Chapter 15
Elimination of Bitterness of Bitter Peptides by Squid Liver Carboxypeptidase C. Kawabata, T. Komai, and S. Gocho Technical Research Center, T. Hasegawa Company, Limited, 335-Kariyado, Nakahara-ku, Kawasaki 211, Japan
Molecular and enzymatic properties of serine carboxypeptidase (EC.3.4.1.6.1, CPase Top), isolated and refined from the common squid (Todarodes pacificus) liver, were studied. It was found that this enzyme reacts well at the C-terminal position of peptides having hydrophobic amino acids. Because of this property, it was anticipated that this enzyme would have the effect of eliminating bitterness of some peptides. This enzyme was used on bitter peptides prepared by hydrolysis of proteins with pepsin and trypsin. It was found that this CPase Top can eliminate bitter peptides prepared from soy protein and corn gluten.
Carboxypeptidase has been studied by many workers. It has been reported that this enzyme exists in many kinds of various microorganisms, plants, and animals (7, 2, 3). Because it is quite useful in the food industry, we studied how to produce it inexpensively. We found that this enzyme occurs in the liver of the common squid (Toardes pacificus) and determined its characteristic features. We found this enzyme to be useful in eliminating the bitterness of bitter peptides, which is described in this paper. MATERIALS AND METHODS Materials and Chemicals. N-acyl peptides were purchased from SIGMA Chemical Co. (St. Louis, MO, U.S.A.), and Peptide Institute Inc. (Osaka, Japan). Soy protein (New Fujipro) was purchased from Fuji Oil Co., Ltd. (Osaka, Japan). Casein (ALACID 720) was purchased from New Zealand Dairy Board (Wellington, New Zealand). Corn gluten (Gluten meal) was purchased from Nihon Shokuhin Kakou Co., Ltd. (Tokyo, Japan). Pepsin (EC.3.23.1)(Pepsin (1:10000), Trypsin (EC.3.4.21.4) (Trypsin Type IX), and L-Glycyl-L-Leucine (Gly-Leu) were purchased from SIGMA Chemical Co. Common squid (Toardes pacificus) livers were purchased from Nakamura Gyogyou-bu Co., Ltd. (Aomori, Japan).
0097-6156/%/0637-0167$15.00/0 © 1996 American Chemical Society
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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS
Preparation of Enzyme. Separation and purification of the enzyme from the liver of the common squid were as follows: Squid livers were mashed and dispersed in five volumes of distilled water, then acidified to pH 4.0. Much oil was eliminated. This was followed by ultrafiltration, salting out with ammonium sulfate (50% saturation), dialyzing and freeze-drying in vacuo, to yield a crude enzyme. A purified enzyme was obtained from this crude enzyme by using column chromatography. Active fractions were separated by cation exchange resin, Mono S (Pharmacia) and further purified by gel-filtration column of Superdex 75 (Pharmacia). The active fractions were collected as the purified enzyme. Assay of Enzyme Activity. Activity of this enzyme was determined by the increase in ninhydrin color after hydrolysis of Carbobenzoxy-L-glutamyl-L-tyrosine (Z-GluTyr) as substrate at 30°C, pH 3.1. One katal of acid carboxypeptidase activity was defined as the amount of enzyme required to liberate 1 mol of C-terminal amino acid per second. Preparation of Bitter Peptides. The bitter peptides were prepared by hydrolysis of soy protein or corn gluten with pepsin and hydrolysis of casein with trypsin. In the preparation of bitter peptides from soy protein, a 5% suspension of soy protein was hydrolyzed by pepsin [enzyme/substrate ratio = 1/100 (w/w)] at 37°C at pH 2.0. The water soluble portion was dialyzed and then freeze dried to yield bitter peptides. Bitter peptides from corn gluten and casein were prepared in a similar manner. Evaluation of Bitterness and Measurement of Liberated Amino Acids. Bitterness was evaluated sensorially by 5 members of a sensory panel by comparing samples to a standard aqueous bitter standard. The standard for bitterness was a Gly-Leu solution, with 1.0% concentration scored as 10 points and 0.1% as 1. Liberated amino acids were measured by the ninhydrin methods after samples were de-proteinized with trichloroacetic acid solution and represented as /xmol/ml of Leu. RESULTS AND DISCUSSION Enzyme Preparation. The purified enzyme from the squid liver was found to be approximately uniform judging from the SDS-PAGE pattern. This final preparation was purified about 470-fold from squid livers. The molecular weight of this enzyme was estimated at 42,000 by SDS-PAGE and gel filtration. The characteristics of this enzyme were studied with this purified material. Mode of Action of the Enzyme. Synthetic substrate, Z-Gly-Pro-Leu-Gly, was incubated with this purified enzyme at 30°C, pH 3.1. Measurements of the released amino acids were made with an amino acids analyzer (Hitachi, Model L-8500) throughout the time of incubation. From the reaction of the enzyme on the substrate, Gly at the C-terminus was first released. Next released was Leu, which is in the penultimate position. From the sequential release of amino acids from the C-terminus, it was confirmed that the enzyme is a carboxypeptidase, which was named CPase Top.
15. KAWABATA ET AL.
Elimination of Bitterness of Bitter Peptides
169
Characteristics of CPase Top. With further study of the characteristics of CPase Top, it was found that the optimum temperture was 40°C, with it being stable under 45°C, and that its optimum pH was 4.0, with stability in the range of pH 2-6. The isoelectric point was 6.0. The enzyme activity was almost completely inhibited by /7-chloromercuribenzoic acid (PCMB), monoiodoacetic acid, diisopropyl fluorophosphate (DFP), HgCl , and partially inhibited by phenylmethanesulfonyl fluoride (PMSF), Af-tosyl-L-phenylalanyl chloromethylketone (TPCK), chimostatin, and CuS0 . These results indicate that this enzyme is a member of the serine carboxypeptidase family (EC.3.4.16.1). 2
4
Substrate Specificity. The relative hydrolytic activity of CPase Top on various Nacyl-peptides was obtained by reacting at 30°C, pH 3.1 for 20 minutes. The relative activities are shown with activity to Z-Glu-Tyr as 100% (Table I). CPase Top shows a preference for peptides having hydrophobic amino acids as Z-Phe-Leu, Z-Tyr-Phe and Z-Tyr-Phe-Leu. When the penultimate amino acid from the Cterminus was a hydrophobic or bulky amino acid as Phe, Tyr, Leu and Glu, the hydrolysis rate of the sustrate was high. When Gly and Pro were penultimate to the C-terminus, activity decreased. Similarly, the same specificity of changing relative activity was obtained for substrates by amino acids penultimate to the C-terminus from CPase produced from Aspergillus saitoii (1). From this substrate specificity, it was assumed that by releasing a hydrophobic amino acid at the C-terminus of soy peptides and other bitter peptides, this enzyme could eliminate the troublesome bitterness of peptides produced by enzymatic hydrolysis of proteins (4-8) in food processing. To prove the above assumption to be true, bitter peptides were prepared from soy protein, casein, and corn gluten, and studies were conducted to eliminate the bitterness of the bitter peptides by using the crude enzyme from squid liver. Hydrolysis of Bitter Peptides from Soy Protein by CPase Top. Three bitter peptides were incubated with crude CPase Top at 30°C, pH 4.0, with the bitterness and the amount of liberated amino acids being evaluated throughout incubation. When a 1 % bitter peptide solution from soy protein was incubated with crude CPase Top (enzyme/substrate ratio = 1 /dcat/g), the bitterness diminished as reaction time increased. The bitterness was almost completely eliminated after 15 hr (Fig. 1). The amount of amino acids liberated increased with time of incubation. Hydrolysis of Bitter Peptides from Casein by CPase Top. When a 0.5% solution of bitter peptides from casein was incubated with crude CPase Top (enzyme/substrate ratio = 2 /Ltkat/g), most of the bitterness was eliminated after 2 hr, but some bitterness remained even after 15 hr (Fig. 2). The liberated amino acids increased with time similarly as with the soy bitter peptides. Hydrolysis of Bitter Peptides from Corn Gluten by CPase Top. When a 0.5% bitter peptides solution was incubated with crude CPase Top (enzyme/substrate ratio = 2 /ikat/g), bitterness diminished similarly in a short time, and was almost undetectable after 15 hr (Fig. 3). Liberated amino acids increased similarly as in the other bitter peptide studies.
170
BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS Table I
Relative Rates of Hydrolysis for C P a s e Top on Range of Small /V-Acyl-peptide Relative activity(%)
Peptide Z-Phe-Leu Z-Phe-Ala
p
483
p
Z-Tyr-Phe-Leu
219 p
120 114
Z-Phe-Tyr" Z-Phe-Pro
p
12
Z-Gly-Pro-Leu-Gly Z-Tyr-Phe Z-Tyr-Glu
p
309
p
201
p
188
Z-Phe-Tyr-Leu Z-Glu-Phe
p
172
p
150
Z-Glu-Tyr
(100)
Z-Gly-Val
24
Z-Gly-Phe
21
Z-Gly-Met
19
Z-Gly-Leu
17
Z-Gly-Pro-Leu-Gly-Pro
3
Z-Gly-Pro
0
B-Gly-Lys
0 p
Z-Pro-Pro P: Partially insoluble at 30*0, pH3.1 Z : CarbobenzoxyB : Benzoyl-
0
Bitter score Amount of liberated amino acids
Incubation time (hr)
Figure 1. Hydrolysis of bitter peptides from soy protein by CPase Top. Reaction conditions: 30°C, pH 4.0.
15. KAWABATA ET AL.
Elimination of Bitterness of Bitter Peptides
171 0.5
12.5 Bitter score Amount of liberated amino acids
-
0.4
o E =1
7.5 -
-
0.3
o (0 o c
5
-
0.2
10
o
u V) •m •M
°
0)
CO 0)
-
2.5
I
I
5
10
0.1
c D o E < 15
Incubation time (hr)
Figure 2. Hydrolysis of bitter peptides from casein by CPase Top. Reaction conditions:, 30°C, pH 4.0.
12.5
0.5 Bitter score Amount of liberated amino acids
10
-0.4
0.3
co 5
n
0.2
0)
.o
0.1
Z
c D O E < 5
10
15
Incubation time (hr)
Figure 3. Hydrolysis of bitter peptides from corn gluten by CPase Top. Reaction conditions: 30°C, pH 4.0 .
172
BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS
CONCLUSION It has been demonstrated that CPase Top enzyme is useful for eliminating bitterness originating from soy protein and corn gluten. With respect to the bitterness from casein, although the bitterness was not completely eliminated, it was effective in reducing most of the bitterness. It is postulated that the bitterness of peptides is due to the hydrophobic amino acids in the C-terminus position (4). This assumption is necessary for the elimination of bitterness by liberating a hydrophobic amino acid at the C-terminus by the substrate specificity of CPase Top. Because of the high effectiveness of CPase Top to eliminate bitterness, and because it is obtained from the edible parts of squids (Japanese traditonal food, Shiokara), the safety of this enzyme is assured. It is hoped that the development of this method for eliminating the bitterness of bitter peptides will be used widely in the food industry. Acknowledgements. The authors thank Professor Eiji Ichishima, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, and Professor Souichi Arai, Department of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Science, The University of Tokyo, for their most valuable advice and suggestions.
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.
Ichishima, E. Biochem. Biophys. Acta 1972, 258, 274-288. Umetsu, H.; Abe, M.; Nakai, T.; Watanabe, S.; Ichishima, E. Food Chem. 1981, 7, 125-138. Mellors, A. Arch. Biochem. Biophys. 1971, 144, 281-285. Arai, S.; Yamashita, M . ; , Kato, H.; Fujimaki, M. Agr. Biol. Chem. 1970, 34, 729-738. Lalasidis, G.; Sjöberg, L. B. J. Agric. Food Chem. 1978, 26, 742-749. Roland, J. F.; Mattis, D. L.; Kaing, S.; Aim, W. L. J. Food Sci. 1978, 43, 1491-1493. Clegg, K. M . ; McMillan, A. D. J. Food Technol. 1974, 9, 21-29. Umetsu, H.; Matsuoka, H.; Ichishima, E. J. Agric. Food Chem. 1983, 31, 50-53.
