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. 2.1 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) 30 45 sodium trimetaphsphate(stmp 99%) sodium tripolyphosphate(stpp 1%) 1, 1. 20 45 1M NaOH ph 11.5 3 45 1M HCl ph 6.0. 4 40. 100 mesh. RS-4 23.09%, 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
1 Preparation of cross-linking agent mix concentration [(STMP + STPP)/starch), w/w] 5 % 10 % 15 % 20 % STMP(99%, g) 2.475 4.95 5.94 7.425 STPP(1%, g) 0.025 0.05 0.06 0.075 Total(100%, g) 2.5 5 6 7.5 23.35%, 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 30. 105 C 4. (%) = 100 Swelling factor swelling factor Tester and Morrison(1990). 100mg 5ml 70 C 30. 0.5ml blue dextran(5mg/ml). 3,000g 10 620nm. Swelling factor(sf). 357
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-4 0.05g 5ml 95 30 5. 650nm 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. 20 120 10,. 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 20 0.9 Centrifuge, save supematant Assay glucose SDS = (G 120 - G 20 ) 0.9 2 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
(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) 700. 2.4 가교정도별에따른옥수수전분의항비만효과검증 (in vivo) 4 C57BL/6J 50 1 ( ). 3 20% 6, (rendomized complete block design) 10 8 13. 1 week 6 week 6 week Adaptation High-fat diet Experimental diet treatment 3 Overall experimental design AIN-76 semipurified diet (ND) 359
(HFD) 2 5% (CLCS-5) 12% (CLCS-12) 4. pellet (ad libitum). 24 2 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 200 200 200 200 DL-methionine 3 3 3 3 Corn starch 650 150 - - Sucrose 0 150 150 150 CLCS-5 - - 150 - CLCS1-2 - - - 150 Corn oil 50 - - - Beef tallow - 400 400 400 Cellulose 50 50 50 50 Mineral Mix S10022G 35 35 35 35 Vitamin Mix V10037 10 10 10 10 Choline bitartrate 2 2 2 2 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
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
vortex mixture. 37 C 5 550nm. (300mg/dl). Triglyceride(mg/g) = Test Absorbance Calibration Absorbance 300 3 SPSS(Statistical Package for Social Science) one-way ANOVA p<0.05 Duncan s multiple range test. 3. 3.1 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 0 30.15 1.34 a 38.92 CLCS-5 51.3 1.01 0.20 b 33.51 CLCS-10 98.1 0.86 0.13 b 20.25 CLCS-12 99.1 0.86 0.13 b 13.17 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
70 C R 2 (correlation coefficient) R 2 =0.878 ( 3). Cross-link,. Schoch(1964) 4. 50 C 90 C 45%, STMP/STPP 5, 10, 12%. (1996),. 50 40 Solubility (%) 30 20 0% 5% 10% 12% 10 0 50 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
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 58.05 3.6 a 36.46 7.07 a 5.50 4.55 c CLCS-5 49.05 5.46 a 33.96 2.37 a 16.98 3.10 b CLCS-10 33.51 8.03 b 16.17 4.77 b 50.31 4.86 a CLCS-12 28.63 8.2 b 12.69 2.37 b 58.68 6.87 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 (%) 70 50 y = 1.009x - 1.904 60 y = -1.856x + 76.99 40 R 2 = 0.972 50 R 2 = 0.920 40 30 30 20 20 10 0 10 0 10 20 30 40 10 20 30 40 SDS (%) Swelling factor (SF) Swelling factor (SF) RDS (%) 70 60 50 40 30 20 10 y = -1.142x + 12.07 R 2 = 0.983 0 10 20 30 40 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
3.3 가교정도별에따른옥수수전분의구조적특성 X-, X- 6. 15.02, 17.18, 22.73 A,.. 17.18 15.02 22.73 (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... 365
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
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
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) 24.00 0.69 b 32.03 1.73 a 30.00 1.94 a 29.50 3.78 a Final weight(g) 26.52 0.41 c 38.64 1.79 a 34.08 1.02 b 33.61 1.99 b Weight gain(%) 7.11 2.82 b 20.67 0.93 a 7.42 3.11 b 9.47 1.96 b Food intake(g/day) 109.10 7.49 a 94.40 0.28 ab 78.90 1.27 b 98.70 21.35 ab Food efficiency(g gain/g food) 0.016 0.001 c 0.094 0.000 a 0.084 0.001 ab 0.071 0.015 b Tissue weight Liver(g) 0.97 0.04 c 1.58 0.27 a 1.24 0.18 b 1.32 0.34 b Kidney(g) 0.32 0.03 b 0.34 0.02 ab 0.34 0.02 ab 0.34 0.03 a Epididymis(g) 0.24 0.09 ns 0.21 0.01 ns 0.19 0.06 ns 0.20 0.04 ns Fat pad weight Perirenal fat(g) 0.11 0.06 c 0.29 0.08 a 0.26 0.08 a 0.19 0.07 b Epididymis(g) 0.48 0.12 b 2.06 0.35 a 1.82 0.40 a 1.71 0.53 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-5 79.49 19.72, CLCS-12 60.50 8.8. (p<0.05),. 368
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) 333.01 12.78 a 337.81 8.19 a 303.50 22.20 b 294.45 17.65 b Triglyceride(mg/dl) 149.23 9.66 a 98.89 4.63 b 86.76 11.44b c 80.13 8.20 c Total cholesterol(mg/dl) 121.94 5.53 c 155.38 4.43 a 142.56 10.06 b 146.55 9.26 ab HDL cholesterol(mg/dl) 76.30 4.21 b 97.20 2.25 a 89.50 4.28 a 91.90 5.88 a LDL cholesterol(mg/dl) 45.64 1.64 c 58.18 2.25 a 53.06 6.12 b 54.65 4.63 b Total lipid(mg/g liver) 114.13 7.29 b 241.50 15.65 a 79.47 19.72 b 80.60 8.81 b Triglyceride(mg/g liver) 19.22 2.44 b 28.14 2.37 a 32.28 3.41 ab 30.51 3.54 a Total cholesterol(mg/g liver) 1.16 0.36 a 1.07 0.43 a 1.08 0.41 ab 0.73 0.31 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. 4. 4.1 제목 4.2 연구개발의목적및필요성,,, RS-4. % 1) (RS-4), 2) - 3) (in vivo). RS-4 - -. 369
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
R 2 (correlation coefficient) R 2 =0.95. 4) 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, 22.73 A,.. 2),. 1), 6 (HFD), (p<0.05). (ND), (HFD) (CLCS-5, CLCS-12) (p<0.05), (HFD) CLCS-5. 371
(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),. 5. 5.1 목표달성도 %, %, (RS-4). [STMP/STPP, (99:1, w/w)] 5, 10, 12%(w/v) % in vitro, (in vivo) %. 372
( ) (in vitro). ( ),,,. ( ) (in vivo),,,, (in vivo).. 5.2 기여도 373
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