Abstract: This study was carried out at Khartoum State to evaluate the hygiene and the quality of milk after preservation with Lactoperoxidase Enzyme System (LPS). The milk was collected directly from a traditional farm, the collection and examination of samples was done during April- June 2005. The result indicated that non significant differences were found for total bacterial counts, coliform counts and acidity in the treated milk samples. Moreover this study showed that total bacterial counts and the acidity were not affected by temperature (room and refrigerator) after treatment with Lactoperoxidase System (LPS). Also the treatment of raw milk with the LPS revealed non significant differences in acidity due to variation of storage temperature. However the result indicated that there were significant differences in total bacterial and coliform counts (p<0.001) of the raw milk stored at both room and refrigerator temperatures. The raw milk samples revealed the presence of five organisms and they were identified as Escherichia coli, Citrobacter freundii, Yersinia enterocolitica, Staphylococcus aureus and Enterococcus feacalis. However the treated milk (with lactoperoxidase system) revealed the presence of E. feacalis, which take time before it was disappeared (9 days for refrigerated treated milk). Thus the present study encourages the uses of lactoperoxidase enzyme system for preservation of milk where cooling facilities are not available to ensure its safety. However the addition of the enzymes should be done by trained authorized persons.
Introduction
Milk is magnificent medium for growth of microorganisms and therefore risk for quick microbial deterioration of quality is present from time of milking to the time of use in the milk processing plant (IDF, 1994). Measurement of bacterial numbers in milk is of interest because of their direct role in milk spoilage and because they are indicator of poor hygienic production (Harding, 1999).
A growing list of ingredients are being used and investigated for use in fluid milk processing to inhibit microbial activity and many of these components have exhibited successful protection against the proliferation of lactic acid bacteria and molds (Yuan, 2001). He also added that protein and peptide such as lactoperoxidase, lactoferrin, bacteriocins, lysosome and xanthine oxidation, occurring naturally in milk and have antimicrobial properties. FAO/ WHO strongly discourages the preservation of milk by chemical means, except the application of H2O2 at native LPS and in the case of H2O2, it must be completely destroyed before consumption (Ozer et al., 2003).
Lactoproxidase system is based on the activation of the lactoproxidase, thiocyanate and hydrogen peroxide system (naturally present in milk in varying concentrations) to inactivate several vital metabolic bacterial enzymes, consequently blocking their metabolism and ability to multiply (Gaya et al., 1991). Lactoperoxidase system (LPS) is active against both Gram positive and Gram negative microbes to varying extents (Marks et al., 2001). Activation of lactoperoxidase system was investigated as potential alternative method to sustain the safety of milk by inhibiting certain microorganisms with known pathogenic potential like Staphylococcus aureus and Escherichia coli (Kangumba et al., 1997).
Recently FAO has conducted large-scale field trials in Pakistan, Philippines and Cuba, which have shown promising results, however, the application of this method has to follow specific guidelines as spelt out by Codex Alimentarius and IDF (Lambert, 1993). The application of the lactoperoxidase system of milk preservation at collection centers aimed at raising regional awareness to safe, cheap and effective alternative milk preservation method (Codex Alimentarius Commission, 2004).
Most of the consumers in Sudan use raw milk that supplied to them from far away without cooling as the marketing system of milk are weak and on a transitional stage. This milk is contaminated by various organisms (Yagoub et al., 2005), which necessitate the introduction of safe methods of preservation suitable for the local conditions. Hence the present study was carried out as a trial to evaluate the preservation of milk with lactoperoxidase enzyme system, a newly enzyme kits that have been recently approved and introduced to the developing countries by the FAO.
Materials and Methods
Sources of Samples
This study was carried out using bovine milk from a traditional farm in
Khartoum State, Sudan during the period of April to June 2005.
Collection and Transportation of Milk Samples
Milk samples were collected directly from farm in Jabal Awlyaa, Khartoum,
Sudan. The samples were kept in plastic containers with capacity of 5 L. The
samples were transported after one hour of milking to the Microbiological laboratory
(School of Biotechnology, El Neelian University). Part of the samples (treated)
was preserved with lactoperoxidase system, which contains thiocyanate and hydrogen
peroxide. The LPS was obtained from Ministry of Animal Resources, Sudan. The
kits were offered to them by FAO for field trials of the LPS in Sudan. Then
both treated (by the addition of lactoperoxidase) and non treated milk samples
(control; with out addition of the lactoperoxidase) were distributed into 100
sterile bottles. The bottles were grouped into 4 groups that included: control
milk samples that kept at room temperature (CR), control milk samples that persevered
at refrigerator temperature (CC), treated milk samples that kept at room temperature
(TR) and treated milk samples that preserved at refrigerator temperature (TC).
Measuring of Milk Acidity
The acidity of the milk samples was determined; at zero time and then each
hour up to nine hours then daily up to 12 days; according to the method described
in the AOAC (1990). Clot on boiling test of the milk samples were done daily
according to Foley et al. (1974).
Microbiological Examination of Milk Samples
Plate count agar was used for determining total bacterial counts, MacConkey
agar was chosen as a differential selective medium for coliform bacterial counts
and mannitol salt agar was a differential and selective plate medium used to
isolate Staphylococcus aureus.
Serial dilutions from the milk samples were prepared aseptically according to the methods described by Richardson (1985). The incubation was done at 32°C for 24- 48 and 37°C for 24 h for the total bacterial counts and coliform counts, respectively (Richardson, 1985) and 4-7°C for 2-10 days for the psychotropic counts (Ballou et al., 1995).
The developed colonies were counted using manual colony counter and the number of the total bacterial count was calculated as colony forming unit/mL (Christen et al., 1992). Identification of the isolated bacteria was done according to Barrow and Feltham (1993).
Results and Discussion
Table 1 shows that the treated milk samples did not show any significant differences in total bacterial counts, when kept at room temperature (4.22x109±4.4x109cfu mL-1) or refrigerator (2.68x09±4.2x109 cfu mL-1). Also the present study revealed that acidity of treated milk was not significantly different due to storage temperature, when adding the lactoperoxidase enzymes. This might indicated that LPS could be a method of milk preservation when cooling facilities are not available which supported Lambert (1993) and Kangumba et al. (1997). The suppression of bacteria by LPS (Table 1) indicated the antimicrobial effect of LPS, which supported McLay et al. (2002), Florisa et al. (2003) and Dufour et al. (2004).
The acidity of raw milk initially was 0.209% and it was observed to show approximately constant levels until it increased to 0.265% after 9 days of storage (Table 2), which was due to the action of lactoperoxidase system. This supported the previous reports of McLay et al. (2002), Florisa et al. (2003) and Dufour et al. (2004). Similarly non significant variations were found for the coliform counts in the treated milk samples that kept at room temperature (2.32x109±2.9x109cfu mL-1) and refrigerator (1.40x109± 2.4x109cfu mL-1). This result might be due to powerful effect of the antimicrobial effect of the lactoperoxidase system, which supported Gaya et al. (1991), Florisa et al. (2003) and Garcia -Graells et al. (2003). On the other hand highly significant variations (p<0.001) were observed for total bacterial counts (1.17x1010±2.0x109cfu mL-1 and 1.83x109±2.9x109cfu mL-1) and coliform counts (4.23x109±1.7x109cfu mL-1 and 1.29x109±1.9x109cfu mL-1) for the non treated (control) milk samples kept at room and refrigerator temperature, respectively (Table 1). This could be due to the effect of cooling (IDF, 1994). This might indicate the usefulness of applying the use of LPS for preservation of milk especially where cooling facilities are not available as was stated before by Lambert (1993). The higher counts of both total and coliform bacteria obtained during this result might be due to the effect of bacterial growth in high temperature as reported by Yuan (2001). However Durfour et al. (2004) reported that two hours exposure of combined LPS resulted in decrease of numbers of viable bacterial attached to the stainless steel. Similarly the present study revealed non significant differences for acidity, when comparing the milk samples that were kept at refrigerator and room temperature, which indicated that this milk contain higher load of bacteria.
Table 2 shows the effect of LPS on the keeping quality on milk during storage, as the total bacterial count at the beginning of the storage period was 9.63x109 cfu mL-1 and it decreased to 3.0x106cfu mL-1 after 10 days of storage. Also coliform count at the beginning of the storage revealed 4.05x109 cfu mL-1 and it decreased to 1.0x106 cfu mL-1.
| Table 1: | Comparison of the microbial loads and acidity of the control raw milk and lactoperoxidase treated milk samples |
| *** = p< 0.001, NS = Non Significant, SD = Standard Deviation, Sig. = Significant | |
| Table 2: | Effect of lactoperoxidase enzymes system on the keeping quality of raw milk |
| Different superscript letter(s) on the same column are significantly different (p<0.05) | |
This might be due to the killing and inhibition effect of lactoperoxidase system on the growth of organisms as reported previously (Gaya et al., 1991; Florisa et al., 2003; Garcia et al., 2003; Durfour et al., 2004; Elliot et al., 2004).
Isolation of Bacteria from Raw Milk
The microorganisms, which were isolated from raw milk include E. coli,
Citrobacter freundii, Yersinia enterocolitica, Enterococcus feacalis
and Staphylococcus aureus. This was supported Yagoub et al. (2005)
who reported that the most predominant bacteria in raw milk samples investigated
in Khartoum North were Staphylococcus aureus (30%), Citrobacter spp.
(21.43%), Shigella spp. (20%), E. coli (14.28/%), Enterobacter
spp. (12.86%) and Salmonella spp. (1.43%). However the treated milk
sample revealed the presence of Enterococcus feacalis. This result explain
the reduction of the total number of bacteria that was found be reduced in case
of lactoperoxidase treated milk (Table 1). This indicated
that the presence of E. coli, Citrobacter freundii, Yersinia
enterocolitica and Staphylococcus aureus in milk were inhibited by
the addition of LPS, which agreed with Kangumba et al. (1997) and Elliot
et al. (2004). However the present study observing a degree of resistance
of Enterococcus feacalis to lactoperoxidase, since the inhibition occurred
after prolong exposure to treatment (9 days for refrigerated treated milk).
This might be because LPS is proved to have greatest activates against Gram
negative bacteria (McLay et al., 2002). Hence the lactoproxidase system
can be used to preserve raw milk in countries where refrigeration facilities
are uncommon (Lambert, 1993). Moreover FAO in collaboration with SIDA (Sweden)
is planning a carry out worldwide testing in about 80 counties worldwide, following
the successful field testing in several countries including Cuba, Pakistan and
Philippine (Codex Alimentarius Commission, 2004).
The present study recommended the uses of LPS; as it proves to be an efficient procedure for preservation of milk; in Sudan especially at the rural areas in order to utilize the large quantities of milk that was unavailable to the consumers. This will also help in providing enough milk for the dairy factories that have just been started in and around the big towns. It is also recommended that fund should be raised from national and international bodies to facilitate the improvement of dairy sector in Sudan. Further research on antimicrobial effect on the different milk borne diseases and spoilage organisms is needed. Also field investigations are highly required to examine the LPS under the different conditions of Sudan.
Acknowledgements
The financial support of the Islamic Development Bank, Jadah (ISDB) is appreciated with thanks. We would also like to thank Dr. Awad El Kareem Abdallah, Head of Animal Production Directorate, Ministry of Animal Resources, Sudan for providing the Lactoperoxidase system enzymes kits.