Effect of the lactoperoxidase system and container smoking on the microbial quality of cows’ milk produced in Kombolcha woreda, eastern Ethiopia

Helen Nigussie and Eyassu Seifu*

Department of Animal Sciences, Ambo University College, PO Box 19, Ambo, Ethiopia
* Department of Animal Sciences, Haramaya University, PO Box 287, Alemaya, Ethiopia
eyassu_seifu@yahoo.com

Abstract

The study was conducted in Kombolcha woreda in eastern Ethiopia to assess the effect of the lactoperoxidase (LP) system in combination with container smoking on the microbial quality of cows’ milk. Milk samples obtained from twenty randomly selected households from four districts were used to examine the effects of four treatments (NT_, control; LPS_, activation of the LP system; CS container smoking; and LPS-CS container smoking plus activation of the LP system) on acid production and microbial load of cows’ milk. All milk samples were tested for titratable acidity, standard plate count and coliform count during storage at an ambient temperature for 24 h.

The titratable acidity, coliform count (CC) and total bacterial count (TBC) in LP activated milk samples (LPS) decreased by 0.13%, 1.73 log units and 1.07 log units, respectively as compared to their respective values in the control (NT) at 7 h of storage. The percent lactic acid (0.20%) in LP activated milk at 7 h of storage was similar to the initial acidity level (0.19%) in the milk. Coliform count and TBC in LPS decreased by 0.57 log and 0.23 log units, respectively at 7 h of storage as compared to the initial count. On the other hand, CC and TBC increased by 1.16 log and 0.84 log units, respectively in the control at 7 h of storage as compared to the initial count. An increase in acid production and microbial count was observed in milk samples kept in smoked containers (CS) at 7 h of storage; however, when container smoking was combined with the LP system (LPS-CS), acid production remained unaltered and microbial counts decreased at 7 h of storage as compared to the initial levels.  

It can be concluded that on-farm activation of the LP system and container smoking combined with the LP system could effectively control microbial growth and extend the shelf life of cows’ milk produced in the study area by at least 7 h during storage at an ambient temperature.  

Keywords: Ambient temperature storage, LP system, on-farm activation, shelf life, traditional preservation

Introduction

Kombolcha woreda (an administrative district that consists of many peasant associations depending on its size) is found in eastern Ethiopia at a distance of 14 km west of Harar town. The woreda has good potential for dairy production and milk is produced in all rural districts of the woreda. Cows and goats are the major dairy animals that produce milk in the woreda. Unlike other parts of Ethiopia, there is huge demand for fluid milk on the market in and around Kombolcha and as a result processing of milk and consumption of fermented dairy products is not as such common in the woreda (Helen 2007). The high demand for fluid milk in the area created marketing opportunity to the farmers and milk is often a regular source of income for the producers in Kombolcha woreda. However, milk is often produced and handled under poor sanitary conditions. As a result, large volume of milk is spoiled in the area due to lack of appropriate milk preservation methods.   

Although milk is a very nutritious food, it is also an important vehicle for transmission of pathogenic microorganisms to human beings unless it is produced and handled under good hygienic conditions. Thus, hygienic production of milk has to get due attention in order to provide more and better quality milk for the general public. The major methods used to safeguard the bacteriological quality of raw milk in industrialized countries are pasteurization and cooling. However, these methods are often not practical in most developing countries for technical and/or economic reasons. Thus, use of alternative milk preservation methods that are safe, cheap and easily applicable under farm conditions is of paramount importance. The lactoperoxidase (LP) system is one such method that helps to minimize microbial proliferation and extend the shelf life of milk. The use of the lactoperoxidase system for preservation of raw milk has been reported by several workers from different countries (Björck et al 1979; Thakar and Dave 1986; Fontheh et al 2005). However, the effectiveness of the system depends on the conditions that prevail in a given area particularly the microbial load of the milk before treatment and the prevailing ambient temperature.  

In many parts of Ethiopia, milk vessels are usually smoked using wood splinters of Olea africana to impart desirable aroma to the milk. Smoking was also found to lower the microbial lad of raw milk (Mogessie and Fekadu 1993). Smoking of milk vessels is the major method that is traditionally used to preserve raw milk in the study area. In Kombolcha woreda, it is hardly possible to find a farmer who delivers milk to the market in unsmoked container. Due to lack of road and transportation systems, some farmers in the study area walk more than 10 km to deliver their milk to the market. Even after arrival at the market, the milk may be kept for several hours at the high ambient temperature on the open market until it is sold. Under these circumstances, the chance of spoilage of milk is very high. Thus, development of a practical method that could help farmers to preserve their milk during storage and transportation to the market would help minimize spoilage of milk at the prevailing high ambient temperature. 

The lactoperoxidase system acts in synergy with other food preservation methods to control the growth of microorganisms in food systems (Zapico et al 1998; García-Graells et al 2002; McLay et al 2002). Thus, the LP system combined with container smoking may inhibit the growth of microorganisms in cows’ milk and thereby extend its shelf life. Use of the LP system in combination with container smoking to control microbial growth in cows’ milk would create a practical and promising opportunity to preserve raw milk at farm level in areas where there are no milk cooling facilities.  

Most of the studies conducted so far on the use of the LP system for preservation of raw milk were conducted at laboratory scale under controlled temperature. However, only few studies have been reported on the use of the LP system at farm level and its effectiveness on raw milk preservation under real life situation during storage at field conditions (Björck et al 1979; Härnulv and Kandasamy 1982; Kumar and Mathur 1989). The objective of this study was, therefore, to assess the effect of the lactoperoxidase system combined with container smoking on the microbial quality of cows’ milk produced in Kombolcha woreda in eastern Ethiopia during storage at an ambient temperature. 

Materials and methods

The study area

The study was conducted in Kombolcha woreda which is located in eastern Ethiopia at a distance of 14 km from Harar town. The woreda comprises 19 Peasant Associations and has a total area of about 46,461 hectares of which 74% is mid highland and 26% is lowland (MoARD 2004). Kombolcha woreda receives an average annual rainfall ranging from 600-900 mm and its altitude ranges from 1600 to 2400 meters above sea level (MoARD 2004). The mean minimum and maximum annual temperatures of the woreda were 14°C to 25°C, respectively (MoARD 2004). The estimated human population (both rural and urban populations) of the woreda was 112,063 (MoARD 2004). The ruminant livestock population of the woreda is estimated to be 216,892 heads (MoARD 2004). Among these, the total cattle and small ruminants’ populations are estimated to be 44,666 and 59,878, respectively. The area is predominantly mixed farming zone where cultivations of food and cash crops such as Chat (Catha edulies) and vegetables are practiced.

Raw milk sampling and arrangement of treatments  

Samples of pooled raw morning cow milk were taken three times at two weeks interval from twenty randomly selected households from four districts. Milk sampling was done within 2 to 3 hours after milking and directly from the owners’ milk vessels. About 100 to 250 ml of milk samples were collected from each participant and these samples were pooled and thoroughly mixed. The average composition of the milk samples was 6.1% w/v fat, 3.7% w/v protein, 16.7% w/v total solids, 10.7% w/v solids-not-fat and 0.92% w/v ash. The milk samples had an average thiocyanate content of 7.38 ppm. 

Representative milk samples (5 liters) collected each time were divided into five portions (of 1 liter each), labeled as NT, LPS, CS, LPS-CS and T₅ and kept in screw capped sterile bottles for microbial quality tests. The first portion of milk (NT) was used as a control, that is, milk without any added preservative. The second portion (LPS) of milk was subjected to activation of the LP system. The third portion of milk (CS) was kept in smoked container in order to determine the effect of container smoking on microbial quality of milk. Container smoking was done using wood splinters of Olea africana at the Haramaya University (HU) dairy laboratory. The milk bottles were inverted over the smoking wood until the smoke dies out. The fourth portion of milk sample (LPS-CS) was subjected to activation of the LP system and kept in smoked bottles to determine the combined effect of the LP system and container smoking on microbial quality of the milk. All the four milk samples were delivered to the laboratory at an ambient temperature (22-23°C). The fifth portion of milk (T₅) was put in a sterile bottle and kept in an ice box and delivered to HU dairy laboratory to determine the initial microbial load of the milk samples used. Additional separate milk samples were taken for chemical analysis. 

All samples were transported to HU dairy laboratory (located 34 km away from Kombolcha) for determination of titratable acidity, total bacterial count and coliform count. Up on arrival at the laboratory, these tests were conducted on the milk samples delivered in an ice box (T₅) to determine the initial acidity and microbial load of the milk. The control milk sample and milk samples subjected to the different treatments were stored at an ambient temperature for a period of 24 h. Samples were taken from these milk samples at 7 h and 24 h of storage for determination of titratable acidity, total plate count and coliform count. All tests were done in duplicate. 

Activation of the lactoperoxidase system  

Activation of the lactoperoxidase (LP) system was done on farm after 2-3 h of milking by addition of 14 ml of freshly prepared solution of 1 mg/ml sodium thiocyanate (General Chemical Division, New York) per liter of milk as a source of thiocyanate (SCN^-) ion. After 1 minute of thorough mixing, 10 ml of freshly prepared solution of 1 mg/ml hydrogen peroxide (BDH Chemicals Ltd., Poole, England) was added into the milk and the mixture was thoroughly mixed for 1 min (IDF 1988).  

Titratable acidity of milk  

Milk acidity was measured by titrating milk samples with 0.1N sodium hydroxide (NaOH) (BDH Chemicals Ltd., Poole, England) solution to a phenolphthalein end point as described by Richardson (1985). Ten ml of milk sample was pipetted into a beaker and then 3 to 5 drops of 1% phenolphthalein (Fluka AG, Buchs, Switzerland) indicator was added into the milk. The milk sample was titrated with the NaOH solution until faint pink color persists. The titratable acidity of the milk was expressed as percent lactic acid and calculated as follows: 

Microbiological tests  

One ml of milk sample was added into sterile test tube having 9 ml of peptone water (Qualigens Fine Chemicals, Private Ltd., India). After thorough mixing, the sample was serially diluted up to 1: 10^-8 dilution level. Total viable bacterial count was determined using Standard Plate Count Agar (Don Whitley Scientific Equipment, Private Ltd., India). Ten to 15 ml of standard plate count agar heated to a temperature of 45°C was pour plated onto duplicate Petri dishes having 1 ml of milk sample and the Petri dishes were rotated to evenly distribute the sample in the agar medium. The plated sample was solidified for 15 minutes in a safety cabinet and incubated for 48 hours at 30°C (Richardson 1985). Coliform count was determined following similar procedure as indicated above for total bacterial count except that Violet Red Bile Agar (Micro Master Laboratories, Private Ltd., India) was used as growth medium. The inoculated plates were incubated at 30°C for 24 hours (Richardson 1985). The estimated colony count was computed by the formula described by IDF (1991). 

Statistical analysis  

Data for total bacterial count and coliform count were log₁₀ transformed before subjected to statistical analysis. The differences in microbial counts and titratable acidity of milk samples subjected to the different treatments at a particular storage period were analyzed by the analysis of variance technique using the General Linear Model (GLM) procedure of SAS (2000). Mean comparison was done using least significant difference (LSD) for variables whose F values were found to be significant. Significant differences were calculated at 5% significance level. 
 

Results  

Effect of the lactoperoxidase system and container smoking on acid development in milk  

The effect of the lactoperoxidase system on titratable acidity in cows’ milk is shown in Table 1. Treatment two (activation of the LP system) significantly (P < 0.05) retarded lactic acid production (0.13%) as compared to the control (NT) and milk samples kept in smoked containers (CS) (0.07%) at 7 h of storage. At 7 h of storage, milk samples treated with LPS and LPS-CS had comparable level of acidity. No significant increase in acid production was observed in LP treated milk samples during 7 h of storage as compared to the initial level of acidity. However, acid production in the control milk sample increased by 68.4% at 7 h of storage as compared to the initial level.  

The level of acidity increased with time in all milk samples after 7 h of storage. However, the level of increase in acidity was significantly lower (P < 0.05) in LPS and LPS-CS as compared to NT and CS at 24 h of storage. Low acid production was observed in milk samples stored in smoked containers (CS) as compared to the control (NT) at 7 h of storage. However, the level of lactic acid in milk samples stored in smoked containers was significantly higher (P < 0.05) than in milk samples treated with the LP system combined with smoking (LPS-CS) both at 7 h and 24 h of storage.              

Effect of the lactoperoxidase system and container smoking on microbial quality of milk 

The effect of the LP system on coliform count (CC) of cows’ milk stored at an ambient temperature is shown in Table 2. The CC in LP activated milk samples (LPS) was significantly (P < 0.05) lower (1.73 log units) than CC in the control (NT) and in milk samples kept in smoked containers (CS) (0.8 log units) at 7 h of storage.

However, at 24 h of storage no significant difference in CC was observed between the LP treated milk samples (LPS), milk samples stored in smoked containers (CS) and the control (NT) (Table 2). On the other hand, container smoking combined with the LP system (LPS-CS) resulted in a significant reduction (1.03 log units) in CC as compared to the control at 24 h of storage (Table 2). Coliform count in LP activated milk samples decreased by 0.57 log units at 7 h of storage as compared to the initial count. However, the CC in the control milk sample increased by 20.7% at 7 h of storage as compared to the initial count. 

The effect of the LP system on total bacterial count (TBC) in cows’ milk stored at an ambient temperature is shown in Table 3.

The TBC in LP activated milk samples (LPS) was significantly (P < 0.05) lower (1.07 log units) than TBC in the control and in milk samples treated with CS (0.63 log units) at 7 h of storage. However, TBC was comparable in LPS and LPS-CS both at 7 h and 24 h of storage. TBC in LP activated cows’ milk decreased by 0.23 log units at 7 h of storage as compared to the initial count. However, the TBC in the control milk sample increased by 11% at 7 h of storage as compared to the initial count.  

A decrease in TBC was observed in milk samples stored in smoked containers (CS) both at 7 h (0.44 log units) and 24 h (0.36 log units) of storage as compared to the control. However, when container smoking was used together with the LP system (LPS-CS), the TBC decreased by 0.87 log units and 0.77 log units at 7 h and 24 h of storage, respectively as compared to the control (Table 3). 

 

Discussion 

The delay in acid development observed until 7 h of storage in LP activated milk samples indicates that under the current condition, activation of the LP system can maintain the quality of cows’ milk in the study area up to 7 h during storage at an ambient temperature. This result is inline with the findings of Thakar and Dave (1986) who reported that activation of the LP system in buffalo milk delayed acid production by 7.5 h during storage at 23°C. Similarly, Kumar and Mathur (1989) reported that titratable acidity remained unaltered up to 6 h in LP-activated buffalo milk during storage at an ambient temperature (30-32°C). Activation of the LP system retarded lactic acid production by 0.21% as compared to the control at 24 h of storage. Since the antimicrobial effect of the LP system depends on the initial microbial load of the milk, the lower lactic acid production observed even after 24 h of storage suggests that under good hygienic milking and handling conditions, activation of the LP system might extend the shelf life of cows’ milk for more than 7 h. Fonteh et al (2005) reported that activation of the LP system can extend the shelf life of cows’ milk produced in Cameroon and stored at ambient temperature (21-23^oC) for at least 9 hours. Extension of the shelf life of milk for about 7 h at field condition is of paramount importance to the farmers who usually do not have milk cooling facilities.  

Container smoking is the most common method that is traditionally used to preserve milk in the study area and it is usually difficult to find farmers who deliver milk to the market in unsmoked containers. The low acid production observed in milk samples stored in smoked containers (CS) in the current study is inline with that reported by Mogessie and Fekadu (1993) who found that the rate of acid production and the amount of acid produced was lower in cow milk samples stored in smoked containers than in milk samples stored in unsmoked containers. Although the level of acid produced in milk samples stored in smoked containers (CS) was lower than the control, the acidity level (0.27%) observed in milk samples stored in CS indicates a significant increase in acid production in milk samples kept in smoked containers during storage for 7 h. On the contrary, when container smoking was used together with the LP system (LPS-CS), acid production was arrested for 7 h. This suggests that the LP system and container smoking have no antagonistic effect on each other and the efficiency of the traditional practice of container smoking used by farmers to preserve milk in the area can be significantly improved if it is used together with activation of the LP system. 

The decrease in coliform count (0.57 log units) observed in LPS at 7 h of storage as compared to the initial count suggests that activation of the LP system can extend the shelf life of cows’ milk stored at an ambient temperature up to 7 h by inhibiting the growth of coliforms in milk. This result is in agreement with the findings of Thakar and Dave (1986) who reported that activation of the LP system resulted in reduction of acid producers count in buffalo milk during storage at 23°C for 7.5 h. The significant (P < 0.05) reduction in CC observed in LPS-CS (container smoking combined with the LP system) at 24 h of storage as opposed to LPS suggests that smoking combined with the LP system may inhibit the growth of coliforms in cows’ milk stored at an ambient temperature for longer period of time than the LP system alone. The initial coliform count (5.60 log units) observed in the present study is much higher than the acceptable level of coliforms in raw cows’ milk. Different reports indicated that initial milk quality and the condition under which the experiment was conducted determine the effectiveness of the LP system for preservation of raw milk (Reiter 1985; Zapico et al 1993; FAO 1999). If the LP system inhibits the growth of coliforms in milk samples that had higher initial CC as observed in the present study, the inhibitory effect of the LP system in cows’ milk with low initial CC would be more and may last for a long time. The better the quality of milk at the time of LP-activation, the longer the extension of its keeping quality (Härnulv and Kandasamy 1982).  

The decrease (0.23 log units) in total bacterial count observed in LP-activated milk (LPS) at 7 h of storage as compared to the initial count suggests that activation of the LP system can maintain the quality of raw milk of cows for at least 7 h during storage at ambient temperature by inhibiting the growth of the general flora. The current result agrees with the findings of Härnulv and Kandasamy (1982) who reported that LP-activated cow milk samples showed viable counts lower than the initial count after 2 h of LP activation during storage at 30°C in Sri Lanka. Similarly, Thakar and Dave (1986) reported that standard plate count showed slight decrease in LP-activated buffalo milk during storage at 23°C for 7 h as compared to the initial count. Although the LP system and smoking combined with the LP system had comparable effect on acid development and microbial load at 7 h of storage, the combined effect of smoking and the LP system at 24 h of storage was always greater than the effect of the LP system alone (Tables 2, 3 and 4).  This suggests that with prolonged storage, smoking combined with the LP system may exert more inhibitory effect than the LP system alone. However, further study is needed to prove this hypothesis. 

 

Conclusion 

• On-farm activation of the LP system and container smoking combined with the LP system can significantly improve the microbial quality and extend the shelf life of cows’ milk up to 7 h during storage at an ambient temperature in the study area.

  
  

• The present study indicated that the LP system can be effectively used together with container smoking to extend the shelf life of raw milk of cows during storage at an ambient temperature.

  
  

• Farmers in the study area can prolong the shelf life of their milk significantly by activating the LP system in addition to their traditional practice of container smoking. 
  
   

Acknowledgements 

This study was sponsored by SIDA/SAREC Project of Haramaya University, Ethiopia. 

 

References

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Received 22 July 2007; Accepted 27 August 2007; Published 5 October 2007

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