Fisheries Science 63(6), 939-944 (1997)

Extractive Components of the Ink

Takaaki Shirai,*1 Naoki Kikuchi,*1 Shingo Matsuo,*1 Hiroshi Inada,*2 Takeshi Suzuki,*1 and Toshiyuki Hirano*1,3 *1Department of Food Science and Technolog y, Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan *2Department of Marine Science and Technology , Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan *3School of Marine Science and Technology , Tokai University, Orido, Shimizu, Shizuoka 424, Japan

(Received September 30, 1996)

An extract was prepared with trichloroacetic acid from the inks of neon flying, boreal clubhook, and boreo pacific gonate and free amino acids, trimethylamine oxide, 5'-ATP and related com pounds, betaines, and other extractive components were analyzed. The squid inks were rich in taurine and hydroxyproline. Higher amounts of aspartic acid, glutamic acid, alanine, leucine, and arginine were detected in the boreal clubhook and boreo pacific gonate squid inks. Each free amino acid of the ink was lower than those of the boreal clubhook and boreo pacific gonate squid inks. A small amount of trimethylamine oxide and large amounts of homarine and glycinebetaine were detect ed in three squid inks. Ethanol extract was prepared to determine the extraction capability. There were some composition similarities between ethanol and trichloroacetic acid extracts. Ethanol was inferior in extraction of inor ganic ions, even in the case of the hot-ethanol extraction. The hot-water extract showed that amounts of glycine, ƒ¿-aminobutyric acid, Cl-, and PO43- were higher than those of the trichloroacetic acid ex tract of the neon flying squid ink.

Key words: squid ink, extractive component, taurine, extraction method

Squidis one of the major marine food resources and pos from June though July 1995 and they were immediately siblythe most important of those that are not yet fully ex frozen at -35•Ž and transported to our laboratory in the ploited,and occur in all oceans and seas. The squid is frozen condition under -18•Ž. These squids were thawed knownto synthesize melanin from tyrosine via 3,4-di at 5•Ž for 2-3 h. Five ink-sacs were removed from five hydroxyphenylalaninein the ink1) and its ink has been half-frozen individuals of neon flying squids and used as traditionally utilized as food products commonly in samples. Ink-sacs were removed from several bodies of Japan.2)On the other hand, squid ink is reported to have boreal clubhook squid and were combined and divided an anti-bacterial activity.3) Recently, the antitumor pep into two samples. Each ink-sac of boreo pacific gonate tideglyconfraction was isolated and purified from squid squid was removed from several squid bodies and were inkby Takaya et al. 4)Squid ink has been utilized as a food combined and used as one sample. materialand is attracting attention in the seafood market in Japan, however, little is known about the food chemis Proximate Composition tryof the extractive components. The squid ink sample was dried at 108•Ž and its The neon flying squid Ommastreohes bartrami is one of moisture was measured. Crude protein was determined by the major commercial squids in Japan and boreo club the Kjeldahl method. Lipid was extracted with the chlo hook Onychoteuthis borealijaponica and boreo pacific roform/methanol mixture of Bligh and Dyer7) and dried in gonate borealis squids are also caught as an air-oven. Ash was measured in an oven at 550•Ž. bycatch of the squid jigging fishery for neon flying squid in the Pacific Ocean.5,6) The inks of these three squids were Preparation of Extracts therefore used as samples in this study and extracts were Three grams of the neon flying squid ink and 1 g each of prepared with ethanol and trichloroacetic acid and the ex the boreal clubhook and boreo pacific gonate squid inks tractive components were estimated. were respectively extracted with 50 ml of 5% trichloroace tic acid in a glass pestle homogenizer cooled with ice. The Materials and Methods homogenate was centrifuged at 8,000 rpm for 15 min at 4•Ž. The residue was washed with 20 ml of 5% trichloro Squid Ink acetic acid and centrifuged twice more. The supernatants Neon flying squid, boreal clubhook squid, and boreo were combined and trichloroacetic acid was removed with pacificgonate squid were jigged in the North Pacific Ocean diethyl ether. Then the lower aqueous layer was evaporat- 940 Shirai et al. ed and diluted to 50 ml with water. This extract was desig Table 1. Proximate composition of the squid inks nated as the trichloroacetic acid extract. (%) Ethanol extract was prepared as follows. The squid ink sample (1-5 g) was homogenized with 50 ml of 80% ethanol in a glass pestle homogenizer cooled with ice and centrifuged at 8,000 rpm for 15 min at 4•Ž. The residue was extracted with 80% ethanol and centrifuged twice more. The supernatants were combined and evaporated to remove ethanol. Then the aqueous solution was defatted with diethyl ether. The lower layer was evaporated and Five individuals of the neon flying squid ink and two individuals each of the boreal clubhook and boreo pacific gonate squid inks were analyzed and values are diluted to 50 ml with water. This extract was named as expressed as means with S.D. and ranges. Carbohydrate was calculated temporarily the ethanol extract. by the difference [100% -(moisture + crude protein + lipid + ash)]. Since squid ink has been traditionally and often utilized with a small amount of liquors in food products and some times cooked, hot-ethanol extraction was also employed. Extractive Components The squid ink sample was homogenized with 80% ethanol Acid, alcohol, heating, etc. are generally known to in a glass pestle homogenizer and then heated under a affect the degree of protein denaturation and extraction reflex condenser in a boiling-water bath for 15 min. The capability physically and chemically.9) Little information homogenate was centrifuged and the residue was washed about the extractive components of squid ink has been with 80% ethanol. The residue was washed with 80% reported yet, so the trichloroacetic acid, ethanol, hot ethanol and centrifuged twice more. The supernatants ethanol, and hot-water extractions were adopted in this were combined and evaporated to remove ethanol. The study to analyze the effects of these deprotein treatments aqueous solution was defatted with diethyl ether and the on squid ink. lower layer was again evaporated. The extract was diluted Table 2 shows the composition of the trichloroacetic to 50 ml with water. This extract was named as the acid extractable components of the three squid inks. Five hot-ethanol extract. individuals of the neon flying squid ink, two samples of Hot-water extraction was also adopted with 3 g each the boreal clubhook squid ink, and one sample of the from five individuals of the neon flying squid ink in the boreo pacific gonate squid ink were analyzed and shown in same manner as the above hot-ethanol extraction substitut the table. Values of the inks of neon flying and boreal club ing water for ethanol. This extract was designated as the hook squids are expressed as means with S.D. and hot-water extract. differences from the means, respectively. Among the free amino acids of the trichloroacetic acid Analysis of Extractive Components extracts shown in Table 2, the inks of neon flying, boreal Free amino acids, 5•Œ-adenosine triphosphate (ATP) and clubhook, and boreo pacific gonate squids were rich in tau related compounds, trimethylamine and its oxide, and rine and hydroxyproline. Higher amounts of aspartic acid, cyclic and ƒÖ-betaines were measured with the previously glutamic acid, alanine, leucine, and arginine were detected reported methods.8) Na+, K+, Cl-, and PO43- were also de in the boreal clubhook and boreo pacific gonate squid termined by the reported methods.8) inks. Each free amino acid of the neon flying squid ink was Extractive nitrogen of the above extracts was deter lower than those of the boreal clubhook and boreo pacific mined by the Kjeldahl method. gonate squid inks. A portion of the extract was dried at 110•Ž and the Trimethylamine oxide was known to be detected in residue was designated as a dry matter. higher amounts in squid muscles,10-13)however, small amounts of less than 40 mg/ 100 g were detected in the Results and Discussion three squid inks in this study. Each amount of glycinebetaine of the neon flying squid Proximate Composition ink among five individuals fluctuated. Large amounts of Five samples of the neon flying squid ink and two sam homarine and glycinebetaine were detected in the squid ples each of the boreal clubhook and boreo pacific gonate inks and these values were of similar levels to or higher squid inks were analyzed and values are expressed as than those of the squid muscles amounting to 60 means with S.D. and differences from the means, respec 300 mg/ 100 g for homarine and 300-930 mg/ 100 g for tively. Table 1 shows moisture, crude protein, lipid, and glycinebetaine, respectively.10-12) ash of the squid inks. Moisture of the boreal clubhook The squid inks seemed to be rich in Cl-. A large amount squid ink was higher than those of the neon flying squid of PO43- was detected in the boreo pacific gonate squid ink and boreo pacific gonate squid inks. Crude protein of the and this value was almost four times higher than that of boreo pacific gonate squid was higher than those of the the mantle muscle of boreo pacific gonate squid.10) The ink boreal clubhook and neon flying squid inks. of neon flying squid was rich in inorganic ions. High molecular weight sugars were reported to exist in The extractive components of the mantle and arm mus squid ink.1) Carbohydrate was calculated temporarily by cles of boreo pacific gonate squid were analyzed previous the difference [100% -(moisture + crude protein + lipid + ly10) and the features of the composition of these compo ash)] in this study. nents were different from that of its ink in this study. Since squid ink is often utilized in raw seafood products Extractive Components of Squid Ink 941

Table 2. Trichloroacetic acid extractable components of the squid Table 3. Ethanol extractable components of the squid inks inks (mg/ 100 g) (mg/ 100 g)

Five individuals of the neon flying squid ink and two samples of the boreal club hook squid ink were analyzed and values are means with S.D. and ranges. Not Five individuals of the neon flying squid ink and two samples of the boreal club detected. DM and EN recoveries are the sum of extractive components divided by hook squid ink were analyzed and values are means with S.D. and ranges. Not the dry matter and the sum of the nitrogen amounts of the analyzed nitrogenous detected. DM and EN recoveries are the sum of extractive components divided by components by the extractive nitrogen, respectively. Significant difference at the dry matter and the sum of the nitrogen amounts of the analyzed nitrogenous p<0.05 higher + or lower against the trichloroacetic acid extract is in Table 2, components by the extractive nitrogen, respectively.

and ATP related compounds from fish muscle was report Such as salted and fermented "shiokara" products and an ed to be inferior to 5% trichloroacetic acid.") The overall extract that was prepared with ethanol was reported to ade compositions of the ethanol extracts being rich in taurine, quately reproduce the original squid taste,101 the ethanol ex hydroxyproline, homarine, Na+, and Cl for the neon traction was adopted. Table 3 shows the ethanol extracta flying squid ink, in almost all amino acids analyzed, homa ble components of the squid inks. Amounts of some com rine, glycinebetaine, Na+, Cl-, and K+ for the boreal club ponents such as homarine, glycinebetaine, and a few other hook squid ink, and in almost all amino acids analyzed, Components of the ethanol extract were slightly lower than glycinebetaine, and Na+ for the boreo pacific gonate squid those of the trichloroacetic acid extract. ink were respectively similar to those of the trichloroacetic The extraction capability of 80% ethanol in the creatine acid extracts. 942 Shirai et al.

Table 4 shows the compositions of the squid ink compo against the amounts of the components of the ethanol ex nents which were extracted with 80% ethanol heated in a tract or the trichloroacetic acid extract in Table 2. This boiling-water bath for 15 min. In the case of the neon statistical difference is expressed as higher + or lower flying squid ink, five individuals were used in this study against those of the ethanol extract and also higher + + or and amounts are expressed as means with S.D. The differ lower against those of the trichloroacetic acid in ence in the amounts of the components of the neon flying Table 4. squid ink in Table 4 was judged statistically at p<0.05 Among the major extractive components in the table, the amounts of glycine and trimethylamine oxide of the hot-ethanol extract of the neon flying squid ink were higher than those of both the ethanol and trichloroacetic Table 4. Hot-ethanol extractable components of the squid inks acid extracts. The amount of PO43-3 of the hot-ethanol ex (mg/ 100 g) tract was slightly higher than that of the ethanol extract. On the other hand, the amounts of inorganic ions of both the ethanol and hot-ethanol extracts were lower at p<0.05 than those of the trichloroacetic acid. Table 4 shows that 80% ethanol was inferior in extraction of inorganic ions, even in the case of the hot-ethanol extraction. In the case of the boreal clubhook squid ink, almost no difference in the composition of the extractive components was seen between the ethanol and hot-ethanol extracts, however, the amounts of arginine, hydroxyproline, homa rine, glycinebetaine, Cl-, and PO43- of the trichloroacetic acid extract seemed to be higher than those of the ethanol extract. A similar feature in the compositions of the extrac tive components of the squid inks were observed in the boreo pacific gonate squid ink, namely that the amounts of aspartic acid, glutamic acid, lysine, histidine, arginine, hydroxyproline, homarine, glycinebetaine, Cl-, P043 -, and several other components of the trichloroacetic acid extract were higher than those of the ethanol and hot -ethanol extracts. To see the effect of the heat treatment on the extraction capability for squid ink, the hot-water extract was pre pared and the composition of the extractive components of the trichloroacetic acid, hot-ethanol, and hot-water ex tracts of the neon flying squid ink were compared. Table 5 shows the composition of the extractive components of the hot-water extract of the neon flying squid ink. Values were means of five individuals with S.D. and the difference was judged statistically at p<0.05 against the composition of the extractive components of the trichloroacetic acid ex tract of the neon flying squid ink and is expressed as higher + or lower in Table 5. A remarkable feature was that the amounts of glycine, ƒ¿-aminobutyric acid, Cl-, and PO43- were higher than those of the trichloroacetic acid ex tract. The amounts of hydroxyproline, homarine, glycine betaine, and K+ were lower than those of the trichloroace tic acid extract. Compared with the compositions of the ex tractive components of the hot-ethanol extract of the neon flying squid ink, the amounts of hot-water extract seemed to be higher than those of the hot-ethanol extract, on the other hand, the amounts of hydroxyproline and trimethyl amine oxide seemed to be lower. The hot-ethanol extraction for the neon flying squid ink increased the amount of hydroxyproline, on the other hand, heat treatment in the hot-water extraction procedure did not increase the amount of hydroxyproline. Five individuals of the neon flying squid ink and two samples of the boreal club hook squid ink were analyzed and values are means with S.D. and ranges. Not The amounts of several extractive components of the detected. DM and EN recoveries are the sum of extractive components divided by neon flying squid ink for different extraction methods are the dry matter and the sum of the nitrogen amounts of the analyzed nitrogenous shown in Fig. 1. The extraction capability of 80% ethanol components by the extractive nitrogen, respectively. Significant differences at for the neon flying squid ink was inferior to those of p<0.05 higher + or lower against the ethanol extract in Table 3 and higher + + or lower against the trichloroacetic acid are in Table 2. trichloroacetic acid and hot-water. The trichloroacetic Extractive Components of Squid Ink 943

Table 5. Extractive components of the neon flying squid ink by hot-water extraction (mg/ 100 g)

Fig. 1. Several extractive components of the neon flying squid ink by different extraction methods (mg/ 100 g).

homarine, glycinebetaine, Cl-, and P043-. More than 10 mg/ 100 g of trimethylamine that has a fishy smell was found in the inks of boreal clubhook and boreo pacific gonate squids. The neon flying squid ink felt rough and granular. The boreal clubhook squid ink felt smooth and powdery. The boreo pacific gonate squid ink was slightly sticky. The three squid inks were tentatively tasted by five people, who found that the taste bodies of these inks were all strong and the tastes of these squid inks were different from each Five individuals of the neon flying squid ink were analyzed and values are means other. The tastes of squid inks were not similar to those of withS.D. Not detected. DM and EN recoveries are the sum of extractive compo nentsdivided by the dry matter and the sum of the nitrogen amounts of the ana their muscles. The taste of the neon flying squid ink was lyzed nitrogenous components by the extractive nitrogen, respectively. Significant thinner than those of other two squid inks. The inks of differenceat p <0.05 higher + or lower against the trichloroacetic acid extract is boreal clubhook and boreo pacific gonate squids did not in Table 2. have umami and sweetness and both inks had fishy, squid like smells. The sum of extractive components analyzed in this acid extraction was superior in the extraction of glycine study was divided by the dry matter and is expressed as the betaine. dry matter recovery in Tables 2 to 5. The extractive nitro From the compositions of the extractive components of gen recovery was calculated by dividing the sum of the the trichloroacetic acid, ethanol and hot-ethanol, and hot nitrogen amounts of the analyzed nitrogenous compo water extracts, acid and heat coagulation and ethanol nents by the extractive nitrogen. These recoveries for the precipitation for high molecular weight compounds of the three squid inks did not reach 100% in this study. squid ink might affect the composition of the extractive components of the squid inks independently in this study. Acknowledgments We are grateful to the Japan Marine Fishery Glycine, alanine, serine, proline, glutamic acid, and 5 Resources Research Center for collecting squid samples, •Œ-adenylic acid (AMP) that have sweetness or umami were not abundant in the neon flying squid ink. The inks of References boreal clubhook and boreo pacific gonate squids were rich 1) H. Matsue, Y. Takaya, H. Uchisawa, T. Naraoka, J. Sasaki, and it glutamic acid, alanine, AMP, and glycinebetaine. The K. Ishida: New lights upon squid ink. Biosci. Industry, 53, 31-35 ink of boreo pacific gonate squid was rich in amino acids, (1995) (in Japanese). 944 Shirai et al .

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