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Insulin and Erythropoietin Production

Insulin is a protein (polypeptide) discovered in 1921 by Banting with the pancreas being the site of its production. It is made up of 51 amino acids, divided into 2 chains; A and B, bonded by disulfide linkages. Chain A is made up of 21 amino acids with an intra-disulphide linkage, while chain B is made up of 30 amino acids (4).
Why Insulin? Insulin is important in glucose metabolism, and is being used for the treatment of Diabetes mellitus; a metabolic disorder of glucose in the body. Initially, Insulin from animals was used to treat this disorder however nowadays synthesized human Insulin is being used, this is because; it is fast absorbed by the body, it has less allergic reactions, it contains less impurities, and it produces good results (3).
Recombinant process of producing Insulin Synthetic Insulin was first produced in 1983 through genetic Engineering, which involve extraction of the human DNA (1), once extracted, the gene for Insulin is isolated, and enzymes are used to cut it. The gene is then cut using enzymes and put into the plasmid of a vector, where in most cases E. coli plasmid is used. Since Insulin contains two chains, two pieces of DNA are extracted, and the genes for the two chains are linked to ? galactosidase enzyme of the bacteria. The plasmids formed are then inserted into a host cell E. coli and sealed using another enzyme called ligase. And the host on replicating produces the enzymes each containing one of the two chains each. Production is followed by extracting and purifying the chains which are mixed in a reaction to reconstitute the disulphide bridges (1).
ESCHERICHIA COLI AS RECOMBINANT INSULIN HOST Entero-bacillus, gram-negative E. coli is about 1 – 2?m, it can survive in the presence/absence of oxygen, and it also grows in an optimum pH and temperature of 7.0 and 37oC respectively. It utilizes glucose as its major carbon source and can also use other carbon sources like pyruvate, glycerol, acetate, and other sugars. K-12 and B strains are mostly used in the laboratory (20)
Reasons for choosing E. coli Genetic Engineering technologies were developed using E. coli as a role organism, and so, the genetics of E. coli are well known among other microorganisms, as such it’s the most used organism for the production of different proteins (14).
Moreover E. coli has a well known safety and production abilities, stable plasmid, controllable promoter, cheaper and easily cultured (6), E. coli also has fast growth rate, it’s easy to handle, and has well known fermentation skills and the ability to produce high protein content (14). That is why most of the proteins licensed recently by FDA and EMEA, were produced in E. coli (5).
With these, and the fact that Insulin is a simple polypeptide (protein) which does not require glycosylation for its bioactivity and stability, E. coli carrying the plasmids for production of insulin will be used as the host for the production of Insulin
Strain and plasmids: BL21 strain containing the pMYW-A and pMYW-B plasmids and temperature repressor ?-c1857, will be used for insulin production (21).
Growth strategy The various growth strategies that will be used to grow E. coli in order to make it happy and produce the desired product (11) include:
Medium: E. coli needs nutrients like carbon, nitrogen and others; thus a carbon source; glycerol will be provided since it’s cheaper and more soluble than glucose (12), a source of nitrogen in the form of ammonium sulphate will also be provided. However such nutrients in large quantities can inhibit the growth of E. coli, as such a defined medium that contain optimum concentrations 20gl-1 glycerol and 2gl-1 ammonium sulphate will be used (11). The medium will also consist of the following; 3gl-1 KH2PO4, 1gl-1 MgSO4.7H2O, 0.8gl-1 citrate, and 6gl-1 K2HPO4 (23). Some trace elements will also be added to the medium. (23)
Process and culture-strategies: E. coli will be grown submerged in a sterile controlled stirred tank reactor, and fed-batch will be used as the growth strategy so as to avoid accumulation of acetate which can be inhibits its growth, and reduce the production of the insulin (18). The growth strategy will be divided into two; initially batch mode will be used to initiate growth, after which the fed-batch exponential feeding will be used to produce the insulin (21).
After adapting the medium and feeding method, oxygen transfer rates (OTRs) had to be increased through a suitable bioreactor design and over-head pressure (16). Large scale reactors usually reach high ORTs using air and normal aeration pressure, and so the oxygen partial pressure (pO2) will be increased by adding pure oxygen to the air-stream entering the reactor, thus increasing its oxygen transfer rates (16) DO will be maintained at 40% of air saturation and aeration rate at 1vvm. Foaming arising due to large number of cells and high aeration-rates will be solved by use of impellers for stirring simultaneously at 300rpm and the use of antifoam (ucolub N115) (16, 21). The process temperature and pH will be maintained at 30oC and 6.8 respectively so as to avoid partial proteolysis of the insulin protein.
Bioreactor Design: Bioreactor vessel is usually cylindrical and made up of stainless steel. It is composed of impeller for stirring, Air sparger is placed at the bottom of the vessel for introduction of air, it has some inlets for introduction of acid/alkali for pH control and also for introduction of antifoams, nutrients and inoculum; It is also has pH, DO and temperature probes for sensing (22), Microbial activity during fermentation usually produces heat, so the bioreactor design must allow for removal of heat, and this can be achieved by cooling with jackets and coils (16) Bioreactors must also be designed in a way that it can withstand high temperature and pressure and to allow cleaning-up and sterilizing (22).

Growth analysis Temperature, pH, DO, foam, partial oxygen and carbon dioxide pressures, will be analysed on-line, other parameters like biomass, will be analysed by using optical density (OD600) and dry cell weight (offline). Cell viability will be analysed by using flow cytometry, the concentrations of substrates and metabolites by enzymatic methods while insulin will be analysed using electrophoresis methods like SDS-PAGE, and ELISA, while its purity will be determined by HPLC (8).
Limitations/Problems There are several problems that may arise during processing and can limit the use of this organism for Insulin production, these are;
Poor secretion because of the structure of its membrane (and tough cell wall), small amount of foldases, chaperones and increased concentrations of proteases, leading to low productivity (7).
Solutions to this problem include all measures taken to increase quality of secretion and production such as:
Use of secretion systems like the system of ?-haemolysin (7)
co-expression after co-cloning of foldases and chaperones (13)
Improving the rates of gene-expression and using proteases deficient mutants like BL21 (18).
use of E. coli mutants that are deficient of cell-wall (12)
Limited post translational-modifications; including disulfide-linkage formation, which is important for the insulin stability and biological activity (9).
Solutions to this problem include;
Production of insulin with altered amino acid sequences through genetic engineering (9)
Using E. coli mutants to enhance the formation of disulfide linkages e.g. Origami (15)
iii. Exporting proteins into the periplasm which has disulphide bonding mechanisms (19).
Codon biases; due to large quantities of exact transfer-RNAs found in E. coli, the codons in the human-genes are often different from those that are found in this organism. This results in inefficient expression of some of these rare codons by the organism resulting in an unexpected protein synthesis termination or wrong incorporation of the amino acids (12).
This problem can be solved by replacing codons that are rare in the desired gene by codons that are often found in the E. coli and by co-expressing the rare transfer-RNAs (15).
Acetate is usually formed as a by-product, and is inhibitory to growth of the cells (20).
Solution is by using a fed-batch feeding method and by limiting DO level (11).
Another problem is that large proteins are often obtained in an insoluble form (5); forming aggregates called inclusion bodies; IBs (20).
This can be solved by adjustment of temperature, increasing the strength of the promoter, adjusting the number of plasmids, concentrations of the inducer, and the composition of the media (9).
Erythropoietin EPO EPO is a glycoprotein that is produced in the renal cortex of the kidney (10, 11). It has also being shown to be present in the brain, spleen, liver and the lungs (7, 17). It is made up of 165 amino acids of about 18kDa (25), with a number of carbohydrates linked to the polypeptide through O and N glycosidic-bonds giving the glycoprotein a total weight of 34kDa.Two disulphide linkages hold the molecule together (15) and the carbohydrates are responsible for the stability of the glycoprotein in-vivo,and increasing its half-life in the body (24).
Why EPO? EPO functions to regulate the amount of red blood cells (RBC) in the blood by controlling the proliferation and differentiation of its immature cells to mature cells (1, 2, 22,). It is also involved in the growth and formation of blood vessels, and healing of wounds (6), it functions in the brain is not clear, but studies showed the glycoprotein to have some protective effects (18). Because of these functions EPO has being used in the treatment of anaemia caused by kidney failure and other causes (25).
Recombinant production of EPO Despite its importance, EPO in body is found in very small amounts and mostly in the urine (4), as such there is the requirement to produce EPO in large amounts, this leads to the work of isolating the glycoprotein from the urine (12, 21), and was used to identify it’s amino acid sequences, and synthesis of its DNA (9, 12), furthermore the human erythropoietin genes were cloned by Lin et al. (17), and consequently recombinant human EPO (rhuEPO) was produced in 1985 using CHO cells (14, 16).
Chinese -Hamster- Ovary (CHO-Cells) as rhuEPO host: These are epithelial cells derived from the ovary of Chinese hamster (a mammal). They grow well in culture and looks like cobble stones. The cells usually attach to a surface available but can be grown in suspension (20).
CHO cells are grown best at 37oC and at pH 7.4; they are cultured in a suitable complex medium which can support their growth for many generations (20).
CHO cell lines are now available from cell culture collections like the American type culture collection; ATCC. Moreover human EPO expression plasmids are now also commercially available, and are usually used for production of EPO using the CHO cells (27).
Reasons for choosing CHO-cells Karthik et al. (13) showed that CHO-cells are being used extensively in the industries for the production of many proteins, because they have demonstrated, to possess some qualities like:
They can modify biological products post-translationally; Proteins produce in CHO-cells have high glycosylation quality making them compatible and stable (13)
Safety of the product; Studies in 1989 have shown that most viruses do not multiply in CHO-cells (13)
Ability to adapt easily and be grown in suspension (13).
Products can now be purified to contain less contaminant (13).
CHO cells have being used for a long time; as such much data has being accumulated for regulatory reasons (13).
They are easy to manipulate genetically (13).
The isolation of cells deficient in Dihydrofolate-reductase enzymes leads to stable clones’ selection and genes amplification to increase production (13).
With all these, and the fact that EPO is a glycoprotein that requires glycosylation for its stability and activity, recombinant CHO cells are chosen to produce EPO.
Cell lines and plasmids: Cell lines which have the capability of glycosylating proteins (Pro-5), harboring the pGEX-HET-puro expression plasmid, will be used to produce the recombinant human erythropoietin (27).
Growth strategy Medium: Complex culture medium will be provided with;
Glucose as a source of carbon and energy,
Amino acids as source of nitrogen,
Salts will be included to make the solution isotonic
Vitamins and hormones will be added as co-factors
Serum is usually added to the culture medium to enhance the growth of the cell (20), but has the following disadvantages:
It chemicals are not defined and can cause cell growth inconsistency between batches (20)
It is very expensive (20)
The serum may contain proteins which can be difficult to separate and purify from the proteins secreted by the cells during downstream processing (20)
It increases foaming and can be a source of contamination by viruses. (20)
Therefore a serum-free (SF) media (16) will be used for the growth of the E. coli.
Process and culture-strategies: The cells will be grown adherent on micro-carriers in a sterile controlled packed bed reactor, and perfusion method of production where some amounts of the medium is removed and replaced by fresh one and the cells are grown slowly will be used (28); because it was found to improve the glycosylation of the proteins more than fed-batch where there is fast growth of cells, (8). Before, many processes were run in a simple batch method, but nowadays, Perfusion or fed-batch methods are mostly employed and higher products are now realized (22). The production will be carried out in two stages; the growth stage and the production stage. Normally stirring will be kept at 100 to 150 rpm, foaming will be avoided by adding Pluronic F68 (16).Temperature will be maintained at 37oC initially during growth and then reduced to 33oC during production, as was shown to increase the overall protein production, while maintaining the quality of the glycoprotein (3, 26). pH will be kept at 7.1 initially and then reduced to 6.8 (8, 26), by passing CO2 gas to the culture or by addition of concentrated sodium-bicarbonate solution in low quantities, because CO2 is also toxic to the cells and can also affect the production of EPO (20). In order to avoid the depletion of oxygen, the oxygen transfer rates (OTRs) will be increased above its utilization rate, with a constant supply of pure oxygen and air, while DO will be maintained at 20-50% of air saturation (20).
Bioreactor Design: Since the cells are big and fragile, the design of the bioreactor has to be considered. Mammalian cell culture bioreactors are designed with bottoms that are round and are usually made up of glass/stainless steel (20). The impellers are usually marine or pitched blade types fitted at the end of mechanical drives shafts so that both vertical and horizontal mixing are allowed at low stirring-rates (20). Temperature is controlled through coiled pipes or open ended fermenter jacket (20). pH, DO and temperature probes are used for sensing and have both air inlet and outlet for respiration.
Growth Analysis Temperature, pH and DO will be monitored on-line, because cells are immobilized, biomass formed cannot be measured directly therefore it will be monitored by measuring rate of glucose consumed daily and the rate of lactate produced (28) Cell viability by flow cytometry, Glucose, glutamine, and lactate concentrations will be analysed using multi-parameter Bio-analytical system (26); while ammonia formed as waste product of amino acid metabolism, will be analysed by colorimetric assay and by the use of detection-kit (26). EPO formed will be analysed using HPLC to determine its purity and its quality by Isoelectric focusing, SDS, and Bradford assay (26). The activity of EPO will be analysed by bioassay and by the use of protein assay-kit (27)
Limitations/Problems. There are many limitations associated with CHO cells culture processes and they include;
They are fragile and highly sensitive to shear stress caused by agitation and bubble because the cells are large and have only cell membrane (20).
This is usually solved using a suitable bioreactor-design and use of Pluronic F68 (20).
They need a complex medium including serum which can cause problems in the downstream processing and is expensive (20).
Solution to this is by using serum- free media (24, 25).
Low yield of proteins have been produced from these cells, the productivity using the microbes being higher than the use of these cells. They also have slow growth rates (13).
The problem of low productivity and slow growth rates can be solved through selecting cell lines that are better and optimizing cultural-strategies.
Ammonia and lactate are generated during growth and can inhibit growth and also affect glycosylation (8).
Solution is by optimizing the strategies of feeding and by monitoring (8).
Glycosylation differences may arise from the EPO produced in the CHO-cells and the human EPO as seen in the way the two are sialylated terminally, as a result that the CHO-cells are not able to express an enzyme called alpha-2,6, sialyltransferase (27).
Solution is by the use of CHO-cells harboring alpha-2, 6, sialyltransferase-cDNA expression-cassettes (27).
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8. Hewitt C.J., Nebe-von Caron G., Axelsson B., McFarlane C.M, Nienow A.W Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multi-parameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration. Biotech. Bioeng. 70: 381-390. 2000
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13. Kujau, M.J., Hoischen, C., Riesenberg, D., Gumpert, J. Expression and secretion of functional mini-antibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and E. coli: a comparison of the synthesis capacities of L-form strains with E. coli producer strain. Appl. Microbiol. Biotech. 49: 51-58. 1998.
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Prevention Strategies for Escherichia Coli

a) As the snack food is made from meat, there are four types of bacteria that will possibly contaminate it which are Escherichia.coli, Salmonella, Staphylococcus aureus,and Listeria monocytogenes (FSIS, 2015).
Escherichia.coli is a bacterial that is usually present in the intestines of humans and animals. Although, most of them are harmless as they play an important role in the intestinal tract of a healthy individual. However, there are a few of them that are pathogenic as they can result in diarrhea and can be transmitted through the contact of human or animalfaeces. Altogether, there are sixpathotypes that are linked with diarrhea which are as follows Shiga toxin-producing Escherichia.coli (STEC), Enterotoxigenic Escherichia.coli (ETEC), Enteropathogenic Escherichia.coli (EPEC), Enteroaggregative Escherichia.coli (EAEC), Enteroinvasive Escherichia.coli (EIEC), Diffusely adherent Escherichia.coli (DAEC) (CDC, 2015).
However, the Escherichia.coliO157 strain, which is classified under Shiga toxin-producing Escherichia.coli (STEC), is considered the most harmful as it causes bloody diarrhea, kidney failure and can result indeath (Anon., 2015).
It infects its host by producing large amounts of Shiga toxins that damages the intestinal lining severely to cause Hemorrhagic Colitis or otherwise known as bloody diarrhea. It can also cause Hemolytic uremic syndrome which results in kidney injury (Koo, 2014).
Salmonellais a common bacterial that usually cause food poisoning which targets the stomach and intestines. It is normally present in meat, eggs, unprocessed milk and poultry (Bech, 2014).
The symptoms are diarrhea, nausea, fever, stomach cramps, and vomiting which usually last for 4 to 7 days. The incubation period is between 12 and 72 hours upon infection (England, 2014).
However, one must consume Salmonella contaminated food in order to cause infection (FSIS, 2015).
Staphylococcus aureusis a common bacteria found on the skin, nasal passages, throats and hair of healthy individuals.Staphylococcalfood contamination can occur when a person who is infected with the bacteria handles the steakwithout proper sanitary handling procedures. Other possible sources of meat contamination of Staphylococcus aureus can also take place such as the surfaces and equipment that the meat is prepared on (Anon., 2015).
Staphylococcal food poisoning happens when an individual ingest the enterotoxins that are produced in the contaminated meat, often due to the improper heating up of the meat to kill off most of the bacteria (USFDA, 2014).
Listeria monocytogenesis a bacterium that is usually found in soil, water, uncooked and processed meat, cooked or processed food, smoked salmon, vegetables and dairy products (CDC, 2013).
Ingestion of food contaminated with this bacterium can cause Listeriosis which is a serious infection (CDC, 2013).
AlthoughListeria monocytogenescan be killed by proper cooking, the bacteria can still contaminate the cooked meat due to physical hazards such as poor food handling practices and sanitation (FSIS, 2015).
Another potential food safety hazard that I have identify is the use of herbs and spices to marinate the steak. Herbs come from plants and spices are from seeds, bark or roots of plants (Ward, 2010).
Because they are from plants, which contains spore-producing bacteria, this indicates that the bacteria spores is capable of further contaminating raw meat which is in contact with soil and vegetative origin.This would introduce additional microbiological hazards such as Bacillus spp. and spore forming organisms which are able to survive the drying process and continue to germinate to produce toxins.
During the process of handling with this dried snack food, there should be a food safety management system such as The Hazard Analysis and Critical Control Point (HACCP) system. This system is used worldwide by all food businessesto identify and control microbiology activity as well as physical and chemical hazards that might be present during production. It consists of seven principles that govern and maintain food hygiene procedures (ASQ Food, 2014).
Therefore, I would advise her to take note of the possible food safety hazards that have been mentioned and the procedures of manufacturing this dried snack food has to be in compliance to the Hazard Analysis and Critical Control Point (HACCP) system.
b) There are several information that I would like to know as to assist the businesswoman in preparing the dried snack food safely for consumers. The following are the information required:
Where was the meat from?
Normally, the cattle are raised till maturity in a farm where they are given formulated food. During cattle raising, the cattle are injected with antibiotics to prevent diseases. In order to slaughter the cattle legally, a “withdrawal” period is needed from the time the antibiotics is given. This is to give sufficient amount of time for the antibiotic residues to be released from the cattle.
The United States Department of Agriculture Food Safety and Inspection Service (FSIS) would conduct sample testing for antibiotic residues upon slaughtering. Results have shown that the percentages of residue violations are very low and there are no traceable antibiotic residues present in the tissues of the cattle (FSA, 2015).
There are several factors that have to be taken into consideration such as the conditions of the farms that the cattle are living and the food that they consume. For example, improper drainage of faeces, design of feeders and drinkers can contribute to the accumulation of bacterial contamination in the farm which leads to the dirty cattle. The cattle are then infected with diseases such as pneumonia, coccidiosis and salmonellosis. Thus, it is important to have a healthy livestock of cattle to avoid all these problems.
There are two factors that can contribute to the contamination of meat production chain such as direct and indirect contact of contamination. Direct contact can occur when bacteria that are present on the skin of the cattle such as excrement and mud which can contaminate the meat during the slaughtering process. Whereas, indirect contact could occur when the hands, tools and equipment that are used to slaughter the cattle contains bacteria which will then infect the meat (FSA, 2002).
How was the uncooked meat handled?
For proper handling of raw beef, place them in a sealed disposable plastic bag. You can either freeze it at -17.8°C so as to keep it longer (about 6 to 12 months)or refrigerate at 4.4°C but have to consume it within 3 to 5 days (Safety, 2015). The reason why the meat is to be kept at cold temperatures is because this is to inhibit bacterial growth.
Time, temperature and conditions of marinating and cooking the meat?
First, the raw steak is marinated with herbs and spices which will introduce additional microbial hazard such asspore forming bacteria to the meat. Thus, for safe marinating procedures, do not marinate at room temperature as most bacteria are able to grow quickly. Instead, marinate the meat in a cold environment so as to avoid entering the danger zone, which is between 4°C and 60°C. For beef steak, the approximate time taken to marinate would be 2 to 4 hours (Stradley, 2014).
For safe cooking of steak, cook them at a minimum internal temperature of 62.8°C for about 4 – 5 minutes per side and allow the meat to rest for at least 3 minutes before eating. This is done to kill off most of the bacteria that might be present on the meat. However, do not cook the steak partially as this does not destroy most of the bacteria and might multiply (USDA, 2015).
Time and temperature of the drying process?
I would suggest drying the marinated steak strips using an oven at 75°C as there is a constant circulation of dry air and heat source. The temperature is important in drying this snack food because gentle heatingwill not be sufficient as this does not ensure that any bacteria that might still be present on the meat prior to cooking are killed (USDA, 2015).
For example, bacteria such as Bacillus flavother musT have a maximum temperature cardinal value of 72°C and if the meat is cooked to the temperature of 60°C, the bacteria will still be able to survive (Todar, 2008 – 2012).
Thus, they will continue to multiply in numbers and colonize the meat.
Storage of final dried meat product? How long is the meat product stored?
Always ensure to store dried meat in a vacuum packaging as this will minimize oxygen levels which inhibit the growth of aerobic bacteria. It also helps to reduce the oxidation of the meat which will result in meat spoilage. These will extent the shelf life of the snack food (Anon., 2012).
A sealed dried meat product can be stored to a period of 12 months. However, if the dried meat product is made from home, then it can only be kept for about 1 to 2 months (USDA, 2013).
Is water activity taken into consideration?
Water is a requirement for bacteria growth, thus, it must be removed so as to inhibit the growth of microorganisms. It can be reduced by the drying process and the addition of salt to preserve the meat. How the salt works is that it would absorb all the water on the meat by the process called osmosis (Martin R. Adams and Maurice O. Moss, 2008).
Are there hygiene practices done in the preparation of the dried snack food?
To prevent cross-contamination, good hygiene practices and procedures are required in regards to handling the snack food. Food handlers should always wash their hands thoroughly with surfactants such as soap before and after handling with meat products. Always clean and disinfect all utensils, equipment and work surfaces before the preparation of the meat product. And do not mix raw and cooked food. Instead, incorporate the use of separate working and storage areas of raw, cooked and dried meat products (FSA, 2015).
c) I would suggest the use of aerobic plate count and immunomagnetic separation to test the presence of Salmonella spp. and Escherichia.coli O157
In aerobic plate count, dilutions of the food sample are carried out and are then plated onto agar plates which contain suitable growth nutrients to favor the growth of microorganisms. The plates are then incubated at temperatures that bacteria can grow. After the incubation period, the total numbers of bacterial colonies arecounted; therefore, the total number of viable cells can be estimated (Hayes, 1995).
Thelevels of Aerobic Colony Count (ACC) are used as a guidance to determine if the food product is safe for consumption (Agency, 2009). For example, if the Aerobic Colony Count (ACC) levels of bacteria have exceeded the expected level, the product cannot be consumed.
For Salmonella spp., the infectious dose is normally 105 organisms, which is quite a big number. Whereas, as for Escherichia.coli O157, the infectious dose isless than 50 organisms (Inc., 2004).
For Escherichia.coli O157 and Salmonella, Immunomagnetic separation is used as the “gold standard” to detect and isolate the bacteria as it is specific and sensitive. Then targeted organism is then isolated which will undergo serotyping for characterization (Inc, 2004).
According to the guidelines for the microbiological safety of ready-to-eat foods, they are to be cooked and processed properly, especially with the addition of herbs and spices. It also includes bacteria that can possibly be from the source of poor hygiene. Lastly, tests on ready-to-eat foods are required so that food products which are not in compliance with the microbiological food safety criteria can be identified to protect public health (Agency, 2009).
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