UNIVERSITI MALAYSIA TERENGGANU PUSAT PENGAJIAN SAINS DAN TEKNOLOGI MAKANAN

LAB REPORT 2 STM 3103 (FOOD SAFETY MANAGEMENT)

EFFECT OF STORAGE TIME ON THE BACTERIAL COUNT OF VARIOUS FOODS

Prepared by: Group 17 [Bachelor of Food Science (Foodservice & Nutrition)] CHONG HAN HUI (UK28095) CHONG POOI YAN (UK29588) NUR ARIFAH SABRINA BINTI MD ZAKI (UK28094) NURUL NAJIHAH BINTI CHE MOKHTAR (UK28083)

Objective To determine the total plate count of food samples at different storage time.

Introduction When considering growth rates of microbial pathogens, in addition to temperature, time is a critical consideration. Food producers or manufacturers address the concept of time as it relates to microbial growth when a product's shelf life is determined. Shelf life is the time period from when the product is produced until the time it is intended to be consumed or used. Several factors are used to determine a product's shelf life, ranging from organoleptic qualities to microbiological safety. For the purpose of this report, the key consideration is the microbiological safety of the product. The Uniform Open Dating Regulation requires the shelf life of a perishable food product to be expressed in terms of a "sell by" date. The "sell by" date must incorporate the shelf life of the product plus a reasonable period for consumption that consists of at least one-third of the approximate total shelf life of the perishable food product. Numbers of microorganisms can be estimated based on cell counts, cell mass, or activity. A direct count of cells may be made by examination of a known volume of cell suspension under a microscope. This method is rapid and requires minimal equipment. This does not distinguish living cells from dead and may be tedious. The application of certain stains makes visible morphological characteristics of the organism which can aid in identification. Probably the most common method of enumerating cells is the plate count. A known volume of a diluted specimen is added to agar in a petri dish. Assuming dilute solution and that each organism will divide until it develops a visible mass or colony, the colonies can be counted and multiplied by the dilution factor to estimate the number of organisms in the original sample. This method is based on the assumption that a colony forming unit (CFU) is a single organism. Bacterial cell numbers need to be reduced by dilution, because more than 200 colonies on a standard 9 cm plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (Bunnimman & William, 2014). To measure the progress of a culture in a clear broth, changes in turbidity can be measured and related to numbers or organism. Because of these method is easy and rapid; many commercial establishments have found application for this method.

Result Table 1: Counts of colonies for watermelon juice sample at various storage time Food samples Storage time (hours) Colonies / plate dilution dilution dilution

Plate Plate Average Plate Plate Average Plate Plate Average 1 2 1 2 1 2 Watermelon 0 4 6 12 8 119 7.0 65.5 4 0 7 3 5.5 1.5 45 1 0 2 22.5 1.5

Colonies for each dilution was counted. For the plate which not in the range in 30300 colonies, the calculated value is associated with (Est.). For dilution 100 (0 hr): = = = = = CFU/ml (Est.)

For dilution 100 (4 hr): = = = = = CFU/ml

Table 2: Total plate count for various foods at different storage time Food Sample 0 hr Yoghurt 2x103CFU/ml (Est.) 8.1x103CFU/ml Total plate count reported as CFU/g or CFU/ml 4 hr 3x104CFU/ml (Est.) 1.51x104CFU/ml 8 hr 1.0x104CFU/ml (Est.) 2.0x107CFU/ml (Est.) 1.95x108 CFU/ml (Est.) 6.75x102CFU/ml 1.85x102CFU/ml 12 hr 1.5x104CFU/ml (Est.) 3.0x107CFU/ml (Est.) 1.20x108CFU/ml (Est.) 2.04x104CFU/ml 1.01x103CFU/ml

Fresh Fish

Fresh Meat

2.6x105CFU/ml 7.0x101 CFU/ml (Est.) 2.0x10 CFU/ml

3.0X105CFU/ml (Est.) 6.55x102CFU/ml

Watermelon juice

Milk

6.0x10 CFU/ml

Discussion The result of total plate count reported for yoghurt were estimate 2x10 CFU/ml at 0 hour, then estimate 3x104CFU/ml after 4 hours, estimate 1.0x104CFU/ml after 8 hours and estimate 1.5x104CFU/ml after 12 hours. The total plate count of yoghurt within 12 hours was not more than 105 CFU/ml. This result is in the agreement of previous study which state that the total microbial load in yoghurt for 0 days was 6.27x106CFU/ml, 23.86 x 106CFU/ml after 7 days and 35.76x106CFU/ml after 21days. Given that 100g is the daily serving portion of yoghurt, it does contained more than 106CFU/ml of probiotic species such as Lactobacillus acidophilus (Elizabeth et al., 2011). The yoghurt bacteria S.Thermophillus and L. bulgaricus exhibit proteolytic activity (Muhammad, et al, 2009). The activity of Streptococcus thermophillus and Lactobacillus delbruckki sps. Bulgaricus was not completely stopped during the post-activation period, which then produces lactic acid up to the availability of the nutrients present in the yoghurt. (Manjula, et al, 2012). Streptococcus Thermophilus is the predominant microorganism found during storage. Two weeks after production yoghurts typically contain 107 to 109CFU/ml (Hui, 2007). The yoghurt must contain 108 CFU/ml at the time of manufacture, and the organism must be capable of one log increase in growth after culturing into milk at the e the pr u ts she i e (National Yoghurt Association, 2004). According to the USDA specifications for yogurt, low-fat
3

yogurt and nonfat yogurt, it stated that the coliform contained in the yogurt should not more than 10 per gram and not more than 50 per gram for yeast and mold (United Stated Department of Agriculture, 2001). From the fresh fish sample, the total plate counts obtained for different storage time are 8.1 103CFU/ml for 0 hour, 1.51 104CFU/ml for 4 hours, 2.0 107 (Est.)CFU/ml for 8 hours, and 3.0 107 (Est.)CFU/ml for 12 hours respectively. The result showed a gradually increasing of value in bacterial count. Natural microflora of fish flesh is dominated by psychrotophic Gram-negative, rod-shaped bacteria belonging to the genera Pseudomonas, Moraxella, Acinetobacter, Shewanella, Flavobacterium, Vibrionaceae and Aeroemonadaceae, but Gram-positive organisms such as Bacillus, Micrococcus, Clostridium, Lactobacillus and Corynebacterium can also be found in varying proportions (Gram & Huss, 1996). However, at high storage temperatures (15-30C), Aeromonas hydrophila, and E. coli are responsible for spoilage, which is the same temperature as this experiment conducted. According to previous study, there are four treatments of E. coli strains in a raw fish were studied in duplicate number. The first treatment (lag phase) was examined after 3 hours in brain heart infusion BHI at 37C, the second treatment (log phase) after 7 hours, the third treatment (stationary phase) after 24 hours, and the fourth treatment (death phase) after 10 days (Al-Qadiri et al. 2008). This states that the normal viable bacterial counts of E.coli for lag phase is 4.3 log10 cfu/ml, for log phase is 6.90 log10 cfu/ml, stationary phase is 5.0 109CFU/ml, and for death phase is 2.0 108CFU/ml. The spoilage process depends on the fish species and on the handling and storage conditions. According to Food Standards Agency (2010), maximum allowable number of defective sample units of fish for E. coli is must be less than 1.0 1011CFU/ml. From the fresh meat sample, the total plate counts obtained for different storage time are 2.6 105CFU/ml for 0 hour, 3.0 105(Est.)CFU/ml for 4 hours, 1.95 108 (Est.)CFU/ml for 8 hours, and 1.2 108 (Est.)CFU/ml for 12 hours respectively. The result showed a gradually increasing of value in bacterial count, but when the storage time is long until 12 hours the bacterial count become decreases. The microbial growth in meat at 0 hour is passing through lag phase of microbial growth. This is a period of adjustment to a new environment which the fresh meat becomes contaminated for the first time by a number of bacterial cells, these cells adapting to the new atmosphere and nutrients available to them. While from 4 hours to 8 hours, the increasing growth of microbial amount is sign of the microbial undergo exponential phase. This is the phase where bacteria commence actively growth in number by regular cells divisions. At 12 hours storage time, the bacterial growth decline due to the depletion of nutrients and the subsequent accumulation of metabolic waste products and other toxic materials in the media will facilitates the bacterium to move on to the death phase. The ability of reproduction is also unable to carry out in this phase. Individual bacteria begin to die due to the unfavourable

conditions and the death is rapid and at uniform rate. The number of dead cells exceeds the number of live cells (Zwietering, 1990). According to previous study, the initial normal microbial loads for fresh meat are ranged from 7.94 103CFU/ml to 7.94 104CFU/ml (Samelis et al., 2001). This is much lower than our result obtained and the causes maybe due to the cross contamination during handling of the sample. Natural microflora of meat present in the muscle fibers and other parts of the care may be due to slaughtering practices or infection of the animal prior to slaughter, such as Brucella, Salmonella, Streptococcus, and Mycobacterium tuberculosis, also certain anaerobic bacteria may be present. Achromobacter and Pseudomonas are predominant in meat held at temperatures. Also the presence of Bacilli, Staphylococci, and lactobacilli may contribute to surface slime. However, recent reports reveal that E. coli O157:H7 and other serotypes of enterohemorrhagic E. coli are responsible for human disease in other parts of the world as well (Tarr, 1994). According to Food Standards Agency (2010), the microbial standard for Salmonella in fresh meat is absence in 25g, and less than 1.0 CFU/ml for E. coli, less than 31 CFU/ml for Enterobacteriaceae and less than 3162 CFU/ml for aerobic colony count. From the watermelon sample, the total plate counts obtained for different storage time are 7.0 101(Est.)CFU/ml for 0 hour, 6.55 102CFU/ml for 4 hours, 6.75 102CFU/ml for 8 hours, and 2.04 104CFU/ml for 12 hours respectively. The result showed a gradually increasing of value in bacterial count. There are few natural microflora present in watermelon which are E. Coli O157:H7, Salmonella Enteritidis, L. monocytogenes, and Pseudomonas (Mosqueda-Melgar et al., 2008). According to the research paper of Nwachukwu et al. (2008), the total bacterial count for sliced watermelons that were collected randomly from different street vendors are ranged between 0.1 - 2.3 105CFU/ml, and the total fungal count was ranging between 0.2 2.8 105CFU/ml. These values are close to the total plate count for sample stored 12 hours. This means that bacterial needs a long period of time to contaminate watermelon. The microbiological standards for watermelon juice from food act or regulation are safe production, packing; processing, distribution and handling of watermelons depend upon a myriad of factors and the diligent efforts and food safety commitment of all parties throughout the distribution chain. No single resource document can anticipate every food safety issue or provide answers to all food safety questions. These guidelines are not intended to replace other food safety programs, but are meant to be used in conjunction with them to address food safety hazards potentially known to affect the watermelon supply chain (Morrissey, 2008). Based on the result obtained in table 2, the total plate count for milk at 0 hour of storage time is 2.0x10 CFU/ml, 6.0x10 CFU/ml after 4 hours, 1.85x102CFU/ml after 8 hours and 1.01x103CFU/ml after 12 hours. The increment of colonies plate counts in the milk sample showed the relationship that the longer the storage time of milk product, the higher the number of colonies plate count of the sample. Raw

milk may contain pathogens derived from the cow or other milk animal such as Campylobacter, Salmonella, Cryptosporidium, E.coli, S.aureus and L.monocytogenes (Roberts, 2003) .The microbial content of unexpired pasteurized milk in this study is unacceptably high as significant amounts of bacteria including coliforms were found in pasteurized milk processed at different dairy plants. Enterobacter species were most frequent present (Anderson, 2011). The greater the microbial load, the more rapidly the milk will decolorize. The predominant psychotropic genera and species will vary but are derived from those present initially in the raw milk. Pseudomonas is the most frequently reported psychrotrophs in stored milk (Robinson, 2002). Pasteurization process reduces the micro flora in raw milk, where usually a high diversity of bacterial species is detected. Lactococcus lactis, Streptococcus thermophillus, Lactobacillus casei and Lactobacillus rhamnosus were the most abundant species (Masoud et al., 2012). Milk is not the most favorable growth medium for L.monocytogenes, since the bacterium is not able to use the proteins and lactose in milk directly as a nitrogen and carbon source (Marshall and Schmidt, 1991). However, L.monocytogenes is able to survive and grow in milk, even at refrigeration temperature (Xanthiakos et al, 2006). However, according to EC, the microbial load limits at the end of milk manufacturing process should be less than 5.0CFU/ml (Commision Regulation (EC), 2005).

Conclusion Effect of storage time plays an important role in the growth of microorganism. As the period of storage time longer, the total bacteria population may decline due to the toxic substances excrete by bacteria themselves or the lack of nutrient content requires for bacteria to grow. Other than that, competitive with others microorganisms may lead to the decline of certain bacterial growth.

References Al-Qadiri, H. M., Al-Alami, N. I., Lin, M., Al-Holy, M., Cavinato, A. G., & Rasco, B. A. (2008). Studying of the Bacterial Growth Phases Using Fourier Transform Infrared Spectroscopy and Multivariate Analysis. Journal of Rapid Methods & Automation in Microbiology, 16: 7389. doi: 10.1111/j.17454581.2008.00117.x Anderson, M. (2011). The microbial content of unexpired pasteurized milk from selected supermarkets in a developing country. Asian Pacific Journal of Tropical Biomedicine, 1:205-211 Bunnimman, R. & William, G. (2014). Plate count. COLOSS honey bee research association. University of Bern Schwarzenburgstrasse. Retrieved from http://www.coloss.org/beebook/II/efb/9/1/1 Burden, D. W. (2008). Guide to the homogenization of biological samples. Retrieved from http://opsdiagnostics.com/notes/ranpri/Homogenization%20Guide%20ver. 1.pdf Commision Regulation (EC) (2005). Microbiological criteria for foodstuff. Official Journal of the European Union. L338/18 Elizabeth, W., Yeung, M., Tong, P.S. (2011). Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus. International Journal of Food Microbiology, 145: 169-175 Food Standards Agency (2010). Food Standards Agency Annual Report 2009-2010. Gram, L. & Huss, H. H. (1996). Microbiological spoilage of fish and fish products. International Journal of Food Microbiology, 33: 121-137 Hui,Y. H. (2007).Handbook of food products manufacturing. (p.668). United States, US: John Wiley & Sons Inc. Publication. Manjula, K., Viswanath, C., & Suneetha, C. (2012). Physio-chemical, sensory and microbial quality of yoghurt fortified with sapota pulp. International Journal of Material Sciences and Chemistry, 1: 4-6 Masoud, W., Vogensen, F. K., Lilevang, S., Sorensen, S. J., Jakobsen, M. (2012). The fate of indigenous micro biota, starter cultures, E.Coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyro sequencing and quantitative real time. International Journal of Food Microbiology, 153, 192-202

Morrissey, B., Bland, B., & Gombas, D. (2008). Watermelon Good Agricultural Practices & Good Handling Practices. Watermelon Guidance Document (Ed.), National Watermelon Association. Retrieved from www.nationalwatermelonassociation.com Mosqueda-Melgar, J., Raybaudi-Massilia, R. M., & Martin-Belloso, O. (2008). Combination of high-intensity pulsed electric fields with natural antimicrobials to inactivate pathogenic microorganisms and extend the shelf life of melon and watermelon juices. Food Microbiol., 25: 479-491 Muhammad, B. F., Abubakar, M. M., Adegbola, T. A. (2009). Effect of period and condition of storage on properties of yoghurt produced from cow milk and soymilk materials. Journal of Dairy Science, 3: 18-24 Nwachukwu, E., Ezeama, C. F., & Ezeanya, B. N. (2008). Microbiology of polyethylene-packaged sliced watermelon (Citrullus lanatus) sold by street vendors in Nigeria. African Journal of Microbiology Reseach, 2: 192-195 Qazi, J.I, Asif, H., & Shahid, R., 2008. Economical Method for Estimation of Bacterial Viable Count. Pakistan J. Zool., 40(4): 289-294 Roberts, D. (2003). Practical food microbiology. (p.42). United Kingdom, UK: Blackwell Publishing Ltd. Robinson, R. K. (2002). Dairy microbiology handbook: the microbiology of milk and milk products. New York, NY: John Wiley and Sons Inc. Samelis, J., Sofos, J. N., Kendall, P. A., & Smith, G. C. (2001). Influence of the Natural Microbial Flora on the Acid Tolerance Response of Listeria monocytogenes in a Model System of Fresh Meat Decontamination Fluids. Appl. Environ. Microbiol., 67(6): 2410-2420. doi: 10.1128/AEM.67.6.24102420.2001 Tarr, P. I. (1994). Escherichia coli O157:H7: overview of clinical and epidemiological issues. J. Food Prot., 57: 632-636 United Stated Department of Agriculture (USDA). (2002, January 19). USDA specifications for yogurt, nonfat yogurt and low-fat yogurt. Retrieved from http://www.ams.usda.gov/AMSv1.0/getfile?dDocName=STELDEV3004551 Zwietering, M. H., Jongenburger, I., Rombouts, F. M., & Van 'T Riet, K. (1990). Modeling of the Bacterial Growth Curve. Appl. Environ. Microbiol. 56(6): 1875-1881

Question 1. In a laboratory experiment, the plates receiving meat samples were incubated at 35C for 48 h, while those containing the cheese sample were incubated at 30C for 3 days. What is the logic for using different incubation conditions for these two foods? Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria. Each sample contains different bacteria that grow in different temperature and needed different time to grow into colony. For example, Campylobacter jejuni grow in meat samples need 2-5 days and 37C- 42C to grow and duplicate into colony. While in the cheese sample, the Listeria monocytogenes in cheese need 3-70 days to grow into colony and it can survive even at refrigerate temperature. Therefore, different incubation conditions are used for incubates meat samples and cheese samples.

2. Microbial counts, as determined in this exercise, are reported as CFU/g (or CFU/ml) rather than cells/g (or cells/ml) for food. Why? The number of viable cells in a sample is assessed from the number of colonies, which develop on incubation of known amount of a sample inoculated on a solid medium. Plate count is one of the familiar methods of measuring colony forming units (C.F.U.) in a sample. A viable count method assumes that a visible colony will develop from each microorganism. However, a single colony may develop from one or from hundreds or even thousands of microorganisms physically associated with each other or distributed at one location during the process of inoculation. Thus each colony develops form one viable unit; therefore viable counts are usually given as number of C.F.U. per unit volume rather than number of microbes (Collins et al., 1995).

3. Compare 2 methods of homogenization of samples with regard to general ease of use and suitability for use in a quality control laboratory. Two method of homogenisation of food sample with regard to general ease of use and suitability for use in a quality control is grinding and shearing. One of the grinding is process used of mortar and pestle to homogenize the sample. Beside used the mortar and pastel we also can used grain mills, coffee grinders, vortexer and also glass homogenizers. This method homogenizes the sample until fine texture. The advantages are grinding a can be used on wet, dry, and frozen samples and also easy to use and relatively inexpensive to purchase like mortar and pestle. Second method is shearing method. These method homogenizers such as blenders work by shearing which is created by a tangential force being applied to the sample. There are several tools that disrupt by shearing, including blenders, rotor-stators, and some of the glass homogenizers. This homogenizer is one of the most widely used tools for homogenizing plant and animal tissues. Rotor-stators are available as handheld units and larger stand supported models. Blenders are an effective first step in reducing the size of samples. Rotor-stator homogenizer had an elative efficiency of 27.6% as, compared to other methods. Sample sizes which can be processed on handheld homogenizers range from less than 1 ml up to 40 L or more. (Burden, 2009)

4. State 2 limitation of total plate count method to determine the microbiological quality of food. A major limitation in total plate count method is selectivity. The nature of the growth medium and the incubation conditions determine which bacteria can grow and thus be counted. Viable counting measures only those cells that are capable of growth on the given medium under the set of conditions used for incubation. Sometimes cells are viable but non-cultivable. Besides that, as the sample is surrounded by a liquid agar for a short period, many organisms intolerant to this elevated temperature of the media may be killed. In addition, since the colonies are surrounded by agar, obligate aerobes may not be able to develop under the conditions of this test.