Hydrogen Sulfide Production by Pseudomonas putrefaciens

Transcription

1 APPLED MCROBOLOGY, Apr. 1974, p Copyright American Society for Microbiology Vol. 27, No. 4 Printed in U.S.A. Hydrogen Sulfide Production by Pseudomonas putrefaciens in Shrimp Experimentally Packed in Nitrogen1 R. M. LAPN AND J. A. KOBURGER Food Science Department, University of Florida, Gainesville, Florida Received for publication 18 December 1973 Shrimp refrigerated in a nitrogen atmosphere develop off-odors not typical of normal spoilage. nvestigations of this phenomenon showed that hydrogen sulfide developed in the headspace gas, and a large percentage of the microbial population present on the shrimp stored in nitrogen was capable of hydrogen sulfide production, in contrast to the flora on shrimp stored in air. The predominant hydrogen sulfide-producing organism, Pseudomonas putrefaciens, was present in low numbers on fresh shrimp but usually reached high numbers by day 8 of nitrogen storage. Further studies revealed that cysteine and cystine were the probable substrates in shrimp utilized by this organism for hydrogen sulfide production. When shrimp sterilized by irradiation were inoculated with P. putrefaciens and incubated in an atmosphere of nitrogen, hydrogen sulfide and the characteristic off-odors developed. Fresh shrimp are extremely susceptible to bacterial and enzymatic degradations which lead to spoilage. After 50 years of research, the most effective preservation technique is rapid freezing of the freshly caught shrimp. Attempts to increase the storage life of the fresh product by the use of chemical additives have generally been discouraging. n a more novel approach to this problem, controlled atmospheric storage has been considered. n a nitrogen atmosphere, however, off-odors not typical of normal spoilage develop and render the product unacceptable Ṫhe pseudomonads appear to be significant contributors to the spoilage of iced seafoods. Generally speaking, however, there has been little information available on the effect of individual species on the ultimate organoleptic quality of iced seafood. n studies reported in a series of papers (5-8), Castell and co-workers reproduced many of the odors characteristic of the earlier stages of fish spoilage. More recently, Miller et al. (17) identified a volatile compound, produced by Pseudomonas perolens in otherwise sterile fish muscle, which is responsible for the musty, potato-like odor that commonly develops in chilled fish muscle. When fresh fish are packed in crushed ice, innumerable air pockets are present, and the eventual spoilage is aerobic; accordingly, most research has involved aerobic spoilage. f air is 1 Florida Agricultural Experiment Stations Journal Series no excluded from the fish, however, as might happen if the fish are packed closely together or against slime-soaked pen boards, anaerobic conditions will eventually exist, and a characteristic foul odor becomes evident even though the fish look and feel fresh (16). Canadians describe fish affected in this way as "bilgy," whereas the term "stinker" is used in Britain (3) Ṫhis investigation was undertaken to characterize and determine the cause of the abnormal spoilage odors, particularly hydrogen sulfide, which develops when shrimp are stored in a nitrogen atmosphere. MATERALS AND METHODS Controlled atmospheric storage. Samples consisted of fresh-headed Penaeus shrimp obtained dockside from various Florida ports. For these studies, three batches of shrimp were obtained from the East coast and two were from the Gulf coast. For aerobic storage, approximately 100 g of shrimp was placed in several sterile Erlenmeyer flasks, and beakers were placed over the flasks as closures. Those 100-g samples to be stored under nitrogen were placed into GasPak (BBL) anaerobic jars that had been specially fitted with an exhaust valve on the top. Long, spinal-tap needles attached to tanks of compressed nitrogen were inserted through the valve to the bottom of the jars, and the atmospheres were allowed to flush for 1 h. When the sensor of a dissolved oxygen meter was placed into the jar, readings indicated that 97% of the oxygen had been replaced after 5 min of flushing. All jars were then incubated at 5 C. Bacterial analyses. At specific intervals, the odor 666

2 VOL. 27, 1974 P. PUTREFACENS N NTROGEN-PACKED SHRMP and appearance of the shrimp were noted, and a subsample of approximately 50 g from each treatment was removed aseptically to a sterile blender jar. Enough sterile buffered diluent was added to make a 1:10 dilution by weight. The mixture was blended at high speed for 2 min. Due to excessive foaming, the next dilution was prepared on a weight-to-volume ratio. All subsequent dilutions were prepared on a volume-to-volume ratio. Standard plate counts were prepared by adding 1-ml volumes of the appropriate, serially diluted, buffered homogenate to two sets of petri dishes. The plates were poured with standard plate count agar (Difco) and allowed to solidify. One set of plates from each dilution was incubated aerobically, and the duplicate set was incubated anaerobically in a BBL GasPak anaerobic jar charged with a disposable hydrogen generator pack. All plates were incubated at 22 C for 5 days. Quantitation of the flora capable of hydrogen sulfide production was performed by adding 1-ml volumes of the appropriate, serially diluted, buffered homogenate to two sets of petri dishes and then pouring with peptone iron agar (PA). Duplicate plates were incubated aerobically and anaerobically for 5 days at 22 C. Colonies producing hydrogen sulfide on PA were readily identifiable because of their intense black appearance on that medium. Since later studies confirmed that the hydrogen sulfide-producing flora was comprised of the same species whether incubated aerobically or anaerobically, all hydrogen sulfide counts referred to within this study will be anaerobic counts. solation and identification of hydrogen sulfideproducing organisms. Throughout the storage studies, hydrogen sulfide-producing organisms were picked from countable dilutions of PA and inoculated into triple sugar iron agar slants (Difco). Organisms were subsequently identified according to the descriptions presented in Bergey's Manual (2) and A Guide to the dentification of the Genera of Bacteria (19). All isolates were stored on nutrient agar slants at 5 C to await biochemical and cytological examination. Staining was done by the Hucker modification of the Gram stain. Motility was observed, with a microscope, in a wet preparation and later the presence of flagella was confirmed by electron microscopy. Biochemical tests were performed at 25 C. Procedures for gelatin hydrolysis, nitrate reduction, and litmus milk reaction were followed according to the Manual of Microbiological Methods (20). The ability to utilize carbohydrates was determined with King OF medium (13). Quantitation of hydrogen sulfide. Figure 1 describes the apparatus used to quantitate the amount of hydrogen sulfide released into the headspace gas over the shrimp stored under various atmospheres. All parts were glass and connected by greased, groundglass joints. With this gas-tight system, the analysis was routinely performed by adding 150 to 200 g of fresh-headed shrimp, similar to those used for bacterial analyses, to a 100-ml, two-necked, round-bottom flask (A). The gas to be investigated, either nitrogen or air from compressed gas tanks, was bubbled slowly through a water trap (B) to reduce the drying effect of the gas stream. The washed gas then entered the shrimp storage flask and forced out any hydrogen sulfide into a zinc acetate solution (C) which trapped the gas as zinc sulfide. The trapping solution was removed at specific intervals and analyzed for hydrogen sulfide according to the spectrophotometric method described in Standard Methods for the Examination of Water and Wastewater (1). RESULTS Effect of nitrogen storage. Figure 2 presents the bacteriological data from fresh shrimp stored in an atmosphere of nitrogen for 17 days at 5 C. The total count of the aerobically stored shrimp increased from 5.2 x 103 organisms per g on day 0 to 1.6 x 109/g on day 17. Organoleptically, these shrimp were unacceptable after 12 days of storage at 5 C. The exposed flesh was dark and liquified, black spotting was heavy at the shell interfaces, and the odor was typical of spoiled shrimp, i.e., putrid, with the associated odor of ammonia. The shrimp refrigerated in a nitrogen atmos- FG. 1. Apparatus for the collection of HS from the headspace of shrimp refrigerated under various atmospheres. (A) Shrimp holding flask; B, water trap; C, absorption flask. LOD ORGANSMS/GRAM FG. 2. Effect of air and N, atmospheres on the total count ( ) and H2S count (-----) of shrimp stored at 5 C. C

3 668 LAPN AND KOBURGER APPL. MCROBOL. phere had an anaerobic count that increased from 1.3 x 103 organisms per g at day 0 to 3.2 x 107/g on day 17. This count was 1.5 log cycles less than the count for aerobically stored shrimp. After 17 days of storage at 5 C, the nitrogen-stored shrimp had not black-spotted, and the exposed flesh had not liquified. They were visibly acceptable. The odor, however, was very objectionable, being atypical of normal spoilage and strongly suggestive of hydrogen sulfide. The off-odors were first apparent after 8 days of refrigerated storage. The off-odors faded completely after several minutes of exposure to the air, and the remaining odor was stale and seaweed-like. The number of organisms capable of hydrogen sulfide production is also plotted in Fig. 2 and was found to be similar for both the aerobically and nitrogen-stored shrimp. The hydrogen sulfide counts for the aerobically stored shrimp increased from less than 100 organisms per g at day 0 to 1.6 x 10 'g after 17 days. This count of organisms capable of hydrogen sulfide production represented less than 0.1% of the total aerobic population. The flora of nitrogen-packed shrimp capable of hydrogen sulfide production after 17 days reached 6.9 x 106 organisms per g and represented 22% of the total flora under nitrogen. Hydrogen sulfide counts were approximately the same for shrimp stored in air and in nitrogen but, since the total counts were much lower for anaerobically packed shrimp, the hydrogen sulfide-producing flora represented a greater proportion of the total flora under nitrogen. Hydrogen sulfide production. The amount of hydrogen sulfide released into the headspace of shrimp refrigerated under nitrogen and air was determined (Fig. 3). Hydrogen sulfide was trapped from the nitrogen-flushed shrimp and by day 16 a total of 1.2 mg of the gas would have accumulated in the headspace if it had not been removed for analyses; however, no hydrogen sulfide was trapped from the headspace of the shrimp flushed with air. solation and identification of hydrogen sulfide-producing organisms. Throughout the storage studies, representative numbers of organisms producing hydrogen sulfide were isolated from the countable PA pour plates. Two different organisms were isolated. Erysipelothrix insidosa was isolated in only a few studies during the early days of storage, but quickly died off and was not isolated after 8 days. The other hydrogen sulfide-producing organism was always present in low numbers in fresh shrimp, but rapidly increased in numbers until it represented a significant percentage of the total population. Characteristics of this organism are listed in Table 1. Based upon these determinations, the organism was identified as Pseudomonas putrefaciens. A basal medium of 0.5% nutrient broth containing 0.5% ferric ammonium citrate, 0.1% potassium dihydrogen phosphate, and 1.5% agar was developed to determine what sulfur us H oo lso1 100*. 54 0o -o AR s DAYS Of STORAGE FG. 3. Effect of N2 and air atmospheres on the release of HS into the headspace of shrimp stored at 5 C. TABLE 1. Characteristics of the common hydrogen sulfide-producing organism from shrimp Gram stain Motility Test or substrate Growth at: 25C + 35C Litmus milk Growth in nutrient broth: 0% NaC + 7% NaCi Gelatin hydrolysis + Nitrate reduction + Oxidase + Pigment Growth on SS agar Result Small gram-negative rods +, single polar flagellum Reduction/proteolysis Tan Oxidation of: (King's medium) Glucose +/- Maltose +/- Sucrose +/- L-Arabinose +/-

4 VOL. 27, 1974 P. PUTREFACENS N NTROGEN-PACKED SHRMP 669 compounds P. putrefaciens could use to produce mended PA for the enumeration of hydrogen hydrogen sulfide. Hydrogen sul[fide was pro- sulfide-producing organisms on haddock fillet. duced from cysteine, cystine, arid thiosulfate. He found that these organisms varied in their The organism failed to produce hydrogen sul- ability to produce hydrogen sulfide on the fide from methionine, elemental sulfur, bisul- surface of PA where the redox potential was fite, sulfite, or sulfate. high. He suggested that the pour plates be Hydrogen sulfide producti )n in sterile overlaid with 5 ml of PA so that all the shrimp inoculated with P. putrrefaciens. Ap- developing colonies would be below the surface proximately 300 g of fresh shrimp was sterilized of the medium at a reduced redox potential. with a dose of 400,000 rads in thee University of PA used in this study was incubated both Florida food irradiator. The shrinip were placed aerobically and anaerobically, and it was obapparatus de- served that the hydrogen sulfide counts were in the hydrogen sulfide-trapping scribed in Fig. 1. Growth from a, 24-h culture of consistently higher and the colonies more ina nutrient agar tensely blackened in the anaerobic plates than P. putrefaciens was washed from; slant into 100 ml of phosphate buiffer which was in the aerobic plates. then mixed with the shrimp. T he atmosphere P. putrefaciens was first isolated and derogen, and the scribed by Derby and Hammer (12) as the cause was continually flushed with nit: zinc acetate-trapping solution was analyzed of surface taint in butter and was named daily for hydrogen sulfide. Uninooculated irradi- Achromobacter putrefaciens by them. The ated shrimp served as a control (rig. 4). After 48 name was later changed to Pseudomonas h, hydrogen sulfide was detectedd in the head- putrefaciens by Long and Hammer (15). King space of the inoculated shrimp. After the ini- (13) described 22 cultures of a hydrogen sulfide- increased producing gram-negative rod isolated from clin- tial lag period, the amount released sharply for 5 days, after whic] h it began to ical specimens and named it "lb." Hugh (per- found in the sonal communication) later showed that King's stabilize. No hydrogen sulfide weas nitrogen-flushed, uninoculated: shrimp. When "lb" organism was P. putrefaciens. Castell et al. the trapping apparatus was disas,sembled at the (9) were the first to report the widespread conclusion of the study, the odoor of the inocu- occurrence of this organism on fishery prodlated shrimp was very similar to) the abnormal ucts. Chai et al. (10) found that P. putrefaciens odors noticed during the latter d ays of nitrogen was one of the major species of spoilage bacteria storage of fresh shrimp. on haddock fillets. There remains some controversy concerning DSCUSSON the taxonomic position of P. putrefaciens. She- hydro- wan (18) described all true group and V P. putrefaciens was the predominant gen sulfide-producing organism isolated from Pseudomonas sp. as producing hydrogen sulfide nitrogen-packed shrimp. Levirl (14) recom- and having guanine/cytosine base ratios of 61 to 63 mol%. Since P. putrefaciens has a guanine/ g cytosine base ratio in the region of 41 to 49 H28 mol%, it cannot belong to the genus,'s xpseudomonas, according to Shewan's classification. n addition, it is facultative rather than -X aerobic, an additional reason for removing it 200 / --ol from the genus Pseudomonas. Future studies -. ~ NOCULMATE lqfwn6 will have to clarify this taxonomic problem P. putrefaciens in pure culture produced hydrogen sulfide in nitrogen-packed shrimp at 5 C and, therefore, appears to be the agent 100 responsible for the hydrogen sulfide off-odors occurring naturally in nitrogen-packed shrimp. S0 Substrate studies indicated that this organism can produce hydrogen sulfide from cysteine and UNCOWULATED cystine. Dabrowski (11) found only trace amounts of these amino acids in the free amino 4 a a12 acid pool of shrimp, and this lack of substrate DAYS OF STORAGE probably accounts for the lag noticed before FG. 4. Production of H2S from St erile shrimp inoc- hydrogen sulfide production takes place in the ulated with a- pure culture of Pseudomonas inoculated, nitrogen-flushed shrimp. During the putrefaciens. lag, proteolytic enzymes are most likely break-

5 670 LAPN AND KOBURGER ing down the tissue proteins and releasing the amino acids. The hydrogen sulfide and other volatiles produced in a reduced environment are responsible for the repulsive off-odors which render the nitrogen-packed shrimp unacceptable after approximately 8 days of refrigeration. This phenomenon is very similar to the one of bilgy fish as reported by Castell (4). Both are characterized by the presence of hydrogen sulfide and developed only under anaerobic conditions. The off-odors are rapidly dissipated when exposed to air. Since P. putrefaciens reaches high numbers in iced fish products also, it is probably responsible for the bilgy fish phenomenon. LTERATURE CTED 1. American Public Health Association Standard methods for the examination of water and wastewater, 13th ed. American Public Health Association, nc., New York.. 2. Breed, R. S., E. G. D. Murray, and N. R. Smith Bergey's manual of determinative bacteriology, 7th ed. The Williams and Wilkins Co., Baltimore. 3. Burgess, G. H. O., C. L. Cutting, J. A. Lovern, and J. J. Waterman Fish handling and processing. Chemical Publishing Company, nc., New York. 4. Castell, C. H "Bilgy" fish. Fisheries Res. Board Can. Progr. Rep. Atlantic Coast Station. 58: Castell, C. H., and M. F. Greenough The action of Pseudomonas on fish muscle. 1. Organisms responsible for odours produced during incipient spoilage of chilled fish muscle. J. Fish. Res. Board Can. 14: Castell, C. H., and M. F. Greenough The action of Pseudomonas on fish muscle. 4. Relation between substrate composition and the development of odours by Pseudomonas fragi. J. Fish. Res. Board Can. 16: Castell, C. H., M. F. Greenough, and J. Dale The action of Pseudomonas on fish muscle. 3. dentification APPL. MCROBOL. of organisms producing fruity and oniony odours. J. Fish. Res. Board Can. 15: Castell, C. H., M. F. Greenough, and N. L. Jenkins The action of Pseudomonas on fish muscle. 2. Musty and potato-like odours. J. Fish. Res. Board Can. 14: Castell, C. H., J. F. Richards, and. Wilmot Pseudomonas putrefaciens from cod fillets. J. Fish. Res. Board Can. 7: Chai, T., C. Chen, A. Rosen, and R. E. Levin Detection and incidence of specific species of spoilage bacteria on fish.. Relative incidence of Pseudomonas putrefaciens and fluorescent pseudomonads on haddock fillets. Appl. Microbiol. 16: Dabrowski, T., E. Kolakowski, and B. Karnicka Chemical composition of shrimp flesh (Parapenaeus spp.) and its nutritive value. J. Fish. Res. Board Can. 26: Derby, H. A., and B. W. Hammer Bacteriology of butter. V. Bacteriological studies on surface taint in butter. owa Agr. Exp. Sta. Res. Bull. 145: King, E The identification of an unusual Gram negative bacteria, p. 14. Center for Disease Control, Atlanta, Ga. 14. Levin, R. E Detection and incidence of specific species of spoilage bacteria on fish.. Methodology. Appl. Microbiol. 16: Long, H. F., and B. W. Hammer Classification of organisms important in diary products.. Pseudomonas putrefaciens. owa Agr. Exp. Sta. Res. Bull. 285: MacCallum, W. A Pen surfaces and odor development in trawler fish holds. Food Technol. 9: Miller, A.,, R. A. Scanlan, J. S. Lee, L. M. Libbey, and M. E. Morgan Volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas perolens. Appl. Microbiol. 25: Shewan, J. M The microbiology of fish and fishery products-a progress report. J. Appl. Bacteriol. 34: Skerman, V. B. D A guide to the identification of the genera of bacteria, 2nd ed. The Williams and Wilkins Co., Baltimore. 20. Society. of American Bacteriologists Manual of microbiological methods. McGraw-Hill Book Company, nc., New York.