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Detection of specific spoilage bacteria from fish stored at low (0°C) and high (20°C) temperatures
International Journal of Food Microbiology, 4 (1987) 65-72
65
Elsevier JFM 00123
Detection of specific spoilage bacteria from fish stored at low (0 o C) and high (20 o C) temperatures Lone Gram, Gunilla Trolle and Hans Henrik Huss Technological Laboratory, Ministry of Fisheries, Technical Unioersity, DK-2800 Lyngby, Denmark
(Received 28 April 1986; accepted 5 September 1986)
The spoilage potential of 309 bacterial strains isolated from fish spoiled at 0 and 20°C was investigated. Gram-negative, non-fermentative, motile rods tentatively identified as Alteromonas were the major spoilage organisms at 0 ° C. These bacteria were also found at 20 o C, but a large number of Gram-negative, fermentative, motile rods belonging to the Vibrionaceae were also identified as spoilage organisms at this temperature. Most of the Vibrionaceae did, however, not produce hydrogen sulphide from thiosulphate but only from the sulphur containing amino acid, L-cysteine. A specific count of fish spoilage organisms at both low and high temperatures could therefore be obtained directly on an Iron Agar containing thiosulphate and cysteine where bacteria capable of forming H2S from either source of sulphur would appear as black colonies. Key words: Fish; Specific spoilage bacteria; Iron agar
Introduction The bacteriology of fish spoiled at chilled temperatures has been extensively studied as reviewed by Liston (1980), H o b b s and Hodgkiss (1982) and others. It is well recognized that the Gram-negative psychrophilic or psychrotrophic organisms are the important agents of spoilage, but the precise classification of these organisms has been rather difficult, uncertain and has varied considerably following new developments in methodology. Spoilage at high ambient temperatures has been studied in fish f r o m tropical waters (Gorzcyka et al., 1985; Baffle et al., 1985) but not in fish originating from temperate waters. A large n u m b e r of bacteria ( 1 0 7 - 1 0 8 / g ) is normally found on spoiling fish, but only part of this flora m a y be classified as active spoilers. In this work the ability to reduce trimethylamine oxide ( T M A O ) and to produce h y d r o g e n sulphide (H2S) are regarded as prominent characteristics of fish spoilage bacteria. Detection Of 'active spoilers' has been attempted b y van Spreekens (1974),
Correspondence address: L. Gram, Technological Laboratory, Ministry of Fisheries, Technical University, DK-2800 Lyngby, Denmark.
0168-1605/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
66 Levin (1968) and Jensen and Schulz (1980). Van Spreekens (1974) used a medium optimal for detecting the pink endopigment of Pseudomonas putrefaciens, while Levin (1968) and Jenson and Schulz (1980) assessed the number of organisms able to produce H2S from sodium thiosulphate. However, the latter medium does not take into account that some spoilage organisms may be able to produce HaS from decomposition of sulphur-containing amino acids and not from thiosulphate. It is a purpose of this study to reassess the use of iron agar (IA) as described by Jensen and Schulz (1980) and compare this medium with IA supplemented with cysteine for the ability to detect the specific spoilage bacteria of fish stored at both low and high temperatures.
Materials and Methods
Fish raw materials Two storage trials were carried out using cod (Gadus morhua) caught in August and November 1985. The fish were gutted immediately after catch, iced and within 20 h brought to the laboratory where most fish on arrival were in rigor mortis. Some of the fish were filleted and vacuum-packed in polyethylene/polyamide bags with EVAL-barriere (Riloten 40/70 from Otto Nielsen Emballage A/S); the oxygen-permeability is 2 ml/m2/24 h at 1 atm. Whole fish and fillets were stored at 20 and 0 ° C (iced). At regular intervals during storage, samples were removed for sensory and microbiological analyses. 4-5 whole fish and 5-6 fillets were taken on each sampling day. Sensory evaluation Fish fillets were heated (80°C/20 rain) in sealed bags and odour, flavour and overall acceptability evaluated by a trained panel of 6 people. A 10-4 hedonic scale was used, with a score of 4 being the limit of acceptability. Microbiological analyses Media Iron agar (IA) was used for determination of total viable counts (TVC) and HzS-producing organisms: 2% peptone (Difco); 0.3% Lab lemco powder (Oxoid); 0.3% yeast extract (Difco), 0.03% ferric citrate; 0.03% sodium thiosulphate; 0.5% NaC1; 1.2% agar. pH was adjusted to 7.4 with 2 N NaOH and the IA sterilized at 121°C for 15 min. When carrying out the experiment in November, iron agars with different sources of sulphur were compared: 1/1 1 with 0.03% sodium thiosulphate (as described above), 1tt 2 with 0.04% L-cysteine (No. C-7755, Sigma) and 1.4 3 containing both 0.03% sodium thiosulphate and 0.04% L-cysteine. When using IA 2 and IA 3, the L-cysteine is added to the solid agar just before melting. When
67 determining bacterial counts, pour plating was used and the plates covered with a thin layer of IA after solidification and incubated at 25 ° C for 3 days. Bacteria able to produce HzS when decomposing thiosulphate a n d / o r cysteine will form black colonies due to precipitation of FeS. The overlay is applied to prevent fading of the black colonies, which happens when FeS is oxidized. Since the FeS-complex is soluble in acid, the effect of pH on the formation of a black precipitate was investigated. Pure cultures were inoculated into IA tubes with pH 7.4 (which is used in IA) and pH 6.8 (which is used in peptone iron agar (PIA, Difco)) (Levin, 1968)) and it was observed that the precipitation was faster and greater in the former. TMAO-medium was used when bacterial strains were tested for their ability to reduce TMAO and produce HES: 2% peptone (Difco), 0.3% Lab Lemco powder (Oxoid), 0.3% yeast extract (Difco), 0.03% ferric citrate, 0,03% sodium thiosulphate, 0.4% NaC1, 0.4% KH2PO4, 0.575% K2HPO4, 0.05% MgSO4, 2 resazurin tablets (BDH Chemicals) and 0.4% agar. pH was adjusted to 6.8 since it has been shown that the TMAO reductase (from Alteromonas spp.) has its maximum activity at this pH (Easter et al., 1982). The medium was sterilized at 121°C for 15 rain. Before use, 0.04% L-cysteine (No. C-7755, Sigma) was added and the medium boiled to expel any oxygen. After cooling to 45°C, 0.5% TMAO, 2H20 (Sigma) was added. The medium was poured into tubes and after inoculation covered with a thin layer of sterile paraffin oil or soft paraffin. The tubes were incubated at 25 o C for 3 days. If TMAO is reduced, the redoxindicator in the medium will change from red to yellow, and a black precipitate of FeS will be formed if H2S is produced from thiosulphate a n d / o r cysteine. A sterile fish juice was used when bacterial strains were tested for their ability to produce off-odours: fresh fish muscle was ground and mixed well with distilled water (1 : 2). The mixture was heated to 70°c to precipitate the majority of proteins, filtered and sterilized by boiling for 15 min.
Procedures Samples for microbiological analyses were prepared by homogenizing 50 g of fish in 50 ml of 0.1% peptone saline using a Colworth Stomacher 400. When samples were taken from whole fish, part of the belly flaps was used. 1 ml of the primary 1 : 1 suspension was withdrawn and decimal dilutions were prepared in 0.1% peptone saline. Isolation of spoilage flora. 10-20 black and 10-20 white colonies were randomly selected from IA plates poured on the day the fish were considered just unacceptable (score < 4). Black colonies were isolated from plates with 10-50 black colonies and white colonies from plates with 30-100 colonies. The strains were grown in veal infusion broth (Difco) and IA. The following reactions and biochemical tests were used for characterization of the spoilage flora: Gram-reaction was tested by the KOH-method (Gregersen, 1978). Phase contrast microscopy was used for determination of shape and motility. Cytochrome-oxidase was tested by the method of Kovacs (1956) and catalase formation with 3% H202. Glucose metabolism was investigated by the O/F-test of
68
Hugh and Leifson (1953). On the basis of these criteria, the strains were tentatively identified following Dainty et al. (1979). Furthermore, the strains were tested for their ability to reduce TMAO to TMA and to produce H2S. Gram-negative, motile rods which were catalase and oxidase positive, unable to attack glucose fermentatively but able to reduce TMAO were tentatively identified as Alteromonas putrefaciens (Lee et al., 1977). The strains isolated in November were also tested for their ability to produce off-odours when inoculated in a sterile fish juice.
Results
Fish stored at 0 o C as either whole fish or vacuum-packed fillets passed the limit of acceptability after 9-10 days of storage whereas fish stored at 20°C were rejected after only 1 day. At rejection time, total viable counts (TVC) had reached levels of 6 x 106-108 C F U / g and the number of H2S-producing organisms varied from 5 x 106 to 8 x 107 C F U / g . At both storage temperatures, the number of H2S-producing bacteria at rejection constituted a larger percentage, i.e. 70-80% of the total count in vacuum-packed fillets as compared to 40-50% for whole fish. Investigation of bacterial strains isolated as either black or white colonies from fish spoiled at 0 ° C showed that all 'black' isolates (94) were potential spoilage organisms due to their ability to reduce TMAO and produce H2S (Table I). Furthermore, 75 of the 94 strains were grown in a sterile fish juice and all produced spoilage off-odours described as fishy, rotten or cabbage-like. 91 'black' isolates were tentatively identified as Alteromonas putrefaciens and 3 as Vibrionaceae due to their fermentative glucose metabolism.
TABLE I Spoilage potential and biochemical characterization of bacterial strains isolated from fish spoiled at 0 o C Medium: Source of sulphur: Appearance: No. of strains isolated Prod. o f ' o f f - o d o u r s ' i n fish juice TMAO-reduction H2 S-production (TMAO-media) Gram reaction Shape (r = rods) Motility Catalase Oxidase Glucose metabolism (HL) fermentative oxidative none
Iron agar I $2O 2 -
Iron agar 2 Cysteine
Iron agar 3 $202- + cysteine
Black
White
Black
White
Black
White
58 39/39 58 58 58 58 r 58+ 58 + 58+
44 1/24 0 0 41 - , 3 + 44 r 2 3 + , 21 44 + 37+, 7-
20 20/20 20 20 2020 r 20+ 20 + 20+
20 2/20 0 0 20 20 r 4+, 1620 + 18+, 2-
16 16/16 16 16 16 16 r 16+ 16 + 16+
20 0/20 0 0 18 - , 2 + 20 r 2+, 1820 + 18+, 2-
0 27 31
3 24 17
3 2 15
4 2 14
0 4 12
3 8 9
69 TABLE II Spoilage potential and biochemical characteristics of bacterial strains isolated from fish spoiled at 20°C Medium: Source of sulphur: Appearance: No. of strains tested Prod. of off-odour in fish juice TMAO-reduction HE S-production (TMAO-media) Gram reaction Shape (r = rods, c = cocci) Motility Catalase Oxidase Glucose metabolism (HL) fermentative oxidative none
Iron agar 1 $203
Iron agar 2 Cysteine
Iron agar 3 $2032- + cysteine
Black
White
Black
White
Black
White
50 10/10 50 50 50 50 r 50+ 50 + 50+
49 3/10 27 30 48 - , 1 + 49 r 35+, 1448 +, 1 38+, 11-
9 9 9 9 99r 9+ 9+ 9+
7 0 0 0 3-, 4+ 3 r, 4 c 2+, 5 7+ 6+, 1 -
10 10 10 10 1010 r 10+ 10 + 10+
6 0 0 0 66r 6+ 6+ 5+, 1 -
2 8 40
33 1 15
1 2 6
2 0 5
4 2 4
2 0 4
N o n e of the strains isolated as white colonies f r o m fish stored at 0 ° C c a u s e d a n y changes in the T M A O m e d i u m , regardless of the source of s u l p h u r used in the p r i m a r y I A plates. T h r e e of these isolates p r o d u c e d , however, very w e a k o f f - o d o u r s w h e n i n o c u l a t e d in fish j u i c e ( T a b l e III, c o l u m n 'B'). These were t e n t a t i v e l y i d e n t i f i e d as Moraxella b e i n g G r a m - n e g a t i v e n o n - m o t i l e , coccoid r o d s which were negative in H u g h a n d Leifson's m e d i u m , catalase positive a n d oxidase negative. T h e m a i n p a r t of the n o n - s p o i l a g e o r g a n i s m s (white colonies) were G r a m - n e g a tive species (Moraxella, Pseudomonas, V i b r i o n a c e a e , E n t e r o b a c t e r i a c e a e a n d Acinetobacter) whereas only 5 were G r a m - p o s i t i v e b a c t e r i a (Lactobacillus, C o r y n e f o r m s ) . O f the 69 ' b l a c k ' isolates f r o m fish s p o i l e d at 2 0 ° C , all were ative spoilers c a p a b l e of reducing T M A O a n d p r o d u c i n g H 2 S ( T a b l e II). 29 of these were also g r o w n in fish j u i c e a n d characteristic spoilage o f f - o d o u r s developed. Seven fermentative strains b e l o n g e d to V i b r i o n a c e a e whereas the m a j o r i t y (62 strains) b e i n g n o n - f e r m e n t a t i v e were classified as Alteromonas putrefaciens. A total of 62 white colonies were investigated a n d 30 strains, all isolated f r o m I A 1 r e p r e s e n t e d b a c t e r i a which r e d u c e d T M A O a n d / o r p r o d u c e d H 2 S in the T M A O m e d i u m ( T a b l e II). Of these organisms, 27 b e l o n g e d to the V i b r i o n a c e a e a n d further investigation b y p o u r p l a t i n g on I A 2 a n d I A 3 showed that 26 c o u l d use L-cysteine b u t n o t t h i o s u l p h a t e as a source of sulphur for H 2 S - f o r m a t i o n ( T a b l e III, c o l u m n s ' A I ' , ' A I I ' a n d ' A I I I ' ) . By c o m p a r i s o n , n o active spoilage o r g a n i s m s were i s o l a t e d as white colonies f r o m the m e d i a where L-cysteine was i n c o r p o r a t e d ( I A 2 a n d I A 3). O f the 23 ' w h i t e ' cultures which were grown in fish j u i c e only 3 i s o l a t e d f r o m I A I p r o d u c e d strong off-odours. A l l 3 were G r a m - n e g a t i v e , m o t i l e r o d s w h i c h were c a t a l a s e positive a n d a t t a c k e d glucose fermentatively. O n e b e l o n g e d t o the V i b r i o n a c e a e (oxidase-positive) a n d one to the E n t e r o b a c t e r i a c e a e (oxidase-nega-
70 TABLE III
Spoilage potential and biochemical characteristics of selected bacterial strains forming white colonies on IA
Origin of strain A*I
AII
AIII
AIV
No. of strains
25
3
2
1
B** 3
Prod. of off-odours in fish juice
NT
NT
2
1
weak,
TMAO-reduction
25
0
2
0
0
H 2S - p r o d u c t i o n ( T M A O - m e d i u m ) H 2 S-production from thiosulphate
24 0 25 25 r
3 0 33r
2 0 22 r
0 0 r
0 0 33r
25 + 25 +
3+ 3+
2+ 2+
+ +
33+
25 +
1 +, 2 -
1 +, 1 -
-
3+
25 0 0
3 0 0
2 0 0
1 0 0
0 0 0
fruity
Gram-reaction Shape
Motility Catalase
Oxidase Glucose metabolism fermentative oxidative none
* A: fish spoiled at 20°C, iron agar 1. ** B: Fish, spoiled at 0 ° C iron agar 1 + 2.
NT: Not tested.
tive) and both produced H2S when decomposing cysteine (Table III, column 'AIII'). The third was also classified as Enterobacteriaceae (based on the oxidase reaction), but was unable to form H2S from either $2032- or cysteine (Table III, column 'AIV'). This was thus the only active spoilage organism isolated which could not be detected as such on any of the media used. The majority of white colonies (57 of 62) belonged to different Gram-negative groups (Vibrionaceae, Enterobacteriaceae Moraxella, Pseudomonas, Acinetobacter and Alcaligenes) and 5 Gram-positive strains were isolated (Micrococcus and coryneforms).
Discussion
Alteromonas putrefaciens, is regarded as the main spoilage organisms of fish and fish products stored at 0°C (Chai et al., 1968; Herbert et al., 1971; Jensen and Schulz, 1980). In the present work, Alteromonas was also identified as the main spoilage organism of fish stored at 0°C. These bacteria also played a dominant role in fish spoilage at higher temperatures (i.e. 20°C), but other spoilage organisms developed as well. Thus, a large number of fermentative, Gram-negative bacteria belonging to the Vibrionaceae were found on fish spoiled at 20°C, which is probably due to their short generation-time (approx. 30 min) at this temperature (Easter et al., 1982). Similar results were obtained by Gorczyka et al. (1985) who
71 found fermentative, Gram-negative bacteria as the main spoilage organisms of rainbow trout at high temperatures (37 o C). The strains which produced characteristic spoilage off-odours were all identified as Aeromonas hydrophila. The bacteriological production of hydrogen-sulphide is a very common cause of spoilage in a variety of foods such as chilled fish (Chai et al., 1968; Herbert and Shewan, 1976), poultry (McMeekin and Patterson, 1975) and meat (Fernandez-Coil and Pierson, 1985). A number of attempts have therefore been made to detect the H2S-producing bacteria and various media have been developed. Several commercial media are available, including peptone iron agar (PIA, Difco) and lead acetate agar (LAA, Difco). These media are based on the detection of H2S-producing bacteria which appear as black/gray colonies due to precipitation of sulphidecomplexes which are formed when HzS produced from thiosulphate reacts with metal ions as Fe E+ or Pb 2+. Levin (1968) investigated the PIA and found that this medium successfully detected the H2S-producing organism, Pseudomonas putrefaciens (now: Alteromonas putrefaciens). Our work has shown that a medium with thiosulphate as the only source of sulphur is not sufficient to detect all fish spoilage organisms. Especially at high temperatures, many fermentative, Gram-negative spoilers (Vibrio/Aeromonas) develop and these organisms remain undetected unless the sulphur containing amino acid, cysteine, is included in the medium. Furthermore, it was noted that the addition of L-cysteine, being a reducing agent, enhanced and the stabilized the blackening of the colonies. A total of 87 white strains isolated from the 3 different IAs were tested for production of off-odours in sterile fish juice and 6 were classified as spoilage organisms (Table III, columns 'AIII', 'AIV' and 'B'). Two strains were, however, capable of decomposing cysteine and would therefore have been detected as spoilers on the cysteine containing medium. One strain produced strong off-odours and weak off-odours were produced by only 3 strains. These organisms, tentatively diagnosed as Enterobacteriaceae and Moraxella are not counted as spoilage bacteria even on the cysteine-supplemented IA. However, in the normal spoilage process of chilled, wet fish the role of Enterobacteriaceae and organisms with only weak spoilage potential is insignificant. The number of black colonies on cysteine containing IA is, therefore, a good indication of the number of spoilage organisms in wet fish. When characterizing pure cultures, it was found that the TMAO-medium with great accuracy can be used to describe the spoilage potential of the organism. Of the 167 strains examined, 163 (approx. 98%) were correctly classified by this medium as 'spoiler' or 'non-spoiler' when compared to the fish juice test. The TMAO-medium is practical, objective and easy to use and this test is therefore preferable to inoculation into fish juice, heat-treated or sterile filtered, or sterile muscle block when handling large numbers of isolates. References Barile, L.E., A.D. MiUa, A. Reilly and A. Villadsen, 1985. Spoilage patterns of mackerel (Rastrelliger faughni Matsui) 2. Mesophilic and psychrophilicspoilage. ASEAN Food J. 1 (3).
72 Chai, T., C. Chen, A. Rosen and R.E. Levin, 1968. Detection and incidence of specific species of spoilage bacteria on fish. II. Relative incidence of Pseudomonas putrefaciens and fluorescent pseudomonads on haddock fillets. Appl. Microbiol. 16 (11), 1738-1741. Dainty, R.H., B.G. Shaw, C.D. Hardinger and S. Michanie, 1979. The spoilage of vacuum-packed beef by cold tolerant bacteria. In: Cold tolerant microbes in spoilage and the environment, (edited by A.D. Russel and R. Fuller), Academic Press, pp. 83-110. Easter, M.C., D.M. Gibson and F.B. Ward, 1982. A conductance method for the assay and study of bacterial trimethylamine oxide reductase. J. Appl. Bacteriol. 52, 357-365. Fernandez-Coil, F. and M.D. Pierson, 1985. Enumeration of hydrogen sulphide-producing bacteria from anaerobically packaged pork. J. Food Protect. 48 (11), 982-986. Gorczyca, E., J.L. Sumner, D. Cohen and P. Brady, 1985. Mesophilic fish spoilage. Food Technol. Austr. 37 (1), 24-26. Gregersen, T., 1978. Rapid method for destinction of Gram-negative from Gram-positive bacteria. Eur. J. Appl. Microbiol. 5, 123-127. Herbert, R.A., M.S. Hendrie, D.M. Gibson and J.M. Shewan, 1971. Bacteria active in the spoilage of certain sea foods. J. Appl. Bacteriol. 34 (1), 41-50. Herbert, R.A. and J.M. Shewan, 1976. Roles played by bacterial and autolytic enzymes in the production of volatile sulphides in spoiling of North Sea cod (Gadus morhua). J. Sci. Food Agric. 27, 89-94. Hobbs, G. and W. Hodgkiss, 1982. The bacteriology of fish handling and processing. In: Developments in Food Microbiology, edited by R. Davies, Applied Science Publishers, London, 1 pp. 71-117. Hugh, R. and E. Leifsons, 1953. The taxonomic significance of fermentative versus oxidative Gram-negative bacteria. J. Bacteriol. 66, 24-26. Jensen, M. and E. Schulz, 1980. Jernagars anvendelse til friskhedsbestemmelse af fersk risk. Dansk Vet. Tidsskr. 63, 8, 154/4, 314-318. Kovacs, N., 1956. Identification of Pseudomonas pyocyanae by the oxidase reaction. Nature 178, 703. Lee, J.V., D.M. Gibson and J.M. Shewan, 1977. A numerical taxonomic study of some Pseudomonas-like marine bacteria. J. Gen. Microbiol. 98, 439-451. Levin, R.E., 1968. Detection and incidence of specific species of spoilage bacteria on fish. I. Methodology. Appl. Microbiol, 16 (11), 1734-1737. Liston, J., 1980. Microbiology in fishery science. Advances in fish science and technology, edited by J.J. Connell and staff of Torry Research Station, Aberdeen, Scotland. Fishing News (Books) Ltd., England. McMeekin, T.a. and J.T. Patterson, 1975. Characterization of hydrogen sulphide-producing bacteria isolated from meat and poultry plants. Appl. Microbiol. 29 (2), 165-169. Van Spreekens, K.J.A., 1974. The suitability of modification of long and Hammer's medium for the enumeration of more fastidious bacteria from fresh fishery products. Arch. Lebensmittelhyg. 24, 213-219.
65
Elsevier JFM 00123
Detection of specific spoilage bacteria from fish stored at low (0 o C) and high (20 o C) temperatures Lone Gram, Gunilla Trolle and Hans Henrik Huss Technological Laboratory, Ministry of Fisheries, Technical Unioersity, DK-2800 Lyngby, Denmark
(Received 28 April 1986; accepted 5 September 1986)
The spoilage potential of 309 bacterial strains isolated from fish spoiled at 0 and 20°C was investigated. Gram-negative, non-fermentative, motile rods tentatively identified as Alteromonas were the major spoilage organisms at 0 ° C. These bacteria were also found at 20 o C, but a large number of Gram-negative, fermentative, motile rods belonging to the Vibrionaceae were also identified as spoilage organisms at this temperature. Most of the Vibrionaceae did, however, not produce hydrogen sulphide from thiosulphate but only from the sulphur containing amino acid, L-cysteine. A specific count of fish spoilage organisms at both low and high temperatures could therefore be obtained directly on an Iron Agar containing thiosulphate and cysteine where bacteria capable of forming H2S from either source of sulphur would appear as black colonies. Key words: Fish; Specific spoilage bacteria; Iron agar
Introduction The bacteriology of fish spoiled at chilled temperatures has been extensively studied as reviewed by Liston (1980), H o b b s and Hodgkiss (1982) and others. It is well recognized that the Gram-negative psychrophilic or psychrotrophic organisms are the important agents of spoilage, but the precise classification of these organisms has been rather difficult, uncertain and has varied considerably following new developments in methodology. Spoilage at high ambient temperatures has been studied in fish f r o m tropical waters (Gorzcyka et al., 1985; Baffle et al., 1985) but not in fish originating from temperate waters. A large n u m b e r of bacteria ( 1 0 7 - 1 0 8 / g ) is normally found on spoiling fish, but only part of this flora m a y be classified as active spoilers. In this work the ability to reduce trimethylamine oxide ( T M A O ) and to produce h y d r o g e n sulphide (H2S) are regarded as prominent characteristics of fish spoilage bacteria. Detection Of 'active spoilers' has been attempted b y van Spreekens (1974),
Correspondence address: L. Gram, Technological Laboratory, Ministry of Fisheries, Technical University, DK-2800 Lyngby, Denmark.
0168-1605/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
66 Levin (1968) and Jensen and Schulz (1980). Van Spreekens (1974) used a medium optimal for detecting the pink endopigment of Pseudomonas putrefaciens, while Levin (1968) and Jenson and Schulz (1980) assessed the number of organisms able to produce H2S from sodium thiosulphate. However, the latter medium does not take into account that some spoilage organisms may be able to produce HaS from decomposition of sulphur-containing amino acids and not from thiosulphate. It is a purpose of this study to reassess the use of iron agar (IA) as described by Jensen and Schulz (1980) and compare this medium with IA supplemented with cysteine for the ability to detect the specific spoilage bacteria of fish stored at both low and high temperatures.
Materials and Methods
Fish raw materials Two storage trials were carried out using cod (Gadus morhua) caught in August and November 1985. The fish were gutted immediately after catch, iced and within 20 h brought to the laboratory where most fish on arrival were in rigor mortis. Some of the fish were filleted and vacuum-packed in polyethylene/polyamide bags with EVAL-barriere (Riloten 40/70 from Otto Nielsen Emballage A/S); the oxygen-permeability is 2 ml/m2/24 h at 1 atm. Whole fish and fillets were stored at 20 and 0 ° C (iced). At regular intervals during storage, samples were removed for sensory and microbiological analyses. 4-5 whole fish and 5-6 fillets were taken on each sampling day. Sensory evaluation Fish fillets were heated (80°C/20 rain) in sealed bags and odour, flavour and overall acceptability evaluated by a trained panel of 6 people. A 10-4 hedonic scale was used, with a score of 4 being the limit of acceptability. Microbiological analyses Media Iron agar (IA) was used for determination of total viable counts (TVC) and HzS-producing organisms: 2% peptone (Difco); 0.3% Lab lemco powder (Oxoid); 0.3% yeast extract (Difco), 0.03% ferric citrate; 0.03% sodium thiosulphate; 0.5% NaC1; 1.2% agar. pH was adjusted to 7.4 with 2 N NaOH and the IA sterilized at 121°C for 15 min. When carrying out the experiment in November, iron agars with different sources of sulphur were compared: 1/1 1 with 0.03% sodium thiosulphate (as described above), 1tt 2 with 0.04% L-cysteine (No. C-7755, Sigma) and 1.4 3 containing both 0.03% sodium thiosulphate and 0.04% L-cysteine. When using IA 2 and IA 3, the L-cysteine is added to the solid agar just before melting. When
67 determining bacterial counts, pour plating was used and the plates covered with a thin layer of IA after solidification and incubated at 25 ° C for 3 days. Bacteria able to produce HzS when decomposing thiosulphate a n d / o r cysteine will form black colonies due to precipitation of FeS. The overlay is applied to prevent fading of the black colonies, which happens when FeS is oxidized. Since the FeS-complex is soluble in acid, the effect of pH on the formation of a black precipitate was investigated. Pure cultures were inoculated into IA tubes with pH 7.4 (which is used in IA) and pH 6.8 (which is used in peptone iron agar (PIA, Difco)) (Levin, 1968)) and it was observed that the precipitation was faster and greater in the former. TMAO-medium was used when bacterial strains were tested for their ability to reduce TMAO and produce HES: 2% peptone (Difco), 0.3% Lab Lemco powder (Oxoid), 0.3% yeast extract (Difco), 0.03% ferric citrate, 0,03% sodium thiosulphate, 0.4% NaC1, 0.4% KH2PO4, 0.575% K2HPO4, 0.05% MgSO4, 2 resazurin tablets (BDH Chemicals) and 0.4% agar. pH was adjusted to 6.8 since it has been shown that the TMAO reductase (from Alteromonas spp.) has its maximum activity at this pH (Easter et al., 1982). The medium was sterilized at 121°C for 15 rain. Before use, 0.04% L-cysteine (No. C-7755, Sigma) was added and the medium boiled to expel any oxygen. After cooling to 45°C, 0.5% TMAO, 2H20 (Sigma) was added. The medium was poured into tubes and after inoculation covered with a thin layer of sterile paraffin oil or soft paraffin. The tubes were incubated at 25 o C for 3 days. If TMAO is reduced, the redoxindicator in the medium will change from red to yellow, and a black precipitate of FeS will be formed if H2S is produced from thiosulphate a n d / o r cysteine. A sterile fish juice was used when bacterial strains were tested for their ability to produce off-odours: fresh fish muscle was ground and mixed well with distilled water (1 : 2). The mixture was heated to 70°c to precipitate the majority of proteins, filtered and sterilized by boiling for 15 min.
Procedures Samples for microbiological analyses were prepared by homogenizing 50 g of fish in 50 ml of 0.1% peptone saline using a Colworth Stomacher 400. When samples were taken from whole fish, part of the belly flaps was used. 1 ml of the primary 1 : 1 suspension was withdrawn and decimal dilutions were prepared in 0.1% peptone saline. Isolation of spoilage flora. 10-20 black and 10-20 white colonies were randomly selected from IA plates poured on the day the fish were considered just unacceptable (score < 4). Black colonies were isolated from plates with 10-50 black colonies and white colonies from plates with 30-100 colonies. The strains were grown in veal infusion broth (Difco) and IA. The following reactions and biochemical tests were used for characterization of the spoilage flora: Gram-reaction was tested by the KOH-method (Gregersen, 1978). Phase contrast microscopy was used for determination of shape and motility. Cytochrome-oxidase was tested by the method of Kovacs (1956) and catalase formation with 3% H202. Glucose metabolism was investigated by the O/F-test of
68
Hugh and Leifson (1953). On the basis of these criteria, the strains were tentatively identified following Dainty et al. (1979). Furthermore, the strains were tested for their ability to reduce TMAO to TMA and to produce H2S. Gram-negative, motile rods which were catalase and oxidase positive, unable to attack glucose fermentatively but able to reduce TMAO were tentatively identified as Alteromonas putrefaciens (Lee et al., 1977). The strains isolated in November were also tested for their ability to produce off-odours when inoculated in a sterile fish juice.
Results
Fish stored at 0 o C as either whole fish or vacuum-packed fillets passed the limit of acceptability after 9-10 days of storage whereas fish stored at 20°C were rejected after only 1 day. At rejection time, total viable counts (TVC) had reached levels of 6 x 106-108 C F U / g and the number of H2S-producing organisms varied from 5 x 106 to 8 x 107 C F U / g . At both storage temperatures, the number of H2S-producing bacteria at rejection constituted a larger percentage, i.e. 70-80% of the total count in vacuum-packed fillets as compared to 40-50% for whole fish. Investigation of bacterial strains isolated as either black or white colonies from fish spoiled at 0 ° C showed that all 'black' isolates (94) were potential spoilage organisms due to their ability to reduce TMAO and produce H2S (Table I). Furthermore, 75 of the 94 strains were grown in a sterile fish juice and all produced spoilage off-odours described as fishy, rotten or cabbage-like. 91 'black' isolates were tentatively identified as Alteromonas putrefaciens and 3 as Vibrionaceae due to their fermentative glucose metabolism.
TABLE I Spoilage potential and biochemical characterization of bacterial strains isolated from fish spoiled at 0 o C Medium: Source of sulphur: Appearance: No. of strains isolated Prod. o f ' o f f - o d o u r s ' i n fish juice TMAO-reduction H2 S-production (TMAO-media) Gram reaction Shape (r = rods) Motility Catalase Oxidase Glucose metabolism (HL) fermentative oxidative none
Iron agar I $2O 2 -
Iron agar 2 Cysteine
Iron agar 3 $202- + cysteine
Black
White
Black
White
Black
White
58 39/39 58 58 58 58 r 58+ 58 + 58+
44 1/24 0 0 41 - , 3 + 44 r 2 3 + , 21 44 + 37+, 7-
20 20/20 20 20 2020 r 20+ 20 + 20+
20 2/20 0 0 20 20 r 4+, 1620 + 18+, 2-
16 16/16 16 16 16 16 r 16+ 16 + 16+
20 0/20 0 0 18 - , 2 + 20 r 2+, 1820 + 18+, 2-
0 27 31
3 24 17
3 2 15
4 2 14
0 4 12
3 8 9
69 TABLE II Spoilage potential and biochemical characteristics of bacterial strains isolated from fish spoiled at 20°C Medium: Source of sulphur: Appearance: No. of strains tested Prod. of off-odour in fish juice TMAO-reduction HE S-production (TMAO-media) Gram reaction Shape (r = rods, c = cocci) Motility Catalase Oxidase Glucose metabolism (HL) fermentative oxidative none
Iron agar 1 $203
Iron agar 2 Cysteine
Iron agar 3 $2032- + cysteine
Black
White
Black
White
Black
White
50 10/10 50 50 50 50 r 50+ 50 + 50+
49 3/10 27 30 48 - , 1 + 49 r 35+, 1448 +, 1 38+, 11-
9 9 9 9 99r 9+ 9+ 9+
7 0 0 0 3-, 4+ 3 r, 4 c 2+, 5 7+ 6+, 1 -
10 10 10 10 1010 r 10+ 10 + 10+
6 0 0 0 66r 6+ 6+ 5+, 1 -
2 8 40
33 1 15
1 2 6
2 0 5
4 2 4
2 0 4
N o n e of the strains isolated as white colonies f r o m fish stored at 0 ° C c a u s e d a n y changes in the T M A O m e d i u m , regardless of the source of s u l p h u r used in the p r i m a r y I A plates. T h r e e of these isolates p r o d u c e d , however, very w e a k o f f - o d o u r s w h e n i n o c u l a t e d in fish j u i c e ( T a b l e III, c o l u m n 'B'). These were t e n t a t i v e l y i d e n t i f i e d as Moraxella b e i n g G r a m - n e g a t i v e n o n - m o t i l e , coccoid r o d s which were negative in H u g h a n d Leifson's m e d i u m , catalase positive a n d oxidase negative. T h e m a i n p a r t of the n o n - s p o i l a g e o r g a n i s m s (white colonies) were G r a m - n e g a tive species (Moraxella, Pseudomonas, V i b r i o n a c e a e , E n t e r o b a c t e r i a c e a e a n d Acinetobacter) whereas only 5 were G r a m - p o s i t i v e b a c t e r i a (Lactobacillus, C o r y n e f o r m s ) . O f the 69 ' b l a c k ' isolates f r o m fish s p o i l e d at 2 0 ° C , all were ative spoilers c a p a b l e of reducing T M A O a n d p r o d u c i n g H 2 S ( T a b l e II). 29 of these were also g r o w n in fish j u i c e a n d characteristic spoilage o f f - o d o u r s developed. Seven fermentative strains b e l o n g e d to V i b r i o n a c e a e whereas the m a j o r i t y (62 strains) b e i n g n o n - f e r m e n t a t i v e were classified as Alteromonas putrefaciens. A total of 62 white colonies were investigated a n d 30 strains, all isolated f r o m I A 1 r e p r e s e n t e d b a c t e r i a which r e d u c e d T M A O a n d / o r p r o d u c e d H 2 S in the T M A O m e d i u m ( T a b l e II). Of these organisms, 27 b e l o n g e d to the V i b r i o n a c e a e a n d further investigation b y p o u r p l a t i n g on I A 2 a n d I A 3 showed that 26 c o u l d use L-cysteine b u t n o t t h i o s u l p h a t e as a source of sulphur for H 2 S - f o r m a t i o n ( T a b l e III, c o l u m n s ' A I ' , ' A I I ' a n d ' A I I I ' ) . By c o m p a r i s o n , n o active spoilage o r g a n i s m s were i s o l a t e d as white colonies f r o m the m e d i a where L-cysteine was i n c o r p o r a t e d ( I A 2 a n d I A 3). O f the 23 ' w h i t e ' cultures which were grown in fish j u i c e only 3 i s o l a t e d f r o m I A I p r o d u c e d strong off-odours. A l l 3 were G r a m - n e g a t i v e , m o t i l e r o d s w h i c h were c a t a l a s e positive a n d a t t a c k e d glucose fermentatively. O n e b e l o n g e d t o the V i b r i o n a c e a e (oxidase-positive) a n d one to the E n t e r o b a c t e r i a c e a e (oxidase-nega-
70 TABLE III
Spoilage potential and biochemical characteristics of selected bacterial strains forming white colonies on IA
Origin of strain A*I
AII
AIII
AIV
No. of strains
25
3
2
1
B** 3
Prod. of off-odours in fish juice
NT
NT
2
1
weak,
TMAO-reduction
25
0
2
0
0
H 2S - p r o d u c t i o n ( T M A O - m e d i u m ) H 2 S-production from thiosulphate
24 0 25 25 r
3 0 33r
2 0 22 r
0 0 r
0 0 33r
25 + 25 +
3+ 3+
2+ 2+
+ +
33+
25 +
1 +, 2 -
1 +, 1 -
-
3+
25 0 0
3 0 0
2 0 0
1 0 0
0 0 0
fruity
Gram-reaction Shape
Motility Catalase
Oxidase Glucose metabolism fermentative oxidative none
* A: fish spoiled at 20°C, iron agar 1. ** B: Fish, spoiled at 0 ° C iron agar 1 + 2.
NT: Not tested.
tive) and both produced H2S when decomposing cysteine (Table III, column 'AIII'). The third was also classified as Enterobacteriaceae (based on the oxidase reaction), but was unable to form H2S from either $2032- or cysteine (Table III, column 'AIV'). This was thus the only active spoilage organism isolated which could not be detected as such on any of the media used. The majority of white colonies (57 of 62) belonged to different Gram-negative groups (Vibrionaceae, Enterobacteriaceae Moraxella, Pseudomonas, Acinetobacter and Alcaligenes) and 5 Gram-positive strains were isolated (Micrococcus and coryneforms).
Discussion
Alteromonas putrefaciens, is regarded as the main spoilage organisms of fish and fish products stored at 0°C (Chai et al., 1968; Herbert et al., 1971; Jensen and Schulz, 1980). In the present work, Alteromonas was also identified as the main spoilage organism of fish stored at 0°C. These bacteria also played a dominant role in fish spoilage at higher temperatures (i.e. 20°C), but other spoilage organisms developed as well. Thus, a large number of fermentative, Gram-negative bacteria belonging to the Vibrionaceae were found on fish spoiled at 20°C, which is probably due to their short generation-time (approx. 30 min) at this temperature (Easter et al., 1982). Similar results were obtained by Gorczyka et al. (1985) who
71 found fermentative, Gram-negative bacteria as the main spoilage organisms of rainbow trout at high temperatures (37 o C). The strains which produced characteristic spoilage off-odours were all identified as Aeromonas hydrophila. The bacteriological production of hydrogen-sulphide is a very common cause of spoilage in a variety of foods such as chilled fish (Chai et al., 1968; Herbert and Shewan, 1976), poultry (McMeekin and Patterson, 1975) and meat (Fernandez-Coil and Pierson, 1985). A number of attempts have therefore been made to detect the H2S-producing bacteria and various media have been developed. Several commercial media are available, including peptone iron agar (PIA, Difco) and lead acetate agar (LAA, Difco). These media are based on the detection of H2S-producing bacteria which appear as black/gray colonies due to precipitation of sulphidecomplexes which are formed when HzS produced from thiosulphate reacts with metal ions as Fe E+ or Pb 2+. Levin (1968) investigated the PIA and found that this medium successfully detected the H2S-producing organism, Pseudomonas putrefaciens (now: Alteromonas putrefaciens). Our work has shown that a medium with thiosulphate as the only source of sulphur is not sufficient to detect all fish spoilage organisms. Especially at high temperatures, many fermentative, Gram-negative spoilers (Vibrio/Aeromonas) develop and these organisms remain undetected unless the sulphur containing amino acid, cysteine, is included in the medium. Furthermore, it was noted that the addition of L-cysteine, being a reducing agent, enhanced and the stabilized the blackening of the colonies. A total of 87 white strains isolated from the 3 different IAs were tested for production of off-odours in sterile fish juice and 6 were classified as spoilage organisms (Table III, columns 'AIII', 'AIV' and 'B'). Two strains were, however, capable of decomposing cysteine and would therefore have been detected as spoilers on the cysteine containing medium. One strain produced strong off-odours and weak off-odours were produced by only 3 strains. These organisms, tentatively diagnosed as Enterobacteriaceae and Moraxella are not counted as spoilage bacteria even on the cysteine-supplemented IA. However, in the normal spoilage process of chilled, wet fish the role of Enterobacteriaceae and organisms with only weak spoilage potential is insignificant. The number of black colonies on cysteine containing IA is, therefore, a good indication of the number of spoilage organisms in wet fish. When characterizing pure cultures, it was found that the TMAO-medium with great accuracy can be used to describe the spoilage potential of the organism. Of the 167 strains examined, 163 (approx. 98%) were correctly classified by this medium as 'spoiler' or 'non-spoiler' when compared to the fish juice test. The TMAO-medium is practical, objective and easy to use and this test is therefore preferable to inoculation into fish juice, heat-treated or sterile filtered, or sterile muscle block when handling large numbers of isolates. References Barile, L.E., A.D. MiUa, A. Reilly and A. Villadsen, 1985. Spoilage patterns of mackerel (Rastrelliger faughni Matsui) 2. Mesophilic and psychrophilicspoilage. ASEAN Food J. 1 (3).
72 Chai, T., C. Chen, A. Rosen and R.E. Levin, 1968. Detection and incidence of specific species of spoilage bacteria on fish. II. Relative incidence of Pseudomonas putrefaciens and fluorescent pseudomonads on haddock fillets. Appl. Microbiol. 16 (11), 1738-1741. Dainty, R.H., B.G. Shaw, C.D. Hardinger and S. Michanie, 1979. The spoilage of vacuum-packed beef by cold tolerant bacteria. In: Cold tolerant microbes in spoilage and the environment, (edited by A.D. Russel and R. Fuller), Academic Press, pp. 83-110. Easter, M.C., D.M. Gibson and F.B. Ward, 1982. A conductance method for the assay and study of bacterial trimethylamine oxide reductase. J. Appl. Bacteriol. 52, 357-365. Fernandez-Coil, F. and M.D. Pierson, 1985. Enumeration of hydrogen sulphide-producing bacteria from anaerobically packaged pork. J. Food Protect. 48 (11), 982-986. Gorczyca, E., J.L. Sumner, D. Cohen and P. Brady, 1985. Mesophilic fish spoilage. Food Technol. Austr. 37 (1), 24-26. Gregersen, T., 1978. Rapid method for destinction of Gram-negative from Gram-positive bacteria. Eur. J. Appl. Microbiol. 5, 123-127. Herbert, R.A., M.S. Hendrie, D.M. Gibson and J.M. Shewan, 1971. Bacteria active in the spoilage of certain sea foods. J. Appl. Bacteriol. 34 (1), 41-50. Herbert, R.A. and J.M. Shewan, 1976. Roles played by bacterial and autolytic enzymes in the production of volatile sulphides in spoiling of North Sea cod (Gadus morhua). J. Sci. Food Agric. 27, 89-94. Hobbs, G. and W. Hodgkiss, 1982. The bacteriology of fish handling and processing. In: Developments in Food Microbiology, edited by R. Davies, Applied Science Publishers, London, 1 pp. 71-117. Hugh, R. and E. Leifsons, 1953. The taxonomic significance of fermentative versus oxidative Gram-negative bacteria. J. Bacteriol. 66, 24-26. Jensen, M. and E. Schulz, 1980. Jernagars anvendelse til friskhedsbestemmelse af fersk risk. Dansk Vet. Tidsskr. 63, 8, 154/4, 314-318. Kovacs, N., 1956. Identification of Pseudomonas pyocyanae by the oxidase reaction. Nature 178, 703. Lee, J.V., D.M. Gibson and J.M. Shewan, 1977. A numerical taxonomic study of some Pseudomonas-like marine bacteria. J. Gen. Microbiol. 98, 439-451. Levin, R.E., 1968. Detection and incidence of specific species of spoilage bacteria on fish. I. Methodology. Appl. Microbiol, 16 (11), 1734-1737. Liston, J., 1980. Microbiology in fishery science. Advances in fish science and technology, edited by J.J. Connell and staff of Torry Research Station, Aberdeen, Scotland. Fishing News (Books) Ltd., England. McMeekin, T.a. and J.T. Patterson, 1975. Characterization of hydrogen sulphide-producing bacteria isolated from meat and poultry plants. Appl. Microbiol. 29 (2), 165-169. Van Spreekens, K.J.A., 1974. The suitability of modification of long and Hammer's medium for the enumeration of more fastidious bacteria from fresh fishery products. Arch. Lebensmittelhyg. 24, 213-219.