Microbiology

Fungal species frequently spoiling fresh tomatoes belong to genera such as Alternaria, Cladosporium, Botrytis, Rhizopus, Mucor, Colletotrichum and rarely to Fusarium, Penicillium e Aspergillus. As a consequence, the presence of mycotoxins actually regulated at an European level (patulin, aflatoxins, ochratoxin A, zearalenone, fumonisins, tricotecens) is not considered as a risk to human health in tomato products. On the contrary, recent studies registered their contamination by Alternaria toxins (tenuazonico acid, alternariol, alternariol monomethyl ether) on such products up to high concentrations.

1. a) Penicillium expansum is considered as the main responsible for patulin production in fresh fruits, but it is not included among associate mycobiota of fresh tomatoes. In fact, it is the main patulin producer in pomaceous fruits (appples, pears) and, to a lesser extent, in stone fruits (peaches, apricots, cherriwes, plums).

Anyway, being tomatoes considered as a fruit by the European directive No. 2012/12/UE, the only tomato product where a threshold limit has been established by the European Union is tomato juice, where the maximum admissible value of such toxin is 50 µg/kg.

1. b) Aflatoxins, ochratoxin A. As for patulin, the aflatoxin- (Aspergillus Flavi) and ochratoxins- (Penicillium verrucosum, Penicillium nordicum, Aspergillus ochraceus, Aspergillus carbonarius) producing species are not included among the associated mycobiota of fresh tomatoes. With regard to this, studies carried out at the SSICA registered a progressive reduction of aflatoxins during a 90-days incubation at room temperature in artificially inoculated tomato products (purees, pastes).

For aflatoxins and ochratoxin A, a threshold limit has not been established at a European level in tomato products.

1. c) Fusarium-toxins. Some species of Fusarium such as equiseti, F. moniliforme (verticillioides), F. oxysporum and F. solani can occasionally spoil fresh tomatoes, although they are not considered as the main responsible of decay on such fruits. Furthermore, literature data have not still reported information concerning the production of zearalenone, deoxynivalenol and fumonisins by Fusarium species in fresh products. For these reasons, Fusarium-toxins are not considered relevant on tomato products. Consequently, the presence of such phytopatogenic fungal species is just addressed by means of preventing interventions on crops.

For Fusarium-toxins, a threshold limit has not been established at a European level in tomato products.

1. d) Alternaria- Tomatoes can be considered as an elective substrate for the growth of Alternaria species (A. alternata, A. arborescens species-group, A. tenuissima species-group) and for the resulting production of tenuazonic acid (TA), alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) e tentoxin (TEN).

For Alternaria-toxins, a threshold limit has not been still established at a European level on any food.  Anyway, the Bavarian health and food safety authority has set a maximum admissible level of 500 µg/kg for tenuazonic acid in sorghum- and millet-based infant foods.

Patulin is not normally present in lemon juice and citrus products in general. In fact, Penicillium expansum is the main cause of spoilage and patulin contamination of fresh fruits such as apple, pear and, to a lesser extent, stone fruits (peach, apricot, cherry, plum) and grapes, while it is not the main cause of citrus fruit spoilage.

Furthermore, the synthesis of patulin by Penicillium expansum is inhibited by the orange and lemon essential oils.

In a study conducted at SSICA on clear apple juices, apricot nectars and tropical fruit it was observed that patulin decreases significantly during 1, 2 and 3 months of storage respectively. In the presence of sulfur groups, patulin reacts rapidly, forming adducts with low or no toxicity.

First of all, the cause of the fungal growth is often the bad preservation of the product already opened by the consumer. In any case, “acute phenomena” or immediate and specific injuries to internal organs, which can be caused by accidental ingestion of industrial beverages in which the presence of fungal mycelium, only after consumption, is detected, are not documented in humans. (a presence which, considering the type of product and the container, can visibly manifest itself with dimensions that at most reach a few centimetres).

It is believed that any abdominal and retching symptoms, sometimes reported by consumers immediately after the accidental ingestion of such products, are due to causes of another nature and/or to the conditioned nervous reflex mechanism.

Aliciclobacilli are gram-positive, sporogenic, acidophilic, strictly aerobic thermotolerant bacteria; their natural habitat is the soil and terrestrial or watery acid thermal springs. The spores are able to germinate at a low pH value. The species responsible for the spoilage of fruit products is Alicyclobacillus acidoterrestris: it does not produce gas and does not change the pH of the product, but the metabolites produced after growth cause the formation of a “medicine like” aroma which spoils its sensory properties (polyphenols, especially guaiacol). This microbial species has a heat resistance of 2-3 minutes at 95°C. The heat treatments imparted to non-concentrated tomato products are generally sufficient to inactivate these spores. However, in recent years there have been several cases of spoilage of non-concentrated tomato products by aliciclobacilli. Spoilage was suggested by consumers who perceived anomalous odours and flavours (“off”) of the product. It was verified that Alicyclobacillus acidocaldarius is responsible for the spoilage of these products. It does not produce guaiacol, a metabolite indicated in the bibliography as the main cause of the “off-flavour” in fruit juices. Moreover, heat-resistance tests showed that the spores of the strains isolated from tomato products were much more resistant (D105 variable from 5 to 15 min) compared to the spores of the A. acidoterrestris: as the heat treatments applied to tomatoes are more drastic than those applied to fruit, the spores of the A. acidocaldarius are thus the only ones able to germinate and grow in tomato products. In order to reduce the risk of spoilage by these micro-organisms in the finished product, the high quality of the raw material, adequate washing and good hygienic working conditions are of fundamental importance. It is also essential to perform a rapid cooling after heat treatment and store the products at temperatures no higher than 25°C to prevent the germination of the surviving spores.

What is the meaning of the presence of spore-forming thermophilic bacteria in fruit products?

Spore-forming thermophilic bacteria develop optimally at a temperature of 55°C; they have been subdivided into optional and obligatory thermophiles according to whether the minimum growth temperature falls or not in the thermal growth range of mesophilic bacteria. The obligate thermophilic bacteria are defined stenothermophilic if unable to multiply at 37°C; optional thermophiles are called eurithermophiles and can grow even at temperatures equal to or below 37°C. The spoilage of preserved foods caused by thermophilic bacteria is generally due to inadequate cooling after heat treatment and/or storage at high temperature. Spore-forming thermophilic bacteria responsible for spoiling food preserved foods are attributable to the species Bacillus coagulans, Geobacillus stearothermophilus (formerly Bacillus), Thermoanaerobacterium Thermosaccharolyticum (formerly Clostridium), Alicyclobacillus spp. Since the thermal treatments applied to preserved foods, whether acid or not, definitely inactivate their vegetative cells, the possibility that they spoil preserved foods is limited to the possibility that their spores (the spore is a biological structure resistant to heat) are capable of germinating (revitalizing) and developing into cells capable of active multiplication. This possibility is, however, a function of the pH values of the foods. In fact, there are pH limit values below which germination is not possible; they are, with very rare exceptions reported in the literature, for G. stearothermophilus pH equal to 5.1, for T. Thermosaccharolyticum pH equal to 4.3, for B. coagulans pH equal to 4.2 and for Alicyclobacillus spp. pH equal to 2-2.5. The thermal inactivation of these spores, however, is not always possible; the first two thermophilic bacteria mentioned above produce spores of such thermal resistance as not to be inactivated even with the usual thermal sterilization processes applied to non-acid preserved foods. It is necessary, in fact, to reach lethality values equal to F121=28-32 minutes to obtain a reasonable probability of microbiological stability at high cooling and/or storage temperatures. For the microorganisms B. coagulans and Alicyclobacillus, on the other hand, by performing heat treatments referable to F100 with variable duration depending on the pH of the product, it is possible to obtain microbiological stability at ambient temperatures higher than the average of temperate climates. From what has been reported, it is possible to state that the search for spore-forming thermophilic bacteria in high-acid preserved foods with low pH values, such as fruit products, is justified only with respect to the micro-organisms belonging to the genus Alicyclobacillus. These bacteria can be defined as optional thermophiles and spoil the product without producing gas but producing bad-smelling substances such as guaiacol; they, being strict aerobes, develop only in preserved fruit products which, due to the way they are packaged, contain sufficient amounts of oxygen in the head space of the package and/or in the product itself. It should also be noted that the search for spores of the bacteria B. coagulans, G. stearothermophilus and T. Thermosaccharolyticum in preserved fruit products can be successful as they, being of telluric origin, are present in varying degrees in the raw materials and the use of culture media at pH values close to neutral allows the germination otherwise impossible in products due to their low pH value. These findings have no diagnostic value with regard to the spoilage of the product.

Preserved food products are divided into two types depending on whether their pH value is higher than 4.6 or lower/equal to 4.6. Different thermal treatments are given to the two groups so differentiated; in particular, microbiological stabilization or sterilization treatments are applied depending on whether the pH values are, respectively, lower/equal to 4.6 or higher than 4.6. This is due to the different growth ability, depending on the pH value, of the pathogenic bacterium Clostridium botulinum, responsible for lethal intoxications due to neurotoxins. In fact, scientific data show that the minimum pH limiting the C. botulinum spore germination is equal to 4.6. This bacterium is therefore not capable of multiplying, in the form of spores, at lower values; moreover, the vegetative cells of C. botulinum, as well as the cells of all the other microorganisms present in food, are certainly inactivated by the microbiological stabilization heat treatment because cell resistance to heat is very low. In preserved foods, which have been given a microbiological stabilization heat treatment, with a pH lower than/equal to 4.6, the growth ability of C. botulinum and, therefore, the production of lethal neurotoxins is inhibited. The low pH value (and the high acidity) of these preserved foods is, by itself, sufficient to inhibit spore outgrowth and the growth of the afore-mentioned bacterium.

For naturally non-acid vegetable products, therefore, correct acidification with acidity correctors such as citric acid, acetic acid, lactic acid, etc. is of fundamental importance. This technological operation that also includes the continuous control of the pH value before packaging in order to ensure homogeneity. The control of preserved foods with pH values ≤ 4.6 is performed by the test method called “Microbiological stability”. According to the method, the sample is analyzed after incubation at 30°C for 14 days in order to verify the presence of spoilage microorganisms, possibly present in the product already heat treated and cooled, even at concentration of a single cell per container, regardless of the capacity of the latter. Therefore, the analysis of “microbiological stability” is aimed at verifying the presence of microorganisms capable of growing at the aforementioned pH values (depending on the product: lactic bacteria, fungi, enterobacteria, B. coagulans); it is carried out in qualitative mode, using exclusively selective media for these microorganisms. Therefore, qualitative or quantitative analyses are not carried out on general culture media such as PCA, TSA, SA, TSC, OPSP etc., since in these occurs growth of bacterial spores of the genus Bacillus and/or Clostridium, generally present in the raw materials, not inactivated by the heat treatments given to preserved acid foods in order to obtain their microbiological stability. The heat treatments imparted to preserved foods with a pH lower than/equal to 4.6, in fact, are not intended to inactivate all the spores present in the raw materials but only those capable of germinating (turning back into metabolically active cells) even at those values of pH and spoil the product. The spores not inactivated by heat treatments do not germinate and, therefore, a bacterial flora capable of spoiling the food does not grow, due to its acidity and pH values. These microbial structures remain, therefore, in such preserved foods in silent or dormant form but grow only when they come into contact with general culture media whose pH values are significantly higher than those of preserved acid foods. This analytical finding has no significance for the purpose of microbiological stability of preserved foods having pH ≤ 4.6.

The initial microbial flora of spices is the same as other agricultural products harvested in similar soil and climatic conditions. This flora is therefore made up of micro-organisms typical of the soil and plants in which they have grown and which survive the drying process. External microbial contamination processes due to insects, dust and, sometimes, processing water can obviously occur. It is also true, as reported by scientific studies, that the total count decreases during storage as long as it is carried out in hygienic as well as temperature and humidity suitable conditions. In general, the risk of food spoilage and infection and/or poisoning represented by the use of spices as food ingredients should be carefully evaluated in the context of the use of the spices themselves. In this regard, an important organization such as the ICMSF (International Commission on Microbiological Specifications for Foods) considers the spices in the “Case 2” of the Sampling Plans:

Type of danger: they are not a direct health hazard; Conditions in which the food is handled and consumed: they do not cause changes in the extent of the danger. The hazards and risks are therefore considered not high; only the “total microbial count” analysis is required. The values of this analysis as reported in scientific publications are always high (up to 10E7 cfu/g) also for spices usually produced in western countries under controlled conditions. Referring more specifically to the finding of bacteria belonging to the Enterobacteriaceae family, it must be pointed out that it cannot be considered an exclusive index of faecal contamination or fouling as many species of these microorganisms are normal components of the microbial flora typical of vegetable raw materials not contaminated, moreover, by excrements of any origin. Most of these species do not show any pathogenicity to humans. Furthermore, the drying processes to which spices are subjected as a fresh product cause an increase in cell concentration per gram due to the significant reduction in the total quantity of water contained at the origin. The presence of total coliforms (a group in any case belonging to Enterobacteriaceae) is no longer considered, for the reasons mentioned above, as an index of faecal contamination. A more reliable indicator of faecal contamination is represented by the E. coli species, as it has a certain intestinal origin. The baking processes to which the products of which the analyzed spices may be an ingredient are subjected, are a very effective means of reducing the total count of the product. Moreover, this cooking process significantly reduces the humidity and water activity values by not allowing, in fact, any microbial growth starting from the possibly residual microbial count, if preserved by the environmental humidity through correct packaging.

One of the causes of spoilage of preserved foods is contamination after heat treatment (or re-contamination). This phenomenon consists in the penetration of microorganisms, generally conveyed by water, during or after the cooling of the packages. In this phase of the production cycle, in fact, the contact of the package, very hot, with cold water results in such a degree of vacuum that it can withdraw, through permanent (due to packaging defects) or temporary (micro leaks) leaks, fluids from the external environment. The possibility of spoilage depends on the degree of vacuum inside the can, the concentration of microorganisms in the cooling water, the type of microorganisms, o the shape and size of the leaks, the contact time. Different types of microorganisms can cause spoilage of the re-contaminated products: bacteria and fungi vegetative cells, spores of aerobic and anaerobic mesophilic bacteria, sometimes pathogenic bacteria. In any case, the degree of contamination of cooling water is particularly important; the higher the microbial concentration in the water, the greater the probability of product spoilage. Water must therefore meet certain microbiological requirements; moreover, even in the case of supplies of water that has already been purified (from the aqueduct), recycling generally carried out during production causes a significant increase in the concentration of microorganisms. In order to make the likelihood of spoilage of the packages as low as possible, and therefore find the least possible number of spoiled packages, it is necessary to resort to disinfection treatments to inactivate the largest possible number of microorganisms. For this purpose, chemical substances are used such as chlorine gas, ozone, sodium hypochlorite or physical means such as U.V. Moreover, food manufacturing companies are required to use drinking water that complies with legal requirements at all stages of production.

Preserved acid vegetable foods (with pH values lower than 4.6) can be spoiled exclusively by lactic acid bacteria, B. coagulans, enterobacteria, butyric clostridia, thermophilic clostridia, yeasts and moulds. Differing types of microorganisms are not able to grow in such products due to low pH values and high acidity. Therefore, the microbiological analysis called stability, in which the sample is analysed after incubation at 30°C for 14 days, is aimed at verifying the presence of the aforementioned microorganisms using exclusively selective culture media for them. The analysis called “total microbial count” (which requires the use of general culture media such as PCA, TSA, etc.) is not carried out, since in these media growth occurs of bacterial spores of the genus Bacillus, however present in all raw materials. In fact, the heat treatments given to acid preserved foods do not provide for the inactivation of all the species of spores of the genus Bacillus (similarly for the spores of the genus Clostridium), since most of them are however incapable of germinating and spoiling the product due to its acidity and pH values. Therefore, these microbial structures (spores) remain in these preserved foods in silent or dormant form but grow only when they come into contact with general culture media. Therefore, the analysis called “total microbial count” has no meaning referring to the commercial stability of the product; however, this analysis is not even used to determine the commercial sterility of products with pH> 4.6, which envisages completely different operating methods and culture media. Furthermore, there is no maximum limit of cfu/g relating to the determination of “total microbial count” neither officially nor unofficially.

What is the meaning of the presence of sulphite reducing clostridia in acidified products?

The presence of cells and spores of sulphite reducing clostridia in food raw materials is common and frequent, as is that of other sporogenic bacteria. The normal practices of preservation and processing of agri-food products such as pasteurization, sterilization, refrigeration, freezing, fermentation, salting, pickling, drying, etc. have the specific purpose of inactivating microbial cells and spores, preventing spore outgrowth or inhibiting cell multiplication. In the specific case of food raw materials preserved in acetic acid, the high concentrations of acid used excellently perform the task of preservation since acetic acid has the effect of inactivating most microbial cells and of preventing spore outgrowth, also due to the decrease in the pH of the products.

In particular, as regards pathogenic sulphite reducing clostridia, it should be noted that:

• the growth of Clostridium botulinum is inhibited by NaCl concentrations equal to or greater than 10% and pH values lower than 4.6;
• the growth of Clostridium perfringens is inhibited by NaCl concentrations equal to or greater than 5% and pH values below 5.0.

The counting of sulphite-reducing clostridia in acidified foods therefore reveals almost exclusively the spores of these pathogenic and non-pathogenic bacteria (eg Clostridium sporogenes) which do not have the ability/possibility to germinate and give rise to product spoilage.

Therefore the presence of spores in a raw material does not cover a particular meaning of pathogenicity and the possibility of growth depends exclusively on the transformation practices following the washing of the acidified product.

Moreover, the presence, even small, of sulphite-reducing clostridia in acidified raw materials will always be found since the practices preceding the acidification phases (sorting, cutting, washing, etc.) cannot determine their complete removal from the product.

It should also be noted that the industrial processes following washing tend:

• in the case of acidified products with pH lower than 4.6 and thermally stabilized, to inactivate the microbial cells and prevent spore germination;
• in the case of non-acidified, but sterilized products, to inactivate both the cells and the mesophilic microbial spores.

The correctness and the effect of the production processes of preserved foods are controlled and established through the application of “Good Manufacturing Practices” and HCCP System.

It consists of a laboratory simulation of what happens to a product during production, distribution and handling. It involves the inoculation, under controlled environmental conditions, of a significant number of micro-organism under study in order to assess the risk of food-borne. Basically two types of MCT are known:

• Process MCT – It involves the inoculation, under controlled environmental conditions, of a significant number of the microrganism being studied in the raw material and the evaluation of what happens to the microrganism during the production process. It can provide validation of production processes
• Product MCT – It involves the inoculation, under controlled environmental conditions, of a significant number of the microrganism under study in the finished product, the packaging of the product under the marketing conditions and the evaluation of what happens to the micro-organism during shelf-life. It can provide a scientific justification for the correct positioning of RTE products in the food categories for Listeria monocytogenes provided for in Reg. 2073/2005.

The execution of an MCT is preceded by a planning phase, during which a series of considerations relating to the specific food being tested are processed. The factors examined in this preliminary phase are:

• the production process and its effects on the trend of the microbial population in the finished product
• the microbiological profile of the raw materials and the finished product
• identification of the risks of food poisoning and spoilage of the product
• types and characteristics of micro-organisms potentially dangerous for the product
• growth capacity of micro-organisms potentially dangerous for the product.

In the food sector, are there emerging fungal species with regard to resistance to the heat treatments given to beverages?

Yes. From the most recent literature data, some strains belonging to the Paecilomyces genus have been to be proved responsible for spoilage cases of thermally treated beverages. Although the tests to ascertain their heat-resistance have not given satisfactory results (RA Samson, ES Hoekstra and JC Frisvad, in Introduction to Food- and Airborne Fungi, 7th ed., CBS, Utrecht, 2004; E. Pieckova and RA Samson, J. Ind. Microbiol. Biotechnol., 24, 227 (2000), it is assumed that the particular structures with which these molds are equipped (abundant chlamydospores and thick-walled hyphae) have allowed them to develop a certain resistance to heat.

The fungal spoilage of beverages can be attributed to heat-resistant moulds or, in most cases, to non-heat resistant moulds. In the first case, the fungal spores are present upstream in one or more ingredients used for the preparation of the product and are not inactivated by the thermal treatment applied. In the second case, contamination occurs after the heat treatment and is due to recontamination of the container, if the shape of the cap does not allow the hermetic closure of the bottle. In particular, recontamination after heat treatment takes place because, in the presence of nutrients and water or just moisture, the mold spores present in the atmosphere can germinate and their hyphae penetrate the bottle by apical growth through the defective points between the cap and the neck. The necessary condition for this penetration to take place is that in the closure defect there is a veil or a continuous column of liquid. The recontamination following the heat treatment and the lack of hermetic closure between the cap and the bottle can be evaluated through a careful analysis of the different closure points of the bottle (neck of the bottle, cap, stopper) and of the product. In this case, when the bottle is opened, all the bottles may have drops of product along the thread and either at these points or inside the product one or more non-heat resistant fungal species is detected.

Yes, even after many months from production. Flexible or semi-rigid containers can have defects such as breaks or micro-holes, due to incomplete welding, which can cause the microbial contamination of an acid product. The penetration of microorganisms can take place quickly, within a few days, especially if the microorganisms capable of contaminating or spoiling the product are mobile, or longer (several days, weeks or more), if the product is contaminated by non-mobile microorganisms such as mycetes. This latter possibility becomes relevant if the product is subjected to handling due to long transport and a long storage period.

In this case the moulds become the main cause of spoilage of semi-finished acid products, in which they can also produce large quantities of mycelium, which grows above all on the surface of the product, without necessarily causing the swelling of the bag. Moulds, in fact, can behave like mobile microorganisms, thanks to their typical growth, which occurs by apical growth of the hyphae (the hyphae are the constitutive cells of the mycelium and have a tubular shape).

In particular, they can also grow in the presence of little moisture and dirt traces: their spores, always present in the dust, in the organic substance exposed to the air and in the air itself, after depositing on the surfaces of the containers, can germinate and penetrate through the defective points of the container. The necessary assumption, for every type of penetration, is however that in the welding defect there is a veil or a continuous column of liquid. The interruption of the veil of liquid, due to the presence of air, oil or other inclusions, can prevent the penetration of the microorganisms.

The contamination of flexible containers can take place:

1-due to imperfect closure, due to wrinkles or inclusion of organic material;

2-through holes due to inadequate movements

The precautions to be taken for semi-finished products of acid products can be:

1. a) quick external drying of the bags or pouches
2. b) adequate transport to the secondary packaging (drum), to avoid breakage of the bag.

Yes. The cause is attributable to the growth inside the aitchbone of “molds” belonging to one of these two species: Penicillium commune and Penicillium solitum which are very frequent in the air of confined environments, can grow in the presence of high concentrations of salt (up to 17.5%) and on the product during ageing at RH higher than 85%. Such species, during their growth, which occurs due to excess of free water inside the iliac bone, are able to produce the volatile compound which is responsible for the defect and which spreads also in the muscular portion, it is reabsorbed, more or less widely and intensely according to the extent of the fungal spoilage.

Yes. It is important to keep in mind some important standard of behaviour:

1. a) always remove the sources of environmental contamination from fungal spores, for example hams on which an extended fungal proliferation is observed, conditions that can cause the increase of environmental contamination by “mould”. It has been calculated that the level of contamination of the aitchbone must be less than 100 fungal spores/aitchbone;
2. b) during the salting phase, keep the saline solution saturated on the ham surface, especially near the aitchbone;
3. c) keep the thigh during the resting, pre-ageing and ageing phases at U.R. environmental levels below 85%.

Can the growth of undesired moulds, in particular those dangerous to human health, be avoided on the surface of traditional salami and other sausages?

Yes. The control of surface moulding can be practiced by using selected starter fungal cultures, commercially available, or by naturally favouring (with specific suitable measures, on a case-by-case basis) the growth of autochthonous species recognized as safe through targeted studies. Commercial starters are made up of selected fungal strains, capable of guaranteeing the product a pleasant appearance and good technological and sensory characteristics, of not producing antibiotics or toxic metabolites such as mycotoxins. The product itself, immediately after bagging/filling, can be inoculated with the suspension in water of the conidia of the starter culture, by spraying or immersion in the suspension itself. Among the starter cultures declared safe for health and functional to the technological ageing processes applied to meat, that of Penicillium nalgiovense, initially isolated from the surface of meat sausages as a “domesticated species”, is currently the one most used in Italian industrial productions. Penicillium chrysogenum is also a starter permitted by Italian legislation. However, its use has not always met the expectations, because some strains, initially characterized by white conidiation (typical of “domesticated”, commercial strains), return to produce conidia of green colour, typical of the species of origin or “wild”.

No, in most cases. Growth of fungal mycelium on casings is mainly considered an indicator of a good maturation process in fermented meats. For this reason, until recently surface moulding has been left to chance. Nevertheless, if technological problems occur (e.g. wrong thermo-hygrometric parameters registered during maturation), the final aspect of cased meats can be altered by anomalous pigmentations of the surface or off-flavors, due to growth of undesirable species. Such problems mainly cause financial losses to food producers. On the contrary, if toxigenic moulds occur, problem is not always immediately detectable, since some of these moulds can be characterized by a pale pigmentation. Among the species associated to fermented meats, toxigenic isolates mainly belong to Aspergillus westerdijkiae (only recently separated from Aspergillus ochraceus) and Penicillium nordicum, which at present are considered as the main responsible for ochratoxin A (OTA) production in fermented and dry-cured meats. With regard to this, studies carried out at the SSICA on salami allowed us to register a progressive partial reduction in OTA content during maturation process, probably due to the detoxifying action by microorganisms native or inoculated into meat.

Based on these considerations, the identification of the prevailing species should be periodically carried out on finished products, in order to detect and eradicate those fungal species capable to cause aspect defects or to produce undesired or toxic metabolites, not attributable to a clearly detectable defectiveness. With regard to this, it should be taken into consideration that during maturation process, the use of surface fungal starter such Penicillium chrysogenum and Penicillium nalgiovense is currently allowed (D.M. 28 Dicembre 1994. Gazzetta Ufficiale Repubblica Italiana, N. 89, 4-5, 15.04.1995), in order to reduce the growth of undesired or toxic fungal species.

By the term biological indicator (IB) a system is meant which is composed of a microorganism and a carrier on which it, or its spores, is deposited. The IBs are used both in the pharmaceutical and in the food fields to monitor and prove the efficacy of different sterilization processes, both chemical and physical in microbial inactivation. More in detail, IBs can be defined as “standardized preparations” of microorganisms with known characteristics such as:

• Defined population: name of the strain and identification code of the collection to which it belongs, purity
• High resistance to the inactivating agent
• Easy to identify
• Non-pathogenic, undemanding and easy to handle

• To act as a support for the microorganism to convey the target micro-organism to come into contact with the inactivating agent
• To be inert with respect to the target microorganism and the sterilization process
• To be suitable in shape and size for the purpose for which it is used
• To be easy to recover

As carriers, ampoules (self-contained) strips of different materials such as aluminium, steel, PVC or, in the case of packaging validation, the packaging itself can be used.

Lethal treatment – Treatment able to determine, during the production process, the necessary reduction in the number of pathogenic microorganisms in a food, in order to obtain a safe product for human consumption.

Post-lethal treatment – Treatment applied to the product with the aim of reducing or eliminating the level of pathogens deriving from its exposure, in a post-lethal treatment phase, to a post-lethal environment.

A product is considered to be exposed to a post-lethal environment when, after having been subjected to a lethal treatment, it can undergo microbial recontamination in the production plant area.