The keeping quality of LPS-activated milk in the western highlands of Cameroon

F A Fonteh, A S Grandison*, M J Lewis* and A T Niba

Department of Animal Sciences. The University of Dschang. P.O. Box 447 Dschang Cameroon.
fontehflorence@yahoo.com
*School of Food Biosciences, The University of Reading, Whiteknights.
P.O. Box 226 Reading, RG6 6AP. United Kingdom.

Abstract

The potentials of applying the lactoperoxidase system (LPS) in extending the shelf life of raw milk at ambient temperatures was investigated in the western highlands of Cameroon. Raw milk was LPS-activated by adding various concentrations (ppm) of thiocyanate and peroxide and denoted as 0:0, 7:10 ppm, 10:10 ppm and 20:20 ppm. The keeping quality of the activated milk samples was assessed by the alcohol stability and clot-on-boiling tests, pH changes and titratable acidity.

The milk in all the treatments remained fresh during the first 12 hours but the control was spoiled by the 15^th hour. There was a continuous drop in pH values matched by a steady rise in titratable acidity. For all parameters measured, 20:20ppm was the last treatment to spoil, suggesting that the shelf life of milk increases with increasing concentrations of thiocyanate and peroxide.

With small amounts of thiocyanate (20 ppm) and peroxide (20 ppm) the shelf life of raw milk can effectively be extended under Cameroonian conditions by approximately 9 hours without refrigeration. Thus LPS-activated milk can be stored for as long 21 hours, allowing sufficient time for its appropriate disposal.

Key words: lactoperoxidase system, milk, preservation, quality

Introduction

Cattle rearing in most of Cameroon is a tradition, a way of life particularly for the Fulani and Foulbé tribesmen who make up the majority of the cattle rearers in Cameroon. Cattle are reared mainly in the western and northern parts of the country. The predominant local breeds are the Zebus (White Fulani and Red Fulani), Akou, Nd'ama, and Goudali which all belong to the Bos indicus family. The cattle population is estimated at 4 to 5 million (Kameni et al 1999). Because of the relatively low genetic performances (in milk and meat production) of the local breeds, efforts were made to improve overall milk and beef production by the introduction of exotic germplasm. The total domestic dairy production is estimated at 50 000 tonnes per annum. However, milk consumption estimates show a consumption which is twice the per capita domestic production; the difference being met by dairy imports (Kameni et al 1999; Tambi 1991). Milk production within the country is at its peak during the rainy season, when there are abundant natural pastures for the cows to graze on. However, it is also during this period that a lot of the milk is lost through spoilage because it cannot be sold quickly enough. Some of the main factors that contribute to accelerated spoilage of the milk include limited accessibility to markets, inadequate refrigeration facilities and an initially high microbial load of the milk produced (Fonteh 2001; Kameni et al 1999).

With the present low level of domestic production and the population increasing at a rate of about 3% per year (National Institute of Statistics 2001), there is an urgent need to develop strategies towards improving domestic milk production. However, it is imperative that before increasing production, the current milk produced does not spoil before it reaches the consumer. Simple techniques such as the lactoperoxidase system (LPS) can be applied, which could significantly prolong the shelf life of raw milk. Upon activation, the LPS exerts antimicrobial activities which result in prolonging the shelf life of milk (Bjorck 1975). The success of this technique has been reported by several workers in many developing countries where refrigeration facilities are absent (Ridley and Shalo 1990; Kumar and Mathur 1989; Kamau and Kroger 1984; Harnulv and Kandasamy 1982). These studies however suggest that the extent to which raw milk can be preserved using this technique largely depends on the conditions that prevail in the given area, particularly the microbial load of the milk before treatment, the ambient temperature as well as the substrate concentrations applied to activate the LPS. Although the International Dairy Federation recommends the use of 10 ppm thiocyanate and 10 ppm hydrogen peroxide as safe levels for activating the LPS in milk (IDF 1988), optimal dosages specific for each area seem worthy of study (Ridley and Shalo 1990; Kamau and Kroger 1984). Consequently, different concentrations of substrates have been recommended for optimising the efficacy of the LPS in different countries. No such study has so far been conducted in Cameroon.

The objective of this study therefore was to investigate the potential for the application of the LPS in the preservation of raw milk under Cameroonian conditions.

Materials and methods

Collection site

Three farms in the Western province of Cameroon were identified for the study. This area falls within the sudano-guinean zone (latitude 5-7^oN, longitude 8-12^oE). The average annual temperature varies between 16 and 27^oC while the relative humidity is 40-97% (Teguia et al 1997). There are two main seasons: the rainy season (late March to October) and the dry season (November to early March) and the mean annual rainfall is about 2000mm. Three farms were chosen and four cows (2 Red Fulani and 2 Goudali) per farm selected for the study. Bulk milk (8 litres) was collected from the selected cows in each farm, transported under ice and immediately LPS-activated in the Feed Analysis Laboratory of the University of Dschang. All samples arrived in the laboratory within 2 hours after milking. Milking was by hand into clean sterilised cans. The experiment was carried out during the rainy season (August). The experiment was repeated per farm, within one week's interval.

Activation of the LPS

The modified IDF method (IDF 1988) was used to administer four different treatments to bulk milk from each farm as follows:

0:0 2 litres of milk were not LPS-activated and served as the control.

7:10ppm: To 2 litres of milk was added 20 mg of sodium thiocyanate (equivalent to 7 ppm SCN^-), mixed thoroughly by use of a magnetic stirrer for at least 5 minutes, followed by the addition of 60 mg of sodium percarbonate (liberating 10 ppm of H₂O₂) and subsequently mixed for another 5 minutes.
10:10ppm:  To two litres of raw milk, 28 mg of sodium thiocyanate was added, mixed, followed by the addition of 60 mg of sodium percarbonate and subsequently mixed.
20:20ppm: To two litres of milk, 56 mg of sodium thiocyanate was added and mixed followed by addition of 120 mg of sodium percarbonate.
The corresponding quantities of thiocyanate and peroxide liberated from these compounds in each treatment are presented in Table 1.

The samples were all kept at room temperature (which varied between 21.5 and 23.3^oC) for 24 hours. All samples were assayed at 0 hour, 12 hours and every three hours thereafter for the next 12 hours. After each assay, all the vessels used for transporting and treating milk were washed and sterilized (by soaking for 15 minutes) using a sodium hypochlorite solution.

Keeping quality tests

The pH was monitored with the use of a pH meter type C925 (Consort, Dendermonde, Belgium). The pH probe was dipped into 10 ml of the milk samples and the values were recorded after the readings had stabilised (about 2 minutes). The pH meter was standardised weekly using pH buffers 4 and 7 respectively. A fall of 0.4 units from the original pH value indicated the onset of spoilage (Davis 1959).

The titratable acidity test was carried out by mixing 1 ml of 0.5% phenolpthalein solution with 10 ml milk and titrating against 0.1 N sodium hydroxide solution (BDH, Poole, UK) until a faint pink colour that persisted for at least 5 seconds was observed. Percentage lactic acid was calculated as follows:

The sample was considered spoiled when when the percentage lactic acid reached 0.18 % or higher (Harding 1995; Rowlands et al 1950). The clot-on-boiling test was conducted by boiling 10 ml of milk in a test tube for 5 minutes. Any distinct clots observed while gently tilting the tube indicated a positive response and marked the end of the shelf life of the milk (Harding 1995). For the alcohol stability test, 5 ml of milk was mixed with 5 ml of 68 % (v/v) ethyl alcohol in a petri dish and mixed by swirling gently. Any flocculation noticed indicated a positive response and the end of shelf life (Hammer and Babel 1957).

Analysis of variance was used to evaluate changes in pH and lactic acid levels in the various samples during storage, and means were separated using Duncan's multiple range test (Steel and Torrie 1980).

Results

As shown in Figure 1, there was very little change in the pH values among the four treatments during the first 12 hours after the milk was LPS activated.

At 0 hour, the average temperature of the milk for all experiments was 10^oC (because it was transported under ice) but had stabilised to room temperature by the 12^th hour. At the 12^th hour of sampling, statistical analysis revealed that the pH values for  0:0, 7:10ppm and 10:10ppm were not significantly different  from each other but were significantly higher than for 20:20ppm (Table 2). After this lag period, there was a rapid fall in the pH value for the control. Considering a change of 0.4 or more units in the original pH value as indicative of the onset of spoilage, it can be seen that the control sample ( 0:0) got spoiled before the 18^th hour of testing at room temperature. However, the treated samples remained good for a longer time. The 7:10 ppm  and the 10:10 ppm samples were spoiled by the 21^st hour whereas the 20:20 ppm samples spoiled only after 24 hours of storage at room temperature. Beyond 18 hours of storage, significant differences  were observed when the average means for the various treatments were compared with each other throughout the rest of the storage period (Table 2). After the 12^th hour, there were significant changes in the pH values within each sample as time progressed, the fastest change being observed in the control ( 0:0). In addition, the interaction between time and substrate concentration was most evident in the control, whose pH value at the end of the storage period (6.39) was significantly lower than for other samples respectively (Table 2).

There was a slight but steady increase in acid level in the control sample during the first 12 hours of storage whereas this change was not obvious in any of the treated samples (Figure 2).

In fact, statistical analysis showed no significant differences (P>0.05) in mean values among the three treated samples at this sampling time (Table 3). Nevertheless, acidity increased gradually and continually in all samples after 12 hours of storage. Treatment 2_0:20ppm was the slowest in acid development, where the mean acid values for this sample during the first 15 hours of storage were not significantly different (P>0.05) from each other (Table 3).

The sharpest rise in acid level was observed in the control while the slowest sample in acid development was that containing the highest concentrations of thiocyanate and peroxide (20:20ppm). At the 18^th hour, the lactic acid content in 7:10ppm and 10:10ppm were not significantly different from each other but significant differences were observed among all samples thereafter (Table 3). With a "cut-off" point at 0.18 % lactic acid, it can be seen that the control spoiled after 12 hours of storage at room temperature. The greatest interaction between time and substrate concentration was again observed in the control, whose acid level at the end of storage (0.35%) was significantly higher (P<0.05) than that in the other samples (Table 3).

The control sample was the first to test positive for the clot-on-boiling test at the 15^th hour of storage (Table 4).

Thereafter, the treated samples progressively tested positive with increasing thiocyanate concentration; the 20 ppm SCN^- sample being the last to test positive after the 21^st hour. Both samples containing 7:10 and 10:10 ppm (thiocyanate:peroxide) produced similar results during the storage period. All treatments had spoiled by the 24^th hour of storage.

Results from the alcohol stability test (Table 5) show that the control and the 7:10 ppm samples were both spoilt by the 15^th hour of storage.

The 20:20 ppm sample was the last to spoil after 18 hours of storage. With this test, all samples were spoiled by the 21^st hour of storage.

Discussion

The pH of the treated samples experienced a slower drop during the entire storage period. This trend was similar to that previously reported (Ewais et al 1985). The best sample was that activated with 20 ppm thiocyanate and 20 ppm peroxide which became spoilt after the 24^th hour of storage. Therefore, activating milk at this rate will extend the shelf of milk by at least 9 hours at room temperature (21 - 23^oC). The results also suggest that increasing the concentration of thiocyanate and peroxide will lead to a longer shelf life of raw milk. The same observation has been reported by earlier workers (Kumar and Mathur 1989; Thakar and Dave 1986) who however observed that increasing the level of the substrates did not bring about a proportional enhancement of the shelf life of milk. They attributed this non-proportionality to the effect of the limiting concentration of the oxidation products of thiocyanate as well as a leveling of the kinetic rate of their formation.

The average initial level of lactic acid in the milk was high (0.16 %). This implies that the initial lactic acid producing bacteria population was high, reflecting the substandard hygienic conditions under which the milk was collected. Little or no acid development was detected in the treated samples during the first 12 hours of storage. A similar observation was reported 8 hours after the LPS was activated in raw milk at a 25:15 thiocyanate:peroxide ratio (Kumar and Mathur 1989). The longest shelf life was the 20:20 ppm (thiocyanate:peroxide) treated sample, which remained good beyond 21 hours of storage at room temperature. Other studies reported that a much shorter shelf life (9 hours) was obtained after using the same concentration levels (Chakraboty et al 1986). This was most probably due to the higher ambient temperature (37^oC) reported in that area of study.

Based on pH variations and acid development throughout the storage period, the interactive effect of time and substrate concentration was most felt in the control ( 0:0) and least evident in 20:20ppm. Therefore, this interactive effect seems to diminish with increasing substrate concentration.

A progressive increase in shelf life with increasing substrate concentration was observed in the treated samples when the clot-on-boiling test was employed. Using the same test, some workers (Kumar and Mathur 1989; Harnulv and Kandasamy 1982) have shown that by activating with 25:15 ppm (thiocyanate:peroxide), buffalo milk can be preserved for 13 hours at 18^oC whereas the shelf life could be extended to 19 hours if the substrate concentration was increased to 70:30 ppm.

Most of the treatments tested positive with the alcohol stability test earlier than with the clot-on-boiling test. For example, the 7:10 ppm treatment tested negative with the clot on boiling test at the 15^th hour but tested positive with the alcohol stability test at the same hour. The same observation was made for the 20:20 ppm treatment at the 21^st hour. These observations support the view that the alcohol stability test is more sensitive than the clot-on-boiling test as reported in the literature (Kumar and Mathur 1989; Harnulv and Kandasamy 1982).

Conclusions

• Preservation of raw milk at the farm level is a problem to rural farmers due to lack of refrigeration facilities. This constitutes a major obstacle to the development of the dairy industry in Cameroon.

• Due to the relatively low ambient temperatures that prevail in the Western Highlands of the country, this problem can be alleviated by the application of the LPS, which is an easily applicable technique.

• Results from this study indicate that both the storage time and the concentration of thiocyanate/peroxide added to LPS-activated milk have very significant independent and interactive effects on the keeping quality of the milk at room temperature, although their interactive effect appears to decrease with increasing substrate concentration.

• Furthermore, the results have demonstrated that raw milk activated with 20 ppm thiocyanate and 20 ppm peroxide can remain fresh at ambient temperatures (21 - 23^oC) for at least 18 hours as opposed to the untreated milk whose shelf life under the same conditions was only 12 hours. This increases the shelf life of milk by about 50 %, thus allowing sufficient time for it to be stored (especially evening milk), transported and sold still in its fresh state in the local markets.

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