World Journal of Dairy & Food Sciences 6 (1): 61-66, 2011 ISSN 1817-308X © IDOSI Publications, 2011
The Inhibition of Some Foodborne Pathogens by Mixed Lab Cultures During Preparation and Storage of Ayib, a Traditional Ethiopian Cottage Cheese 1
Anteneh Tesfaye, 2Tetemke Mehari and 3Mogessie Ashenafi
1
Department of Biology, Addis Ababa University, P.O. Box: 1176, Addis Ababa, Ethiopia Department of Chemistry, Addis Ababa University, P.O. Box: 1176, Addis Ababa, Ethiopia 3 Institute of Pathobiology, Addis Ababa University, P.O. Box: 1176, Addis Ababa, Ethiopia
2
Abstract: The antagonistic impact of mixed lactic cultures against foodborne pathogens (Escherichia coli, Salmonella Typhimurium DT104 and Staphylococcus aureus) were evaluated during preparation and storage of ayib. Souring of milk reduced the count of the test pathogens by 4 log units at 48 h. The test pathogens were reduced to # log 4 and # log 0.8 cfu/g at day 9 while ayib was stored at refrigeration and ambient conditions, respectively. Complete elimination of the test organisms was achieved at day 7 when dipped ayib was kept at ambient conditions. Dipping ayib was observed to improve its safety. Key words: Ayib % Lactic acid bacteria % Antagonism % Foodborne pathogens INTRODUCTION
of antimicrobial substances by the fermenting organisms. However, some enteropathogens have been reported to survive and grow in fermented milks [4]. Isolation of Staph. aureus from ayib collected from open market in Awassa was reported by Ashenafi [2]. Salmonella heidelberg was associated with consumption of contaminated cheese at a restaurant [5]. Although low pH is expected to inactivate bacterial pathogens, acid adaptation was reported to promote the survival of different foodborne pathogens in different types of cheeses [6, 7]. Use of pediocin producing Lactobacillus plantarum (L. plantarum) culture to combat Listeria contamination in red smear cheese was suggested by Loessner et al. [8]. The inhibition of E.coli 0157: H7 during the souring of milk in the course of preparation of ergo and ayib and during storage of the two products was shown by Tsegaye and Ashenafi [9]. Pravez et al. [10] suggested the potential benefits of consuming fermented dairy products containing viable lactic acid bacteria (LAB). Workers have also indicated that cheese can serve as a vehicle for provision of probiotic organisms when compared with the other more acidic dairy products [11]. LAB with probiotic properties can be incorporated into cheese as starters to cheese milk or to the curd before hooping [12].
Ayib is a traditional Ethiopian cottage cheese produced by slowly heating naturally soured milk until a distinct curd mass forms and floats over the whey. Ayib is a popular milk product consumed by the various ethnic groups in the country [1]. Temperature of ayib preparation could vary between 40°C to 70°C without any significant impact on composition and yield. However, temperature above 80°C imparts a cooked flavor to the product. Ashenafi [2] suggested that curd preparation at 70°C produced a wholesome product with low microbial count. During preparation of ayib, the high initial count of microorganism in milk, which increases during the fermentation process, is shown to decrease by the combined action of cooking and low pH [3]. The presence of high microbial load of ready-to-consume ayib is believed to be introduced from plant parts used for packaging and imparting flavor; and from handlers, too [2]. The safety of cheeses with respect to food-borne diseases is of great concern around the world and especially true in developing countries, where production of milk and various dairy products often take place under unsanitary conditions [1]. Generally, fermented food products are usually considered safe because of the low pH and production
Corresponding Author: Mogessie Ashenafi, Institute of Pathobiology, Addis Ababa University, P.O. Box: 1176, Addis Ababa, Ethiopia, E-mail:
[email protected].
61
World J. Dairy & Food Sci., 6 (1): 61-66, 2011
The aims of this study were to assess the antagonistic effect of defined mixed LAB cultures with probiotic potential against some foodborne pathogens during preparation and storage of ayib; and the possible use of ayib as a carrier of potentially probiotic LAB by dipping ayib preparations into mixed LAB cultures.
Counting was done at 0, 12, 24, 36, 48, 60 and 72 h. Plates were incubated at 32°C for 24 to 48 h. The pH was measured during sampling. Soured milk was defatted by churning with a shaker at 100 RPM for 1 h. The floating fat was removed with sterile spoon. Defatted soured milk samples were separately cooked at 50°C and 70°C in a water bath for 55 min. The curd (ayib) was separated from the whey after cooling cooked solid mass to room temperature and by filtering through sterile cheese cloth. Counting of pathogens and pH determination of cooked curd was done at 24 h intervals [9].
MATERIALS AND METHODS Bacterial Strains: E. coli ATCC 25922, Staph. aureus ATCC 25923 and S. Typhimurium DT104, were used as test foodborne pathogens in this study. The LAB strains were recovered from locally fermented dairy products, ayib and ergo; and traditional low-alcoholic beverages, borde and shamita. LAB isolates were identified to species and subspecies level using API 50CHL kit and selected based on their in vitro and in vivo probiotic qualities (data not included). Briefly, colonies of pure LAB cultures were transferred into API 50CHL medium (Biomeriuex, Marcy I’Etoile, France) and homogenized. Homogenized suspensions were filled into 50 wells of API 50CHL strips and covered with mineral oil and incubated at 30 to 32°C for 48 h. Identification of LAB strains using the biochemical profile was done using software database (V5.0) provided by the company. The nine LAB isolates were arranged into four mixed LAB cultures based on their fermentative characteristics (Table 1).
Survival of the Test Pathogens During Storage of Readyto-consume Ayib: Ayib was prepared by cooking pasteurized milk fermented with the various mixed starter cultures. The test pathogens were separately inoculated in duplicates into 200 g of cooled ayib in sterile stomacher bags to give initial inoculum level of log 6 cfu/g mixed thoroughly and incubated at ambient condition. Separately, ayib samples was similarly processed and stored at refrigeration condition. Phosphate buffered saline (PBS) inoculated with the test pathogens served as control. Enumeration of pathogens was done at 24 h intervals for 9 days. When counts were log 10 cfu/g in yoghurt at 4°C for over 15 days. Generally, fermented food products or live microbial food supplements are understood to have probiotic effect [23]. The need to consume at least log 5 cfu/g probiotic organisms daily with food to achieve the beneficial effects
5.
6.
7.
8.
65
Ashenafi, M., 1992. The Microbiology of Ethiopian Ayib. In Applications of biotechnology to traditional fermented foods. Report of an Ad Hoc Panel of the Board on Science and Technology for International Development, ed., Steinkraus, K. Washington, D.C.). National Academy Press, pp: 71-74. Ashenafi, M., 1990. Microbiological Quality of 'Ayib', a Traditional Ethiopian Cottage Cheese. International J. Food Microbiol., 10: 263-268. Ashenafi, M., 2006. The Microbiology of Ethiopian Foods and Beverages: a review Ethiopian J. Biological Society, 5: 189-245. Caro, I. and M.R. Garcia-Armesto, 2007. Occurrence of Shiga Toxin-Producing Escherichia coli in a Spanish Raw Ewe's Milk Cheese. International J. Food Microbiol., 116: 410-413. Fontaine, R.E., M.L. Cohen, M.T. Martin and T.M. Vernon, 1980. Epidemic Salmonellosis from Cheddar Cheese: Surveillance and Prevention. American J. Epidemiol., 111: 247-253. Ramsaran, H., J.C. Brunke, A. Hill and M.W. Griffiths, 1998. Survival of Bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in Soft Cheeses. J. Dairy Sci., 81: 1810-1817. Vernozy-Rozand, C., C. Mazuy-Cruchaudet, C. Bavai, M.P. Montet, V. Bonin, A. Dernburg and Y. Richard, 2005. Growth and Survival of Escherichia coli O157:H7 during the Manufacture and Ripening of Raw Goat Milk Lactic Cheeses. International J. Food Microbiol., 105: 83-88. Loessener, M., S. Guenther, S. Steffan and S. Scherer, 2003. A Pediocin-Producing Lactobacillus plantarum Strain Inhibits Listeria monocytogenes in Multispecies Cheese Surface Microbial Ripening Consortium. Applied and Environmental Microbiol., 69: 1854-1857.
World J. Dairy & Food Sci., 6 (1): 61-66, 2011
9.
10.
11.
12.
13.
14.
15.
Tsegaye, M. and M. Ashenafi. 2005. Fate of Escherichia coli 0157:H7 During the Processing and Storage of Ergo and Ayib, Traditional Ethiopian Dairy Products, International Food Microbiol., 103: 11-21. Pravez, S., K.A. Malik, S. A. Kang and H.Y. Kim, 2006. Probiotics and Their Fermented Food Products are Beneficial for Health. J. Appl. Microbiol., 100: 1171-1185. Songisepp, E., P. Hutt, P. Elias, T. Brilene, M. Zilmer and M. Mikelsaa, 2004. A New Probiotic Cheese with Antioxidative and Antimicrobial Activity, Dairy Sci., 87: 2017-2023. Gardiner, G., R.P. Ross, J.K. Collins, G. Fitzgerald and C. Stanton, 1998. Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains. Applied and Environmental Microbiol., 64: 2192-2199. Goepfert, J.M., N.F. Olson and E.H. Marth, 1968. Behavior of Salmonella typhimurium During Manufacture and Curing of Cheddar Cheese. Applied and Environmental Microbiol., 16: 862-866. Abdella, M., H. Beeker and G. Terplan, 1996. Comparative Studies on the Detection of Salmonellae in Ethiopian Cottage Cheese (Ayib) Using Different Culture Methods. Archiv für Lebensmittel-Hygiene, 47: 83-90. Ercolini, D., V. Fusco, G. Blaiotta, F. Sarghini and S. Coppola, 2005. Response of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus to the Thermal Stress Occurring in Model Manufactures of Grana Padano Cheese. J. Dairy Sci., 88: 3818-3825.
66
16. Arocha, M.M., M. McVey, S.D. Loder, J.H. Rupnow and L. Bullerman, 1992. Behavior of Hemorrhagic Escherichia coli O157:H7 During the Manufacture of Cottage Cheese. J. Food Protection, 55: 379-381. 17. Spano, G., E. Goffredo, L. Beneduce, D. Tarantino, A. Dupuy and S. Massa, 2003. Fate of Escherichia coli O157:H7 During the Manufacture of Mozzarella Cheese. Letter of Applied Microbiol., 36: 73-76. 18. Spahr, U. and K. Schafroth, 2001. Fate of Mycobacterium avium subsp. paratuberculosis in Swiss Hard and Semi-Hard Cheese Manufactured from Raw Milk. Applied and Environmental Microbiol., 67: 4199-4205. 19. El-Gazzar, F. E. and E.H. Marth, 1992. Salmonellae, Salmonellosis and Dairy Foods: A Review. J. Dairy Sci., 75: 2327-2343. 20. Bachrouri, M., E.J.Quinto and T.M. Mora, 2002. Survival of Escherichia coli O157:H7 During Storage of Yoghurt at Different Temperatures. J. Food Sci., 67: 1899-1903. 21. Cheng, C.M. and C.W. Kaspar, 1998. Growth and Processing Conditions Affecting Acid Tolerance in Escherichia coli O157:H7. Food Microbiol., 15: 157-166. 22. Haddadin, M.S.Y., S.S. Awaisheh and R.K. Robinson, 2004. The Production of Yoghurt with Probiotic Bacteria Isolated from Infants in Jordan. Pakistan J. Nutrition, 5: 290-293. 23. Fuller, R., 1989. Probiotics in Man and Animals. J. Appl. Bacteriol., 66: 365-369. 24. Lee, Y.K. and S. Salminen, 1995. The Coming Age of Probiotics. Trends in Food Sci. and Technol., 6: 241-245.
