TABLE OF CONTENT
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION AND RECOMMENDATION
The study was conducted in Kucha district of Gamo zone southern Ethiopia to evaluate chemical composition and microbial quality of butter and cottage cheese collected from small holder farmers. One hundred twenty grams (120g) of each fresh butter and cottage cheese samples were aseptically collected from smallholder farmers using sterile container for chemical composition and microbial quality analysis. Each parameter was analyzed in duplicates following standard laboratory procedures and the average value was taken to avoid false positive results. The study indicated that butter had average values of 18.90±0.58, 81.09±0.58, 5.00±0.06, and 81.33±0.70, respectively for moisture, total solids, pH and fat contents; while for cheese the average of 41.60±0.64, 4.07±0.06, 18.49 ±0.54, 1.46±0.17, and 2.38±0.16, respectively for moisture, pH, protein, fat and ash contents were found. Butter had average counts of 6.883×10cfu/g, 5.925×10cfu/g, and 4.681×10cfu/g, respectively for total bacteria, enterobacteriaceae and coliforms. Also in cheese their counts were 8.14×10cfu/g, 7.162×10 cfu/g and 6.608×10cfu/g, respectively. The studied Butter and cheese were contaminated by varies bacterial species mainly Escherichia coli, Salmonella species, Enterococcus faecalis, Enterobacter aurugenosa, Staphylococcus aureus, Shigella species, Klebsiella species, Vibrio parahaemolyticus; but their occurrences were varied with agro-ecologies and sample types. From this finding it could be understood that both the chemical composition and microbial quality of butter and cheese were affected by agro-ecological effects and processing and handling variations. Therefore, it needs the combined effort of different stakeholders to improve the quality of dairy products through the improvement of milk production and handling techniques in the current study area.
Key words: butter, cottage cheese, chemical composition, microbial quality, Agro-ecology
Milk produced in different parts of the country under traditional production systems is either consumed directly or processed into various traditionally produced more shelf stable dairy products mainly naturally fermented milk ( ergo ), soured skimmed milk ( arera ), butter ( kibe ) and cottage cheese ( ayib). The average values of total solids, fat and ash contents of butter produced in rural farmers in southern Ethiopia were 85.84±1.02, 81.53±1.00 and 0.16±0.004, respectively and also 17.2% moisture, 1.3% protein, 0.1% carbohydrate, 0.024% calcium and 0.0015% iron (Mekdes, 2008).
The cottage cheese was reported to have pH value ranging from 3.7 to 4.6 and lower pH of cheese may contribute to its long shelf life (Seifu et al., 2013). The pH value of cheese made from cow milk sample is 4.3 and the value is higher than other species (Sadia et al., 2016). The cottage cheese made in small holder farms had around 75% moisture and higher moisture content in cheese is due to the longer coagulation time, which leads to the accumulation of moisture content. The mineral in final product is characterized by the ash content of cheese and it might be influenced by the strength of the brine solution used during cheese preparation (Khan et al., 2007). Cottage cheese produced traditionally contains about 1.23% ash in the central highlands (Zelalem et al., 2007). The cottage cheese prepared from cow milk is 2.6% fat (Sadia et al., 2016). One of the chief elements that define the specific body, texture and flavor of cheese is fat (Khan et al., 2007). Preserving of cheese caused reduction in protein content due to its degradation. As a result of this, water soluble components are formed, which lead to loss of protein in the preserving solution (Talib et al., 2009).
The microorganisms predominantly encountered in the dairy industry are bacteria, yeasts, moulds and viruses. Some of the bacteria (lactic acid bacteria) are useful for milk processing, causing milk to sour naturally. Bacteria can cause spoilage of milk and poor yields of products; and microbial analysis of milk and milk products includes tests such as total bacterial count, yeasts and moulds and coliform estimation (Oliver et al., 2005). Zelalem (2010) reported that the average total bacterial counts ranged from 6.18 cfu/g in butter samples collected from Selale area to 7.25 cfu/g in samples from Sululta.
According to food and drug association (FDA, 2015) the acceptable limit of total bacterial count of butter is 7.49 cfu/g and the presence of high variability among samples depending on the sources were also reported. Spoilage occurs when butter is stored at room temperature for a long time mainly by putrefactive microorganisms. Microorganisms having lipolytic activity are highly responsible for disorders such as rancidity or bad flavor (Wondu, 2007).
Coliforms as hygiene indicator can be used as important criteria for determination of microbiological quality of butter and coliform counts in butter is ranging from 1.92 to 4.5 cfu/g. The presence of coliform groups in dairy products indicates the probability of fecal contamination of the dairy product. These differences could be attributed to the wide variation in hygienic handling during milking, processing, storage and transport to market (Zelalem, 2010).
Enterobacteriaceae counts of greater than 4cfu/g of butter samples were also reported from different parts of the country (Zelalem et al., 2007).
Cottage cheese samples collected from an open market in Awassa had high counts of aerobic mesophilic bacteria (over 8 cfu/g) and the sources of contamination could be from handlers, milk vessels used for packaging and possibly herbs used for flavor impartation (Ashenafi, 2006). Study conducted by Eyassu (2013) revealed that, total viable bacterial count ranging from 5.4 to 7.8 cfu/g for ‘metata Ayib’ (Ethiopian semi hard cheese) in West Gojjam. The bacterial contamination by Klebsiella species, Escherichia coli, Enterobacter and Klyuvera species were reported to be found in Ethiopian cottage cheese (Seifu et al., 2013). As reported by Ashenafi (2006), the safety of cheese with respect to food borne diseases is a great concern around the world and in developing countries. This is especially true in Ethiopia where cottage cheese typically produced in small dairy farms under poor hygienic conditions is commonly consumed (Alehilign, 2004). On the other hand, there is limited information documented on the chemical composition and microbial quality of butter and cottage cheese in the current study area, from now understanding and evaluating the situation is vital for improved interventions on processing protocols and hygiene conditions and practices of the postharvest product quality at both local and national level. Therefore, this study was conducted to evaluate the chemical composition and microbial quality of butter and cottage cheese and to determine the isolates which contaminate these products.
MATERIALS AND METHODS
The study was conducted in small holder farmers in kucha district of Gamo zone, southern Ethiopia.
One hundred twenty grams (120g) of each fresh butter and cheese samples were aseptically collected from selected households using sterile container, transported and stored at +4C refrigerator for next laboratory analysis.
Chemical composition analysis of butter and cheese samples
The pH value of butter and cheese samples was determined by measuring the samples with digital pH meter, according to Leclercq-Perlat et al. (1999). The moisture content of butter was determined according to the standard procedure of International Livestock Research Institute (ILRI, 1995). Whereas the moisture content of cheese was determined according to Gobbetti et al. (1999), drying 5 gram sample in oven at 102±2°C for 2 hours. Ash content of butter and cheese samples was analyzed using muffle furnace (SX-5-12) according to the method described by International Dairy Federation (IDF) as cited by Gobbetti et al. (1999). The fat content of butter and cheese samples was extracted by petroleum ether using the Soxhlet apparatus as described by the Association of Official Analytical Chemists (AOAC, 2005). The total nitrogen content of cheese samples was determined according to Kjeldahl procedures (Parvaneh, 1996) and multiplication of total nitrogen by the conversion factor of 6.38 gave the protein content (Olerta et al., 1999).
Microbiological quality analysis of butter and cheese samples
Twenty-five grams of each butter and cheese sample was thoroughly mixed in 225 ml of 0.1% peptone water for the initial dilution. Appropriate serial dilutions up to seventh dilution were then prepared by aseptically transferring 1ml of the previous dilution into 9 ml of 0.1% peptone water.
Total Bacteria Count
Total bacterial count was performed according to National Standard Methods Standard Operational Procedure of Food (NSMSOPF, 2005) for milk and milk products. A serial dilution of 10- up to 10- was prepared using sterile peptone water. One ml of each serially diluted butter and cheese samples were taken and inoculated onto the sterile petridish plates. After autoclaving and cooling of the media at 45C in water bath, 15 ml of standard plate count (SPC) agar was poured onto the plates which contained inoculum, and then the plates were inverted and incubated at 37°C for 24-48 hours. Plates which contain between 30 to 300 colonies were selected. Then, the colony counts were undertaken according to Marth (1978) by using digital colony counter.
One milliliter of each serially diluted butter and cheese samples were inoculated on to the sterile petridish plates. After boiling and cooled to 45C in a water bath, 15 ml of violet red bile lactose agar (VRBLA) was poured on to the plates which contained inoculum and then the plates were inverted and incubated at 32C for 24-48 hours. Characteristic colonies, which were dark red with a diameter of at least 0.5 mm, were counted. Average counts were calculated from selected plate counts of between 15 and 150 colonies (Richardson, 1985).
Zero point one milliliter (0.5 ml) of each serially diluted butter and cheese samples were inoculated on to sterile petridish plates. Then, 15 ml of molten tempered violet red bile glucose agar (VRBGA) was poured on to inoculated petridish plates after autoclaving and cooling of the media. Then the plates were inverted and incubated at 30°C for 48 hours. Colonies which produce purple red with a diameter of 0.5 mm or greater and sometimes surrounded by a red zone of precipitated bile containing 15 to 150 colonies were recorded. Five suspected colonies from the highest dilution were selected and sub-cultured on to a nutrient agar plates and incubated at 37°C for 24 hours. Ten to fifteen colonies obtained from nutrient agar plates were concerned for morphological identification of enterobacteriaceae (NSMSOPF, 2005).
Bacterial species identification
Gram positive and negative organisms were categorized by gram stain using colonies from standard plate count agar. Gram negative rod organisms were undertaken for oxidase test then cultured on nutrient agar plate. Gram positive organisms were sub cultured on 5% Sheep blood agar base and Mannitol salt agar. Colonies growing on blood agar were examined for hemolytic activity and sub cultured on bile esculin agar and nutrient agar (for catalase test). Tube coagulase test was undertaken for identification of Staphylococcus and Micrococcus species. Gram negative and oxidase positive organisms were sub-cultured on triple sugar iron agar to confirm the carbohydrate fermentations. Thiosulfate citrate bile salts sucrose agar was used for the selective isolation of Vibrio species. Other species were also identified by culturing on selective media and undertaking biochemical tests according to Bergey’s manual of determinative bacteriology IDF chart.
The data were analyzed using SPSS (2016), version 20 statistical software. The means of quantitative data between agro-ecologies were compared by employing one-way analysis of variance. Pearson correlation coefficients were used to realize the relationships between factors of importance. The number of microorganisms (colony forming units) per gram of butter and cheese samples were calculated as average count per plate x the dilution factor (IDF, 1987) and log10 transformed values were analyzed for mean comparison. Level of significance was considered at p <0.05. The results were presented using tables, means and standard error of means. The statistical model used for analyzing data was:
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RESULTS AND DISCUSSION
Chemical Composition of Butter and Cottage Cheese
Chemical composition of butter
The chemical composition of butter samples in current study is shown in Table 1. Except from ash percentage, there was significant difference ( p <0.05) between agro-ecologies on the chemical composition of butter, where the average moisture, pH, and fat contents were significantly higher ( p <0.05) in midhighland agro-ecology and the reverse was true for the total solids content in lowland agro-ecology. This variation might be attributed to the fixed effects of agro-ecology on the milk composition due to the difference in altitude which affects the type of feed, and its quality as well as availability; and also the variation in management practice of lactating cows, churning mechanism, individuality of lactating animals, health care, lactation stage, parity, age, milking, milk handling, storage and processing practices which were varies from household to household.
The overall mean of total solids (81.09±0.58), fat (81.33±0.70) and ash (0.37±0.04) composition of butter found in current study were comparable with the compositions of butter reported from southern Ethiopia contained mean value of 85.84±1.02, 81.53±1.00 and 0.16±0.004, respectively for total solids, fat and ash (Mekdes, 2008). Similarly butter from traditional production system contained mean value of 17.2% moisture, 81.2% fat and 0.2% ash (Ashenafi, 2006). Related literature also indicated that butter from traditional production system had an average moisture content ranged from 20-43% (Yonad, 2009). The high level of moisture in butter may have an influence on its microbiological and physicochemical quality since the presence of water in butter can activate lipases, stimulate the growth of microorganisms and cause the hydrolysis of triglycerides when stored at room temperature (Ronholt et al., 2013).
The food value of butter depends on its butter fat content. The fat content of butter is reduced by the incorporation of excess water and most countries protect the consumer by prescribing a legal limit for water content (Zelalem et al., 2007). Butter is one of the primarily fat sources and an important source of dietary energy. Besides fats, butter also contains small proportions of proteins, milk sugar and water which make it a suitable substrate for microorganisms (Mahendra et al., 2016).
The pH level is considered to be the best indicator of food quality and their protection during production and storage (Razzaq, 2003). The pH might be affected by the enzymatic action (Khan et al., 2007). The pH plays an important role in taste and shelf life of product.
The mineral in final product is characterized by the ash content and it might be influenced by the inclusion of some minerals like table salt during processing and preservation (Khan et al., 2007). Also the mineral content of feed consumed by dairy cow and found in water used for cleaning milk vessels can influence the mineral content of dairy products.
Table 1. Chemical composition of butter samples
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Relationship between butter chemical composition variables
The relationship between butter chemical compositions variables are indicated in Table 2. The Pearson correlation coefficients indicated that total solids content of butter had moderate positive linear relationship with pH, which suggests that the higher total solids content corresponds with the lower moisture content, thereby reducing the pH, which can inactivates the growth and multiplication of certain microorganisms and resulted for better quality of the product. On the other hand, the moisture content of butter had strong and moderate negative relationship with total solids and pH, respectively. This also indicates that the higher moisture content of the butter, the reverse is its total solids content, resulted for decrease in pH and limited spoilage and better shelf life of butter. From the current finding, it could be noticed that butter quality could influenced by the relationship between its composition variables and their relation could be maintained through employing appropriate handling, storage and processing practices throughout the production and processing chain.