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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 42, No. 1, pp. 39~44 (2010) The Korean Society of Food Science and Technology š ww ƒ k x³* ww tw Preparation of Branched-chain Amino Acid (BCAA)-enriched Hydrolysates from Corn Gluten Yong Il Chung, In Young Bae, and Hyeon Gyu Lee* Department of Food and Nutrition, Hanyang University Abstract The process of the preparation of branched-chain amino acid (BCAA)-enriched hydrolysates from corn gluten was optimized through the parameters of pre-treatment (heating and cellulosic hydrolysis), hydrolysis method (acid, protease, and microbe plus protease), concentration, and spray drying condition. The protein yield of corn gluten was increased by heating and cellulase treatments. Among three different hydrolysis methods, the combined use of microbes and protease was the most effective in terms of free amino acid (FAA) and BCAA content of the corn gluten hydrolysates. In addition, the FAA and BCAA content in the hydrolysates prepared by microbial and enzymatic combined treatment were improved by a concentration process. Spray drying conditions for the preparation of the powder from the hydrolyzed reactant were an inlet temperature of 185 o C, outlet temperature of 80 o C, and the use of maltodextrin as an anticaking agent. Thus, this study established an economical process for preparation of value-added hydrolysates of excellent productivity and quality, in terms of high BCAA content and product stability. Key words: corn gluten, branched-chain amino acid, microbial and enzymatic combined hydrolysis, concentration process, spray drying l w l w ù(60% ), ü y ûš, lysine y w w û sƒš. w ƒw w w t w. ù ù w ƒš. p, rk (branchedchain amino acid, BCAA) w ƒ. rk tw BCAA w w w(1). w, g g wz y x g wkš š š (2,3). rk w w» w w vw. *Corresponding author: Hyeon Gyu Lee, Department of Food and Nutrition, Hanyang University, Seoul , Korea Tel: Fax: hyeonlee@hanyang.ac.kr Received June 17, 2009; revised September 30, 2009; accepted September 30, 2009 l» rk w w», z w (g )wƒ. ƒw ƒww œ, ƒš. ù, œ w jw yw 3-MCPD(3- chloro-1,2-propanediol) 2,3-DCP(2,3-dichloro-1-propanol)ƒ (4)., zw ƒw w w, ü ƒw œ ww. w z w û ƒw w BCAA w û (5). wr, w (g) w z w w. ù w vw y BCAA w w w(6)., l ƒw w œ w w zœ¾ BCAA w wk œ vw., l œ, ƒw,» w y yw mw l ƒ w œ ywš w. mw, t t y wì BCAAƒ twš w l ƒw y w. 39

2 40 w twz 42 «1y (2010) ƒw w l ()gvdg (Incheon, Korea) w. x w z Trichoderma reesei Celluclast TM (700 U/g), Bacillus licheniformis Alkalase TM (2.4 U/g), Aspergillus oryzae Flavourzyme TM (1,000 U/g) Novozymes(Begsvaerd, Denmark) t w. Bio-Rad Co.(Hercules, CA, USA) t,»k p w. l ƒw l ƒw (a) ƒw wš, ky wz w,» w l w ; (b), z (g) w ƒww ; (c)» w, k mw w ; (d)» w yw w. l l 125 o C q w 8 ³w w g.» l 10 nwš, 50 C o 100 rpm z, w z ww. l 5 ƒwš, Celluclast w TM 0.5%(w/w) ƒw 50 o C, ph w. l ƒw ƒw 35% HCl l yww z, 1/3j ƒwš, 105 C 48 o ww. ƒwƒ óù 15 k z, 35 C¾ o þƒwš, 50% NaOH w ey(ph 10 )wš, 35% HCl w y(ph 4.9 )w. zƒw l 5 ƒw z, 45 o C, ph 7 Alkalase TM Flavourzyme TM yww z w 4% ƒw 72 ww. w (g)w l 20% xk g(1 10 w 2 2% casein ww potato dextrose(pd) wš 40 C 3 w w o Aspergillus oryzae z w w., Aspergillus oryzae 30 C o 2 wš, ƒ 20%(w/w), 5%(w/w)ƒ 50 C ƒw o 45 C w o, yw z( Alkalase TM Flavourzyme ) TM w 0.5%(w/w) ƒw z, 45 C 72 o (g) w w. œ w œ mw ƒw v lv(pilot filter press,, Incheon, Korea) w 2 wš, 2 (Rotary vacuum evaporator system N-11, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) w z, vlv w 1 w šx wš,»»n mw k(pilot electrodialysis acilyzer 02, Astom Co., Tokyo, Japan)w.»(Pilot» KL-8,, Chungnam, Korea) w yw, t t ù w yš, rw,. w» atomizer rpm w 18,000 rpm šj š, inlet o C, outlet o C y mw. œ p w 105 o C ƒ, Kjeldahl, Soxhlet s, z 550 o C zy w w. (Model NS-3P, Merbabu Trading Co., Ltd., Osaka, Japan) w dw. wš 0.5 g ƒw q w 50 ml 350 ml AccQ-Tag 100 ml yww z, 55 o C 10 yw. y 10 ml AccQ-Tag C 18 f( mm, Millipore Co., Milford, MA, USA) HPLC l (Waters 1525 Binary Pump, Waters 717 plus Autosampler, Waters 474 Fluorescence detector, Waters Co., Milford, MA, USA) w M p ùp 10% p p 1% ph 5.02 (eluent A) mp 4:6 yww (eluent B) x 1.0 ml/min w. s wš Bio-sil SEC-125 f( mm, Bio-Rad Co., Hercules, CA, USA) HPLC l (Waters 510 HPLC pump, Waters 486 Tunable absorbance detector, Waters Co., Milford, MA, USA) w M NaCl w 0.05 M ùp (ph 6.8) w 1.0 ml/min w. t p (670 kda), (158 kda), ù(44 kda), (17 kda), k B12 (1.35 kda) t(bio-rad Co., Hercules, CA, USA) w. ƒw Su (7) s(fn) (TN) ƒƒ Kjeldahl s w d w. š w y s ƒš, ƒœ w ky ky wz w w z ƒ y z š (8). l 60%(w/w) w š,, x, 20-25%(w/w) w. l z w wš zw l ƒw jš, w z Celluclast TM ww(table 1). l z wz w 63.66% 70.92% 11% ƒw. l w w k y wz ƒƒ 150%¾ z ƒ g Chae (9,10) û. w w w, w z

3 ƒ 41 Table 1. Effect of pre-treatment on protein content of corn gluten Pre-treatment Total nitrogen Total protein Corn gluten Heat treated corn gluten Heat and cellulase treated corn gluten x»w ƒ. l ƒw BCAA wy Table 2 cellulase, z z (g) w ƒw w ƒw BCAA w w. z w ƒw w, 20%, BCAA 20-24%, w BCAA 0-3% ƒw v yw. w, ƒw ƒw z (g )-z w ƒw BCAA w ƒƒ 61%, 31% 31%, 4%¾ ùkû. ù, w BCAA z ƒw (g)-z w ƒw ƒƒ 42% 26%¾ w. ù BCAA w wš, ƒw (g)» p z w ƒ ƒ yw., (g)-z w w z ƒš, BCAAƒ tw l ƒw yw. - œ ƒw BCAA wy m (g) z ƒw w œ mw l ƒw BCAA w w Table 3., ƒw wš, 24 w z vlv w w w (11.48% 14.40%) BCAA(2.56% 5.30%) w ƒƒ 25% 100% ƒw. p, BCAA leucine w 5.14% 15.97% 3 w.» w x y ƒw y t t y(, ) w, rwš,.» inlet outlet ƒ ûù, atomizer rpm ww Table 3. Effect of concentration process on free amino acid (FAA) and branched-chain amino acid (BCAA) contents of the corn gluten hydrolysates produced by microbial and enzymatic combined treatment Hydrolysates With concentration Without concentration FAA BCAA ASP THR SER GLU PRO GLY ALA CYS MET VAL ILE LEU TYR PHE HIS LYS ARG Total ƒ caking»ù š w k., inlet outlet ƒ k ƒ w(11)., ƒw y w» yw t t w wùƒ. Table 4, inlet outlet ƒ w w wš, ƒw. w, y xkw inlet outlet ƒ ƒƒ 185 o C 80 o C w w š, š, ƒ š w j» x ƒ ww p ùký yw(fig. 1)., y w ƒ w ù w w caking w w(12). ù t ww š ƒ» lactose, dextrin, maltodextrin, gum arabic w,,, w (Table 5). w 4 š, maltodextrin dextrin lactose gum arabic,, Table 2. Changes of free amino acid (FAA) and branched-chain amino acid (BCAA) contents of corn gluten hydrolysates produced by various proteolytic methods Concentration With cellulase treatment Without cellulase treatment Acid Protease koji+protease Acid Protease koji+protease FAA BCAA BCAA/FAA

42 w twz 42 «1y (2010) Table 4. Establishment of spray drying condition of the corn gluten hydrolysates by microbial and enzymatic combined treatment Inlet temp. ( o C) 180 185 190 Outlet temp.

4 42 w twz 42 «1y (2010) Table 4. Establishment of spray drying condition of the corn gluten hydrolysates by microbial and enzymatic combined treatment Inlet temp. ( o C) Outlet temp. ( o C) Moisture content Characterization of powder Color (OD at 500 nm) Appearance Wet Wet Wet Bright but wet Bright and dry Dark Dark Dark Dark, ƒ caking z. w y 2% maltodextrin š wš, inlet 185 o C, outlet 80 o C, 18,000 rpm ƒ k t. l w œ y l ƒ w», w wj 50% w. w, zœ w w w j ƒw w. p, z w œ z ƒw w ƒ š, z w w 55% w (13,14). ù, w (g ) z w w l ƒww 132%¾ w yw(table 6). w, ƒw z z w ( zœ ƒ z w 4%) w 0.5% w., w l ƒw œ z, w. œ p, l ky wz w zw wœ BCAA w» w w» w y mw rk p w, 5.0%, 62.5%, ky 26.5%, 0.0%, z 6.0% ùkû. Table 7 Fig. 1. Microscopic characterization of spray-drying powder obtained from the corn gluten hydrolysates by microbial and enzymatic combined treatment.

5 Table 5. Effect of anticaking agents on moisture absorption of spray-drying powder of the corn gluten hydrolysates by microbial and enzymatic combined treatment Anticaking agents Granularity 1) Color 2) Flowability 3) Hygroscopicity 4) Lactose Dextrin Maltodextrin Gum arabic ) Granularity: ++ coarse, +++ fine, ++++ very fine 2) Color: + dark, ++ normal, +++ bright 3) Flowability: ++ bad, +++ good, ++++ very good 4) Hygroscopicity: ++ high, +++ normal, ++++ low Table 6. Comparison of protein utilization (product yield) among corn gluten hydrolysates prepared by various hydrolysis process Process Total nitrogen Product yield ü BCAA w 40.7% š, valine 11.5%, isoleucine 14.8%, leucine 14.4% w. w, s 300 Da w 7.25%, Da 84.75%, 700-1,000 Da 8.0% wš (data not shown). ƒ 43 Dosage of enzyme (%, basis of protein) Microbe Protease Microbe+Protease Table 7. Amino acid composition of spray-dried powder of the corn gluten hydrolysates by microbial and enzymatic combined treatment Amino acid Total amino acid Free amino acid BCAA ASP THR SER GLU PRO GLY ALA CYS MET VAL ILE LEU TYR PHE HIS LYS ARG Total , ƒw, xy œ yw BCAA w ƒ rk yw. l z ky wz w 11% ƒw. ƒw w w g z yww k ƒw w BCAA w. w, ƒ w mw BCAA w 100% w. w ƒw y w» š w, inlet 185 o C, outlet 80 o C, 18,000 rpm 2% maltodextrin ƒ k t. ƒw y œ mw 32%¾ wš, BCAA w 41% l ƒw w., l ƒw w y mw BCAA ƒ tw ƒw t t y yw. w, w (g) z w w l ƒw w z w BCAA w ùkü ƒw., w l ƒw œ z, w. 1. Wagenmakers AJM. Muscle amino acid metabolism at rest and during exercise: Role in human physiology and metabolism. Exerc. Sport Sci. Rev. 26: (1998) 2. Yamaguchi M, Nozaki O, Ito M, Furukawa Y. Effect of corn peptide administration on plasma amino acid concentration and alcohol metabolism in stroke-prone spontaneously hypertensive rats. J. Clin. Biochem. Nutr. 22: (1997) 3. Yamaguchi M, Nishikiori F, Ito M, Furukawa Y. The Effect of corn peptide ingestion on facilitating alcohol metabolism in healthy men. Biosci. Biotech. Bioch. 61: (1997) 4. Hamlet CG, Jayaratne SM, Matthews W. 3-Monochloropropanel,2-diol (3-MCPD) in food ingredients from UK food products and ingredients suppliers. Food Addit. Contam. 19: (2002) 5. Kubota K. Production of protein seasoning solution. Japan patent (1994) 6. Poonam N, Dalel S. Solid-state (substrate) fermentation systems and their applications in biotechnology. J. Basic Microb. 34: (1994) 7. Su NW, Wang ML, Kwok KF, Lee MH. Effect of temperature and sodium chloride concentration on the activities of proteases and amylases in soy sauce koji. J. Agr. Food Chem. 53: (2005) 8. Kim JK, Park HK, Chung KS, Kim HS, Son HS, Chung JW. Process for preparing soymilk from soymilk residues or tofu residues. Korean Patent Publication No (1994) 9. Chae HJ, In MJ, Lee JD. Production of a protein supplement from soymilk residues by combined use of enzymes and microorganisms. Agric. Chem. Biotechnol. 41: (1998) 10. Chae HJ, Han MS, In MJ. Study on utilization of vegetable byproduct from food processing by enzyme treatment. J. Korean Soc. Appl. Biol. Chem. 47: (2004) x

6 44 w twz 42 «1y (2010) 11. Knut F, Martina K. Influence of spray drying conditions on functionality of dried whole egg. J. Sci. Food Agr. 82: (2002) 12. Jose MA, Guy L, Marcus K. Effect of water content on the glass transition and caking of fish protein hydrolyzates. Biotechnol. Progr. 9: (1993) 13. Apar DK, Özbek B. A kinetic study on corn gluten hydrolysis. Chem. Eng. Technol. 32: (2009) 14. Apar DK, Özbek B. Hydrolysis and solubilization of corn gluten by Neutrase. J. Chem. Technol. Biot. 82: (2007)

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14.531~539(08-037).fm G Journal of the Korea Concrete Institute Vol. 20, No. 4, pp. 531~539, August, 2008 š x y w m š gj p { sƒ z 1) * 1) w w Evaluation of Flexural Strength for Normal and High Strength Concrete with Hooked