Determination of Total, Viable Cells and Enterobacteraceae in Categorized Milk Powder
Imran Rashid Rajput,
S. A. Fazlani,
The present study was conducted to examine the microbiological quality of commercial milk powders. A total of 30 dried milk powders, 10 each of Skim Milk Powder (SMP), Semi Skim Milk Powder (SSMP) and Full Cream Milk Powder (FCMP) purchased from market of Hyderabad, Sindh were evaluated for microbiological quality characteristics, like Total Viable Count (TVC), thermoduric count and Enterobacteraceae Count (EbC). Total viable count, (6.1 x 103±7.2 x 102cfu/g) and Enterobacteraceae count, (2.3 x 103±2.6 x 102cfu/g) were significantly (p<0.05) higher in FCMP compared to SMP (3.7 x 103±9.8 x 102 and 1.7 x 103±1.7 x 102cfu/g, respectively) and SSMP (3.5 x 103±4.4 x 102 and 1.5 x 103±1.0 x 102cfu/g, respectively). The overall average concentration of TVC (4.43 x 103±4.8 x 102cfu/g) in dried milk powder was recovered lower (11.28 folds) compared to Pakistan Standard Institution (PSI) and/or Indian Standard Institution (ISI) (5.0 x 104 cfu/g) and the overall average count of Eb (1.84 x 103±1.2 x 102) were detected higher, (18.4 folds) compared to ISI standard respectively. Although TV count were within the range of standard of specification (PSI/ISI), but the counts of, Eb indicates the unhygienic condition of dried milk powders with higher risk level for human health.
Milk is a major part of food consumption and plays a prominent role in the Pakistani diet and comes second to cereals in the level of per capita consumption (Anonymous, 2008). No doubt, the milk powders are generally considered as product of good microbiological quality with no risk of spoilage, but several factors may contribute to change its physical and chemical properties which reduce shelf-life and thus its commercial value (Cousins et al., 1987). Although the micro-organisms in dried milks cannot grow due to its low moisture content and do not play any direct role in their spoilage. But their occurrence in these products is of great significance and serves as an index of hygienic standards maintained during production, processing and handling. As these powders find their application in dairy industry, for processed cheese or for reconstitution purposes, the presence of micro organisms even in low numbers may cause potential hazards and/or defects in the derived products (Yadav et al., 1993). The milk provides a highly nutritious substrate that can support the wide variety of bacteria as well as yeast and molds for their growth and reproduction (Phillips and Griffiths, 1990). The contamination role of bacteria during the production of milk powder has been well documented. With this many studies have provided evidence of vegetative growth during the manufacturing of milk powders. The other contamination source in milk powders are reuse of by products such as buttermilk and permeate from milk ultra filtration ingredients added to the process such as lactose and recycle loops in manufacturing plants (Hill and Smythe, 2004). As these powders find their utilization in dairy sector, for yoghurt, tea, ice-cream and cheese making or for reconstitution purposes, the presence of micro organisms even in low numbers may cause potential hazards and/or defects in the derived products (Yadav et al., 1993). Since, still no work has been found on any aspects of milk powders in the province of Sindh. Thus, present study has been designed for determination evaluation quality of milk powders.
MATERIALS AND METHODS
Collection of milk powder samples: A total of thirty samples of milk
powders i.e 10 from each category (skim, semi skim and full cream) were collected
in a sterilized sample bottles from the randomly selected milk powder shops
of Hyderabad and brought to the Laboratory of Dairy Microbiology, Department
of Dairy Technology. Faculty of Animal Husbandry and Veterinary Sciences, Sindh
Agriculture University Tandojam, for the evaluation of microbial quality characteristics.
However, among the thirty samples, six samples showed either spreaded colonies
and/or heavily contaminated. Thus rejected and not included in the present study.
Preparation of diluent
Peptone water: Peptone water (Oxoid, Ltd. England) (15 g) was dissolved
in distilled water (1 L) and distributed in bottle (90 ml) and/or in test tubes
(9 ml). The bottles/tubes were capped or plugged with cotton and autoclaved
(121oC) for 15 min. The sterilized diluents were stored at (4-8oC)
Preparation of test samples: Milk powder (10 g) was diluted in warm (45oC) sterile diluents I-e peptone water solution (90 ml) to make primary dilution (10-1). Then a series up to 10-5 dilution was prepared by transferring primary dilution (1 ml) into test tube containing sterile diluents (9 ml) to obtain 10-2 dilution and repeating the operations with sterile diluents (9 ml) using the 10-2 and further dilutions to obtain 10-3, 10-4 and/or 10-5.
Enumeration of total viable count (colony count technique at 30oC): Total viable counts were enumerated according to the method of International Dairy Federation (IDF, 1991). Pre prepared test sample (1ml) of 10-3, 10-4 and/or 10-5 dilutions (section-3.9.1) was transferred into sterile petri dishes in duplicate through sterile graduate pipette and/or dispensing pipette (1000 μl) with sterile plastic tips and warm (45±1oC) sterile plate count agar medium (15 ml) was mixed with inoculum. The mixture was allowed to solidify and incubated (30oC) for 72±2 h.
Enumeration of Enterobacteraceae counts (colony count technique at
37oC): Enterobacteraceae counts were enumerated according
to the method of British Standard Institute (BSI, 1993). Pre prepared test sample
(1 ml) of 10-1, 10-2 and/or 10-3 dilution (section
3.9.1) was transferred into sterile petri dishes through dispensing pipette
(1000 μl) with sterile plastic tips and warm (45±1oC)
sterile violet red bile agar medium (15 ml) was mixed with inoculum. The mixture
was allowed to solidify and incubated (37oC) for 24±2 h.
||Number of colonies obtained
||Sum of colonies counted on all the dishes retained
||Number of dishes retained in the first dilution
||Number of dishes retained in the second dilution
||Dilution factor corresponding to the first dilution
Total Viable Count (TVC): Total viable count of Skim Milk Powder (SMP)
Semi Skim Milk Powder (SSMP) and Full Cream Milk Powder (FCMP) was evaluated
and the results are presented in Fig. 1. A wide variation
was observed in TV counts in all types of dried milk powders examined in the
present study. The concentration of TV count in SMP, ranged between 1.2 x 103
to 9.2 x 103 cfu/g and averaged 3.7 x 103±9.83
x 102 cfu/g. While in case of SSMP, the TV counts were observed in
between 1.8 x 103 to 5.3 x 103 cfu/g with mean value of
3.5 x 103±4.36 x 102 cfu/g.
shows minimum, maximum and mean values of total viable counts (cfu/g)
in skim milk, semi skim milk and full cream milk powders
SE± = 1058
LSD (0.05) = 2201
SMP = Skim Milk Powder
SSMP = Semi Skim Milk Powder
FCMP = Full Cream Milk Powder
cfu = Colony Forming Unit
Where ever, TV count in FCMP, varied between 2.9 x 103 to 9.6 x
103 cfu/g and averaged 6.1 x 103±7.23 x 102
cfu/g. Moreover, the results of statistical analysis (Analysis of Variance)
showed significant difference (p<0.05), in TV counts in SMP, SSMP and FCMP
(Appendix-II). It was further observed that TV count of FCMP (6.1 x 103±7.23
x 102 cfu/g) was significantly (p<0.05). While there was no significant
difference (p>0.05) in TV counts observed between SMP and SSMP. The concentration
of TV counts was lower in SMP (13.55 folds), SSMP (14.24 folds) and in FCMP
(8.21 folds) compared to Indian Standards Institute, ISI and Pakistan Standard
Institution I-e < 5.0 x 104 cfu/g. While the overall concentration
of TV counts of all types of milk powders were lower (2.25 folds) compared to
ISI standard < 5.0 x 104 cfu/g. While the overall average
concentration of TV counts in FCMP, SSMP and SMP was (11.28 folds) lower than
PSI/ISI standard 5.0 x 104 cfu/g (Table 1).
Enterobacteraceae Count (EBC): SMP, SSMP and FCMP were evaluated
for Enterobacteraceae count and the results are depicted in Fig.
2 and Appendix-VII. TPS counts varied greatly in all types of dried milk
powders examined in the present study. The concentration of Enterobacteraceae
count in SMP ranged between 1.1 x 103 to 2.6 x 103 cfu/g
and averaged 1.7 x 103±1.6 x 102 cfu/g. While in
case of SSMP the Enterobacteraceae counts were observed in between 1.1
x 103 to 1.9 x 103 and averaged 1.51 x 103 ±
1.03 x 102 cfu/g. Enterobacteraceae count in FCMP, varied
between 1.2 x 103 to 3.1 x 103 cfu/g and averaged 2.3
x 103 ±2.60 x 102cfu/g.
Viable Counts (cfu/g) in different dried milk samples compared to ISI
and PSI standards
|a = Observed Values, x = (Standard Value of PSI/ISI = <50000
cfu/g), ISI = Indian Standards Institute, PSI = Pakistan Standard Institute
Graph shows minimum, maximum and mean values of Enterobacteraceae
count (cfu/g) in skim milk, semi skim milk and full cream milk powders
SE± = 267
LSD (0.05) = 554
SMP = Skim Milk Powder
SSMP = Semi Skim Milk Powder
FCMP = Full Cream Milk Powder
cfu = Colony Forming Unit
Moreover, the results of statistical analysis (AOV) showed significant difference
(p<0.05), in Enterobacteraceae counts in SMP, SSMP and FCMP (Appendix-VIII).
Further LSD comparison of means (0.05) observed that Enterobacteraceae
b count of FCMP (2.3 x 103±2.6 x 102 cfu/g) was
significantly (p<0.05), higher than SMP (1.7 x 103±1.6
x 102 cfu/g). SSMP (1.51 x 103 ±1.03 x 102
cfu/g). While there was no significant difference (p>0.05), in Enterobacteraceae
counts observed between SMP and SSMP), higher than SMP (3.7 x 103±9.83
x 102 cfu/g) and SSMP (3.5 x 103±4.36 x 102
cfu/g). The concentration of Enterobacteraceae counts was higher in SMP
(17 folds), SSMP (15.1 folds) and in FCMP (23 folds) compared to that of Indian
Standards Institute (ISI, 1975) I-e < 1.0 x 102 cfu/g.
While the average concentration of Enterobacteraceae counts of all types
of milk powders were observed (18.4 folds) higher in contrast to ISI standard
<1.0 x 102 cfu/g (Table 2).
counts (cfu/g) in different dried milk samples compared to ISI standards
= Observed Values, x = (Standard Value of ISI (1975) = <1.0
x 102 cfu/g), ISI = Indian Standards Institute
Present study has been conducted to assess the general hygienic quality of
dried milks (full fat, low fat and fat free milk powders) and the extent of
microbes have been observed. Although the microorganisms in dried milk owing
to their low moisture content can not grow and thus do not play any direct role
in their spoilage, their occurrence in these products is of great significance
they server as an index of hygienic standards maintained during Production,
Processing and handling (Yadav et al., 1993). However, in dairy industry,
these powders have significant use in the production of dahi, yoghurt, ice-cream,
tea and/or reconstitution purpose and presence of microorganisms may cause defects
in the derived products. In the present study the total viable count of FCMP
(6.09 x 103±7.23 x 102 cfu/g) was significantly
(p<0.05) higher than SMP (3.69 x 103±9.83 x 102
cfu/g) and SSMP (3.51 x 103±4.36 x 102 cfu/g).
While, there was no significant difference (p>0.05) in TV counts observed
between SMP and SSMP. It is of interest to point out that total viable count
(4.43 x 103±4.8 x 102 cfu/g) observed in the dried
milk is lower than reported by (Rueckert et al., 2005; Khaskheli, 1998;
ISI, 1975 and PSI, 1982) i.e 5.6x104±4.3x103, 8.7x103,
1.0x104 and 5.0x104 respectively. The vegetative cells
normally killed at 80oC for 10 mins. But the present study manifested
their presence in milk powders. Because, their ability to attach with stainless
steel and folded surface (Flint et al., 2006). Once they attach to the
surface, vegetative cells grow with spores by forming biofilms. This biofilms
is not completely removable by CIP system but can be decreased; the remaining
contaminants present on folded stainless steel transfer into final product,
i.e powders milk (Parker et al., 2001).
Enterobacteraceae count of FCMP (2.3 x 103 ±2.60 x 102 cfu/g) was significantly (p<0.05) higher than SMP (1.7 x 103±1.69 x 102 cfu/g) and SSMP (1.51 x 103±1.03 x 102 cfu/g). While there was no significant difference (p>0.05), in Enterobacteraceae counts observed between SMP and SSMP. The mean value (1.84 x 103±1.25 x 102) obtained in present study is lower than reported by Taha et al. (1972) i.e 13 x 106. It is the general concept that Enterobacteraceae are not present in powder milk because they grow at 30-37oC and can not survive during processing, it is true hypothesis and proved by various researches. But, mostly Enterobacteraceae. The powder milk is packed hygienically in large sterilized containers and bags. However, transportation some damaged containers and bags have been observed, probably they can contaminate the milk powders. When milk powder reaches up to whole seller it has been re-packed in and/or small bags and containers which are not sterilize. Further the retailers decrease the size of the bags 1 Kg and/or 2 Kg to sell the consumers. During these practices no measures of hygienic conditions are adopted which may increase the Enterobacteraceae concentration in powder milk.
1: Anonymous, 2007. Pakistan economic survey, 2007-2008. Economic Advisory Wing, Ministry of Finance, Govt of Pakistan, Islamabad.
2: BSI., 1993. Microbiological Examination of Food and Animal Feeding Stuffs: Enumeration of Enterobacteriaceae. British Standards Institution, London
3: Cousins, C.M., A.J. Bramley and R.K. Robinson, 1987. Microbiologia de la leche cruda. Microbiol. Lactol., 1: 109-150.
4: Flint, S., J.L. Drocourt, K. Walker, B. Stevenson M. Dwyer, I. Clarke and D. McGill, 2006. A rapid, two-hour method for the enumeration of total viable bacteria in samples from commercial milk powder and whey protein concentrate powder manufacturing plants. Int. Dairy J., 4: 379-384.
5: Hill, B.M. and B.W. Smythe, 2004. Thermophilic spores in milk powder destined for use in UHT and retort sterilization processes. Proceedings of the New Zealand Microbiological Society Conference.Palmerston North, New Zealand: New Zealand Microbiological Society.
6: IDF., 1991. Enumeration of Microorganism in Milk and Milk Products. International Dairy Federation, Brussels, Belgium
7: Gupta, B.V., 1993. A Comprehensive Dairy Microbiology. Metropolitan Book Co. Pvt. Ltd., USA., pp: 685-699
8: Khaskheli, M., 1998. Some aspects of the production and quality improvements of fermented milk/cereal mixture (Kishk). Ph.D. Thesis, University of Glasgow, UK.
9: Parker, E.L., R.S. Ronimus, N. Turner, S. Poudel, A. Ruckert and H.W. Morgan, 2001. A RAPD-based comparison of thermophilic bacilli from milk powders. Int. J. Food Microbiol., 85: 45-61.
10: Phillips, J.D. and M.W. Griffiths, 1990. Pasturized Dairy Products: Constraints Imposed by Environmental Contamination. Wiley, USA., pp: 387-456
11: PSI, 1982. Specification for Milk Powder. Pakistan Standards Institution, Karachi, Pakistan
12: Rueckert, A., R.S. Ronimus and H.W. Morgan, 2005. Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus. J. Applied Microbiol., 99: 1246-1255.
13: Taha, S., M. Naguib and A. Ghani, 1972. Dried milk microbiology. J. Milk Field Technol., 36: 559-559.
14: Yadav, J.S., S. Grover and V.K. Batish, 1993. Microbiology of Dried Milks: A Comprehensive Dairy Microbiology. 1st Edn., Metropolitan Book Co. Pvt. Ltd., New Delhi, India, pp: 315-349