Manufacturing efficiencies and microbial properties of butter and Ayib - Ethiopian cottage cheese

Zelalem Yilma, G Loiseau* and B Faye**

Dairy Technology, Ethiopian Institute of Agricultural Research, Holetta Research Center, PO Box 2003, Addis Ababa, Ethiopia, e-mail: zeyilmak@yahoo.fr
*CIRAD-AMIS agro Alimentaire, Bat 16, TA 40/16, 73 rue J.F. Breton, 34398, Montpellier, Cedex 5, France
**CIRAD-EMVT, 34398 Montpellier, Cedex 5, France

Abstract

Four butter-making methods (3 traditional: clay pot - CP, 'mesbekia' - M - a wooden agitator with three to five finger like projections at one end, and a combination of the two - CP+M; and one improved internal wooden agitator fitted to the traditional clay pot - IWA) were evaluated for their efficiencies in terms of fat recovery and churning time at 12 smallholder milk producers in the central highlands of Ethiopia.

About 21 litres of milk was needed to produce one kg of butter (83% total solids - TS). An average churning time of 222 min was recorded for the three traditional methods with a mean fat recovery of about 90%. Churning time was significantly reduced by 65% for the IWA as compared to the average of the 3 traditional methods. The effect of holding Ayib (Ethiopian cottage cheese) in whey for different time length (1 h, 2 h and overnight) on Ayib yield was compared. The mean Ayib-making time and temperature was respectively 48 min and 57^oC. Weight of Ayib recovered significantly increased with increasing length of time Ayib stayed in whey before separation, amounting to 1266, 1475 and 1776 g per 9.2 litres of buttermilk for 1 h, 2 h and overnight, respectively. About 6 litres of buttermilk was needed to produce one kg of Ayib (20.4% TS). Besides, aerobic mesophylic bacterial count (AMC), counts of enterobacteria, and coliform bacterial count (CC) were performed. Average AMC, counts of enterobacteria and CC of butter samples were 8, 5.3 and 3.8 log cfu/g, respectively, while the counts for Ayib samples were 7.9, 5.1 and 4.4 log cfu/g, respectively. Enterobacter, Escherichia, Klebsiella and Klyuvera were the genera identified, while Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Klebsiella gr. 47 are the species commonly isolated from both products.

The knowledge on traditional butter- and Ayib-making efficiencies and their microbial properties is essential to increase yield and improve quality and preservation of the products.

Keywords: Ayib, butter, efficiency, Ethiopia, microbial quality, milk processing, smallholder

Introduction

Butter and Ayib (Ethiopian cottage cheese) are important in the Ethiopian diets. Butter is made by churning Ergo (naturally fermented whole milk) using traditional churns such as clay pot and bottle gourd. It is composed of 80% fat, 16% moisture, 2% salt and 2% non-fat milk solids (O'Mahony and Ephraim 1985a). For butter-making, Ergo is filled to about half of the capacity of a churn. The churn is then agitated after covering and securely tying it with materials such as a false banana (Enset edulis) leaf, a piece of skin or hide. After butter granules have coalesced into large grains, the churn is rotated on its base to help collect butter grains and form lumps of butter in the center. The butter is then skimmed off, kneaded in cold water and washed to remove visible residual buttermilk (FAO 1990). Ayib, a cottage type soft cheese, is made by heating buttermilk (a by-product of butter-making) in a clay pot or other material on a low fire at a temperature between 40-50^oC (FAO 1990; O'Connor 1994). When the curd and the whey separate, the heating is stopped and the contents of the pot are allowed to cool. After draining off the whey using materials such as muslin cloth, the cheese curd (Ayib) is kept in a clean bowl or pot until it is served with different dishes (FAO 1990).

Churning efficiency is measured in terms of the time required to produce butter granules (O'Mahony 1988) and by the amount of milk fat recovered in the form of butter Brännäng and Persson 1990). Although unavoidable, processing losses should not exceed 5% of the total fat available in sour whole milk (O'Connor et al 1993; O'Mahony and Peters 1987). Though different from place to place and even from household to household (Fekadu and Abrahamsen 1994a; Yilma 1999), traditional milk processing methods generally give low yields of final products per unit of milk, are labor intensive and time consuming (O'Connor et al 1993; O'Mahony and Peters 1987). Centrifugal separation, on the other hand, is quick and efficient leaving less than 0.1% fat in the separated milk (O'Connor 1994) but unaffordable to most smallholder producers.

Their capacity to produce desirable flavor and physical characteristics in many dairy products make certain microorganisms important in the dairy industry. However, a number of microorganisms are not only capable of causing off-flavor and physical defects in dairy products but also represent an important source of disease transmission to humans and animals. This later is the case when products are contaminated by pathogens or microbial toxins (Walstra et al 1999). This problem can be severe in countries like Ethiopia where most of the milk produced is marketed to consumers without being pasteurized and where there is no functional official quality control standard. Moreover, in Ethiopia 98% of the annual milk is produced by subsistence farmers who live in rural areas (Reda 1998) where cooling and other facilities needed for dairy industry are not sufficient.

In spite of the aforementioned prevailing situation, there is limited information documented on the efficiency of traditional milk processing technologies and the microbial properties of the products, while understanding them is crucial in making improvement interventions and is a prerequisite in the market orientation process of the dairy sector in the country. This study was, therefore, conducted to evaluate the efficiency of butter-making and Ayib-making methods under smallholder farmers' management condition in the central highlands of Ethiopia and provide basic information on the microbial properties of these products.
 

Materials and methods

Study location and participating farmers

The study was carried out at Holetta (altitude: 2400 masl; annual rainfall: 1100 mm; av. Temperature, min.: 6°C max.: 24 ^oC) and Selale (altitude: 2500 - >3000 masl; annual rainfall: 1200 mm; av. Temperature, min.: 6°C max.: 21^oC) in the central highlands of Ethiopia that possess a high potential for dairy production. The seasons of a year in these areas can broadly be categorized into dry (October to May) and wet (June to September). A total of 12 smallholders, 6 from each site, were selected to take part in the study based on their interest to be involved, availability of the raw material milk and that they practice processing milk into various products. Smallholder producers in these study refer to those possessing <10 cows and processing milk using locally available traditional technologies.

Processing efficiency

Unadulterated milk, collected from participating farmers and cooperative centers was mixed and sampled for later chemical analysis. Ten litres of milk was used for each butter-making method and all the four butter-making methods were tested at each household. The milk, after adequate fermentation according to farmers' viewpoint, was processed at each household. The buttermilk was used as a raw material for Ayib-making. Ergo was sampled after it was stirred until a uniform state was achieved. Sampling of butter and Ayib was performed right after recovery. Samples of fresh whole milk were preserved using potassium dichromate (O'Connor 1994). All samples were then brought to the laboratory from the farm in an ice box for chemical analysis. Samples were kept at +4°C for up to 24 h until the analysis was performed.

Butter making

Three traditional smallholder butter-making methods and one improved processing method developed by International Livestock Research Institute (ILRI) were considered as treatments and compared for their efficiency. These were: 1) sour milk was agitated by placing the churn (clay pot - CP) on a mat on the floor and rolling it back and forth, 2) sour milk was stirred with "Mesbekia" - M (a stick with three to five finger like projections at one end) by inserting it in the sour milk inside the clay pot and using the palms of both hands to rotate the stick. In this case the clay pot is not moved, 3) sour milk was initially stirred for about three minutes with M as in 2 and then agitated by rolling the sour milk in the clay pot back and forth until milk fat is recovered in the form of butter as in 1 (CP+M) and 4) internal wooden agitator (IWA) fitted to the traditional clay pot. The combined performance of the three traditional methods referred as "Traditional" in the forthcoming discussion was compared with the IWA.

The following formula was used to calculate percent fat recovered (O'Mahany and Ephraim 1985a):

Fat recovery = [(fat in whole milk - fat in buttermilk)/fat in whole milk] x 100

Time elapsed for butter recovery was obtained by subtracting the time of recovery of the product from the time of starting the processing. Weight of butter recovered was recorded using a spring balance (5g sensitivity) after draining the liquid product using a muslin cloth and rinsing with water.

Ayib making

After Ayib was made by heating the buttermilk, it was allowed to cool in the whey and precipitate for 1 h, 2 h and overnight before separation. These time lengths Ayib stayed in whey before separation, referred as TASW later, were selected for comparison of the yield based on results of a survey study conducted prior to the current experiment. Some farmers reported to get maximum yield after keeping it in whey for an hour after its recovery, while others reported to get increased yield and shelf-life by keeping it in whey overnight. The first Ayib-making temperature was measured after 15 minutes of cooking the buttermilk. Temperatures were then recorded at 5-minute intervals until the end of the cooking (when adequate coagulation was achieved). Averages of stabilized temperatures were considered as Ayib-making temperature. Weight of Ayib recovered was measured using a spring balance (5g sensitivity) after separating it from the whey using a muslin cloth.

Physico-chemical tests

Determination of % fat and % total solids (TS) was performed following standard procedures (O'Connor 1994). Acidity of the products was determined by titrating 10 ml of milk with N/9 NaOH to a phenolphthalein end point (Robinson 1981). Milk density was measured using a lactometer and a thermometer. For cow's milk an approximate estimation of density can be interpreted as follows: Density <1.028 (diluted milk), between 1.028 and 1.033 (normal milk) and between 1.033 and 1.037 (skimmed milk) (Lambert 1988).

Microbial analysis

A total of 80, butter 40 and Ayib 40, samples were collected aseptically in sterile bottles, kept in an icebox and transported to laboratory within 8 h of sampling for analysis. Aerobic Mesophylic Bacterial Count (AMC), counts of Enterobacteriaceae and Coliform Count (CC) were considered for both products. Standard conventional laboratory procedures were followed for the analyses. Each of the analyses was made in duplicates and for each of them there was a control. All the analyses were performed within 24 h of sampling. For AMC dilutions were selected so that the total number of colonies on a plate was between 30 and 250, while for CC dilutions were selected for plate counts between 15 and 150. For Enterobacteriaceae dilutions were selected so that counts would be within the range between 20 and 200 colonies per plate (Richardson 1985). Twenty-five g of butter and Ayib were thoroughly mixed in 225 ml of 0.1% peptone water (Oxoid, UK) for the initial dilution. Appropriate serial dilutions were then made by aseptically transferring 1 ml of the previous dilution into 9 ml of 0.1% peptone water (Oxoid, UK). Peptone water and media prepared for each test, except Violet Red Bile Agar (VRBA) (Oxoid, UK), which was brought to the boil to dissolve completely, were autoclaved for 15 min at 121°C.

Aerobic Mesophylic Bacterial Count (AMC)

After autoclaving as mentioned earlier, Standard Plate Count Agar (PCA) (Oxoid, UK) was cooled to 45^oC in a water bath. AMC was made by incubating surface plated appropriate decimal dilutions of butter and Ayib in the PCA medium at 32^oC for 48 h (Richardson 1985).

Coliform Count (CC)

Appropriate decimal dilutions of butter and Ayib were surface plated on Violet Red Bile Lactose Agar (VRBA) (Oxoid, UK) and counts were made after incubating the plats at 32^oC for 24 h. Typical dark red colonies (> 0.5 mm in diameter) were considered as coliforms. This was confirmed by transferring up to five typical colonies from each plate to tubes of 2% Brilliant Green Lactose Bile Broth (BGLBB) (Oxoid, UK). Gas production after 24 h of incubation at 32°C was considered sufficient evidence for the presence of coliforms (Richardson 1985).

Counts of Enterobacteriaceae

Zero point one ml of the required dilution was surface plated on appropriately marked duplicate Petri plates of Violet Red Bile Glucose (VRBG) Agar (Oxoid, UK), which were then incubated at 35^oC for 48 h. Colonies that were rose-colored and surrounded by a halo of purple precipitate were counted as presumptive Enterobacteriaceae per g (Szabo 1997). Up to 10 well isolated typical colonies per plate were picked and re-streaked to purify on nutrient agar plats and incubated at 35^oC for 24 h. These were transferred on nutrient agar slats and incubated at 35^oC for 24 h. This was then followed by Gram-staining, oxidase test, catalase test and API 20 E biochemical identification test (BioRad, France).

Data collection and statistical analysis

Efficiency evaluation

Data collected on the main dependent variables: Per cent fat recovered, weight of butter recovered and churning time (for butter-making) and weight of cheese recovered and chemical composition of Ayib (for Ayib-making) and; the main independent variables: butter-making method, site and season (butter-making) and site, season and TASW (Ayib-making) were analyzed. General Linear Model (GLM) of the Statistical Analysis System was used for the analysis of the data (SAS 2001). Means within the same category were separated using the Least Significant Difference (LSD) grouping.

Microbial analyses

The number of microorganisms (colony forming units) per gram of butter and Ayib samples was calculated as average count per plate x the dilution factor (IDF 1987). Log 10 transformed values were analyzed using the General Linear Model (GLM) for least squares means in SAS using a fixed effect model. The Least Significant Difference (LSD) test was used to separate the means and differences were considered significant when P<0.05 (SAS 2001).
 

Results

Butter making

Among the traditional butter-making methods, fat content in buttermilk was low, fat recovery was high (Table 1) and churning time was long (Table 2) for M. Acidity of sour milk was about 5% higher at Holetta than at Selale (Table 2).

 

Fat recovery was higher at Selale (Table 2). Butter-making method affected significantly (P<0.05) % TS of butter (Table 1). Butter produced at Selale had 3.42% higher TS content than that of Holetta (Table 1). Per cent TS was higher for CPT than the other treatments (Table 1). Fat content of buttermilk was 35% and 23% higher at Holetta and during the wet season, respectively (Table 1). Fat recovery didn't differ significantly between the traditional and IWA (Table 3). Churning time was 65% shorter for the IWA than for the traditional methods (Table 3).

Fermentation time was shorter during the dry season than the wet season (Table 2).

Ayib making

An average of 6 litres of buttermilk was needed to produce 1 kg of Ayib (2.29% fat, 1.23% ash, 20.42% TS). The 6% higher Ayib-making temperature at Holetta explains the significantly lower (27%) Ayib-making time at Holetta than at Selale (Table 4).

The results of Ayib-making time and temperature were, however, not consistent between seasons. Moreover, these parameters differed between households within the same site and season. Ayib yield in terms of weight increased with increasing TASW, where highest values were recorded for overnight TASW (Table 4). Average Ayib yield was about 26% of the buttermilk (23% for 1 h to 30% for overnight) (Table 4). Overall mean percent TS and fat content of Ayib was 20.42 and 2.29, respectively (Table 5).

Microbial analysis

For both butter and Ayib samples, comparable values of AMBC, CC and counts of Enterobacteriaceae were recorded (Figure 1).

The genera identified in both products include Klebsiella, Escherichia, Enterobacter and Klyuvera in their order of abundance. Escherichia coli was the dominant isolate identified (Table 6).

 

Discussion

Butter making efficiency

The higher fat content in buttermilk within the traditional methods might be attributed to the long churning time and/or the mechanism of churning that allows the incorporation of large volumes of air. A short churning time corresponds with low churning efficiency (Fekadu and Abrahamsoen 1994b). An earlier report indicated 1.4% fat for buttermilk using the traditional method and 1.1% using IWA (O'Connor et al 1993). Loss of fat in the buttermilk may not be considered as a loss as far as its nutritional value is concerned if consumed by the family members. However, considerable financial loss can occur if the buttermilk is converted to cottage cheese and marketed. This is because the price of cheese per a given weight is only around 11% of that of butter (Yilma 1999). Although, the reason why acidity of sour milk was higher at Holetta than at Selale is unknown, acidity of >0.6% reportedly did not influence fat recovery (O'Connor 1994). The higher fat recovery at Selale might be attributed to the combined effect of low churning temperature and high fat in whole milk. This result is consistent with a previous report (O'Connor 1994). The higher TS content of butter at Selale (Table 1) might be due to the higher percent fat recovery. However, higher values (0.83%) (Fekadu and Abrahamsen 1994b) and (1.4%) (O'Mahony and Peters 1987) were reported for buttermilk. These results show considerable efficiency variabilities of smallholder milk processing techniques.

Percent fat recovery less than that obtained for the traditional method in this study was reported in the central Ethiopia (O'Mahony and Ephraim 1985b). Fat recovery between 78.6 and 81.1% in Southern Ethiopia was also reported Fekadu and Abrahamsen 1994b). The shorter churning time for IWA as compared to the traditional clay pot might be due to the internal agitator fitted to the traditional clay pot that facilitates the incorporation of large volumes of air in the milk and fast formation of lumps of butter. The way the internal agitator fitted to the traditional clay pot operates also induces increased degree of agitation, which is reported to be associated with reduced churning time (O'Connor 1994). This result agrees with reports of earlier works (Fekadu and Abrahamsen 1994b; O'Connor et al 1993; O'Mahony and Ephraim 1985a). We observed that about 21 litres of milk were needed to produce 1 kg of butter, which is in agreement with previous reports (Fekadu and Abrahamsen 1994b). The significantly shorter fermentation time during the dry season than during the wet season might be attributed to higher ambient temperatures, which activate lactic acid producing bacteria that are responsible for the fermentation of lactose to lactic acid.

Ayib making

Ayib making time and temperature differences might have occurred because of the variation on cooking temperatures used for Ayib-making and on the decision made on the breakpoint of adequate coagulation of casein. Some farmers used a higher intensity of fire that resulted in fast coagulation of casein, while others used a lower intensity that resulted in slow coagulation of casein. A similar speculation has been suggested (Fekadu and Abrahamsen 1994a). However, these differences may not have apparent effects on yield. Higher Ayib-making temperatures between 70 and 90^oC for instance were reported not to affect Ayib yield, but gave the product a cooked flavor (Ashenafi 1990). The speculation for the increased Ayib yield with TASW could be that by letting the Ayib stay in the whey longer before separation, the temperature decreases and small particles of Ayib that were intermingled in the whey will have enough time to precipitate and merge together increasing the yield. An earlier report indicated Ayib yield to account for 18% of the buttermilk (O'Mahony and Ephraim 1985a), which is lower that the result of the present study (26%). Ayib produced in different parts of Ethiopia generally has high moisture content contributing to its poor keeping quality. Percent fat and protein contents of 6.2 and 14.6, respectively (ILCA 1992) and percent fat content of 1.9 (Fekadu 1994) were reported.

Microbial qualities of butter and Ayib

The microbial qualities of both products were generally poor as compared to international standards. The maximum limit of AMC, which is commonly employed to indicate the sanitary quality of food, for raw milk intended for processing is 10⁵ cfu/ml and for that intended for direct human consumption is 5x10⁴ cfu/ml (Bodman and Rice 1996; Council Directive 92/46 EEC 1992). Coliforms, which are associated with fecal contamination, are killed by pasteurization. When present in milk, they are regarded as "indicators" of post-pasteurization contamination as a result of poor sanitation (Jayarao et al 2004). The maximum limit of coliforms for soft cheese made from heat-treated milk and butter is 10⁵ cfu/g and 10 cfu/g, respectively (Council Directive 92/46 EEC 1992). In the EU, the upper limit for Enterobacteriaceae for pasteurized milk and other pasteurized liquid dairy products is <5 cfu/ml determined at the end of the manufacturing process. The value for cheeses made from heat treated milk is 10³ cfu/g, while the value for cheeses made from raw milk is 10⁵ cfu/g (CEC 2005). Traditional milk equipment are reported to be often porous and therefore a reservoir for many organisms and difficult to clean (O'Connor 1994). Results of the microbial analysis underline the presence of bacterial contamination of public health importance. Enterobacter sakazakii identified in butter samples for instance is associated with severe diarrhea and necrotizing enterocolitis in enfants, immuno-compromised patients and neonates (£28 days). Outbreaks of sepsis and meningitis in all age groups caused by Enterobacter sakazakii were also reported (FAO/WHO 2004; INFOSAN 2005). Klebsiella pneumoniea and Klebsiella oxytoca isolated from both products are associated with illnesses such as systemic infections, necrotizing enterocolitis and sever diarrhoea in infants (FAO/WHO 2004).

In conclusion we observed that IWA improved churning time by 65%, decreased losses of fat in buttermilk by 13%, increased fat recovery by 1.1% and increased weight of butter by 3.6%. Twenty-one litres of milk was needed to produce 1 kg of butter (83% TS), while 6 litres of buttermilk was needed to produce one kg of Ayib (2.29% fat, 1.23% ash, 20.42% TS). The longer the Ayib stayed in whey the better the yield of Ayib. Traditional methods need to be improved to reduce processing time and increase recovery of final products per unit of raw material used. The isolation and identification of different species of enteric bacteria in substantial concentrations underlines the problem of hygiene and contamination during the handling of dairy products from farm to table. Standard sanitary practices coupled with cooling facilities are important in limiting contamination. Where cooling facilities are not easily available, both producers and distributors should work together to create awareness on hygienic handling of dairy products during production, processing, storage and distribution.
 

Acknowledgements

The financial support of International Foundation for Science (IFS), the French embassy in Ethiopia and Ethiopian Institute of Agricultural Research is gratefully acknowledged.
 

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Received 11 February 2007; Accepted 26 March 2007; Published 6 July 2007

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