이현규한양대학교식품영양학과 1.,,.,,. (RS, Resistant starch), RS-1, RS-2, RS-3 RS-4 4.,,,,. RS-4 (physicochemical), (physiological). % 1) (RS-4), 2) -, 3) (in vivo)
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1 이현규한양대학교식품영양학과 1.,,.,,. (RS, Resistant starch), RS-1, RS-2, RS-3 RS-4 4.,,,,. RS-4 (physicochemical), (physiological). % 1) (RS-4), 2) -, 3) (in vivo). 355
2 가교결합정도에따른옥수수전분의이화학적특성 RS-4 sodium trimetaphosphate sodium tripolyphosphate, porcine -amylase(a-3176, Type VI: From porcine pancreas), maltose, 3,5-dinitrosalicylic acid(dns) Sigma Chemical Co.(St. Louis, MO, USA). RS-4 25g 50ml 5% sodium sulfate(1.25g) sodium trimetaphsphate(stmp 99%) sodium tripolyphosphate(stpp 1%) 1, M NaOH ph M HCl ph mesh. RS %, 5% Starch 50 g + 70 ml D.W. Na 2SO 4 (10%, sb) STMP/STPP Stirring for 1 hr at 45 C ph 11.5 (1 M NaOH) Stirring for 3 hrs at 45 C Neutralization ph 6.5 (1 M HCI) Centrifuging 3,000g, 15 min (4 times) Drying at 40 C Grinding Cross-linked starch (RS4) 1 Flow diagrams of RS type 4 preparation procedures. 356
3 1 Preparation of cross-linking agent mix concentration [(STMP + STPP)/starch), w/w] 5 % 10 % 15 % 20 % STMP(99%, g) STPP(1%, g) Total(100%, g) %, 10% 23.50%, 12% 23.97%. Kaur (2006) peak viscosity. (25%, w/ w) starch pasting cell(ar1000, TA Instruments, New Castle, DE, USA) controlled-stress rheometer. RVA 0~2 50 C, 2~10 95 C, 10~12 95 C, 12~20 50 C, 20~22 50 C. (degree of cross-linking). Degree of cross-linking = (A-B)/A 100 A: the peak viscosity of the control sample (without STMP/STPP) B: the peak viscosity of the cross-linked starch Schoch 0.5g 40ml 50 C, 60 C, 70 C, 80 C, 90 C 30 shaking 3,000rpm C 4. (%) = 100 Swelling factor swelling factor Tester and Morrison(1990). 100mg 5ml 70 C ml blue dextran(5mg/ml). 3,000g nm. Swelling factor(sf). 357
4 SF = 1 + [(7,700/W)((A S - A R )/ A S )] A S : absorbance of the supernatant A R : absorbance of reference(without starch) RS-4 past clarity Reddy Seib(1999). RS g 5ml nm clarity. 2.2 가교정도별에따른옥수수전분의 in vitro 소화율변화측정 Englyst (1992) ( 2). 30mg( ) 2ml micro tube 0.75ml sodium acetate buffer(ph 5.2) 0.75ml 37 C shaking incubator ,. 3,5-dinitrosalicylic acid(dns). (%) 37 C shaking incubator 20 glucose (mg) 0.9, (%) 120 glucose Starch (30 mg. db) Sodium acetate buffer, ph 5.2 (0.75 ml) Glass beads (d=5 mm) Equilibrate for 10 min at 37 C Pancreatin/amyloglucosidase (0.75 ml) Shake at 37 C and 250 rpm Boiling for 10 min G 20 min G 120 min Centrifuge, save supematant Assay glucose RDS = G Centrifuge, save supematant Assay glucose SDS = (G G 20 ) The schematic diagram for the measurement of RDS (Rapid Digestible Starch), SDS (Slowly Digestible Starch), RS (Resistant starch) by digestion test using pancreatin and amyloglucosidase 358
5 (mg) 0.9. (%). 2.3 가교정도별에따른옥수수전분의구조적특성 X- (XRD) X-ray diffractometer(d/max-1200, Rigaku, Japan) (2 ) 5~40.. Analysis parameter: 50kV and 90mV, CuK radiation =0.154nm, nickel filter. (SEM) image specimen stub gold-paladium coating SEM(Scanning electron microscope, JSM-6330 F., JEOL, Ltd., Tokyo, Japan) 가교정도별에따른옥수수전분의항비만효과검증 (in vivo) 4 C57BL/6J 50 1 ( ). 3 20% 6, (rendomized complete block design) week 6 week 6 week Adaptation High-fat diet Experimental diet treatment 3 Overall experimental design AIN-76 semipurified diet (ND) 359
6 (HFD) 2 5% (CLCS-5) 12% (CLCS-12) 4. pellet (ad libitum) C 12.,,. (food efficiency ratio, FER). (g) (EFR) = (g) 2 Composition of experimental diets (g/kg diets) Ingredient ND Experimental groups 1) HFD CLCS-5 CLCS-12 Casein DL-methionine Corn starch Sucrose CLCS CLCS Corn oil Beef tallow Cellulose Mineral Mix S10022G Vitamin Mix V Choline bitartrate Total calories(kcal/kg) 3,592 5,542 5,542 5,542 Total(g/kg) 1,000 1,000 1,000 1,000 1) ND, Normal diet; HFD, High fat diet; CLCS, High fat diet containing with cross-linked corn starch with different degree of cross-linking. 360
7 4 dry ice. 2 3,000rpm 20. (,, ) 0.9%. -70 C. total lipid(tl), triglyceride(tg), total cholesterol(tc), high density lipoprotein cholesterol(hdl) (Olympus AU400,Tokyo, Japan). Low density lipoprotein cholesterol(ldl) Friedewald (Friedewald et al., 1986). LDL = Total cholesterol - [HDL cholesterol + ( triglyceride 5 )] Folch(1957)., 1g 10ml Chloroform Methanol mixture(2:1, v/v) 2ml (tissue homogenizer) 2,500rpm 30. chloroform dry oven, 1ml. kit(asan kit, AM202-K). 0.02ml 0.02ml ethanol 3ml vortex mixture. 37 C 5 500nm. (300mg/dl). Total cholesterol(mg/g) = Test Absorbance Calibration Absorbance 300 Triglyceride kit(asan kit, AM157S-3). 0.02ml 0.02ml ethanol 3ml 361
8 vortex mixture. 37 C 5 550nm. (300mg/dl). Triglyceride(mg/g) = Test Absorbance Calibration Absorbance SPSS(Statistical Package for Social Science) one-way ANOVA p<0.05 Duncan s multiple range test 가교결합정도에따른옥수수전분의이화학적특성, 3 5% 12% 51.3% 99.1%. Reddy Seib(1999) 30.15% (STMP/STPP) ( 3).., 95 C (Morikawa & Nishinari, 2000; Kaur et al., 2006). 3 Degree of cross-linking, paste clarity and swelling factor of native and cross-linked corn starch prepared with different level of STMP/STPP Degree of cross-linking(%) Paste clarity(% T650) Swelling factor Control a CLCS b CLCS b CLCS b All values are means of triplicate determinations standard deviation. a-b Values with different subscripts within the same column are significantly different among samples at =0.05 level by Duncan's multiple range test. 362
9 70 C R 2 (correlation coefficient) R 2 =0.878 ( 3). Cross-link,. Schoch(1964) C 90 C 45%, STMP/STPP 5, 10, 12%. (1996), Solubility (%) % 5% 10% 12% C 60 C 70 C 80 C Temperature ( C) 90 C 4 Solubility of native and cross-linked corn starch prepared with different levels of cross-linking with the mixture STMP/STPP 3.2 가교정도별에따른옥수수전분의 in vitro 소화율 RDS SDS RS 4. 4 RDS Native, 5%, 10%, 12% 58.05%, 49.05%, 33.51%, 28.63%, SDS 363
10 36.46%, 33.96%, 16.17%, 12.69%. RS 5.50%, 16.98%, 50.31%, 58.68%. RDS SDS RS.,,,. (R 2 =0.983) (R 2 =0.972) (R 2 =0.920) ( 5). 4 RDS, SDS, and RS contents derived from native and cross-linked corn starch prepared with different level of STMP/STPP Starch fraction(%) RDS SDS RS Control a a c CLCS a a b CLCS b b a CLCS b b a a-c Values with different subscripts within the same column are significantly different among samples at =0.05 level by Duncan's multiple test. RS (%) y = 1.009x y = x R 2 = R 2 = SDS (%) Swelling factor (SF) Swelling factor (SF) RDS (%) y = x R 2 = Swelling factor (SF) 5 Linear regression of swelling factor and starch fraction (RDS, SDS and RS) of crosslinked corn starch prepared with different levels of cross-linking with the mixture STMP/STPP. 364
11 3.3 가교정도별에따른옥수수전분의구조적특성 X-, X , 17.18, A, (A) (B) (C) (D) 6 X-ray diffraction patterns (XRD) of native and crosslinked corn starch prepared with different levels of crosslinking with the mixture STMP/STPP. (A) Native, (B) CLCS-5, (C) CLCS-10, (D) CLCS
12 7 Scanning electron microphotograph (SEM) of native and cross-linked corn starch prepared with different levels of cross-linking with the mixture STMP/STPP. (A) Native, (B) CLCS-5, (C) CLCS-10, (D) CLCS-12(Right: 200 ; Left: 500, bar = 10µM). 366
13 3.4 가교정도별에따른옥수수전분의항비만효과검증 (in vivo),,, 5. (ND). 6 (HFD). (HFD) (p<0.05), CLCS-5 CLCS-12., de Decker (1993), younes (1995) Type II 2.5%. 6 (ND), (HFD) (CLCS-5, CLCS-12) (p<0.05), CLCS-12. (HFD) CLCS-12, (HFD) (CLCS-5, CLCS-12) (p<0.05),.,, (p<0.05). 5 (HFD) (CLCS-5, CLCS-12) (ND) (p<0.05),... (p<0.05). (HFD) CLCS-5 CLCS-12 (p<0.05),.. 367
14 5 Body weight, food intake, tissue and fat pads weight of C57BL/6J mice fed high-fat diet after 6 weeks of treatment with experimental diets ND HFD CLCS-5 CLCS-12 Initial weight(g) b a a a Final weight(g) c a b b Weight gain(%) b a b b Food intake(g/day) a ab b ab Food efficiency(g gain/g food) c a ab b Tissue weight Liver(g) c a b b Kidney(g) b ab ab a Epididymis(g) ns ns ns ns Fat pad weight Perirenal fat(g) c a a b Epididymis(g) b a a a Initial weight: mean body weight at beginning the experimental diets. Weight gain (%) = [(Final weight (g) - initial weight (g))/initial weight (g)] 100 Energy intake (kcal) = mean food consumption (g) dietary metabolize energy (kcal) Food efficiency = mean body weight gain (g)/mean food intake (g) a-c Values with different subscripts within the same column are significantly different among samples at =0.05 level by Duncan s multiple range test.,,, HDL- ( 6)., CLCS-12 CLCS-5, HDL- LDL- CLCS-5. 6,, (p<0.05)., CLCS , CLCS (p<0.05),. 368
15 6 Lipidic parameter in serum and liver of C57BL/6J mice fed high-fat diet after 6 weeks of treatment with experimental diets ND HFD CLCS-5 CLCS-12 Serum Liver Total lipid(mg/dl) a a b b Triglyceride(mg/dl) a b b c c Total cholesterol(mg/dl) c a b ab HDL cholesterol(mg/dl) b a a a LDL cholesterol(mg/dl) c a b b Total lipid(mg/g liver) b a b b Triglyceride(mg/g liver) b a ab a Total cholesterol(mg/g liver) a a ab b a-c Values with different subscripts within the same raw are significantly different among samples at =0.05 level by Duncan s multiple range test 제목 4.2 연구개발의목적및필요성,,, RS-4. % 1) (RS-4), 2) - 3) (in vivo). RS
16 4.3 연구개발내용및범위 (RS) : DNS : (SEM) : X- :,, (RVA) (in vivo),, : 4.4 연구개발결과및활용에대한건의 [STMP/STPP, 1:99 (w/v)] 5.0~20.0%(w/v),,. 1) (0 12%) 0% 99.1%. 2) 70 C 90 C 45%, STMP/STPP 5, 10, 12%. 3) Swelling factor, 370
17 R 2 (correlation coefficient) R 2 = ) RS-4. 1) (SDS) (RS) RDS Native, 5%, 10%, 12% 58.05%, 49.05%, 33.51%, 28.63%, SDS 36.46%, 33.96%, 16.17%, 12.69%. RS 5.50%, 16.98%, 50.31%, 58.68%. RDS SDS RS. 1) X-ray 15.02, 17.18, A,.. 2),. 1), 6 (HFD), (p<0.05). (ND), (HFD) (CLCS-5, CLCS-12) (p<0.05), (HFD) CLCS
18 (HFD) (CLCS-5, CLCS-12) (p<0.05). 2) (HFD) (CLCS-5, CLCS-12) (ND) (p<0.05),. (HFD) CLCS-5 CLCS-12 (p<0.05), (CLCS-5, CLCS-12) (p<0.05). 3),,, HDL-, CLCS-12 CLCS-5, HDL- LDL- CLCS-5. 4),. (p<0.05), 목표달성도 %, %, (RS-4). [STMP/STPP, (99:1, w/w)] 5, 10, 12%(w/v) % in vitro, (in vivo) %. 372
19 ( ) (in vitro). ( ),,,. ( ) (in vivo),,,, (in vivo) 기여도 373
20 6.,,, Aparicio-Saguilan, A., Flores-Huicochea, E., Tovar, J., Garcia-Suarez, F., Gutierrez-Meraz, F., Bello-Perez, L. A. (2005). Resistant starch-rich powders prepared by autoclaving of native and linterized banana starch: Partial characterization. Starch/Stärke, 57, Bjorck, I., Gunnarsson, A., & Ostergard, K. (1989). A study of native and chemically modified potato starch. Part II: Digestibility in the rat intestinal tract. Starch/Stärke, 41, Carmona-Garcia, Roselis, Sanchez-Rivera, M. M., Méndez-Montealvo, G., Garza-Montoya, B., & Bello-Pérez, L. A. (2009). Effect of the cross-linked reagent type on some morphological, physicochemical and functional characteristics of banana starch (Musa paradisiaca). Carbohydrate pol ymers, 76, Chung, H. -J., Shin, D. -H., & Lim, S. -T. (2008). In vitro starch digestibility and estimated glycemic index of chemically modified corn starches. Food Research International, 41, Chung, H. -J., Woo, K. -S., & Lim, S. -T. (2004). Glass transition and enthalpy relaxation of cross-linked corn starches. Carbohydrate pol ymers, 55, Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, S33-S50. Folch, J., Lees, M., & Stanley, G. H. S. (1956). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, Han, J. A., & BeMiller, J. N. (2007). Preparation and physical characteristics of slowly digesting modified food starches. Carbohydrate pol ymers, 67, Hood, L. F., & Anderson, V. G. (1976). In vitro digestibility of hydroxypropyl distarch phosphate and unmodified tapioca starch. Cereal Chemistry, 53, Hoover, R., & Sosulski, F. (1986). Effect of cross-linking on functional properties of legume starches. Starch/Stärke, 374
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22 early Indica rice starch. Starch/Stärke, 58, Spence, K. E., & Jane, J. (1999). Chemical and physical properties of ginko (Ginko biloba) starch. Carbohydrate pol ymers, 67, Tester, R. F. & Morrison, W. R. (1990). Swelling and gelatinization of cereal starches I. effects of amylopectin, amylose, and lipids. Cereal Chemistry, 67, Woo, K. -S. (1999). Cross-linked, RS4 type resistant starch: preparation and properties. PhD Thesis. Kansas State: University Manhattan, KS. Woo, K. -S., & Seib, P. A. (2002). Cross-linked resistant starch: preparation and properties. Cereal Chemistry, 79, Wurzburg, O. B. (1986). Cross-linked starches. In O. B. Wurzburg (Ed.), Modified starches: properties and uses (pp ). Florida, USA: CRC press. Xie, X. J., Liu, Q., & Cui, S. W. (2006). Studies on the granular structure of resistant starches (type 4) from normal, high amylose and waxy corn starch citrates. Food Research International, 39, Zobel, H. F. (1988). Molecules to granules: A comprehensive starch review. Starch/Stärke, 40,
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