MILLER and SPINELLI: EFFECT OF PROTEASE INHIBITORS ON PROTEOLYSIS 



inhibitors were purchased from Sigma Com- 

 pany. Dibasic phosphate peroxides were pre- 

 pared in our laboratory according to the method 

 of Nakatani and Katagiri (1970). The potato 

 extract was prepared in our laboratory accord- 

 ing to the method of Melville and Ryan (1972). 



Test for the Presence of Peroxides 

 or Bromates 



The following method of measuring peroxides 

 and bromates was adapted from two methods, 

 that of Price and Lee (1970) and that of the Asso- 

 ciation of Official Analytical Chemists handbook 

 (1975): 



4 ml H 2 



1 ml of oxidant standard or 1 g fish 

 1 ml saturated KI 



1 ml 0.001 M ammonium molybdate in 1 N 

 H2SO4. 



Shake for 1 min, titrate to a light yellow with 

 0.1 N sodium thiosulfate, and add a few drops of 

 1% starch; continue titrating to the end point. 

 Both hydrogen peroxide and potassium bromate 

 liberate iodine by oxidation; therefore, this 

 method can be used to indicate the presence of 

 either one. Quantification was determined by 

 comparison with a known standard expressed in 

 milliequivalents. 



RESULTS AND DISCUSSION 



Tests with Blended Fish 



Blended fish was used to test a variety of 

 potential inhibitors which are listed with 

 concentrations and results in Table 1. The 

 enzyme inhibitors tested included trypsin inhib- 

 itors from four sources: soybeans, lima beans, 

 turkey egg white, and chicken egg white. We also 

 tested crude potato extract which has been 

 shown to contain several protease inhibitors 

 (Melville and Ryan 1972; Ryan et al. 1974; 

 Bryant et al. 1976; Hass et al. 1976). None of the 

 tested enzyme inhibitors caused significant in- 

 hibition in concentrations that would be suitable 

 for use in food systems. 



From the remaining potential inhibitors 

 which included metal chelators, oxidizers, and 

 sulfhydryl binding compounds, we found hydro- 

 gen peroxide, potassium bromate, dibasic phos- 

 phate peroxides, iodoacetate, and N-ethylmaleim- 



TABLE 1.— Protease inhibitors. 



'increased proteolysis 

 change ±. 



+, decreased proteolysis — , no signficant 



ide to warrant further investigation. The reaction 

 with iodoacetate and N-ethylmaleimide indicated 

 that we were dealing with a thiol enzyme. 



Tests with Ground Fish 



Both hydrogen peroxide (H2O2) and potas- 

 sium bromate (KBrOa) are currently being used 

 in the U.S. food industry to impart desired func- 

 tional and organoleptic properties to the foods to 

 which they are added. For example, KBr0 3 is 

 used in breadmaking to improve the physical 

 properties of the dough (Tsen 1968). H2O2 has 

 been used as a preservative in dairy products 

 (Cuq et al. 1973) and as a bleaching agent in some 

 fish products (Sims et al. 1975; James and 

 McCrudden 1976). The dibasic phosphate perox- 

 ides have been used as a stablilizer for H2O2 in 

 various food products such as soy products, meat, 

 fish, and cereals (Pintauro 1974). 



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