Preservation of Raw Camel Milk by Lactoperoxidase System Using Hydrogen Peroxide Producing Lactic Acid Bacteria

This study was conducted to investigate the effect of lactic acid bacteria (LAB) activated lactoperoxidase system (LPs) on keeping quality of raw camel milk at room temperature. Camel milk samples were collected from Errer valley, Babile district of eastern Ethiopia. The level of hydrogen peroxide (H 2 O 2 ) for activation of LPs was optimized using different levels of exogenous H 2 O 2 . Strains of LAB (Lactococcus lactis 22333, Weissella confusa 22308, W. confusa 22282, W. confusa 22296, S. Infatarius 22279 and S. lutetiensis 22319) with H 2 O 2 producing properties were evaluated, and W. confusa 22282 was selected as the best strain to produce H 2 O 2 . Storage stability of the milk samples was evaluated through the acidification curves, titratable acidity (TA), total bacterial count (TBC) and coliform counts (CC) at storage times of 0, 6, 12, 18, 24 and 48 hours. The LP activity and the inhibitory effect of activated LPs were evaluated by growing E. coli in pasteurized and boiled camel milk samples as contaminating agent. Results indicated that


Introduction
Camels are important source of livelihood for millions of people living in the arid and semi-arid areas of many parts of the world, providing food, cash income and transport and have significant cultural values to the pastoral communities [1]. Camels are mainly kept for milk production and can produce milk for a longer period of time even during dry season [2] [3]. Camel milk has been traditionally consumed raw or in the form of fermented milk at household level for years with only limited amount being sold [4]. The changes in life styles such as fast-growing population, intensified growth of small towns and commercialization of pastoral products in the lowlands increased the demand for camel milk [5]. This condition opened market opportunities for the pastoral communities and peri-urban milk producers [6] [7].
However, camel milk is usually transported without cooling facilities for long distances to reach to the consumers or processing point which increased concerns over the microbiological quality of the milk [8]. Microbial growth is a major concern of public health as some can potentially cause milk-borne illness [9].
Milk with high levels of microbial contamination is not safe for direct consumption or it cannot be processed into different dairy products [10]. Therefore, prevention of quality loss through inhibition of bacterial growth during collection, transportation and storage of raw milk is of paramount importance. Several preservation techniques including cooling, heat treatment, acidification and addition of chemicals have been used at different levels from production to processing to prevent growth of spoilage and pathogenic microorganisms in foods [11].
Cooling of fresh milk during collection and transportation is widely used in most parts of the world especially in the developed countries [12].
In the areas where cooling facilities are unavailable to preserve raw milk due to economic and technical reasons, the International Dairy Federation [13] and Joint Food and Agricultural and World Health Organizations [14] had developed a method to increase the storage stability of the milk. The method is based on activating natural antibacterial system in raw milk which consists of lactoperoxidase (LP), thiocyanate (SCN -) and hydrogen peroxide (H 2 O 2 ). The LPs is commonly activated by exogenously increasing the concentrations of the thiocyanate and H 2 O 2 [15]. However, consumer awareness and concern regarding chemical additives and the demand for safe foods have led to find alternatives in food preservation [16]. In this regard, an emerging preservation technique is demanded via activating the LPs by lactic acid bacteria (LAB). Lactic acid bacteria are capable of producing several metabolites including organic acids, H 2 O 2 and bacteriocins, which have antagonistic effect to a wide range of microorgan-isms [17].
Hydrogen peroxide producing LAB was reported to inhibit growth of spoilage and pathogenic microorganisms [18]. This effect, however, might be due to the hydrogen peroxide produced or by the activated LPs from the production of H 2 O 2 in the milk. Previous work [19] indicated some strains of LAB (Lactococcus lactis 22333, W. confusa 22308, W. Confusa 22282, W. confusa 22296, S. Infatarius 22279 and S. lutetiensis 22319) isolated from camel milk can produce H 2 O 2 . However, the effect of these strains on the LPs activation properties is not evaluated. Besides, there is no information on the use of the H 2 O 2 producing LAB to activate the LPs and extend the keeping quality of raw camel milk. Therefore, this study was conducted to evaluate the effect of LAB activated LPs on the storage stability of raw camel milk at ambient temperature.

Collection of Milk Samples
The milk samples were collected from Errer valley, Babile District of eastern Ethiopia, about 30 km from Harar city. Errer valley is located at 9˚14'N latitude and 42˚14'E longitude at an altitude of 1300 -1600 m.a.s.l. The milk was collected in clean and sterilized plastic containers from four households of five lactating camels at different parities and stage of lactations. The samples were pooled and packed under icebox and transported to Haramaya University Dairy Technology Laboratory. Milk samples were collected three times for each experiment and analysis was done in duplicates. The milk samples to be preserved with LPs were activated within 2 hrs of collection, as the indigenous antimicrobial activity in the freshly drawn milk is usually used up within 2 -3 hrs due to suboptimal levels of the thiocyanate ion and hydrogen peroxide in the milk according to Codex Alimentarius Commission.

Determination of Thiocyanate Concentration
The thiocyanate concentration naturally present in camel milk samples was determined by spectrophotometer (3605, Jenway) at an absorbance of 460 nm, after deproteinisation of the milk with trichloroacetic acid (TCA) (Sigma-Aldrich T6399) and addition of ferric nitrate to form a ferric complex (orange to orange-red). Four millilitres (4.0 ml) of milk was mixed with 2.0 ml of 20% TCA solution. The mixture was mixed well and allowed to stand for 30 minutes. The solution was filtered through a filter paper (Whatman No. 40). One and half (1.5 ml) of the clear filtrate was then mixed with 1.5 ml of the ferric nitrate reagent and the absorbance was measured at 460 nm using spectrophotometer. The thiocyanate concentration was calculated from a standard curve prepared using known concentrations of sodium thiocyanate (Alfa Aesar 33388) [13].
confusa 22282, W. confusa 22296, S. infatarius 22279, S. lutetiensis 22319) were obtained from Technical University of Denmark (DTU), Copenhagen. The strains were originally isolated from camel milk from Babile area of eastern Ethiopia and were characterized by their H 2 O 2 production properties [19]. The strains were maintained on de Mann Rogosa Sharpe (MRS) agar and allowed to grow on Prussian blue (PB) agar, in MRS broth (Sigma-Aldrich 6966) and in pasteurized camel milk for detection and quantification of the H 2 O 2 produced.
The test organisms previously grown on MRS agar were also inoculated into MRS broth at room temperature for 72 hours and centrifuged (Sigma  3-30KS) at 10,000 rpm for 15 minutes at 4˚C. Protenase K enzyme (Sigma-Aldrich P6556) (5 mg/ml) was added to cell free extract solution to exclude the antimicrobial effect of bacteriocins, and pH was adjusted to 7.0 by means of 0.1N NaOH to reduce the effect organic acids. The supernatant was filtered through 0.2 mm pore size cellulose acetate filter. Twenty-five millilitres (25 ml) of supernatant of broth cultures of the test organisms was measured into a 100 ml flask to which 25 ml of dilute H 2 SO 4 was added. This solution was then titrated with 0.1N Potassium permanganate (KMnO 4 ). Each millilitre of 0.1 N KMnO 4 used was assumed to be equivalent to 1.701 mg of H 2 O 2 . The decolourization of the sample was regarded as the end point. The volume of H 2 O 2 produced was calculated according to AOAC [20].

Preparation of Inoculum and Determination of Acidification
Curves Fresh camel milk was divided into glass containers of 30 ml and boiled for 30 minutes at 90˚C. The samples were cooled to room temperature and inoculated with single colonies of LAB from agar plates. The bacterial cultures were incubated at 30˚C for 18 hrs and the mother culture was sealed and frozen at −20˚C until used [19].
Fresh camel milk samples of 200 ml were pasteurized in autoclavable bottles at 64˚C for 30 min. The samples were cooled to room temperature and inoculated with 1% of the mother culture. The acidification curves were followed using iCinac (Alliance Instruments, Frepillon, France). Calibrated and disinfected iCinac (pH) probes were inserted into the milk samples to ferment in a water bath at room temperature for 48 hours while the instrument continuously measures pH for every minute [19].

Effect of Lactoperoxidase System Activation on the Storage Stability of Camel Milk
An optimization experiment for the level of exogenous H 2 O 2 to activate LPs was conducted using TBC and titratable acidity, considering 30 ppm to be used as a positive control in this experiment. Weissella confusa 22282 was selected for activation of the LPs due to its better H 2 O 2 production in MRS broth and reduction in acidification rate in pasteurized camel milk. Mother culture of the strain was prepared by inoculating the strain into MRS broth for 72 hours at 30˚C and activating culture was prepared by growing the culture from the MRS broth in skim milk at a rate of 100 mg/L at 22˚C for 16 hours [21]. The culture was inoculated into raw camel milk at rate of 1% and shelf life of the milk samples was evaluated through the pH (acidification curves), TA, TBC and CC at storage times of 0, 6, 12, 18, 24 and 48 hours. Raw milk with no addition and 30 ppm H 2 O 2 were used as negative and positive controls respectively ( Table 1). The TBC was done by pour plate method using standard plate count (SPC) agar (Sigma-Aldrich 70152). Plates with colonies ranging from 30 -300 were counted and expressed as colony forming units per millilitre (cfu/ml) according to IDF [22]. The titratable acidity was determined by titrating the milk sample with 0.1 N NaOH (Himedia MB09) using a phenolphthalein indicator to an end-point of faint pink colour [23]. Coliform count was done by pour plate method using violet red bile agar (VRBA) (Sigma-Aldrich 70188). Plates with 15 to 150 cfu/mL were used for determining total coliform counts [22].

Growth of Escherichia coli in Lactoperoxidase System Activated Camel Milk
The activity of lactoperoxidase in the milk and the effect of activated

Statistical Analysis
General linear model (GLM) procedure of SAS version 9.0 was employed to determine the significance between treatment means at a particular storage period.
Mean separations were done using least significant difference (LSD) for variables whose F values were significantly different. Significant differences were calculated at 5% significance level. Descriptive statistics was used to calculate means and standard deviations of chemical compositions and amount of H 2 O 2 produced.

Thiocyanate Concentration of Camel Milk
The natural thiocyanate content in the present study was 22.34 ± 5.11 mg/L that falls within the ranges reported earlier (9.74 to 32.9 mg/L) [26] and (9.7 to 36.4 mg/L) [27] and higher than the level found (6.04 mg/L) [28] from Erer valley of eastern Ethiopia. Natural thiocyanate content of 7.38 was found from cow milk in Kombolchadistrict, eastern Hararghe of Ethiopia [29]. The higher thiocyanate content in the present study compared to the finding from the same area [28] might be due to differences in analytical measurements, feeding system and environmental conditions. Several factors could affect the thiocyanate concentration of milk such as age of the animal, health of the animal, species of animal, breed, lactation stage and nutritional condition among which the kind of feed supplied plays a major role [30]. Thiocyanate content might also vary among season of the year where the level in summer was higher than the thiocyanate concentration in winter [31]. Camels in the Erer valley of Babile area spend during the day outdoors browsing different types of plants including herbaceous plants, shrubs, shoots, cacti and different types of acacia trees. Acacia trees and shrubs are expected to have high contents of cyanogenic glycosides which are a precursor of thiocyanate in plants [32].
The SCN value in the current study is higher than the concentration (15 ppm) required for the activation of the LPs according to Codex Alimentarius Commission [33]. The present finding is therefore showed that LPs in camel milk can be activated by natural SCN content in the milk with the addition of only desired amount of H 2 O 2 [27].  (Table   3). All the strains except L. lactis 22333 were observed to grow yielding a deep blue colour around the colonies on PB agar due to reaction of H 2 O 2 to hexacyanoferrate (III) and iron (III) [34]. The overall results of H 2 O 2 indicated that W. confusa 22282 was the promising strain to use for activation of the antimicrobial system in raw camel milk.        effect due to the presence of higher levels of other indigenous antimicrobial components including the H 2 O 2 . It was reported that activation of LPs in camel milk decreased the multiplication of total bacteria for more than 12 hours of storage [40]. Other reports indicated that the activated LPs in milk had bacteriostatic effect against a mixed raw milk flora dominated by mesophilic bacteria [14].

Effect of Lactoperoxidase System Activation on Growth of Escherichia coli
The activation of LPs by W. confusa 22282 and exogenous H 2 O 2 in pasteurized camel milk remarkably reduced the growth rate of E. coli population compared their respective population in boiled milk throughout the storage period ( Figure  3). This result indicated that the role of LP enzyme in boiled milk might be impaired in catalyzing the oxidation of thiocyanate by H 2 O 2 . The LPs improves keeping quality of milk pasteurized at 72˚C/15 s compared to milk heated to 80˚C/15 seconds [41]. Similar findings [42] was reported that activation of LPs extended the keeping quality of pasteurized milk (72˚C/15s) inoculated with Pseudomonas aeruginosa, S. aureus and S. thermophilus while the milk heated at 80˚C/5s and activated with the LPs had no effect on growth of these organisms. Moreover, this finding is supported by recommendation by FAO/WHO [8] that heating milk for 15 seconds at 80˚C, completely inactivates enzymatic activity of the milk might be because of the destruction of indigenous inhibitory components by the higher temperature. Indigenous enzymes are heat-labile and can easily be destroyed under the boiling conditions [43].
Similarly, the rate of E. coli growth in the LAB and exogenous H 2 O 2 activated LPs in pasteurized camel milk was considerably lower than the rate in non-activated pasteurized milk (Figure 3). Reduction in microbial spoilage of LPs activated   pasteurized cow milk inoculated with E. Coli compared to the non-activated one was also reported [44]. An inhibition of the growth of E. coli for 24 hours [45] and a nearly total inhibition of E. coli O157:H7 [21] was also reported in cow milk and in commercial fermented milk and traditional Madila activated with LPs, respectively.
No remarkable difference was observed in retarding E. coli population between the W. confusa 22282 activated and H 2 O 2 activated LPs in the pasteurized camel milk except a better retarding effect in the LAB activated samples toward the end of the storage period, which might be due to continuous production of H 2 O 2 in the milk (Figure 3). The W. confusa 22282 treated boiled milk had shown slight reduction compared to the untreated boiled milk samples might be because of the effects of residual LP in the milk [42] or due to production of metabolites by the LAB [46]. Metabolites produced by LAB were reported to affect the growth of E. coli, S. aureus, Salmonella spp. in food items [47]. On the other hand, the H 2 O 2 treated boiled milk shown remarkable reduction in E. coli counts compared to the untreated boiled samples. This clearly indicated that H 2 O 2 had antimicrobial effect by itself as supported by other finding [48].

Conclusion
The current study showed that LAB can produce H 2 O 2 to activate the natural antimicrobial system in the milk. The optimization experiment indicated that W. confusa 22282 can produce sufficient amount of H 2 O 2 in MRS broth. The activation of LPs by W. confusa 22282 as a source of H 2 O 2 in raw camel milk significantly (P < 0.05) reduced the rate of lactic acid development, TBC and CC compared to the non-activated samples at 12, 18, 24 and 48 hours of storage at room temperature. Similarly, the activation of LPs by W. confusa 22282 in pasteurized and boiled camel milk samples inoculated with E. coli as contaminant showed that the LPs activated pasteurized milk remarkably reduced the E. coli population compared to the W. confusa 22282 treated boiled milk throughout the storage period. The rate of growth of the E. coli population in the W. confusa 22282 activated LPs in pasteurized milk was also considerably lower than the rate in non-activated pasteurized milk. This indicated that pasteurization (63˚C/30 minutes) cannot destroy the enzyme LP and the storage stability of the pasteurized milk samples was therefore due to the activation of the antimicrobial system in the milk. Generally, the present study showed that it is possible to activate the natural antimicrobial system in camel milk using H 2 O 2-producing LAB (W. confusa 22282) as a source of H 2 O 2 in the presence of appropriate concentrations of natural thiocyanate in the milk. Therefore, it can be concluded that biological activation of LPs by W. Confusa 22282 in camel milk can substitute the chemical activation method provided that the milk has sufficient inherent thiocyanate.
for financing this research project. Haramaya University is highly appreciated for providing facilities to conduct the research.