FISHERY BULLETIN: VOL, 69, NO. I 



PROTEOLYTIC ENZYME ACTIVITY IN 



WHOLE AND EVISCERATED FISH 



Presented in this experiment are data on the 

 effect of time, temperature, and pH on the rate 

 of proteolytic enzyme activity in samples of 

 whole and eviscerated comminuted fish. 



MATERIALS AND METHODS 



Fish 



Hake, Merluccius productus, and Pacific her- 

 ring, Ciupea harengus pallasi, were used in this 

 study. Hake samples were obtained from both 

 coastal waters (outside hake) and Puget Sound 

 (inside hake). Herring were obtained from 

 Bellingham Bay. All samples were obtained 

 fresh and were immediately frozen and held at 

 —20° C. 



Preparation of Samples 



To insure sample homogeneity, lots of fish 

 were partially thawed and then ground in a Ho- 

 bart grinder' through a Vs-inch plate. The 

 ground fish was mixed well and divided into 

 portions of approximately 50 g each. These 

 were frozen to — 40° C in a plate freezer and 

 stored at —20° C. 



Sufficient comminuted fish was prepared to 

 permit running all pH and temperature vari- 

 ables on subsamples from the same lot. Samples 

 of herring and outside hake were prepared from 

 both whole and eviscerated fish. With inside 

 hake, autolj-lic rates were measured only on 

 whole fish. 



METHODS 



Fifty grams of comminuted fish were allowed 

 to thaw at room temjierature and then were 

 mixed well with 100 ml of H2O to form a slurry. 

 The pH of the slurry was adjusted to the de- 

 sired level with 0.1 M HCl and then sufficient 

 water was added to give a final volume of 250 

 ml. After dilution, the sample was placed in 

 a water bath. 



' The use of trade names is merely to facilitate de- 

 scriptions; no endorsement is implied. 



Aliquots of the slurry were taken (1) imme- 

 diately after the final dilution was made, (2) 

 when the sample reached the desired temper- 

 ature, and (3) at intervals of 10, 20, 30, and 60 

 minutes after the sample reached the desired 

 temperature. These aliquots were mixed im- 

 mediately with an equal volume of 10% TCA, 

 allowed to stand for 30 min, and then filtered. 

 The nonprotein nitrogen (NPN) content of the 

 filtrates was determined by the procedure of 

 Lowry, Rosebrough, Farr, and Randall (1951) 

 and is presented in terms of optical density 

 (OD) measurements at 500 m^. 



Rates of proteolytic enzyme activity at pH 

 6.0, 5.0, 4.5, 4.0, and 3.0 were measui'ed at tem- 

 peratures of 30°, 40°, 50°, 60°, 70°, and 80° C. 

 Controls consisted of samples in which no pH 

 adjustment had been made. 



RESULTS AND DISCUSSION 



The results of the above experiments are 

 shown in Figures 1 through 5. 



In whole herring (Fig. 1), no significant de- 

 gree of proteolytic enzyme activity was observed 

 at pH levels higher than pH 5.0 over the temper- 

 ature range studied. At pH 4.0-4.5, maximum 

 activity was found at 40° C, and at pH 3.0, max- 

 imum activity was found at 30° C. 



Under these experimental conditions, only 

 negligible proteolytic enz.\'me activity was ob- 

 served in eviscerated herring .samples (Fig. 2) 

 indicating that in herring proteolytic enzymes 

 are essentially of visceral origin. 



Figures 3 and 4 show the results of the ex- 

 periments using whole inside and outside hake. 

 At 30° C, the rate of proteolytic activity in both 

 control samples was negligible. As the pH was 

 lowered, the rate of activity increased. At 40° C, 

 the rate increased at all iiH levels, and these 

 rates remained elevated until the temperature 

 exceeded 60° C. In the.se two samples, differ- 

 ences were observed in the pH of optimum ac- 

 tivity at various tenijiei-atures. These variations 

 were attributed to difl'erences in the age, feeding 

 habits, etc., of the two populations. 



The eflE'ects of pH and temperature on the rate 

 of enzyme activity in eviscerated outside hake 



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