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Key Topics
- Requirement
- Solution
- A. Identify the different reasons for microbiological testing of the named product. [20 marks]
- B. Explain the approach you would take in determining the organisms you would include in the test regime.
- C. Identify and characterize the organisms you would test for, with your rationale for their inclusion and an indication of when you would test.
- D. Produce outline microbiological laboratory procedures for three of the organisms you have selected, including a pathogen, a quality indicator and an indicator of poor hygiene practice.
- References
Requirement
Microbiological Assessment on ready to Eat Salad
Solution
A. Identify the different reasons for microbiological testing of the named product. [20 marks]
Microbial testing is conducted to check the level of safety of any product (Mossel et al, 1995). It tries to look for any possible signs of contamination in the products that are being sold to the public. The ready to eat salad, as mentioned in the case, is the combination of various things such as single cream, fortified wheat flour, asparagus, pasteurised free range egg, and few others. All these elements have different resistance towards changing temperature and preservation techniques. Any deformed procedure of upkeep can result in spoiled salad. For instance, milk may sustain for less time in comparison to the oil. Each of the combination responds differently and spoilage of anyone can lead to bad tasting salad.
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RTE Salad is ready to consume and, therefore, it requires proper care when being processed, or preserved for future use (Christian et al, 2008). Moreover, packaging and distribution of the same cannot be conducted like other products. This requires proper management of the conditions under which these are being transferred. If the proper packaging is not considered then it might get impacted from the microorganisms. Therefore, it is suggested that the ready to eat salads should be sent for distribution under proper condition.
The reason behind conducting the microbial test on the salads is to know whether the product has been infected by any harmful microorganisms. If the product contains any microorganisms that are pathogenic then it must be brought into notice as it can harm the individual consuming the product. Moreover, the test of the product helps in understanding the shelf life that is left for the product. It gives the organization a fare idea into the days they can keep the product for customer use.
If the toxins that automatically generates from the combined components may result into ingestion in the consumer of that product. Moreover, it is imperative to understand that how much impact the toxicity will have on the human digestion and the level of toxicity appropriate for the consumption (Berrada et al, 2006). The test should be conducted to understand these things. The test will also help in understanding the level of tolerance these pathogens will demonstrate and the impact the same can have when someone consumes the salad. Therefore, it is suggested that the organization should conduct thorough testing program at certain intervals. The periodic testing will help in understanding any occurrence of any new kind of organisms in the salad and the period that are taken for such organism to occur and the necessary steps that can be taken to mitigate such issues.
The testing of the product allows the company to withdraw the product well on time before it results in any kind of issue. Timely withdrawal protects company from any consumer backlash and, also, protects them from any law suits that results when any organizations involve in selling products that are spoiled or not suitable to sell. Such timely actions can only be possible if the regular testing is being conducted. Moreover, it allows the company to understand the period till which the product will be suitable for the self and based on such understanding the company can implement procedures where the product will be removed from the shelf.
The presence of microorganisms in the salad leads to the reduction in the nutritional value of the same as the microbes present in the salad tend to destroy certain important components necessary for maintaining the nutritional value of the product.
Citing all the reasons mentioned above, it can be stated that the organizations should involve in regular microbiological testing of the RTE Salad. The testing should mainly focus on the components that are more susceptible to changing physical conditions. The organization should involve in testing on different interval to maintain the freshness of the product when it reaches the consumers hand also to remove the product well on time from the shelf.
B. Explain the approach you would take in determining the organisms you would include in the test regime.
This section details the approach that has been taken to determine the organisms that will be included in the test regime of the RTE Salad. The RTE salad will be procured from the local market under the proper preservation. The research has considered around 50 g of the test sample of RTE Salad will be brought to the test centre and will be kept under proper condition till the it is examined for the concerned objective. The content of the RTE salad is the mixture of various ingredients. The source of Cows milk in the salad is single cream, tam valley cheddar cheese, unsalted butter, and full fat soft cheese. Wheat flour in the salad is the source of the many nutrients such as Calcium Carbonate, Iron and others. The vitamins present in the salad is mainly comes from the presence of Asparagus in them. The fat content of the salad is sourced from the pasteurised free range egg, Channel Islands’ cows’ milk and rapeseed oil. The source of proteins in the salad is the poached and smoked salmon, and corn flour. Moreover, salt and water content in the salad can be attributed as the source of mineral and ions.
The research will be conducted for the RTE to determine the aerobic colony count which will help in understanding perceptiveness and effectiveness of the microbial. Aerobic Colony Count (ACC) is the count of the viable bacteria which depends on the counting colonies growth in the nutrient agar plate. It will be employed to RTE salads to understand the sanitary quality of the salad. The incubation condition which will be used for the salad is 30 degree Celsius for the time period of 48 hours. The second examination will on the “Indicator Organism” which refers to the selected surrogate markers. It will help in understanding the quality of the food in terms hygiene is concerned. The presence of E. coli in the in the salad will be the indicator of the quality level. E. coli is considered as the common surrogate indicator and the presence of faecal content will be the proper indicator. If the total number of E. coli present in the food is substantial, then it can be stated that the salad has no proper cleanliness and has been handled or stored improperly. The third thing to be examined is the “Specific pathogens”. It refers to the bacteria that lead to conditions such as food poisoning. The mechanisms that are involved in the production are the toxins that affect the intestinal function. The impact of the food poisoning varies from the cases to cases. It can be caused by S. aureus (symptoms are vomiting and nausea) or Salmonella spp. and Campylobacter spp. (symptoms are diarrhoea and dehydration) which leads to paralysis and sometimes death.
There are two possible methods that have been considered. They are direct methods and indirect methods. Mentioned below are the explanations of these methods.
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Total Population Count - Direct Methods: This process can be conducted manually by observation of cells in a specialised counting chamber slide viewed under the phase contrast microscopy. The process can also be conducted in an electronic machine depending on the availability of the same in the lab. The value that will be collected from these methods will be reported as the total bacteria per millilitre. The problem that has been associated with the techniques is that bacteria that are too small are difficult to count and is difficult to see with phase contrast. There are other issues where the bacteria clump together and make the counting difficult. And it can be counted as one bacterium in spite of the presence of many of them. However, one of the areas that are important is that it counts even the dead bacteria that are present in the sample. And it is tried that the process is conducted as soon as possible so that it does not give way to increase in the bacteria count.
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Total Population Count – Indirect Methods: This process focus on the turbidity of a culture quantitatively with a spectrophotometer or qualitatively against a known turbidity standard such as McFarland’s. The measure that has been selected for this study is the McFarland’s method. This procedure of population counting is used to standardize the bacterial inoculum for antimicrobial sensitivity testing. The increase in turbidity reflects the increase in the number of the bacteria. As the focus will be to look at the actively growing culture in exponential (log) growth, then the turbidity of the culture is a good estimate of the viable population count. However, if the culture has entered in the lag phase, then the total counts have to carefully consider as it will increase significantly.
C. Identify and characterize the organisms you would test for, with your rationale for their inclusion and an indication of when you would test.
The organisms that would be tested and the rationale behind the inclusion of the same have been detailed below:
E. coli: This bacterium is found in the lower intestine of the warm blooded organisms (Doyle, 1991). Most of them are found to be harmless, however, few of them leads to food poisoning to the hosts. If it is present in edible foods then the company needs to call back the product from customer sale as it loses its fitness for consumption. The E. coli is usually brought into division by serotype. The division depends on the major antigens which forms the part of the lipopolysaccharide layer (Gallagher et al, 2003). The evolution of E coli takes place through the normal process without any requirement of special condition. Virulent strains leads to certain diseases in human beings. It leads major cramps in the abdomen and also to diarrhoea which some cases becomes bloody under the 24 hour period. It is considered as major source of production of proteins. The growth of the E coli are found to be identical at the 5.5 or be it 7.5. It starts to decrease when the pH value decreases. The suitable range that has been identified for the development of the E coli has been identified as 4.0 to 4.5. The metric of growth that has been identified here is related to the dependency of the interaction level of the pH with the level or metrics of the growth of E coli (Rhee et al, 2003). For instance, if the level of stress increases then it might lead to the growth in pH which might eventually impact the level of the E coli (Leyer et al, 1995). Moreover, the formation of acid or any deviation leads to the growth or fall in the number of E coli depending on the various conditions. The decrease in pH value normally leads to the fall in the level of E coli. The acidic content of the salad if left for a while might increase which in turn will lead in increased number of E coli content which harm more comparatively.
V. Cholerae: Vibrio Cholerae has been identified as bacteria that lead to the disease called cholera. Cholera most often leads to death or severe issues if not detected and treated well in time (Goulet et al, 2001). The symptoms associated with the cholera are almost similar to that of food poisoning. If the user consumes food or water that contains this bacterium then the user might get ill, beginning with gastro issues. The shape of this bacterium is like a comma with a small unique tail (Candrian, 1995). The bacteria have been identified as the direct reason for the cholera in the people. It is from the Vibrio genus those are identified to cause issues related to food. The habitat of the bacteria is the contaminated water. The entry of such bacteria to the human body takes place by consuming food that is infected with the same (Anonymous, 1998). The bacteria that survive the acids present in the stomach transfers itself to the intestine and leads to some serious gastrointestinal issues. The use of PCR (polymerase chain reaction) has been suggested to identify such bacteria. The PCR methods depend on the extraction of DNA from the contaminated foods (Bieche et al, 1999). The method requires the researcher to modify the food matrix that is being tested and expects few extra hours while the process continues.
Salmonella sp.: These bacteria are closely associated to food poisoning in human beings. All the group of the Salmonella species belongs to the Salmonella genus. The bacteria impact the intestines of the humans. There are three major serovars of Salmonella. They are: Typhimurium, Typhi and Enteritidis. The first type of bacterium leads to typhoid fever. It leads to fatal diseases most of the time. The symptoms that succeed form the entry of this bacterium are nausea, fever and even death. The second type of bacterium is the common cause of food poisoning which will be main concern of this paper as all the study orientation of the paper is towards the food poisoning in general. This bacterium, however, has been considered less or not fatal than the S Typhi. The symptoms that follow this bacterium are diarrhoea, vomiting, cramps in the abdomen which usually takes 6-7 days to be cured. However, if the age of the person is beyond sixties and if their immune system is not strong, then this bacterium might lead to fall in the health to death. The third type of the bacterium has most resemblance to the second type as per the diseases are concerned.
D. Produce outline microbiological laboratory procedures for three of the organisms you have selected, including a pathogen, a quality indicator and an indicator of poor hygiene practice.
This section outlines the laboratory procedures that are required to test the organisms explained above along with a pathogen. The method that has been adopted is that of the PCR which focus on ruling out the presence of any negative samples in the salad and then identifying the present Ecoli in it. The duration that has been identified for the same is three days and the screening method for the same has been stated to use modified form of Buffered Peptone Water. It will be added with pyruvate that contains anti-microbial reagents. The test will be focused on the samples to give real time result while screening the sample through the PCR screening procedure. The equipment that will be used to conduct the test are mentioned below:
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- Sterile glass jars
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- Micro centrifuge tubes
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- Vortex mixer
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- Sterile tongs
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- Latex gloves
The reagents or the media that will be applied to conduct the test will be the modified buffered peptone water. Moreover, the real time PCR will include some additional reagents such as OmniMix-HS. Along with that, the use of Tellurite Cefixime has been considered for the test.
The sample preparation includes two steps. One is transferring the sample salad into the indicated jar and then weighing the same and then mixing the sample using stomacher at the constant speed for around 2 minutes. Then the sample will be incubated for enrichment.
The next step considers the use of real time screening PCR. It also focuses on collecting the data on real time basis. The paper suggests validation of the platforms that need to be used while enriching the culture.
There are two steps that are considered for the test. One is preparing DNA template and the other is PCR Controls. Under the DNA template preparation there are several steps that can be taken to move the activity ahead. The initial step is to transfer 1gm of enriched sample to micro centrifuge tube and the centrifuging the same for three minutes approximately. The next step that succeeds is the removal of the supernatant from the sample. The next step stated earlier was PCR Controls where the positive control achieved considers the template that has been prepared from E coli. However, if there is no presence of any internal amplification control then incorporation of a reaction tube is suggested.
For the samples that are placed for overnight enrichment should be found positive after the assessment in the real time PCR. It requires confirmation on the cultural front. However, the samples those will not be screened through PCR, the five step procedure can be conducted. In this procedure, the initial step is to dilute the sample that has been kept and then incubating the plate at 37 degree centigrade. The next step of screening consists of the colonies by taking the portion of each colony.
The detection of Salmonella and the pathogens follow almost similar procedure that considers preparation of test portion and then isolation of the organism followed.
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Mossel, David Alexander Antonius, et al. Essentials of the microbiology of foods: a textbook for advanced studies. John Wiley & Sons, 1995.
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Christison, C. A., D. Lindsay, and A. Von Holy. "Microbiological survey of ready-to-eat foods and associated preparation surfaces in retail delicatessens, Johannesburg, South Africa." Food Control 19.7 (2008): 727-733.
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Berrada, H., Soriano, J. M., Pico, Y., & Manes, J. (2006). Quantification of Listeria monocytogenes in salads by real time quantitative PCR. International journal of food microbiology, 107(2), 202-206.
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Anonymous, 1998. EN ISO 11290-2. Microbiology of food and animal feeding
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stuffs—Horizontal method for the detection and enumeration of Listeria
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monocytogenes: Part 2. Enumeration. International Organisation for
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Standardisation, Geneva.
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Bieche, I., Laurendean, I., Tozlu, S., Olivi, M., Vidaud, D., Liderau, R., Vidaud,
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M., 1999. Quantitation of MYC gene expression in sporadic breast tumors
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with a real-time reverse transcription-PCR assay. Cancer Research 59,
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2759–2765.
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Rhee, M.S., Lee, S.Y., Dougherty, R.H., Kang, D.H., 2003. Antimicrobial
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effects of mustard flour and acetic acid against Escherichia coli O157:H7,
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Listeria monocytogenes, and Salmonella enterica serovar Typhimurium.
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Applied and Environmental Microbiology 69, 2959–2963.
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Gallagher, L., Ebel, E.D., Krause, J.R., 2003. Draft FSIS Risk Assessment for
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Listeria. Ready-to-eat meat and poultry products. Food Safety and
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Inspection Service, Washington D.C.
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Goulet, V., de Valk, H., Pierre, O., Stainer, F., Rocourt, J., Vaillant, V., Jacquet,
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C., Desenclos, J.C., 2001. Effect of prevention measures on incidence of
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human listeriosis, France, 1987–1997. Emerging Infectious Diseases 7,
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Doyle, M. P. (1991). Escherichia coli O157: H7 and its significance in foods. International journal of food microbiology, 12(4), 289-301.
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Leyer, G. J., Wang, L. L., & Johnson, E. A. (1995). Acid adaptation of Escherichia coli O157: H7 increases survival in acidic foods. Applied and Environmental Microbiology, 61(10), 3752-3755.
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Candrian, U. (1995). Polymerase chain reaction in food microbiology. Journal of Microbiological Methods, 23(1), 89-103.
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