Microbiological Guidelines: Support for Interpretation of Microbiological Test Results of Foods

By Collective

Food plays an essential part in everyday life.

Food should be tasty, healthy, sustainable and preferably not too expensive. But food should also be safe and with sufficient guarantees on maintaining good quality aspects until the end of shelf life. The various actors in the food supply chain have an interest in verifying the expected quality and safety by means of microbiological analyses of food. Measurement brings knowledge and microbiological guidelines help in the decision-making process for judging the acceptability of food or food production processes.
The present handbook provides microbiological guidelines and current applicable EU legal criteria (status 1.1.2018) for a wide range of food categories (dairy, meat, seafoods, plant-based foods, bakery products, composite foods, shelf-stable food, water) and subcategories therein, based upon the type of food processing and intrinsic characteristics of the foods. This book can be consulted to provide quick answers on the expected microbiological contamination of foodstuff. It can help in interpretation of test results in assessing good (hygienic) practices in the production of food, determining the shelf life and ensuring food safety. The handbook also presents definitions of the wide variety of foodstuffs available and some reflections on, in particular, food safety issues or the on-going debate for some food items in assessing microbial quality.

This book provides crucial information about food safety, for the use of students and professionals.

EXTRACT

"First we eat, then we do everything else"
M.F.K. Fisher

Food plays an important part in everyday life. But when being a food scientist or in the food business, food gets to be an even bigger part of your life. Our team at the Food Microbiology and Food Preservation research group (FMFP-UGent) at Ghent University during its academic tasks in education, research, scientific activities at committees, but also in interaction with many food companies and stakeholders in the food supply chain in projects or contract work, has built up considerable expertise on the microbiological analysis of a large variety of foodstuffs. Being situated in Ghent, and thus close to Brussels, the heart of Europe, we intrinsically have to understand and deal with legal EU criteria or action limits. The latter is the reason why this book is mainly oriented towards inclusion or making reference to EU legal microbiological criteria for foodstuffs as well.

ABOUT THE AUTHORS

The main author, Prof. Mieke Uyttendaele, leads, together with Prof. Frank Devlieghere, the Food Microbiology and Food Preservation Research Group (FMFP-UGent) at Ghent University, Belgium. Her teaching and research area covers aspects of microbiological analysis of foods, food safety and food hygiene. She has built over twenty years of experience by executing, initiating and coordinating various projects in this research discipline dealing with sampling and testing to collect baseline data on the microbial contamination of foods, looking into the virulence of food-borne pathogens, elaborating challenge testing to study the behavior of food-borne pathogens. All this information serves as an input for quality assurance and microbial risk assessment to support food safety decision-making and setting microbiological criteria. She was/is the promotor of more than 25 Ph.D students (including EU and non-EU citizens).
Throughout her career, Prof. Uyttendaele has published more than 270 peer reviewed scientific papers, authored several book chapters and presented at numerous international Conferences/Workshops. Throughout the years she has also used her scientific expertise in interpretation of test results for analyses obtained in routine monitoring or analysis executed at the food service lab at FMFP-UGent.

• Language: English
• Publisher: Die Keure Publishing
• Release date: Apr 4, 2018
• ISBN: 9789048632787

Book preview

Belgium

PART I

Microbiological parameters to be used in testing of foods

1. Microbiological quality indicators / spoilage organisms

1.1. Introduction

Only some specific groups of microorganisms are relevant to be used to evaluate the microbiological quality of food products. In most cases, foods in which defined (specific spoilage causing) microorganisms have been proliferating to high levels will show organoleptic defects such as acidity, slime formation, discoloration, gas accumulation (swollen food package) or off-odors and changes in taste due to the presence of high levels of volatile and non-volatile organic compounds. The major causes of early spoilage are:

a highly contaminated raw material;

an insufficient heat treatment and/or an insufficiently (fast) cooling of the product;

a post-contamination of the product during or after processing (including heat treatment) due to a lack of hygiene or a failure in the production process or inadequate control of these processes;

non-respect of the cold chain during transport and further storage of the food product.

Foods can be spoiled by different groups of spoilage organisms. The rate and type of spoilage depends on the level and the type of the initial microbial contamination as well as the intrinsic (pH, aw, preservatives), extrinsic (temperature, relative humidity, presence of gasses, type of packaging) and implicit (interactions between microorganisms) factors of the food product. The proliferation of spoilage organisms is accompanied with the production of spoilage metabolites such as acids (e.g. lactic acid, acetic acid), alcohols (e.g. ethanol), sulphur containing compounds (e.g. H2S, methyl sulphide), amines (e.g. ammonium, trimethylamine), esters (e.g. ethyl acetate), short chain fatty acids (e.g. butyric acid), etc. To determine the shelf life of a food product, it is important to know which and how many spoilage causing microorganisms are present. In many cases, the combination of intrinsic, extrinsic and implicit factors will lead to the selection of specific spoilage organisms (SSO) during storage. This combination of factors will not only determine the type of SSO’s but also the type of metabolism which is used by them. Therefore, also the type of metabolites which are produced and the corresponding sensorial defects will depend on the combination of intrinsic, extrinsic and implicit factors. So, despite similar microbial counts, two identical food products can show a different type of microbial spoilage when stored under different conditions and thus result in sensorial deviations occurring in one product that are not present in the other one.

The most important spoilage indicators for foods are:

Total aerobic or anaerobic colony count

Aerobic or anaerobic sporeforming bacteria and/or spores

Lactic acid bacteria

Yeasts and moulds

1.2. Total colony count

The total colony count refers to the colony count of all vegetative cells and spores of bacteria, yeasts and moulds that are able to grow at the set time and temperature of incubation in or on an agar plate (with an agreed upon composition of the agar medium). Usually in legislation and in many microbiological guidelines or specifications, one refers to the mesophilic aerobic colony count (counting colonies after 3 days of aerobic incubation of inoculated Plate Count Agar (PCA) pour plates at 30 °C). However, most of the microbiological unstable products are stored under refrigeration conditions. It is therefore recommended, when the microbiological quality of chilled food products needs to be determined, to use the psychrotrophic aerobic colony count (4-5 days of incubation of inoculated PCA at 22 °C). Psychrotrophic (cold tolerant) microorganisms represent these group of microorganisms that have the ability to grow in refrigerated food products and are thus mainly responsible for spoilage of these chilled foods. The psychrotrophic microorganisms are to be considered as a subgroup of the mesophilic microorganisms. Strict mesophilic microorganisms have an optimal temperature of 30-37 °C but their minimum temperature for growth is ca. 7 °C making it not possible for them to cause spoilage of refrigerated products. Therefore, the aerobic mesophilic count is not suitable for the estimation of the microbial quality of refrigerated food products and the enumeration of the psychrotrophic aerobic count is preferred to assess the overall microbiological quality of these chilled foods. Psychrotrophic microorganisms are also to be differentiated from the psychrophilic microorganisms. The latter have an obligate lower optimum temperature of 10-15 °C with a minimum temperature for growth below the freezing point and a maximum growth temperature at ca 25 °C. Psychrophilic microorganisms are to be determined through incubation at 7 °C during 7-10 days, but most of them will also still grow at 22 °C. Psychrotrophic microorganisms are able to multiply at refrigeration temperatures (0-7 °C), but their growth at low temperatures is somewhat retarded in comparison to growth at their optimal temperature (20-30 °C). Ideally, the aerobic psychrotrophic count, them being the dominant micro-biota in refrigerated foods, is therefore to be determined through incubation at 22 °C during 4-5 days.

From the above, it is clear that one ‘total’ colony count does not exist, and the colony count determined is always biased by the time, temperature (and atmosphere) of incubation and the agar medium used. Different types of colony counts can be used for microbial analysis of foods. Specific subgroups of microorganisms are defined depending on the incubation conditions (Figure 1). The following subgroups of colony count are deemed to be most appropriate as an indication of initial quality and presence of spoilage causing microorganisms of the food product:

mesophilic colony count: 3 days at 30 °C (for food products which are shelf stable or stored at room temperature);

psychrotrophic colony count: 4-5 days at 20-22 °C (for food products which are recommended to be stored under refrigerated conditions i.e. ≤ 7 °C);

psychrophilic colony count: 7-10 days at 5-7 °C (for food products which are stored on ice or in super cooling (just above the freezing point));

thermotrophic colony count: 1-2 days at 45 °C (for products which are to be stored at elevated ambient temperatures or subjected to these higher temperatures during storage or transport);

thermophilic colony count: 1-2 days at 55 °C (used for canned products in which extremely heat resistant spores might survive but are only able to germinate and grow at elevated temperatures, e.g. in tropical regions or subjected to these high temperatures during storage or transport).

Figure 1: response of microorganisms as a function of storage temperature (Mossel et al., 1995).

PP = psychrophiles, PT = psychrotrophes, ME = mesophiles, TT = thermotrophes, TP = thermophiles, R = response.

Next to temperature and time, also the incubation atmosphere will determine which subgroup of microorganisms will grow on the agar medium and thus be determined in a colony count. When agar plates are incubated under aerobic conditions (in air) the colony count will refer to an aerobic colony count. However, in the latter condition, the growth is not restricted to strict aerobic microorganisms, but all microorganisms which are able to grow under aerobic conditions will be counted, including the facultative anaerobic germs.

For the determination of the anaerobic colony count one incubates the plates under anaerobic conditions (often in an air-tight closed jar in which all O2 is removed or air is replaced by 100 % N2). Anaerobic incubation allows the growth of strict anaerobic bacteria such as Clostridia. However, also facultative anaerobic germs (e.g. lactic acid bacteria, Bacillus) will to some extent grow anaerobically (although more likely to form pin point colonies). Therefore, the ‘total’ colony count cannot be considered as the sum of the aerobic and the anaerobic count as there is a clear overlap in bacterial subgroups between both types of colony counts observed.

For the determination of the anaerobic colony count, it is preferred and standard method to make use of dedicated media (e.g. Reinforced Clostridial Agar (RCA)) containing reducing compounds which lower the redox potential of the medium (e.g. cysteine) and as such promote the growth of anaerobic or facultative anaerobic bacteria. The determination of anaerobic colony counts of fresh meat and fish is typically performed on RCA.

Fresh marine fish and fishery products contain germs which are adapted to the marine environment and often need specific compounds present in this environment to proliferate. For this reason, colony counts on fresh fish, crustaceae and molluscs from marine origin should preferably be performed on a dedicated agar medium such as Marine Agar (MA) or Long and Hammer Agar (LHA) containing specific marine salts and minerals, to avoid an underestimation of the total count when enumerated on a general medium such as PCA.

1.3. Aerobic or anaerobic sporeforming bacteria and/or spores

The presence of spores is an indication of the quality of heat-treated food products. Potentially, these spores are able to germinate and multiply during the further processing or storage of the analyzed food product.

Sporeforming spoilage bacteria or food pathogens belong generally to the genus Bacillus (often specified as aerobic sporeformers, although most of them are also able to germinate and multiply under anaerobic conditions) or Clostridium (often specified as anaerobic sporeformers). Also other related sporeforming genera such as Geobacillus or Alicyclobacillus are food relevant sporeformers.

Examples of food relevant species of the Bacillus genus are Bacillus subtilis, B. pumilus, B. circulans, B. cereus, B. coagulans, B. polymyxa, B. licheniformis, B. macerans while Clostridium perfringens, C. botulinum, C. thermosaccharolyticum, C. pasteurianum, C. nigrificans, C. sporogenes, C. putrefaciens and C. butyricum are examples of food relevant species of clostridia. Two other important sporeforming species that might cause food spoilage are (high heat resistant) Geobacillus stearothermophilus and (high acid tolerant) Alicyclobacillus acidoterrestris.

Pasteurized food products, like cooked chilled foods (also referred to as Refrigerated Foods with Extended Durability or REFPEDs), are usually processed in order for a minimum heat treatment to be achieved. The current minimum heat treatments guarantee food safety based on a defined number of decimal reductions (D-values) of a specific target organism. These heat treatments are considered safe harbors (in Dutch: pasteurisatiebaremas). This implies that they can be readily used as a processing step, without the need for extensive information about the product’s properties or the initial microbial contamination. Safe harbors are usually based on a set of worst case assumptions such as the D-value of the most heat resistant pathogen and the presence of high counts of the pathogen in the product prior to pasteurization. In addition the producers must ensure that all parts of the product are processed sufficiently. These worst-case assumptions are the reason that safe harbors are significantly fail-safe.

A pasteurization can be designed to inactivate the most heat resistant vegetative bacterial pathogen, i.e. generally agreed upon to be Listeria monocytogenes. To achieve this a P70-value of 2 minutes is set to ensure a 6 log10 reduction of L. monocytogenes (a P0-value expresses the effect achieved by the complete non-isothermal heat treatment on the inactivation of L. monocytogenes as the equivalent time in minutes at 70 °C; for more information refer to ‘terminology’ in the interludium).

A pasteurization can be designed to inactivate all vegetative cells (including spoilage microorganisms) i.e. generally agreed upon to be the group of fecal streptococci. To achieve this a P70-value of 40 (heat treatment at 70 °C for 40 minutes or equivalent) is set to accomplish a 12D reduction of fecal streptococci.

A pasteurization can also be designed to inactivate the spores of psychrotrophic Clostridium botulinum strains (type E and non-proteolytic type B and F). These psychrotrophic strains are more heat sensitive than other C. botulinum strains (i.e. the mesophilic more heat resistant Cl. botulinum type A and proteolytic type B and F strains aimed to be destroyed in a canning process). The mesophilic Cl. botulinum strains will have no ability to grow upon refrigerated storage of the pasteurized cooked chilled foods. The main concern, and target organisms in pasteurization of these cooked chilled foods are thus the psychrotrophic Cl. botulinum strains because of their ability to grow at refrigeration temperatures (temperature > 3.3 °C). A heat treatment that achieves a 6D reduction of these non-proteolytic_psychrotrophic C. botulinum is thus the main goal in the production for cooked chilled foods (REPFEDs). This goal is achieved by heating the core of the product for 10 minutes at 90 °C (P90 = 10) or an equivalent non-isothermal process (for more information on the terminology for quantification of thermal inactivation, refer to the interludium).

By using a 10 minutes at 90 °C pasteurization process (or equivalent non-isothermal heat treatment), it may happen that spores of psychrotrophic B. cereus strains survive. In addition, a great deal of these type of cooked chilled foods will because of organoleptic reasons not be able to withstand a P90 = 10-pasteurisation. This thus needs further reflection on the control of microbiological quality of incoming raw material, product composition including the use of organic acids or other preservative agents, storage conditions (time, temperature, atmospheric conditions) and increased efforts to communicate to end-users on the need to respect the indicated shelf life duration and cold chain (usually recommended to be ≤ 5 °C) to prevent germination, outgrowth and toxin production of these psychrotrophic Bacillus cereus spores occasionally present. The safety of these minimal processed foods, in particular if for sensorial reasons not being able to respect a safe harbor heat treatment is thus to be guaranteed by means of a comprehensive risk assessment, dedicated risk management and sampling and testing for verification of the well-functioning of the food safety management system in place (Daelman, 2013).

Bacterial spores are usually resistant towards a 10 min 80 °C heat treatment. So, for the determination of the number of spores in a food product, 10 ml of the sample (when liquid) or 10 ml of the primary dilution (for a solid sample) is heated during 10 min at 80 °C (temperature is monitored via a thermometer in a parallel 10 ml suspension) before plating. This heat treatment enables the killing of all vegetative cells while the bacterial spores will survive. After the heat treatment, the suspension is immediately cooled down on ice, further diluted if necessary and pour plated on PCA or RCA for the determination of respectively the aerobic and anaerobic spore count. Incubation on PCA or RCA happens under respectively aerobic and anaerobic circumstances. When heated spores come into contact with the medium and are placed at ideal growth temperatures, they will germinate, multiply and as such form colonies. The incubation temperature and time can be varied, as was the case for the total colony count, in order to determine specific subgroups of spores such as psychrotrophic, psychrophilic, mesophilic, thermotrophic or thermophilic spores. For more information on the enumeration methods of sporeforming microorganisms and spores, one is referred to Part IV, section 3.2. on microbial parameter and analytical method.

Interludium: Some terminology on quantification of thermal inactivation

1. Classic log-linear (D-/z-) approach

The traditional approach to pasteurization or sterilization uses two parameters to quantify the time-temperature combination required for inactivation.

The first parameter is the D-value or decimal reduction time. This time is defined as the time in minutes needed to destroy 90 % of microorganisms present in the product. The DT -value (D-value at temperature T (°C)) is usually determined by survivor studies, in which the log of the number of surviving microorganisms is plotted versus the heat treatment time at constant temperature. The primary model is given in Equation 1, with N0 and N(t) the bacterial count respectively at the beginning and at time t.

Note that this equation describes the same behavior as the first order model for bacterial inactivation (Equation 2).

The second parameter to determine the time-temperature combination needed for inactivation is the z-value. This value is determined by plotting the log of the DT -value in function of temperature (Figure 2). From figure 2 it can be seen that for any two values of D and the corresponding temperatures T, the z-value can be determined.

Figure 2: Thermal death time curve (DT values as a function of changing heating temperature) to obtain z-value

The secondary model is given in Equation 3 (Bean et al., 2012). With Dref the decimal reduction time at a given reference temperature Tref (°C) and zT the temperature increase (or decrease) needed to reduce (or augment) the D-value with a factor 10.

The D-/z- approach is fairly rudimentary, but it is widely used in the food industry as a generally accepted and practical system. However, the log-linear graphs to determine D-values are best-case scenarios; a number of bacterial inactivation curves will deviate significantly from these semi-log curves. Two commonly occurring deviations are ‘shoulders’ and ‘tails’. In the case of a shoulder, the initial inactivation is slower, resulting in a less steep inactivation curve in the beginning. In the case of tailing, the initial inactivation follows the log-linear approach but flattens out at a certain point. Causes for tailing may be the method of thermal treatment (e.g. insufficient mixing), a heterogeneous population, heat adaptation or the presence of both vegetative cells and spores. When the D-value is determined for mild temperatures (e.g. 60 °C) the vegetative cells will be inactivated, but the spores will survive, resulting in tailing.

When the inactivation curve has a shoulder or tail, the log linear (D-/z-) approach does not give a good fit, alternative models may be used such as a log logistic or Weibull model. Geeraerd et al. (2005) developed a Microsoft® excel add-in for end-users in the food industry. The add-inn can fit nine model types to user-specific data.

2. P and F-values

The D-values discussed above are determined at a specific temperature; hence the DT notation. In order to calculate the heating time required at a certain temperature (T °C) to achieve a certain number of log reduction, it is sufficient to multiply the DT -value with the number of required reductions. The z-value enables the calculation of the D-value at different temperatures.

To determine the heating time needed to achieve a certain inactivation at a different temperature, the lethal rate can be used. The lethal rate is the time needed to achieve an equivalent heat treatment, compared to Tref , at a different temperature (Equation 4). With Tref the reference temperature (e.g. 90 °C), T the heating temperature (e.g. 85 °C) , and t the heating time at Tref. As an example, if z is 9 °C, then 1 minute at 85 °C corresponds to 0.28 min at 90 °C. Note that the unit of L depends on the unit of t (Equation 4).

However, the D-/z- approach can only be used for isothermal heating processes, something that is unlikely given these batch weights and volumes common in industrial food production.

If the heating process is non-isothermal the inactivation can be approximated by integrating the legal rate over the thermal treatment. A calculation that can be approximated by summating the lethal value between measuring intervals (Δt), with Ti the temperature during the measuring interval (Equation 5).

However, this method of extrapolation is preferably not extended outside the range of tested temperatures. Even more important, it should not be taken beyond the limit of biological logic. For example, if temperature decreases, then at some point, temperature no longer has an inactivating but a growth stimulating effect. An example: a L. monocytogenes strain with D70°C = 0.33 and z = 7.5 °C, requires 0.33 minutes at 70 °C to achieve a 1 log reduction. Following the definition of z-value, 3.3 minutes at 62.5 °C will give the same reduction. And theoretically, 33,000 minutes at 32.5 °C will still give the same reduction. However, in reality this temperature will no longer inactivate L. monocytogenes, but allow it to grow.

The intensity of a heating process is expressed as P- and F- values being process values, with F-values used for sterilization and P-values for pasteurization.

A P-value is usually written as: Pz Tref, with Tref the reference temperature and z the z-value of the target organism. An example is given in equation 6.

The P⁷.⁵70 = 2 means that the (non-isothermal) heat treatment applied was equivalent (i.e. had the same lethal rate) to an isothermal treatment of 2 minutes at 70 °C.

For the most common sterilization or pasteurization values, the z-value or even the Tref is no longer included in the abbreviation. It is generally understood that F0 relates to sterilization at 121.1 °C. In theory this does not apply to pasteurization processes, which are flexible in temperature, although also for P-values the z-value of the target organism is nowadays rarely mentioned in superscript, but P-values indicated as e.g. P90, P70 or not even noting the temperature and indicated as P0 (usually 70 °C assumed as temperature).

1.4. Lactic acid bacteria

Lactic acid bacteria are a large group of Gram-positive bacteria and occur as rods as well as cocci. Lactic acid bacteria are acid tolerant and are able to develop at low pH. They have an anaerobic metabolism but are aerotolerant making growth ideal in an environment of 5-6 % O2 (microaerophilic). They are however able to proliferate efficiently in aerobic as well as anaerobic environments. Lactic acid bacteria grow within a broad temperature range (psychrotrophic to thermotrophic), are very nutrient demanding and at the same very competitive. The presence, especially of sugars positively influences their growth. They are ubiquitously spread in the environment, present in the intestinal tract, the sexual system and the skin glands of animals but also on plant materials and in/on the soil. Increasingly, they are also found in (food) production and processing environments, especially in places which are moist, refrigerated and not often/thoroughly cleaned and disinfected. Once they contaminate the food, they are able to multiply very fast, also under refrigeration temperatures, and also in modified atmosphere packed foods with or without CO2, and are as such substantially affecting the shelf life of pre-packed food products with a prolonged shelf life under refrigeration. They are often present (in initial low numbers) in these type of foods, due to post-contamination during food processing. Recent research studies have also shown that these type of lactic acid bacteria, contaminants from the food processing environment, on these foods are often not able (or only very slow) to multiply at 30 °C, the standard incubation temperature for a mesophilic colony count of lactic acid bacteria and thus these type of spoilage bacteria are underestimated if not using a psychrotrophic colony count of lactic acid bacteria at 22 °C (Pothakos, 2013). Enumeration of psychrotrophic lactic acid bacteria is thus preferred in assessing the (initial) microbiological quality of these refrigerated pre-packed foods.

Psychrotrophic lactic acid bacteria often belong to the genus Leuconostoc or Lactococcus and are causing spoilage through acidification, slime formation, gas formation and/or production of off-odors. They are very often introduced into the processing environment through incoming raw materials. Once introduced, they are very fast distributed (within some hours) throughout processing environments, especially when this environment is wet. They are able to form biofilms, and thus may become persistent and establish themselves as an in-house microbial contaminant, although they are susceptible to the classical disinfectants used in disinfection of food processing environments, and thus thorough adherence to cleaning and disinfection is an effective approach to avoid these type of spoilage lactic acid bacteria re-occurring and causing (economic) damage for the food business.

If high numbers of lactic acid bacteria are found in a food product it is recommended that the food product is subjected to sensorial analysis before rejection of the food under consideration. There are subgroups of lactic acid bacteria producing only lactic acid (i.e. homofermentative lactic acid bacteria) and these will upon proliferation only mildly acidify the product. A sensorial deviation due to growth of these homofermentative lactic acid bacteria will usually only be detectable when the numbers exceed 5x10⁷ cfu/g or higher. When heterofermentative lactic acid bacteria are present, they will produce, next to lactic acid, also other metabolites (e.g. acetic acid, ethanol, CO2, slime, …) leading to spoilage. Sensorial deviations can then occur at already lower levels of lactic acid bacteria colony counts. Lactic acid bacteria are also used as starter cultures (fermentations) and can therefore be found at high levels in fermented products without any sign of spoilage. This is for example the case in fermented meat products (salami), dairy products (yoghurt, cheese) and vegetable products (olives, etc.). In summary, lactic acid bacteria often represent beneficial bacteria in foods, which often merely acidify the food, and thus if lactic acid bacteria are the dominant population in the food product under consideration, often higher numbers can be tolerated without adverse quality perception. If thus, high numbers of lactic acid bacteria are encountered in the food, the food product should only be judged unacceptable if indeed unacceptable sensorial deviations are established.

The classical taxonomy of the lactic acid bacteria was based on physiological and morphological properties. Molecular techniques have given new insights in this classification which led to some shifts in the taxonomy as well as the definition of some new genera. The most important lactic acid bacteria genera in the microbial ecology of food are described below. The most important spoilage genera within the group of lactic acid bacteria are Lactobacillus, Leuconostoc, Carnobacterium, Lactococcus, Pediococus and Weissella.

Lactobacillus

Lactobacillus species are abundantly present in food products and will often cause spoilage. Sometimes the spoilage is mild, but obligate heterofermentative strains may cause pronounced spoilage. Some species are very acid tolerant and are therefore known to spoil acid products such as acid sauces (e.g. Lactobacillus fermentum, brevis and plantarum). Lactobacillus is also often used as a starter culture of for example fermented meat products (e.g. Lactobacillus plantarum, sakei, curvatus) and dairy products (e.g. Lactobacillus bulgaricus).

Leuconostoc

Leuconostoc typically occurs on plant materials. However, in the last decade, Leuconostoc species has often been found to be cold tolerant, colonizing processing plants and being responsible for fast spoilage of different types of pre-packed, refrigerated food products (fresh meat, meat products, cooked shrimps, multi-ingredient ready-to-eat salads, …).

Lactococcus

Lactococcus are non-motile cocci and are sometimes found to spoil refrigerated food products (e.g. Lactococcus piscium). Lactococcus lactis subsp. cremoris is often used as a starter culture in fermented dairy products.

Carnobacterium:

This genus contains at present 11 species, from which C. divergens and C. maltaromaticum are most frequently found in food products. They are tolerant for freezing/thawing and high pressure and are able to multiply at low temperatures. They are increasingly associated with spoilage of anaerobically pre-packed refrigerated food products.

Pediococcus

This genus contains several spoilage strains (e.g. from beer and wine) but some Pediococci species are also used as a starter culture of fermented meat products (Pediococcus pentocaceus and acidilactici).

Weissella

Weissella is causing heterofermentative spoilage of refrigerated food products. It often produces slime and sometimes H2O2, causing a typical green discoloration of meat and meat products. The most prevalent species is Weissella viridescens.

Streptococcus

Streptococci have complex nutritional demands. Within the Streptococci, opportunistic pathogens such as S. pneumoniae have been described. S. thermophilus is important as a starter culture in combination with L. bulgaricus for production of yoghurt and cheese.

Enterococcus

Enterococcus species have many applications in foods. Several of them are used as a starter culture in fermented meat, fish, dairy and vegetable products, and thus elevated numbers can be expected to be present in these. However, Enterococcus faecalis and Enterococcus faecium also have a track record of being used as hygiene indicators, although nowadays some strains are also used in their function of starter culture. In some cases Enterococcus species can also cause spoilage.

Tetragenococcus and Vagococcus are two genera of the lactic acid bacteria which are of less importance in foods.

The medium most often used for the determination of lactic acid bacteria is the De Man Rogosa Sharpe (MRS) medium. Because some psychrotrophic strains, most often belonging to the Leuconostoc or Lactococcus genus, have difficulties to grow at 30 °C but grow better at 22 °C, it is important for refrigerated food products that the psychrotrophic lactic acid bacteria are determined to assess microbiological quality (colony count thus after incubation for 4-5 days at 22 °C) instead of the mesophilic lactic acid bacteria (incubation at 30 °C for 72h). Moreover, it is important to realize that some lactic acid bacteria may not show the ability to grow on MRS agar due to the presence of relative large amounts of acetate being present in the MRS agar medium. This is especially the case for Carnobacterium. In the latter case a modified MRS medium without acetate should be used.

1.5. Yeasts and moulds

Moulds and yeasts belong to the kingdom Fungi. Moulds are characterized by mycelial growth, through which they reproduce via production of spores. Yeasts, on the other hand, multiply via knot formation or cleavage (of budding cells) and therefore can form a colony from a single cell.

Yeasts and moulds are omnipresent in the environment in large numbers and can be transferred to food products via contact with insufficiently cleaned equipment or by air. Yeasts and moulds usually dominate the microflora of a food product when the conditions are less favorable for bacterial growth i.e. at low pH or low aw values. These conditions are typical for sour, sweet, dry and fermented foods such as fermented dairy products, fruits, fruit drinks and soft drinks. Although food products with a neutral pH and a high aw are normally spoiled by bacteria, they can also be spoiled by yeasts. This occurs when these foods are initially heavily contaminated with yeasts, which is for example sometimes the case on leafy greens. Outgrowth of yeasts and moulds can lead to a decrease in the sensorial quality of food products. In addition to these spoilage phenomena, it should be noted that yeasts and moulds are desirable in the production process of some types of food products such as some (fermented) meat products, cheeses, alcoholic beverages and bakery products.

With regard to moulds, visual spoilage in the form of visible mycelia is an indicator of mould growth. Given the specific nature by which moulds grow, namely the outgrowth of multicellular mycelium followed in time by the formation of spores, it is pointless to determine mould counts at the end of shelf life. Potential spoilage by moulds at that time can be determined by visual inspection. Due to the fact that moulds are strict aerobes, they are not relevant for products that are packaged in oxygen-free (anaerobic) atmospheres. Visible outgrowth of moulds is only possible in the presence of oxygen.

Unlike moulds, the growth of yeasts is often less conspicuous (visually) and consequently often inadequately taken into account. Yeast growth is accompanied by the production of several types of metabolites such as alcohols, organic acids and esters. In addition, pectinolytic enzymes can be produced that affect the texture of fruits and vegetables (soft rot) and may eventually lead to deterioration of the visual quality. For other types of food products, high outgrowth of yeasts can also be accompanied by visual defects. Significant changes in the sensorial properties of foods are generally observed when yeasts reach counts of 3x10⁵-10⁶ cfu/g.

Important yeast species related to the spoilage of foods include Candida spp., Debaryomyces spp., Rhodotorula spp., Debaryomyces spp., Trichosporon spp., Cryptococcus spp., Pichia spp., Kluyveromyces spp., Saccharomyces spp. and Zygosaccharomyces spp. Zygosaccharomyces spp. can grow at a greatly reduced aw (Z. rouxii) or very acid pH (Z. bailii) values.

Moulds which are often associated with food spoilage include Penicillium spp., Botrytis spp., Rhizopus spp., Alternaria spp., Cladosporium spp., Geotrichum spp., Mucor spp., Aspergillus spp., Eurotium spp. and Fusarium spp. Amongst these are species that are also pathogenic to humans as they produce mycotoxins e.g. Aspergillus flavus (aflatoxins), Penicillium expansum (patulin). It should be noted that mycotoxins are not produced at aw values < 0.83 (Table 1).

Table 1: Minimum aw for growth and toxin production of mycopathogenic moulds.

Source: Erkmen and Bozoglu (2016).

Certain yeasts and moulds can grow at very low water activities. These are generally known as osmophilic yeasts and xerophilic moulds. As examples, the min. aw for the growth of the osmophilic yeast Zygosaccharomyces rouxii and the xerophilic mould Xeromyces bisporus is 0.60. Osmophilic yeasts are usually the cause of spoilage of high-sugar foods, including jams, molasses, corn syrup, flavored syrups and toppings, honey, concentrated fruit juices, chocolate candy with soft fillings, etc. Many of the common spoilage yeasts in the group belong to the genus Zygosaccharomyces. Osmophilic yeasts are of no public health significance, but are of economic importance to the food industry due to causing spoilage in usually assumed shelf-stable foods.

Xerophilic moulds are important shelf life limiting factors for certain food products such as sweets and bakery products which have a low aw (< 0.90). Following the classification proposed by Pitt and Hocking (2009), xerophilic moulds are distinguished from other spoilage moulds as follows:

a xerophilic mould is a mould that is capable of growing out at aw values lower than 0.85.

a xerophilic mould is a mould that grows to a greater diameter on 25 % Glycerol Nitrate Agar (G25N, aw = 0.95) than on Czapek Yeast Extract Agar (CYA, aw = 0.99) and Malt Extract Agar (MEA, aw = 0.99) after 7 days at 25 °C.

The maximum growth rate for non-xerophilic moulds (such as Mucor and Fusarium spp.) occurs at aw ≥ 0.98. Their growth rates decrease when the aw is lowered. In comparison, xerophilic moulds typically show weak or no growth at high aw values (> 0.98) but show good growth from aw 0.95 and lower (therefore the greater diameter on G25N). A few important representatives of xerophilic moulds with regards to foods are Wallemia spp., Eurotium spp. and Xeromyces bisporus. The min. aw for growth of xerophilic moulds is also much lower than those of non-xerophilic moulds (see Table 2).

Table 2: Minimum aw values for xerophilic and non-xerophilic moulds associated with food products with low aw values.

Source: Samson et al. (2010).

It also happens that some osmophilic yeasts and xerophilic moulds are so well adapted to growth at low aw values that they can no longer grow (reproduce) at higher aw values. These are known as extreme osmophilic yeasts or extreme xerophilic moulds. Their inability to grow at high(er) aw values means that they are not taken into account by classical methods used to isolate and/or enumerate yeast and moulds. For the determination of osmophilic yeasts and xerophilic moulds, specific media must be used with a reduced aw which are incubated for a long period of time. For more information on the enumeration methods of xerophilic moulds and osmophilic yeasts, one is referred to Part IV, section on Methods.

Note that sometimes the term ‘xerotolerant’ is used instead of xerophilic. Xerotolerant refers to those moulds which tolerate low aw values but do not prefer them (= do not need low aw values for growth to occur). For example, isolates of Wallemia sebi and Eurotium amstelodami can grow within a short period of time (10 days) on media with a low aw (i.e. 50 % sucrose = aw 0.87) but are also able to grow at aw 0.95.

Mycelia (hyphae) and ordinary conidia (spores) are generally not heat resistant, typically being inactivated by pasteurization temperatures as low as 60 °C. However, certain moulds are known to be heat resistant by virtue of their ability to produce ascospores. These fungi belong to the class Ascomycetes and include species belonging to the Byssochlamys, Neosartorya, Talaromyces and Eupenicillium genera. To date, ascospores are widely accepted as the most heat resistant structures of filamentous fungi (Dijksterhuis, 2007). The ascospores of heat resistant moulds (HRMs) usually occur in groups of eight located within a sac known as an ascus (Figure 3). Asci with 16 or 32 ascospores occur less often. The asci are often enclosed in cleistothecia, with each cleistothecium consisting of clusters eight asci.

Figure 3 Illustration of the arrangement of ascospores into ascus and asci into a cleistothecium.

Source: Simbarashe Samapundo, FMFP-UGent.

The heat resistance of ascospores differs widely depending on species, strain, age, heating medium, pH, growth conditions, etc. (Tournas, 1994). Although not yet fully elucidated, the heat resistance of HRMs is in part attributed to the unusually very thick cell walls of the ascospores and their arrangement into protective asci and cleistothecia (Figure 3). Examples of D-values of some common HRMs are shown in Table 3.

Table 3: D-values of selected heat resistant moulds (HRMs).

HRMs are of great importance to high acid food products, i.e. food products, as they survive the thermal treatments (pasteurization) employed and subsequently germinate and cause spoilage. According to Tournas (1994), product recalls in the order of millions of dollars occur every year due to damage caused by HRMs to fruit and fruit products. In addition to spoilage, some HRMs are of public health concern as they can produce a number of toxic secondary metabolites, such as byssotoxin A, byssochlamic acid, the carcinogen, and patulin.

1.6. Specific Spoilage Organisms (SSO)

The microbial spoilage of food products can occur due to the proliferation of several groups of microorganisms such as Gram-negative proteolytic bacteria, Gram-positive non-sporeforming bacteria (e.g. lactic acid bacteria, Brochothrix thermosphacta, micrococcaceae), sporeforming bacteria, (osmophilic) yeasts and (xerophilic) moulds. After processing, the food will contain a large variety of microorganisms. Due to the specific combination of intrinsic, extrinsic and implicit factors occurring during storage of foods, specific groups of microorganisms will start to proliferate and the microbial variety will decrease. Therefore, at the end of the shelf life, the microbiome will be dominated by one or some strains of a specific group of microorganisms. This group of microorganisms are called the Specific Spoilage Organisms (SSO) of that specific food product. It is important that these SSO’s are part of the microbial parameters which are chosen to be used for analysis and judgement of the microbiological quality at the end of the shelf life of a food product. If one aims to identify or detection prior established particular specific spoilage organisms, special culture media may need to be developed (e.g. mimicking the exact food composition or based upon an extract of the food to prepare agar medium) or alternative molecular techniques or instrumental methods could be considered and may be useful. It is recommended, in order to link the specific spoilage organism indeed to spoilage and elaborate an effective approach to inhibit the SSO’s growth or its potential for spoilage, that if the SSOs are identified, to collect more information on the metabolites produced by the SSO, the substrates and metabolic pathways being used by the SSO and the behavior and competitiveness of the SSO within the microbial community of the food under consideration. An example of such an approach to determine and characterize SSOs is described in some PhD studies elaborated at FMFP-UGent (Noseda, 2012; Pothakos, 2013).

References and further reading

BEAN, D., BOURDICHON, F., BRESNAHAN, D., DAVIES, A., GEERAERD, A., JACKSON, T., MEMBRE, J., POURKOMAILIAN, B., RICHARDSON, P. and STRINGER, M., 2012, Risk assessment approaches to setting thermal processes in food manufacture, ILSI Europe Report Series, 2012, 1-40.

BEUCHAT, L. R., 1986, "Extraordinary heat resistance of Talaromyces flavus and Neosartorya fischeri ascospores in fruit products", Journal of Food Science 51, 1506-1510.

CONNER, D. R. and BEUCHAT, L. R., 1987, "Efficacy of media for promoting ascospore formation by Neosartorya fischeri, and the influence of age and culture temperature on heat resistance of ascospores", Food Microbiology 4, 229-238.

DAELMAN, J., 2013, "Quantitative microbiological exposure assessment of Bacillus cereus in cooked-chilled foods", PhD Dissertation submitted in fulfillment of the requirements for the degree of doctor (PhD) in Applied Biological Sciences, Faculty of Bioscience Engineering, Ghent University.

DEVLIEGHERE, F., RAJKOVIC, A., SAMAPUNDO, S., UYTTENDAELE, M., JACXSENS, L., VERMEULEN, A. and DEBEVERE, J., 2015, Food Microbiology and Analysis, Course Ghent University.

DIJKSTERHUIS, J., 2007, Heat resistant moulds. In: Food Mycology: a multifaceted approach to fungi and food in DIJKSTERHUIS, J., SAMSON, R. A. (eds), CRC Press, Taylor & Francis Group, Boca Raton, FL, US, pp. 101-117.

ERKMEN, O. and BOZOGLU, T. F., 2016, Food Microbiology: Principles into practice. Volume 1: Microorganisms related to foods, food borne diseases, and food spoilage, John Wiley & Sons Ltd.

GEERAERD, A., VALDRAMIDIS, V. P. and VAN IMPE, J., 2005, Ginafit, a freeware tool to assess non-loglinear microbial survivor curves, International Journal of Food Microbiology 102, 95-105.

KOTZEKIDOU, P., 1997, "Heat resistance of Byssochlamys nivea, Byssochlamys fulva and Neosartorya fischeri isolated from canned tomato paste", Journal of Food Science 62, 410-412.

MICHENER, H. D. and KING, A. D., 1974, "Preparation of free heat-resistant ascospores from Byssochlamys asci", Applied Microbiology 27, 671-673.

MOSSEL, D. A. A., CORRY, J. E. L., STRUIJK, C.B. and BAIRD, R. M., 1995, Essentials of the microbiology of foods: a textbook for advanced studies, John Wiley & Sons Ltd., ISBN 0 47193036 9.

NOSEDA, B., 2012, Volatile spoilage markers indicating bacterial spoilage in modified atmosphere packaged fish and fishery products, PhD Dissertation submitted in fulfillment of the requirements for the degree of doctor (PhD) in Applied Biological Sciences, Faculty of Bioscience Engineering, Ghent University.

PITT, J. I. and HOCKING, A. D., 2009, Fungi and food spoilage, Springer.

POTHAKOS, V., 2013, Psychrotrophic lactic acid bacteria (LAB) as a source of fast spoilage occurring on packaged and cold-stored food products, PhD Dissertation submitted in fulfillment of the requirements for the degree of doctor (PhD) in Applied Biological Sciences, Faculty of Bioscience Engineering, Ghent University.

SAMSON, R. A., HOUBRAKEN, J., THRANE, U., FRISVAD, J. C. and ANDERSON, B., 2010, Food and Indoor Fungi, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.

SCOTT, V. N. and BERNARD, D. T., 1987, "Heat resistance of Talaromyces flavus and Neosartorya fischeri from commercial fruit juices", Journal of Food Protection 50, 18-20.

TOURNAS, V., 1994, Heat-resistant fungi of importance to the food and beverage industry, Critical Reviews in Microbiology 20, 243-263.

2. Hygiene indicators

2.1. Indicator organisms to serve as an indication of good ‘process hygiene’

The term indicator organisms has been suggested by Ingram (1977) (cited in Mossel et al., 1995) for those microorganisms whose presence in given numbers point to failure to comply with applying Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP), or Good Hygienic Practices (GHP). Usually these ‘good practices’ aim to reduce the negative impact of stages in food processing prone to the increase of microbial contamination of foods, or aim at avoiding the spread or further growth of microorganisms if contamination has occurred. As such these ‘indicator’ organisms are mainly used to monitor process hygiene. Exceeding the set threshold limits indicates evidence of poor hygiene, inadequate processing (insufficient heat-treatment or any other non-thermal treatment to accomplish inactivation of microorganisms), process failure or post-process contamination of foods. In some situations these indicator organisms are selected to serve as an objective measurement indicating fecal contamination instead of less effective visual evaluation performed by inspectors.

2.1.1. Selection of indicator organisms

Several bacteria can be selected as (process) hygiene indicators. The indicators typically consist of Enterobacteriaceae, coliform bacteria, enterococci or Escherichia coli, (Baylis and Petitt, 1997; Mossel et al., 1998; Busta et al., 2003; Suslow et al., 2003), but also coagulase positive staphylococci or aerobic colony count are sometimes used to evaluate hygienic working conditions or failures in control measures. Escherichia coli is commonly used to provide evidence of potential fecal contamination in certain foods.

Enterobacteriaceae versus coliforms versus thermotolerant coliforms versus E. coli

The family Enterobacteriaceae comprises a large group of Gram-negative, non-spore-forming bacteria. Enterobacteriaceae ferment a variety of carbohydrates, but their ability to produce acid and gas from the fermentation of D-glucose is one characteristic that remains an important diagnostic property and is commonly used as a basis for their detection and enumeration.

Some members of the Enterobacteriaceae (e.g. Enterobacter spp., Citrobacter spp., Klebsiella spp. and Escherichia including E. coli) can be recognized using methods that exploit their ability to rapidly ferment lactose (usually within 24-48 h) producing acid and gas. The latter are collectively termed coliform bacteria. But also other species including Hafnia alvei and strains belonging to genera such as Buttiauxella, Leclercia, Pantoea, Serratia, Yersinia, etc. may rapidly ferment lactose and are thus recognized by conventional culture methods as coliforms. Bacteria outside the Enterobacteriaceae, notably Aeromonas spp., can also ferment lactose and these can be falsely detected as coliforms using some methods if no additional confirmatory tests are performed. Consequently the group of coliforms are usually defined by the method used. Currently there is no taxonomic basis for this coliform group and there is no consensus view of which genera or species should be included (Baylis et al., 2011).

Historically, coliforms were the most common indicator group used as a hygiene indicator in the milk and dairy industry. Milk and dairy products are lactose-containing food for which the number of Enterobacteriaceae is assumed (and has been shown) to coincide with the number of coliforms. Also water treatment to achieve potable water has a long track record of using coliform testing to control effectiveness of treatment procedures (including chlorination). Within the EU, process hygiene criteria now mentioned in EU Regulation 2073/2005 on microbiological criteria of foods have moved towards testing for Enterobacteriaceae instead of coliforms, also to monitor process hygiene of milk and dairy products. The reason for this being mainly that coliforms are an ill-defined taxonomic group defined by the method used and this lack of good definition in the coliform group can present problems for international trade. Still in many EU member states, coliform counts are still used to assess the (premium labelled) quality of raw milk or in the framework of self-checking systems to assess hygiene at the farm. Testing water for coliforms has remained in the EU, not least because specific guidelines and regulations demand coliform testing. However, if testing of coliforms is still in use, the majority of the labs will be using alternative rapid methods, often using chromogenic media not needing further confirmation steps to determine coliforms count.

It needs to be highlighted that when testing foods for Enterobacteriaceae or coliforms as indicator organisms for unhygienic food processing, the significance of the results obtained must be put into context with the type of food under examination and the stage in the food chain selected for sampling (Baylis and Petitt, 1997).

Their ubiquitous distribution means that it is inevitable that some members of the Enterobacteriaceae will enter the food chain, and if insufficient heat or other non-thermal inactivation is present during food processing, different numbers of Enterobacteriaceae counts are expected to occur in these foods. This is especially important with foods