isms with known proteolytic characteristics were 

 further studied: Bacillus subtilis (18 strains) 

 and Rhizopus oligosporus (4 strains) from var- 

 ious culture collections. 



Two-stage fermentations were developed 

 for B. subtilis and R. oligosporus. The initial 

 stages, at moderate pH for optimum cell growth, 

 were followed by (a) fermentation at pH 10 for 

 optimum activity of the alkaline protease of B. 

 subtilis or (b) fermentation of pH 3 for opti- 

 mum activity of the acid protease of R. oligos- 

 porus. 



Samples of product were prepared for nu- 

 tritional evaluation at the NCFPC using five 

 selected strains of microorganisms. Menhaden 

 was fermented in a 26-liter fermenter under 

 optimal conditions. Most of the samples had 

 low protein concentrations and high ash con- 

 tents resulting from pH adjustments. Three of 

 the samples had lipid contents below 1%. In 

 general yields of soluble product and protein 

 recoveries were poor and the direct use of micro- 

 organisms for production of totally soluble prod- 

 ucts does not appear to be an economic practi- 

 cality. 



Studies on lipid stabilization were restricted 

 to developing a package and tray fermentation 

 method of co-fermentation of fish with tempeh 

 or the tempeh fungus R. oligosporus. The re-' 

 suits were not very promising. Cakes composed 

 of fish-soybean mixtures were not porous as are 

 the soybean cakes used in the normal tempeh 

 fermentation, and diflliculties in maintaining 

 moisture controls and the degree of aeration 

 were encountered. 



MIT contract: solubilization of FPC. — Per- 

 sonnel of the Department of Nutrition and Food 

 Science, Massachusetts Institute of Technology, 

 have, under contracts with the National Marine 

 Fisheries Service, investigated methods for the 

 solubilization of solvent-extracted fish protein 

 concentrate (FPC). An alkaline solubilization 

 method and the use of proteolytic enzymes in 

 both batch and continuous processes have been 

 studied. The objective was to make FPC more 

 suitable for use in food products such as protein 

 beverages. 



Alkaline solubilization . — An alkaline pro- 

 cess in which FPC was solubilized by a 20-min 

 reaction at pH 12.5 and 95°C has recently been 



reported by Tannenbaum, Ahem, and Bates 

 (1970). The degree of proteolysis was limited 

 by the short time period, but extensive racemiza- 

 tion of constituent amino acids did take place 

 and probably had a detrimental effect on the 

 nutritive value of the product. 



The use of the alkaline-process product in 

 several food products was the subject of a sec- 

 ond paper (Tannenbaum, Bates, and Brodfeld, 

 1970). It could be used without further treat- 

 ment in a number of foods, but off-flavor and 

 color reduction were necessary when it was used 

 in protein beverage formulas. This was eflfected 

 by passing the non-acid precipitable fraction 

 through charcoal followed by recombination of 

 the fractions. 



Proteolytic enzymes . — Investigations of 

 proteolytic enzymes for the solubilization of FPC 

 were divided into two phases. The initial phase 

 consisted of batch studies with a number of dif- 

 ferent enzyme preparations. The information 

 gained in the batch studies was used in the sec- 

 ond phase, a study of continuous reactor in which 

 ultrafiltration membranes could be used to allow 

 removal of soluble products of hydrolysis while 

 retaining the enzyme (also soluble) for contin- 

 uing reaction with added substrate. 



Batch studies . — In batch studies the rela- 

 tive activities of various proteolytic enzymes 

 were determined with respect to FPC solubiliza- 

 tion. Pronase (a mixture of proteolytic enzymes 

 from Streptomyces griseus), pancreatin, and 

 Bacillus subtilis protease (Monzyme) were 

 found to be effective in varying degrees. Meth- 

 ods were devised, or adapted, in order to assay 

 proteolytic activity, particularly the stability of 

 the enzyme during the course of FPC proteolysis 

 in order to estimate enzyme degradation and en- 

 zyme inhibition. The proteolytic activity of all 

 enzymes decreased during the course of FPC 

 proteolysis. At 34 °C and after 8 hr of hydrol- 

 ysis, Pronase degradation was estimated to be 

 approximately 30%. At the same time Pronase 

 was inhibited by the products of hydrolysis by a 

 factor of 60%. The influence of pH, tempera- 

 ture, substrate concentration, and enzyme con- 

 centration on FPC proteolysis by Pronase, pan- 

 creatin, Monzyme, and enzyme combinations was 

 studied. 



11 



