Evaluation of Lactoperoxidase system as raw milk preservative at different storage temperature conditions in the central highlands of Ethiopia

Firew Kassa, Zelalem Yilma, Getnet Assefa, Tadesse Bekele, Yohannes Gojam, Rahel Nebiyu and Binyam Kassa

Ethiopian Institute of Agricultural Research, Holetta Agricultural Research Center
P. O. Box 2003, Addis Ababa, Ethiopia
eshokas@gmail.com

Abstract

Lactoperoxidase system (LPs) was evaluated at different storage temperature conditions as raw cows’ milk preservative at Holetta Agricultural Research Center (On – station) and at milk collection centers at Degem and Girar Jarso districts of Selale area (On – farm). The milk collected from research center and collection centers was either treated with LPs or untreated (control). For the on – station trail, both LPs treated and untreated milk was kept at room temperature, in cold water, in incubator adjusted at 30°C and in refrigerator adjusted at 10°C. For the on – farm trial LPs treated and untreated milk was kept at room temperature and in cold water. Alcohol and clot – on – boiling tests were employed to detect milk deterioration.

LPs treatment has resulted in increased milk shelf life. Its effect for both on – station and on – farm trials tended to be more efficient as storage temperature decreases. LPs treatment has extended milk shelf life by 6, 7.5, 12.5 and 74 hours, under storage temperature conditions of incubator (30°C), room temperature (22.5oC), cold water (20.0°C), and refrigerator (10oC) respectively as compared to LPs untreated milk. The results of on – station and on – farm trails showed that alcohol test detected milk deterioration earlier than clot – on – boiling test for both LPs treated and untreated milk samples. LPs can be applied to extend the shelf life of milk. This may possibly bring multiple advantages for smallholder dairy farmers in order to keep evening milk overnight without cooling facilities and to deliver it in the following morning at the collection centers.

Key words: LPs treatment, alcohol test, clot-on-boiling test, shelf life

Introduction

Lactoperoxidase (LPs) is an enzyme naturally found in milk. One of its unique biological functions is an antibacterial effect in the presence of hydrogen peroxide (H₂O₂) and thiocyanate. Both of these substances are naturally present in milk in varying concentrations. The natural bacteriostatic effect of Lactoperoxidase system lasts at least one hour after milking (Chamberlain 1993). For continued effect, the system has to be activated. This activation can be achieved by adding about 10 parts per million (PPM) (5 PPM is naturally present) of thiocyanate (preferably in powder form) to the raw milk to increase the overall level to 15 PPM (Bennet 2000). 

Although its concentration in raw milk varies with breed, age, lactation stage, feeding and the health state of the milking animals (Korhonen 1977 and Korhonen et al 1977), the enzyme lactoperoxidase is present in bovine and buffalo milk in relatively high concentrations. It oxidizes thiocyanate ions in the presence of hydrogen peroxide (H₂O₂). By this reaction, thiocyanate is converted into hypothiocyanous acid (HOSCN). At the pH of fresh milk HOSCN is dissociated and exists mainly in the form of hypothiocyanate ions (OSCN^-). This agent reacts specifically with free sulphydryl groups, thereby inactivating several vital metabolic bacterial enzymes, consequently blocking their metabolism and ability to multiply. As milk proteins contain very few sulphydryl groups and those present are relatively inaccessible to OSCN^-, the reaction of this component in milk is quite specific and is directed against the bacteria present in the milk (Yamada 1998; Claesson 1999).  

As milk contains the required nutrients for growth, contaminating bacteria if once get access in to the milk, could rapidly multiply and render it unsuitable for further processing and/or unfit for human consumption. Yet, bacterial growth can be retarded by refrigeration, thereby slowing down the rate of deterioration of milk. However, in the traditional milk production system, which accounts for about 97% of the country’s annual milk production (Getachew 2003), refrigeration facilities are not available and when available they are not affordable.  

Although the few dairy enterprises currently operating in and around Addis Ababa depend on the traditional sector for the majority of their milk supply, they are operating below their capacity mainly due to the shortage of milk supply. This does not imply the gap between the production and the demand for milk, as only 5% of the milk produced in the rural areas are marketed (Getachew 2003). In these areas, where the traditional sector dominates, the possibilities of selling fresh milk are limited and market distances are beyond product durability. It is therefore important to look for alternative methods for retarding bacterial growth in raw milk during collection and transportation to the dairy processing plant. In spite of these prevailing situations, there is an opportunity to increase dairying and there is also a trend of increasing milk production especially in the highlands. With well coordinated disposal system, the utilization of milk produced at small-scale level can therefore be improved. To this effect, the use of LPs can be the right spatial and temporal alternative for increasing the shelf life of raw milk during collection and transportation to dairy processing plants. Therefore this study was conducted to study the effect of LPs treatment on the shelf life of cow’s milk stored at different storage temperature conditions.

Materials and methods

Study area 

The study was conducted at Holetta Agricultural Research Center (on-station), altitude: 2400 masl; annual rainfall: 1100 mm; average temperature: Minimum and maximum 6^oC and 24^oC, respectively. The on-farm study was also conducted around Selale area, altitude: 2500-3000 masl; annual rainfall: 1200 mm; average temperature: minimum and maximum 6^oC and 21^oC, respectively.   

Experimental procedure 

For the on-station study, 40 liters of milk was collected from the dairy herd at Holetta Agricultural Research Center. The collected milk was thoroughly mixed and equally distributed in to two separate containers. One half (20 L) of the milk was treated with LPs while the other half (20 L) was maintained LPs untreated. Each of the LPs treated and LPs untreated milk was then divided in to four equal volumes (5L each) and stored at different temperature conditions namely at room temperature, in cold water, in incubator adjusted at 30^oC and in refrigerator adjusted at 10^oC. The room temperature and cold water conditions were ambient temperature conditions of Holetta, while the incubator (30^oC) and refrigerator (10^oC) conditions were supposed to represent ambient temperatures of different sites in the central highlands of Ethiopia.  

For the on-farm study, 20 liters of milk was collected from each of the milk collection centers at Degem and Grar Jarso districts (Selale area) where 10 liters of milk was treated with LPs while the remaining 10 liters were untreated. Each of the LPs treated and LPs untreated milk containers were then divided in to two equal volumes (5 L) in different containers where one part was stored at room temperature, while the second half was kept in cold water (Table 1).

Activation procedure 

Fourteen mg of NaSCN was added per liter of milk. The milk was then mixed to ensure an even distribution of the SCN^- and plunged for about 1 minute. This was followed by the addition of 30 mg of Sodium percarbonate per liter of milk. The milk was then stirred for another 2-3 minutes to ensure the complete dissociation of Sodium percarbonate and the even distribution of the hydrogen peroxide in the milk. As suggested by Yamada (1998), the activation of the LPs was carried out within 2-3hours from the time of milking.  

Sampling and measurements 

Milk was sampled at the interval of an hour for the two quality tests considered namely alcohol test and clot-on-boiling tests. For alcohol test, 75% Ethanol was mixed with equal volume of milk, while for clot on boiling test 10ml of milk was heated (O’Connor 1994). In both cases milk coagulation indicated quality deterioration and considered to be a break point for shelf life. Temperature and pH were measured and recorded after each sampling for the quality tests. Both the on-station and on-farm experiments were carried out in four replicates. Data collected on shelf life, storage temperature and final pH were analyzed using multivariate analysis of GLM procedures of (SPSS version 13).

Results and discussion

The use of LPs treatment has improved the shelf life of milk as compared to the LPs untreated milk (Table 2 – 5) stored in similar storage conditions including refrigerator, cold water, room temperature and incubator. LPs treated milk under the storage condition of incubator (30^oC) kept fresh for 6 more hours than LPs untreated milk under alcohol test as quality check. The effect of LPs treatment was more important under cold water and refrigerator storage conditions. LPs treated milk stored in cold water storage condition stayed fresh for 12.5 more hours while milk stored under refrigerator storage conditions kept fresh for 74.0 more hours than LPs untreated milk samples. Similarly, the pH values of the milk with LPs treatment and colder storage temperature conditions dropped slowly as compared to that stored in 30^oC. This explains that LPs treatment could be more effective as temperature decreases. Fonteh et al (2005) has also reported the shelf life of LPs treated milk stored in ice box (10^oC) kept fresh for 12 hours without considerable drop in pH while the LPs untreated milk stored at room temperature (21 – 23^oC) got spoiled only after additional 3 hours of storage. Other report also indicated that the LPs treated milk stored in refrigerator condition had 5 to 6 days of shelf life (Bjork et al 1979). 

Under the room temperature storage condition (21.5 – 22.0^oC), the LPs treated milk was kept fresh for additional 7.5 and 8 hours respectively than the LPs untreated milk using alcohol test and clot-on-boiling test as a quality indicator (Table 2 and 3). Similar results were reported by Taye (2000). This implies that LPs treatment improves the shelf life of milk under relatively moderate ambient temperature conditions. Claesson (1994) and Patel and Sannabhadti (1993) have also reported the treatment of LPs has increased the shelf life of raw milk stored at ambient temperature (30^oC) for about 7 to 8 hours. Likewise, Helen and Eyasu (2007) has reported the increment of shelf life of LPs treated milk for 7 additional hours with container smoking in the field study at Kombolcha, Ethiopia.  

As compared to the other storage conditions, the milk stored in incubator had the lowest shelf life with or without LPs treatment. However, the LPs treatment has still extended the shelf life of milk for additional 6 hours as compared to LPs untreated one stored in similar conditions of incubator (30^oC). This indicates that though the effect of LPs treatment was more efficient at low storage temperature conditions, using the LPs system at moderate or elevated tropical temperatures can extend the shelf life of milk. This can represent multiple advantages to the farmers including extra time until milk is delivered to the collection points and related economic benefits.  

On the other hand, the extended shelf life of the LPs treated milk stored in refrigerator (10^oC) as opposed to that stored in incubator (30^oC) explains that LPs treatment is more efficient under low temperature storage conditions. The LPs treated milk kept in refrigerator storage condition, stayed fresh for 167 additional hours than that stored incubator temperature (30^oC). This illustrates that storage temperature can have marked role for the improvement of shelf life of milk in addition to the use of LPs treatment. Similarly Bjork et al (1979) has reported the shelf life of LPs treated milk stored in refrigerator ended after five to six days. However for the case of smallholder farmers where using refrigerators is unaffordable or impractical for several reasons, LPs treatment can be a good alternative to extend the shelf life of the milk until they deliver it to the market place such as milk collection points.  

In general, the pH values of the LPs treated milk samples were dropped slowly while the untreated milk showed faster drop of the pH. This indicates that the LPs untreated milk shows quicker quality deterioration in terms of shelf life than the LPs treated milk. The acidity of LPs untreated milk kept in incubator adjusted at 30^oC dropped by 0.85 pH units at the stop as compared to the refrigerator storage condition. The rate of the drop for milk kept in incubator was also the fastest of all the storage conditions suggesting quicker quality deterioration. Similarly, Fonteh et al (2005) have reported quicker pH drop of LPs untreated milk kept at ambient temperature of 21 – 23^oC. Similarly Kumar and Mathur (1989) have reported the increase of titratable acidity of milk stored in similar conditions of elevated temperatures. The higher storage temperature of incubator has resulted in faster drop of pH and hence reduced shelf life of milk (Table 2 and 3). This suggests the incidence of faster metabolic activities and related rapid proliferation of lactic acid bacteria and possibly other milk spoiling mesophilic microorganisms at elevated temperature conditions of about 30^oC. In both LPs Treated and untreated milk the pH under refrigeration condition was closest to the pH of fresh milk. This also shows that there might be limited prevalence of microbial metabolic activities at lower temperature conditions (10^oC) which contributed to the longer shelf life of raw milk.

From the on-station study it was also noted that alcohol test has detected milk deterioration 1, 5, 5.5 and 2.2 hours earlier than that of clot – on – boiling test for LPs treated milk (Tables 2 and 3) for incubator, refrigerator, room temperature and cold water storage conditions respectively. Similarly, alcohol test detected milk coagulation 42 minutes to 7 hours earlier than clot on boiling test for LPs untreated milk (Tables 2 and 3) in incubator, refrigerator, room temperature and cold water storage conditions.  As reported by O’Connor (1994) alcohol test is more sensitive in detecting milk acidity than the clot – on – boiling test. Conversely, in addition to suggesting the effectiveness of alcohol test for faster identification of acid development in milk than clot on boiling test, it also explains that the LPs treatment extends the shelf life of fresh milk for more hours. Moreover, the average final pH values of LPs untreated milk under alcohol and clot – on – boiling test were lower than the pH values of LPs treated milk. The results also suggest faster acid development in LPs untreated milk as compared to the milk under LPs treatment (Tables 2 – 5).  

For the on-farm study, only two temperature conditions were considered which include keeping the milk container at ambient temperature conditions of room temperature and cold water (Table 4 and 5). As in the case of the on-station study the use of LPs treatment resulted in improvement of the shelf life of raw milk than LPs untreated milk. The LPs treated milk stored under room temperature had additional 6.5 hours shelf life as compared to the LPs untreated milk stored in similar condition when evaluated under alcohol test (Table 4). Moreover, the LPs treatment has resulted in improvement of shelf life for the milk stored in cold water for additional 6.8 hours than the LPs untreated one. This explains that the use of LPs treatment improves the shelf life of fresh milk in the farmers’ condition where there are no cooling facilities. Thus, use of LPs may enable the dairy farmers to deliver their evening milk in the next morning at the collection centers without loss in the quality. The results from various field studies also indicated significant improvement in the quality of raw milk where the shelf life of un–refrigerated and LPs treated milk was prolonged for additional three to four hours over the LPs untreated milk (Lambert, 1993).  

The shelf life of LPs treated milk stored in room temperature also showed improvement of 6 hours as compared to the LPs untreated milk when evaluated under clot-on-boiling test. Similarly, the shelf life of milk stored in cold water had 6.3 hours advantage over the LPs untreated one (Table 5). Bjork et al (1979) has also reported similar results and additional preservation time of raw milk can be seen as essential input for the safe delivery of milk with appropriate quality for the collection and processing centers. This means that more milk can be transported to milk collection centers decreasing milk loses and increasing the income generated at farm and producer level. This further implies that large quantity of milk can be collected preserved, and transported from areas where there is lack of dairy infrastructure and processed at milk collection centers and appropriate processing plants. 

Apart from the improvement of shelf life for LPs treated milk than the LPs untreated one, the overall keeping quality of both LPs treated and LPs untreated milk was produced on – station was better as compared to that of on – farm condition. This might be attributed to the difference in the quality of the milk which is related to the hygienic practices used during milking and subsequent handling of milk. Moreover, as it was observed during the field trials, milk was more prone to contamination at on – farm conditions than on – station. This is mainly due to the shortage or absence of facilities such as clean water and limited knowledge of hygienic production and handling of milk and milk products. As indicated by Claesson (1999), lactoperoxidase system is less efficient when applied to poor hygienic quality milk. Moreover various reports (Fonteh, et al. 2005) have also showed that the length of LPs treated milk shelf life and its acid developing rate depends on its initial quality. Therefore, for increased efficiency of LPs treatment, it is a prerequisite to produce milk of good hygienic quality.

Conclusion and recommendation

• Lactoperoxidase treatment at the recommended level of inclusion can extend the shelf-life of raw milk depending on the storage temperature condition. Moreover, LPs treatment could be more efficient when applied to good quality milk and kept in cool places such as cold water storage conditions. The treatment of LPs system can be applied at farm level where relatively large quantities of milk are produced and more importantly it can be practiced at the small scale milk collection centers where hundreds of liters of milk are collected daily and processed using traditional and manual processing facilities while part of the milk is transported to the bigger and modern processing plants. Training users on how to use the LPs system and planning for its possible supplying options could be useful for farmers producing larger volumes of milk, milk collection centers and cooperatives that channel milk for the large processing plants. Additional works of this type focusing on hygienic practice and microbiological aspect could help to exploit the full benefit of the system in smallholder farmers’ conditions.

References

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Received 14 April 2012; Accepted 20 March 2013; Published 2 April 2013

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