INDICATOR MICROORGANISMS in food microbi
- 2. Purpose of microbiological criteria Microbiological criteria are used to distinguish between acceptable and unacceptable products or between acceptable and unacceptable food processing and handling practices Types of microorganisms present Numbers of microorganisms Safety presence or absence of pathogenic microorganisms or their toxins the number of pathogens the expected control or destruction of these agents
- 3. Indicator organisms Determine the microbiological quality Food safety risk of a food Indicator organisms require a positive relationship between the occurrence of the indicator organism and the likely presence of a pathogen or toxin has been established (indicator present pathogen present) Microbiological criteria used to determine: 1. Safety of food 2. Adherence to good manufacturing practices (GMPs) 3. Keeping quality (shelf life) of foods 4. Utility (suitability) of a food or ingredient for a particular purpose Microbiological criteria - ensuring the safety and quality of foods- which in turn elevates consumer confidence It also provides guidelines for control of food processing systems and critical control point in microbiological hazard (HACCP) systems
- 4. Factors to establish microbiological criteria Evidence of a hazard to health based on epidemiological data or a hazard analysis Microflora of the food and the ability of the food to support microbial growth The effect of processing on the microflora of the food Potential for microbial contamination and/or growth during processing, handling, storage, and distribution The category of consumers at risk The state in which the food is distributed The potential for abuse at the consumer level Spoilage potential, utility, and GMPs The manner in which the food is prepared for ultimate consumption Methods available to detect and/or quantify the microorganism(s) and toxin(s) of concern The costs/benefits associated with the application of the criterion
- 5. Role of Microbiological Criteria for Foods and Food Ingredients (1985 report) Microbiological criterion type of microorganism group of microorganisms or toxin produced by a microorganism Either not be present at all Present in only a limited number of samples Present as no less than a specified limit A statement describing the identity of the food or food ingredient A statement identifying the contaminant of concern An analytical method to be used for the detection, enumeration, or quantification of the contaminant of concern A sampling plan Microbiological limits considered appropriate to the food and commensurate with the sampling plan
- 6. Two types of criteria Mandatory criterion is one that may not be exceeded, and food that does not meet the specified limit- some action required (rejection, destruction, reprocessing or diversion) Advisory criterion permits acceptability judgments to be made, and it should serve as an alert to deficiencies in processing, distribution, storage, or marketing
- 7. Standard- Definition A microbiological criterion that is part of a law, ordinance, or administrative regulation A standard is a mandatory criterion Failure to comply constitutes a violation of the law, ordinance, or regulation and will be subject to the enforcement policy of the regulatory agency having jurisdiction Wherever possible it should contain limits only for pathogenic microorganisms of public health significance in the food concerned Limits for non-pathogenic microorganisms may be necessary when the methods of detection for the pathogens of concern are cumbersome or unreliable Standards based on fixed numbers of nonpathogenic microorganisms may result in the recall or down grading of otherwise wholesome food Penalty provisions could be applied when a lot is rejected
- 8. Guidelines A microbiological criterion often used by the food industry or regulatory agency to monitor a manufacturing process Guidelines function as alert mechanisms to signal whether microbiological conditions prevailing at critical control points or in the finished product are within the normal range Hence, they are used to assess processing efficiency at critical control points and conformity with Good Manufacturing Practices A microbiological guideline is intended to increase assurance that the provisions of hygienic significance have been achieved It may include microorganisms which are not of direct public health significance
- 9. Specifications A microbiological criterion that is used as a purchase requirement whereby conformance becomes a condition of purchase between buyer and vendor of a food ingredient A microbiological specification may be advisory or mandatory applied at the establishment at a specified point during or after processing to monitor hygiene It is intended to guide the manufacturer and is not intended for official control purposes The Codex use of “specification” only refers to end products and does not include raw materials, ingredients, or foods in contractual agreements between two parties
- 10. Sampling plans A sampling plan includes both the sampling procedure and the decision criteria regarding the disposition of a lot of product based on the results of the sampling plan To examine a food for the presence of microorganisms, a representative sample is examined by defined methods A representative sample is examined by defined methods A lot- quantity of product produced, handled, and stored within a specified time period under uniform conditions Impractical to conduct microbiological analysis on the entire lot statistical concepts of population probability and sampling must be used to determine the number and size of sample units from the lot and to provide conclusions drawn from the analytical results The sampling plan is designed so that inferior lots, within a specified level of confidence, are rejected
- 11. Types of sampling plans Variables- frequency distribution of microbes Attributes- when microbes not homogenously distributed throughout food/ target organism in low level- e.g pathogenic organism/ acceptance or rejection at points where there is lack of knowledge about food processing (e.g. port or point of entry)
- 12. Attributes sampling plan Two-Class Plans The concentration of microorganisms of the sample units tested to a particular attribute class depending on whether the microbiological counts are above or below some pre-set concentration, represented by the letter m Presence/absence sampling plans The decision criterion is based on 1. the number of sample units tested, n 2. the maximum allowable number of sample units yielding unsatisfactory test results, c For example, when n =5 and c =2 in a two-class sampling plan designed to make a presence/absence decision on the lot (i.e., m=0), the lot is rejected if more than two of the five sample units tested are positive. As n increases for the set number c, the stringency of the sampling plan also increases Two-class plans are applied most often in qualitative (semiquantitative) pathogen testing, where the results are expressed as the presence or absence of the specific pathogen per sample weight analyzed
- 13. Three-Class Plans Three-class sampling plans use the concentration of microorganisms in the sample units to determine levels of quality and/or safety Counts above a pre-set concentration M for any of the n sample units tested are considered unacceptable, and the lot is rejected The level of the test organism acceptable in the food is denoted by m This concentration in a three-class attribute plan separates acceptable lots (i.e., counts less than m) from marginally acceptable lots (i.e., counts greater than m but not exceeding M) Counts above m and up to and including M are not desirable but the lot can be accepted provided the n number of samples that exceed m is no greater than the preset number, c Thus, in a three-class sampling plan, the food lot will be rejected if any one of the sample units exceeds M or if the number of sample units with contamination levels above m exceeds c Similar to the two-class sampling plan, the stringency of the three-class sampling plan is also dependent on the two numbers denoted by n and c The larger the value of n for a given value of c, the better the food quality must be to have the same chance of passing, and vice versa
- 14. Establishing limits Microbiological limits, as defined in a criterion, represent the level above which action is required Levels should be based on knowledge of the raw materials and the effects of processing, product handling, storage, and end use of the product Limits should also take into account the likelihood of uneven distribution of microorganisms in the food the inherent variability of the analytical procedure the risk associated with the microorganisms the conditions under which the food is expected to be handled and consumed Microbiological limits should include sample weight to be analyzed, method reference, and confidence limits of the referenced method where applicable
- 15. How to determine shelf life of a food Determined by the number of microorganisms initially present As a general rule, a food containing a large population of spoilage microbes will have a shorter shelf life than the same food containing fewer numbers of the same spoilage microorganisms Relationship between total counts and shelf life is not absolute Some types of microorganisms have a greater impact on the organoleptic characteristics of a food than others due to the presence of different enzymes acting upon the food constituents In addition to the effect of certain levels and/or types of spoilage microorganisms, changes in perceptible (able to see) quality characteristics will also vary depending on the food and the conditions of storage, such as temperature and gaseous atmosphere All of these parameters need to be considered when establishing limits for the microbiological criteria used to determine product quality and/or shelf life
- 16. Foods produced and stored under GMPs may be expected to have a different microbiological profile than those foods produced and stored under poor conditions The use of poor-quality materials, improper handling, or unsanitary conditions may result in higher bacterial counts in the finished product However, low counts in the finished product do not necessarily mean that the product was produced under acceptable GMPs Processing steps such as heat treatments, fermentation, freezing, or frozen storage can reduce the counts of bacteria Ground beef- high microbial counts even under the best conditions of manufacture- growth of psychrotrophic bacteria during refrigeration Limits set for microbiological criteria used to assess adherence to GMPs require a working knowledge of the types and levels of microorganisms present at the different processing steps to establish a relationship between the microbiology of the food and adherence to GMPs
- 17. HACCP Microbiological criteria- HACCP development There are 7 principles of HACCP: 1. Identify the hazards 2. Determine the critical control points (CCPs) 3. Establish critical limit(s) 4. Establish a system to monitor control of the CCP 5. Establish the corrective action to be taken when monitoring indicates that a particular CCP is not under control 6. Establish procedures for verification to confirm the HACCP system is working effectively 7. Establish documentation concerning all procedures and records appropriate to these principles and their application
- 18. The process consists of (i) hazard identification, (ii) hazard characterization, (iii) exposure assessment (iv) risk characterization Examples of Food Safety Objective (FSO) include the following: 1. The number of Listeria monocytogenes organisms in ready-to-eat foods may not exceed 100 CFU per g 2. Salmonella spp. must not be detected in powdered infant formula in 60 25-g subsamples of the production lot 3. Aflatoxin concentration in peanuts should not exceed 15 µg/kg
- 19. Indicators of microbiological quality Estimation of a product for indicator microorganisms provide simple, reliable, and rapid information process failure, postprocessing contamination, contamination from the environment the general level of hygiene under which the food was processed and stored Usually provide information in a shorter time than that required for isolation and identification of specific microorganisms or pathogens Used to check product quality or to predict shelf life of the food Loss of quality- not limited to one microorganism – but variety of microorganisms- due to the unrestricted environment of the food Then we determine the counts of groups of microorganisms most likely to cause spoilage in that particular food
- 20. Aerobic plate count (APC) or Standard plate count (SPC) Determine “total” numbers of microorganisms in a food product APC- screen for anaerobic, thermoduric, mesophilic, psychrophilic, thermophilic, proteolytic, and lipolytic (modify method a bit) SPC- for dairy products APC- microbiological criteria used to i) Monitor foods for compliance with standards or guidelines set by various regulatory agencies ii) Monitor foods for compliance with purchase specifications iii) Monitor adherence to GMPs
- 24. APC drawbacks 1. The “plate count” is based on the assumptions that every cell forms one colony and that every colony originates from one cell The ability to form a colony depends on - the physiological state of the cell, the medium used for enumeration, the incubation temperature and time, and the number of cells present 2. Only measure live cells and therefore would not be of value, e.g. to determine the quality of raw materials used for a heat processed food 3. Little value in assessing organoleptic (sense organs testing) quality since high microbial counts generally must be present prior to organoleptic quality loss 4. Because different bacteria vary in their biochemical activities, quality loss may also occur at low total counts, depending on the predominant microbes present 5. Takes several days With any food, specific causes of unexpected high counts can be identified by examination of samples at control points and by plant inspection Reliable interpretation- requires knowledge of the expected microbial population at the point in the process or distribution at which the sample is collected If counts are higher than expected, this will point to the need to determine why there has been a violation of the criterion
- 25. Viable but non culturable Salmonella, Campylobacter, Escherichia, Shigella, and Vibrio species, and other genera, can exist in a state where they are viable but cannot be cultured This differentiation of vegetative cells into a dormant “viable but nonculturable” (VNC) state is a survival strategy for many nonsporulating species The VNC state is morphologically different from that of the “normal” vegetative cell During the transition to the VNC state, rod-shaped cells shrink and become small spherical bodies which are not spores Changes in membrane fatty acid composition occur in Vibrio during entry into the VNC state It takes from 2 days to several weeks for an entire population of vegetative cells to become VNC
- 26. Direct Microscopic count (DMC) Estimate of both viable and nonviable cells in samples containing a large number of microorganisms (i.e., >105 CFU/ml) Drawbacks Not differentiate between live and dead cells (unless a fluorescent dye such as acridine orange is employed) It requires that the total cell count exceed 105cells/ml, use of the DMC is of limited value as part of microbiological criteria for quality issues The use of DMC as part of microbiological criteria for foods or ingredients is restricted to a few products such as raw, non-grade A milk, dried milks, liquid and frozen eggs, and dried eggs
- 27. Other methods Howard mold count- count mold in canned fruits and tomato products, vegetable canneries Yeast and mold count Heat-resistant mold count Thermophilic spore count Indirect methods Testing for metabolic products produced by the microorganisms Measure LPS concentration (gram negative) Measurement of ATP Dye reduction time- higher population faster reduction
- 28. Indicators of foodborne pathogens and toxins Risk assessment to determine the potential hazards and their significance to consumers Public health officials and the dairy industry responded to widespread outbreaks of milk- borne disease occurring in the early 1900s in the United States By imposing controls on milk production, developing safe and effective pasteurization procedures, and setting microbiological criteria, the safety of commercial milk supplies was greatly improved Often food processors alter the intrinsic or extrinsic parameters of a food (nutrients, pH, water activity, inhibitory chemicals, gaseous atmosphere, temperature of storage, and the presence of competing microbes) to prevent growth of undesirable microorganisms If control over one or more of these parameters is lost, then there may be a risk of a health hazard For example, in the manufacture of cheese or fermented sausage, a lactic acid starter culture is relied upon to produce acid quickly enough to inhibit the growth of Staphylococcus aureus to cell numbers that would be potentially harmful
- 29. Microbes having low infective dose - presence significant public health risk For such microbes, the concern is not whether the pathogen is able to grow in the food but that the microorganism could survive for any length of time in the food Foods having intrinsic or extrinsic factors sufficient to prevent survival of pathogens or toxigenic microorganisms of concern may not be candidates for microbiological criteria related to safety For example, the acidity of certain foods, such as fermented meat products, might be assumed sufficient for pathogen control The hazard associated with a food is determined by (i) the type of microorganism expected to be encountered (ii) the expected conditions of handling and consumption after sampling The stringency of sampling plans for foods is based: i. the hazard to the consumer from pathogenic microorganisms and their toxins or toxic metabolites ii. The potential for quality deterioration to an unacceptable state, and it should take into account the types of microorganisms present and their numbers
- 30. Foodborne pathogens can be grouped into one of three categories based on the severity of the potential hazard severe hazards moderate hazards with potentially extensive spread, moderate hazards with limited spread Potential hazard for extensive spread/ secondary spread to other foods - environmental contamination and cross-contamination within processing plants and food preparation areas, including homes Beef- E. coli O157:H7- One or a few contaminated piece(s) of meat can lead to widespread contamination of product during processing, such as grinding to produce ground beef Cross contamination- improperly stored with ready to eat foods Lowest risk group (moderate hazards, limited spread) are found in many foods, usually in small numbers Illness usually occur when foods contain large numbers of the pathogen(C. perfringens) Illness occur when large numbers to produce sufficient toxin (S. aureus)
- 31. Indicator testing Tests for indicator microorganisms suggests possibility of a microbial hazard E. coli in drinking water- indicates fecal contamination- the potential presence of enteric pathogens Jay suggested: An indicator should ideally meet the following criteria: i. be easily and rapidly detectable ii. be easily distinguishable from other members of the food flora iii. have a history of constant association with the pathogen whose presence it is to indicate iv. always be present when the pathogen of concern is present v. be a microorganism whose number ideally should correlate with those of the pathogen of concern vi. possess growth requirements and a growth rate equaling those of the pathogen vii. have a die-off rate that at least parallels that of the pathogen and, ideally, persist slightly longer than the pathogen of concern viii. be absent from foods that are free of the pathogen except perhaps at certain minimum numbers
- 32. Buttiaux and Mossel suggested additional criteria: i. Ideally the bacteria selected should demonstrate specificity, occurring only in intestinal environments. ii. They should occur in very high numbers in feces so as to be encountered in high dilutions iii. They should possess a high resistance to the external environment, the pollution of which is to be assessed iv. They should permit relatively easy and fully reliable detection even when present in low numbers
- 33. Fecal Coliforms and E. coli Contamination of a food with E. coli- risk other enteric pathogens may be present in the food The fecal coliforms have a higher probability of including microbes of fecal origin than do coliforms which are comprised of bacteria of both fecal and nonfecal origin However, many fecal coliform bacteria are not E. coli and are indigenous to vegetation and plant materials and so are not of fecal origin Fecal coliforms- not a reliable indicator of fecal contamination E. coli is the most widely used indicator of fecal contamination The failure to detect E. coli in a food- does not assure the absence of enteric pathogens Raw foods- small numbers of E. coli can be expected because of the close association of these foods with the animal environment and the likelihood of contamination of carcasses from fecal material, hides, or feathers during slaughter-dressing procedures
- 34. E. coli in a heat-processed food- indicates either process failure or postprocessing contamination from equipment or employees or from contact with contaminated raw foods In the case of refrigerated ready-to-eat products- coliforms are recommended as indicators- with regard to reintroduction of pathogens from environmental sources and maintenance of adequate refrigeration Thermal processing- coliforms- usually the processing environment, resulting from inadequate sanitation procedures and/or temperature control Coliforms are present in higher numbers than E. coli and the levels of coliforms do not increase over time when the product is stored properly (so recommended method for criteria) Other coliforms- Enterobacter, Klebsiella, Citrobacter, etc
- 35. Enterococci Sources Fecal material- both warm-blooded and cold-blooded animals Plants sources Enterococci- salt tolerant (grow in the presence of 6.5% NaCl) resistant to freezing E. faecalis and E. faecium-heat resistant- usually survive pasteurization temperatures
- 36. Hurdle technology Hurdle technology is a method of ensuring the safety of foods by eliminating or controlling the growth of pathogens, making the food safe for consumption and extending its shelf life through the application of a combination of technologies and approaches
- 37. Homeostasis and Hurdle Technology Instead of setting one parameter to the extreme limit for growth, hurdle technology “deoptimizes” a variety of factors For example, a limiting water activity of 0.85 or a limiting pH of 4.6 prevents the growth of foodborne pathogens Hurdle technology might obtain similar inhibition at pH 5.2 and a water activity of 0.92 Hurdle technology assaults multiple homeostatic processes In acidic conditions, cells use energy to pump out protons In low-water-activity environments, cells use energy to accumulate compatible solutes
- 38. Maintenance of membrane fluidity also requires energy When the energy needed for biosynthesis is diverted into maintenance of homeostasis, cell growth is inhibited When homeostatic energy demands exceed the cell’s energy producing capacity, the cell dies. Hurdle technology can encompass the use of antimicrobial agents (e.g., nisin) and technology including the use of ozone and the application of irradiation in conjunction with shifts in pH and water activity to inhibit microbial growth