Article pp.153-160 du Vol.27 n°2 (2007)

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FOCUS : JSMTV

Microbial ecosystems of meat and meat products

J. Labadie

1 – INTRODUCTION

Meat products like other processed food products are contaminated by many microbial species which grow more or less rapidly according to the kind of processing involved and to the physico-chemical parameters of the products. In fact, for microbiologists, workshops are true ecosystems as they always fit to the exact definition of any kind of ecosystem. Hence an ecosystem is the dynamic association of two permanently interacting components:

– An environment with a defined spatio-temporal dimension : the biotope.

– A community of living organisms with peculiar characteristics. An ecosystem is a permanently changing unit, the modifications are depending of the energy fluxes. So it is not necessary to have expertise in ecology or in meat technology to understand that food processing plants and workshops are true ecosystems as well as a forest, a lake, an atoll.

However, according to the kind of processing chosen for meat and meat products, two or three main ecosystems can be characterized: those influencing the storage life of fresh meats, those influencing cured and fermented meats and those influencing heated or cooked meat products. In this paper, the two first will only be discussed as the third one frequently involves micro-organisms which are not meat specific.

2 – MICROBIAL ECOSYSTEMS INFLUENCING THE STORAGE LIFE OF FRESH PRODUCTS

Before the examination of the nature and composition of this ecosystem, it is necessary to understand how microbial contaminations occur during the first steps of processing meats. It is generally admitted that, at slaughter, muscles are sterile, or eventually poorly contaminated, but to a very limited extent. It is the processing, in a broad sense, which will introduce the microbial flora on the surface or inside the muscles. These microbial flora are themselves coming Laboratoire de Microbiologie – INRA de Clermont-Theix – 63122 Saint-Genès-Champanelle – France.

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from natural ecosystems which are highly diverse: Soil, plants, water, mammal digestive tracts, skin, etc. If one take into consideration the fact that these ecosystems represent an enormous amount of micro-organisms in terms of quan- tity and biodiversity, it is clear that meats should be heavily contaminated.

Fortunately, it is not observed because the slaughtering process and the following operations such as carcass splitting and deboning, muscle cutting, meat grinding and/or conditioning are generally carried out in conditions which con- siderably limit the introduction of microbial flora on the processing lines and on meats. More over, chilling combined with cleaning and disinfection procedures select micro-organisms which are able to resist, survive and/or grow on tiny quantities of meat juice or residues remaining on the surfaces. Hence, these microbial flora are always specific spoilage organisms (SSO), BJORKROTH et al.

(1998), the properties of which are well fitted to grow on meat, and generally in the cold. Figure 2 shows what happens in general inside a package containing a food product during its storage life in the cold.

TVC SSO Metabolites

Shelf life

Storage time

Log (cfu/g) Conc. of metabolites

Minimal spoilage level

Chemical spoilage index

Figure 1

Evolution of the spoilage micro-organisms during the storage of a food product.

TVC: Total Viabel counts. SSO: Specific Spoilage Organisms From DALGAARD (1995).

Antimicrobial Belt

pH Value

Low Water Activity

Preservatives Heat Treatment

Hygiene Practices

Packaging Conditions

Chilled Storage

>> From Raw Material to Finished Product >>

Figure 2

The hurdles used to inhibit or to kill the spoilage micro-organisms. LEISTNER (1992).

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The different hurdles to the development of micro-organisms have been introduced into the “hurdle concept” by Leistner at the beginning of the 80^ties. This concept outline the fact that the more the processing the more the hurdles to the microbial growth. Figure 2 shows the principles of the concept. The scheme although simple and trivial, clearly indicates that the microbial flora which is able to overcome the hurdles is perfectly fitted to the conditions applied.

Meat products are very numerous and it would be fastidious to inventory all those consumed by human beings. However, it is possible to distinguish two main categories those which are packaged in the air and merely stored at chilled temperatures and those packaged under vacuum or modified atmospheres. The first category involves products preserved at different temperatures but in the same atmosphere, i.e. the air we are all breathing the composition is always the same. On the contrary the second category involves many different atmospheres including many gazes under different pressures. CO₂, N₂, Oxygen, etc. are generally used in many combinations inside package the permeability could be highly variable. Such a huge number of potential packaging conditions could be the cause of highly diverse microbial flora existing and growing. In fact, the micro-organisms identified at the end of storage life are not very numerous and are generally the same. The main reasons of such a limitation within the bacteria really capable of growth in meats, are the meats themselves in terms of support and source of usable substrates for bacteria. It is the combination of packaging parameters with the meats as support for growth which select the typical flora growing on meats. Muscles and meat are solid supports, with a peculiar chemical composition containing, after the rigor mortis, a few glucose, high quantities of lactate and iron, and a pH generally lower than pH 6.0. For micro-organisms meat resemble a culture medium already used by bacteria. Figure 3 shows the different low molecular substrates which could be used by micro-organisms. It is clear that such substrates in combination with the conditions chosen for storage are a kind of bottleneck for most of bacteria which contaminate the muscles.

Concentrations (mg/g)

Figure 3

Concentrations of low molecular weight substrates for bacteria into beef meat, before and after the rigor mortis. FISHER et AUGUSTINI (1977).

Composés pre post

Créatine phosphate 3.0

Créatine 4.5 6.5

Adenosine triphosphate 3.0

Inosine monophosphate 0.2 3.0

Glycogène 10.0 1.0

Glucose 0.5 0.1

Glucose 6 phosphate 1.0 0.2

Acide lactique 1.0 9.0

Acides aminés 2.0 3.5

Dipeptides (anserine, carnosine) 3.0 3.0

pH 7.2 5.5

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3 – CHILLED MEAT PRODUCTS STORED IN AIR

For these products, some aerobic bacterial species are dominating the spoilage flora. For all meat packaged in air containing bags, the dominant bacteria are Pseudomonas species. It is interesting to notice that many food products stored in air, milk, vegetables, fish, are also spoiled in air by Pseudomonas species, however it does not mean that the same species are colonizing the food products in general. In meats, Pseudomonas fragi is dominating all the products after several days of storage, (LABADIE, 1999). This species which was only identified as belonging to the non pigmented Pseudomonas, grow more rapidly in meat and meat products at low temperatures (even up to 25°C) than Pseudomonas fluorescens. The generation time are 7.6h and 8.2h respectively, for P. fragi and P. fluorescens. ( LEBERT et al., 1998). This difference is sufficient to explain the dominating role of the first species in meats. What is the meaning of such difference in terms of fitting to an ecological niche. In fact it is estab- lished since the work of CHAMPOMMIER et al. (1996), that the lack of pigmenta- tion results of the impossibility to synthesize siderophores (green fluorescent) which are the iron transporters for many bacteria. Iron is very important for aerobic metabolism, and P. fragi which needs great quantities of iron (twice as for P. aeruginosa, CHAMPOMMIET et al., 1996) import this metal inside the bacterial cells by other routes which are not using siderophores. Thus, haemoglobin, aerobactin, transferin, lactoferrin are imported by P. fragi. As iron is present in great quantities in muscles, this species by saving the energy which is used by other Pseudomonas to synthesize sidérophores, takes an advantage and grow more rapidly. Other hypotheses was proposed by LABADIE (1999); one of the most interesting was the possibility by many strains to excrete proteases, inside blebs of membranes, in response to the presence of specific meat peptides.

These proteases could help the bacteria to penetrate meat tissues and to colo- nize the entire muscles. The production of inducible protease by a P. fragi strain was observed by THOMPSON et al. (1985); in P. aeruginosa, a pathogenic spe- cies taxonomically very close to P. fragi, a similar induction of proteolytic enzymes was also observed. (KADURUGAMAWA and BEVERIDGE, 1995).

For another emblematic species only isolated from meat and fish, Bro- chothrix thermosphacta, it is in first analysis more difficult to explain its pres- ence by an ecological advantage given by meats, and meat products. Its natural ecological niche was tentatively searched by TALON (1984) in different places, slaughterhouses, plants, soil, skin of animals, etc. No clear conclusion was possible after this work, but the isolation from the ground of one new Brochothrix species, B. campestris with several isolates of B. thermosphacta indicated that B. thermosphacta certainly survive and grow on the surface of the ground.

B. thermosphacta grow rapidly in meat and meat products at low temperatures.

This bacterium is psychrotrophic and psychrophilic to some extent as its opti- mum temperature for growth is close to 20°C and it is able to grow at 0°C. This property is certainly very important to explain its growth in meat products, in combination of other properties not completely demonstrated as meat specific, that is the induction of a peptidase and a glycerol ester-hydolase (GARDNER, 1982) by meat and fat, and the use of glucose and glutamate (GILL, 1977) for growth. Unfortunately this bacterium is poorly studied, and it is difficult to go

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further simple hypotheses. It is likely that meat and fish allow specific potentiali- ties to be expressed, in combination with its peculiar composition, and the psychrotrophic character of Brochothrix thermosphacta.

4 – PRODUCTS STORED IN VACUUM AND IN MODIFIED ATMOSPHERES

Vacuum packaging, and gas packaging combined with low temperatures are highly selective for the microbial flora present on meat surfaces. Vacuum packaged meats, and particularly beef meats, are highly stable in the cold, because of the low oxygen concentrations which drastically limit the microbial growth but the lactic acid bacteria which could reach 10⁷-10⁸/g at the end of storage life. Other bacteria are able to grow but they generally grow very slowly. Hence, vacuum packaged meats could be stored during 3 or 4 weeks at 0°C, without any organoleptic problems. Apart from the MA stored meats containing pure CO₂, with or without oxygen scavengers which allow 4 weeks storage lives at 2°C, the MA containing two (CO₂, O₂) or three gases (CO₂, O₂, N₂) only allow limited display lives. For instance, an atmosphere containing 66% O₂, 25%

CO₂, 9% N₂, only permit a two weeks display life, (Christopher, Smith, Dill, Car- penter and Vanderzant 1980). This short life is due to the presence of oxygen necessary to maintain an attractive colour but which in counterpart allow the growth of Pseudomonas spp, B. thermosphacta and Psychrobacter spp. Apart from the CO₂, or on the contrary its absence, the other factors explaining the selectivity of this storage process, are once again the psychrotrophy of the bacterial species dominating the microbial flora. This is particularly true for bacteria always present but which constitute a very limited part of the microbial flora, Enterobacter spp, Hafnia spp, Citrobacter spp. These Enterobacteriaceae are growing as they are able to slowly grow at low temperatures but they are not meat specific spoilage bacteria as they are often present on other many chilled food products (BLIXT and BORCH, 1996).

The microbial ecosystems of meat stored under vacuum or modified atmospheres are dominated by lactic acid bacteria, the species of which are really meat specific. For instance, Lactobacillus sakei, Lactobacillus curvatus, Leucon- ostoc gelidum, Leuconostoc carnosum, Carnobacterium piscicola, are all meat or muscle specific bacteria (they could be isolated from fish muscle, or fish). All these species are psychrotrophic, but all of them prefer meat (or fish) to grow.

The fact that important quantities of non proteolytic lactic acid bacteria can grow on protein rich substrates containing low carbohydrates, could be surpris- ing. During many years, it was not possible to understand why L. sakei is able to dominate the bacterial flora present on vacuum packaged meats. Since the publication of its genome sequence in 2005 (CHAILLOU et al., 2005), and the comparison of its genes with those of other genomes of lactic acid bacteria, it is possible to propose hypotheses explaining why L. sakei is linked to its meat substrates. First of all a real psychrotrophy which was pointed out by REUTER

(1980) as a common feature of all the meat lactobacilli, is certainly linked to the presence of many genes involved in adaptation to cold. For instance, 4 genes coding for small proteins, the Csps (Cold shock proteins) already described in

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other bacteria, (HEBRAUD et al., 1994) are present into the genome of L. sakei.

Only three of these genes are present into the genome of L. plantarum which is colonizing similar ecological niches of L. sakei. One of these genes is present into the genome of the gut lactic acid bacterium L. acidoplilus which survive and multiply in an ecological niche the temperature of which is stable (37°C).

Meat is, as already outlined, a medium containing very small quantities of glucose for growth of bacteria, and the existence inside the genome of L. sakei of only a few PTS systems which are sugars transporters is certainly an indication that L sakei is linked to meat as main substrate. However, this bacterium is possessing genes which render possible the use of ribose and IMP, components which are present in greater quantities in meats. For instance, the quantities of ribose in meats are 30 times those of glucose. More over, as L. sakei is able to degrade the amino acid arginin and the polymer glycogen into meats, its seems perfectly fitted to use components able to insure the production of energy.

The genome analysis (CHAILLOU et al. 2005) of L. sakei also revealed the presence of 40 genes involved in the resistance to oxidation and to stress in general. L. sakei is certainly one of the species possessing the best weapons to fight oxidation into meat products. As L. sakei also possess a catalase containing an haeminic group which could be of meat origin, it seems also well fitted to eliminate H₂O₂ produced by other bacteria which are in competition with it for meat substrates. L. sakei also possess the genes which makes possible the importation of osmoprotectants such as glycine betaine by specific systems.

This ability certainly explain why this lactic acid bacteria is surviving and multi- plying in cured products such as dry fermented sausages and hams.

It is probable that some of the genes and operons identified into the L. sakei genome are shared by other lactic acid bacteria growing in meat and meat products, Leuconostoc gelidum, Leuconostoc carnosum, Carnobacterium pisci- cola. All theses genes must however be identified before any comparison between these bacterial species.

5 – THE MICROBIAL ECOSYSTEMS OF CURED PRODUCTS

Cured products the microbial flora are influencing the flavour and aroma are mainly the fermented products and particularly, dry sausages and many similar products. One of the main difference existing between fresh and fermented products is the fact that the dominating micro-organisms exert opposite effects.

In fresh products, the micro-organisms are always responsible of spoilage. On the contrary, in fermented sausages, the dominant bacteria, the lactic acid bacteria are producing lactic acid and many end products of the metabolism, which are necessary i) to give the products their texture ii) to give the products their typical aroma. The bacteria involved in processing of dry sausages are belonging to two genera, Lactobacillus and Staphylococcus. The main species are L. sakei, L. curvatus, L. plantarum, Staphylococcus xylosus, S. carnosus, S. suc- cinus, S. warneri, S. equorum. However, to be sure that the dominant bacteria of fermented products are those which produce lactic acid and flavours, it is

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often necessary to introduce inside the batters at the beginning of processing, some selected starters.

The main processing operations which are used to process dry sausages are the followings:

– Addition, inside the batters of sugars, salt, and spices.

– Introduction of selected starters.

– Fermentation of the batters at 23°C. This fermentation step is necessary to decrease the pH and to favour the development of the typical sausage colour.

– A ripening-drying process of several weeks is following the fermentation process. This time is important to allow the colour to be stabilized, and to allow the production of flavours resulting from the microbial metabolism.

The drying process generally results in the decreasing of most spoilage flora.

It is clear that such a processing really build up the quality of the fermented sausages.

Sometimes, however these processing steps are not all set up, noticeably when traditional products are processed. In these products starters and sugars are not introduced into the batter at the beginning of processing. The microbial flora which is responsible of fermentations are only those contaminating the mixtures of meat and fat. Workshops themselves could also be the source of starters for these sausages. In reality this is not the case (TALONet al., 2006) and fermentations are only due to micro-organisms already present on the car- casses of animals. Finally, the conditions which are chosen during the initial fermentation step and the ripening-drying steps are those which allow the starters to become dominant in the microbial flora. In such conditions, traditional products could have too high pH, at the end of the ripening, that is pH 6.0-6.2. How- ever, in general the dominant microbial flora is mainly composed of Lactobacillus sakei and Staphylococcus spp ( S. xylosus, S. equorum, S. sapro- phyticus). The only problems encountered by traditional products are due to poor hygiene practices during the processing combined with products of high pHs. In these products important quantities of Enterobacteriaceae and spoilage bacteria could be still present at the end of ripening (TALON et al., 2006).

Since the addition of meat starters inside the batters of sausages, their quality greatly improved and reach high standards in terms of hygiene and organoleptic quality. This is mainly explained by the fact that selected starters of meat origin are well adapted to such meat products and are always dominating the microbial flora.

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6 – CONCLUSIONS

Meat is a source of nutrients for micro-organisms which select, in combination with other parameters necessary for the processing, microbial species well adapted to the meat as a substrate for growth. It would be interesting for all the bacteria growing on meat, and particularly the lactic acid bacteria, to determine the properties which explain their fitting for meat in general, or those which explain their ecological advantages on meats. These properties could be used ton select, among the strains or species, those which could be used as new starters or which could inhibit the spoilage or pathogenic flora.

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