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Effect of lactoperoxidase enzyme system and storage temperature on the keeping quality of sheep milk

M S A Saad, I E M El Zubeir and A M A Fadel Elseed*

Department of Dairy Production, Faculty of Animal Production, University of Khartoum.
Khartoum North, Postal Code 13314, Sudan
ibtisammohamed@hotmail.com
* Department of Animal Nutrition, Faculty of Animal Production, University of Khartoum,
Postal code 13314, Sudan, Fax: +249 185 321246

Abstract

This study was conducted to estimate the effect of lactoperoxidase enzyme system (LPS) on the shelf life of sheep milk. Bulk sheep milk samples were collected and transported to the laboratory, where the milk samples were divided into two parts: One part was preserved with LPS and the other was kept as control. The two parts (treated and control) were further subdivided into three portions each and stored at temperatures of 37C, 25C and 7 C. Chemical analysis was carried out to determine fat, protein, lactose and solid non fat contents. Similarly clot on boiling test, titratable acidity and microbial content were done.

Sheep milk contained 6.59% fat, 4.11% protein, 10.05% SNF, 5.28% lactose and 0.21% acidity. The shelf life of sheep milk samples was 6-7 hours, 7-8 hours and 9-10 days forthe control samples kept at 37 C, 25 C and 7 C, respectively, and were 8-9 hours, 9-11 hours and 15-16 days for LPS treated milk samples. The result showed highly significant (P≤ 0.001) effect of LPS on the shelf life, acidity and microbial content of sheep milk. However, the data indicated that the treated milk samples did not show any significant differences in acidity, TBC, coliform and psychrotrophic counts after preservation with LPS. The LPS can be used in marketing sheep milk for added commercial values. Good hygienic practices in milk production are important to the efficacy of the LPS and to the microbiological quality of the raw milk.

Keywords: LPS preservation, microbiological quality, sheep milk, shelf-life


Introduction

Most of the consumers in Sudan use raw milk supplied to them from far away without cooling as the marketing system of milk are weak and on a transitional stage (Elmagli and El Zubeir 2006). 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.

The natural antimicrobial system present in milk, the lactoperoxidase system, has been used to preserve raw milk quality in areas where it is not possible to use refrigeration for economic reasons (Seifu et al 2005). Lactoperoxidase system is active against both Gram positive and Gram negative microbes to varying extents (Marks et al 2001, FAO/WHO 2005, El Zubeiret al 2006). FAO/WHO (2005) strongly discourages the preservation of milk by chemical means, except the application of H2O2 as native LPS and, in the case of H2O2, it must be completely destroyed before consumption (Ozer et al 2003). The lactoperoxidase is a natural bacterial defense system through the oxidation of thiocyanate ions (SCN-) by hydrogen peroxide (Tenovuo 2002, FAO/WHO 2005). The lactoperoxidase system has been found useful in prolonging the shelf life of raw milk in countries where refrigeration facilities are absent and its efficiency has been proven (El Zubeir et al2006). 

The lactoperoxidase enzyme is present in the milk of all mammals, although there is a variation at the species and even at the individual animal level (Fonteh et al 2002). The application of the lactoperoxidase system for milk preservation at collection centers aimed at raising regional awareness to safe, cheap and effective alternative milk preservation method (Codex Alimentarius Commission 2004). El Zubaier (2012) evaluated the hygiene quality of raw cow milk after preservation with LPS. The study showed that significant (P≤0.001) differences were found for total viable bacteria and coliform counts of the cow’s milk stored at both room and refrigerator temperature. The tested thiocyanate and H2O2 mixtures showed no relevant antimicrobial effect. However, by adding lactoperoxidase enzyme, the mixtures became not only an effective bactericidal (Streptococcus mutans and sanguinis) but also a fungicidal (Candida albicans) agent (Welk et al 2009).  FAO/WHO (2005) found that good hygienic practices in milk production are critical to the efficacy of the lactoperoxidase system and to the microbiological quality of the milk.

Sheep milk is highly nutritious as it has high total solids, fat and protein (Awaura and Akinsoyinu 2010). Hence the present study was initiated with the objectives of assessment of chemical composition (fat, protein, lactose and solids non fat) and acidity of sheep milk. It also aimed at improvement of the shelf life of sheep milk by using lactoperoxidase enzyme system.


Material and methods

Source of samples

Bulk sheep milk samples (5 batches) were collected from a farm in Shambat, Khartoum State during the period of June to August 2010. The samples were transported immediately after milking to the microbiology laboratory, Department of Dairy Production, University of Khartoum. The milk samples were divided into two parts. One part of the samples was preserved with lactoperoxidase enzyme system (LPS), while the other was kept as a control. The LPS was obtained from Ministry of Animal Resources which is provided to them by FAO in order to test the efficiency in the field. The two parts (treated and control) were further subdivided into three, the first one was kept at 37C, the second was kept at 25 C and the third was kept in the refrigerator (6 2 C).

Chemical analysis
Chemical composition

The milk constituents (fat, protein, lactose and SNF) and physical characteristics (density and freezing point) of the milk samples were examined using Milk Analyzer Lactoscan 90 (Aple Industries Services –La Roche Sur Foron, France) according to the manufacturer instructions. The details of the methods were described previously by AbdElrahman et al (2009).

Determination of shelf life of milk

The acidity of milk samples was determined according to AOAC (1990) and the clot on boiling test of the milk samples was done, according to Foley et al. (1974). They were at zero time and then each hour up to 6 hours, then daily up to 16 days.

Microbiological examination 
Culture media

All media were obtained in a dehydrated form and were prepared according to the manufacturer instructions.

Plate count agar (Scharlau Chemie S.A. Batch 15599 Barcelona, Spain, European Union), was selected to determine total viable bacteria and psychrotrophic bacteria counts (Houghtaby et al   1992). MacConkey agar (Scharlau Chemie S.A. Batch 15455 Barcelona, Spain, European Union), was used to determine the coliform count (Harrigan and MacCane 1996).  Potato dextrose agar (Hi Media Laboratories Pvt. Ltd. Mumbai – 400 086, India) was used to determine the total yeast and moulds counts (Frank et al 1992). 

Sterilization and dilution

The media, distilled water and tips were sterilized in the autoclave at a temperature of 121C for 15 minutes. Glassware such as Petri- dishes, test tubes, pipettes, and flasks were sterilized in hot air oven at 170C for 2 hours (Marshall 1992). One ml of the milk sample was transferred with sterile pipette into 9 ml ringer solution in a test tube and mixed thoroughly. Using another sterile pipette one ml of prepared dilution was transferred into the second tube containing 9 ml. The process was repeated to make ten fold dilutions from 10-1to10-8 (Houghtaby et al 1992). 

Plating technique

For total bacterial, psychrotrophic and coliform counts, one ml of each sample was transferred into two sterile Petri- dishes, then 15-20 ml of the selected media at 45- 46 C were poured under sterile condition into each plate. The medium was mixed immediately and shaked for 5-10 seconds. For yeast and moulds, 0.1 ml of diluted sample were spread over prepared dried plates using sterile bent glass rod, then the cultured plates were incubated at 32C 1 for 48 hours, 35 C 2 for 24 hours, 25 C for 5 days and at refrigeration temperature for 10 days for total bacterial, coliform, yeast and moulds and psychrotrophic bacteria, respectively (Marshall 1992). 

Counting

Developed colonies on plates were counted using colony counter. According to Marshall (1992), the plate containing 25- 250 cfu were enumerated for total bacterial and psychrotrophic count while the plate containing 15- 150 colony forming units (cfu) were enumerated for coliform count and yeast and moulds count.  

Statistical analysis

Data of present study were analyzed statistically using factorial design. The analysis of variance (ANOVA) tests were carried out by using the general linear model procedure of the Statistix (Version 8.0). The Least Significant Difference (LSD) was used to determine the differences between means.


Results and Discussion

The chemical composition of sheep milk

The mean values of fat, protein, lactose and SNF content of raw sheep milk were 6.59 0.02%, 4.11 0.07%, 5.28 0.02% and 10.05 0.04%, respectively (Table 1). The result of ewe’s chemical composition obtained during this study agreed with the report of Abdalla and Daffalla (2010). However it revealed lower mean for fat content than that reported by Park (2006) who found that fat and SNF contents were 7.9% and 12.0%. These variations might be due to the differences in nutrition (Bencini and Pulina1997). They reported that nutrition affects the total milk production as well as the quality of the milk and that the concentration of fat in the milk is correlated with the concentration of fiber in the diet. Similarly, it supported the previous report of Doepel and Lapierre (2006).The present results disagreed with Jandal (I996) who found that fat and protein contents were 7.62% and 6.21%, and with that obtained by Blench (2001) who found the range of 3.9–4.7% for lactose content. These variations might be due to differences of breeds which supported Bencini (2002) who reported that the genotype of the sheep can affect the composition of the milk. 

Shelf life of sheep milk samples

Table 1 showed that the mean of acidity were 0.21 0.004%. The result showed non significant effect of interaction between LPS and storage temperature for titratable acidity, while highly significant (P≤0.001) variation were found for acidity if control milk samples and LPS treated milk samples that kept at both 25 C and 37 C. The result showed reduction in the acidity for LPS treated sheep milk samples until 9 days in the refrigerator storage which was due to the action of lactoperoxidase system (Figure 1). This supported El Zubeir et al (2006) who reported that the acidity of LPS treated bovine milk was initially 0.209% and it was observed to show approximately constant levels until it increased to 0.265% after 9 days of storage.

Figure 1: Effect of LPS on the titratable acidity of sheep milk stored at different temperatures

 The present findings are also in support to Florisa et al (2003). Table 3showed that the treated milk samples did not show any significant differences in acidity for milk stored at different storage temperatures. This might indicate that the lactoperoxidase system is an acceptable chemical method for raw milk preservation, especially in rural areas where refrigeration facilities are absent to farmers (Lambert 1993, Elliot et al 2004, El Zubeiret al 2006).

Table 1: Chemical and microbial properties of fresh raw sheep milk

Measurements

Mean  Standard error

Minimum

Maximum

Fat (%)

6.59   0.024

5.33

7.33

Protein (%)

4.11    0.07

3.5

4.85

Lactose (%)

5.28    0.02

4.59

5.85

Solids non fat  (%)

10.05 0.04

8.64

11.15

Titratable acidity (%)

0.21 0.004

0.20

0.22

Total bacterial count (cfu/ml)

8.461074.42106

1.08107

6.51108

Coliform count (cfu/ml)

6.2105 3.9104

5.1104

9.1105

Psychrotrophic count (cfu/ml)

5.941075.36106

4.58107

7.05107

Yeast and mould counts (cfu/ml)

5.6105 4.4104

3.4104

7.8105

The present study showed that the samples of sheep milk stored at different temperatures and treated by LPS showed higher shelf life than non treated (control) sheep milk samples (Table 2). This might indicate the usefulness of applying LPS for preservation of milk especially where cooling facilities are not available which supported Codex Alimentarius Commission (2004). The present result showed a shelf life of 15-16 days, which was lower than that reported by El Zubeir (2012) who stated that lactoperoxidase treatment prolongs the camel milk shelf life for 11-19 days at refrigeration temperature. This was in accord to Seifu et al (2005) and El Zubeir et al (2006). The shelf life of sheep milk samples was affected significantly (P≤0.001) by LPS, storage temperature and the interaction between temperature and LPS. This supported FAO/WHO (2005) which stated that the inhibitory effect of LPS treatment is dependent on the storage temperature of LPS treated milk. The result was in support to El Zubeir (2012) who found highly significant (P≤0.001) effect of LPS as a milk preservative.

Table 2:  The effect of lactoperoxidase enzymes system on the shelf life of sheep milk samples

Storage temperatures

 

Control samples

LPS treated samples

37 C

Range (hours)

6 – 7

8 – 9

Mean SE

6.57 0.14

8.12 0.14

25 C

Range (hours)

7 – 8

9 - 11

Mean SE

7.53  0.14

9.69 0.14

7 C

Range (days)

9 – 10

15 – 16

Mean SE

9.5 0.14

15.6 0.14

Microbiology of LPS treated sheep milk

The mean counts of total bacteria, coliform bacteria, psychrotrophic bacterial and yeast and moulds of sheep milk samples were 8.46107 4.42106cfu/ml, 6.2105 3.9104cfu/ml,5.94107 5.36106 cfu/ml and 5.6105 4.4104cfu/ml, respectively (Table 1). On the other hand, the total bacterial count of treated milk samples was not significantly different due to storage temperature. The result revealed highly significant (P≤0.001) variations in microbial load by LPS. This might be due to powerful effect of the LPS as an antimicrobial, which supported Elliot et al (2004), El Zubeir et al (2006); El Zubeir (2012).The suppression of yeast and moulds by lactoperoxidase system (Figure 3) indicated the antimicrobial effect of LPS, which supported Welk et al  (2009) who reported that LPS became not only an effective bactericidal but also a fungicidal agent.

Table 3 revealed that the treated milk samples did not show any significant differences for TBC, coliform and psychrotrophic counts of sheep milk samples kept at different temperature when adding the LPS. Moreover the effect of LPS on the keeping quality of milk samples during storage at 37 C and 25 C revealed that total bacterial count, coliform count, psychrotrophic bacterial count and yeast and moulds counts decreased after six hours for LPS treated samples and showed lower values compared to those of fresh milk samples. Moreover the data were significantly different (P≤0.05) when compared to the control samples (Table 4).

Table 3: Interaction between LPS and temperature on the acidity and microbial contents of sheep milk samples

Measurement

Means SE

Sample

7 C

25 C

37 C

Acidity (%)

Control

0.231 0.003 c

0.232   0.004  c

0.244 0.004 a

Treated

0.221 0.003 b

0.223 0.004  b

0.225 0.004 b

Total bacterial count

(cfu/ml)

Control

1.7810 85.61106  b

1.851081.25107  b

2.40108 1.25107  a

Treated

7.03107 5.6106  c

7.72107 1.25107 c

7.83107 1.25107  c

Coliform count

(cfu/ml)

Control

1.07106 2.31105 a

1.22105.15105  ab

1.3810 5.15105  ab

Treated

3.94 105 2.31105  b

5.98105 5.15105  b

5.61105   5.15105 b

Psychrotrophic bacterial Count (cfu/ml)

Control

1.36108 5.33106  c

1.55108 1.19107  a

1.8110 1.19107 a

Treated

3.54107 5.33106  b

3.88107 1.19107  b

3.97107 1.19107  b

Yeast and moulds counts

(cfu/ml)

Control

7.73105 3.73104  b

8.65 105   8.34104  b

1.22108.34104  a

Treated

1.52 105 3.73104  c

1.66105 8.34104  d

2.68 105 8.34104  d

 The result revealed that the growth of TB, coliform bacteria, psychrotrophic bacteria and yeast and moulds for LPS treated milk samples stored at refrigerator reduced throughout the storage period. On the other hand, the TBC, coliform bacteria,psychrotrophic bacterial count and yeast and moulds countsof the control milk samples was increasing throughout the shelf life of milk (Figure 1, Figure 2 and Figure 3).

Figure 2: Effect of LPS on the psychrotrophic and total bacterial counts of sheep milk stored at refrigerator


Figure 3:  Effect of LPS on the coliform and yeast and moulds counts of sheep milk stored at refrigerator

The results of psychrotrophic bacterial and coliform counts for treated samples at refrigerator decreased gradually before disappearing (Figures 2 and Figure 3). This might be because LPS proved to have powerful activities against Gram negative bacteria as reported previously by Marks et al (2001) and El Zubeir et al (2006). Hence the present study supported the previous recommendations that LPS could be a method of milk preservation under tropical hot environment that lack the refrigeration facilities (Florisa et al 2003, Garcia et al 2003; El Zubeir 2012).


Conclusion


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Received 3 May 2013; Accepted 9 May 2013; Published 2 June 2013

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