Hans Henrik Huss

Detection of specific spoilage bacteria from fish stored at low (0°C) and high (20°C) temperatures

Abstract 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°C, but a large number of Gram-negative, fermentative, motile rods belonging to the Vibrionaceae were also identifieas spoilage organisms at this temperature. Most of the Vibrionaceae did, however, not produce hydrogen sulphide from thio-sulphate 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.

Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres

Microbial growth, sensory and chemical changes and composition of gas atmosphere were studied in vacuum packed (VP) and modified atmosphere packed (MAP) cod fillets stored at 0 degree C. Contrary to previous studies, coccobacilli and pleomorphic Gram-negative microorganisms (2-4 by 2-5 microns) and not Shewanella putrefaciens were found most likely to be the main spoilage organisms. These microorganisms, which may be Photobacterium phosphoreum, can explain the short shelf-life extension of VP and MAP fish products compared to meat products. It is suggested that they may inhibit the typical H2S-producing fish spoilage bacteria, S. putrefaciens, as the maximum concentration of H2S-producing bacteria found in MAP fish products is very low. Compared to VP, a shelf-life extension of 6-7 days was obtained with 48% CO2 in MAP. However, with pure CO2 the shelf life was only extended by 2-3 days. Poor texture and high drip loss indicated that the shelf life of these fillets was limited by chemical reactions and not only by microbial activity.

Growth and activity of Shewanella putrefaciens isolated from spoiling fish

A total of 101 cultures of H2S-producing organisms isolated from spoiling cod was studied. All cultures were identified as Shewanella putrefaciens. Two groups were distinguished on the basis of pattern of trimethylamine oxide reduction determined by conductance measurement, generation time at 25 degrees C and salt tolerance. The S. putrefaciens cultures were further characterized in a number of model experiments in order to examine the spoilage activity under various conditions. A good correlation between bacterial counts, detection time in conductance measurements and production of trimethylamine and off-odour was found. There were no differences in spoilage potential between strains of S. putrefaciens, and neither initial level of inoculum nor batch of cod used as substrate influenced the levels of bacterial count corresponding to a certain production of trimethylamine and off-odour.

Microbiological quality and shelf life prediction of chilled fish

A number of storage experiments have been carried out with whole cod and vacuum-packed cod fillets stored in ice. The microbiological quality of the fish was determined on the basis of detection time estimated rapidly by conductance assays in a TMAO-containing medium at 25 degrees C. Detection time and sensory data have been incorporated into a predictive linear model to estimate the remaining shelf life of the products. It is concluded that the shelf life of iced whole cod can be predicted using this model but not that of vacuum-packed fillets because of the greater variability of bacterial activity in packaged fish.

The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus).

A total of 177 bacterial cultures isolated from Lake Victorian Nile Perch (Lates niloticus) were investigated. The flora on newly caught Nile perch consisted of organisms belonging to the genera Moraxella, Alcaligenes, Acinetobacter, Pseudomonas, Aeromonas, Micrococcus and other Gram-positive organisms. 39% were identified as Gram-positive species and 61% were negative in the Gram-reaction. Three cultures out of 53 investigated caused weak rotten off-odours in sterile fish broth and one culture, an Aeromonas spp. produced strong rotten, fishy, hydrogen sulphide off-odours. From Nile perch spoiled at ambient temperature, 15 of the 42 strains isolated caused rotten, fishy, hydrogen sulphide off-odours. These specific spoilage bacteria were all identified as Aeromonas and all reduced trimethylamine oxide to trimethylamine and produced hydrogen sulphide. From spoiled iced Nile perch, 74 out of 82 (90%) of the bacteria isolated were identified as Pseudomonas. A small proportion of these (13 out of 74) produced off-odours in sterile fish broth resembling the spoiling fish. These specific spoilers could not be separated from the non-spoilers based on biochemical activities used in classical taxonomy. While the Pseudomonas spp. isolated did not produce trimethylamine or H2S, a few of the remaining isolates (two Shewanella putrefaciens and five Aeromonas spp.) did produce these compounds. The role of Shewanella putrefaciens in the iced spoilage of Nile perch was, however, insignificant, since they only very late in the storage reached numbers where their spoilage could be detected.

Heat resistance of Listeria monocytogenes in vacuum packaged pasteurized fish fillets

The heat resistance of two strains of Listeria monocytogenes in sous-vide cooked fillets of cod and salmon was investigated. Fish sticks of 5 g were inoculated, vacuum-packed and heated at different combinations of time and temperature (58-80 degrees C). Time-temperature combinations allowing survival and time-temperature combinations at which the bacteria were destroyed, were used to determine D- and z-values. D-values were in the range of what has been published for other food products. D60-values were between 1.95 and 4.48 min depending on the strain and the fish. Both strains were one-four-times more heat resistant in salmon than in cod, showing the importance of the heating menstruum. This difference may be due to the higher fat content in salmon as compared to cod. Z-values were calculated to be 5.65 and 6.4 degrees C, respectively, for the two strains. The suitability of methods for heat resistance experiments and the survival of L. monocytogenes in sous-vide cooked fish fillets are discussed.

Antibiotic resistance in bacteria isolated from three freshwater fish farms and an unpolluted stream in Denmark

Abstract Resistance profiles against two antibiotics, oxytetracycline and oxolinic acid, were determined for 296 bacterial strains isolated from three fish farms (202 strains) and from an unpolluted stream (94 strains) in Denmark. Resistance against oxytetracycline (25 μg/ml) was seen amongst 15% (29 strains) from fish farms and for 6% (6 strains) from an unpolluted stream. Oxolinic acid resistance was also more prevalent amongst fish farm strains where 27% (53 strains) grew in the presence of 12.5 μg/ml oxolinic acid. Only 16% (15 strains) of isolates from the unpolluted stream were resistant against oxolinic acid. Bacteria from the fish farms may spread with the water circulation and, although the resistance frequencies found in this study were not statistically different, the use of chemotherapeutics may add to the environmental resistance pool. Two types of inhibition curves were observed. One showed instant inhibition at a given concentration of antibiotic. The other curve showed a slow decrease in growth with increasing concentration of antibiotic. Six percent (13 strains) and 4% (4 strains) from fish farms and an unpolluted stream were resistant to both antibiotics. The isolates from the fish farms and the unpolluted stream were identified as Vibrionaceae, Enter-obacteriaceae, Alcaligenes spp., Acinetobacter spp., Pseudomonas spp., Moraxella spp., Moraxella-like and Shewanella putrefaciens. Fifteen percent were Gram-positive organisms of which one half were resistant to oxolinic acid. Resistance profiles for the Gram-negative bacteria were, in general, independant of species or groups and no difference was seen between isolates from three different trout farms exposed to different environmental stress.

Characteristics and antagonistic activity of lactic acid bacteria isolated from chilled fish products

In fish products, contrary to other foods, the commercial use of starter cultures is relatively unexploited. In this study, 61 isolates of lactic acid bacteria from lightly preserved, chilled fish products were characterized with special emphasis on their antagonistic activity and their possible use as starter cultures. The strains were phenotypically identified and characterized with regard to gas production, carbohydrate fermentations, production of off odours, H2O2-production, antagonistic activity and growth at 2, 5 and 10 degrees C. Growth at 10 degrees C was detected for all strains within 4 days. At 5 degrees C, 90% of the isolates were growing within 3-9 days. At 2 degrees C only 33% of the strains were growing within 9 days. Fourteen strains were selected for further testing of antagonistic activity against spoilage- and pathogenic bacteria at different pH in a disc assay. Two strains, a Leuconostoc spp. (V 6) and a Lactobacillus plantarum (LKE 5), differed from the other lactic acid bacteria by a very wide inhibitory spectrum for V 6 and by very distinct inhibition zones for LKE 5. The mechanisms of their inhibitory effects are discussed.

Growth and histamine production by Morganella morganii under various temperature conditions

Abstract The generation times (G) for Morganella morganii were determined using absorbance measurements (at 0–10°C) or conductance measurements (at 15–25°C). Growth was rapid at 15–25°C (G = 2.6 at 15°C and G = 1.08 h at 25°C) but at the lower temperatures growth was greatly reduced. Growth and histamine production by M. morganii in histidine-containing broth (HDB) and in mackerel (packed in air-permeable plastic bags) was studied. It was found that large amounts of histamine are formed at low temperatures (0–5°C), where no growth takes place, following storage at higher temperatures (10–25°C). Measurements of conductance can be used to separate between samples with no or only insignificant content of histamine ( 50 ppm) which had shorter DT.

Antagonistic activity of two strains of lactic acid bacteria against Listeria monocytogenes and Yersinia enterocolitica in a model fish product at 5°C

The antagonistic activity of lactic acid bacteria against one strain of Listeria monocytogenes, Scott A and one strain of Yersinia enterocolitica serotype O:3 was studied by using either a strain of a Leuconostoc spp. or a Lactobacillus plantarum strain. Each of the two potential pathogenic, psychrotrophic bacteria were inoculated with either of the two lactic acid bacteria in sterile shrimp extracts containing citric acid (pH 5.8 and 6.0) and in some experiments also 3% (w/v) NaCl. The samples were incubated at 5 degrees C. Antagonistic effect was obtained with both lactic acid bacteria against both pathogenic organisms, with the effect depending on inoculation levels of the pathogenic organism, the lactic acid bacteria strain and media composition.