MATERIALS AND METHODS

Study site: This study was conducted between January to June 2006 at the Department of Food Science and Technology, The University of Georgia, Griffin GA, USA.

Preparation of milk for wara processing: Pasteurized whole milk was purchased from a store at Griffin Georgia. The milk was maintained at 4°C in a cooler and transported to the laboratory where it was stored at 4°C until use. Three liters of this milk each was put in two sterile pots for wara soft cheese processing (Fig. 1).

Samples, packaging and storage: Cheese from the above processing was divided into 16 pieces of 10 g each and divided into two equal groups for 28 and 4°C storage. Four in each group was inserted aseptically into a vacuum bag and sealed with a vacuum machine. The remaining four in each group is then aseptically inserted into sterile nylon bags which are not vacuum packaged; these four served as the control at each storage temperature. The two groups were then stored at the respective temperatures. Another group of cheese was stored in whey at the same storage temperatures as the vacuum packaged cheese.

Fig. 1:	Flow chart of wara processing

Microbiological sampling: Sampling was done every other day for a period of 5 days for cheese stored in whey and 21 day storage for vacuum packaged cheese. At each sampling day, one bag each of vacuum packaged cheese, one nylon packaged cheese and whey cheese containing 10 g cheese each was used. The pH was taken using a VWR scientific model 1800 electrode pH meter.

The samples were then homogenized within the bags with 10 mL of phosphate buffered saline with Seward Stomacher Lab System on the first sampling day. On subsequent sampling days cheese was homogenized with 20 mL of phosphate buffered saline. Serial dilution was made with 0.1% peptone water for ease of counts. Appropriate dilutions were surface plated on MacConkey agar (Becton, Dickinson and Company) for Enterobacteriaceae counts, potato dextrose agar (PDA) for yeast and molds counts, tryptose soy agar (tsa) for total aerobic bacteria, psychrotrophic and anaerobic bacteria counts. Plates for Eenterobacteriacea, molds and yeast and aerobic bacteria were incubated aerobically at 37°C while psychrotrophic bacteria plates were incubated at 6-7°C for 5-7 days. Plates for anaerobic bacteria were incubated anaerobically at 37°C in a GasPak system.

Statistical analysis: The study was performed in two replicates, each with appropriate duplications. All microbiological data were transformed into Log₁₀ CFU mL^-1 or Log₁₀ CFU g before comparison of means. Analysis of data was accomplished using the Fisher’s least significant difference (Glimm et al., 1997) of means of bacterial populations calculated with the General Linear Model (GLM) procedure of SAS based on a 95% confidence level (SAS, 2000).

RESULTS AND DISCUSSION

In wara cheese stored in whey, general the population of total aerobes, anaerobes, molds and yeasts counts increased with storage days except on two occasions in whey stored wara cheese.

Table 1:	Microbial counts and pH of Calotropis procera and Lemon juice cheese stored in whey
C.P = Calotropis procera cheese ; D= storage days, Values followed by same letters in a same row are not significantly different with respect to storage temperature.

Enterobacteriacea counts were undetectable through out storage and pH dropped with extension of storage period in whey stored cheese (Table 1). In vacuum packaged wara cheese molds and yeast counts which were initially undetectable became detectable by the 4 to the 7 day of storage and then remained undetectable to the end of the 21 day storage period. Enterobacteriacea counts were undetectable throughout storage except on few occasions on days 7 and 11 of storage. pH which decreased from 5.65-6.14 on day 1 to 4.97-6.22 on day 11 also decreased from 5.23- 6.26 on day 14 to 4.84- 6.17 on day 21 (Table 2). A significance difference occurred between storage temperatures and C. procera and Lemon juice processed cheese.

Some types of whey cheeses are supposed to be consumed within a short time upon manufacture (e.g., Ricotta, Requeijão and Manouri), whereas others bear a longer shelf life (e.g., Gjetost, Mysost and Myzithra). Whey cheeses are significantly different from one another in terms of chemical composition, which is mainly due to variations in the source and type of whey, as well as to the processing practices followed (Pintado et al., 2001).

Table 2:	Microbial counts and pH of vacuum packaged Calotropis procera cheese and lemon juice cheese for 21 day storage at 28 and 15°C
CP = vaccum Calotropis cheese; Values followed by same letters in a same row are not significantly different with respect to storage temperature(p<0.05).

Generally the populations of total aerobes and anaerobes, increased with extension of storage time at 15 and 28°C and the effect of storage temperature on total aerobes and anerobes was significant (p<0.05) in this study. The populations of total aerobes and anaerobes increased more rapidly in wara cheese stored at 28°C as compared to 15°C. The populations of these two groups of microorganisms were significantly different in cheese samples stored at 28°C compared to the samples stored at 15°C (p<0.05). This is probably because the temperature of 28°C is more close to the optimal growth temperatures of these organisms. Lower storage temperatures such as 15°C slow down microbial metabolic activities therefore, retarding the proliferation of microbial cells (Jay, 2000). Enterobacteriacea were undectected throughout storage and molds and yeast counts were undetec until the 5 day of storage in whey cheese. This probably due to a low level contamination of of these microbes in the raw milk used for processing. The slight decrease in pH along the storage days is likely due to increase in the number acid–producing bacteria.

The effect of vacuum packaging was greatest on molds and yeast and enterobactericea counts which were undetec by the end of 21 day storage. Similar report were made in Requeijao a traditional Portuguese whey cheese which was packaged under vacuum, Yeasts were severely inhibited. Packaging under vacuum decreased yeast growth rates and also reduced the populations attained at the end of the exponential phase of growth, resulting in a shelf-life extension of the Stracchino cheese of over 28 days (Sarais et al., 1996). A similar reduction in coliform counts along storage days of cheese has earlier been reported (Alalade and Adeneye, 2006). This report is similar to entero bacteriacea counts, which were undetec in this study. Similarly an extension in shelf-life was reported in vacuum packaged gas flushed bananas (Chauhan et al., 2006). The inhibition of microbes in vacuum packaged cheese is because air is evacuated from gas-impermeable pouches followed by sealing (Jay, 2000). But the count of total aerobes despite the vacuum packaging is an indication that wara is porous and still has some air stored within its matter thereby encouraging multiplication of aerobes. The presence of anaerobes throughout the storage is expected because the wara was stored in an anaerobic condition. These findings are particularly important in terms of improving wara and other dairy products. Moisture content and pH of whey cheeses are usually high and favor microorganism growth (molds, yeasts, lactic acid bacteria and Enterobacteriaceae accounting for the dominant microflora in these cheeses). A positive correlation was earlier reported between coliform bacterial count and moisture content (Alalade and Adeneye, 2007). The use of lemon juice as a sanitizer and bacteriocidal agent against pathogens has been reported (Sengun and Karapinar, 2005; Sengun and Karapinar, 2004) this explains the reason for the significant difference in all counts between the Calotropis procera and lemon juice processed cheese.

CONCLUSION

Our findings elucidated the limitation of shelf-life duration in ‘wara’ cheese in a developing country (Nigeria). Overall our data indicate that vacuum packaging suppressed molds, yeast and enterobacteriacea. The use of lemon juice in wara processing also suppressed all the microbes in this study. This study suggests that ‘wara’ cheese (as well as other whey cheeses and dairy products) could be vacuum packaged to reduce the risks of food poisoning bacteria and extend the shelf-life of these products.

Adequate packaging of whey cheeses should be provided, and legislation should be prepared to fix standard characteristics of each type of whey cheese.

ACKNOWLEDGMENT

This study was funded in part by FY-2005/2006 Junior Staff Development Fulbright Scholarship of the United States Institute of International Education.