Establishment of Microbiological Criteria
5.7 Components of Microbiological Criteria for Foods
According to the Codex Alimentarius Commission (CAC 2013) an MC consists of:
• the purpose of the MC
• the food or process to which the MC applies
• the specified point in the food supply chain where the MC applies
• the microorganism(s) and the reason for their selection
• the microbiological limits (m, M) and/or other limits considered appropriate to the food
• a sampling plan defining the number of samples to be taken (n), the size of the analytical unit and where appropriate, the acceptance number (c)
• depending on its purpose, an indication of the statistical performance of the sampling plan
• analytical methods and their performance parameters
• action to be taken when the MC is not met
When applying an MC for assessing products, it is essential, in order to make the best use of money and resources, that only appropriate tests be applied to those foods and at those points in the food sup-ply chain that offer maximum benefit in providing consumers with foods that are safe and suitable for consumption. The MC should be economically feasible for food businesses to execute. Consideration also needs to be given to sampling; particularly the type of sample, sampling strategy (e.g., random, stratified) and the sampling frequency.
5.7.1 Microorganisms and Their Toxins/Metabolites of Importance in a Particular Food
Microorganisms and/or their toxins/metabolites of concern include:
• bacteria, viruses, yeasts, molds and algae;
• parasitic protozoa and helminths; and
• microbial toxins/metabolites.
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According to the Codex Alimentarius Commission, this includes the markers of microbiological pathogenicity (e.g., virulence-related genes or plasmids) or other traits (e.g., anti-microbial resistance genes) where/when linked to the presence of viable cells (CAC 2013).
The microorganisms included in a specific MC should be widely accepted as relevant to the par-ticular food and/or process technology, either as pathogens, as indicator organisms or as spoilage microorganisms. Microorganisms whose significance in the specified food is doubtful should not be included in an MC. For example, the mere finding, with a presence-absence test, of certain microor-ganisms known to cause foodborne illness (e.g., Bacillus cereus, Clostridium perfringens, Staphylococcus aureus and Vibrio parahaemolyticus) does not necessarily indicate a threat to public health. These bacteria typically require growth in a food before they are considered a significant hazard.
Where pathogens can be detected directly and reliably (e.g., performance of sampling plans), con-sideration should be given to testing for them in preference to testing either directly or indirectly for indicator organisms. Testing for an indicator microorganism may be useful as discussed below.
5.7.1.1 The Use of Indicator Organisms
Indicator organisms are frequently used to examine foods or ingredients. An important use of indict-ors is to verify process control and identify opportunities for process improvements. Most of the considerations used to set MC are applicable for indicators; however, indicators are not used solely for pathogenic concerns. Microorganisms, their cellular components, or their metabolic products used as indicators may indicate:
• the possible presence of a pathogen or toxin (e.g., S. aureus for potential enterotoxin and/or exces-sive human handling in cooked crab meat)
• the possibility that faulty practices occurred during production, processing, storage and/or distribu-tion (e.g., Enterobacteriaceae in pasteurized milk)
• the suitability of a food or ingredient for a desired purpose (e.g., Escherichia coli in nuts for ice cream)
• an estimate of the keeping quality of perishable foods during the expected conditions of handling and storage (e.g., yeast in yogurt)
• the possibility of changes in the food through fungal activity that would result in a less acidic food, thus making the food potentially more hazardous
• the effectiveness of cleaning and disinfection (e.g., ATP for residual soil)
Indicator microorganisms and agents can be divided into indicators of potential human tion, fecal contamination, survival of a pathogen or spoilage organism, or post-processing contamina-tion. Examples of microbial indicators that can be analyzed quantitatively or qualitatively include aerobic bacteria, coliforms, Enterobacteriaceae, E. coli, yeasts, molds, proteolytic bacteria and ther-mophilic bacteria. Examples of cellular components that can be used include ATP, ribonucleic acid (RNA), endotoxins (e.g., limulus lysate test for cellular polysaccharides) and various enzymes (e.g., thermonuclease). Examples of metabolic products used as indicators include hydrogen sulfide (early putrefaction), carbon dioxide (spoilage by Zygosaccharomyces bailii), lactic acid (certain meat prod-ucts including ham), ethanol (in fruit juice), diacetyl (in fruit juice or beer) and ergosterol (mold in grain).
Some of the characteristics of an ideal indicator organism include the following:
• presence indicates potential for spoilage, faulty practice or faulty process
• easily detected and/or quantified
• survival or stability (including inactivation kinetics) similar to or greater than the hazard or spoil-age microorganism
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• growth capabilities (requirements) similar or faster than the hazard or spoilage microorganism
• identifiable characteristics of indicator are stable
• methods are rapid, inexpensive, reliable, sensitive, validated and verified with positive control
• quantitative results have a correlation between indicator concentration and level of hazard or spoil-age microorganism
• results are applicable to process control
It must be recognized that indicators often represent a compromise that is less than the ideal of being able to test for the microorganism(s) or toxin(s) of concern. However, indicators offer consider-able advantages that ensure their continued use. To fulfill their purpose as indicators it is essential to select indicators that provide the best information for the intended purpose with the least amount of compromise.
In practice, indicators seldom, if ever, prove the presence or the absence of a target microorganism;
they merely indicate the possibility. For purposes of process control, the absence or low numbers of an indicator can verify that a process is under control, thus there is a lower likelihood that the unac-ceptable target microorganism is present. However, a pathogen may be present independent of an indicator. Thus, combining indicator tests that provide information on conditions that reduce the like-lihood of the occurrence of a pathogen with periodic verification testing for the pathogen is appropri-ate in many situations. Indicators can be very useful, but their selection and application must be done with care and a thorough understanding of how to accurately interpret the analytical results.
5.7.2 Microbiological Limits
Microbiological limits may be established as a basis to assess the safety or quality of a food and should be compatible with any POs that have been established for a specific food. Limits should be based on microbiological data appropriate to the food and should be applicable to a variety of similar products.
The process of establishing limits for use as standards should include collecting and analyzing data from a variety of operations to determine what can be expected for foods produced under acceptable conditions of GHP and HACCP. These data can then be used to establish limits that can be met by all who operate under acceptable conditions. Alternatively, the limit can be made more stringent if improvement is deemed necessary in a certain segment of industry to reduce the likelihood of a hazard.
This assumes operators can adapt by making practical modifications. If, however, the technology does not exist or is not affordable, then the more stringent limit will fail and the desired improvement will not be achieved. The process of establishing MC, and other acceptance criteria, should be transparent and allow input from all interested parties.
Microbiological limits relate only to the specified time and place of sampling and not to the pre-sumed number of microorganisms at an earlier or a later stage. Because GHP aims at producing foods with microbiological characteristics significantly better than those required by public health consider-ations, a numerical limit in a guideline may be more stringent than in a standard or end product specification.
In the establishment of microbiological limits, any changes in the microbiota likely to occur during storage and distribution (e.g., decrease or increase in the numbers) should be taken into account. The risk associated with the microorganisms and the conditions under which the food is expected to be handled and consumed should also be considered. These considerations are discussed in Chap. 8.
Microbiological limits should also take account of the likelihood of uneven distribution of microor-ganisms in the food (see Chaps. 6 and 7) and the inherent variability of the analytical procedure (see Chap. 9).
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Whether foods are acceptable or not is defined in a criterion by the:
• microbiological limit(s)
• number of samples examined
• size of the analytical unit
• number of units that should conform to the limits
If a criterion requires the absence of a particular microorganism, the number and size of each ana-lytical unit should be indicated. It should be recognized that no feasible sampling plan can ensure the absence of a particular microorganism in the entire contents of the lot.
The microbial population in many foods produced under GHP and HACCP is generally not explic-itly expressed because they are as low as is reasonably achievable. In some cases, the levels cannot be measured due to the technical problems involved. For instance, a lot of canned food that has received a “bot cook” will most probably not contain surviving spores of C. botulinum in 1010 or 1011/g of product. Likewise, enteric pathogens are not likely to be found in many kilograms of pasteurized products. Setting limits for pathogens in processed foods in which a validated kill step is included during processing should not be an arbitrary activity, but should be done only if there is a need to detect contaminated product.
Limits for use in purchase specifications are best established from data collected during normal production when the operation is under control. It is not uncommon for a company to establish more stringent criteria for its own use to ensure compliance with customer and regulatory requirements.
In sampling by attributes procedures, microbiological limits, m and M, define the presence/absence or concentration of a microorganism, microbial toxin or metabolite that differentiates acceptable from unacceptable sample units of food, and c is the maximum allowable number of defective or marginally acceptable sample units (see Chap. 7). In a two-class plan, m separates acceptable units from defective units, while in a three-class plan; m separates acceptable units from marginally acceptable units. The limit m may be considered by those establishing the criterion to be acceptable and attainable through application of GHP and/or HACCP. In a three-class plan, M separates marginally acceptable units from unacceptable units (see Chap. 7).
In sampling procedures not based on attributes, the microbiological limits (m and M) are replaced by alternative limits. For example, for variables plans, the acceptable microbiological quality limit (V) and the maximum proportion (p0) of the lot that can be accepted with concentrations above the limit (V) (see Chap. 7).
5.7.3 Sampling Plans, Sampling Procedures and Handling of Samples Prior to Analysis
Sampling plans should include the sampling procedure and the decision criteria to be applied to the result, based on examination of a prescribed number of sample units and subsequent analytical units of a stated size by defined methods. A well designed sampling plan defines the probability of detecting microorganisms in a lot, but no sampling plan can ensure the absence of a particular microorganism from an entire lot. Sampling plans should be administratively and economically feasible.
In particular, the choice of sampling plans should take into account the:
• risks to public health associated with the hazard (severity and likelihood of occurrence of the hazard)
• susceptibility of the target group of consumers (very young or old, immune- compromised, etc.)
• heterogeneity of distribution of microorganisms where variables sampling plans are employed
• randomness of sampling
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• acceptable quality/safety level (i.e., percentage of non-conforming or defective sample units tolerated)
• desired statistical probability of accepting or rejecting a non-conforming lot
The information needed for the first two points could be obtained through a risk assessment; how-ever, good epidemiological data may suffice. For many applications, two- or three-class attribute sampling plans may be useful. In general, the greater the risk, the more stringent (e.g., greater number of samples) should be the sampling plan. For a detailed discussion of establishing sampling plans, see Chaps. 6, 7 and 8.
The time between taking the field samples and analysis should be as short as reasonably possible and the conditions during transport to the laboratory (e.g., temperature) should not allow an increase or decrease in the numbers of the target microorganisms. By controlling these conditions the results should reflect, within the limitations given by the sampling plan, the microbiological conditions of the food. For samples taken by regulatory authorities as part of official controls, consideration should be given to maintaining the chain of evidence by ensuring that the integrity and identity of the sample is maintained and the handling procedure is recorded. For further details see Chap. 9.
5.7.4 Microbiological Methods
The choice of microbiological method can have a significant impact on the quantitative and qualitative results generated. Accordingly, MC must specify the method used. The microbiological methods specified should be reasonable with regard to complexity, availability of media and equipment, ease of interpretation, time required and costs. Where possible, only methods for which the reliability (e.g., specificity, sensitivity, reproducibility) has been statistically established by comparative or collabora-tive studies in several laboratories, i.e., validated methods, should be used. Reference methods estab-lished by international standards organizations like ISO, AOAC and CEN fit these criteria. Moreover, preference should be given to methods that have been validated for the commodity of concern.
Alternative methods to reference methods like rapid methods, can be used, providing that they have been validated against a reference method in keeping with an internationally recognized validation standard (e.g., ISO 16140). Additional information on methods and their reliability is provided in Chaps. 9 and 10.
5.7.5 Reporting
The test report should provide complete information to identify the sample, the sampling plan, the test method and, if appropriate, the interpretation of the results.
