Proteolytic Activity of Microorganisms Isolated from Freshwater Fish

Transcription

1 APPLIED MICROBIOLOGY, Jan. 1968, p Copyright 1968 American Society for Microbiology Vol. 16. No. 1 Printed in U.S.A. Proteolytic Activity of Microorganisms Isolated from Freshwater Fish NURIA KAZANAS U.S. Bureau of Commercial Fisheries Technological Laboratory, Ann Arbor, Michigan Received for publication 5 September 1967 A raw fish-juice was prepared and sterilized tough the use of ttco y-irradiation. It was evaluated for suitability in an agar medium for testing the proteolytic activity of bacteria isolated from fish. Microorganism proteolytic activity was also detected by conventional methods with skim milk-agar. We tested 1,145 isolates from fresh and spoiling irradiated (0.0, 0.3, and 0.6 Mrad) yellow perch fillets for proteolytic activity, by the use of both media. Most isolates that showed proteolytic activity exhibited this activity in both media. A few isolates showed proteolytic activity only in one medium or the other. Proteolysis was found mainly among bacteria isolated from nonirradiated perch fillets. Nonproteolytic organisms were slightly more abundant than were proteolytic ones toughout refrigerated storage (6 days); the latter constituted 48% of the total organisms. Irradiation eliminated essentially all proteolytic bacteria when the fillets were stored at 1 C. However, some proteolytic bacteria survived for a few days after irradiation when the fillets were stored at 5 C. During our studies (3, 4) on the preservation of proteolytic activity of bacteria isolated from fish, freshwater fish, bacteria were isolated from irradiated and nonirradiated fresh yellow perch proteolytic organisms in fresh and spoiling irradi- and to report (at a later date) on the incidence of (Perca flavescens) fillets when the fillets were ated and nonirradiated fish. spoiled at refrigerator temperatures. Inasmuch as these organisms were associated with the spoilage MATERIALS AND METHODS of fish, we wanted to gain a better understanding Raw fish-juice. Whole yellow perch obtained from of their relation to spoilage. Lake Michigan were caught either by gillnet or by Microorganisms growing at low temperature trawl and were immediately frozen [about 20 lb and exhibiting proteolytic activity have been assumed to be primarily responsible for fish spoilage needed, the raw fish-juice was prepared from the (9.07 kg) per stock] for as long as 2 months. When (7). The conventional method of determining the above stock as follows. Fish were eviscerated and proteolytic activity of microorganisms has employed skim milk, gelatin hydrolysis, or both. For ground while in a semifrozen state and were pressed. skinned while still frozen. Flesh and bones were a better evaluation of the proteolytic activity of For the latter operation, a Carver laboratory press, microorganisms isolated from fresh and spoiling model B, was used. The pressed juice was first passed raw fish, a fish substrate simulating the natural tough a supercell prefilter pad, and then tough a composition of fresh, raw fish is also desirable. double thickness of Whatman no. 40 filter paper on It was therefore necessary for us to develop a a Buchner funnel. The juice was allotted in 100-mi fish substrate that would be routinely available portions and irradiated to a dose of 3 Mrad by means of the "0Co source at the Phoenix Memorial and that could be easily sterilized. To Laboratory of The University of Michigan. The juice was simulate natural conditions and to preserve the native state held in ice during irradiation. Finally, this juice was of labile constituents as much as possible, we employed in the preparation of raw perch-juice used cold sterilization as the method. Although medium. filtration sterilization could accomplish cold sterilization, the various manipulative difficulties that evaporated skim milk (White House brand), con* Skim milk medium. Commercially prepared sterile would be involved in filtering fish-juice removed centrated twofold, was used. A 30% skim milk-agar this method from consideration. medium was prepared by adding 15 ml of the skim We undertook the present study to develop this milk to 85 ml of sterile Nutrient Agar (Difco) held medium, to evaluate its suitability for testing at 42 C; it was then poured into petri dishes. 128

2 VOL. l16, 1968 PROTEOLYTIC ACTIVITY OF MICROORGANISMS 129 Bacterial stocks. The 1,145 organisms tested for proteolytic activity were isolated from fresh and spoiling nonirradiated and irradiated (0.3 and 0.6 Mrad) yellow perch fillets during the investigation of application of 60Co y-rays to the extension of refrigerated shelf-life of freshwater fishery products stored at either 1 or 5 C (3, 4). These organisms were obtained from six separate studies on the preservation of shelf-life. The isolates represented several species of the genera Pseudomonas; the Acomobacter-Alcaligenes group; Brevibacterium, Micrococcus, Flavobacterium, Bacillus, Sarcina, Microbacterium, Corynebacterium, Lactobacillus, Vibrio, Aeromonas; and a few yeasts. Preliminary evaluation of raw fish-juice medium. To obtain a suitable concentration of fish-juice in the medium for detecting bacterial proteolysis, we tested a number of the organisms on several concentrations of the fish-juice contained in 1.5% agar. The concentration of raw fish-juice in the final medium ranged from 9 to 50%. Both ingredients were held at 42 C before they were mixed and poured into petri dishes. Only those bacterial isolates from yellow perch known to have a positive or negative proteolytic activity on 30% skim milk medium were employed in this preliminary fish-medium evaluation (Table 1). Proteolytic assay. Eight to 10 isolates (18- to 24- cultures) were streaked on plates of either fish juiceagar or skim milk-agar medium, and were incubated at 20 C for 24 to 48. Caseinase or fish-protein protease activity possessed by the isolates could be an adaptive process that is elaborated only as needed. Thus, stimulation of these extracellular enzymes may occur under repeated transfers to fresh substrate. Therefore, those isolates showing negative activity in either milk-agar or fish extract-agar were retransferred to fresh milk-agar or fish extract-agar. If proteolytic activity was not demonstrated after repeated transfers, the plates were further reincubated at 30 and 37 C, since these temperatures seemed more suitable for proteolytic activity for some of the isolates. Proteolytic activity was demonstrated by a clearing zone in the medium surrounding the bacterial growth. RESULTS AND DISCUSSION The raw fish-juice medium as finally composed contained 100 ml of sterile fish extract, which was added to 1 liter of sterile 1.5% agar. Both ingredients were held at 42 C prior to mixing. The selection of this mixture was primarily justified on the basis that all strains tested grew well on it, and their proteolytic enzymes produced a zone of clearing in the medium around or underneath the growth (Fig. 1). Higher concentrations of raw fish-juice in the medium delayed recognition of the enzyme activity or it was not detected at all (Table 1). Comparatively, the zone of clearing was usually larger in the milk medium. Two batches of irradiation-sterilized medium, prepared at different dates, were observed for sterility while under refrigeration (1 C). Superficial microbial growth was observed embedded in one batch after approximately 20 days. This growth consisted of many tiny pink colonies that were actively proteolytic after reisolation in raw fish juice-agar and skim milk-agar media, and incubation at 20 C. Microscopic examination of these cultures revealed cocci occurring in tetrads. On the basis of the unusually high resistance to irradiation, colony pigmentation, bacterium morphology, and proteolytic activity, these organisms resembled Micrococcus radiodurans isolated from cans of meat (1) and other sources (5). Pigmented coccus strains with similar resistance properties have also been isolated from haddock tissue (2). Growth was not observed in the second batch of medium after 62 days of refrigeration. Irradiation to 3 Mrad did not accomplish FIG. 1. Clearing zones produced by proteolytic organisms on skim milk- and raw fish juice-agar media. A. Skim milk-agar medium. B. Raw fish juice-agar medium.

3 130 KAZANAS APPL. MICROBIOL. TABLE 1. Proteolytic activity of bacteria in various concentrations of irradiated raw perch-juice in agar, incubated at 20 C Genus 9% Proteolytic activitya in agar with indicated concn of raw perch-juice 18% 24% 30% 50% Skim milk-agar (18-20 ) Corynebacterium... Sarcina... Flavobacterium... Flavobacterium... Bacillus... Pseudomonas... Pseudomonas i a Symbols:, very proteolytic;, proteolytic; i,,traces of proteolysis; -, no proteolysis observed. permanent sterility of the medium. Apparent sterility in the medium, however, was observed for about 20 days. Inasmuch as testing for microbial proteolytic activity was done on the medium within 24 to 48 after the fish-juice was irradiated, the latent microbial growth did not have any apparent interference with the test. Isolates representing the various genera commonly found in the freshwater fish and plated on the irradiation-sterilized fish-extract medium exhibited abundant growth in 24 at 20 C in all fish-juice concentrates prepared. Pugsley et al. (6) observed a postirradiation destruction effect when Escherichia coli was inoculated onto the surface of nutrient agar plates previously exposed to X irradiation. Solberg and Nickerson (8) reported inhibition rather than destruction of the microorganisms when these were inoculated onto the surface of chicken meat immediately after the meat was irradiated. Both authors demonstrated, however, that the effect dissipated as the time between exposure and inoculation increased. These findings were later confirmed (9) with irradiated chicken. When the chicken was inoculated as late as 25 after substrate-irradiation, only about 1 logarithm reduction in approximately a 104 inoculum was produced. Sydow and Tear (10) also found that irradiated synthetic ±4 ±4 4 TABLE 2. Bacterial isolatesa exhibiting proteolytic activity from nonirradiated and irradiated (0.3 and 0.6 Mrad) perch fillets held at various incubation temperatures Nature of activity No. of isolates exhibiting activity when incubated at indicated temp 20 C 30 C 37 C Proteolytic in both media b, 9c 5c Proteolytic in skim milk medium only Proteolytic in raw fish-juice medium only...9 a Total isolates tested, 1,145. b Skim milk-agar medium. c Raw fish juice-agar medium. liquid medium had a bactericidal effect on a radiosensitive marine Pseudomonas sp. taken from exponentially growing cultures. The indirect toxic effects on the medium, however, were found to decrease slowly with time. Whether bacterial inhibition would also occur in raw fish-juice inoculated immediately after being irradiated cannot be determined from this study, since the irradiated fish-juice was not only diluted in the

4 VOL. 16, 1968 TABLE 3. PROTEOLYTIC ACTIVITY OF MICROORGANISMS 131 Number of organisms isolated from irradiated and nonirradiated yellow perch fillets exhibiting activity on either perch or skim milk medium Isolates Organisms isolated after indicated irradiation 0.0 Mrad 0.3 Mrad 0.6 Mrad No. % No. % No. % Total isolates testeda Total proteolytic /932b c/ c/ Total pigmented / / / Proteolytic-pigmented / / /10 60 Pigmented proteolytic of total proteolytic. 259/ / /6 100 final preparation of the medium, but it was also inoculated within 24 to 48 after being irradiated. Under these conditions, raw-fish juice-agar medium appeared suitable for testing proteolytic activity of bacteria isolated from fish. From a total of 1,145 isolates tested, 474 showed proteolytic activity in either medium. Of these, 420 were proteolytic in both media; an additional 45 were proteolytic only in the skim milk, and 10 others only in the raw fish-juice medium (Table 2). This result might indicate that an organism producing caseinase does not necessarily produce fish proteases or vice versa; however, this inference requires further investigation. The majority of the proteolytic organisms (93.2%) showed proteolytic activity at an incubation temperature of 20 C. An additional 5.5 and 1.3% showed proteolytic activity in either medium at 30 and 37 C, respectively. Proteolytic activity was found mainly among bacterial isolates from nonirradiated fish (Table 3) during storage of the fillets at either 1 or 5 C. Their outgrowth while in storage is presented in Fig. 2. None of the genera Aeromonas, Acomobacter-Alcaligenes sp., Microbacterium, or Lactobacillus showed proteolytic activity. It is noteworthy (Table 3) that more than half of the pigmented isolates showed proteolytic activity; further, these organisms in turn comprised 55% of the total proteolytic flora. Sarcina and Micrococcus sp., which comprised a large number of these pigmented organisms, were found to be actively proteolytic. All Flavobacterium, but only a few pigmented Brevibacterium, showed proteolytic activity on both media. Kazanas (3) demonstrated earlier that, with the exception of Flavobacterium and Brevibacterium, most of the pigmented organisms in nonirradiated fillets were no longer detectable after the 3rd day of fillet LAII! 1 I1 1-_ Ir -a\4;pvjqqq ; lti at; nx1ar > r microorganisms isoialea auring a totai ot six snelf-life preservation studies ( Of fillets stored at either 1 or 5 C. b Number of positive organisms per number of organisms tested. c Pigmented yeast and some bacteria surviving irradiation on fillets stored at 5 C. STORAGE TIME(DAYS) FIG. 2. Relationship of total numbers of proteolytic and nonproteolytic organisms growing in fresh yellow perch fillets stored at I C. storage at 1 C. Flavobacterium, which had reached counts of 107, was no longer detected after the 5th day, but pigmented Brevibacterium was found in large numbers at the time these fillets were scored as unacceptable. Irradiation eliminated essentially all proteolytic bacteria in fillets stored at 1 C (Table 3). The few proteolytic bacteria found from irradiated fish were those isolated from fillets that were stored at a higher temperature (5 C). These were a few Pseudomonas, Bacillus, and Brevibacterium that appeared to have survived in the fillets for only a few days after irradiation. Although yeasts isolated from irradiated and nonirradiated fish were not tested for proteolytic activity, a few pink yeasts resembling Rhodotorula sp. and found among the bacteria isolated in TPY

5 132 KAZANAS' medium were tested. These organisms were actively proteolytic, but were not isolated from the fillets after the 8th day of storage (3). It has been reported (3) that Acomobacter- Alcaligenes sp. and yeasts predominate in 0.3 and 0.6 Mrad-irradiated fillets at the time of spoilage and obscure the isolation of minority groups that may also be present. In this study, all Acomobacter-Alcaligenes sp. tested were nonproteolytic on both media. By-products other than tough proteolytic activity would then appear as the contribution of these genera toward spoilage. Inasmuch as all yeasts were not tested for proteolytic activity, and the only yeast tested was the proteolytic pink species, it would not be justifiable to assume that by-products from microorganisms other than by proteolytic action would be the only microbial contribution toward spoilage of fish irradiated at 0.3 Mrad or higher. AcKNowLEDrMENTs This investigation was conducted under contract AT(11-1)-1283 from the U.S. Atomic Energy Commission. Technical assistance was received from Sharon McKenzie in the microbiological assays and from Hilding G. Olson in the operation of the 60Co source at the Phoenix Memorial Laboratory, University of Michigan. APPL. MicRoBIoL. LITERATURE CITED 1. ANDERSON, A. W., H. C. NORDAN, R. F. CAIN, G. PARRISH, AND D. DUGGAN Studies on a radio-resistant micrococcus. I. The isolation, morphology, cultural characteristics and resistance to gamma radiation. Food Technol. 10: DAVIS, N. S., G. J. SILVERMAN, AND E. B. MA- SUROVSKY Radiation-resistant, pigmented coccus, isolated from haddock tissue. J. Bacteriol. 86: KAZANAS, N Effect of -a-irradiation on the microflora of freshwater fish. II. Generic identification of aerobic bacteria from yellow perch fillets. Appl. Microbiol. 14: KAZANAS, N., J. A. EMERSON, H. L. SEAGRAN, AND L. L. KEMPE Effect of y-irradiation on the microflora of freshwater fish. I. Microbial load, lag period, and rate of growth on yellow perch (Perca flavescens) fillets. Appl. Microbiol. 14: KRABBENHOFT, K. L., A. W. ANDERSON, AND P. R. ELLIKER Ecology of Micrococcus radiodurans. Appl. Microbiol. 13: PUGSLEY, A. T., T. H. ODDIE, AND C. E. EDDY Action of X-rays on certain bacteria. Proc. Roy. Soc. (London) 118B: SHEWAN, J. M., G. HOBBS, Am W. HODGKISS The Pseudomonas and Acomobacter groups of bacteria in the spoilage of marine white fish. J. Appl. Bacteriol. 23: SOLBERG, M., AND J. T. R. NICKERSON A biological after-effect in radiation processed chicken muscle. J. Food Sci. 28: SOLBERG, M., AND J. T. R. NICKERSON Growth support potential of irradiated chicken for Escherichia coli and Staphylococcus aureus. Food Technol. 17: SyDow, E., AND J. TAR The effect of ionizing radiation on a marine Pseudomonas sp. I. Effects of irradiated liquid glucose-salt medium on populations in exponential-growth phase. Intern. J. Radiation Biol. 7:11-19.