Livestock Research for Rural
Development 27 (7) 2015
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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
1
Zambia National Service Headquarters P.O. Box 32251, Lusaka, Zambia,
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 2.053.47% 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 37oC 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
Table 1: Descriptive statistics-Milk composition and bacteriology
Variables
N Minimum Maximum
Ultra high
Density (g/cm3)
11
1.023
1.030
temperature
Butterfat (%)
11
3.020
3.600
Solid not fat (%)
11
7.230
9.110
Freezing point (oC)
11
-0.480
-0.597
Total bacteria count
11
0
0
(cfu/ml)
Total coliform count
11
0
0
(cfu/ml)
Pasteurized
Density (g/cm3)
7
1.017
1.031
Butterfat (%)
7
3.19
3.91
Solid not fat (%)
7
8.13
9.23
Freezing point (oC)
7
-.534
-.607
Total bacteria count
7
2,000
38,000
(cfu/ml)
Total coliform count
7
0
188
(cfu/ml)
Mean
1.027
3.420
8.359
-0.550
SD
.002
.186
.628
.397
0
0
0
0
1.027
3.59
8.64
-.566
.005
.236
.496
.332
16,586
12,868
66
72
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).
Table 2: T-test results comparing pasteurized and UHT milk with
regards to BF, SNF and FP
Independent variables
t
df
Sig. (2 tailed)
Butterfat (%)
-1.791
16
.092
Solid not fat (%)
-1.008
16
.328
Freezing point (oC)
-0.913
16
-0.375
Correlation coefficient test results showed that there was correlation between FP and density
and between FP and SNF (Table 3).
Table 3: Correlation results between FP and density and between FP
and SNF
Density(g/cm3 )
Solid not fat (%)
Pearson Correlation
-.724**
-.998**
Sig. (2-tailed)
.001
.000
N
18
18
**. Correlation is significant at the 0.01 level (2-tailed).
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.
Table 4: Test results for presence of ARs in
pasteurized and UHT
N
Positive
Percent
UHT
11
1
9.1
Pasteurized
7
2
28.6
N = number of samples
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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