was prepared and hydrolysed in one of these 

 runs. The press cake hydrolysate and the autol- 

 ysates of raw fish were partially soluble, whole 

 slurry products which had relatively high fat 

 contents but could be produced at low cost. Ma- 

 terial balances, proximate and amino acid anal- 

 yses, and determinations of PER were obtained 

 for each process. 



The hake hydrolysates were equivalent to ca- 

 sein as a supplement to wheat flour protein but 

 inferior as a sole source of protein. By hydrol- 

 ysis of alewife a totally water-soluble product 

 was finally obtained which was statistically 

 equivalent to casein as a sole source of protein. 



NMFS Contract Research. 



Columbia contract: fish fermentation. — Un- 

 der NMFS contracts with the Marine Biology 

 Division of Lamont-Doherty Geological Observa- 

 tory, Columbia University, investigations into 

 the direct use of microorganisms in the biologi- 

 cal treatment and preservation of fish were 

 made. 



Screening of microorganisms . — Over 600 

 diflferent microorganisms were screened for 

 suitability for fish fermentation. These included 

 microorganisms from the culture collection at 

 Lament Observatory and from major culture 

 collections (e.g., American Type Culture Col- 

 lection) . Many others were isolated especially 

 for the studies from cheeses and other fermented 

 foods, from fish and fish products, and from fish 

 processing equipment. 



The isolated microorganisms were used in 

 pure culture fermentations with a lyophilized 

 powder, prepared from whole menhaden, as the 

 substrate. The various cultures were evaluated 

 in terms of lipolytic activity (hydrolysis of men- 

 haden oil), proteolytic activity (hydrolysis of 

 gelatin and of casein), indole and ammonia pro- 

 duction (negative factors), and also by subjec- 

 tive organoleptic tests (aroma). 



A majority of the microorganisms that were 

 screened produced aromas that were unpleasant 

 to varying degrees. Eight yeasts and molds 

 were selected for further investigation, on the 

 basis of organoleptic characteristics primarily. 

 A fungus, Geotrichum candidum, and a yeast, 

 Candida lipolytica, were determined to be most 

 promising for menhaden fermentation. Both 



microorganisms utilized the lipids and nonpro- 

 tein nitrogen contained in menhaden and ef- 

 fected a reduction in fat content and a net in- 

 crease in protein content of the ferment while 

 yielding a product with a neutral to pleasant 

 aroma. These results have been published 

 (Burkholder et al., 1968). 



Additional studies . — Another strain of G. 

 candidum, No. PSM-179, was later isolated from 

 a Peruvian fish meal plant and found to have 

 exceptional lipolytic activity. The two strains 

 of G. candidum, plus C. lipolytica were studied 

 in more detail. The eifects of aeration, temper- 

 ature, initial pH, and time on the growth and 

 lipase production of the cultures were deter- 

 mined. Maximal lipase production and growth 

 of G. candidum was achieved after 4 days with 

 mild agitation in a 0.15m phosphate-buffered 

 medium at pH 7.1 and 28°C. With C. lipolytica, 

 maximal lipase production was obtained in 24 hr 

 and declined with additional fermentation. 



The very active lipase of the PSM-179 strain 

 of G. candidum was studied in detail. Deter- 

 mination of the subcellular distribution of the 

 lipase showed that most of the lipolytic activity 

 was associated with the culture medium, indi- 

 cating that the enzyme is extracellular. 



The incorporation of radioactively labeled 

 triglyceride from the substrate by PSM-179 was 

 also studied. Using labeled triolein as substrate, 

 it was found that this strain of G. candidtlm uti- 

 lized both oleic acid and glycerol as carbon 

 sources. Both of the products of lipolysis were 

 incorporated into the cellular material but a 

 higher percentage of the glycerol was utilized. 



Proteolytic microorganisms . — The use of 

 proteolytic microorganisms to produce a nutri- 

 tious, water-soluble FPC was also studied. A 

 total of 82 new proteolytic microorganisms were 

 obtained from various culture collections and 

 screened to determine relative proteolytic activ- 

 ities. Part of these plus microorganisms from 

 earlier screening tests were selected for further 

 proteolytic fermentation screening experiments. 

 Frozen menhaden, rather than a lyophilized 

 powder, was the substrate used. 



One strain of C. lipolytica plus a bacterium 

 and a yeast (unidentified) were selected for 

 further studies on the basis of soluble solids and 

 protein determinations. Also two microorgan- 



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