Journal of Applied Bacteriology Symposium Supplement 1992,73, 103s-1145

The relationship between the phenotypic properties of bacteria from chill-stored meat and spoilage processes R.H. Dainty and B.M. Mackey’ MATFORSK, Norwegian Food Research Institute, As, Norway and ’Agricultural and Food Research Council, lnstitute of Food Research, Reading Laboratory, UK

1. Introduction, 103s 2. The microflora of chill-stored meat, 103s 2.1 Storage in air, 103s 2.2 Storage in vacuum packs, 105s 2.3 Storage in modified gas atmospheres, 106s 3. Factors influencing growth of microbes on meat,

106s 3.1 Storage in air, 106s

1. INTRODUCTION

After dressing, red meat carcasses typically carry between lo2 and lo4 bacteria/cm2. These are derived from hide, faeces and gut contents and the hands and instruments of slaughtermen. The initial flora is very diverse, although it is mainly mesophilic, with micrococci, staphylococci, Bacillus spp., coryneforms, Enterobacteriaceae, flavobacteria, pseudomonads, lactic acid bacteria and Brochothrix thermosphacta likely to be present (Dainty et al. 1983). These organisms are distributed on lean, fat and connective tissues each of which can support growth. However, growth on lean tissue is considered to be of over-riding importance in spoilage and has therefore been the subject of most attention. Lean tissues provide a rich variety of readily available sources of energy, carbon and other nutrients (Table 1) at p H values highly conducive to growth, i.e. typically in the range pH 5.5-6.5. The higher values ( > p H 6.0) result ,

Table 1 Typical chemical composition (%, w/v) of lean red meat post-rigor

Protein Lipid L( +)-lactic acid Amino acids Creatine Anserine/carnosine Water

20 3 0.9 0.4 0.5 0.3 75

ATP products Glycogen Sugars Sugar phosphates Nicotinamide Nucleotides

3.2 Storage in vacuum packs, 107s 3.3 Storage in modified gas atmospheres, 1 0 8 s 4. Spoilage chemistry, 1 0 8 s 4.1 Storage in air, 109s 4.2 Storage in vacuum packs, 1 1 0 s 4.3 Storage in modified gas atmospheres, 1 1 IS 5. Conclusions, 112s 6. References, 112s

from stress-induced depletion of glycogen in the live animal that leads in turn to low concentrations of glucose and lactic acid in the meat post-mortem. This combination of high p H and low glucose has important consequences for spoilage processes under certain storage conditions. Despite this ideal environment, not all of the bacteria initially present grow during storage ; meats invariably develop a characteristic flora whose composition can, in large measure, be predicted from a knowledge of temperature, meat p H and gaseous atmosphere. In the last 10 years significant advances have been made in the classi-. fication and methods of identification of all the major types of bacteria comprising these associations. This has allowed a rational choice of organisms to be made for meat inocu-, lation studies designed to establish the factors affecting growth of microbes on meat and their role in spoilage processes. I n reviewing these advances, it is our intention to establish how well we are able to explain spoilage in terms of known phenotypic properties of bacteria that are typically isolated from meat stored in air, vacuum packs or in modified gas atmospheres.

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Correspondmte t o : Dr R.H. Dainty, MATFORSK, Norwegian Food Research Institute, Osloveien I , N-1430 As, Norway.

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2. THE MICROFLORA OF CHILL-STORED MEAT 2.1 Storage In air

In traditional practice carcasses are ‘conditioned’ by refrigerated storage for up to 2 weeks to allow the meat to become tender, and are then butchered into joints or cuts

104s R . H . DAINTY AND B . M . MACKEY

7

Table 2 Microbes present on aerobically spoiled chilled meat

L

----

Characteristics

Organism

Gram-negative, motile, oxidase (+)

Pseudomonas fragi (biotypes 1 and 2) lundensis Juorescens

spp., e.g. putidu Pseudomonas fragi (biotypes 1 and 2) (formerly Moraxella) Psychrobacter immobilis (formerly Moraxella-like) Acinetobacter (johnsonii) Enterobacteriaceae

+

non-motile, oxidase ( )

non-motile, oxidase ( -) facultative anaerobes

Brochothrix thermosphacta Lactic acid bacteria

Gram-positive 12 Time ( d )

Fig. 1 Schematic diagram of microbial growth on red meat stored chilled in air. --, Lactic acid bacteria; -. . . -, Brochothrix therrnosphacta; .. , Enterobacteriaceae;- -, Pseudornonas spp. ~

that are stored unwrapped in air or in plastic trays covered with an oxygen-permeable film. Under these conditions shelf-life is limited by microbial growth that leads to offodours when numbers reach around 107/cm2 and to visible slime at about I08jcm2. The storage flora is dominated by psychrotrophic, aerobic, Gram-negative motile and non-motile rods referred to in earlier literature as Pseudomonas, Acinetobacter, Moraxella and Moraxella-like but now identified as Pseudornonas, Acinetobacter and Psychrobacter species. Development of a typical flora is shown schematically in Fig. 1 and a more detailed description is given in Table 2. Both Shaw & Latty (1982, 1984) and Molin & Ternstrom (1982, 1986), who studied the motile rods, found three major groups of pseudomonads, only two of which, Pseudomonas fragi and Ps. Juorescens, could be identified with known species. The properties of the organisms that comprised the third groups described by both sets of workers were very similar and were subsequently proposed as a new species, Ps. lundensis (Molin el a / . 1986). Both groups of workers also recognized sub-groups within the Ps. fragi cluster, only one of which, cluster 2 of Shaw & Latty and cluster A of Molin & Ternstrom, contained type/reference strains of Ps. fragi. When Shaw's terminology is used Ps. .frqi cluster 2 strains are typically the dominant kinds found on meat, followed by Ps. fragi cluster 1 strains, Ps. lundensis and Ps. Juoiescens biotypes I and 111 (Table 3). Storage temperatures between 0" and I O T , storage time, type or source of meat did not radically affect these relative incidences. Pseudomimas spp. typically account for > 50'% of the flora and sometimes up to 90'%.

In a taxonomic study of the non-motile rods Shaw & Latty (1988) showed that the oxidase-positive strains could be identified with Ps. fragi biotypes 1 and 2 (previously Moraxella), and Psychrobacter immobilis (previously Moraxella-like) and the oxidase-negative strains with Acinetobacrer spp. (probably johnsonii). The incidence of these organisms has been poorly studied. Erobo et al. (1985) detected Psy. immobilis and Acinetobacter spp. at ca 1% each in ground beef, while Shaw & Latty (1988) found that Psy. immobilis comprised up to 50% of the flora on some fat surfaces and Acinetobacter spp. comprised up to 10% of the total flora of some meat samples. Brochothrix thermosphacta and cold-tolerant Enterobacteriaceae (e.g. Serratia liquefaciens, Enterobacter agglomerans, Hafnia alvei) also occur in aerobically-stored meat but Table 3 Relative proportion of Pseudornonas spp. on red meats stored chilled in air % of total pseudomonads in study of

Pseudomonas fragi biotype 1 frugi biotype 2 lundensis

Shaw & Latty

Molin & Ternstrom

1982

1984

1982

31

7

49 12*

jluorescens

(biotypes I and 11)

* Reported as cluster 3. Reported as B,, B, and B, .

1Reported as A.

1986

1st

50

73

11*

14

5

13

L3$ 19 13

BACTERIA FROM CHILL-STORED M E A T AND SPOILAGE PROCESSES 105s

rarely account for more than a small proportion of the total flora. These organisms appear to be more prevalent on pork and lamb, particularly on fat surfaces; storage at 5°C rather than 1°C tends to favour their growth (e.g. Barlow & Kitchell 1966; Enfors et al. 1979; Blickstad & Molin 1983).

Table 4 Lactic acid bacteria associated with red meats stored chilled in vacuum packs or modified gas atmospheres

Lactobacillus sake curvatus bavaricus

2.2 Storage in vacuum packs

The shelf-life of fresh meat may be greatly extended by sealing under vacuum in bags that have a low permeability to atmospheric gases. Continued respiratory activity of meat tissues dramatically reduces the oxygen content to below 1% while COz tension increases to about 20% (v/v). During storage under these conditions growth of Gramnegative bacteria is severely restricted and the flora typically becomes dominated by Gram-positive organisms (Fig. 2), the so-called ‘atypical streptobacteria’ of earlier studies (Kitchell & Shaw 1975; Hitchener et a/. 1982). Clarification of their taxonomic position has occurred in several stages (Holzapfel & Gerber 1983; Shaw & Harding 1984, 1985; Collins et al. 1987) and the major groups currently recognized together with other types of lactic acid bacteria found on meat are summarized in Table 4. Carnobacterium is a new genus proposed by Collins et al. (1987) to accommodate strains unable to grow on acetate agar pH 5.6 (so-called non-aciduric) and producing L( +)lactic acid, acetic acid, ethanol, CO, and formic acid in an atypical heterofermentation (Holzapfel & Gerber 1983 ; but see de Bruyn et al. 1987). Production of formic acid rather

/ I

I 6 Time (weeks)

Fig. 2 Schematic diagram of microbial growth on red meat stored chilled in vacuum packs. __ ,Lactic acid bacteria; -. . .-,

Brochothrix thermosphacta; Pseudomonas spp.

‘.. ,Enterobacteriaceae; - - -,

Carnobacterium diverged piscicolat

Leuconostoc carnosum gelidum mesenteroides subsp. mesenteroides

Lactococcus rafinolyticus

~~~~

* Formerly Lactobacillus divergens.

t Lactobacillus carnis (Shaw & Harding 1985) is synonymous with Carnobacterium (formerly Lactobacillus) piscicola.

than just CO, often makes gas detection difficult in the standard heterofermentation test. The Lactobacillus spp., i.e. Lact. curvatus, Lact. sake and Lact. bavaricus, are all classical homofermenters producing DL-lactic acid and able to grow freely on acetate agar (so-called aciduric). It is of interest that none of the homofermenters isolated from meats by Borch & Molin (1988) could be assigned to any of these species. Shaw & Harding (1989) have also clarified the taxonomy of the meat leuconostocs. The majority belong to one of three species, Leuconostoc mesenteroides subsp. mesenteroides and two previously undescribed species given the names Leuc. gelidum and Leuc. carnosum. The only other genus of lactic acid bacteria that has been identified on vacuumpacked meats is Lactococcus and the majority of strains can be assigned to the species Lactococcus raflnolyticus (Schillinger & Lucke 1987). Table 5 summarizes the results of the few studies from which the relative incidence of the various types on normal p H meat can be determined. Despite some variation, lactobacilli and carnobacteria appear to be most common but there are many reports of leuconostocs being dominant. More detailed studies, with appropriate media to account for the different p H sensitivities of some of the species, or gene probes, are required before definite conclusions can be reached. Growth of other organisms, including Broch. thermosphacta, Pseudomonas spp. and Enterobacteriaceae may also occur from time to time, at levels of up to 103-10h/ cm’, but their occurrence is not a consistent feature of the spoilage flora (Dainty et al. 1983). Pork and iamb again appear to be particularly prone to suffer growth of Broch. thermosphacta and Enterobacteriaceae (Blickstad & Molin 1983; Gill & Harrison 1989) while high initial contamination levels can also cause problems with enterobacteria on beef (Gill & Penney 1988). If meat of high pH (

The relationship between the phenotypic properties of bacteria from chill-stored meat and spoilage processes.

Journal of Applied Bacteriology Symposium Supplement 1992,73, 103s-1145 The relationship between the phenotypic properties of bacteria from chill-sto...
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