Wudpecker Journal of Agricultural Research Vol. 1(11), pp. 459 - 465, December 2012 Available online at http://www.wudpeckerresearchjournals.org 2012 Wudpecker Research Journals ISSN 2315-7259 Full Length Research Paper

Hygienic and microbial quality of raw whole cow’s milk produced in Ezha district of the Gurage zone, Southern

Ethiopia

Abebe Bereda1, Zelalem Yilma2 and Ajebu Nurfeta3

1Department of Animal Science, College of Agriculture and Natural Resource Sciences, Debre Berhan University P.O.Box 445, Debre Berhan, Ethiopia.

2Heifer International, East Africa Dairy Development Country Program Mobilization Coordination Office, Addis Ababa, Ethiopia.

3School of Animal and Range Sciences, College of Agriculture, Hawassa University, P.O.Box 5, Hawassa, Ethiopia.

Accepted 23 September 2012

The objective of the study was to assess the handling and hygienic quality of raw whole milk in Ezha district of the Gurage zone, Southern Ethiopia. A total of 120 randomly selected milk producing households were interviewed to assess the milk production, hygienic conditions, utilization and processing of milk and milk products. In addition, a total of 40 raw milk samples were aseptically collected and tested between February and March 2010 for microbial analysis from two agro-ecologies (Dega and Woina Dega). Average counts of Aerobic Mesophilic Bacterial Count (AMBC), Coliform Count (CC) and Entrobacteriacea Count (EBC) for milk sampled from producers were 9.82

log cfu/mL, 4.03 log

cfu/mL, 4.15 log cfu/mL), respectively. Mean CC significantly differed between the two agro-ecologies. Generally, the mean values of AMBC, CC and EBC observed in the current study were above maximum acceptable limits. The milk produced in the study area should be heat treated and adequate sanitary measures need be taken at all stages of milk handling so that milk of acceptable quality can be produced and reaches the consumer. Key words: Smallholder, milk, microbial quality, milk hygiene, Ethiopia.

INTRODUCTION Milk and milk products have important role in feeding the rural and urban population of Ethiopia owing to its high nutritional value. Milk is produced daily, sold for cash or readily processed. It is a cash crop in the milkshed areas that enables families to buy other foodstuffs and significantly contributing to the household food security. Given the long tradition of using milk and milk products by the Ethiopian societies, there is no doubt that increasing smallholder dairy production and productivity would bring about a conspicuous impact on improving the welfare of women, children and the nation's population at *Corresponding author E-mail: ajebu_nurfeta@yahoo.com.

large (MOA, 1998). The safety of dairy products with respect to food-borne

diseases is a great concern around the world. This is especially true in developing countries where production of milk and various milk products takes place under unsanitary conditions and poor production practices (Mogessie, 1990). The microbial content of milk is a major feature in determining its quality (Beyene, 1994). It shows the hygienic level exercised during milk production and handling, that is cleanliness of the milking utensils, condition of storage, manner of transport as well as the cleanliness of the udder of the individual animal (Coorevits et al., 2008).

The number and types of micro-organisms in milk immediately after milking are affected by factors such as

lack of knowledge about clean milk production, use of unclean milking equipment and lack of potable water for cleaning purposes contributing to the poor hygienic quality of raw milk (Bekele and Bayileyegn, 2000). Milk from a healthy udder contains few bacteria but it picks up many bacteria from the time it leaves the teat of the cow until it is used for consumption or further processing. These micro-organisms are indicators of both the manner of handling milk from milking till consumption and the quality of the milk. Milk produced under hygienic conditions from healthy animals should not contain more than 5 × 105 bacteria per millilitre (mL) of milk (O’Connor, 1994).

In Ethiopia, in general and in the study area in particular milk and milk products are important for family consumption and as a source of income through sale of products such as butter and Ayib - Ethiopian cottage cheese. Consequently, the products must be of high hygienic quality. Though in less developed areas especially in hot tropics, the production of products of safe and high quality is important, the prevailing situation is far from the ideal condition (DeGraaf et al., 1997). Poor hygiene, practiced by handlers of milk and milk products, may lead to the introduction of pathogenic micro-organisms into the products. Since they do not undergo further processing before consumption, these foods may pose risk to the consumers.

Therefore, provision of milk and milk products of good hygienic quality is desirable from consumer health point of view (Zelalem, 2010). There is limited or no work undertaken so far to understand the hygienic practices during production and the microbiological quality of raw milk in Ezha districts of Gurage zone, which is essential to make improvement interventions. The aim of this study was, therefore, to assess the hygienic quality of raw cow’s milk produced in Ezha districts of Gurage zone. MATERIALS AND METHODS Study area The study was conducted between February and March 2010 in Ezha districts of the Gurage zone, which is located at 200 km Southwest of Addis Ababa. The altitude of the district ranges from 1800 to 3098 meters above sea level and receives an annual rainfall of 900-1600 mm with the mean minimum and maximum annual temperatures of 5 and 38oC, respectively (EDARDO, 2009). Data collection

This study had two parts: Survey and microbial analysis. For the survey, random sampling procedure was employed to select sample kebeles (lowest administrative unit) and households for the study. The district was first stratified as Dega (cold highland) and Woina Dega (medium temperature and altitude) agro-ecologies. From a total of 28 Kebeles located in the district two Kebeles were randomly selected from each of the two agro-ecologies. A total of 30 households per Kebele that own at least one local milking cow

Bereda et al. 460 were randomly selected to assess milk production, hygienic conditions, utilization and processing of milk and milk products. Following this assessment, a total of 40 (10 households × 4 kebeles) samples of raw milk were collected for the microbiological analysis (Table 1). Samples of fresh whole milk were collected aseptically following the procedure described by Richardson (1985). After through mixing, milk samples were taken in sterile bottles of about 125 mL capacities for microbial analysis. The samples were transported to Holetta dairy microbiology laboratory in an icebox and kept in refrigerator until the time of analysis. Each analysis was made in duplicates. The analysis was performed within 36 hours after sampling (Alganesh et al., 2007). Microbial analysis Aerobic mesophilic bacterial count Milk samples were homogenized and serially diluted by adding 1mL of the test portion into 9 mL of 0.1% sterile peptone water. Dilutions were made so that plate counts range between 30 and 300 colonies (Richardson, 1985). Appropriate dilutions were placed on Petri dishes and pour plated with 10 to 15 mL molten plate count agar (about 45°C) (Oxoid, UK) and allowed to solidify for 15 minutes and incubated for 48 hours at 37°C. Finally, counts were made using a colony counter. The plate counts were calculated by multiplying the count on the dish by 10

n, in which n stands for the number of

consecutive dilutions of the original sample (Van den Berg, 1988). Coliform count After vortexing the sample portion, appropriate decimal dilutions were made by transferring 1 mL of the sample into 9 mL of 0.1% peptone water for initial dilution and by transferring 1 mL of the previous dilution into 9 mL of peptone water. After surface plating the appropriate dilution in duplicates on Violet Red-Bile Agar (VRBA), Petri dishes were incubated at 32°C for 24 hours and counts were made on typical dark red colonies normally measuring at least 0.5 mm in diameter on uncrowned plates (Richardson, 1985). Total entrobacteriaceae count (EBC) After vortexing the sample portion, appropriate decimal dilutions were made by transferring 1 mL of the sample into 9 mL of peptone water for initial dilution and by transferring 1 mL of the previous dilution into 9 mL of peptone water. The 0.1 mL of the required dilution was surface plated on petri dishes of Violet Bile Glucose (VRBG) Agar (Oxoid, UK), which were then incubated at 32°c for 48 hrs. Colonies that were rose-colored and surrounded by a halo of purple precipitate were counted as presumptive Entrobacteriaceae (Richardson, 1985). Data analysis Survey data collected were analyzed using descriptive and inferential statistics such as means, frequency distribution and percentage using SPSS software (ver.13). Microbiological counts were transformed into logarithmic values (log10 cfu mL-1) and these transformed values were analyzed using the General Linear Model for least square means in SPSS using fixed effect model. ANOVA test was used to see the mean difference between bacterial counts from different sampling sources

461 Wudpecker J. Agric. Res.

Table 1. Milk sampling layout for microbial analysis.

Agro-ecology Kebeles Sampled Households Dega

Kokara 10 Gedeb 10

Woina Dega

Dasene 10 Yegobet 10

Total 40 RESULTS AND DISCUSSION Housing and cleaning practices According to the current study the majority of the interviewed households (90.8%) shared the same house with their animals, while 9.2% of the households used separate houses for their cows (Table 2). The purposes of housing in the study area were to protect cattle from theft, wild animals (predators) and extreme unfriendly weather conditions. The cattle housing and its purposes observed in this study were similar with other reports (Asaminew, 2007; Derese, 2008; Asrat, 2009).

Clean, dry and comfortable bedding condition is important to minimize the growth of pathogenic microorganisms. As observed in the present study 66.7% of the respondents used grass and cereal straw as bedding material for their animals. The remaining households (23.3%) did not use any bedding material at all (Table 2). Teats and udders of cows inevitably become soiled while they are laying in stalls or when they are allowed to stay in muddy barn yard. Used bedding has been shown to harbor large numbers of microorganisms (Murphy and Boor, 2000). Practices that expose the teat end to these organic bedding sources, wet and muddy pens increase the risk of occurrence of mastitis and milk contamination (Ruegg, 2006).

About 47% of the respondents clean the barn three times a week, while 39% clean two times and only 11.7% of them reported to clean daily (Table 2). Contrary to this study, Zelalem (2010) reported that about 87% of the respondents cleaned their barn on daily basis, while few (9%) of them cleaned only once or twice a week in the Ethiopian highlands. In general, providing proper shelter for animals has not been given the required attention. Housing conditions in many of households were dirty and unclean. This may have a negative impact on the quality of milk and milk products produced and processed. Proper and clean housing environment is a prerequisite to produce milk and milk products of acceptable quality (Asaminew, 2007). Milking practices In the study area all of the interviewed households

practiced hand milking. Cows in the study area are usually milked twice a day except few (5%) households in the Dega area who reported once daily milking (Table 3). The practices of milking found in this study were similar with other reports (Lemma et al., 2004; Asrat, 2009). However, milking operation is only limited to once or twice per day during the last stage of lactation around Wolayta zone (Ayantu, 2006; Rahel, 2008).

Dairying is a labour-intensive farm activity. In the current study, dairying offers more opportunities for females to be closely involved in the daily management than other family members. Overall, milking operation is mainly carried out by housewives (about 99.2%) (Table 3). Ayantu (2006), Rahel (2008), Derese (2008) and Haile et al. (2012) also reported a similar situation. Husbands are also involved in milking of cows next to housewives and daughters with their level of involvement. Therefore, this result indicates the necessity of gender education in the district on hygienic production and subsequent handling of milk and milk products in order to produce milk and milk products of not only acceptable but of high quality. The benefits of such practice are two folds: consumers will have access to wholesome products and producers fetch better income from the sale of quality products.

Equipment used for milking, processing and storage determine the quality of milk and milk products. All of the interviewed households in the study area used plastic jars as milking utensil. The use of plastic and traditional containers can be a potential source for the contamination of milk by bacteria, because this allows the multiplication of bacteria on milk contact surfaces during the interval between milkings. This is mainly due to the difficulty of removing all milk residues from traditional containers that are porous by nature with the common cleaning systems. Producers need, therefore, to pay particular attention for the type as well as cleanliness of milk equipment. Milking equipment should be easy to clean. Aluminum and stainless steel equipment are mostly preferred. Milk hygienic practices Cleaning the udder of cows before milking is important since it could have direct contact with the ground, urine,

Bereda et al. 462

Table 2. Types of housing, bedding and frequency of barn cleaning in the two agro-ecologies.

Variables

Agro-ecology Overall (n=120) Dega (n=60) Woina Dega (n=60)

Types of housing (%) Same house with family 86.7 95 90.8 Separate housing 13.3 5 9.2 Bedding used (%) Grass 53.3 61.7 57.5 Cereal straw 5 - 2.5 Both 10 23.3 16.7 Not at all 31.7 15 23.3 Frequency of cleaning barn (%) Daily 21.7 1.7 11.7 Twice per week 45 33.3 39.1 Three times per week 28.3 65 46.7 Four times per week 5 - 2.5

n= Number of observations

Table 3. Milking practices in Ezha district. Variable

Agro-ecology Overall (n=120) Dega (n=60) Woina Dega (n=60)

Frequency of milking per day (%) Two times per day 95 100 97.5 Once a day 5 - 2.5 Milking utensils (%) Plastic jar 100 100 100 Nickel 10 - 5 Milker (%) Housewives 98.3 99.2 99.2 Daughters 43.3 6.7 25 Sons 8.3 4.2 Husbands 16.7 15.8 15.8

n= Number of observations dung and feed refusals while resting. However, all respondents did not use udder washing before milking (Table 4). Lack of washing udder before milking can impart possible contaminants into the milk. The current study is in agreement with other reports (Lemma et al., 2004; Fayo, 2004; Derese, 2008). Contrary to this study, Haile et al. (2012) reported that 82.5% of the small size farm owning households in Hawassa city are practicing pre milking udder washing. FSA (2006) reported that cleaning of the udder before milking is important to remove both visible dirt and bacteria from the outer surface of the udder. Unless properly handled, milk can be contaminated by microorganisms at any point from production to consumption. Producers should therefore make udder washing a regular practice in order to minimize contamination and produce good quality milk.

Production of milk of good hygienic quality for consumers requires good hygienic practices (clean milking utensils, washing milker’s hands, washing the

udder and use of individual towels) during milking and handling, before delivery to consumers or processors (Getachew, 2003). In the study area, the majority of the respondents practiced washing of their milk utensils (87.5%) and milker’s hands (71.6%) before milking. However, the cleaning is not efficient and utensils are not properly dried. Milkers dip their fingers in the milking vessel to moisten teats of the cows with the intention of facilitating milking. However, such practice may cause microbial contamination of the milk from the milker’s hand.

The sources of water available to farmers used for different purposes (to clean milk equipment and hands) varied in the districts (Table 4). The majority of the respondents (57%) had access to river water followed by pipe water (35.5%) and hand dug well water (7.2%). However, the quality of both river and hand dug well waters used for cleaning may not be of the required standard thus can contribute to the poor quality of milk in

463 Wudpecker J. Agric. Res.

Table 4. Milk handling practices of households in Ezha district.

Variable

Agro-ecology Overall (n=120) Dega (n=60) Woina Dega (n=60)

Milk hygienic practice (%) Wash udder before milking - - - Wash hand before milking 95.0 48.3 71.65 Wash milking utensil with water 80 95 87.5 Source of water for cleaning milk utensils (%)

Tap water 10 61 35.5 Hand dung well water 3.3 11 7.2 River water 86.7 28 57.3 Filtering milk after milking (%) Enset fiber filtering 15 16.7 15.8 Plastic sieve filtering 21.7 21.7 21.7

n= Number of observations the area. It is, therefore, important to heat treat water from river and hand dug wells intended for cleaning purpose.

Another milk treating practice in the district was filtering milk after milking before pouring into bulk container to remove any dirty material. About 16 and 22% of the respondents filter milk using Ensete Ventricosum fiber and plastic sieve, respectively (Table 4). Asrat (2009), on the other hand, reported no practice of milk filtering before mixing with the previous lot or use for other purposes. Microbial quality of raw whole milk Aerobic mesophilic bacterial count (AMBC) The mean Aerobic mesophilic bacterial count was not significantly different (P>0.05) between Dega and Woina Dega (Table 5). The average AMBC of milk samples were 9.82 log cfu/mL. The overall mean AMBC observed in the current study was higher than the maximum acceptable limits given for raw milk intended for processing (1.0 × 105 cfu/mL) and direct human consumption (5.0 ×104 cfu/mL) (Bodman and Rice, 1996). This high level of contamination of milk might be due to initial contamination originating from the udder surface, quality of cleaning water, milking utensils and materials used for filtering the milk. The most frequent cause of high AMBC is poor hygienic practices during milking. Milk residues on equipment surfaces provide nutrients for growth and multiplication of bacteria that contaminate milk of subsequent milkings. Cows with mastitis (streptococcal and coliforms) and failure to cool milk rapidly to < 4.4°C and extremely hot and humid weather can also contribute to high standard plate count in raw milk.

The total bacteria counts of milk observed in current study is similar with the value (9.10 log cfu/mL) reported by Zelalem (2010) for milk samples collected from different parts of Ethiopia. This value is higher than total bacteria counts of milk in different part of Ethiopia, 6.36 log/cfu/mL in Wolayta zone (Asrat, 2010), 108 cfu/mL in most of the dairy cooperatives operating in Ethiopia (Francesconi, 2006) and 7.6 log cfu/mL in Eastern Wollega (Alganesh et al., 2007).

Generally, the microbial qualities of milk in the current study are poor compared to bacteriological established standards of dairy products. As indicated by John (1995) the plate count of grade A raw milk should be less than 2 × 105 cfu/mL, between 2 × 105 cfu/mL to 1 × 106 cfu/mL for grade B and greater or equal to 1 × 106 cfu/mL for grade C milk in USA. This implies that the sanitary conditions in which milk has been produced and handled are substandard subjecting the product to microbial contamination and multiplication. It is indicated that total bacterial count is a good indicator for monitoring the sanitary conditions practiced during production and handling of raw milk (Chambers, 2002). Coliform count (CC) The coliform count differed significantly (p<0.05) between Dega and Woina Dega (Table 5). Such differences might be attributed to differences in the hygienic conditions such as the quality of cleaning water, practice of cleaning the barn and personnel hygiene followed by producers. The overall coliform count observed in the current study (4.03 log cfu/mL) is comparable with the value (4.03 log cfu/mL) reported for milk samples collected from cows kept under traditional condition in the Wolayta zone (Rahel, 2008). However, higher values of 4.84 logcfu/mL in milk samples collected in the Bahir Dar milkshed

Bereda et al. 464

Table 5. Mean (+S.E) microbial counts of cow milk in the two agro-ecologies. Variable

Mean counts (Log cfu/mL) Overall mean (n=120)

Dega(n=60) Woina Dega (n=60)

AMBC 10.01+0.11 9.63+0.11 9.82+0.81 EBC 4.11+0.16 4.18+0.16 4.15+0.11 CC 3.89+0.12a 4.14+0.13b 4.03+0.09 pH 6.3+0.3a 6+0.1b 6.15+0.58

Row mean bearing with different superscripts letters are significantly different from each other (p<0.05). AMBC = Aerobic mesophilic bacterial count, CC = Coliform count, EBC=Enterobacteriacea count, S.E= Standard error.

(Derese, 2008) and 4.49 log cfu/mL in milk samples in the West Shewa zone of Oromia region (Asaminew, 2007) were reported.

The overall values of coliform counts observed in the current study were much higher when compared with the recommended values given by the American Public Health Service: < 100 cfu/mL for Grade A milk and 101-200 cfu/mL for Grade B milk (WHO, 1997). Generally, the presence of high numbers of coliforms in milk indicates that the milk has been contaminated with fecal materials and it is an index of hygienic standard used in the production of milk, as unclean udder and teats can contribute to the presence of coliforms from a variety of sources such as poor herd/farm hygiene, use of improperly washed milking equipment, unsanitary milking practices, contaminated water and cows with subclinical or clinical coliform mastitis can all lead to elevated coliform count in raw milk (Jayarao et al., 2004). Enterobacteriaceae count (EBC) The average Enterobacteriaceae count was not significantly different (P>0.05) between Dega and Woina Dega (Table 5). Mean Enterobacteriacea count in this study was 4.15 log cfu/mL, which is low compared with selected Ethiopian traditional dairy product (5.2 log cfu/mL) and raw milk (5.7 log cfu/mL) reported by Zelalem et al. (2007) in the central highlands of Ethiopia. However, the mean Enterobacteriacea count in this study was much higher than the maximum limit for Enterobacteriaceae count (<1 cfu/mL) of pasteurized milk (Council Directives 92/46 EEC, 1992). On the other hand, the overall Enterobacteriacea count observed in this study was higher than the maximum acceptable limit (105 cfu/g) set for cheeses made from raw milk for European Union (CEC, 2005). Enterobacteriaceae are commonly found in either dry or humid environments and are good indicators of the contamination of equipment caused by environmental sources (Zink, 1995). Members of this family are sensitive to thermal treatments and sanitizers, and should be monitored in food environments.

Milk pH The mean pH value of milk observed in the current study was 6.15 (Table 5). The pH of the milk from Woina Dega was significantly (P < 0.01) lower than from Dega one. The milk pH values observed from the present study was less from pH of cow’s fresh milk which varies between 6.6 and 6.8 (Van den Berg 1988). Rahel (2008) also reported a higher pH value of 6.49 for milk produced in the Wolayta zone under traditional practices. The lower pH could be due to rapid fermentation (by lactic acid bacteria) of milk kept under high ambient temperature. Moreover; milk is produced under poor hygienic conditions and is not kept at cold temperature. The main contaminants are usually lactic acid producers which cause rapid souring. Although, lactic acid has an inhibitory effect on pathogenic bacteria, fermentation cannot be considered as a sole means to provide a safe milk product (Heeschen, 1994). Conclusion The quality of milk samples collected in Ezha district was generally below standards. This is mainly due to lack following strict hygienic practices during milk production and subsequent handling. Moreover, all the respondents reportedly used plastic jars as milk container while milking, which can be a potential source for the contamination of milk by bacteria. The Aerobic Mesophilic Bacterial, Coliform and Enterobacteriaceae counts obtained in the present study were higher than acceptable limits. This calls for the need of training of milk producers on the potential causes of contamination and public health risk of consuming contaminated raw milk. It is essential to heat treat milk intended for direct consumption as well as the water used for udder washing and cleaning of milk and milk products handling equipment. Acknowledgments The study was made possible through the financial support of the Debre Berhan University under Ministry of

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