfu/ml which is higher than acceptable standard of 5 cfu/ml, while UHT treated milk did not contain any coliform. Three brands of milk (16.6%) were found with added water containing 10.6, 13.6 and 33 % respectively. Unacceptable presence of coliform, antibiotic residue and added water in heat treated milk sample is indicative of health hazards to consumers and lack of monitoring by regulatory authority. There is need to test and monitor processed milk available to consumers periodically, educating farmers on the need of strict observance of antibiotic withdrawal period and imposing stiffer penalty on milk adulteration.

Key words: processed milk, composition, bacterial count, antibiotic residue, Zambia

Introduction

Milk is a translucent white liquid, produced by the mammary glands of mammals with high nutritional value, providing the primary source of nutrition for young mammals before they are able to digest other types of food. Raw cow milk is composed of approximately 87.2 % water, 3.7 % fat, 3.5% protein, 4.9% lactose 0.7% ash and has pH of 6.8 (Olatunji 2012). Because of high nutritive composition and a pH which is close to neutral, milk is an ideal medium for the growth of micro-organisms. Once they enter milk, most of the microorganisms spoil the milk in question and thereby reduce its shelf-life, nutritive value and taste. Other microorganisms are pathogenic to humans and can transmit disease if the milk is not properly and adequately heat treated. Unlike meat and meat products, consumers are less likely to subject milk to any subsequent heating before consumption, thus contaminated milk with pathogenic microorganisms is potentially more dangerous.  In a study in South Africa, 87 % of the milk samples were found to be unfit for human consumption on the basis of minimum standards prescribed by act (More 2003) and in Zambia, Kasase et al (2005) found 39 % of the pasteurized milk not suitable for human consumption. This makes it vital that at all times, it is ensured that milk and milk products are wholesome, safe, fresh, clean and free from contamination with spoilage and pathogenic micro-organisms meeting the acceptable standard (Bille and Kaposao 2012). Over 90% of all reported cases of dairy related illnesses continue to be of bacterial origin where at least 21 milk borne and potentially milk borne diseases being recognized. 

Pasteurization means heating milk at 72°C for15 seconds by ‘high temperature short-time’ (HTST) or heating at 65°C for 30 minutes by ‘long-time low temperature’ (LTLT) methods, respectively and cooling to 4-5°C. Ultra-High-Temperature (UHT) means heating milk at 135-145°C for 2-4 seconds and promptly cooling to room temperature. These processes are applied to liquid foods such as milk to render them safe for human consumption, improve texture and flavour and to prolong shelf life by eliminating microorganisms and enzymes that tend to spoil the foods. Pasteurization causes minimal physical, chemical and organoleptic changes in milk. Such milk, if properly processed and handled, can have a shelf-life exceeding 14 days for pasteurized products stored in a refrigerator and 6-9 months for UHT products stored at room temperature (Bille and Kaposao 2012).

The predominant spoilage micro-organisms found in pasteurized and chilled milk are gram negative psychrotrophic or psychrophilic bacteria. The common species belong to the genus Pseudomonas, Flavobacterium and Alcaligenes, as well as some members of the coliforms group (Vanderzant and Splittstoesser 1992). When heat treated chilled milk becomes spoiled, it can be detected organoleptically as it curdles with sweet or bitter taste, low acidic flavour and rancid taste and has bad smell due to the activities of psychrotrophic bacteria. The rate of microbial growth and quality deterioration of products is influenced by the number and types of bacteria in the freshly pasteurized products and the storage temperatures (Berg 1988).

The common pathogenic bacteria that are transmitted through milk consumption are Mycobacterium bovis, which causes tuberculosis (Benson 2005). This occurs mostly in poor countries because of the poor health status of animals, hygiene and sanitation and absence of tuberculin testing and elimination of tuberculosis (TB) reactor cows. However, this bacterium can easily be destroyed by the right pasteurization temperature. There is only one study available from Zambia on microbiological quality of three brands of pasteurized milk (Kasase et al 2005).

Hygienic quality is important from public health view point. Milk whose quality has not been tested may constitute a serious public health hazard. It might be contaminated with pathogens and food poisoning bacteria (Fakudze and Dlamini 2001). Consumer’s milk whose quality is not monitored could also become potential health hazard from antibiotic residues contamination (Hillerton et al 1999). The liberalized market, smallholder dairy development efforts and demand of processed milk has recently been increased tremendously in Zambia. This has resulted in a number of milk processing companies operating in Zambia in the last 10 years. Therefore this necessitates investigation of the quality of processed milk to ascertain public health safety.

The purpose of this study was to assess the compositional and microbial quality of pasteurized and Ultra High Temperature (UHT) treated milk purchased randomly from supermarkets in Lusaka, Zambia.

Materials and methods

The study was conducted during January to February 2014. A total of 18 different brands of heat treated milk in 500 ml UHT sachets, tetra packs and plastic bottles produced by different milk processing companies were purchased randomly from supermarkets in Lusaka on the day of milk delivery to avoid any microbiological change in milk due to temperature fluctuation while in the custody of supermarket. Out of these 18 brands, 11 were UHT treated milk and 7 were pasteurized milk. These milk brands were assigned the brand codes 1 to 18. The milk was transported to the University of Zambia, Public Health Laboratory in an ice packed cooler box for analysis. Compositional analysis and bacteriological counts were initiated within 04 hours of sample collection. 

Compositional and Freshness Analysis of Milk Samples

The 18 brands of heat treated milk  samples were analyzed for composition using a LactiCheck Ultrasonic Milk Analyzer (Page & Pedersen International Ltd, USA) for butter fat (BF) content, protein, lactose, density, solid not fat (SNF), freezing point, pH and added water. Approximately, 20 ml of each milk sample was thoroughly mixed  in  a test tube, taken into the sample cup, placed below the aspiration tube of the Lacti Check as per manufacturer’s instructions and the results were noted after one minute. All milk samples were tested for freshness using 72% alcohol and clot on boiling test.

Antibiotic Residues (ARs) in the milk brands

Two different tests were applied to test the 18 brands of milk for antibiotic residues (ARs). These were Copan Milk Test 100 (Copan Diagnostics Inc., USA) and Betastar Combo Test (Neogen Corporation, USA).

Copan Test 100 (Copan Diagnostics Inc., USA)

This is a qualitative test for detecting the presence of ARs in milk. In this test, Bacillus stearothermophilus var. calidolactis spores are enclosed within an agar based gel matrix containing nutritive substances and a pH indicator. When milk sample which is free from ARs is added and incubated at 64°C for 3 hours, the bacterial spores within the test kit media germinate and produce acid which contributes to a pH drop. The pH drop causes a colour change from purple to yellow. However, if ARs are present, the spores will not germinate and no acid produced and hence the colour of the media remains unchanged, that is purple.

Beta Star Combo Test (Neogen Corporation, USA)

This is a rapid detection assay for both beta-lactam and tetracycline antibiotics. The test employs binding reagents linked to gold particles and has 2 stages. In stage 1, preliminary incubation of the binding reagents with milk containing antibiotics result in the interaction of the antibiotics with binding reagents. In stage 2 the solution is transferred onto an immunochromatographic medium which has got three lines by which signal development occurs. Line 1 on medium captures all tetracycline binding reagents that have not interacted with tetracycline antibiotic during the preliminary incubation. Line 2 on the medium serves as a control line to ensure proper function of the test itself and also serves as a reference comparison for lines 1 and 3. Line 3 on the medium captures all the beta-lactam binding reagents that have not interacted with any beta-lactam antibiotic during the preliminary incubation. To interpret the results, the intensities of the antibiotic test lines (lines 1 and 3) are compared to the control line (line 2). When the intensity of the test line is greater than or equal to the control line, the milk sample is negative for the presence of the antibiotic. When the intensity of the test line is less than the intensity of the control line, the milk sample is positive for the antibiotic.

Bacteriological Count

In each of the 18 brands of milk, TBC and coliform counts were determined. Total bacterial count (TBC) was determined using the standard plate count (SPC) method where one ml of milk from each milk sample was  used to make three serial dilutions of 1:10. 1:100 and 1:1,000. From each dilution, 1 ml was plated in duplicates on standard plate count agar and incubated for 48 hours at 32°C. Then using a colony counter, bacteria (or clusters) that grew and became visible colonies were counted and expressed as number of colony forming units per milliliter (cfu/ml) of milk. Determination of coliform colony forming units per ml was carried out on Violet Red Bile Glucose Agar, incubated at 32°C for 48 hours then counted the colonies using a colony counter. The milk was also cultured for presence Staphylococcus spp, Salmonella spp and Clostridium perfringes using selective media at 37^oC for 24 hours. Staphylococcus medium and xylose lysine deoxycholate agar were used for Staphylococcus spp and Salmonella spp respectively, while Clostridium Perfringes Agar was used for Clostridium perfringes (Billie et al., 2009).

Statistical analysis

Using SPSS version 20, the following statistical tests were conducted:

• Independent samples t tests to establish whether there were any statistical significant differences between UHT and pasteurized milk regarding BF, SNF at a significance level of 0.05
  
  
• Correlation coefficient tests to determine association between FP and milk density and between FP and SNF at significance level of 0.01

Results

Milk Composition

Tables1 shows the results of descriptive statistics for milk compositional and bacteriological tests regarding the 18 brands of heat treated (pasteurized and UHT) milk purchased from supermarkets in Lusaka, Zambia

Clot on boiling and alcohol tests were all negative for both pasteurized and UHT milk. Two brands of UHT milk (18%) were found adulterated with water containing 10.6 and 13.6% and one brand of pasteurized milk (14%) was found adulterated with water containing 33%.

Results showed that SNF for 4 brands (36.4%) of UHT milk and 3 brands (42.9%) of pasteurized milk respectively, were below recommended standards of 8.3%. For BF, 1 brand (9.1%) of UHT milk and 1 brand (14.3%) of pasteurized milk was below recommended standard of 3.2%. These 2 brands of milk were also low in SNF showing evidence of adulteration with water.

Independent samples t test results showed that there was no statistical difference between UHT and pasteurized milk with regards to BF, SNF and FP (Table 2).

Correlation coefficient test results showed that there was correlation between FP and density and between FP and SNF (Table 3).

Bacteriological Count

Total bacteria count (TBC) and coliform count on all the 18 brands of heat treated milk (pasteurized and UHT) are summarized in Tables 1. All the 18 milk brands conformed to the recommended standards of TBC load while 3 brands (16.6 %) of pasteurized milk did not conform to the recommended standards on TCC. Clostridium perfringenes, Salmonella spp and Staphylococcus spp were not detected in all the 18 brands of milk.

Antibiotic Residues (ARs)

It was found that 9.1% and 28.6% of UHT and Pasteurized milk brands respectively, were contaminated with ARs (Table 4). Overall, 3 out of 18 milk samples (16.7%) were positive for ARs. Those samples positive on Copan Milk Test were also found positive on Beta Star Combo test.

Discussion

Raw milk is commonly contaminated with large numbers of micro-organisms including potential food borne pathogens. Therefore lapses in Good Manufacturing Practices (GMP) and Good Hygiene Practices (GHP) programs in milk processing plants especially inadequate pasteurization or post contamination can result in pasteurized milk being contaminated with these micro-organisms which can harm consumers (Kasase et al 2005). In Zambia, the law stipulates that heat treated milk should have TBC not exceeding 50,000 cfu/ml and coliform count not more than 5 cfu/ml (Food and Drugs Act 2001 of the Laws of Zambia). In accordance with this requirement, all the 11 UHT milk brands and 7 pasteurized brands were found within the acceptable TBC limit. However four samples (22 %) of the pasteurized milk brands were found to be contaminated with coliforms. Coliforms could have contaminated the milk after processing during packaging or due to use of poor quality packaging material. Higher TBC and coliform count has been reported in heat treated milk in Swaziland (Fakudze and Dlamini 2001) and in South Africa (More 2003). However in a recent study of the hygienic quality of seven brands of heat treated milk in Namibia by Bille and Kaposao (2012), found all samples within the acceptable standard of that country indicating good hygienic practices by producers and processors. The acceptable low total bacterial count in pasteurized milk in our study could be attributed to new processing companies coming into market using improved quality of computerized pasteurization machines and equipment and renovation of old machine to meet HACCP requirements. There is also continuous training programme for smallholder farmers in place towards hygienic production of milk and incentive for low bacterial count in raw milk. Furthermore the milk samples were collected immediately they were delivered to supermarket to avoid effect of poor refrigeration or temperature fluctuation on growth of bacteria. However in a similar study by Kasase et al (2005) in Zambia found 39 % of the pasteurized milk having higher TBC than acceptable Zambian standard. Clostridium perfringenes, Salmonella spp and Staphylococcus spp were not detected in all the 18 brands of milk and our findings are similar to the findings of Kasase et al (2005).

Water is the most common adulterant in milk which is often added to milk by unscrupulous milk dealers who want to increase the volume in order to earn easy money. Addition of water to milk reduces the nutritive value of milk, and if contaminated, it poses a health risk to consumers (Kandpal et. el 2012). Freezing point (FP) test is the recognized international reference test for added water to milk. Freezing point (FP) of milk is taken as constant so the FP test is used to assess whether or not water has been added to milk. Two brands (18.1%) of UHT milk and one brand (14.2%) of pasteurized milk was found to be adulterated by adding 10.60, 13.60 and 33 % water which warrants regulatory authority to monitor the quality from time to time to check adulteration and impose stiffer penalty towards malpractices. Study found a positive co-relationship with addition of water and freezing point.

The legal requirement of SNF in processed full cream milk for sale to consumers in Zambia is not less than 8.3%. Three UHT milk sample contained SNF less than 8.3 and two of these three samples had added water in the milk. The density of these milk samples was also less than 1.025 while one pasteurized milk contained 7.37 SNF below the recommended level and this milk had added water the reason for low SNF. 

The BF content of UHT milk ranged from 3.02 to 3.60 % fat and that of pasteurized milk 2.91 to 3.74% indicating 11.11 % of the milk samples below legal standard of 3.2 % fat. This indicates removal of fat from the milk.

The presence of antibiotic residue in milk is undesirable because they may result in hypersensitivity, tissue damage and antimicrobial resistance in human (Katz and Brandy 2000, de Zayas et al 2004, Karimuribo et al 2015). Contrary to the recommended standards which require that milk should contain no ARs (The Food and Drug Act 2001 of the Laws of Zambia), out of the 18 heat treated brands of milk, 3 samples (16.6 %) were positive for ARs, Fakudze and Dlamini (2001) reported about 50% of the processed milk contained antibiotic residue in Swaziland. Medeiros et al (2004) and Tetzer et al (2005), both studies from Brazil, Mokhtari et al (2013) from Iran and Karimuribo et al (2015) from Tanzania reported antibiotic contamination in heat treated milk by 43%, 33.3%, 32.9% and 12.5% respectively. Kunda (2015) in a just ended study in Zambia reported 30.1% antibiotic contamination in raw milk, a possible linkage of presence of antibiotic residue in processed milk in Zambia.

It is worth to mention that pasteurization does not eliminate the residues of antibiotics present in raw milk (Moats 1988, Loksuwan 2002, de Oliveira et al 2012). Antibiotic residue in milk can cause allergic reactions that can occur in some consumers who are allergic to certain antibiotics and also organisms may develop resistance (de Oliveira et al 2012). Consumption of milk containing antimicrobial residues may pose health risks that include allergic reactions such as anaphylaxis, while some may lead to development of aplastic anaemia (Katzs and Brady, 2000). Further, these antimicrobial residues may give rise to public health concerns due to the development of antimicrobial resistance in intestinal bacterial populations (de Zayas et al 2004).

Our results highlight the need for state public health authorities to set a maximum residual level for antimicrobial agents in milk and establish monitoring programmes to determine antimicrobial residues in milk. In Zambia any one can purchase antibiotics even without a prescription and farmers use them indiscriminately and are often unaware of their withdrawal period. There is need for farmers to be educated on use of antibiotics to treat cows and to observe the withdrawal period after they treat their cows with antibiotics. Further, antibiotics should only be sold on prescription from veterinary personnel. This is the first time antibiotic residues study in milk and compositional quality of milk in Zambia has been attempted and the study indicates the presence of antibiotic residue in milk.

Conclusion and recommendations

• Unacceptable presence of coliforms and added water in pasteurized milk found in the study, indicated existence of lapses in Good Manufacturing Practices (GMP) and Good Hygiene Practices (GHP) in the milk processing plants in Zambia.

  
  

• The presence of antibiotic residue in milk requires education of milk producers on withdrawal period and government regulatory authority to establish the antibiotics maximum residual limit (MRL) standards.

  
  

• There is need for periodical monitoring of milk quality before it is sold to consumers.

Acknowledgements

The first author wishes to express sincere thanks to the Government of Republic of Zambia and Zambia National Service, especially Command and Training Branch, for the financial and logistical support rendered towards this study.

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Received 28 April 2015; Accepted 2 June 2015; Published 2 July 2015

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