EIA

EIA also known as Enzyme -Linked ImmunoSorbent Assay function based on the detection and measurement of primary antibody-antigen binding reaction.

Relevant antibody bound to a solid phrase (polystyrene microtiter plates), heat-treated broth culture (from food) is then added to the well. Unbound antigen washed away; antibody has a specific shape, it will only bind to one particular kind of antigen due to its key-and-lock principle. And only when antigens are bounded to the antibody on the polystyrene microtiter plates, will there be positive results. After the antigen is immobilized the enzyme labelled antibody (conjugate), sometimes known as detection antibody is then added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. And incubated, after incubation, unbound conjugate washed away. Specific substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic signal is subsequently added to show the reaction via enzymatic reactions. Followed by incubation at room temperature; to allow enzymatic reactions to take place. Reaction ceased by addition of sulfuric acid. Results read through photometer or other optical devices at specific wavelength.
ELISA tests can be categorized into indirect, sandwich or capture. Indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample, whether it is from the serum of an immunized animal or the cell supernatant from growing hybridoma clones. Sandwich ELISA is used to determine the antigen concentration in unknown samples. Competitive ELISA is used when two “matched pair” antibodies are not available for experiment target. Different ELISA tests have different procedures however, the principles behind these tests are similar.

DNA Probe

Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all living organisms. Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from DNA. As DNA and RNA of organisms are specifically different between each other, by identifying the DNA and RNA of organisms can then be used to isolate and identify. Foodborne pathogen can be detected when DNA or RNA of the food samples are complementary to the nucleotide sequence in the probe. If DNA or RNA of food samples match that of the DNA probe, a complex is formed and thus a positive results.

New DNA probe assays for detection of Salmonella, Listeria, E. coli and Staphyococcus aureus use non-isotopically labelled DNA probe to detect specific ribosomal RNA targets of organisms. Test samples will first undergo lysis, using alkaline or enzymatic reagents which cause the cells to burst and die. Probes are then added to the test samples to form probe-target complexes; in solution form. These complexes formed and hybridized will be captured onto polystyrene sticks to separate the desired components from the unwanted cellular and sample debris. Hybridization is a process of combining complementary, single-stranded nucleic acids into a single molecule. The single molecule thus produced would then be detected after hybridization by its intrinsic properties (e.g., fluorescence) or through recognition by a specific antibody. If detection is by intrinsic properties, the polystyrene sticks will be incubated with solution containing an anti-fluroescein antibody-horseradish peroxidase conjugate and a mixture of tetramethyl benzidine/hydrogen peroxide. After reaction ceased, results can then be obtained by determining the absorbance of samples at a specific wavelength (usually 450nm) using photometer instrument or other optical devices.

HPLC - Principles

The basic operating principle of HPLC is to force the analyte through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.

GC/MS

Gas Chromatograph/Mass Spectrometer is also known informally as GC/MS.

A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are many types of mass spectrometers and sample introduction techniques which allow a wide range of analyses. This discussion will focus on mass spectrometry as it's used in the powerful and widely used method of coupling Gas Chromatography (GC) with Mass Spectrometry (MS).

Mass Spectrometer

MS measures the mass-to-change ratio (m/z) of ions that have been produced from the sample.


Sample -> Inlet -> ionization source -> analyzer -> Ion detector -> data system

From ionization source to ion detector, it's taken place in vaccum system.

Unionized molecules and fragments are pumped out of the ionization source. Ions are passed into an analyzer where they are separated according to their mass-to-change ratio. Ion strike an ion detector, where they produce an electrical signal that is recorded and plotted by data system.

The stages within the mass spectrometer are:

1. Producing ions from the sample
2. Separating ions of differing masses
3. Detecting the number of ions of each mass produced
4. Collecting the data and generating the mass spectrum

The technique has several applications, including:

* identifying unknown compounds by the mass of the compound molecules or their fragments
* determining the isotopic composition of elements in a compound
* determining the structure of a compound by observing its fragmentation
* quantifying the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative)
* studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum)
* determining other physical, chemical, or even biological properties of compounds with a variety of other approaches

Toxin Detection

Toxic Metals
Rapid microwave digestion and acid digestion systems, and analysis using atomic absorption spectrophotometers and inductively coupled plasma-mass spectrometer

Polychlorinated Biphenyls (PCBs)
Gas chromatograph (GC)

Chloropropanols
Gas chromatograph / mass spectrometer (GC/MS)
Acrylamide
Liquid chromatograph / mass spectrometer / mass spectrometer (LC/MS/MS)

antibiotics, growth promotants and other veterinary drugs
Microbial inhibition assay for antibiotics
Enzyme-linked immunosorbent assay for beta-agonists, antibiotics and growth promotants
High performance liquid chromatography or LC/MS/MS for nitrofurans, coccidiostats and other antibiotics

Local Authority on GM Food

GMAC - Genetic Modification Advisory Committee
(Information adapted from www.gmac.gov.sg)

Roles & Responsibility
The Genetic Modification Advisory Committee was established in Singapore in April 1999 to oversee and advise on the research and development, production, use and handling of Genetically Modified Organisms (GMOs) in Singapore.

The objective of this committee was to ensure public safety while allowing for the commercial use of GMOs and GMO-derived products by companies and research institutions, in compliance with international standards.

Flow Chart for Evaluation, Approval and Registration of Genetically Modified Organisms (GMOs) Related to Agriculture
http://gmac.gov.sg/guidelines/agriculture_appendix_3.html

ADI

What is Acceptable Daily Intake?
The Acceptable Daily Intake (ADI) is an estimate by JECFA of the amount of a food additive,expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk(standard man - 60 Kg) (WHO Environmental Health Criteria document N° 70, Principles for the Safety Assessment of food Additives and Contaminants in Food, Geneva, 1987). The ADI is expressed in milligrams of the additive per kilogram of body weight.
For this purpose, "without appreciable risk" is taken to mean the practical certainty that injury will not result even after a life-time's exposure (Report of the 1975 JMPR, TRS 592, WHO, 1976). The ADI is established over lifetime. A body weight of 60 kg is usually taken to represent the average weight of the population (Report of the 1988 JECFA , TRS 776 sec. 2.2.3. WHO, 1989). However, in some countries, and especially in the developing ones, a 50 kg body weight would better represent the average body weight of the population.

How To Establish ADI?
Before discussing different approaches used in estimating food additive intake, the methods of establishing an ADI need to be reviewed.
Groups of animals (e.g. rats) are given daily diets containing different levels of the additive under examination. For example, levels of the additives in the diet could be: 0.1%, 1%, 2%, 5%. If a toxic effect is found at the 2% level and a "no toxic effect" at 1% level, the 1% level (expressed in mg/kg body weight) will be
the "no-observed-effect level", and it is from this level that the extrapolation to humans is done. In this case, the no-observed-effect level lies between the 1% and 2% levels, and if no toxicological evaluations are done at intermediary levels (1.25%, 1.50%, 1.75%) the choice of the 1% level as the no-observed-effect level introduces
already a first safety factor. The extrapolation from the no-observed-effect level to an ADI is often done by using a safety factor of 100 (10 x 10) which assumes that humans are 10 times more sensitive than experimental animals and that there
is a 10-fold variation in sensitivity within the human population. This safety factor of 100 is based on the experience and common sense of toxicologists and therefore cannot be compared to a physical value such a-s the boiling point of a pure substance. More information regarding the no-observed-effect level and the use of safety factors can be found in "Principles for the Safety Assessment of Food Additives and contaminants in Food".(Environmental Health Criteria No 70, WHO, Geneva 1987, p. 77-79).
Estimations of intake may be sequentially calculated starting with the simplest TMDI and proceeding to more refined EDI if necessary. When precise data on consumption of foodstuff exist, they should be used. When such precise data do not exist, approximations can be adequate to support a safe use. A hypothetical figure based upon extreme theoretical cases such as the TMDI can give adequate assurance of safety in use if such figure is lower than the ADI. However, if the ADI is exceeded, using this approach, before a decision is made a search would have to be made for data which approximate the actual intake (the TMDI can be improved by taking into account intake of special population groups).

What is TMDI?
The Theoretical Maximum Daily Intake (TMDI) is calculated by multiplying the average per capita daily food consumption for each foodstuff or food group by the legal maximum use level of the additive established by Codex standards or by national regulations and by summing up the figures.
The TMDI gives only a rough indication of the dietary intake of a food additive since it does not take into consideration the food habits of special populations groups, and it assumes that:
(a) all foods in which an additive is permitted contain that additive;
(b) the additive is always present at the maximum permitted level;
(c) the foods in question containing the additive are consumed by people each day of their lives at the average per capita level;
(d) the additive does not undergo a decrease in level as a result of cooking or processing techniques;
(e) all foods permitted to contain the additive are ingested and nothing is discarded.

Procedures For ELISA

-Relevant antibody bound to a solid phrase (polystyrene microtiter plates)
-Heat-treated broth culture (from food) added to the well
-Unbound antigen washed away
-Enzyme labeled antibody (conjugate) is added
-Incubated
-After incubation, unbound conjugate washed away
-Specific substrate added – to show the reaction via enzymatic reactions
-Incubation at room temperature
-Reaction ceased by addition of sulfuric acid
-Results read through photometer at specific wavelength

Toxins Found In Flour/Starch

Natural Toxins
-Amines and monoamine oxidase inhibitors with chemicals neurotransmitters like tyramine, tryptamine, serotonin, adrenaline, noradrenaline, dopamine
-Proteinase inhibitor like alpha amylase and trypsin inhibitors
-Lectins
-Coeliac sprue (gluten enteropathy)
-Solanum alkloids - esp glycoalkaloid
-Isoflavone glycosides
-Coumestans, lignans

Polycyclic Aromatic Hydrocarbons
-PAHs - present in low level in cereals mostly due to aerial deposition

Heavy Metals
-Cadmium
-Chromium
-Nickel

Pesticides

Mold Toxin
-Citreoviridin
-Ochratoxin A
-Ergot Alkaloids
-Aflatoxins
-Fusaria toxins (Including T2 toxin [type A trichothecene], Diacetoxyscirpenol [type A trichothecene], Deoxynivalenol [type B trichothecene], zearalenone, fumonisins, moniliformin, fasaric acid)

Baterial Toxins
-Bacillus spp.
-Miscellaneous Enterobacteriaceae
-Listeria
-Clostridium botulinum
-Clostridium perfringens

Transgenic Crops

How are Transgenic Crops Made?

For many years plant breeding entailed the selection of the finest plants to get the best crops. In those days, variation occurred through induced mutation or hybridization where two or more plants were crossed. Selection occurred through nature, using a “selection of the fittest” concept, where only the seeds best adapted to that environment succeeded. For example, farmers selected only the biggest seeds with non-shattering seed heads, assuming these to be the best. Today, scientists can not only select, but also create crops by inserting genes to make a seeds bare any trait desired.

In order to make a transgenic crop, there are five main steps: extracting DNA, cloning a gene of interest, designing the gene for plant infiltration, transformation, and finally plant breeding (see Figure 1).



To understand this process, one must first known a bit about DNA (deoxyribonucleic acids). DNA is the universal programming language of all cells and stores their genetic information. It contains thousands of genes, which are discrete segments of DNA that encode the information necessary to produce and assemble specific proteins. All genes require specific regions in order to be utilized (or expressed) by a cell. These regions include (see Figure 2):

1. A promoter region, which signals where a gene begins and it used to express the gene;

2. A termination sequence, which signals the end of a gene;

3. And the coding region, which contains the actual gene to be expressed.

All these regions together allow a gene to create a protein. Once a gene is transcribed into a protein, it can then function as an enzyme to catalyze biochemical reactions or as a structural unit of a cell, both of which will contribute to the appearance of a particular trait in that organism.

All species are capable of turning DNA into protein through a process known as translation. This capability makes it possible to artificially put genes from one organism into another-a process generally termed transgenics. But just isolating random DNA and inserting it into another organism is not practical. We must first know what particular segments of DNA, and in particular what genes, to insert. Unfortunately, with reference to producing new crops, not much is known about which genes are responsible for increased plant yield, tolerance to different stresses and insects, color, or various other plant characteristics. Much of the research in transgenics is now focused on how to identify and sequence genes contributing to these characteristics.

Genes that are determined to contribute to certain traits then need to be obtained in a significant amount before they can be inserted into another organism. In order to obtain the DNA comprising a gene, DNA is first extracted from cells and put into a bacterial plasmid. A plasmid is a molecular biological tool that allows any segment of DNA in be put into a carrier cell (usually a bacterial cell) and replicated to produce more of it. A bacterial cell (i.e. E. coli) that contains a plasmid can put aside and used over and over again to produce copies of the gene the researcher is interested in, a process that is generally referred to as “cloning” the gene. The word “cloning” referring to how many identical copies of the original gene can now be produced at will. Plasmids containing this gene can be used to modify the gene in any way the researcher sees fit, allowing novel effects on the gene trait to be produced (see Figure 1).

Once the gene of interest has been amplified, it is time to introduce it into the plant species we are interested in. The nucleus of the plant cell is the target for the new transgenic DNA. There are many methods of doing this but the two most common methods include the “Gene Gun” and Agrobacterium method.

The “Gene Gun” method, also known as the micro-projectile bombardment method, is most commonly used in species such as corn and rice. As its name implies, this procedure involves high velocity micro-projectiles to deliver DNA into living cells using a gun [1]. It involves sticking DNA to small micro-projectiles and then firing these into a cell. This technique is clean and safe. It enables scientists to transform organized tissue of plant species and has a universal delivery system common to many tissue types from many different species1. It can give rise to un-wanted side effects, such as the gene of interest being rearranged upon entry [1] or the target cell sustaining damage upon bombardment. Nevertheless, it has been quite useful for getting transgenes into organisms when no other options are available.

The Agrobacterium method involves the use of a soil-dwelling bacteria known as Agrobacterium tumefaciens, which has the ability to infect plant cells with a piece of its DNA. The piece of DNA that infects a plant is integrated into a plants chromosome through a tumor-inducing plasmid (Ti plasmid), which can take control of the plant’s cellular machinery and use it to make many copies of its own bacterial DNA. The Ti plasmid is a large circular DNA particle that replicates independently of the bacterial chromosome [1] (see Figure 3).

The importance of this plasmid is that it contains regions of transfer DNA (tDNA), where a researcher can insert a gene, which can be transferred to a plant cell through a process known as a floral dip. A floral dip involves dipping flowering plants into a solution of Agrobacterium carrying the gene of interest, followed by the transgenic seeds being collected directly from the plant [1]. This process is useful in that it is a natural method of transfer and therefore thought of as a more acceptable technique. In addition, Agrobacterium is capable of transferring large fragments of DNA very efficiently without substantial rearrangements, followed by maintaining high stability of the gene that was transferred [1]. One of the biggest limitations of Agrobacterium is that not all important food crops can be infected by this bacteria [1].

http://www.scq.ubc.ca/?p=518

Economic Advantages of GM Foods

Greater Profits, Lower Cost Input because of:
* Resistance to pests or weeds with a lower need for pesticides or herbicides
* Improved characteristics of the plant such as being more nutritious, less sensitive to transport or storage
* Less labour and application of machinery in agriculture
* Enlarged areas cultivated with plants adapted to extreme conditions, floods or droughts.
* The higher cost for seeds and licences are offset by higher yields.
* improved yields with lower need of expensive pesticides
* lower crop losses due to pests and weeds
* less labour and machinery
* plants which are adapted to extreme conditions, floods or droughts.
* Bioremediation is the use of bacteria to clean up contaminated land. Micro-organisms and plants can be modified to enhance their capacity to, for example, absorb heavy metals or radioactive elements and leave the soil safe. Reducing the need to clean up contaminated lands which can be very difficult and costly.

Safety Issues For GM Foods

Allergenicity - The allergenicity potential of the new protein expressed on the transgene inserted into the plant is a major food safety concern. Most traits introduced into GM crops result from the expression of one or more protein that may possess allergenic properties. Crops modified for insect resistance have been shown to have the potential for allergic responses. The allergenicity potential of GM food has often been difficult to establish with existing methods as the transgenes transferred are frequently from sources not eaten before, many have unknown allergenicity or there may be a potential for genetic modification process to result in increase of an allergen already present in the food. If the GM food contains genes, which are known to have a relation to allergenicity, a series of special chemical and immunological tests are required to identify the proteins, which cause the allergenic reaction. These in vitro tests have to be confirmed by in vivo tests. In any case, the new food should be labelled to inform the consumer that traces of an allergenic protein could be included.

Antibiotic Resistance - Potential for Gene transfer: Concern has been expressed on the possibility of transfer of GM DNA from the plant to gut microflora of humans and animals. Of importance have been the antibiotic resistant genes that are frequently used as selection markers, in the genetic modification process. Such genes have the potential to adversely affect the therapeutic efficacy of orally administered antibiotics.

Nutritional composition - Genetic modification of plants may result in alteration in nutritional composition which in turn may affect the nutritional status of the consumer or population groups. Currently developed plants with improved nutritive value include
GM rice with enriched vitamin A and GM soyabean and rapeseed with modified fatty acid. The impact of such intended modification in nutrient level in crop plants can affect nutritional status of the individual. There is also the potential for unexpected alteration in nutrients as it was observed in the case of GM rice (accumulation of xanthophylls, increase in prolamines). Such changes can affect nutrient profiles resulting in nutritional imbalances in the consumer.

Levels of antinutrients in GM Food
An antinutrient is a substance that interferes the uptake of nutrients. Antinutrients found in conventional foods include trypsin inhibitors and phytic acid. High levels of trypsin inhibitors are often found in raw cereals and legumes, especially soybean. Trypsin is an enzyme produced in the gut and digests proteins. Trypsin inhibitors interfere with the action of trypsin, affecting the digestion of proteins in the gut and subsequent absorption of nutrients, resulting in malnutrition. They are easily inactivated by heat and so may not be of concern as long as foods containing them are heat treated. Phytic acid is naturally present in plants such as soybeans and canola. It reduces the uptake of phosphorous, calcium, magnesium and zinc from food. However, phytic acid also appears to protect against some forms of cancer, so this beneficial effect needs to be weighed against the antinutritional effect.

Toxicity Potential - Various toxicants are known to be inherently present in different plants. Genetic engineering has the potential to alter such constituents or produce newer toxicants. Crops developed for pest resistance and herbicide resistance are particularly focussed for toxicity concern. The case of GM potatoes experiencing Galanthus nivalis
lectin gene for insecticidal properties is an example of the potential of GM foods to cause toxicity. In a group of rats fed with GM potato damage to immune systems and stunted growth was observed and the experiment had generated considerable controversy.

The Stability of Inserted Gene -

Other Unintended Effects -

Safety Issues for GM Foods

i) the inserted gene may itself have adverse effects;
ii) the inserted gene may code for a protein that is toxic to human beings or produces an allergic reaction
iii) the inserted gene may alter the way existing genes in a plant or animal express themselves, which may in turn increase the production of existing toxins or switch on the production of previously silent genes;
iv) the inserted gene may alter the behaviour of a micro-organism, which is carrying it to make it potentially harmful;
v) the inserted gene may be transferred from a micro-organism which is carrying it to other micro-organisms, in the human gut or respiratory tract or to animals or humans;
vi) the consumption of a GM micro-organism may alter the balance of existing
micro-organisms in the human gut.

Process of Gene Modification (GM Animals)

Process of GM:
1. identification of the gene interest;
2. isolation of the gene of interest;
3. transfer and modification of gene
3. amplifying the gene to produce many copies;
4. associating the gene with an appropriate promoter and poly A sequence and insertion into plasmids
5. multiplying the plasmid in bacteria and recovering the cloned construct for injection (aka recombinant-DNA formation, or gene splicing);
6. transference of the construct into the recipient tissue, usually fertilized eggs;
7. integration of gene into recipient genome;
8. expression of gene in recipient genome; and
9. inheritance of gene through further generations.

(adapted from http://www.fao.org/docrep/006/Y4955E/y4955e06.htm)

GM Food (Introduction)

Genetically Modified (GM) foods are produced from genetically modified organisms (GMO) which have had their genome altered through genetic engineering techniques. The general principle of producing a GMO is to insert DNA that has been taken from another organism and modified in the laboratory into an organism's genome to produce both new and useful traits or phenotypes. Typically this is done using DNA from certain types of bacteria.

Interesting Facts To Know About Gram +ve VS Gram -ve Microbes

Gram +ve Microbes


Gram -ve Microbes


Gram negative cells tend to be more susceptible to heat and disinfectants as compared to Gram positive. Furthermore, Gram-positive rods tend to have spores while Gram negative bacteria do not. The Gram reaction is a reproducible characteristic of a species and therefore is an additional tool for bacterial identification.

Vacuum packaging can extend the shelf life of some products. This procedure inhibits the Gram-negative bacteria that require oxygen for growth; thus the production of foul odors is delayed. The growth of Gram-positive bacteria, that are also present in the vacuum-packaged product, is a little slower and their metabolic products are not as objectionable. Thus vacuum packaging does not stop spoilage, it only alters the spoilage rate and the nature of spoilage flora. Vacuum packaging, however, carries with it a serious potential hazard because if the product is temperature abused, Clostridium botulinum will be able to grow.

Gram-negative bacteria, such as Pseudomonas, tend to produce more foul odors than Gram-positive bacteria, such a Bacillus or Streptococcus species. Off odors (incipient spoilage) are usually detected when bacterial numbers reach 106 to 107 CFU/G of the product.

Information for Different Ingredient Group

Below are the information needed for Template 4, identification of predominant microbes in our group of ingredient. In addition, other important information regarding critical limits etc.

1. Meat and poultry products
1.1. Types of products
Raw meat and poultry products consist of raw products; shelf-stable, raw-salted and salted-cured products (salt pork, dry-cured bacon, country ham); perishable raw-salted and salted-cured products (fresh sausage, chorizo, bratwurst, Polish and Italian sausage); marinated products; and raw breaded products. Ready-to-eat products include perishable cooked uncured products (cooked roast beef, cooked pork, cooked turkey); perishable cooked cured products (franks, bologna, ham, and a variety of luncheon meats); canned shelf-stable cured products (Vienna sausages, corned beef, meat spreads, small canned hams, canned sausages with oil and water activity [aw] <0.92, dried beef, and prefried bacon); perishable canned cured products (ham and other cured meats); shelf-stable, canned uncured products (roast beef with gravy, meat stew, chili, chicken and spaghetti sauce with meat); fermented and acidulated sausages (German and Italian style salamis, pepperoni, Lebanon bologna, and summer sausage); and dried meat products (jerky, beef sticks, basturma, and other dried meats). Because of the complexity of the product/processing matrices, product parameters (moisture protein ratio, aw, and pH) and processing schedules are needed to ascertain whether ready-to-eat products require time/temperature control for safety or are shelf stable.

1.2. Microbial concerns
Red meats and poultry come from warm-blooded animals and, as such, their microbial flora is heterogeneous, consisting of mesophilic and psychrotrophic bacteria. These bacteria include pathogenic species from the animal itself and from the environment, and bacterial species introduced during slaughter and processing of raw products. Raw meat and poultry have an aw >0.99 and a pH range of 5 -7, which is an optimal combination for microbial growth. When red meats and poultry are cooked or processed and subsequently refrigerated, the bacterial load from the raw tissue is greatly reduced, leaving only spore-formers, enterococci, micrococci, and some lactobacilli. In addition, environmental post-processing pathogen contamination can occur and the reduction in competitive bacterial flora may allow for pathogen growth. Some products are shelf stable because they received either a botulinum cook or a lesser cook in combination with other controls, such as acidity or other additives (for example, spaghetti meat sauce and Sloppy Joe mix).

1.3. Pathogens of concern
The principal pathogens of concern are Staphylococcus aureus, enterohemmorrhagic Escherichia coli (ruminants), Salmonella spp. (all meats), Listeria monocytogenes (all meats), Campylobacter jejuni/coli (poultry), Yersinia entercolitica (pork), and Clostridium perfringens and Clostridium botulinum (mainly processed products). There is a particular concern when these species are present and/or can grow in cooked products without competition.

1.4. Effects of processing
Meat and poultry products require a wide array of control measures in their processing. Cured meats and some sausage products utilize additives such as salt, nitrate, nitrite, and sugars with processing procedures such as cooking and smoking. Salt, for example, may restrict bacterial flora to salt-tolerant species. Smoking and/or cooking will destroy many vegetative cells. However, the processing environment and product handling and packaging may introduce microorganisms, including pathogens, into the packaged product that also must be considered.

While some canned products may be processed as "commercially sterile", others are canned "semi-preserved" and must be stored under refrigeration. Some products utilize a secondary control such as acidity and are shelf stable though not necessarily "commercially sterile." Specific labeling for refrigeration is required on the semi-preserved products that require refrigeration as a control. Pickled products depend on a low pH, absence of oxygen, and the lack of a fermentable sugar to inhibit the growth of most bacteria. Acid-tolerant species may develop, such as certain lactobacilli, and if air is available, certain yeast and molds may grow. The activity of lactic acid bacteria in fermented sausages is desirable and is an integral part of the process control for achieving the desired pH for these products.

Because of the complexities of products and processing, the USDA Food Safety and Inspection Service (FSIS) has provided guidelines for product parameters in its "Food Standards and Labeling Policy Book" (USDA 1996, with change 98-01). The FSIS guidelines include product specifications such as "meat sticks and cheese", along with general topic categories such as for example "Sausage - Shelf Stable"; "Moisture Protein Ratio -MPR;" and "Moisture Protein Ratio - pH." These policies must always be considered in conjunction with process controls under the HACCP Rule, 9 C.F.R. 417. A product processed in the retail environment is not covered by this rule; however, the variance requirements of the Food Code should require that meat and poultry products have equivalent product specifications for shelf stability and process records documenting control of hazards.

There is substantial history of safety of meat and poultry products that meet these criteria. In addition to the above criteria, certain combinations of pH, aw, and /or other factors can be used to prevent pathogen level increase when meat products are held at ambient temperatures. Products processed in the retail environment and exempt from the HACCP Rule should also follow these guidelines and maintain records documenting control of hazards.

1.5. Time/temperature control
Unless the specific product parameters referenced in the previous section are met, meat and poultry products must be considered as requiring time/temperature control. Raw meat and poultry products currently require safe-handling instruction labeling that includes a time/temperature control provision. For ready-to-eat foods, product parameters and processing schedules are needed to ascertain whether temperature control for safety is required. Post-processing contamination is also an important consideration and should not be overlooked. Because meat offers a rich nutrient media for microbial growth, products that incorporate meat and poultry as ingredients, such as meat salads and meat pastries, also must be considered as requiring time/temperature control.

2. Cereal grains and related products
2.1. Types of products
Cereal grains and related products include baked goods (breads, muffins, cakes, pastries, cookies, biscuits, bagels, and so on), frozen and refrigerated dough, breakfast cereals (cold cereal, oatmeal, grits, and so on), refrigerated or dry pasta and noodles, and cooked grains (for example, rice). Some products, such as baked goods, have a long history of safe storage at room temperature; others, such as rice, require time/temperature control after preparation.

2.2. Pathogens of concern
Grains and milled products are raw agricultural commodities; therefore, a variety of microorganisms, including mold, yeast, coliforms and other bacteria, occur naturally. Grains and milled products are dried to inhibit mold growth during storage, a process that easily controls growth of bacterial pathogens. Therefore, while organisms such as Salmonella spp. may be present, the prevalence and levels are low (usually <1%). Raw ingredients used to prepare dough products (for example, eggs, dairy products, meats) may introduce Salmonella spp., and need to be considered when analyzing potential hazards. Staphylococcus aureus may present a potential hazard for certain raw dough, such as pasta dough processed at warm temperatures for extended periods of time (days); however, yeast leavened dough and cookie dough control the organism through competitive inhibition and low aw , respectively. Bacillus cereus presents a concern in cooked rice.

2.3. Effects of processing
Baking, boiling, steaming, or frying are the methods used to cook the cereal-grain products. The temperatures required to achieve product quality easily destroy vegetative pathogens that may be present. These temperatures are needed to properly set the starch structure and/or to rehydrate dry products. Baking and frying not only destroy vegetative pathogens such as S. aureus and Salmonella spp., but they also remove moisture from the product-especially at the exterior surface. This dehydrated surface inhibits the growth of most bacteria; thus, mold is the primary microbial mode of failure for baked goods. When stored at room temperature, baked and fried products typically continue to lose moisture to the atmosphere, further reducing the potential for pathogen growth. Thus, baked and fried cereal-grain products such as cakes, breads, muffins, and biscuits have a long history of safe storage at room temperature despite having an internal aw of approximately 0.94-0.95 (but may be as high as 0.98).

While boiled or steamed cereal products achieve temperatures lethal to vegetative pathogens during the cooking process, these products increase in aw to levels that support the growth of many microbial pathogens. Thus, time/temperature control is required to assure the safety of these products. For example, numerous B.cereus outbreaks have been associated with fried rice prepared using boiled rice that was held for hours at room temperature.

2.4. Time/temperature control
Although baked and fried cereal-grain products (for example, cakes, breads, muffins, and biscuits) have a high aw, a number of reasons may justify their shelf-stability: they have a long history of safe storage at ambient temperature; processing temperatures and moisture reduction, especially on the surface, preclude the growth of pathogens; and they are often formulated to include ingredients that enhance product safety and stability so as to permit distribution without temperature control for limited periods of time. Ingredients that are used to enhance safety and stability include humectants to reduce aw (sugars and glycerine), preservatives (calcium propionate, potassium sorbate, sorbic acid), acids to reduce pH (vinegar, citric acid, phosphoric acid, malic acid, fumaric acid), spices with antimicrobial properties (cinnamon, nutmeg, garlic), and water-binding agents to control free water (gums, starches). The primary mode of spoilage of baked goods is mold growth, which is visible and alerts the consumer to avoid consumption, further reducing the risk of illness due to spoiled product. These characteristics plus their long history of safe storage at room temperature would allow these products to be stored at ambient temperature. Boiled or steamed cereal products, such as rice, require time/temperature control after preparation due to the increase in aw.

Dough is frequently used to enrobe other food ingredients. Careful consideration must be given to these combination products to accurately assess the need for time/temperature control. For example, egg and dairy ingredients baked inside a pastry, such as cream-cheese croissant, will receive sufficient heat treatments to destroy vegetative pathogens and may therefore be stable at room temperature with water activities above 0.86. However, if the filling is injected after the baking process, as in the case of a cream-filled éclair, the potential for contamination must be assessed. Meat and vegetable-filled cereal products with high water activities (>0.94) and neutral pH generally require time/temperature control because the baking process can activate spore formers such as C. botulinum that are present in these ingredients.

Aw Activity of Food To Support Different Microbial Growth

Information for Template 4. To identify the microbes that can grow in dried instant ramen and soup powder.

Concerns For Vacuum-packed and Refrigerated Teriyaki Chicken

C. botulinum and C. perfringen are an obligate anaerobes where vegetative cells can be killed at 80degreeC in few seconds time however, being spore-forming bacteria, the endospore formed can survive the high heat treatment and will result in foodborne illness when conditions become favorable for its growth during storage. In Instant Teriyaki Chicken Ramen, the teriyaki chicken are vacuum-packed to extend its shelf-life however, no one preservation method is foolproof, the vacuum condition makes it an ideal condition for the growth of C. botulinum and C. perfringens. To help better ensure food safety, the storage condition of the final product will be kept at refrigerated condition, a temperature range of which will not support the growth of these two microbes.

Yet on the other hand, the refrigerated condition can still promote the growth of Listeria which is found in the environment and can grow at a wide temperature range, and grow as at as low as 0degreeC so, the main preventive control is to empoly aanitary safeguards to prevent reintroduction of pathogens. Chief among these is Listeria monocytogenes.

The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) chartered by the U.S. Department of Agriculture (USDA) and the Department of Health and Human Services (HHS) recently commented on the microbial safety of refrigerated foods containing cooked, uncured meat or poultry products that are packaged for extended refrigerated shelf-life and are ready-to-eat or prepared with little or no additional heat treatment. The Committee recommended guidelines for evaluating the ability of thermal processes to inactivate L. monocytogenes in extended shelf-life refrigerated foods. Specifically, it recommended a proposed requirement for demonstrating that an ROP process provides a heat treatment sufficient to achieve a 4 decimal log reduction (4D) of L. monocytogenes.

Product Recall Decision Tree

Taken from http://www.nzfsa.govt.nz/processed-food-retail-sale/recalls/guidance/index.htm

Potential Hazards Classifications

Physical hazards:
Glass
Metal
Other foreign matters

Chemical hazards:
Allergens
Animal drugs residues
Cleaning compound
Residues
Illegal residues/pesticides in raw material, packaging material, shipping containers
Natural toxins
Unapproved food additives
Overdosage of food additives
Antibodies/hormones

Biological hazards:
Cross-contamination (post processing)
Pathogen in raw materials, during storage
Parasites
Pests

Food Intoxication VS Food Infection

Food Intoxication
Clostridium botulinum
Clostridium perfringens
Bacillus cereus
Staphylococcus aureus
Aspergillus sp.


Food Infection
Listeria monocytogenes
Vibrio cholerae
Vibrio parahaemolyticus
Vibrio vulnificus
Salmonella sp.
Shigella sp.
Escherichia coli
Yersinia enterocolitica
Campylobacter jejuni
Plesiomonas shigelloides

Microbial Concerns For Seafood Products

Bacterial Pathogens associated with raw and processed seafood

* Salmonella spp.
* Clostridium botulinum
* Listeria monocytogenes
* Vibrio cholerae O1
* Vibrio cholerae non-Ol
* Vibrio parahaemolyticus and other vibrios
* Vibrio vulnificus

Parasites that are sometimes found in raw seafood

* Anisakis sp. and related worms
* Diphyllobothrium spp.
* Nanophyetus spp.
* Eustrongylides sp.
* Acanthamoeba and other free-living amoebae
* Ascaris lumbricoides and Trichuris trichiura

Viruses that sometimes contaminate raw seafood

* Hepatitis A virus
* Hepatitis E virus
* Rotavirus
* Norwalk virus group
* Other viral agents

Natural Toxins that are sometimes found in seafood

* Ciguatera poisoning
* Shellfish toxins (PSP, DSP, NSP, ASP)
* Scombroid
* Tetrodotoxin (Pufferfish)

For processed fishcake it's those in bold that's more of a concern.

HACCP Decision Tree

Adapted from http://vm.cfsan.fda.gov/~comm/nacmcfp.html

Introduction

Ensuring food safety is detrimental, especially for food industries. If mishandling of food result in foodborne disease outbreaks, not only will the company reputation suffers, large sum of loss and most importantly innocent consumers will suffer too. In serious cases, it may even resulted in deaths. Hence, it is really important that food undergo strict sampling and control in mid of processing to minimize possible contamination that may result in foodborne diseases.

HACCP is one of the standard to help ensure food safety which takes each process into consideration and from there identify the critical control points of which may make or break a product safety. In fact to guarantee food safety is everyone's responsibility. From the food producer to middlemen to the consumers. If at any part of the chain, one mishandles the food, possible foodborne disease outbreak may take place. Often, as consumers, they neglected their roles in ensuring food safety themselves. For instance, consumers' abuse where they leave a supposedly-to-be-kept-frozen food out in the danger temperature zone (5-60°C) for more than 4 hours and they then consume the food, resulting in foodborne disease and point their fingers at the company.

Hence, it is everyone's responsibility to ensure food safety. Everyone has their own roles to play. If everyone were to play their roles well, eating is a great pleasant!