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Effect of Enzyme Supplements on the Production of Fish Sauce from Male Capelin (Mallotus villosus)
Can. Ins(. Food Sci. Technol. J. Vol. 19, No.1, pp. 28-33, 1986
RESEARCH
Effect of Enzyme Supplements on the Production of Fish Sauce from Male Capelin (Mallotus villosus) N. Raksakulthai, Y.Z. Lee l and N.F. Haard 2 Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AlB 3X9
Un camite d'experts, compose d'individus descendants du sudest asiatique, a juge que la sauce au poisson capelan additionnes d'hepatopancreas de calmar etait fortement acceptable et preferable au produit commercial fabrique aux Phillipines. L' analyse de regression a donne une correlation positive (r 2 =0.745) entre I' evaluation de preference comme variable dependante et les acides amines libres totaux comme variable independante. Dans Ie cas des acides amines libres totaux, I'hydrophobicite moyenne des acides amines libres, Ie rapport des residus acides aux residus basiques, et I'azote non acide amine camme variables independantes, Ie r2 de l'analyse de regression multiple fut 0.792.
Abstract Male, inshore capelin and salt (4: I w/w) were used to prepare fish sauce. Addition of proteolytic enzymes (fungal protease, pronase, trypsin, chymotrypsin, squid protease) or squid hepatopancreas to minced and salted capelin inceased the rate of protein solubilization during the first month of the fermentation. After 13 months, the free amino acid content of fish sauce prepared from mince was 4.4070 compared to only 2.7% for sauce prepared from round fish and 6.3% for sauce prepared from mince containing hepatopancreas. Supplementing mince with fungal protease, pronase, or trypsin resulted in somewhat lower yields of free amino acid, 3.3, 3.3 and 3.6%, respectively. Alanine was the most abundant free amino acid in all samples (11.9-13.0 mole %) except pronase supplemented mince (15.6 mole % leucine) and hepatopancreas supplemented mince (11.5 mole % glutamic). Preference analysis of capelin fish sauce by a panel, comprised of individuals of S.E. Asian descent, showed that the product supplemented with squid hepatopancreas was highly acceptable and preferable to a commercial product from the Philippines. Regression analysis with preference score as the dependent variable and total free amino acids as the independent variable showed a positive correlation (r 2= 0.745). With total free amino acids, average hydrophobicity of free amino acids, ratio of acidic to basic residues, and non-amino acid nitrogen as independent variables, the r2 for multiple regression analysis was 0.792.
Introduction Various sauces or pastes are prepared from salted fish, fish parts or other marine and freshwater organisms. One of the most popular of such products in S.E. Asia, and some other parts of the Far East, is fish sauce; known as nuoc-mam in Vietnam, nampla in Thailand, patis in the Philippines, shotturu in Japan and ngapi in Burma. According to Saisithi et al. (1966), fish sauce is part of the daily diet of more than 250 million people. Because of the growing population of Canadians and Americans of Vietnamese ancestry, there would appear to be a sizable market for this product in N. America. In 1984 more than 41,000 metric tonnes of inshore capelin(Mallotus villosus) were harvested by Newfoundland fishermen and the roe containing females were sold to Japanese buyers. The male capelin ( "" 20,000 tonnes in 1984) were not utilized for food export; although an unknown amount of male capelin was used to manufacture fish meal at a return of less than $100/tonne. The present study was undertaken to determine the suitability of male inshore capelin as a raw material for the manufacture of fish sauce. Preliminary studies in 1981-82 showed that fish sauce, prepared by mixing round capelin and salt (4:1 w/w) and holding for 6-12 mo, contained a relatively low content of soluble nitrogen. For this reason, the influence of mincing the capelin prior to salting and the addition of proteolytic enzymes in this process was
Resume De la sauce au poisson fut preparee a partir de capelan cotier male et de sel (4:1 pip). L'addition d'enzymes proteolytiques (protease fongique, pronase, trypsine, chymotrypsine, protease de calmar) ou d'hepatopancreas de calmar a du capelan hache et sale augmenta Ie taux de solubilisation des proteines au cours du premier mois de la fermentation. Apres 13 mois, la teneur en acides amines libres de la sauce au poisson faite de hachis fut 4.4%, par rapport a seulement 2.7% pour la sauce faite de poisson entier et 6.3% pour celie faite de hachis contenant de l'hepatopancreas. Les hachis additionnes de protease fongique, de pronase ou de trypsine furent quelque peu moins riches en acides amines libres, soit respectivement 3.3, 3.3 et 3.6%. L' alanine fut I' acide amine Ie plus abondant dans tous les echantilIons (11.9-13.0 mole %) sauf dans Ie hachis additionne de pronase (15.6 mole % de leucine) et Ie hachis additionne d'hepatopancreas (11.5 mole % d'acide glutamique). I Present
address: Department of Poultry Science, University of British Columbia, Vancouver, B.C. 2To whom correspondence should be addressed.
Copyright
i>
1986 Canadian Institute of Food Science and Technology
28
investigated. Earlier, it was reported that the hepatopancreas of squid (ll/ex illecebrosus) was a rich source of proteolytic enzymes which aid the development of desirable taste in fermented squid or herring (Lee et al., 1982). Hepatopancreas is a by-product of processing dried or tube frozen squid in Newfoundland. Accordingly, the influence of squid hepatopancreas on the production of fish sauce from male capelin was also investigated.
Materials and Methods Preparation of fish sauce Inshore capelin were harvested in Outer Cove, Newfoundland by handnet in 1982 and held at 4°C for approximately 12 h. The males were separated and approximately 50 kg of whole, male fish was ground to pass a 6 mm plate with a Hobart 7 horsepower meat grinder. Salt was mixed with the mince so that it was 25070 based on fish weight and the mixture was left for approximately 2 h until a uniform brine slurry was formed. Samples of slurry (1000 g) were packed in glass 1 L jars. Duplicate 1000 g samples were supplemented with 33 mg of either bovine pancreas trypsin, type 1; bovine alpha- chymotrypsin, type I-S (Sigma Chemical Co., St. Louis, MO); pronase, analytical grade (Boehringer Mannheim Canada Ltd., Dorval, PQ); fungal protease 31,000 (Miles Laboratories, Elkart, IN); or 80 mg(protein) squid hepatopancreas enzyme extract (Lee et al., 1982); or 25 g (weight) squid hepatopancreas. Duplicate 1000g samples of salted round capelin or salted mince were also packed in 1 L glass jars. The jars were sealed with lids and stored at ambient temperature (20-25°C) for 11 mo. The fish sauce was recovered by filtration of the brined mince through Whatman # 1 paper and subsequently stored in sealed 500 mL glass jars at ambient temperature. Fish sauces were analyzed and evaluated by a sensory panel 2 to 3 mo after filtration. On the basis of results obtained in 1982-83, additional lots of fish sauce were prepared from minced capelin, with and without 2.5% squid hepatopancreas, in 1983-84.
Initial rate of fermentation The rate of formation of products from protein hydrolysis in brined capelin was determined during the first month by mixing 0.75 mL aliquots of brine with 0.75 mL 20% trichloroacetic acid (TCA), centrifuging for 4 min at 12,000 x g, dilution of 0.1 mL of the resulting supernatant with 0.9 mL distilled water, centrifuging the diluant for 4 min at 12,000 x g and determining the A280nm of the resulting supernatant with similarly treated 25% NaCI as the blank.
Model 121MB analyzer using the method described in the Beckman bulletin 121M-TB-013. Total amino acids were determined after hydrolysis of sauce in 6N HCI at 1l0°C for 24 h. These analyses were performed on the final product obtained 13 mo after initiation of the preparation of fish sauce. The free amino acid content of the samples which had been supplemented with enzymes was also analyzed 33 mo after the initiation of the experiment. The sampling times of 13 and 33 mo were chosen because the times are more or less representative of unaged (13 mo) and aged (33 mo) fish sauce. Average hydrophobicity (HOaJ of free amino acids was calculated by Bigelow's method (1967). The ratio of acidic to basic free amino acids was calculated from the molar ratios of acidic residues (aspartic, glutamic, taurine, threonine, serine, proline) and basic residues (l-methyl-histidine, 3-methyl-histidine, lysine, arginine, and histidine). Amino acid data was presented as mM (millimoles amino acid per L of sample) or as mg% (mg amino acid per 100g samples).
Sensory evaluation Fish sauces prepared in 1982-83 were evaluated for preference by a panel of 4 individuals of S.E. Asian descent (Indonesia, Malaysia, Philippines, Korea) using a nine point hedonic scale as described by Larmond (1977). Fish sauces prepared in 1983-84 were evaluated by hedonic scale and by ranking of overall quality twice by a panel of 5 Vietnamese-Canadians. Samples were presented to panelists in partitioned booths with standardized lighting. The results were analyzed for statistical significance by the methods described by Larmond (1977).
Regression Analysis The relationship between sensory panel results and chemical analyses was evaluated by multiple regression using a Lisa microcomputer and LisaCalc software.
Results and Discussion Influence of fermentation aids on initial rate of fermentation The formation of non-protein nitrogen (NPN) in fish sauce brine occurred rapidly during the first week
.... squid HP
." chy.try.
•
Amino acids and related substances The free amino acid content and other ninhydrin positive components (i.e. alpha-aminobutyric acid, ornithine, ethanolamine, anserine, carnosine, creatinine, o-phosphoethanolamine, urea, methionine sulfoxide, methionine solfone, glutamine, asparagine. sarcosine, citrulline, L-a-aminoadipic acid, L-a-aminon-butyric acid, cystathionine, l3-aminoisobutyric acid and homocitrulline) were determined with a Beckman Can. Insl. Food Sci. Technol. J. Vol. 19, No. I, 1986
trypsin
(> squid E
10
20
30
+
pronase
•
F. prot.
o
control
40
Time,days
Fig. I. Formation of protein hydrolysis products in fish sauce prepared from minced capelin, 0 control; • squid HP; ~trypsin; .chymotrypsin; <> squid extract; + pronase; .F. prot.
Raksakulthai et al. / 29
Table I. Content of free amino acids in capelin fish sauce. Treatment Free amino acids mM mg010 None, round capelin 216 2729 Mince, no enzyme 352 4432 + Fungal Protease 262 3278 + Pronase 273 3256 + Trypsin 284 3606 + Chymotrypsin 374 4777 + Squid Protease 380 4777 + Squid Hepatopancreas 496 6298 IData are average of one analysis for duplicate samples of 13 month
and more gradually during the subsequent 4 w of fermentation (Figure I). Addition of proteolytic enzymes or squid hepatopancreas to mince resulted in formation of more TeA soluble protein than occurred in the brine of control mince. Samples with 3.3 mg 070 of chymotrypsin, trypsin, squid protease or pronase formed about twice as much NPN as the untreated mince; samples supplemented with 2.5% squid hepatopancreas formed about 3 times as much NPN in the brine as did the control samples. Fungal protease was apparently less effective in accelerating the hydrolysis of capelin mince than were the other enzyme preparations. The squid protease fraction employed in this study contains cathepsin e and other contaminating proteases (Hameed and Haard, 1985).The hepatopancreas from squid is also a rich source of cathepsins B, D, E and other unidentified proteases (Inaba et al., 1976; Leblanc and Gill 1982). The data indicate that addition of enzymes to minced capelin increases the progress of the initial stage of the fermentation. Fish sauce prepared with minced Ikanbilis (Stolephorus sp.) and salt in a ratio of 3:2 formed more total nitrogen after 21 d at 33°e when supplemented with 0.8% bromelain (1.25 fold), 2.75% papain (1.2 fold) or 2.5% ficin (l.1 fold) (Beddows and Ardeshir, 1979). Fermentation of mackerel, sardine or shrimp with 25% salt at 37°C for I mo with 0.2-0.5% added enzyme increased the yield of total soluble nitrogen about 1.05 fold with trypsin, 1.1 fold with bromelain and 1.15 fold with papain (Ooshiro et al., 1981). Aiding Anchovy bagoong fermentation (3 parts Fish: I part salt) with 0.5% papain increased the yield of crude protein in the 28 d brine by about 1.1 fold (Guevara et al., 1974). The yield of amino type nitrogen in brined horse mackerel (4: 1) held at 37°e was increased by 1.4 fold by addition of 0.2% Bioprase (Muraymam et al., 1962). Several of these workers reported that the sensory characteristics of fish sauce prepared with added enzyme was somewhat different from that of sauces prepared without a fermentation aid.
Free amino acids and related components The meaty flavour of fish sauce appears to be determined by free amino acids and amines; although ammonia and other volatile bases contribute to the ammonia-like aroma and volatile organic acids contribute to the "cheese" aroma of the product (Dougan and Howard, 1975). Thirteen mo after salting, the free amino acid content of capelin fish sauces ranged 30 / Raksakulthai et al.
Major amino acids l mole % alanine (12.0) > glutamic (11.3) > leucine (9.4) > lysine (9.2) alanine (11.9) > glutamic (11.2) > leucine (9.8) > lysine (9.2) alanine (13.0) > glutamic (12.0) > leucine (10.8) > lysine (7.8) leucine (15.6) > alanine (15.0) > glycine (11.9) > valine (11.3) alanine (13.0) > lysine (11.0) > leucine (11.0) > glutamic (8.6) alanine (12.9) > leucine (12.9) > lysine (9.7) > glutamic (8.7) alanine (12.2) > leucine (10.6) > lysine (9.7) > glutamic (9.4) glutamic (11.5) > alanine (10.5) > asp (10.1) > leucine (9.6) old product.
from 2729 mg% to 6298 mg% (Table 1). The free amino acid content of sauce prepared from round capelin was 62% of that formed in sauce prepared from mince. Fish sauces prepared from mince supplemented with proteolytic enzymes contained from 3278 to 4777 mg% free amino acids and on this basis the final product was not especially favored by these enzyme supplements. However, fish sauce prepared with squid hepatopancreas contained 6298 mg% free amino acids, an apparent improvement of 1.42 fold over minced controls and 2.3 fold over fish sauce prepared from round capelin. Storage of the filtered fish sauces for an additional 20 mo (aging) resulted in the formation of additional free amino acids (Table 2). The free amino acid contents of 13 mo old sauces prepared in 1983-84 were 3040 mg% for minced controls and 6630 mg% for mince containing 2.5% squid hepatopancreas. The lower content of free amino acids in 1983-84 sauce prepared from mince compared to that prepared in 1982-83 may relate to differences in endogenous proteolytic activity which is known to vary greatly with feed intake of capelin (Gildberg, 1978). Fish sauce produced in Thailand is required to have an amino acid nitrogen content of 950 mg% (Anonymous, 1970). The amino acid nitrogen content of Nuoc-mam ranges from 450 mg% for ordinary quality grade sauce to 900 mg% for first quality grade product and of commercial patis ranges from 904 to 994 mg% for special and extra grades (Uyenco et al., 1963). Based on an approximate nitrogen content of free amino acids of 16%, for samples analyzed in this study, the amino acid nitrogen content of capelin fish sauces were 437 mg% from round capelin, 709 mg% from minced capelin and 1008 mg% from minced capelin supplemented with squid hepatopancreas. Estimation of free amino acid nitrogen by the method used in S.E. Asian countries, which is based on the difference between formaldehyde nitrogen and ammoniacal nitrogen, would give somewhat higher values than by direct estimation from free amino acids because of the contribution of peptide alpha-amino groups to formaldehyde nitrogen. The amino acid content of fish sauce hydrolyzates, i.e., total amino acids, prepared from minced capelin ranged from 6976 mg.% or 1116 mg% amino acid nitrogen, to 9810 mg% (hepatopancreas), representing 1570 mg% amino acid nitrogen. These data were used to determine the free amino acids as percentage of total amino acids in fish sauce (Table 2). J. Ins!. Can. Sci. Technol. Aliment. Vol. 19. No. I, 1986
Table 2. Characteristics of free amino acids from capelin fish sauce and commercial products. Free Amino Acids I 13 Months Fish Sauce
Treatment
mg%
Capelin, whole None Capelin, mince None + fungal prot. + pronase + trypsin + chymotry. + squid prot. + squid pan.
2729 4431 3278 3256 3606 4777 4777 6298
Commercial
mg% 5609 5280
Thailand Philippines
AlB
33 Months
HO. v
2.92 891 3.00 903 2.53 969 14.90 1290 2.11 966 1.97 1165 2.67 958 2.60 935 Free Amino Acids AlB 3.00 1.80
% Total
mg010
AlB
HO.v
34 65 44 40 45 49 68 64
5280 4814 5190 4936 5638 8680
2.76 6.44 2.37 2.31 2.79 3.24
953 1021 1015 1126 940 887
010 Total
70 59 65 51 81 89
HO. v 831 970
IData for one analysis and representative of results obtained with second experiment; AlB is molar ratio of acidic to basic amino acid residues; HO.vis Bigelow's average hydrophobicity of amino acid residues; % total amino acids is free amino acids in mg010 divided by total amino acids in mg%. The latter data was obtained by amino acid analysis after hydrolysis of fish sauce in 6N HCI as described in Methods.
16 , . . . - - - - - - -
12
,
D
+ Hepatopancreas
•
Control
Amino Acid
Fig. 2. Free amino acid composition of fish sauce prepared from minced capelin with and without squid hepatopancreas as a fermentation aid.
The percentage of total amino acids which was free in hepatopancreas treated sauce was 64070 at 13 mo and 89070 at 33 mo. For 13 mo old samples containing commercial enzyme supplements, the percentage of total amino acids present as free amino acids was apparently lower than in the control. The protease
fraction obtained from squid hepatopancreas appeared to be more effective in improving the ratio of free to total amino acids than it was in improving the total content of amino acids in the products. The results indicate that, with the processing conditions employed in this study, addition of squid hepatopancreas to minced capelin yields a fish sauce which exceeds the standards set for first grade products in S.E. Asian countries. The main free amino acids in capelin fish sauce prepared from mi.nce were alanine (11.9070), glutamine (11.2070), leucine (9.8070), lysine (9.2070), aspartic (8.3070),glycine (7.1070), valine (6.9070), serine (6.3070), taurine (5.1070), threonine (5.1070) and isoleucine (4.4070). The mole percent of the major free amino acids identified in 13 mo old sauce prepared from mince and mince supplemented with hepatopancreas is illustrated in Figure 2. With the exception of pronase, the addition of proteolytic enzymes to mince did not greatly alter the spectrum of major amino acids formed (Table 1). In pronase treated samples, the ratio of acidic to basic amino acids was high, due to the low recovery of basic. Amino acids, and the average hydrophobicity of the free amino acids was much higher than other samples (Table 2).
Table 3. Content of Ninhydrin reactive substances in capelin fish sauce. Treatment Ninhydrin Positive Components! mM mg% (% Control)
Major Components mole %
None 64 385 (108) NH 3 (63.9) > citruline (14.4) > ethanolamine (6.9) 68 356 (100) NH 3 (72.4) > citruline (17.8) > ethanolamine (6.5) Mince (Control) + Fungal Protease 54 290 (81) NH 3 (73.1) > citruline (20.2) > ethanolamine (4.2) + Pronase 169 1562 (439) ethanolamine (64.6) > ornithine (22.4) > citru!' (8.6) + Trypsin 61 346 (97) NH 3 (67.8) > citruline (18.9) > ethanolamine (6.9) + Chymotrypsin 76 494 (139) NH 3 (68.3) > citruline (21.2) > carnosine (5.1) + Squid Protease 65 380 (107) NH 3 (69.4) > citruline (22.5) > ethanolamine (5.6) + Squid Hepatopancreas 89 494 (139) NH 3 (69.7) > citruline (14.1) > ethanolamine (7.2) ICompounds analyzed: glycerophosphoethanolamine phosphoethanolamine urea, methioninesulfoxide*, asparagine, glutamine*, sarcosine*, alpha-amino adipic acid, citruline*, alpha-amino-n-butyric, homocitruline, cystathionine*, gamma-glutamyl-e-Iysine, J3-aminoisobutyric, homocystine, gamma-aminobutyric*, ethanolamine*, ammonia*, ornithine*, anserine*, carnosine*, glutathione, glucosamine, galactosamine. Compounds with* were present in some or all of samples. Data are for duplicate analyses of 13 month old fish sauce preparations. Can. InSf. Food Sci. Technol. J. Vol. 19. No. J, 1986
Raksakulthai et al. I 31
Table 4. Sensory characteristics of capelin and commercial fish sauce. Sample Preference Score' Mince capelin 6.00a,b + fungal protease 5.25 + pronase 6.25a,b + squid protease 6.25a,b + trypsin 6.50a,b + chymotrypsin 6.50a,b + squid hepatopancreas 7.00a Commercial product 2 6.75 IValues followed by "a" are not significantly different from control (P < 0.1); values followed by "b" are not significantly different from control (P < 0.2), n = 4. 2Rufina brand fish sauce from Philippines.
tathionine, gamma-aminobutyric, anserine, and carnosine, were present at low concentration in some but not all samples. Fish sauce prepared with pronase was unusual with respect to its high content of these compounds, notably ethanolamine and ornithine. The unusual free amino acid composition and the high content of non-amino acid nitrogen compounds for pronase treated fish sauce may indicate the intervention of salt tolerant microorganisms in this fermentation (Fujii and Sakai, 1984b). Commercial patis contained 637 mg070 non-amino acid nitrogen consisting of 80070 ammonia, 13070 citrulline, 3070 ethanolamine and 3070 ornithine.
Sensory Evaluation The relationship between amino acid composition and quality of fish sauce is not understood at this time. Some authors have suggested that fish sauce flavour is largely influenced by the mono-amino acid fraction (Amano, 1962), although there appears to be no evidence to substantiate this assertion. It would appear that the glutamic acid content of fish sauce plays an important role in taste since it is well recognized that glutamate contributes to "umami" or delicious taste in fish and other seafoods (Yamaguchi, 1979) and it is a major component of capelin fish sauce. According to Fujii and Sakai (l984a), the major amino acids of Japanese fish sauce (shotturu) are glutamic> valine > glycine> aspartic > alanine; the major free amino acids of Nuoc-mam are reported to be alanine> glutamic > isoleucine > leucine and lysine (Amano, 1962); and those identified in anchovy fish sauce are alanine > valine > leucine > lysine (Miyazawa et al., 1979). The analysis of commercial, patis from the Philippines showed the same major free amino acids as capelin fish sauce, i.e., alanine> leucine> lysine > glutamic> valine.
Other Nitrogen Compounds With the exception of pronase treated samples, the capelin fish sauces contained between 300 and 500 mg070 of non-amino acid nitrogen compounds (Table 3). Ammonia accounted for about 70070 of this fraction and citrulline made up 15-20070 of the nitrogen compounds identified. Ethanolamine and ornithine were present in all of these samples; other compounds, i.e. methionine sulfoxide, glutamine, sarcosine, cys-
Table 5. Preference Scores of capelin and commercial fish sauces. Sample Preference Score' Rank Totals1,3 Minced Capelin Commercial patis 2 Minced Capelin + hepatopancreas Capelin + hepatopancreas Commercial Capelin
5.3a 5.6a
Relationship between chemical composition and sensory preference Regression analysis of the data collected from 9 samples of capelin fish sauce with preference score as the dependent variable showed a coefficient of determination (r 2) of 0.745 with total free amino acids as the independent variable (Table 6). Multiple regression analysis with total amino acid content, ratio of acidic to basic amino acid residues, average hydrophobicity of free amino acids and non-amino acid content as independent variables showed that all of these factors could better account for the preference scores than free amino acids alone. The regression equation, which had an r 2 of 0.792 is shown in footnote 2 of Table 6. The results of this analysis are more or less consistent with previous suggestions that more total free Table 6. Regression analysis of preference scores as a function of amino acid and ninhydrin in positive components. Dependent variable(yl) Independent variable (X)I r2 preference score
8.2 12 21 27
lValues followed by "a" are not significantly different (p < 0.05); panel composed of 5 Vietnamese; n = 10.; capelin sauces prepared in 1983-84. 2Rufina brand patis from Philippines. 3Capelin + hepatopancreas was significantly better (p < 0.05) than the other two samples; n = 10.
32 / Raksakulthai et al.
Capelin fish sauce samples were evaluated for preference and compared to a commercial fish sauce by experienced consumers of this type of product. The results indicate that commercial product and hepatopancreas supplemented capelin sauce were preferable to sauce made from minced capelin with no added enzyme (Table 4). Capelin sauce prepared with fungal protease was rated lower in overall quality than the control capelin sauce. All other treatments were not significantly different (p < 0.2). Additional lots of capelin sauce prepared in 1983-84, minced and supplemented with hepatopancreas, were analyzed for preference by ranking and hedonic scoring. The capelin sauce prepared with hepatopancreas proved to be clearly superior to the unsupplemented capelin sauce and the commercial control (Table 5).
HOail) 0.090 0.088 non-amino acid N (2) acidic/basic free amino acids (3) 0.001 0.745 total free amino acids (4) 1,4 0.780 2,4 0.773 1,2,4 0.791 1,3,4 0.789 1,2,3,4 0.792 2 IFrom regression analysis of data for 9 different samples of fish sauce. 2y = 0.001 (X,) + 0.007 (X 2) - 0.033 (X 3) + 0.007 (X 4 ) + 2.45. J. lnsl. Can. Sci. Techno!. Aliment. Vol. '9, No. '. '986
amino acids are associated with superior grade product; and further suggest that a low acidic/basic ratio, a high H0 a" and a high content of non-amino acid nitrogen compounds are associated with high preference scores. Overall, the analysis suggests that the amount and type of free amino acids and the amount of ammonia and other nitrogenous compounds contribute to the acceptability of capelin fish sauce. Additional study with fish sauce prepared from other fish and by different methods, e.g. salt content and temperature, is necessary to determine the broader applicability of the regression analysis. Using the equation shown in Table 6 the calculated preference score for commercial patis was 7.17, somewhat higher than the actual preference scores of 6.75 and 5.60 by the two panels (Tables 4,5).
Conclusion An excellent quality fish sauce can be prepared from inshore male capelin when the fermentation is carried out at a minced fish: salt ratio of 4: 1 at 20-25°C and 2.5070 squid hepatopancreas is used as a fermentation aid. Addition of various other enzyme preparations to minced capelin increases the initial rate of protein hydrolysis but does not yield a product containing a much higher content of free amino acids. The overall acceptability of capelin fish sauce by individuals of S.E. Asian descent appears to be related to the amount and type of free amino acids and other non-amino acid nitrogen compounds.
Acknowledgment The study was supported by an operating grant from the Natural Sciences and Engineering Research Council Canada. We greatly acknowledge the assistance of Mr. D. Hall and Mrs. S. Banfield for assistance with amino acid analyses.
References Amano, K. 1962. The influence of fermentation on the nutritive value of fish with special reference to fermented fish products of South-East Asia. In: Fish in Nutrition. E. Heen and R. Kreuzer (Eds.). p. 180. Fishing News Book. London. Anonymous. 1970. Standards for local fish sauce, Thai Industrial Standards Institute, Ministry of Industry, Bangkok, Thailand. Beddows, e.G. and Ardeshir, A.G. 1979. The production of fish protein solution for use in fish sauce manufacture. I. The use of added enzymes. J. Food Techno!. 14:603.
Can. Ins/, Food Sci. Techno!. J. Vol. 19. No.1. 1986
Bigelow, e.e. 1967. On the average hydrophobicity of proteins and the relationship between it and protein structure. J. Theor. Bio!. 16:187. Dougan, J. and Howard, G.E. 1975. Some flavouring constituents of fermented fish sauce. J. Sci. Food Agric. 26:887. Fujii, T. and Sakai, H. 1984a. Chemical composition and micro flora of fish sauce "shotturu". Bull. Jap. Soc. Sci. Fish. 50: 1061. Fujii, T. and Sakai, H. 1984b. Chemical and microbiological analyses of putrid fish sauce "shotturu". Bull. Jap. Soc. Sci. Fish. 50:1067. Gildberg, A. 1978. Proteolytic activity and the frequency of burst bellies in capelin. J. Food Technol. 13:409. Guevara, G., Matias, V.C., Dela Pena, P.O. 1972. Fish fermentation with the use of papain. The Phil. J. Fish. 10:30. Hameed, K.S, and Haard, N.F. 1985. Isolation and characterization of cathepsin C from Atlantic short finned squid, IIIex i1lecebrosus. Compo Biochem. Physiol.82B:24 I. Inaba, T., Shindo, N. and Fujii, M. 1976. Purification of cathepsin B from squid liver. Agric. BioI. Chern. 40: 1159. Leblanc, E.L. and Gill, T.A. 1982. Comparative study of proteolysis in short-finned (II/ex il/ecebrosus) and long-finned (Loligo paeli Leseur) squid. Compo Biochem. Physio!. 73B:201. Larmond, E. 1977. Laboratory methods for sensory evaluation of food. Publication 1637, Canada Department of Agriculture. Lee, Y.Z., Simpson, B.K. and Haard, N.F. 1982. Supplementation of squid fermentation with proteolytic enzymes. J. Food Biochem.6:127. Miyazawa, K, Le, C.V., Ito, K. and Matjunuoto, F. 1979. Studies on fish sauce. Hiroshima University. J. Fac. Appl. BioI. Sci. 18:55. Murayawa, S., Calvez, D.L. and Nitayachin, P. 1962. Study on the production of fish sauce - I. Effect of commercial proteolytic enzyme on the production of fish sauce. Bull, Tokai Reg. Fish. Lab. 32:155. Ooshiro,Z., Ok, T., Une, H., Hayashi, S. and Itakura, T. 1981. Study 0,11 use of commercial proteolytic enzymes in production of fish sauce. Mem. Fac. Fish., Kagoshima Univ. 30:383. Saisithi, P., Kasemsarn, Bung-Orn, Liston, J. and Dollar, A.M. 1966. Microbiology and chemistry of fermented fish. J. Food Sci. 31:105. Uyenco, V., Lawas, I., Briones, P.R. and Taruc, R.S. 1953. Mechanics of bagoong (fish sauce) and patis (fish sauce) processing. In: Indo-Pacific Fisheries Council Proceedings, Food and Agricultural Organization of the United Nations, Bangkok. p. 210. Yamaguchi, S. 1979. The umami taste. In: Food Taste Chemistry J.C. Boudreau (ED.). pp 33-51. American Chemical Society Symposium Series 115.
Submitted May 13, 1985 Revised November 5, 1985 Accepted November 18, 1985
Raksakulthai et al. / 33
RESEARCH
Effect of Enzyme Supplements on the Production of Fish Sauce from Male Capelin (Mallotus villosus) N. Raksakulthai, Y.Z. Lee l and N.F. Haard 2 Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AlB 3X9
Un camite d'experts, compose d'individus descendants du sudest asiatique, a juge que la sauce au poisson capelan additionnes d'hepatopancreas de calmar etait fortement acceptable et preferable au produit commercial fabrique aux Phillipines. L' analyse de regression a donne une correlation positive (r 2 =0.745) entre I' evaluation de preference comme variable dependante et les acides amines libres totaux comme variable independante. Dans Ie cas des acides amines libres totaux, I'hydrophobicite moyenne des acides amines libres, Ie rapport des residus acides aux residus basiques, et I'azote non acide amine camme variables independantes, Ie r2 de l'analyse de regression multiple fut 0.792.
Abstract Male, inshore capelin and salt (4: I w/w) were used to prepare fish sauce. Addition of proteolytic enzymes (fungal protease, pronase, trypsin, chymotrypsin, squid protease) or squid hepatopancreas to minced and salted capelin inceased the rate of protein solubilization during the first month of the fermentation. After 13 months, the free amino acid content of fish sauce prepared from mince was 4.4070 compared to only 2.7% for sauce prepared from round fish and 6.3% for sauce prepared from mince containing hepatopancreas. Supplementing mince with fungal protease, pronase, or trypsin resulted in somewhat lower yields of free amino acid, 3.3, 3.3 and 3.6%, respectively. Alanine was the most abundant free amino acid in all samples (11.9-13.0 mole %) except pronase supplemented mince (15.6 mole % leucine) and hepatopancreas supplemented mince (11.5 mole % glutamic). Preference analysis of capelin fish sauce by a panel, comprised of individuals of S.E. Asian descent, showed that the product supplemented with squid hepatopancreas was highly acceptable and preferable to a commercial product from the Philippines. Regression analysis with preference score as the dependent variable and total free amino acids as the independent variable showed a positive correlation (r 2= 0.745). With total free amino acids, average hydrophobicity of free amino acids, ratio of acidic to basic residues, and non-amino acid nitrogen as independent variables, the r2 for multiple regression analysis was 0.792.
Introduction Various sauces or pastes are prepared from salted fish, fish parts or other marine and freshwater organisms. One of the most popular of such products in S.E. Asia, and some other parts of the Far East, is fish sauce; known as nuoc-mam in Vietnam, nampla in Thailand, patis in the Philippines, shotturu in Japan and ngapi in Burma. According to Saisithi et al. (1966), fish sauce is part of the daily diet of more than 250 million people. Because of the growing population of Canadians and Americans of Vietnamese ancestry, there would appear to be a sizable market for this product in N. America. In 1984 more than 41,000 metric tonnes of inshore capelin(Mallotus villosus) were harvested by Newfoundland fishermen and the roe containing females were sold to Japanese buyers. The male capelin ( "" 20,000 tonnes in 1984) were not utilized for food export; although an unknown amount of male capelin was used to manufacture fish meal at a return of less than $100/tonne. The present study was undertaken to determine the suitability of male inshore capelin as a raw material for the manufacture of fish sauce. Preliminary studies in 1981-82 showed that fish sauce, prepared by mixing round capelin and salt (4:1 w/w) and holding for 6-12 mo, contained a relatively low content of soluble nitrogen. For this reason, the influence of mincing the capelin prior to salting and the addition of proteolytic enzymes in this process was
Resume De la sauce au poisson fut preparee a partir de capelan cotier male et de sel (4:1 pip). L'addition d'enzymes proteolytiques (protease fongique, pronase, trypsine, chymotrypsine, protease de calmar) ou d'hepatopancreas de calmar a du capelan hache et sale augmenta Ie taux de solubilisation des proteines au cours du premier mois de la fermentation. Apres 13 mois, la teneur en acides amines libres de la sauce au poisson faite de hachis fut 4.4%, par rapport a seulement 2.7% pour la sauce faite de poisson entier et 6.3% pour celie faite de hachis contenant de l'hepatopancreas. Les hachis additionnes de protease fongique, de pronase ou de trypsine furent quelque peu moins riches en acides amines libres, soit respectivement 3.3, 3.3 et 3.6%. L' alanine fut I' acide amine Ie plus abondant dans tous les echantilIons (11.9-13.0 mole %) sauf dans Ie hachis additionne de pronase (15.6 mole % de leucine) et Ie hachis additionne d'hepatopancreas (11.5 mole % d'acide glutamique). I Present
address: Department of Poultry Science, University of British Columbia, Vancouver, B.C. 2To whom correspondence should be addressed.
Copyright
i>
1986 Canadian Institute of Food Science and Technology
28
investigated. Earlier, it was reported that the hepatopancreas of squid (ll/ex illecebrosus) was a rich source of proteolytic enzymes which aid the development of desirable taste in fermented squid or herring (Lee et al., 1982). Hepatopancreas is a by-product of processing dried or tube frozen squid in Newfoundland. Accordingly, the influence of squid hepatopancreas on the production of fish sauce from male capelin was also investigated.
Materials and Methods Preparation of fish sauce Inshore capelin were harvested in Outer Cove, Newfoundland by handnet in 1982 and held at 4°C for approximately 12 h. The males were separated and approximately 50 kg of whole, male fish was ground to pass a 6 mm plate with a Hobart 7 horsepower meat grinder. Salt was mixed with the mince so that it was 25070 based on fish weight and the mixture was left for approximately 2 h until a uniform brine slurry was formed. Samples of slurry (1000 g) were packed in glass 1 L jars. Duplicate 1000 g samples were supplemented with 33 mg of either bovine pancreas trypsin, type 1; bovine alpha- chymotrypsin, type I-S (Sigma Chemical Co., St. Louis, MO); pronase, analytical grade (Boehringer Mannheim Canada Ltd., Dorval, PQ); fungal protease 31,000 (Miles Laboratories, Elkart, IN); or 80 mg(protein) squid hepatopancreas enzyme extract (Lee et al., 1982); or 25 g (weight) squid hepatopancreas. Duplicate 1000g samples of salted round capelin or salted mince were also packed in 1 L glass jars. The jars were sealed with lids and stored at ambient temperature (20-25°C) for 11 mo. The fish sauce was recovered by filtration of the brined mince through Whatman # 1 paper and subsequently stored in sealed 500 mL glass jars at ambient temperature. Fish sauces were analyzed and evaluated by a sensory panel 2 to 3 mo after filtration. On the basis of results obtained in 1982-83, additional lots of fish sauce were prepared from minced capelin, with and without 2.5% squid hepatopancreas, in 1983-84.
Initial rate of fermentation The rate of formation of products from protein hydrolysis in brined capelin was determined during the first month by mixing 0.75 mL aliquots of brine with 0.75 mL 20% trichloroacetic acid (TCA), centrifuging for 4 min at 12,000 x g, dilution of 0.1 mL of the resulting supernatant with 0.9 mL distilled water, centrifuging the diluant for 4 min at 12,000 x g and determining the A280nm of the resulting supernatant with similarly treated 25% NaCI as the blank.
Model 121MB analyzer using the method described in the Beckman bulletin 121M-TB-013. Total amino acids were determined after hydrolysis of sauce in 6N HCI at 1l0°C for 24 h. These analyses were performed on the final product obtained 13 mo after initiation of the preparation of fish sauce. The free amino acid content of the samples which had been supplemented with enzymes was also analyzed 33 mo after the initiation of the experiment. The sampling times of 13 and 33 mo were chosen because the times are more or less representative of unaged (13 mo) and aged (33 mo) fish sauce. Average hydrophobicity (HOaJ of free amino acids was calculated by Bigelow's method (1967). The ratio of acidic to basic free amino acids was calculated from the molar ratios of acidic residues (aspartic, glutamic, taurine, threonine, serine, proline) and basic residues (l-methyl-histidine, 3-methyl-histidine, lysine, arginine, and histidine). Amino acid data was presented as mM (millimoles amino acid per L of sample) or as mg% (mg amino acid per 100g samples).
Sensory evaluation Fish sauces prepared in 1982-83 were evaluated for preference by a panel of 4 individuals of S.E. Asian descent (Indonesia, Malaysia, Philippines, Korea) using a nine point hedonic scale as described by Larmond (1977). Fish sauces prepared in 1983-84 were evaluated by hedonic scale and by ranking of overall quality twice by a panel of 5 Vietnamese-Canadians. Samples were presented to panelists in partitioned booths with standardized lighting. The results were analyzed for statistical significance by the methods described by Larmond (1977).
Regression Analysis The relationship between sensory panel results and chemical analyses was evaluated by multiple regression using a Lisa microcomputer and LisaCalc software.
Results and Discussion Influence of fermentation aids on initial rate of fermentation The formation of non-protein nitrogen (NPN) in fish sauce brine occurred rapidly during the first week
.... squid HP
." chy.try.
•
Amino acids and related substances The free amino acid content and other ninhydrin positive components (i.e. alpha-aminobutyric acid, ornithine, ethanolamine, anserine, carnosine, creatinine, o-phosphoethanolamine, urea, methionine sulfoxide, methionine solfone, glutamine, asparagine. sarcosine, citrulline, L-a-aminoadipic acid, L-a-aminon-butyric acid, cystathionine, l3-aminoisobutyric acid and homocitrulline) were determined with a Beckman Can. Insl. Food Sci. Technol. J. Vol. 19, No. I, 1986
trypsin
(> squid E
10
20
30
+
pronase
•
F. prot.
o
control
40
Time,days
Fig. I. Formation of protein hydrolysis products in fish sauce prepared from minced capelin, 0 control; • squid HP; ~trypsin; .chymotrypsin; <> squid extract; + pronase; .F. prot.
Raksakulthai et al. / 29
Table I. Content of free amino acids in capelin fish sauce. Treatment Free amino acids mM mg010 None, round capelin 216 2729 Mince, no enzyme 352 4432 + Fungal Protease 262 3278 + Pronase 273 3256 + Trypsin 284 3606 + Chymotrypsin 374 4777 + Squid Protease 380 4777 + Squid Hepatopancreas 496 6298 IData are average of one analysis for duplicate samples of 13 month
and more gradually during the subsequent 4 w of fermentation (Figure I). Addition of proteolytic enzymes or squid hepatopancreas to mince resulted in formation of more TeA soluble protein than occurred in the brine of control mince. Samples with 3.3 mg 070 of chymotrypsin, trypsin, squid protease or pronase formed about twice as much NPN as the untreated mince; samples supplemented with 2.5% squid hepatopancreas formed about 3 times as much NPN in the brine as did the control samples. Fungal protease was apparently less effective in accelerating the hydrolysis of capelin mince than were the other enzyme preparations. The squid protease fraction employed in this study contains cathepsin e and other contaminating proteases (Hameed and Haard, 1985).The hepatopancreas from squid is also a rich source of cathepsins B, D, E and other unidentified proteases (Inaba et al., 1976; Leblanc and Gill 1982). The data indicate that addition of enzymes to minced capelin increases the progress of the initial stage of the fermentation. Fish sauce prepared with minced Ikanbilis (Stolephorus sp.) and salt in a ratio of 3:2 formed more total nitrogen after 21 d at 33°e when supplemented with 0.8% bromelain (1.25 fold), 2.75% papain (1.2 fold) or 2.5% ficin (l.1 fold) (Beddows and Ardeshir, 1979). Fermentation of mackerel, sardine or shrimp with 25% salt at 37°C for I mo with 0.2-0.5% added enzyme increased the yield of total soluble nitrogen about 1.05 fold with trypsin, 1.1 fold with bromelain and 1.15 fold with papain (Ooshiro et al., 1981). Aiding Anchovy bagoong fermentation (3 parts Fish: I part salt) with 0.5% papain increased the yield of crude protein in the 28 d brine by about 1.1 fold (Guevara et al., 1974). The yield of amino type nitrogen in brined horse mackerel (4: 1) held at 37°e was increased by 1.4 fold by addition of 0.2% Bioprase (Muraymam et al., 1962). Several of these workers reported that the sensory characteristics of fish sauce prepared with added enzyme was somewhat different from that of sauces prepared without a fermentation aid.
Free amino acids and related components The meaty flavour of fish sauce appears to be determined by free amino acids and amines; although ammonia and other volatile bases contribute to the ammonia-like aroma and volatile organic acids contribute to the "cheese" aroma of the product (Dougan and Howard, 1975). Thirteen mo after salting, the free amino acid content of capelin fish sauces ranged 30 / Raksakulthai et al.
Major amino acids l mole % alanine (12.0) > glutamic (11.3) > leucine (9.4) > lysine (9.2) alanine (11.9) > glutamic (11.2) > leucine (9.8) > lysine (9.2) alanine (13.0) > glutamic (12.0) > leucine (10.8) > lysine (7.8) leucine (15.6) > alanine (15.0) > glycine (11.9) > valine (11.3) alanine (13.0) > lysine (11.0) > leucine (11.0) > glutamic (8.6) alanine (12.9) > leucine (12.9) > lysine (9.7) > glutamic (8.7) alanine (12.2) > leucine (10.6) > lysine (9.7) > glutamic (9.4) glutamic (11.5) > alanine (10.5) > asp (10.1) > leucine (9.6) old product.
from 2729 mg% to 6298 mg% (Table 1). The free amino acid content of sauce prepared from round capelin was 62% of that formed in sauce prepared from mince. Fish sauces prepared from mince supplemented with proteolytic enzymes contained from 3278 to 4777 mg% free amino acids and on this basis the final product was not especially favored by these enzyme supplements. However, fish sauce prepared with squid hepatopancreas contained 6298 mg% free amino acids, an apparent improvement of 1.42 fold over minced controls and 2.3 fold over fish sauce prepared from round capelin. Storage of the filtered fish sauces for an additional 20 mo (aging) resulted in the formation of additional free amino acids (Table 2). The free amino acid contents of 13 mo old sauces prepared in 1983-84 were 3040 mg% for minced controls and 6630 mg% for mince containing 2.5% squid hepatopancreas. The lower content of free amino acids in 1983-84 sauce prepared from mince compared to that prepared in 1982-83 may relate to differences in endogenous proteolytic activity which is known to vary greatly with feed intake of capelin (Gildberg, 1978). Fish sauce produced in Thailand is required to have an amino acid nitrogen content of 950 mg% (Anonymous, 1970). The amino acid nitrogen content of Nuoc-mam ranges from 450 mg% for ordinary quality grade sauce to 900 mg% for first quality grade product and of commercial patis ranges from 904 to 994 mg% for special and extra grades (Uyenco et al., 1963). Based on an approximate nitrogen content of free amino acids of 16%, for samples analyzed in this study, the amino acid nitrogen content of capelin fish sauces were 437 mg% from round capelin, 709 mg% from minced capelin and 1008 mg% from minced capelin supplemented with squid hepatopancreas. Estimation of free amino acid nitrogen by the method used in S.E. Asian countries, which is based on the difference between formaldehyde nitrogen and ammoniacal nitrogen, would give somewhat higher values than by direct estimation from free amino acids because of the contribution of peptide alpha-amino groups to formaldehyde nitrogen. The amino acid content of fish sauce hydrolyzates, i.e., total amino acids, prepared from minced capelin ranged from 6976 mg.% or 1116 mg% amino acid nitrogen, to 9810 mg% (hepatopancreas), representing 1570 mg% amino acid nitrogen. These data were used to determine the free amino acids as percentage of total amino acids in fish sauce (Table 2). J. Ins!. Can. Sci. Technol. Aliment. Vol. 19. No. I, 1986
Table 2. Characteristics of free amino acids from capelin fish sauce and commercial products. Free Amino Acids I 13 Months Fish Sauce
Treatment
mg%
Capelin, whole None Capelin, mince None + fungal prot. + pronase + trypsin + chymotry. + squid prot. + squid pan.
2729 4431 3278 3256 3606 4777 4777 6298
Commercial
mg% 5609 5280
Thailand Philippines
AlB
33 Months
HO. v
2.92 891 3.00 903 2.53 969 14.90 1290 2.11 966 1.97 1165 2.67 958 2.60 935 Free Amino Acids AlB 3.00 1.80
% Total
mg010
AlB
HO.v
34 65 44 40 45 49 68 64
5280 4814 5190 4936 5638 8680
2.76 6.44 2.37 2.31 2.79 3.24
953 1021 1015 1126 940 887
010 Total
70 59 65 51 81 89
HO. v 831 970
IData for one analysis and representative of results obtained with second experiment; AlB is molar ratio of acidic to basic amino acid residues; HO.vis Bigelow's average hydrophobicity of amino acid residues; % total amino acids is free amino acids in mg010 divided by total amino acids in mg%. The latter data was obtained by amino acid analysis after hydrolysis of fish sauce in 6N HCI as described in Methods.
16 , . . . - - - - - - -
12
,
D
+ Hepatopancreas
•
Control
Amino Acid
Fig. 2. Free amino acid composition of fish sauce prepared from minced capelin with and without squid hepatopancreas as a fermentation aid.
The percentage of total amino acids which was free in hepatopancreas treated sauce was 64070 at 13 mo and 89070 at 33 mo. For 13 mo old samples containing commercial enzyme supplements, the percentage of total amino acids present as free amino acids was apparently lower than in the control. The protease
fraction obtained from squid hepatopancreas appeared to be more effective in improving the ratio of free to total amino acids than it was in improving the total content of amino acids in the products. The results indicate that, with the processing conditions employed in this study, addition of squid hepatopancreas to minced capelin yields a fish sauce which exceeds the standards set for first grade products in S.E. Asian countries. The main free amino acids in capelin fish sauce prepared from mi.nce were alanine (11.9070), glutamine (11.2070), leucine (9.8070), lysine (9.2070), aspartic (8.3070),glycine (7.1070), valine (6.9070), serine (6.3070), taurine (5.1070), threonine (5.1070) and isoleucine (4.4070). The mole percent of the major free amino acids identified in 13 mo old sauce prepared from mince and mince supplemented with hepatopancreas is illustrated in Figure 2. With the exception of pronase, the addition of proteolytic enzymes to mince did not greatly alter the spectrum of major amino acids formed (Table 1). In pronase treated samples, the ratio of acidic to basic amino acids was high, due to the low recovery of basic. Amino acids, and the average hydrophobicity of the free amino acids was much higher than other samples (Table 2).
Table 3. Content of Ninhydrin reactive substances in capelin fish sauce. Treatment Ninhydrin Positive Components! mM mg% (% Control)
Major Components mole %
None 64 385 (108) NH 3 (63.9) > citruline (14.4) > ethanolamine (6.9) 68 356 (100) NH 3 (72.4) > citruline (17.8) > ethanolamine (6.5) Mince (Control) + Fungal Protease 54 290 (81) NH 3 (73.1) > citruline (20.2) > ethanolamine (4.2) + Pronase 169 1562 (439) ethanolamine (64.6) > ornithine (22.4) > citru!' (8.6) + Trypsin 61 346 (97) NH 3 (67.8) > citruline (18.9) > ethanolamine (6.9) + Chymotrypsin 76 494 (139) NH 3 (68.3) > citruline (21.2) > carnosine (5.1) + Squid Protease 65 380 (107) NH 3 (69.4) > citruline (22.5) > ethanolamine (5.6) + Squid Hepatopancreas 89 494 (139) NH 3 (69.7) > citruline (14.1) > ethanolamine (7.2) ICompounds analyzed: glycerophosphoethanolamine phosphoethanolamine urea, methioninesulfoxide*, asparagine, glutamine*, sarcosine*, alpha-amino adipic acid, citruline*, alpha-amino-n-butyric, homocitruline, cystathionine*, gamma-glutamyl-e-Iysine, J3-aminoisobutyric, homocystine, gamma-aminobutyric*, ethanolamine*, ammonia*, ornithine*, anserine*, carnosine*, glutathione, glucosamine, galactosamine. Compounds with* were present in some or all of samples. Data are for duplicate analyses of 13 month old fish sauce preparations. Can. InSf. Food Sci. Technol. J. Vol. 19. No. J, 1986
Raksakulthai et al. I 31
Table 4. Sensory characteristics of capelin and commercial fish sauce. Sample Preference Score' Mince capelin 6.00a,b + fungal protease 5.25 + pronase 6.25a,b + squid protease 6.25a,b + trypsin 6.50a,b + chymotrypsin 6.50a,b + squid hepatopancreas 7.00a Commercial product 2 6.75 IValues followed by "a" are not significantly different from control (P < 0.1); values followed by "b" are not significantly different from control (P < 0.2), n = 4. 2Rufina brand fish sauce from Philippines.
tathionine, gamma-aminobutyric, anserine, and carnosine, were present at low concentration in some but not all samples. Fish sauce prepared with pronase was unusual with respect to its high content of these compounds, notably ethanolamine and ornithine. The unusual free amino acid composition and the high content of non-amino acid nitrogen compounds for pronase treated fish sauce may indicate the intervention of salt tolerant microorganisms in this fermentation (Fujii and Sakai, 1984b). Commercial patis contained 637 mg070 non-amino acid nitrogen consisting of 80070 ammonia, 13070 citrulline, 3070 ethanolamine and 3070 ornithine.
Sensory Evaluation The relationship between amino acid composition and quality of fish sauce is not understood at this time. Some authors have suggested that fish sauce flavour is largely influenced by the mono-amino acid fraction (Amano, 1962), although there appears to be no evidence to substantiate this assertion. It would appear that the glutamic acid content of fish sauce plays an important role in taste since it is well recognized that glutamate contributes to "umami" or delicious taste in fish and other seafoods (Yamaguchi, 1979) and it is a major component of capelin fish sauce. According to Fujii and Sakai (l984a), the major amino acids of Japanese fish sauce (shotturu) are glutamic> valine > glycine> aspartic > alanine; the major free amino acids of Nuoc-mam are reported to be alanine> glutamic > isoleucine > leucine and lysine (Amano, 1962); and those identified in anchovy fish sauce are alanine > valine > leucine > lysine (Miyazawa et al., 1979). The analysis of commercial, patis from the Philippines showed the same major free amino acids as capelin fish sauce, i.e., alanine> leucine> lysine > glutamic> valine.
Other Nitrogen Compounds With the exception of pronase treated samples, the capelin fish sauces contained between 300 and 500 mg070 of non-amino acid nitrogen compounds (Table 3). Ammonia accounted for about 70070 of this fraction and citrulline made up 15-20070 of the nitrogen compounds identified. Ethanolamine and ornithine were present in all of these samples; other compounds, i.e. methionine sulfoxide, glutamine, sarcosine, cys-
Table 5. Preference Scores of capelin and commercial fish sauces. Sample Preference Score' Rank Totals1,3 Minced Capelin Commercial patis 2 Minced Capelin + hepatopancreas Capelin + hepatopancreas Commercial Capelin
5.3a 5.6a
Relationship between chemical composition and sensory preference Regression analysis of the data collected from 9 samples of capelin fish sauce with preference score as the dependent variable showed a coefficient of determination (r 2) of 0.745 with total free amino acids as the independent variable (Table 6). Multiple regression analysis with total amino acid content, ratio of acidic to basic amino acid residues, average hydrophobicity of free amino acids and non-amino acid content as independent variables showed that all of these factors could better account for the preference scores than free amino acids alone. The regression equation, which had an r 2 of 0.792 is shown in footnote 2 of Table 6. The results of this analysis are more or less consistent with previous suggestions that more total free Table 6. Regression analysis of preference scores as a function of amino acid and ninhydrin in positive components. Dependent variable(yl) Independent variable (X)I r2 preference score
8.2 12 21 27
lValues followed by "a" are not significantly different (p < 0.05); panel composed of 5 Vietnamese; n = 10.; capelin sauces prepared in 1983-84. 2Rufina brand patis from Philippines. 3Capelin + hepatopancreas was significantly better (p < 0.05) than the other two samples; n = 10.
32 / Raksakulthai et al.
Capelin fish sauce samples were evaluated for preference and compared to a commercial fish sauce by experienced consumers of this type of product. The results indicate that commercial product and hepatopancreas supplemented capelin sauce were preferable to sauce made from minced capelin with no added enzyme (Table 4). Capelin sauce prepared with fungal protease was rated lower in overall quality than the control capelin sauce. All other treatments were not significantly different (p < 0.2). Additional lots of capelin sauce prepared in 1983-84, minced and supplemented with hepatopancreas, were analyzed for preference by ranking and hedonic scoring. The capelin sauce prepared with hepatopancreas proved to be clearly superior to the unsupplemented capelin sauce and the commercial control (Table 5).
HOail) 0.090 0.088 non-amino acid N (2) acidic/basic free amino acids (3) 0.001 0.745 total free amino acids (4) 1,4 0.780 2,4 0.773 1,2,4 0.791 1,3,4 0.789 1,2,3,4 0.792 2 IFrom regression analysis of data for 9 different samples of fish sauce. 2y = 0.001 (X,) + 0.007 (X 2) - 0.033 (X 3) + 0.007 (X 4 ) + 2.45. J. lnsl. Can. Sci. Techno!. Aliment. Vol. '9, No. '. '986
amino acids are associated with superior grade product; and further suggest that a low acidic/basic ratio, a high H0 a" and a high content of non-amino acid nitrogen compounds are associated with high preference scores. Overall, the analysis suggests that the amount and type of free amino acids and the amount of ammonia and other nitrogenous compounds contribute to the acceptability of capelin fish sauce. Additional study with fish sauce prepared from other fish and by different methods, e.g. salt content and temperature, is necessary to determine the broader applicability of the regression analysis. Using the equation shown in Table 6 the calculated preference score for commercial patis was 7.17, somewhat higher than the actual preference scores of 6.75 and 5.60 by the two panels (Tables 4,5).
Conclusion An excellent quality fish sauce can be prepared from inshore male capelin when the fermentation is carried out at a minced fish: salt ratio of 4: 1 at 20-25°C and 2.5070 squid hepatopancreas is used as a fermentation aid. Addition of various other enzyme preparations to minced capelin increases the initial rate of protein hydrolysis but does not yield a product containing a much higher content of free amino acids. The overall acceptability of capelin fish sauce by individuals of S.E. Asian descent appears to be related to the amount and type of free amino acids and other non-amino acid nitrogen compounds.
Acknowledgment The study was supported by an operating grant from the Natural Sciences and Engineering Research Council Canada. We greatly acknowledge the assistance of Mr. D. Hall and Mrs. S. Banfield for assistance with amino acid analyses.
References Amano, K. 1962. The influence of fermentation on the nutritive value of fish with special reference to fermented fish products of South-East Asia. In: Fish in Nutrition. E. Heen and R. Kreuzer (Eds.). p. 180. Fishing News Book. London. Anonymous. 1970. Standards for local fish sauce, Thai Industrial Standards Institute, Ministry of Industry, Bangkok, Thailand. Beddows, e.G. and Ardeshir, A.G. 1979. The production of fish protein solution for use in fish sauce manufacture. I. The use of added enzymes. J. Food Techno!. 14:603.
Can. Ins/, Food Sci. Techno!. J. Vol. 19. No.1. 1986
Bigelow, e.e. 1967. On the average hydrophobicity of proteins and the relationship between it and protein structure. J. Theor. Bio!. 16:187. Dougan, J. and Howard, G.E. 1975. Some flavouring constituents of fermented fish sauce. J. Sci. Food Agric. 26:887. Fujii, T. and Sakai, H. 1984a. Chemical composition and micro flora of fish sauce "shotturu". Bull. Jap. Soc. Sci. Fish. 50: 1061. Fujii, T. and Sakai, H. 1984b. Chemical and microbiological analyses of putrid fish sauce "shotturu". Bull. Jap. Soc. Sci. Fish. 50:1067. Gildberg, A. 1978. Proteolytic activity and the frequency of burst bellies in capelin. J. Food Technol. 13:409. Guevara, G., Matias, V.C., Dela Pena, P.O. 1972. Fish fermentation with the use of papain. The Phil. J. Fish. 10:30. Hameed, K.S, and Haard, N.F. 1985. Isolation and characterization of cathepsin C from Atlantic short finned squid, IIIex i1lecebrosus. Compo Biochem. Physiol.82B:24 I. Inaba, T., Shindo, N. and Fujii, M. 1976. Purification of cathepsin B from squid liver. Agric. BioI. Chern. 40: 1159. Leblanc, E.L. and Gill, T.A. 1982. Comparative study of proteolysis in short-finned (II/ex il/ecebrosus) and long-finned (Loligo paeli Leseur) squid. Compo Biochem. Physio!. 73B:201. Larmond, E. 1977. Laboratory methods for sensory evaluation of food. Publication 1637, Canada Department of Agriculture. Lee, Y.Z., Simpson, B.K. and Haard, N.F. 1982. Supplementation of squid fermentation with proteolytic enzymes. J. Food Biochem.6:127. Miyazawa, K, Le, C.V., Ito, K. and Matjunuoto, F. 1979. Studies on fish sauce. Hiroshima University. J. Fac. Appl. BioI. Sci. 18:55. Murayawa, S., Calvez, D.L. and Nitayachin, P. 1962. Study on the production of fish sauce - I. Effect of commercial proteolytic enzyme on the production of fish sauce. Bull, Tokai Reg. Fish. Lab. 32:155. Ooshiro,Z., Ok, T., Une, H., Hayashi, S. and Itakura, T. 1981. Study 0,11 use of commercial proteolytic enzymes in production of fish sauce. Mem. Fac. Fish., Kagoshima Univ. 30:383. Saisithi, P., Kasemsarn, Bung-Orn, Liston, J. and Dollar, A.M. 1966. Microbiology and chemistry of fermented fish. J. Food Sci. 31:105. Uyenco, V., Lawas, I., Briones, P.R. and Taruc, R.S. 1953. Mechanics of bagoong (fish sauce) and patis (fish sauce) processing. In: Indo-Pacific Fisheries Council Proceedings, Food and Agricultural Organization of the United Nations, Bangkok. p. 210. Yamaguchi, S. 1979. The umami taste. In: Food Taste Chemistry J.C. Boudreau (ED.). pp 33-51. American Chemical Society Symposium Series 115.
Submitted May 13, 1985 Revised November 5, 1985 Accepted November 18, 1985
Raksakulthai et al. / 33