Development of Spoilage Microbiota

The conditions under which the animals are reared and slaughtered determine the level, extent and type of contamination. Possible sources of contamination include the abiotic environment in contact with the animal (air, soil, water, feeds), the animal itself (hides, intestinal tract, faeces) and the processing equipment including utensils and humans. Contamination may also vary according to specific characteristics of each animal, its geographic origin as well as the season of the year.

The micro-organisms that usually dominate the initial microbiota of fresh carcasses are Gram-negative rods (mainly pseudomonads) and micrococci (mainly Kocuria spp. and Staphylococcus spp.). Furthermore, Gramnegative bacteria such as Acinetobacter spp., Alcaligenes spp., Moraxella spp. and Enterobacteriaceae, and Gram-positive species including spore-forming bacteria, lactic acid-producing bacteria and Brochothrix thermo-sphacta, as well as yeasts and moulds, may also be present in small numbers.

Growth and development of the spoilage microbiota of fresh meat is governed, as in the case of all foodstuffs, by

(i) intrinsic parameters of the meat, such as pH and buffering capacity, water activity, Eh and poising capacity, presence of antimicrobial compounds and nutrient composition,

(ii) type and extent of processing,

(iii) extrinsic factors such as temperature, relative humidity and the composition of the gaseous atmosphere,

Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece e-mail: [email protected]

F. Toldra (ed.), Safety of Meat and Processed Meat,

Food Microbiology and Food Safety, DOI 10.1007/978-0-387-89026-5_3,

© Springer Science+Business Media, LLC 2009

(iv) implicit factors, including antagonism and synergism and

(v) interactive effects of the above mentioned factors, other than that expected from their individual action.

All these ecological determinants influence the establishment of a particular microbial association and determine the rate of attainment of a maximum population known as the 'ephemeral (specific) spoilage micro-organisms' (E(S)SO), i.e. those that are able to adopt various ecological strategies. The latter are developed as a consequence of environmental determinants and allow the micro-organisms to proliferate and eventually dominate an environmental niche. Thus, in raw meat of low and high pH that is stored aerobically at cold temperatures, Pseudomonas spp. and Shewanella putrefaciens are considered to be the main spoilage bacteria (Garcia-Lopez, Prieto, & Otero, 1998). On the other hand, B. thermosphacta and lactic acid bacteria dominate during storage of meat under vacuum or other modified atmospheres (Stanbridge & Davies, 1998).

Apart from the imposed environmental conditions, microbial interactions play an equally important but still not fully exploited role in the development of the microbial association (Nychas, Drosinos, & Board, 1998; Tsigarida, Boziaris, & Nychas, 2003). Study of these interactions is important in understanding spoilage. Gram and Dalgaard (2002) reported that Pseudomonas spp. could inhibit the growth of S. putrefaciens due to its ability to produce side-rophores. Moreover, competition for nutrients (e.g. glucose), metabiosis (production of a favourable environment) and cell-to-cell communication (quorum sensing) could also affect the physiological attributes of the organisms under the imposed ecological determinants (Drosinos & Board, 1994; Drosinos & Nychas, 1997; Lambropoulou, Drosinos, & Nychas, 1996). Indeed, it has been reported that the chemical changes occurring in naturally contaminated fish and meat were found to be significantly different from those on sterile muscle tissue when it was individually inoculated with the ephemeral spoilage micro-organisms (Koutsoumanis & Nychas, 1999; Tsigarida & Nychas, 2001). Studies in co-culture model systems were found to be helpful in simplifying the natural food ecosystem and providing an insight into possible interactive behaviours during the development of potential ephemeral spoilage micro-organisms. Furthermore, they may prove themselves useful in identifying metabolites that may be further used as a unique chemical spoilage index (Tsigarida et al., 2003).

The contribution of nutrients to either antagonistic or synergistic interactions has also been the case of intensive study. The principal carbon source, namely glucose, has been found to be metabolized more rapidly by the obligate aerobic strains of pseudomonads, in comparison to the facultative anaerobic strains of B. thermosphacta and oxidative strains of S. Putrefaciens (Tsigarida et al., 2003). Although their growth rate was not affected by co-culturing with either Shewanella spp. or B. thermosphacta, an acceleration of glucose consumption was evident. It was concluded the pseudomonads can play a syn-trophic role for Brochothrix spp. This observation is of great importance since B. thermosphacta has a much greater spoilage potential than lactobacilli and can be important in both the aerobic and anaerobic spoilage of muscle foods. On the other hand, a typical antagonistic interaction that affects the selection of spoilage flora is evident in the case of pseudomonads and S. putrefaciens. It is well established in the literature that the inhibitory effect of the former bacterium over the latter is attributed to the ability of Pseudomonas spp. to produce siderophores (Gram & Dalgaard, 2002). However, in this case, competition for glucose seems also to play a critical role in Pseudomonas spp. dominance. Another example of the interactive properties of Gram-negative spoilage microbiota is their ability to produce chemical communication signals such as acylated homoserine lactones (AHLs). It has been shown that these AHL compounds can be found in wide range of foods including fish, meat and vegetable products (Smith, Fratamico, & Novak, 2004) in concentration proportional to the growth of Gram-negative bacteria. The role of AHLs in muscle food spoilage is currently unknown, but several phenotypes (pectinolytic, lipo-lytic, proteolytic and chitinolytic activities) potentially involved in spoilage of different foods have been linked to AHL regulation in several bacteria (Gram & Dalgaard, 2002). Elucidation of their role in muscle food spoilage will be an important area for future research.

All the physicochemical changes that occur in fresh meat take place in its aqueous phase. There are three classes of substances that are utilized by spoilage microbiota:

(i) compounds involved in the glycolytic pathway (e.g. glucose, glucose-6-P)

(ii) metabolic products (e.g. lactate)

(iii) nitrogen energy sources (e.g. amino acids, proteins)

The low-molecular-weight compounds, especially carbohydrates and their intermediate catabolic products, are preferentially utilized by the meat micro-biota as energy source. Depletion of these substrates will inevitably lead to an amino acid degrading metabolism in, at least, some bacterial species (Table 3.1).

Glucose and lactate (the second most preferred energy source) along with their oxidative products (e.g. gluconate, gluconate-6-P) have been proposed to serve as spoilage indicators. This is particularly evident in the case of meat stored under aerobic conditions where pseudomonads are the major spoilage micro-organisms. Pseudomonads catabolize sequentially D-glucose and L- and D-lactic acid, with the oxidation of glucose and glucose-6-P via the extracellular pathway leading to a transient accumulation of D-gluconate and an increase in gluconate-6-P concentration. Furthermore, it has been shown that the sum of free amino acids along with the water-soluble protein content increased during storage and this corresponded well with colony counts, particularly in meat samples with relatively high glucose concentration (Nychas & Arkoudelos, 1990; Nychas & Tassou, 1997). In addition, the rate of free amino acid increase under aerobic conditions was higher than under modified atmosphere storage. These observations could have a commercial importance, since spoilage is usually associated only with post-glucose utilization of amino acids by pseudo-monads (Gill, 1986).

Table 3.1 Order of substrate utilization during growth of major muscle spoilage bacteria

under aerobic condition (based

on

Nychas,

Skandamis,

Tassou,

& Koutsoumanis, 2008;

Nychas, unpublished data)

Substrate

A

B

C

D

E H

Glucose/glucose-6-P

1

1

1

1

11

Lactate

2

2

2

2

Pyruvate

3

3

Gluconate/gluconate-6-P

4

4

Propionate

5

Formate

Ethanol

6

Acetate

7

Amino acids

5

8

2

3

Ribose

3

Glycerol

4

A: Pseudomonas spp.; B: S. putrefaciens; C: B. thermosphacta; D: Enterobacter spp. E: Hafnia alvei; H: lactic acid bacteria.

Based on Gill (1986), Nychas et al. (1998), Ellis and Goodacre (2001), Nychas (unpublished)

A: Pseudomonas spp.; B: S. putrefaciens; C: B. thermosphacta; D: Enterobacter spp. E: Hafnia alvei; H: lactic acid bacteria.

Based on Gill (1986), Nychas et al. (1998), Ellis and Goodacre (2001), Nychas (unpublished)

The key chemical changes associated with the metabolic activities of pseu-domonads have been the subject of intensive study. The effect of pseudomo-nads' growth on various substances in sterile meat block, meat juice and gel cassette system during storage at 0, 4-5, 10 and 25°C is shown in Table 3.2 (Drosinos & Board, 1994, 1995; Tsigarida & Nychas, 2001; Tsigarida et al., 2003; Roca & Olsson, 2001). The identification of the molecules acting as precursors for the production of specific catabolic products during growth of Gram-negative bacteria in broth, model system (gel cassette or sterile meat) and in naturally spoiled meat has also been thoroughly investigated and the results

Table 3.2 Metabolic activity of pseudomonads in sterile meat block, meat juice and gel cassette system at 0, 4-5, 10 and 25°C (Drosinos, 1994; Nychas et al., 1998; Tsigarida et al.,

2003)

Substrate

P.fragi

P. fluorescens

Pseudomonas spp.

d-Glucose

+

+

+

Glucose-6-P

+

-

+

d-Gluconate

+

+

+

Gluconate-6-P

+

-

+

l-Lactic acid

+

+

+

d-Lactic acid

+

+

nd

Pyruvate

+

+

nd

Acetic acid

+

+

+

Formic acid

nd

nd

+

1-Propanol

nd

nd

+

Amino acids

+

+

nd

Creatine

+

-

nd

Creatinine

+

-

nd

Ammonia

+

+

+

are summarized in Table 3.3 (McMeekin, 1982; Dainty, Edwards, & Hibbard, 1985; Dainty, Edwards, Hibbard, & Marnewick, 1989; Edwards & Dainty, 1987; Edwards, Dainty, Hibbard, & Ramantanis, 1987; Stutz, Silverman, Angelini, & Levin, 1991; Schmitt & Schmidt-Lorenz, 1992b; Jackson, Acuff,

Table 3.3 Factors affecting the production of end products of Gram-negative bacteria (e.g. Pseudomonas spp., Shewanella putrefaciens, Moraxella) when inoculated in broth, model system (gel cassette or sterile meat) and in naturally spoiled meat in comparison with sterile meat (Tsigarida et al., 2003; Nychas et al., 1998; Nychas, unpublished) Conditions studied

Table 3.3 Factors affecting the production of end products of Gram-negative bacteria (e.g. Pseudomonas spp., Shewanella putrefaciens, Moraxella) when inoculated in broth, model system (gel cassette or sterile meat) and in naturally spoiled meat in comparison with sterile meat (Tsigarida et al., 2003; Nychas et al., 1998; Nychas, unpublished) Conditions studied

End product

Broth

Model food

Meat

Sterile meat

Factors

Gluconate

+

+

+

ND

Glucose and oxygen

Gluconate-6-P

+

+

+

+

(limitation)

Lactic acid

+

+

+

+

Acetic acid

+

+

+

ND

Formic acid

+

+

+

NAD

Sulphides

NAD

+

+

+

Temperature and substrate

Dimethylsulphide

NAD

+

+

(glucose) limitation

Dimethyldisulphite

NAD

+

+

Methyl mercaptan

NAD

+

+

Methanethiol

NAD

+

+

High pH

Hydrogen sulphide

NAD

-/ +

+

ND

Dimethyltrisulphide

NAD

+

+

Methyl esters

NAD

+

+

Glucose

(acetate)

Ethyl esters (acetate)

NAD

+

+

Glucose

Acetone

NAD

+

+

ND

2-Butanone

NAD

+

+

ND

2-[2-butoxyethoxy]-

NAD

+

ND

ethanol, acetate

Acetoin/diacetyl

NAD

+ /-

+

ND

Diethyl benzene

NAD

+

+

ND

1-Butanol

NAD

ND

+

ND

Trimethylbenzene

NAD

+

+

ND

Toluene

NAD

+

+

+

ND

Butanal

NAD

NAD

+

+

ND

Hexane

NAD

+

+

ND

2,4-Dimethylhexane

NAD

+

+

ND

Methyl heptone

NAD

+

+

ND

2-Methylbutanal

NAD

+

+

ND

Methanol

NAD

+

+

ND

Ethanol

NAD

+

+

ND

2-Methylpropanol

NAD

+

+

ND

2-Methylbutanol

NAD

+

+

ND

3-Methylbutanol

NAD

ND

+

ND

Propanol-1

NAD

+

+

+

ND

Ammonia

NAD

+

+

Glucose

NAD: not available data; ND: not determined

Vanderzant, Sharp, & Savell, 1992; Lasta, Pensel, Masana, Rodriguez, & Garcia, 1995; Roca & Olsson, 2001; Tsigarida et al., 2003; Tsigarida & Nychas, 2001). Moreover, a wide range of volatile compounds are produced during growth of spoilage microbiota in naturally contaminated samples of meat stored chilled in air (McMeekin, 1977; Dainty et al., 1985, 1989; Molin & Tenstrom, 1986; Edwards & Dainty, 1987; Stutz et al., 1991; Jackson et al., 1992; Lasta et al., 1995; Tsigarida & Nychas, 2001; Vainonpaa et al., 2004) and are presented in Table 3.4.

The increase in D-gluconate concentration inevitably led to the proposition of a new 'hurdle' regarding the extension of meat shelf life. This new hurdle was the addition of glucose in meat and its concomitant transformation to gluco-nate (Gill, 1986; Lambropoulou et al., 1996) with a simultaneous decrease of the pH value due to the accumulation of oxidative products. A selective determinant on meat ecosystem may be offered by this transient pool of gluconate and the inability of the taxa participating in the microbial association to utilize this additional energy source (Nychas et al., 1998). Indeed, the addition of carbohydrates, and especially glucose, has already been suggested as a factor able to delay spoilage particularly in dark, firm, dry (DFD) meat (pH > 6.0), primarily due to the fact that the glucose content affects not only the cell density attained at the onset of spoilage (Gill, 1986) but also themetabolic products produced by the microbiota (Nychas & Arkoudelos, 1990). Meat with DFD characteristics spoils more rapidly than meat of normal pH (pH 5.5-5.8).

Pseudomonas fragi was found to catabolize creatine and creatinine under aerobic conditions and release ammonia in the growth medium, resulting in pH increase. Ammonia can also be produced by many micro-organisms,

Table 3.4 Major volatile microbial metabolites detected in naturally contaminated samples of meat stored chilled in air

1-Undecene

Benzaldehyde

Iso-pentyl formate

1,4-Heptadiene

Butane

Methanethiol

1,4-Undecadiene

Cadaverine

Methanol

2-Butanone

Crotonate

Methyl ethyl ketone

2-Methyl butanol

Diacetyl

Methyl mercaptan

3-Methyl butanal

Diaminopropane

Methylthioacetate

3-Methyl butanol

Dimethylsulphide

n-Heptanoate

3-Methyl-2-butenoate

Dimethyltrisulphide

n-Hexanoate

3-Methylbutanoate

Ethanol

n-Hexanoate

4-Methyl-benzaldehyde

Ethyl acetate

n-Octanoate

4-Heptanol

Hexane

n-Propanoate

Acetaldehyde

Hydrogen sulphide

n-Propanoate

Acetoin

Iso-butanoate

Acetone

Iso-butyl acetate

Agmartine

Iso-pentyl acetate

Ammonia

Iso-propyl acetate

Based on Nychas et al. (1998); Nychas et al. (2007); Nychas (unpublished)

Based on Nychas et al. (1998); Nychas et al. (2007); Nychas (unpublished)

including pseudomonads during their amino acid metabolism. Ethanol, acetone, propan-2-ol, dimethylsulphide, propan-1-ol, ethyl acetate, 2,3-butandione, acetic acid, diacetyl, hexane, heptane, pentanol, heptadiene, acetoin, octane and 2,3-butandiol are other volatile compounds that have also been detected in spoiled meat (Nychas et al., 1998).

Enterobacteriaceae family can also play a role in spoilage, provided the meat ecosystem favours its growth. They preferentially utilize glucose and glucose-6-P as carbon sources and degradation of amino acids occurs only after their depletion (Gill, 1986). Moreover, some members of this family produce ammonia, volatile sulphides, including H2S, and malodorous amines from amino acid metabolism.

Gram-positive bacteria associated with meat storage ecosystems, apart from B. thermosphacta, include various Lactobacillus, Carnobacterium, Leuconostoc, Lactococcus and Weissella species. It has been reported that oxygen tension, glucose concentration and initial pH have a major influence on the physiology of these micro-organisms, and hence on end-product formation (Nychas et al., 1998). B. thermosphacta has a much greater spoilage potential than lactobacilli in both aerobic and anaerobic spoilage of meat. During aerobic growth, it utilizes glucose and glutamate but no other amino acid (Gill & Newton, 1977). Additionally, during its aerobic metabolism in media containing glucose, ribose or glycerol as the main carbon and energy source, a mixture of end products including acetoin, acetic, iso-butyric and iso-valeric acids, 2,3-butanediol, diacetyl, 3-methylbutanal, 2-methylpropanol and 3-methylbutanol, is produced (Dainty & Hibbard, 1980). The precise proportion of these end products is affected by the glucose concentration, pH and temperature (Nychas et al., 1998).

Dr. Atkins New Diet Revolution

Dr. Atkins New Diet Revolution

Wanting to lose weight and dont know where to start? Dr Atkins will help you out and lose weight fast. Learn more...

Get My Free Ebook


Post a comment