보안과제 ( ), 일반과제 ( o ) 107069-03
. 2010 5
.. 1.,.,,. in-vitro in-vivo gel probiotics. (Dioscorea batatas) ( ) ( ) ( ), 600. 80%,,,,.,,,,,,
( ),.,..,,,,,.,. glucomannan, mucin, choline, arginine,.... 4311 ton. 70%. 10 a (1,000 m 2 ) 370, 24 920, 1. - 3 -
, 50 33 6000,. 50... 2.., Disocorea batatas.,.,,,.,,,,,,,.,,, glucomannan,,..,,,,, formulation - 4 -
..,., Nigeria stew.,,,.,.. 1. 1) (1) (2) 2) (1) (2) 3) (1) (2) - 5 -
(3) (4) 2. 1) (1) (2) 2) (1) (2) 3) (1) (2) (3) (4) 3. (In vivo test) 1) - 2) - 3) - - 6 -
. 1.,,. 1% 60, 90 in-vitro, 8 newtrase protamex., protamex. Hexane, chloroform, ethylacetate, butanol ethanol, HMG-CoA reductase inhibition bile acid binding capacity ethylacetate ethanol, ethanol. Ethanol, 30,000 dalton HMG-CoA reductase inhibition bile acid binding capacity, gel filtration 29,000 dalton 16,000 dalton. 2. 33 in-vitro, Lactobacillus acidophilus, Enterococcus flavescens, Enterococcus faecalis var. liquefaciens, Lactococcus lactis, Lactobacillus casei 5. Lactobacillus acidophilus., ethanol butanol, ethanol. Ethanol, 30,000~50,000 dalton gel chromatograpy 66,000 dalton. - 7 -
3...,,.. - 8 -
. 1. : 1. Myung-Ki Lee, et al. Inhibition of Browning and Preference Improvements of Dioscorea batatas through the Addition of Sugar Alcohols and Organic Acids. J Food Sci Nutr. 14: 220-2005 (2009) 2. : 8 2008 2009 2010 3. : 9. 4 :. 2008... 5 : Mouse. 2008... 5 : Mouse. 2008.. - 9 -
. 4 :. 2009... 3 : Lactobacillus acidophilus. 2009... 6 :. 2010... 5 :. 2010... 5 :. 2010... 5 :. 2010.. 4. : 1 ( ) - : - : - 10 -
SUMMARY. Title of Research Development of products using functional components and hematocele improvement effect from Dioscorea batatas. Objectives and Significance of Research 1. Objectives and significance of research For the aims to broaden the utilization of Dioscorea batatas, this study developed enzyme hydrolysate and fermented product manufacturing methods, and performed separation and purification of elements that potentially have functional effects on improvement blood stream. Furthermore, we developed diversity processed food with these functional elements to increase the Dioscorea batatas consumption. Products development was performed with raw materials that having functionality evidenced by in-vitro and in-vivo experiments. As a long-term market circulation product which can preserved for a long period, Dioscorea batatas chocolate was developed, and as a short-term market circulation products which can be easily prepared for substitute meal and have a probiotic benefits, beverage, pottage, and sweet jelly were developed. These applications on processed food promote consumption and commercialization of Dioscorea batatas. Dioscorea batatas, belongs to Dioscoreaceae family, is grown in mountain field and grassland which is hermaphrodites, has both male and female reproductive organs, proliferate themselves and occurring various heritable variations. Approximately, 600 species of Dioscoreaceae family have been reported. More than 80% of Dioscorea batatas domestically produced in Gyeongsang province, and therefore advanced studies of unique quality properties, preference, uniformity, stable preservation of Dioscorea batatas are required for promoting as a new indigenous crop. Dioscorea batatas is used in Asia as the herbal medicine, which is utilized after
processing such as uncooked or steamed-and-dried its periderm after peeling. There have been several reports of beneficial effects on anti-oxidative and glucose regulating, anti-cancer, obesity, immune-regulation, intestinal function, and lipid metabolism. Sinnongbonchokyung, Chinese medical book, says pharmacological effect of old Dioscorea batatas is placed after wild ginseng. Therefore, it seems to be a worthwhile subject to investigate biological functionality and nutritional superiority of Dioscorea batatas and develop high value food material as well as processed foods, in terms of academical, technical, and economical aspects. A. Technical aspects Dioscorea batatas is eaten raw, steamed and cooked, or grated Dioscorea batatas served with the york, fried and coated with syrup. Lately, its starch used as a energy source and healthy food in the US. Thus, it has become important to investigate particular active constituent for improving blood flow and develop a new food as a high valuable material. Variety elements such as glucomannan, one of mucous polysaccharides, mucin, choline, arginine in Dioscorea batatas have been reported have various bioactive benefits include lowering blood cholesterol. However, few studies have been conducted to support these functional superiorities scientifically. Therefore, there are a lot of studies required to establish technology for increasing effective use of Dioscorea batatas. B. Economic and industrial aspects Dioscorea batatas grows over the central-south region, and some species even grows in north area in Korea. Almost 4,311 tons of Dioscorea batatas produced in last year(2008), and half of them are from Andong in Gyeongbuk province of Korea. In terms of producing area, it is over 70% of total area in Korea. Farmers make 3.7 million won per 10 a (1,000 m 2 ) on average in last year, currently more than 920 farm households growing Dioscorea and sell after cleaning and cutting process. Beneficial effects of Dioscorea batatas became known to the world, more than 50 kinds of processed products such as powder, tea, and soup, are exported to the US and - 12 -
Southeast Asia, which worth of more than 336,000 dollars, through Dioscorea Processing Plant in Andong Nonghyup. As well as, record 5 billion won in domestic sales rewards for their effort of product development and improvement of species of Dioscorea as a regional specialty. As above, economical value of Dioscorea is drastically rising that could revive local economy and increase export by effective utilization. 2. Current domestic and international technology A. Preliminary research a. Domestic preliminary research Dioscorea batatas is a perennial herbaceous plant belonging to Dioscoreaceae family grown over the country mountain. Most studies on Dioscorea batatas are performed with variety cultivated in fields, and few studies on functional aspects found in the literature. Some of main components contained in Dioscorea batatas include glucomannan, mucin, arginine, and choline, are known to effective for diabetes, pulmonary tuberculosis, disinfection, antidote, rheumatism. Scientific research, however, has not been sufficiently investigated. Thus, we attempt to purify the active components and establish the functional effects of Dioscorea batatas. We expecting effective extraction of bioactive compounds based on advanced information on how to separate functional component from ginseng, ballon flower, and lanceolata. b. International preliminary research In Japanese cuisine, Dioscorea is eaten raw and grated, after only a relatively minimal preparation. The raw vegetable is starchy and bland, mucilaginous when grated, and may be eaten plain as a side dish, added to noodle, or using for confectionary source. In China, fried Dioscorea eaten coated with sweet syrup. Dioscorea batatas is a main carbohydrate source for West African, particularly in Nigeria, it is a gourmet food served as hot stew. Recently in America, Dioscorea starch - 13 -
is a good source of energy which is easily digest, and use as an ingredient in bread, biscuit, and soup, especially for children and the infirm. As described, Dioscorea batatas is consumed as a simply process food, without a consideration of the functionality on blood flow improvement. So, more efforts needed to develop products that can maximize the health benefits of Dioscorea batatas.. Scope and Contents of Research 1. Blood stream improvement effects of isolated functional material using hydrolysis enzymes from Dioscorea batatas. 2. Blood stream improvement effects of isolated functional material using lactic acid bacteria from Dioscorea batatas. 3. Development of processed foods using Dioscorea batatas and their functional materials. IV. Conclusion and recommendations 1. Blood stream improvement effects of isolated functional material using hydrolysis enzymes from Dioscorea batatas. To develop functional material from Dioscorea batatas, observed optimal concentration, temperature and time using 8 types of carbohydrate and protein hydrolysis enzyme. As a result, the optimal condition difference for 8 type of each enzyme, but mainly in the 1% of concentration, 60 and 90 min of enzyme reaction condition, especially protein enzymatic hydrolysates (newtrase, protamex) showed the most blood stream improvement effects. The result of animal experiments using enzymatic hydrolysates, protamex hydrolyzate had the highest blood cholesterol-lowering effect. So we conducted solvent separation using protamex hydrolyzate. Hexane, chloroform, ethylacetate, butanol and ethanol solvent fractionation carried out, and HMG-CoA reductase inhibition and bile acid binding capacity were measured. The ethylacetate fraction and ethanol fraction were higher their effects, through the animal experiments, - 14 -
ethanol fraction was finally selected. The ultrafiltration of ethanol fraction, 30,000 dalton in size that HMG-CoA reductase inhibition and bile acid binding capacity were superior to, gel filtration through confirm the final result of molecular size less than 29,000 dalton and 16,000 dalton, respectively. 2. Blood stream improvement effects of isolated functional material using lactic acid bacteria from Dioscorea batatas. Fermented Dioscorea batatas used by 33 species of lactic acid bacteria and observed improvement of in-vitro blood flow effects, fermentation materials of Lactobacillus acidophilus, Enterococcus flavescens, Enterococcus faecalis var. liquefaciens, Lactococcus lactis, Lactobacillus casei had a good activity of HMG-CoA reductase inhibition and bile acid binding capacity, respectively. And animal experiment, the effect of cholesterol control in the Lactobacillus acidophilus fermented material was excellent, therefore we selected. For Separation of the active components from fermented material, we enforced solvent fractionation. And ethanol and butanol fractions were the best improvement in the blood flow effect and animal experiments were selected for the final fraction of ethanol fraction. Ethanol fraction was separated by ultrafiltration method, 30,000~50,000 dalton was excellent for the improvement in the blood flow effect. The final step, separated by gel chromatograpy, and substance were found to be less than 66,000 dalton. 3. Development of processed foods using Dioscorea batatas and their functional materials. For increase consumption promotion and expandation of processed food on Dioscorea batatas, we development various kinds of products. First, as a long-term storage capabilities in a convenient product, chocholate (using protamex hydrolysate) and sweet jelly (using lactic acid bacteria fermented material) prepared. The raw material of chocolate and jelly, can used not only functional components but the raw, powder and steamed Dioscorea batatas for take advantage of a wide variety of manufacturing methods and materials that were standardized basis. Second, for spread as a convenient - 15 -
meals, prepared by fermented beverage and portage. Almost products had a good activity of control blood flow effect in the animal experiments. The serum cholesterol level showed similar with normal fat diet group. Although these products evaluated a good sensory properties. - 16 -
CONTENTS Chapter 1. Introduction 32 Chapter 2. Materials and Methods 35 1. Development powder processing of Dioscorea batatas 35 A. Materials 35 B. General ingredients analysis of Dioscorea batatas 35 C. Change of ingredients in Dioscorea batatas by dry procedure 37 D. Effect of additives for antibrowning and improvement of sensory properties 38 2. Determination optimal condition of functional components isolation from Dioscorea batatas hydrolysate 40 A. Manufacturing of Dioscorea batatas hydrolysate 40 B. Optimization for enzyme hydrolysis of Dioscorea batatas 40 3. Determination optimal condition of functional components isolation of fermented Dioscorea batatas 43 A. Manufa cturing of fermented Dioscorea batatas 43 B. Preperation and optimization of fermented Dioscorea batatas 43 4. Functional properties of enzyme hydrolysate and fermented product in selected optimum conditions 45 A. DPPH radical scavenging effect 45 B. Fibrinolytic effect 45 C. HMG-CoA reductase inhibition activity 46 D. Bile acid binding capacity 46 5. Purification of functional components and stabilization on blood stream improvement - 17 -
activity 49 A. Solvent fractionation of hydrolysate Dioscorea batatas 49 B. Solvent fractionation of fermented Dioscorea batatas 50 C. Ultrafiltration of solvent fraction 50 D. Gel chromatography 52 E. In-vitro investigation of blood stream improvement activity 52 F. Quantitative analysis of charbohydrates from components 52 G. Quantitative analysis of protein from components 53 H. Quantitative analysis of lipid from components 53 I. Molecular weight observation of components 54 K. Stabilization of functional fractions 6. Development of processed products using Dioscorea batatas 55 A. Manufacture of Dioscorea batatas wine 55 B. Development of long term storage products 57 1) Manufacture of Dioscorea batatas chocolate 57 2) Manufacture of Dioscorea batatas sweet jelly 62 C. Development of short term storage products 70 1) Manufacture of Dioscorea batatas fermented beverage 70 2) Manufacture of Dioscorea batatas pottage 76 D. Quality characteristics of products 81 E. Storage stability of processed products F. Research for application of developed functional food 83 7. In-vivo experimentation of blood stream improvement activity 84 A. In-vivo experimentation of hydrolysate and fermented Dioscorea batatas 84 B. In-vivo experimentation of functional fraction from Dioscorea batatas 85 C. In-vivo experimentation of processed products 86 D. Determination of blood stream improvement activity 87-18 -
Chapter 3. Results and Discussion 93 1. Development powder processing of Dioscorea batatas 93 A. General ingredients analysis of Dioscorea batatas 93 B. Change of ingredients in Dioscorea batatas by dry procedure 94 C. Effect of additives for antibrowning and improvement of sensory properties 96 2. Determination optimal condition of functional components isolation from Dioscorea batatas hydrolysate 100 A. Effect of temperature and type on hydrolytic enzyme 100 B. Effect of concentration and type on hydrolytic enzyme 103 C. Effect of time on hydrolytic enzyme 107 3. Determination optimal condition of functional components isolation of fermented Dioscorea batatas 111 A. ph, titratable acidity, total bacteria and lactic acid bacteria of fermented Dioscorea batatas 111 B. Sugar, glucose and reducing sugar content of fermented Dioscorea batatas 112 4. Functional properties of enzyme hydrolysate and fermented product in selected optimum conditions 121 A. Fibrinolytic effect and HMG-CoA reductase inhibition effect of hydrolysate 121 B. Bile acid binding capacity of hydrolysate 123 C. Fibrinolytic effect of fermented Dioscorea batatas 125 D. HMG-CoA reductase inhibition effect of fermented Dioscorea batatas 126 E. Bile acid binding capacity of fermented Dioscorea batatas 127 5. Purification of functional components and stabilization on blood stream improvement activity 131 A. Blood stream improvement activity of hydrolysate 131 B. Blood stream improvement activity of solvent fraction from hydrolysate 133-19 -
C. Blood stream improvement activity of fermented Dioscorea batatas 139 D. Blood stream improvement activity of solvent fraction from Dioscorea batatas 141 E. Blood stream improvement activity of ultrafiltration components 145 F. Quantitative analysis and blood stream improvement activity of gel chromatography components 145 G. Stabilization of functional fractions 6. Development of processed products using Dioscorea batatas 176 A. Physicochemical properties of Dioscorea batatas wine addition with rice 176 B. Physicochemical properties of Dioscorea batatas wine addition with oriental herbs 179 C. Quality properties of chocolate 181 D. Quality properties of sweet jelly 184 E. Quality properties of fermented beverage 190 F. Quality properties of pottage 197 G. Storage stability of processed products H. Research for application of developed functional food 203 7. In-vivo experimentation of blood stream improvement activity 208 A. In-vivo experimentation of hydrolysate and fermented Dioscorea batatas 208 B. In-vivo experimentation of functional fraction from Dioscorea batatas 216 C. In-vivo experimentation of processed products 223 Chapter 4. Conclusion 238 Chapter 5. References 240-20 -
SUMMARY CONTENTS 1 2 1..... 2... 3... 4.. DPPH. (Fibrinolytic activity). HMG-CoA (3-hydroxy-methylglutaryl coenzyme A) reductase. (Bile acid binding capacity) - 21 -
5.,.... gel chromatography.. Gel chromatography. Gel chromatography. Gel chromatography.. 6... 1) 2). 1) 2)... 7. in-vivo.... - 22 -
3 1.... 2.... 3.. ph,,., 4.. HMG-CoA reductase. (Bile acid binding capacity).. HMG-CoA reductase. 5....... Gel chromatography. - 23 -
6........ 7. in-vivo... 4 5-24 -
1 Dioscorea batatas Dioscorea japonica, mannan, steroid saponin phenanthrene.,,,,,,,,,,,,,.,,.,., Nigeria stew.,,,.,.,.,,,.,,,,,,,.,,, - 25 -
glucomannan,,.,,..,,.,,,, ㆍ,, formulation.. - 26 -
2 1..,,. 2007, 1~2mm. 1.. 1) AOAC 105., 2 g 105 dry oven 3 5 30. 1 2. 2) (, 2005). 2 g 550 600., 200. 3) AOAC. (N) 2 3 mg 0.5 g - 27 -
10 ml. 1 2... 4). 2 10 g, 100 10 5 2 3.. 8, 98~100 1.. 5) 100,,,. 6) phenol-sulfuric acid method. Sample 1mL 10% phenol solution 1mL 10 H 2 SO 4 5mL 30. 470nm. Glucose standard curve (%). 7) Sample 1mL 1mL 10 Boiling 1mL 520nm. Glucose stnadard curve (%). 8) (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a(, Redness) - 28 -
b(, Yellowness). L=97.57, a=0.00, b=1.79.. 50, 55, 60 12 2 200 mesh... 1). erythritol, maltitol, sorbitol, xylitol(mitsubishi, Japan), mannitol(dae-jung, Korea), citric acid(sigma, USA), fumaric acid malic acid(dae-jung, Korea), tartaric acid(kanto, Japan), succinic acid(hayashi, Japan). 2~3 mm erythritol, maltitol, mannitol, sorbitol xylitol 10%, citric acid, fumaric acid, malic acid, succinic acid tartaric acid 0.2% 60 (HK-DO1000F, Korean Eng. Corp, Korea) 16, (18%)., (HMF-985, Hanil, Korea) 1 200 mesh. 2), (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a (, Redness) b(, Yellowness). L=97.57, a=0.00, b=1.79. - 29 -
. Schoch Kim., (%) 0.5 g 40 ml 30 30 120 rpm 4 20 2,000 g 30,., phenol-sulfuric acid, (g/g). Water solubility = Weight of total glucose in supernatant sample (g) Sample weight (g) 100 Swelling power = Weight of swollen sample (g) Sample weight (g) (100-%water solubility) 100 3) 30.. (brightness),,,. likert 9. 4) 3, statistical Analysis System(SAS) Duncan's multiple range test. 2.. 100 g 1.5 10 homogenizer. - 30 -
100 ml shaking water bath (40, 50, 60, 70 ). %(v/w). 100 15 6,000 g 30.. 1). 100 ml 1%(w/w) 40, 50, 60, 70 3.,.,,,. 2) Table 1 1%(v/v), shaking water bath 3.,,,. 3) 0.05, 0.1, 0.5, 1.0, 2.0% (w/w).,,,. 4).,,,. - 31 -
Table 1. Temp ( ) Optimal ph Alcalase 55~70 6.5~8.5 Bacillus licheniformis,,, Neutrase 45~55 5.5~7.5 Bacillus amyloliquefaciens Flavourzyme 50 5.0~7.0 Protamex 35~60 5.5~7.5 (N- ) Aspergillus oryzae Bacillus amyloliquefaciens, Bacillus licheniformis,,, 3.. 33 (Table 2) 33 Enterococcus 5, Lactobacillus 14, Lactococcus 2, Leuconostoc 6, Pediococcus 2, Weissella 3 KCTC MRS (Difco, USA) 30 2. 200 mesh 1.5 1. 4.5, 2%(v/v) 37 8, 1.. 37, 6,000 g, 10 30 0.45 um syringe filter 1.8 ml eppepdorf tube -20. 1 ph,,,,,,. - 32 -
Table 2. No Strain Scientific name 1 KCTC 3638 Enterococcus casseliflavus 2 KCTC 3195 Enterococcus faecalis var. liquefaciens 3 KCTC 3552 Enterococcus flavescens 4 KCTC 3102 Enterococcus hirae 5 KCTC 3641 Enterococcus malodoratus 6 KCTC 3102 Enterococcus mundtii 7 KFRI 150 Lactobacillus acidophilus 8 KFRI 238 Lactobacillus amylophilus 9 KCTC 3102 Lactobacillus bervis 10 KFRI 1030 Lactobacillus bifermentans 11 KFRI 346 Lactobacillus casei 12 KFRI 1182 Lactobacillus collinoides 13 KFRI 654 Lactobacillus curvatus 14 KCTC 1047 Lactobacillus delbrueckii subsp. delbrueckii 15 KCTC 3112 Lactobacillus fermentum 16 KCTC 3602 Lactobacillus maltaromicus 17 KFRI 481 Lactobacillus pentosus 18 KCTC 1048 Lactobacillus plantarum 19 KCTC 3594 Lactobacillus reuteri 20 KCTC 3205 Lactobacillus sanfranciscensis 21 KFRI 684 Lactococcus lactis 22 KCTC 201 Lactococcus lactis subsp. cremoris 23 KCTC 3524 Leuconostoc carnosum 24 KCTC 3526 Leuconostoc citreum 25 KCTC 3102 Leuconostoc gelidum 26 KCTC 3528 Leuconostoc lactis 27 KCTC 3530 Leuconostoc mesenteroides subsp. dextranium 28 KCTC 3100 Leuconostoc mesenteroides subsp. mesenteroides 29 KFRI 832 Pediococcus pentosaceus 30 KCTC 3507 Pediococcus pentosacius 31 KCTC 3807 Weissella cibaria 32 KFRI 1184 Weissella confusa 33 KCTC 3531 Weissella paramesenteroides - 33 -
4.. DPPH DPPH Blois (1958) DPPH(1,1-Diphenyl-2-picrylhydrazyl) radical radical 517 nm., 0.1 mm DPPH soln (99.8% Methanol), 517 nm 0.94~0.97. 0.1 mm DPPH soln (99.8% Methanol) 3 ml 1 ml 30 517 nm. DPPH radical. scavenging effect (%) = [1 - Absorbance of sample517nm Absorbance of control517nm ] 100. (Fibrinolytic activity) Fibrin( ) Astrup. 50 mm (ph 7.4, 0.15M NaCl ) Fibrinogen 0.3% 5 ml 2% agarose ( ) 5 ml. thrombin(100nih unit/ml) 0.1 ml petri-dish 30~60 Fibrin plate. fibrin plate pasteur pipette 5 mm 7 20 ul 37 12, plasmin (1.0 unit/ml).. (%) = plasmin 100-34 -
. HMG-CoA (3-hydroxy-methylglutaryl coenzyme A) reductase HMG-CoA reductase Kleinsek (1989). 1 ml Cuvette 20 ul, 0.5 um ph 7.0 100 ul, DTT(20 mm)100 ul, NADPH(3 mm) 100 ul, 100 ul 37 HMG-CoA(3 mm) 100 ul 340 nm 5. DMSO. (%) = 1 - (sample O.D -sample blank O.D) (control -control blank O.D) 100. (Bile acid binding capacity) Bile acid binding capacity Camire (1993)., 0.1 g 5 ml 0.1 N-HCl 2 ml 37 1 1 N-NaOH ph 7.0 cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid 31.25μ mol/ml 0.1 M phosphate buffer (ph 7.0) 4 ml porcine pancreatin 10 mg/ml 0.01 M phosphate buffer (ph 7.0) 5 ml 37 1. 1.33 M phosphoric acid 2 ml 26,890 g 10 0.01 M phosphate buffer (ph 7.0) 5 ml vortex mixer. 1N-NaOH ph 7.0. 0.28 ml 3 ml test reagent(nitroamide dinucleotide, nitro blue tetrazolium salt, diaphorase, 3a-hydroxysteroid dehydrogenase) 0.5 ml. sample blank test reagent 3a-hydroxysteroid dehydrogenase. control control blank, control blank dehydrogenase, control 0.5 ml 37 5. 1.33 M phosphoric acid 0.1 ml 530 nm bile - 35 -
acid. bile acid Fig. 1. Fig. 1. 5.. 100g 9, Fig. 2 1% 60 shaking water bath 90. 8,000 rpm, 4 30. n-hexane, chloroform, ethyl acetate, n-butanol ethanol. 50 ml 100 ml, 20 ml 40 ml 5. 30 ml - 36 -
. Fig. 2. Protamex. 100g 9 Lactobacillus acidophilus 1% ( 1.7 10 7 cfu/g ). 4 37 incubator 8000 rpm 30... n-hexane, chloroform, n-butanol, ethyl acetate ethanol 5 separated funnel 40 ml 80 ml. 5 20 ml, 30 ml.. (ultrafiltration, UF), size-cut off. - 37 -
. 15 ml micro centrifuge tube membrane cell (ultrafree-15, Millipore) 3000 xg. 300,000 dalton, 300,000 dalton, 100,000~300,000 dalton, 100,000 dalton, 50,000~100,000 dalton, 50,000 dalton, 30,000~50,000 dalton, 30,000 dalton, 10,000~30,000 dalton, 10,000 dalton. Fig. 3.. gel chromatography (gel filtration chromatography). Sephedex G-75(3-80 kda). (φ2.8 50 cm), buffer(5 ml) loading (ph 7.0). fraction collector 6 ml.,,, HMG-CoA reductase, (Bile acid binding capacity). - 38 -
. HMG-CoA reductase inhibition bile acid binding capacity, compound.. Gel chromatography -. 0.5 ml 5% phenol 1 ml 5 ml 20 470 nm.. Gel chromatography BCA Lowry Folin BCA. BCA(bicinchoninic acid) 4% CuSO 4 50:1, Standard Working Reagent(SWR). 1 well, SWR 142 ul 8 ul 3 7 30. 562 nm Calibration Curve, Curve., bovine serum albumin.. Gel chromatography Folch., 2 g chloroform/methanol(2:1, v/v) 20 ml 30 stirring (Whatman No.2). 20mL. Buchner funnel, round bottom flask rotary vacuum evaporator. Folch chloroform/methanol(2:1, v/v) 20 ml. - 39 -
Chloroform chloroform. Chloroform evaporator,.. Sepadex G-75 column. Sepadex G-75column(2.8 50cm) 100mM Tris-HCl buffer(ph 8.3) blue dextran void volume(vo). column elution volume(ve), Ve/Vo. Somatostatin(1.6 kda), Aprotinin(6.5 kda), Cytochrome C(12.4 kda), Carbonic Anhydrase(29 kda), Albumin(66 kda). * Total bed volume( ) ; column volume Elution volume( ) ; volume Void volume( ) ; volume Inner volume( ) ; volume... 24 ( : 30 cm). 500 ml 500 ml, 1. - 40 -
10 420 nm. 6.. 1) 3~6, 100~120 1 hr. 1~4, 1~2 (HMF-985, Hanil, Korea). 0.1~2%, 0.5~3%, 0.5~1% 25±2 24 hr.,,,, 25±2 10 days. 60~70 10~30 min.. - 41 -
(, ) (100~120, 1~2hr) 1 (25±2, 24hr) 2 (25±2, 10 days) Fig. 4.. 1) (1).,,, - 42 -
,.,. 50%. 40~42 2 5 Tempering 29 Table 3. (2.5 2.5 2 cm) 4 35 min. Table 3. Ingredients Conc. 5% 10% 15% 20% 25% 30% 1) EH-D 50 100 150 200 250 300 White chocolate 950 900 850 800 750 700 Total 1000 1000 1000 1000 1000 1000 1) EH-D: enzyme hydrolysate of Dioscorea batatas (2),,.. 40~42 25 Tempering 29 Table 4. (2.5 2.5 2cm) 4 35 min. - 43 -
Table 4. Conc. Ingredients 5% 10% 15% 20% 25% 30% 1) S-D 50 100 150 200 250 300 White chocolate 950 900 850 800 750 700 Total 1000 1000 1000 1000 1000 1000 1) S-D: Steamed Dioscorea batatas (3). 40~42 25 Tempering 29 Table 5. (2.5 2.5 2cm) 4 35 min. Table 5. Conc. Ingredients 1% 3% 5% 7% 10% 1) P-D 10 30 50 70 100 White chocolate 990 970 950 930 900 Total 1000 1000 1000 1000 1000 1) P-D: Powder of Dioscorea batatas (4),.,,, 30 likert 9. (5). - 44 -
50%. 10%, 15% 3%, 20% 20% 5%. 45~50 27 Tempering 32, (2.5 2.5 2cm) 15 min. 40~42 25 Tempering 29 Table 6. 10%, 15% 3% (2.5 2.5 2cm), 20% 20% 5% 2/3 1/3 4 35 min. *tempering : tempering. - 45 -
Table 6. Ingredients Conc. 1) EH-D 2) S-D 3) P-D 10% 20% 15% 20% 3% 5% EH-D 100 200 - - - - S-D - - 150 200 - - P-D - - - - 30 50 White chocolate 900 800 850 800 970 950 Dark chocolate - - - Total 1000 1000 1000 1000 1000 1000 1) EH-D: enzyme hydrolysate of Dioscorea batatas 2) S-D: Steamed Dioscorea batatas 3) P-D: Powder of Dioscorea batatas 2) (1) 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%. 40% Table 7. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 30% 10 0 gel 10%,. (2.5 2.5 2cm). - 46 -
Table 7. Ingredients 0.5% 1.0% 1.5% 2.0% 2.5% 3.0% Agar powder 0.5 1.0 1.5 2.0 2.5 3.0 1) LF-D 59.5 59 58.5 58 57.5 57 Sugar 30 30 30 30 30 30 Oligosachharide 10 10 10 10 10 10 Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas - 47 -
(2) 30%, 35%, 40%, 45%, 50%. 10 %, Table 8. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 100 gel 10%,. (2.5 2.5 2cm). Table 8. Ingredients 30% 35% 40% 45% 50% Agar powder 2 2 2 2 2 1) LF-D 68 63 58 53 48 Sugar 20 25 30 35 40 Oligosachharide 10 10 10 10 10 Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (3) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 100 gel 10%,. (2.5 2.5 2cm). - 48 -
(4) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 100 gel 10%,. (2.5 2.5 2cm). (5) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 100 gel 10%,. (2.5 2.5 2cm). Table 11., Ingredients Concentration (%) 10 20 30 1) NF-D / 2) NP-D / 3) S-D 10 20 30 Water 40 30 20 Agar powder 2 2 2 Granulated sugar 38 38 38 Oligosaccharide 10 10 10 Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas - 49 -
(6) Table 12. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 100 gel 10%,. (2.5 2.5 2cm). Table 12. Ingredients 3% 5% 7% P-D 3 5 7 Water 47 45 43 Agar powder 2 2 2 Sugar 38 38 38 Oligosaccharide 10 10 10 Total 100 1) P-D: Powder of Dioscorea batatas (7), 10%, 20%, 30%, 5%,,,. 2%.,,, 48%( 14%, 8%, 8%, 8%, 10%), 2%. Table 13. - 50 -
10%, 20%, 30%, 5% (HMF-985, Hanil, Korea) 50 mesh. 2% 30 min, 100. gel 10%, (2.5 2.5 2cm). 50 mesh 30 min, 100 heating 10% (2.5 2.5 2cm) Fig. 5. - 51 -
Table 13. Ingredients 5) WH-SJ 6) SK-SJ 7) CB-SJ 8) CS-SJ 9) PE-SJ 1) LF-D 10 10 10 10 10 2) NP-D 30 30 30 30 30 3) S-D 20 20 20 20 20 4) P-D 5 5 5 5 5 Water 40 40 40 40 40 Agar 1 1 1 1 1 Xanthan gum 1 1 1 1 1 Sugar 38 38 38 38 38 Oligosaccharide 10 10 10 10 10 whey powder 5 - - - - skim milk - 5 - - - cocoa butter - - 5 - - Citrus syrup - - - 5 - plum extract - - - - 5 Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas 5) WH-SJ, 6) SK-SJ, 7) CB-SJ, 8) CS-SJ and 9) PE-SJ means added with whey powder, skim milk, cocoa butter, citrus syrup, plum extract in sweet jelly, respectively. *Each sweet jelly included a Dioscorea batatas products( 1)-5) ) respectively.. 1) (1). ( - 52 -
70%, 30% ) 200 g 800 ml 60 2 hr. 10 Brix., 60 6 hr 100 30. starter Lactobacillus acidophilus( 1 10 5-6 CFU/mL) 1% 37 24-48 hr.. Table 14. Ingredients Control Malt-B Sugar-B 1) LF-D 500 500 500 Water 500 500 500 Malt solution (10 Brix) - 100 - Sugar - - 100 1% L. acidophilus 10 10 10 Total 1010 1110 1110 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (2). Lactobacillus acidophilus 1 2 Lactobacilus amylophilus Leuconostoc mesenteroides subsp. mesenteroides Lactobacilus amylophilus Lactobacillus acidophilus. Leuconostoc mesenteroides subsp. mesenteroides. 1. - 53 -
, 2. Lactobacillus acidophilus( 1 10 5-6 CFU/mL), Leuconostoc mesenteroids subsp. mesenteroids( 1 10 5-6 CFU/mL) Lactobacillus amylophilus( 1 10 5-6 CFU/mL) Table 14 1% 37 24-48 hr. 50% 1 10% 1% (1 10 5-6 CFU/mL) 2 L. acidophilus L. amylophilus L. amylophilus Leu. mesenteroides Leu. mesenteroides subsp. mesenteroides subsp. mesenteroides 37, 24hr L. acidophilus 37, 24-48 hr Fig. 6. - 54 -
(3) 10%, 20%, 30%, 40%, 50%. Leuconostoc mesenteroids subsp. mesenteroids( 1 10 5-6 CFU/mL) Lactobacillus amylophilus( 1 10 5-6 CFU/mL) 1% 37 24-48 hr. Table 15. 10% 20% 30% 40% 50% 1) LF-D 100 200 300 400 500 Water 790 690 590 490 390 Granulated sugar 100 100 100 100 100 L. amylophilus 5 5 5 5 5 Leu. mesenteroides subsp. mesenteroides 5 5 5 5 5 Total 1000 1000 1000 1000 1000 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (4),. 20%, 40%, 60%, 20%, 40%, 60%, 5%, 10%, 15%. Leuconostoc mesenteroids subsp. mesenteroids( 1 10 5-6 CFU/mL) Lactobacillus amylophilus( 1 10 5-6 CFU/mL) 1% 37 24-48 hr. (5). 10%, - 55 -
20%, 20%, 5%. 200 g, 200 g, 50 g, 100 g, 100 g 700 ml, 700 ml, 850 ml, 800 ml, 800 ml (HMF-985, Hanil, Korea) 50 mesh 900 ml. 900 ml,,,, vitamin C, L. acidophilus, L. amylohpilus, Leu. mesenteroides subsp. mesenteroides 3 7 24 hr. 1) LF-D 10% 2) NP-D 20% 3) S-D 20% 4) P-D 5% X 5% 5% 0.2% vitamin C 0.1% 0.2% L. amylophilus 0.5% Leu. mesenteroides subsp. mesenteroides 0.5% 37, 24-48hr 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas Fig. 7. 2) (1) - 56 -
. 5 %, 7 %, 10 %, 5 %, 10 %, 15 % Table 16. 5 %. Table 16. Ingredients 5% 7% 10% 1) LF-D 50 50 50 70 70 70 100 100 100 Water 850 800 750 830 780 730 800 750 700 Oligosachharide 50 100 150 50 100 150 50 100 150 Rice powder 50 50 50 50 50 50 50 50 50 Total 1000 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (2)., 10 %, 20 %, 30 %. Table 17. Table 17. Ingredients 10% 15% 20% 1) NP-D 100 100 100 150 150 150 200 200 200 Water 800 750 700 750 700 650 700 650 600 Oligosaccharide 50 100 150 50 100 150 50 100 150 Rice powder 50 50 50 50 50 50 50 50 50 Total 1000 1) NP-D: Not processed Dioscorea batatas - 57 -
(3).,,., Table 18. Table 18. Ingredients 20% 40% 60% 1) S-D 200 200 200 400 400 400 600 600 600 Water 750 700 650 550 500 450 350 300 250 Oligosaccharide 50 100 150 50 100 150 50 100 150 Rice powder - - - - - - - - - Total 1000 1) S-D: Steamed Dioscorea batatas (4).,.. Table 19. - 58 -
Table 19. Ingredients 3% 5% 7% 1) P-D 30 30 30 50 50 50 70 70 70 Water 870 820 770 850 800 750 830 780 730 Oligosaccharide 50 100 150 50 100 150 50 100 150 Rice powder 50 50 50 50 50 50 50 50 50 Total 1000 1) P-D: Powder of Dioscorea batatas (5) 3 2 1 30, (HMF-985, Hanil, Korea) 50 mesh. 150 g, 200g, 30 g, 50 g 700 ml, 700 ml, 820 ml, 800 ml (HMF-985, Hanil, Korea) 50 mesh. 40~50 80%. Table 20. Table 20. Ingredients Conc. 1) LF-D 5% 2) NP-D 15% 3) S-D 20% 4) P-D 3% Dioscorea batatas 50 150 200 30 Water 800 700 700 820 Oligosaccharide 100 100 100 100 Rice powder 50 50-50 Total 1000 1000 1000 1000 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas - 59 -
. 1) (intensity characteristics) (sensory characteristics). 30 Likert 9 1 " (extremely weak)", 9 " (extremely strong)", 1 " (extremely bad)", 9 " (extremely good)". 2) ph 10 ml ph meter (Metrohm 827). 3) 10 ml ph 8.3 0.1 N NaOH lactic acid(%). 4) (PDX-1, Vee-gee corp,. U.S.A) 100 ul. 5) (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a(, Redness) b(, Yellowness). L=97.57, a=0.00, b=1.79. 6) (MB45, Moisture analyzer, OHAUS Corp., Switzerland). 7) (DV- + RV viscometer, Brookfield, U.S.A). - 60 -
8) 0.85% 10 10 ph 5.5 MRS 30 24-48 hr. Lactobacillus amylophilus starch agar (Difco, USA), Leuconostoc mesenteroides subsp. mesenteroides PES. Lactobacillus acidophilus 1, m-lbs starch.. 1),., (bloom). Bloom fat-bloom sugar-bloom, tempering.,, bloom, 10%, 15% bloom. bloom tempering tempering 30, 40, 50, 60 1 bloom. 2) probiotics, - 61 -
. 5, 7.,, 1 7...,,.. 7. in-vivo. 1) 3 ICR mouse male. 10 10 group. group cholesterol pellet free feeding. - 62 -
Table 21. Group Diets Group I Group II cholesterol 2% Group III cholesterol 2% + 5% Group Ⅳ cholesterol 2% + LB. casei 5% Group Ⅴ cholesterol 2% + Lc. lactis 5% Group Ⅵ cholesterol 2% + En. flavescens 5% Group Ⅶ cholesterol 2% + Lb. acidophilus 5% Group Ⅷ cholesterol 2% + protamex 5% Group Ⅸ cholesterol 2% + Newtruse 5% Group Ⅹ cholesterol 2% + En. liqufaciens 5%. 1) 8 SD rat 60 10 6 cage 6. 300 g (22 ) (60%) 12. 3.., cholesterol, lard pellet free feeding. - 63 -
Table 22. Group Diets GroupⅠ: NFD ( 4.3% ) GroupⅡ: HFD cholesterol 2% + 10% GroupⅢ: FEt-HFD cholesterol 2% + 10% + ethanol 10% GroupⅣ: FBu-HFD cholesterol 2% + 10% + butanol 10% GroupⅤ: PEA-HFD cholesterol 2% + 10% + protamex ethyl acetate 10% GroupⅥ: PEt-HFD cholesterol 2% + 10% + protamex ethanol 10%. 1) 4 SD rat (male) 10 9 group 90 7.,,, 22±2, 55±5%, 12 light-dark cycle. AIN-93G (Haraln Teklad, Madison, USA) Table 23. 12,. - 64 -
Table 23. Group Diets Items G1 - G2 ( + 10% lard + 2% cholesterol) - G3 + 10% - G4 + 10% G5 + 10% G6 + 10% G7 + base 10% - G8 + 10% - G9 + 10%. 1) Body weight group body weight g. 2) total Cholesterol total choleaterol kit., total cholesterol. blood serum total cholesterol. Cholesterol ester cholesterol cholesterol H 2 O 2. hydrogen peroxide peroxidase. Cholesterol ester ----------> Cholesterol + fatty acids (by cholesterol esterase) cholesterol + O 2 --------> cholest-4-en-3-ona + H 2 O 2 (by cholesterol oxidase) 2H 2 O 2 + 4-AAP + p-hba------> Colored comp. +4H 2 O (by peroxidase) Serum 20 ul kit 3 ml 37 5-65 -
incubation spectrophotometer 500 nm, endpoint. 3) HDL-cholesterol cholesterol cholesterol ( ) cholesterol. cholesterol cholesterol. HDL (high density lipoprotein) cholesterol. HDL cholesterol. HDL (paraoxonase) LDL PAFAH(platelet activation factor acetylhydrolase). HDL cholesterol. Kit lipoprotein apo-lipoprotein B LDL (Low-Density lipoprotein) HDL (High-Density lipoprotein) cholesterol. HDL level. HDL-CHO assay kit 500 nm control. Serum 100 ul 100 ul 10 3,000 rpm 10 100 ul 3 ml 37 5 spectrophotometer 500 nm, endpoint. 4) LDL-cholesterol LDL cholesterol total cholesterol, HDL cholesterol triglycerides friede-wald. LDL cholesterol= [total cholesterol-(hdl cholesterol + (triglyceride/5))] - 66 -
5) Triglyceride serum complex lipoprotein. triglyceride determination triglyceride glycerol free fatty acid enzymatic akaline hydrolysis release glycerol. + L-a- H 2 O 2 + 4- + ESPT serum 20 ul 3 ml 37 10 spectrophotometer 550 nm, endpoint. 6) GOT GPT GOT,,,,,,,. GPT GPT. GPT.. GPT. GPT., GOT.,. GOT GPT, GPT - 67 -
GOT. Cholesterol vital response GOT, GPT level. GOT L-Aspartic acid + a-ketoglutaric acid ----------->oxaloacetate + L-glutamate MDH Oxaloacetate + NADH + H+ -------------------->L-Malate + NAD+ + H2 GOT kit, GPT kit. 96-well plate sample 10 ul reagent R1+R2 (4:1) 100 ul, microplate reader (Molecular Device thermomax) 340 nm. 1 37 incubation 1 37 kinetic OD. 7) HMG-CoA reductase inhibition 1, 2 HMG-CoA reductase in-vitro. 3 HMG-CoA reductase in-vivo. HMG-CoA reductase Kleinsek. 300 ml KCl, 6 mm EDTA, 15 mm dithiothretol 240 mm potassium phosphate buffer 2 mm NADPH 100 ul, 1 mm HMG-CoA 100 ul 100 ul 37, 240 nm 6.22 mm/cm/l. 1 1 mg protein NADPH pmol. 8) 3 mean+standard deviation, - 68 -
statistical Analysis System(SAS) Duncan's multiple range test. - 69 -
3 1..,,, Table 24., 5.66% 4.70%, 6.57% 5.09%. 12.11% 9.88% 3%, 0.36% 0.15% 2 76.26% 79.23%. L( ) 85.32 84.39 a( ) +1.60, +1.63, b( ) +10.76, +10.34. 0.51% 0.43% 0.14% 0.10%.,. - 70 -
Table 24. Items ( ) ( ) Moisture (%) 86.11 4.70 5.66 Ash (%) 1.04 6.57 5.09 Crude protein (%) 2.31 12.11 9.88 Crude lipid (%) 1.21 0.36 0.15 Carbohydrate (%) 9.34 76.26 79.23 L 78.54 84.39 85.32 Color a -0.87 +1.63 +1.60 b +3.19 +10.34 +10.76 Total sugar content (%) 0.25 0.43 0.51 Total Reducing sugar (%) 0.37 0.14 0.10. Table 25, Table 26. 50, 2 8 6.57%, 12 4.90%. 55 6 8.56% 12 4.78%. 60 6 7.35%, 12 4.62%. - 71 -
Table 25. Sample Dry Temperature ( ) Dry time (hr) 50 55 60 0 87.37 87.37 87.37 2 83.79 57.53 33.78 4 45.52 37.94 19.65 6 18.05 8.56 7.35 8 6.57 5.66 4.70 10 5.77 5.18 4.61 12 4.90 4.78 4.62 8. 50, 55 60. 50 L 82.11 55, 60 L ( ), a( ) 2.03, b ( ) 12.20. Table 26. Sample Dry Temperature ( ) Dry time (hr) 50 55 60 L (Lightness) 82.11 85.32 84.39 a (Redness) +2.03 +1.60 +1.63 b (Yellowness) +12.20 +10.76 +10.34. 1), Table 27. mannitol erythritol Hunter-L 91.66 89.64 (79.41),., tarta- - 72 -
ric acid Hunter-L 87.88. Table 27. sugar alcohol organic acid Sample powder color value L(Lightness) a(redness) b(yellowness) Control 79.41±1.63 3.25±0.40 19.28±1.92 Erythritol 89.64±0.31 1.13±0.08 10.46±0.22 Mannitol 91.66±0.29 0.61±0.04 7.06±0.22 Maltitol 84.63±1.41 1.66±0.06 18.24±0.83 Sorbitol 83.05±0.43 1.34±0.15 16.02±0.43 Xylitol 83.52±2.00 1.28±0.18 12.99±0.55 Citric acid 86.80±0.42 0.92±0.10 12.45±0.28 Fumaric acid 86.48±0.43 0.67±0.05 11.17±0.65 Malic acid 85.70±0.46 0.78±0.06 11.33±0.37 Succinic acid 85.88±0.22 1.01±0.06 12.80±0.20 Tartaric acid 87.88±1.12 0.75±0.17 11.56±0.27, erythritol, tartaric acid,. erythritol maltitol. erythritol. - 73 -
Table 28. sugar alcohol organic acid Sample powder Water solubility (%) Swelling power (g/g) Control 5.94±0.07 4.12±0.02 Erythritol 10.37±0.12 7.58±0.06 Mannitol 6.61±0.03 5.54±0.08 Maltitol 5.61±0.02 5.28±0.06 Sorbitol 6.32±0.10 5.74±0.13 Xylitol 6.91±0.17 5.34±0.12 Citric acid 6.34±0.16 5.07±0.07 Fumaric acid 6.71±0.71 4.88±0.49 Malic acid 6.00±0.10 4.83±0.06 Succinic acid 6.53±0.05 4.26±0.03 Tartaric acid 6.75±0.36 4.75±0.13 2).., 10 : 1 (Table 29). Table 29. Ratio (water : powder) color viscosity taste overall acceptability 1 : 1 4.00±0.82 9.00±0.82 2.75±2.06 3.00±0.82 3 : 1 4.25±1.26 7.00±0.82 2.50±0.58 3.00±1.41 5 : 1 4.50±0.58 6.25±0.50 2.00±0.82 3.00±0.82 7 : 1 6.25±0.50 5.25±0.96 3.50±1.00 5.25±1.26 10 : 1 6.25±0.50 3.25±0.96 5.75±0.50 6.75±1.26 Table 30. mannitol, maltitol, sorbitol erythritol 2. erythritol - 74 -
erythritol. succinic acid, tartaric acid, malic acid. fumaric acid citric acid., ( ). Table 30. Sample powder Sugar alcohol Brightness Color preference Sweetness Sourness Taste preference Control 3.83±2.14 ab 4.67±1.37 a 5.33±1.97 c 3.50±2.07 c 5.83±1.83 bc Erythritol 6.00±2.83 b 5.00±2.83 a 8.00±1.79 a 2.17±1.17 c 7.67±1.63 abc Maltitol 7.33±1.97 ab 6.33±1.97 a 7.00±2.00 a 2.83±1.94 c 7.33±1.75 a Mannitol 7.75±0.50 b 5.25±0.50 a 7.75±0.96 a 1.50±0.58 c 6.75±2.06 abc Sorbitol 6.75±1.71 ab 5.75±0.96 a 5.25±1.71 c 2.50±3.00 c 5.75±2.22 bc Xylitol 3.17±1.47 a 4.33±2.34 a 7.50±2.35 ab 3.00±2.53 c 6.67±2.16 abc Citric acid 5.25±1.26 ab 4.25±2.06 a 2.75±1.50 c 5.00±2.16 a 5.50±0.58 c Fumaric acid 4.67±2.58 ab 5.00±2.10 a 3.00±1.10 c 4.50±1.76 bc 7.33±2.42 bc Organic acid Malic acid 6.17±1.17 ab 5.00±2.53 a 2.83±1.33 c 5.33±2.42 bc 5.17±0.98 c Succinic acid 6.25±1.71 b 5.00±1.83 a 1.00±0.00 c 7.00±0.82 ab 4.25±0.50 c Tartaric acid 6.75±2.36 ab 4.50±2.65 a 2.25±2.50 c 7.00±0.82 a 5.25±0.96 c * Superscript letters indicate significant difference at α=0.05 as determined by Duncan's multiplerange test. 2.. ( ) (AMG, Viscozyme, Pectinex, Celluclast 1.0%(w/w)) 40, 50, 60, 70., (Table 31) (Table 32) Celluclast 2~4-75 -
., AMG Viscozyme, Pectinex, Celluclast. (Table 33) 0.5% Celluclast 1.0~3.6% 2, 50, 60,., AMG. (Table 34) 0.15% 0.3~1.34% 2,. AMG, 60. (Table 35) 60, AMG Viscozyme, Pectinex, Celluclast 40, 60 20%. AMG 30%. 60. Table 31. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before 1.1 1.1 1.1 1.1 40 3.8 2.7 2.0 1.3 50 4.1 2.8 2.3 1.5 60 4.1 3.1 2.6 1.4 70 4.1 2.8 2.6 1.5-76 -
Table 32. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before 0.98 0.98 0.98 0.98 40 3.44 2.42 1.76 1.47 50 3.74 2.61 2.23 1.30 60 3.79 2.88 2.07 1.78 70 3.83 2.53 2.47 1.46 Table 33. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before 0.54 0.54 0.54 0.54 40 3.40 1.71 1.40 1.22 50 3.83 2.44 1.93 0.81 60 3.76 2.34 2.17 1.07 70 3.36 2.24 1.89 1.23 Table 34. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before 0.15 0.15 0.15 0.15 40 1.16 0.29 0.28 0.31 50 1.07 0.30 0.29 0.28 60 1.32 0.29 0.29 0.29 70 0.78 0.30 0.29 0.30-77 -
Table 35. (%) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before 31.15 24.87 26.62 19.25 40 28.90 17.00 15.00 21.78 50 31.98 17.30 17.82 18.47 60 31.98 30.63 23.89 23.50 70 30.38 30.57 27.05 14.33 2.. ( ) (Alcalase, Neutrase, Flavourzyme, Protamex 1.0%(w/w)) 40, 50, 60, 70., (Table 31) (Table 32) Alcalase 2~4., Protamex Neutrase, Flavourzyme, Alcalase. (Table 33) 0.5% Alcalase 1.0~3.6% 2, 50, 60,., Protamex. (Table 34) 0.15% 0.3~1.34% 2,. Protamex, 60. (Table 35) 60, Protamex Neutrase, Flavourzyme, Alcalase 40, 60 20%. Protamex 30% - 78 -
. 60. Table 31. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 1.1 1.1 1.1 1.1 40 3.8 2.7 2.0 1.3 50 4.1 2.8 2.3 1.5 60 4.1 3.1 2.6 1.4 70 4.1 2.8 2.6 1.5 Table 32. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.98 0.98 0.98 0.98 40 3.44 2.42 1.76 1.47 50 3.74 2.61 2.23 1.30 60 3.79 2.88 2.07 1.78 70 3.83 2.53 2.47 1.46 Table 33. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.54 0.54 0.54 0.54 40 3.40 1.71 1.40 1.22 50 3.83 2.44 1.93 0.81 60 3.76 2.34 2.17 1.07 70 3.36 2.24 1.89 1.23-79 -
Table 34. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.15 0.15 0.15 0.15 40 1.16 0.29 0.28 0.31 50 1.07 0.30 0.29 0.28 60 1.32 0.29 0.29 0.29 70 0.78 0.30 0.29 0.30 Table 35. (%) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 31.15 24.87 26.62 19.25 40 28.90 17.00 15.00 21.78 50 31.98 17.30 17.82 18.47 60 31.98 30.63 23.89 23.50 70 30.38 30.57 27.05 14.33. 0.05~2.0%(w/w) 90. (Table 36) (Table 37), Alcalase 1.5. Protamex, 0.5%. Neutrase 0.5%., Neutrase. (Table 38) Alcalase 2 0.05% - 80 -
. Protamex, 0.5% 2.8g/100g Neutrase, Flavourzyme 3., Neutrase 0.5%., Neutrase. (Table. 39), 0.5%. Protamex, 0.5% 1.6g/100g 4, Neutrase, Flavourzyme, Alcalase. (Table 40) 0.05%. Protamex 0.5%. Neutrase 0.5% 2%.,,. Table 36. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 1.1 1.1 1.1 1.1 0.05 2.9 1.4 1.5 1.2 0.1 3.2 1.7 1.9 1.3 0.5 3.8 1.8 1.8 1.3 1 3.8 2.4 1.8 1.4 2 3.7 2.9 2.1 1.3-81 -
Table 37. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 1.80 1.80 1.80 1.80 0.05 3.09 2.18 2.34 2.08 0.1 3.26 2.25 2.07 2.07 0.5 3.76 2.24 2.32 2.04 1 3.78 2.95 2.22 2.05 2 3.68 3.30 2.41 2.07 Table 38. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.95 0.95 0.95 0.43 0.05 2.70 1.17 1.16 0.46 0.1 2.42 1.30 1.13 0.45 0.5 2.92 1.27 1.33 0.71 1 2.92 1.95 1.30 0.59 2 2.60 2.09 1.77 0.67 Table 39. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.21 0.21 0.21 0.21 0.05 0.84 0.31 0.33 0.29 0.1 0.83 0.31 0.31 0.29 0.5 1.57 0.31 0.30 0.29 1 1.54 0.31 0.31 0.30 2 1.75 0.43 0.57 0.32-82 -
Table 40. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before 0.54 0.54 0.54 0.54 0.05 18.80 11.54 19.39 17.13 0.1 23.90 14.23 16.54 18.51 0.5 25.91 19.80 19.39 18.46 1 27.40 20.67 19.46 18.03 2 27.27 27.89 25.14 18.58. 60 30~240., (Table 41) (Table 42). 60~90 1.5 30 2 30. (Table 43). Protamex 60 Neutrase, Flavourzyme, Alcalase 180 1.5. Neutrase 60 180. Flavourzyme, Alcalase 60. (Table 44) 3. (Table 45) 60 90.. - 83 -
60 50%. Table 41. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before 0.33 0.33 0.33 0.33 30 0.33 0.33 0.33 0.34 60 0.40 0.37 0.36 0.33 90 0.41 0.38 0.38 0.37 120 0.50 0.39 0.39 0.36 150 0.53 0.42 0.39 0.37 180 0.61 0.45 0.41 0.36 210 0.63 0.46 0.40 0.36 240 0.64 0.48 0.41 0.36-84 -
Table 42. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before 0.030 0.030 0.030 0.030 30 0.060 0.058 0.058 0.057 60 0.060 0.060 0.060 0.058 90 0.062 0.059 0.059 0.059 120 0.059 0.059 0.059 0.059 150 0.061 0.061 0.061 0.061 180 0.061 0.059 0.059 0.059 210 0.059 0.059 0.059 0.059 240 0.062 0.061 0.061 0.061 Table 43. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before 0.33 0.33 0.33 0.33 30 0.33 0.33 0.33 0.34 60 0.40 0.37 0.36 0.33 90 0.41 0.38 0.38 0.37 120 0.50 0.39 0.39 0.36 150 0.53 0.42 0.39 0.37 180 0.61 0.45 0.41 0.36 210 0.63 0.46 0.40 0.36 240 0.64 0.48 0.41 0.36-85 -
Table 44. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before 0.11 0.11 0.11 0.11 30 0.14 0.11 0.15 0.11 60 0.15 0.13 0.16 0.15 90 0.29 0.28 0.29 0.29 120 0.29 0.29 0.29 0.28 150 0.30 0.29 0.30 0.29 180 0.31 0.29 0.29 0.29 210 0.30 0.29 0.29 0.29 240 0.30 0.29 0.30 0.29 Table 45. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before 0.0 0.0 0.0 0.0 30 5.4 29.1 31.9 42.2 60 53.1 51.4 51.4 54.3 90 55.1 53.1 53.1 52.4 120 54.8 51.1 50.6 49.0 150 56.3 51.5 50.8 49.3 180 57.0 51.7 51.7 47.5 210 57.8 50.3 50.3 47.5 240 55.1 53.0 53.0 46.0 3.. ph,, Table 46 33 ph ph 6.1 2 ph 4.2~5.5, 2 ph. (Table 47) 0.5% 2 0.1~0.5%, 2. (Fig. 8) (Fig. - 86 -
9) 10 6 ~10 7 CFU/mL 2 2.1 10 9 CFU/mL, 1.3 10 9 CFU/mL 2. KFRI 150 (Lactobacillus acidophilus) 2., 2 ph 2 ph.., (Table 48) 1.5 Brix 2 2.0~6.0 Brix. KCTC 3102 (Enterococcus mundtii) 5.7~6.3 Brix KCTC 3195 (Enterococcus faecalis var. liquefaciens), KCTC 3102 (Lactobacillus bervis), KFRI 832 (Pediococcus pentosaceus) 3 4.0 Brix 4 4.0~5.3 Brix. KCTC 3524 (Leuconostoc carnosum), KCTC 3205 (Lactobacillus sanfranciscensis), KCTC 201 (Lactococcus lactis subsp. cremoris), KCTC 3112 (Lactobacillus fermentum), KFRI 238 (Lactobacillus amylophilus), KFRI 150 (Lactobacillus acidophilus) 2.4~3.7 Brix 2. 2.0 Brix. (Table 49) 10.0% 2 26.5~206.1% 2. KCTC 3638 (Enterococcus casseliflavus), KCTC 1048 (Lactobacillus plantarum ) 8.06, 8.95 g/100g 2 202.15, 131.21 g/100g. KCTC 3102 (Lactobacillus bervis), KFRI 238 (Lactobacillus amylophilus), KFRI 481 (Lactobacillus pento ), KCTC 3594 (Lactobacillus reuteri), KCTC 201 (Lactococcus lactis subsp. cremoris) 7.2~9.8 g/100g 6 135.4~180.6-87 -
g/100g. 10.05~70.1 g/100g. (Table 50) 3.0 g/100g. KFRI 238 (Lactobacillus amylophilus) 3.48 g/100g 5, 6 15.4 g/100g. KFRI 481 (Lactobacillus pentosus), KFRI 684 (Lactococcus lactis), KCTC 3507 (Pediococcus pentosacius) 3.5 g/100g 6 10.10 g/100g. - 88 -
Table 46. ph Strain Fermentation time (days) 0 1 2 3 4 5 6 7 KCTC 3638 6.12 5.24 5.11 5.02 4.82 4.67 4.62 4.60 KCTC 3195 6.08 6.10 5.74 5.15 4.95 4.91 4.82 4.80 KCTC 3552 6.09 4.66 4.62 4.60 4.62 4.64 4.62 4.62 KCTC 3102 6.10 5.98 6.18 5.02 4.72 4.51 4.42 4.40 KCTC 3641 6.08 5.72 4.73 4.56 4.46 4.49 4.43 4.37 KCTC 3102 6.08 6.08 5.63 5.50 5.15 4.75 4.65 4.64 KFRI 150 6.08 5.07 5.20 5.00 5.00 4.93 5.00 5.04 KFRI 238 6.16 5.85 5.00 4.61 4.49 4.36 4.41 4.40 KCTC 3102 6.08 5.95 5.68 5.09 5.03 4.95 4.85 4.80 KFRI 1030 6.13 4.61 4.52 4.50 4.50 4.43 4.50 4.52 KFRI 346 6.12 5.51 4.96 4.86 4.79 4.67 4.57 4.45 KFRI 1182 6.13 5.57 4.61 4.52 4.40 4.32 4.30 4.30 KFRI 654 6.10 5.30 5.13 5.06 5.01 4.96 4.89 4.85 KCTC 1047 6.04 4.88 4.35 4.36 4.27 4.23 4.24 4.25 KCTC 3112 6.08 6.01 6.02 5.40 5.05 5.00 4.80 4.72 KCTC 3602 6.15 4.52 4.47 4.44 4.43 4.34 4.34 4.25 KFRI 481 6.20 5.64 4.63 4.45 4.34 4.35 4.37 4.40 KCTC 1048 6.13 5.37 4.44 4.37 4.26 4.21 4.18 4.20 KCTC 3594 6.08 4.87 4.88 4.74 4.82 4.83 4.73 4.50 KCTC 3205 6.10 5.81 5.71 5.54 5.34 5.21 4.95 4.91 KFRI 684 6.08 5.21 4.48 4.42 4.33 4.29 4.29 4.30 KCTC 201 6.13 5.50 4.85 4.73 4.65 4.56 4.61 4.60 KCTC 3524 6.10 5.94 5.60 5.38 5.22 4.99 4.98 4.93 KCTC 3526 6.07 4.64 4.61 4.61 4.60 4.46 4.49 4.46 KCTC 3102 6.04 5.41 5.17 5.04 5.00 4.95 4.71 4.61 KCTC 3528 6.06 4.67 4.65 4.65 4.61 4.58 4.69 4.72 KCTC 3530 6.04 4.75 4.70 4.65 4.44 4.37 4.33 4.34 KCTC 3100 6.16 4.97 4.75 4.71 4.70 4.66 4.66 4.67 KFRI 832 6.20 6.34 5.65 5.28 5.20 4.75 4.57 4.56 KCTC 3507 6.21 5.54 5.33 5.51 5.35 5.11 5.00 4.95 KCTC 3807 6.12 5.32 4.90 4.83 4.75 4.68 4.48 4.40 KFRI 1184 6.03 5.05 4.76 4.60 4.56 4.43 4.43 4.42 KCTC 3531 6.1 4.83 4.71 4.50 4.37 4.33 4.31 4.40-89 -
Table 47. Strain Fermentation time (days) 0 1 2 3 4 5 6 7 KCTC 3638 0.09 0.17 0.23 0.38 0.38 0.41 0.47 0.47 KCTC 3195 0.02 0.05 0.09 0.14 0.18 0.18 0.19 0.19 KCTC 3552 0.09 0.24 0.23 0.25 0.24 0.38 0.41 0.40 KCTC 3102 0.00 0.01 0.02 0.14 0.16 0.20 0.23 0.25 KCTC 3641 0.02 0.14 0.36 0.35 0.35 0.38 0.39 0.40 KCTC 3102 0.02 0.05 0.10 0.11 0.14 0.17 0.17 0.16 KFRI 150 0.09 0.17 0.14 0.16 0.18 0.25 0.23 0.25 KFRI 238 0.09 0.11 0.27 0.34 0.36 0.38 0.36 0.45 KCTC 3102 0.02 0.09 0.09 0.15 0.16 0.17 0.16 0.16 KFRI 1030 0.09 0.19 0.18 0.18 0.25 0.29 0.25 0.25 KFRI 346 0.05 0.23 0.36 0.41 0.41 0.39 0.39 0.45 KFRI 1182 0.05 0.18 0.45 0.46 0.45 0.45 0.49 0.50 KFRI 654 0.09 0.14 0.36 0.20 0.20 0.22 0.21 0.20 KCTC 1047 0.05 0.23 0.41 0.41 0.41 0.41 0.41 0.42 KCTC 3112 0.02 0.06 0.05 0.11 0.15 0.14 0.18 0.20 KCTC 3602 0.09 0.25 0.32 0.27 0.25 0.29 0.30 0.29 KFRI 481 0.05 0.14 0.41 0.41 0.45 0.47 0.47 0.50 KCTC 1048 0.02 0.14 0.41 0.41 0.41 0.41 0.45 0.45 KCTC 3594 0.05 0.27 0.38 0.39 0.41 0.41 0.40 0.41 KCTC 3205 0.02 0.09 0.09 0.09 0.11 0.12 0.14 0.16 KFRI 684 0.05 0.23 0.35 0.36 0.36 0.41 0.41 0.41 KCTC 201 0.09 0.14 0.23 0.23 0.25 0.34 0.23 0.27 KCTC 3524 0.02 0.05 0.12 0.12 0.14 0.14 0.15 0.16 KCTC 3526 0.09 0.20 0.23 0.25 0.21 0.22 0.25 0.29 KCTC 3102 0.02 0.09 0.14 0.16 0.15 0.14 0.16 0.14 KCTC 3528 0.10 0.22 0.29 0.23 0.20 0.23 0.18 0.18 KCTC 3530 0.09 0.19 0.23 0.23 0.29 0.31 0.30 0.30 KCTC 3100 0.09 0.18 0.27 0.29 0.27 0.27 0.27 0.29 KFRI 832 0.00 0.02 0.10 0.13 0.14 0.17 0.20 0.23 KCTC 3507 0.01 0.09 0.09 0.10 0.11 0.15 0.14 0.15 KCTC 3807 0.05 0.18 0.38 0.38 0.39 0.41 0.41 0.41 KFRI 1184 0.09 0.17 0.23 0.27 0.27 0.34 0.32 0.32 KCTC 3531 0.05 0.19 0.41 0.45 0.41 0.41 0.41 0.45-90 -
Fig. 8. - 91 -
Fig. 9. - 92 -
Table 48. Strain Fermentation time (days) 0 1 2 3 4 5 6 7 KCTC 3638 1.2 2.5 2.7 2.7 3.1 2.9 3.0 3.0 KCTC 3195 1.9 3.0 3.8 4.8 5.0 4.8 5.1 5.2 KCTC 3552 1.2 2.1 1.8 1.8 1.9 1.9 2.0 2.0 KCTC 3102 1.7 1.9 2.1 2.4 3.5 4.2 4.7 5.4 KCTC 3641 1.2 1.4 1.7 1.8 1.7 1.8 1.8 1.7 KCTC 3102 1.9 3.1 5.7 6.0 6.3 6.0 6.1 6.2 KFRI 150 1.4 1.6 2.2 2.6 3.4 2.7 2.8 2.8 KFRI 238 1.4 3.0 2.9 3.0 3.1 3.3 3.7 3.7 KCTC 3102 1.8 4.0 3.5 4.0 4.4 4.0 4.1 4.1 KFRI 1030 1.2 1.7 1.6 1.7 1.9 1.8 1.8 1.8 KFRI 346 1.2 1.7 1.6 1.7 1.9 1.8 1.8 1.8 KFRI 1182 1.2 2.1 1.8 1.8 1.9 1.9 2.0 2.0 KFRI 654 1.3 1.5 1.4 1.5 1.7 1.8 1.8 1.8 KCTC 1047 1.4 1.5 1.5 1.7 1.8 1.9 1.8 1.8 KCTC 3112 1.8 2.7 2.4 2.0 2.6 2.3 2.4 2.6 KCTC 3602 1.3 1.6 1.5 1.7 1.8 2.0 1.9 1.9 KFRI 481 1.2 1.7 1.6 1.7 1.9 1.8 1.8 1.8 KCTC 1048 1.3 1.7 1.6 1.9 2.1 2.4 2.1 2.1 KCTC 3594 1.2 2.1 1.8 1.8 1.9 1.9 2.0 2.0 KCTC 3205 1.8 2.6 3.0 3.3 3.2 3.0 3.4 3.2 KFRI 684 1.2 1.4 1.7 1.8 1.7 1.8 1.8 1.7 KCTC 201 1.3 1.7 1.6 2.2 2.4 2.4 2.8 2.8 KCTC 3524 2.0 2.4 3.0 3.0 3.4 3.4 3.4 3.0 KCTC 3526 1.3 1.6 1.5 1.6 1.7 1.7 1.7 1.7 KCTC 3102 2.0 2.4 2.3 3.0 3.0 3.8 4.0 4.7 KCTC 3528 1.3 1.6 1.6 1.6 1.5 1.6 1.7 1.7 KCTC 3530 1.4 1.5 1.5 1.7 1.8 1.9 1.8 1.8 KCTC 3100 1.3 1.6 1.5 1.6 1.6 1.7 1.8 1.7 KFRI 832 1.8 3.4 3.8 3.8 4.9 4.5 4.9 4.9 KCTC 3507 1.7 2.2 2.0 2.0 2.2 2.3 2.3 2.1 KCTC 3807 1.2 1.7 1.9 2.0 2.1 2.1 2.0 2.1 KFRI 1184 1.3 1.7 1.6 1.9 2.1 2.4 2.0 2.0 KCTC 3531 1.4 1.5 1.5 1.7 1.8 1.9 1.8 1.8-93 -
Table 49. Strain Fermentation time (days) 0 1 2 3 4 5 6 7 KCTC 3638 8.95 98.41 202.15 120.14 125.89 132.20 126.87 110.61 KCTC 3195 7.20 33.93 67.71 71.78 78.79 101.50 135.56 95.61 KCTC 3552 8.06 61.87 131.21 68.13 65.47 62.80 60.14 57.48 KCTC 3102 9.88 111.21 124.21 120.47 127.20 150.47 180.65 141.96 KCTC 3641 9.56 35.89 36.73 39.25 55.37 60.56 73.32 64.77 KCTC 3102 10.05 28.50 32.52 33.50 43.18 47.38 55.65 57.06 KFRI 150 10.98 35.14 81.59 103.04 145.09 122.80 122.66 123.64 KFRI 238 9.88 131.12 120.42 125.05 128.97 153.50 183.22 147.90 KCTC 3102 9.56 35.89 36.73 39.25 55.37 60.56 73.32 64.77 KFRI 1030 9.56 15.28 16.13 29.50 25.27 30.36 33.52 34.74 KFRI 346 10.05 28.50 32.52 33.50 43.18 47.38 51.65 53.06 KFRI 1182 11.54 20.84 29.30 26.50 43.18 45.64 43.88 57.03 KFRI 654 10.79 38.32 28.60 26.64 42.76 48.64 51.65 50.76 KCTC 1047 10.07 29.63 38.13 31.68 49.63 48.40 49.65 49.73 KCTC 3112 11.54 20.84 29.30 26.50 23.27 28.64 33.88 35.75 KCTC 3602 10.86 34.02 55.37 38.41 51.87 55.65 57.06 57.76 KFRI 481 7.20 33.93 67.71 71.78 78.79 101.50 135.56 95.61 KCTC 1048 8.06 61.87 131.21 68.13 65.47 62.80 60.14 57.48 KCTC 3594 9.88 111.21 124.21 120.47 127.20 150.47 180.65 141.96 KCTC 3205 9.56 35.89 36.73 39.25 55.37 60.56 73.32 64.77 KFRI 684 10.05 28.50 32.52 33.50 43.18 47.38 55.65 57.06 KCTC 201 7.20 33.93 67.71 71.78 78.79 101.50 135.56 95.61 KCTC 3524 12.48 34.02 53.13 68.13 63.08 87.90 76.54 83.13 KCTC 3526 10.05 28.50 32.52 33.50 43.18 47.38 55.65 57.06 KCTC 3102 11.54 20.84 29.30 26.50 43.18 48.64 43.88 57.06 KCTC 3528 9.28 39.91 26.78 32.10 32.38 41.92 57.76 60.98 KCTC 3530 10.07 29.63 38.13 31.68 49.63 43.88 55.65 57.76 KCTC 3100 11.54 20.84 29.30 26.50 23.27 48.64 43.88 35.75 KFRI 832 10.79 38.32 28.60 26.64 42.76 43.18 55.65 57.76 KCTC 3507 10.07 29.63 38.13 31.68 49.63 49.63 55.65 57.76 KCTC 3807 11.54 20.84 29.30 26.50 23.27 48.64 43.88 35.75 KFRI 1184 12.48 34.02 53.13 68.13 63.08 87.90 76.54 83.13 KCTC 3531 11.54 20.84 29.30 26.50 43.18 48.64 43.88 57.06-94 -
Table 50. Strain Fermentation time (days) 0 1 2 3 4 5 6 7 KCTC 3638 3.50 5.52 5.45 6.43 6.47 5.14 5.16 3.59 KCTC 3195 - - - - - - - - KCTC 3552 3.50 3.60 3.28 2.01 2.50 0.00 0.00 0.00 KCTC 3102 - - - - - - - - KCTC 3641 3.46 3.5 4.48 2.30 1.00 0.95 1.13 1.33 KCTC 3102 3.5 3.74 2.98 2.04 1.85 1.66 1.47 1.28 KFRI 150 3.62 3.70 8.50 10.06 12.09 10.8 10.45 10.71 KFRI 238 3.48 7.98 10.29 12.19 12.87 15.41 15.11 14.6 KCTC 3102 - - - - - - - - KFRI 1030 3.56 3.73 5.86 1.55 2.40 2.18 1.93 2.11 KFRI 346 3.46 3.50 4.48 2.30 1.00 0.95 1.13 1.33 KFRI 1182 3.5 3.74 2.98 2.04 1.85 1.66 1.47 1.28 KFRI 654 3.48 3.54 3.92 3.42 2.92 2.42 2.07 2.11 KCTC 1047 - - - - - - - - KCTC 3112 - - - - - - - - KCTC 3602 3.48 3.62 3.37 1.42 2.07 0.95 1.13 1.33 KFRI 481 3.56 4.17 5.54 5.81 8.13 9.04 11.51 9.16 KCTC 1048 3.48 3.60 0.85 0.97 0.93 0.94 0.10 0.94 KCTC 3594 3.50 3.59 5.56 1.61 1.92 1.85 1.61 1.79 KCTC 3205 3.48 3.62 3.37 1.42 2.07 0.95 1.13 1.33 KFRI 684 3.57 3.77 5.41 4.87 7.01 8.97 10.01 9.16 KCTC 201 3.50 5.52 5.45 6.43 6.47 5.14 5.16 3.59 KCTC 3524 - - - - - - - - KCTC 3526 3.46 3.48 1.12 1.06 1.22 1.13 1.18 1.24 KCTC 3102 - - - - - - - - KCTC 3528 3.46 3.50 4.48 2.30 1.00 0.95 1.13 1.33 KCTC 3530 3.50 3.74 2.98 2.04 1.85 1.66 1.47 1.28 KCTC 3100 3.48 3.60 0.85 0.97 0.93 0.94 0.10 0.94 KFRI 832 3.47 4.95 5.15 6.33 5.97 5.40 5.16 3.59 KCTC 3507 3.57 3.77 5.84 4.87 7.53 9.44 11.11 9.16 KCTC 3807 3.48 3.60 0.85 0.97 0.93 0.94 0.10 0.94 KFRI 1184 3.50 3.59 5.56 1.61 1.92 1.85 1.61 1.79 KCTC 3531 3.46 3.50 4.48 2.30 1.00 0.95 1.13 1.33-95 -
4.. HMG-CoA reductase Table 51 HMG-CoA reductase. 50, 0.5%, 90,,,,, HMG-CoA reductase. 1.6 cm. Table 51. HMG-CoA reductase Fibrinolytic activity (cm) HMG-CoA reductase (%) Control 1.6 14.86 Alcalase - 45.32 Flavourzyme - -11.42 Protamex - 234.96 Neutrase - 32.90 HMG-CoA reductase Protamex 234.96%, Alcalase 45.32%, Neutrase 32.90% Flavourzyme. control HMG-CoA reductase.. (Bile acid binding capacity) 41% cholate, 39% chenodeoxycholat, 15% deoxycholate, 4% ursodeoxycholate, 1% lithocholate. 1 3-96 -
cholic acid sodium salt glycine taurine glycoholic acid sodium salt taurocholic acid sodium salt, 1 7-a-dehydroxylation 2 2 deoxycholic acid sodium salt., ( 1%). feedback,. Table 52.. cholic acid 5.89% deoxycholic acid, taurocholic acid, glycocholic acid. Protamex glycocholic acid 15.91%, deoxycholic acid 12.07%. Neutrase glycocholic acid 9.63% Protamex glycocholic acid., protamex 4 glycocholic acid deoxycholic acid. Table 52. Enzyme Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Control 5.89 0.00 0.00 0.00 Alcalase 5.81 6.89 6.55 4.54 Flavourzyme 7.02 4.56 7.59 4.13 Protamex 6.11 12.07 6.87 15.91 Neutrase 5.74 6.84 6.77 9.63-97 -
. Fibrinolytic activity (Table 53), KFRI 150 (Lactobacillus acidophilus), KCTC 3552 (Enterococcus flavescens), KCTC 3100 (Leuconostoc mesenteroides subsp. mesenteroides) 3, 2, 4. thrombin fibrinogen fibrin. (fibrin clots) plasmin (fibrinolytic enzyme), (thrombosis)., 33.. HMG-CoA reductase Table 54 HMG-CoA reductase. KCTC 3552 (Enterococcus flavescens) 4 281.96% KCTC 3195 (Enterococcus faecalis var. liquefaciens) 4 219.53% KCTC 3552 (Enterococcus flavescens). KFRI 150 (Lactobacillus acidophilus) 4 120.66%, KCTC 3112 (Lactobacillus fermentum) 114.48%, KFRI 654 (Lactobacillus curvatus) 90.1% KCTC 3526 (Leuconostoc citreum) 59.17% HMG-CoA reductase. 4.. - 98 -
Table 55 4. 33. KFRI 150 (Lactobacillus acidophilus) 4 cholic acid 8.12%, deoxycholic acid 9.80%, taurocholic acid 7.60%, glycocholic acid 6.98%. KCTC 3507 (Pediococcus pentosacius) cholic acid 9.58%, deoxycholic acid 8.02%, taurocholic acid 7.41%, glycocholic acid 6.30%. - 99 -
Table 53. Strain Fermentation time (days) 1 2 3 4 5 6 7 8 KCTC 3638 - - - - - - - - KCTC 3195 - - - - - - - - KCTC 3552 - - +++++ - - - - - KCTC 3102 - - - - - - - - KCTC 3641 - - - - - - - - KCTC 3102 - - - - - - - - KFRI 150 - +++++ - - - - - - KFRI 238 - - - - - - - - KCTC 3102 - - - - - - - - KFRI 1030 - - - - - - - - KFRI 346 - - - - - - - - KFRI 1182 - - - - - - - - KFRI 654 - - - - - - - - KCTC 1047 - - - - - - - - KCTC 3112 - - - - - - - - KCTC 3602 - - - - - - - - KFRI 481 - - - - - - - - KCTC 1048 - - - - - - - - KCTC 3594 - - - - - - - - KCTC 3205 - - - - - - - - KFRI 684 - - - - - - - - KCTC 201 - - - - - - - - KCTC 3524 - - - - - - - - KCTC 3526 - - - - - - - - KCTC 3102 - - - - - - - - KCTC 3528 - - - - - - - - KCTC 3530 - - - - - - - - KCTC 3100 - - - +++++ - - - - KFRI 832 - - - - - - - - KCTC 3507 - - - - - - - - KCTC 3807 - - - - - - - - KFRI 1184 - - - - - - - - KCTC 3531 - - - - - - - - - 100 -
Table 54. HMG-CoA reductase Strain Fermentation time (days) 1 2 3 4 5 6 7 8 KCTC 3638 - - - - - - - - KCTC 3195-105.49-219.53 - - - - KCTC 3552 - - - 281.96 - - - - KCTC 3102 - - - - - - - - KCTC 3641-222.72 - - - - - - KCTC 3102 - - - - - - - - KFRI 150-91.38-120.66 - - - - KFRI 238-110.61 - - - - - - KCTC 3102-53.99-23.65 - - - - KFRI 1030 - - - - - - - - KFRI 346-406.86 - - - - - - KFRI 1182-27.15 - - - - - - KFRI 654-171.97-90.01 - - - - KCTC 1047 - - - - - - - - KCTC 3112-119.85-114.48 - - - - KCTC 3602 - -157.9 - - - - - - KFRI 481 - -44.63 - - - - - - KCTC 1048-17.790 - - - - - - KCTC 3594 - - - - - - - - KCTC 3205 - - - - - - - - KFRI 684-138.82 - - - - - - KCTC 201-64.419 - - - - - - KCTC 3524-40.57 - - - - - - KCTC 3526 - - - 59.17 - - - - KCTC 3102 - - - - - - - - KCTC 3528 - - - - - - - - KCTC 3530-378.83 - - - - - - KCTC 3100 - - - - - - - - KFRI 832-7.49 - - - - - - KCTC 3507-203.30 - -81.58 - - - - KCTC 3807-136.45 - -238.1 - - - - KFRI 1184-34.39 - - - - - - KCTC 3531 - - - - - - - - - 101 -
Table 55. Fermentation time (days) Strain 0 2 4 C D T G C D T G C D T G Control 2.64 3.59 3.01 3.30 2.87 2.98 3.01 2.81 2.51 2.98 2.72 2.81 KCTC 3638 - - - - 4.58 5.81 5.95 5.28 - - - - KCTC 3195 2.95 3.42 3.17 3.14 3.70 4.92 4.17 4.18 5.10 6.93 5.83 6.21 KCTC 3552 2.93 3.17 3.15 2.53 - - - - 7.65 8.71 8.82 6.90 KCTC 3528 - - - - 4.57 5.49 5.39 5.38 - - - - KCTC 3641 - - - - 4.86 6.03 5.47 5.73 6.32 7.66 7.89 6.28 KCTC 3630 - - - - - - - - - - - - KFRI 150 2.49 3.16 2.91 3.41 4.96 6.52 6.45 6.15 8.12 9.80 7.60 6.98 KFRI 238 - - - - 3.93 4.69 4.13 3.88 5.94 8.55 6.70 6.12 KCTC 3102 - - - - 3.14 3.92 3.63 4.89 5.45 5.90 6.39 6.19 KFRI 346 - - - - 4.96 6.29 6.29 5.69 - - - - KFRI 1182 - - - - 4.26 5.22 4.96 4.70 - - - - KCTC 1047 - - - - - - - - 5.59 7.30 7.02 6.41 KCTC 3112 - - - - 3.26 4.08 3.85 3.59 6.59 8.11 7.47 7.25 KCTC 3602 - - - - 4.78 6.21 6.15 5.57 - - - - KFRI 481 - - - - 3.99 5.00 4.61 4.33 5.95 7.74 7.45 6.61 KCTC 1048 - - - - 3.63 4.41 4.51 4.19 - - - - KCTC 3594 - - - - 4.47 6.23 5.71 5.37 - - - - KCTC 3205 - - - - 4.78 6.27 6.05 5.68 - - - - KFRI 1030 - - - - 4.04 4.79 4.81 4.96 - - - - KFRI 654 - - - - 3.54 4.77 4.29 3.89 5.44 7.55 7.30 6.79 KCTC 201 - - - - 4.82 6.09 6.04 5.74 - - - - KFRI 684 - - - - 7.45 6.30 5.55 5.32 - - - - KCTC 3524 - - - - 4.15 5.06 3.95 5.35 6.28 7.03 7.27 7.23 KCTC 3526 - - - - - - - - 6.29 7.71 7.58 6.79 KCTC 3527 - - - - - - - - 6.12 6.76 7.18 6.56 KCTC 3528 - - - - - - - - - - - - KCTC 3530 3.21 2.95 3.27 3.17 - - - - - - - - KCTC 3100 - - - - - - - - - - - - KFRI 832 - - - - 3.53 5.08 4.37 4.13 6.53 7.67 6.88 6.71 KCTC 3507 - - - - 3.94 4.34 4.62 4.09 9.58 8.02 7.41 6.30 KCTC 3807 - - - - - - - - 6.33 7.64 6.90 6.50 KFRI 1184 - - - - 4.76 6.19 5.59 5.08 - - - - KCTC 3531 - - - - - - - - - - - - C : Cholic acid, D : Deoxycholic acid, T : Taurocholic acid, G : Glycocholic acid - 102 -
5.. protamex HMG-CoA reductase, bile acid, DPPH. HMG-CoA reductase Table 56.. Bile acid glycocholic acid. plate DPPH 43.2% 2. Table 56. HMG-CoA reductase Sample Inhibition activity (%) Supernatant 84.5 Precipitate 75.1 Table 57. Binding capacity (um) Sample Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Supernatant 16.2 19.6 17.4 19.7 Precipitate 15.2 18.1 17.1 19.1 Table 58. Sample Fibrinolytic activity (cm) Supernatant - Precipitate - - 103 -
Table 59. DPPH Sample DPPH radical scavenging effect (%) Supernatant 43.2 Precipitate 23.5. 1) HMG-CoA reductase Table 60. ethanol 76.5%. 10~30%. Table 60. HMG-CoA reductase Item Solvent Inhibition activity (%) n-hexane 31.0 CHCl 3 34.4 상-상 1) E.A 51.9 n-buoh 36.4 EtOH 76.5 n-hexane 13.9 CHCl 3 15.2 상-침 2) E.A 21.2 n-buoh 13.1 EtOH 20.7 n-hexane 12.7 CHCl 3 12.9 침-상 3) E.A 18.8 n-buoh 15.2 EtOH 33.2 n-hexane 5.3 CHCl 3 6.1 침-침 4) E.A 10.6 n-buoh 4.5 EtOH 5.9 1) 2) 3) 4) - 104 -
2) Bile acid binding capacity Table 61.. ethanol ethyl acetate 4 deoxycholic acid glycocholic acid. Table 61. Item 상-상 1) 상-침 2) 침-상 3) 침-침 4) Binding capacity (um) Solvent Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid n-hexane 14.8 16.2 15.4 15.2 CHCl 3 13.5 15.3 14.1 13.4 E.A 18.5 23.3 22.0 20.2 n-buoh 17.0 20.7 19.4 18.2 EtOH 20.8 25.2 23.1 24.2 n-hexane 8.4 10.9 10.1 8.7 CHCl 3 10.2 11.7 11.0 10.5 E.A 11.3 12.5 12.1 10.8 n-buoh 7.8 8.8 7.9 7.2 EtOH 7.2 7.7 7.7 6.7 n-hexane 8.1 9.6 9.3 8.8 CHCl 3 6.8 10.3 9.6 9.1 E.A 10.9 10.7 10.2 8.6 n-buoh 10.9 13.3 14.0 10.4 EtOH 9.6 18.4 18.1 22.3 n-hexane 7.5 9.1 7.4 5.8 CHCl 3 9.7 12.6 11.6 8.6 E.A 10.5 14.0 12.2 12.0 n-buoh 5.6 6.8 7.6 5.6 EtOH 5.0 6.0 5.5 4.9 1) 2) 3) 4) - 105 -
3) Table 62.. Table 62. 상-상 1) 상-침 2) 침-상 3) 침-침 4) Sample Fibrinolytic activity (cm) n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2) 3) 4) 4) Ethyl acetate 69.2%, Ethanol 50.9% n-butanol 45.1%... - 106 -
Table 63. DPPH 상-상 1) 상-침 2) 침-상 3) 침-침 4) Sample DPPH radical scavenging effect (%) n-hexane - CHCl 3 4.7 E.A 69.2 n-buoh 45.1 EtOH 50.9 n-hexane - CHCl 3 - E.A 4.5 n-buoh - EtOH 6.7 n-hexane - CHCl 3 - E.A - n-buoh 16.9 EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2) 3) 4) - 107 -
. 1) HMG-CoA reductase, bile acid, DPPH.. HMG-CoA reductase 72.3% bileacid 4,. 65.1%. Table 64. HMG-CoA reductase Sample Inhibition activity (%) Supernatant 72.3 Precipitate 9.7 Table 65. Sample Binding capacity (um) Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Supernatant 7.1 8.7 7.3 5.9 Precipitate 5.5 6.2 6.2 5.7 Table 66. Sample Fibrinolytic activity (cm) Supernatant - Precipitate - Table 67. DPPH Sample DPPH radical scavenging effect (%) Supernatant 65.1 Precipitate - - 108 -
. 1) HMG-CoA reductase HMG-CoA reductase inhibition. Table 68. HMG-CoA reductase ethanol (95.2%). Table 68. HMG-CoA reductase Item Solvent Inhibition activity (%) 상 - 상 1) 상 - 침 2) n-hexane 46.6 CHCl 3 60.1 E.A 87.9 n-buoh 84.0 EtOH 95.2 n-hexane 39.8 CHCl 3 49.1 E.A 40.9 n-buoh 39.8 EtOH 47.2 1) 2) 2) Bile acid binding capacity Table 69. cholic acid, deoxycholic acid, taurocholic acid glycocholic acid n-butanol Ethanol. n-hexane. - 109 -
Table 69. Item Solvent Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid n-hexane 6.5 7.9 6.8 6.6 CHCl 3 7.2 8.3 7.9 7.1 상 - 상 1) E.A 8.7 9.9 11.3 9.0 n-buoh 11.8 17.7 13.1 17.6 EtOH 10.3 16.4 9.9 10.1 n-hexane 7.4 7.8 7.2 7.4 상 - 침 2) CHCl 3 6.1 7.0 6.6 6.1 E.A 7.0 7.7 7.8 7.0 n-buoh 4.4 4.9 5.1 4.5 EtOH 3.4 4.1 4.0 3.7 1) 2) 3) Table 70.. Table 70. Sample Fibrinolytic activity (cm) 상 - 상 1) 상 - 침 1) n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2) - 110 -
4) Table 71. Ethanol 68.9%. n-butanol 53.3%. n-hexane 4.2%. n-hexane chloroform, Ethanol 9.1%. Table 71. DPPH Sample DPPH radical scavenging effect (%) 상 - 상 1) 상 - 침 2) n-hexane 4.2 CHCl 3 12.1 E.A 34.4 n-buoh 53.3 EtOH 68.9 n-hexane - CHCl 3 - E.A 5.2 n-buoh 3.6 EtOH 9.1 1) 2). 1) HMG-CoA reductase ethanol HMG-CoA table 72. HMG-CoA reductase 93.4%. 100,000-111 -
5.7~18.9% 30,000~ 50,000 64.4%. ethanol 30,000~50,000. 89.9%. 30,000 66.5% 30,000. Table 72. ethanol HMG-CoA reductase Inhibition activity (%) (dalton) Control* 93.4 89.9 300,000 5.7 4.5 300,000 88.3 82.0 100,000-300,000 18.9 17.0 100,000 73.0 71.3 50,000-100,000 7.3 7.5 50,000 60.1 67.2 30,000-50,000 64.4 9.6 30,000 12.9 65.0 10,000-30,000 9.1 33.6 10,000-5.7 26.7 Control*: ethanol 2) ethanol HMG-CoA reductase 30,000~50,000. 4 deoxycholic acid 30,000-112 -
. HMG-CoA reductase ethanol 30,000~50,000 gel-filtration. Table 73. ethanol bile acid binding capacity Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid Control* 10.6 17.2 9.2 10.1 300,000 3.0 2.8 1.1 0.9 300,000 8.6 14.0 7.8 8.4 100,000-300,000 1.7 3.0 1.1 1.3 100,000 6.9 11.2 7.2 7.0 50,000-100,000 1.2 0.1 1.3 1.8 50,000 6.5 9.4 6.2 5.6 30,000-50,000 5.7 7.7 6.8 5.3 30,000 1.0 1.9 0.7 0.2 10,000-30,000 0.3 0.7 0.3 0.2 10,000 0.0 0.1 0.3 0.1 Control*: ethanol - 113 -
Table 74. ethanol bile acid binding capacity Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid Control* 21.4 26.1 24.0 23.6 300,000 1.2 2.0 1.7 2.2 300,000 19.4 25.0 23.2 18.6 100,000-300,000 4.4 2.5 3.6 3.1 100,000 18.7 22.2 19.2 18.2 50,000-100,000 2.7 1.4 4.7 4.4 50,000 15.8 20.2 15.2 12.2 30,000-50,000 1.5 1.4 1.7 1.4 30,000 14.8 17.8 11.5 11.1 10,000-30,000 4.8 4.8 3.2 4.6 10,000 5.9 8.3 8.0 5.5 Control*: ethanol. Gel chromatography 1) Gel chromatograpy Gel filtration 280 nm O D (Fig. 10, Fig. 11). 50,000~30,000 dalton gel filtration 280 nm Fig. 10.. 10~30 40~50 peak. 30,000 dalton gel filtration Fig. 11. 4 (Ⅰ : 10~45, Ⅱ : 51~60, Ⅲ : 86~100, Ⅳ : 121~125). 4. - 114 -
Fig. 10. Gel chromatograpy Sephedex G-75 (280 nm) * : 탄수화물, 지질, 단백질측정 fraction - 115 -
Fig. 11. Gel chromatograpy Sephedex G-75 (280 nm) * : 탄수화물, 지질, 단백질측정 fraction 2) fraction No. 11~30 41~50, fraction No. 16~28 22 23 45.5%. (Ⅰ : 11~45, Ⅱ : 51~60, Ⅲ : 86~100, Ⅳ : 121~125) fraction, Ⅰ 34~40 15%, Ⅱ 52~58 22% Ⅲ Ⅳ.. - 116 -
Table 75. Gel chromatograpy Fraction number Total carbohydrate (%) 11 7.8 12 8.0 13 8.2 14 8.0 15 8.2 16 10.0 17 11.2 18 15.0 19 16.0 Ⅰ 20 17.0 21 34.0 22 45.5 23 45.9 24 16.8 25 16.5 26 15.5 27 14.2 28 13.2 29 5.5 30 5.4 41 0.9 42 1.5 43 1.5 44 1.6 Ⅱ 45 8.7 46 1.7 47 1.5 48 1.0 49 0.6 50 0.2-117 -
Table 76. Gel chromatograpy Fraction number Total carbohydrate (%) 11 1.2 12 3.3 13 3.5 14 3.6 15 4.4 16 4.6 17 6.2 18 6.2 19 6.3 20 7.0 21 7.1 22 7.3 23 7.5 24 7.6 25 7.7 26 8.0 Ⅰ 27 8.1 28 8.3 29 9.0 30 9.3 31 8.5 32 6.0 33 9.1 34 12.1 35 13.3 36 14.0 37 14.3 38 14.5 39 14.8 40 15.9 41 6.0 42 6.0 43 4.1 44 3.5 45 3.2 continued - 118 -
Ⅱ Ⅲ Ⅳ 51 8.9 52 22.5 53 23.5 54 23.6 55 22.5 56 22.0 57 21.2 58 20.1 59 15.9 60 0.1 85 0.1 86 5.1 87 6.2 88 7.5 89 9.0 90 7.9 91 7.9 92 6.8 93 6.5 94 7.6 95 3.2 96 1.7 97 1.6 98 1.0 99 2.0 100 1.2 121 1.2 122 1.4 123 1.6 124 1.5 125 1.2 3) gel filtration, Ⅰ fraction No. 26~29 10.0~13.2%, Ⅱ 45 11%.. Ⅰ 10% Ⅱ 54~56 15%. Ⅲ Ⅳ 95 123 10.2% 17.2%. - 119 -
Table 77. Gel chromatograpy Fraction number Total protein (%) 11 0.0 12 1.2 13 1.3 14 1.4 15 2.3 16 2.5 17 2.7 18 2.9 19 3.4 20 5.0 Ⅰ 21 6.0 22 6.3 23 6.9 24 7.9 25 9.3 26 10.0 27 11.0 28 12.5 Ⅱ 29 13.2 30 8.0 41 1.2 42 3.5 43 4.6 44 4.5 45 11.0 46 3.0 47 2.8 48 2.5 49 2.0 50 1.0-120 -
Table. 78. Gel chromatograpy Fraction number Total protein (%) 11 0.1 12 0.1 13 0.1 14 1.0 15 1.0 16 1.2 17 1.3 18 2.1 19 2.2 20 3.1 21 3.3 22 4.2 23 4.3 24 5.8 25 5.8 26 6.0 Ⅰ 27 6.0 28 6.1 29 6.3 30 6.5 31 6.8 32 7.9 33 7.8 34 8.0 35 8.0 36 8.1 37 8.2 38 9.0 39 9.1 40 9.2 41 9.2 42 9.4 43 10.0 44 10.3 45 5.1 continued - 121 -
Ⅱ Ⅲ Ⅳ 51 2.1 52 13.9 53 14.9 54 15.9 55 16.0 56 15.2 57 13.1 58 10.9 59 5.9 60 0.0 85 0.2 86 3.3 87 3.5 88 3.0 89 4.0 90 5.5 91 6.2 92 7.9 93 8.0 94 9.8 95 10.2 96 2.2 97 2.0 98 1.9 99 1.5 100 1.2 121 1.0 122 6.5 123 17.2 124 5.4 125 2.3 4), Ⅰ. 23 5.3%. fraction, Ⅰ fraction No.36~37 12.0~13.2%, Ⅱ Ⅲ.. - 122 -
Table. 79. Gel chromatograpy Fraction number Total lipid (%) 11 0.3 12 0.3 13 0.3 14 0.4 15 0.5 16 0.7 17 0.7 18 0.8 19 0.7 Ⅰ 20 0.9 21 3.5 22 4.5 23 5.3 24 2.0 25 1.8 26 1.6 27 1.0 28 1.2 29 0.9 30 1.0 41 0.3 42 0.5 43 0.5 Ⅱ 44 0.5 45 3.0 46 1.0 47 0.5 48 0.4 49 0.2 50 0.0-123 -
Table 80. Gel chromatograpy Fraction number Total protein (%) 11 1.3 12 3.0 13 3.0 14 3.1 15 3.2 16 4.6 17 4.7 18 4.8 19 5.0 20 5.1 21 5.2 22 5.4 23 5.5 24 5.2 25 5.4 26 6.0 27 6.3 Ⅰ 28 6.4 29 7.0 30 8.0 31 8.0 32 8.4 33 8.6 34 8.9 35 9.0 36 12.0 37 13.2 38 6.3 39 6.9 40 8.0 41 8.6 42 8.6 43 7.0 44 6.8 45 6.6 continued - 124 -
51 4.5 52 4.6 Ⅱ Ⅲ Ⅳ 53 4.0 54 4.0 55 4.0 56 4.0 57 3.0 58 2.0 59 1.0 60 0.0 85 0.6 86 0.3 87-0.2 88 0.0 89 0.1 90 0.3 91-0.1 92 0.3 93 0.2 94 0.1 95 0.0 96-0.5 97 0.2 98 0.3 99 0.2 100 0.4 121-0.5 122 0.0 123 0.3 124 0.3 125 0.0 30,000~50,000 dalton 30,000 dalton gel filtration 280 nm 2, 4., Ⅰ 21~25 45%, 13%, 5%. Ⅱ Ⅰ 45 9%, 11% - 125 -
1%. Ⅰ 35~40 15%, 8%, 13.2% Ⅱ 55 23%, 16%. 5%. Ⅲ 95 10%. Ⅳ Ⅲ 17%. Ⅰ Ⅱ Ⅰ,, Ⅱ. Ⅲ Ⅳ. 5) HMG-CoA reductase,, peak fraction HMG-CoA reductase., 10% HMG-CoA reductase, Ⅰ fraction No. 21~22 52%. 4, HMG-CoA reductase, Ⅰ Ⅳ 10~20%. Ⅱ 56 49.0% Ⅲ 95 58.9%. - 126 -
Table 81. Gel chromatograpy HMG-CoA reductase Fraction number Inhibition activity (%) 16 30.2 17 35.0 18 36.1 19 45.8 20 50.2 21 52.1 22 52.6 23 40.0 24 30.2 25 28.5 26 26.0 27 26.8 28 24.0 29 22.0 30 20.1 45 19.0 Table 82. Gel chromatograpy HMG-CoA reductase Fraction number Inhibition activity (%) 34 17.5 35 18.2 36 18.6 37 19.0 38 20.1 39 23.3 40 18.9 52 10.2 53 11.3 54 13.0 55 35.2 56 49.0 57 44.0 58 43.0 59 40.5 91 32.6 92 29.5 93 38.6 94 43.5 95 58.9 123 10.0-127 -
6) 2 HMG-CoA reductase fraction No. 24. 4 Taurocholic acid. 4 HMG-CoA reductase Ⅰ, Ⅳ 5 um. Ⅱ 55~57 Ⅲ 93~95. Table 83. Gel chromatograpy Fraction Binding capacity (um) number Cholic acid Deoxycholic Taurocholic Glycocholic acid acid acid 16 5.5 6.4 5.1 3.5 17 6.6 4.3 8.3 5.4 18 7.3 8.4 11.0 3.9 19 7.3 6.1 10.9 7.6 20 8.9 15.2 19.0 7.7 21 6.5 7.1 20.0 7.9 22 5.0 10.5 25.0 6.6 분획 23 7.2 11.0 26.5 7.1 24 8.7 9.9 11.3 9.0 25 6.0 5.2 3.2 3.2 26 5.2 5.0 3.0 2.2 27 5.1 4.1 2.9 1.0 28 5.3 4.3 2.5 2.4 29 4.0 3.0 1.2 1.0 30 3.5 2.8 1.0 1.0 분획 45 4.5 3.2 1.0 0.9-128 -
Table 84. Gel chromatograpy Binding capacity (um) Fraction number Deoxycholic Taurocholic Glycocholic Cholic acid acid acid acid 34 5.4 4.3 2.8 2.5 35 3.6 3.8 4.2 4.1 36 4.5 5.3 6.5 4.0 분획 37 4.6 6.2 7.1 6.5 38 2.0 1.9 2.3 4.3 39 3.8 3.3 2.9 3.2 40 4.3 4.5 6.2 3.1 52 5.6 6.3 6.4 6.0 53 6.0 7.0 6.3 5.5 54 6.5 6.5 6.7 6.8 분획 55 7.0 6.9 6.3 7.4 56 7.8 10.2 10.0 9.6 57 7.2 9.0 9.2 6.5 58 6.3 3.6 4.1 6.6 59 5.0 3.0 4.6 4.5 91 5.4 5.3 3.3 4.5 92 3.8 3.3 4.5 2.4 분획 93 6.7 6.5 7.2 4.5 94 7.8 8.2 6.5 7.6 95 9.8 8.3 8.7 6.5 분획 123 4.5 5.6 5.4 5.5 7) (Sephadex G-75, ((3-80 kda)). Fig. 12 280nm. gel filtration chromatography molecular weight gel column molecular weight gel column. volume (Table 85). molecular weight Fig. 12 Albumin(66 kda), Carbonic Anhydrase(29 kda), Cytochrome C(12.4 kda), Aprotinin S(6.5 kda), Aomatostatin(1.6 kda). - 129 -
Table 85. Gel filtration Sample Molecular weight (Da) Log molecular weight (ml) Somatostatin 1640.0 3.2 476.0 0.36 Aprotinin 6500.0 3.8 388.0 0.28 Cytochrome C 12400.0 4.1 340.0 0.24 Carbonic Anhydrase 29000.0 4.5 292.0 0.20 Albumin 66000.0 4.8 212.0 0.13 * π 길이 r=2.8 cm, =50 cm 2.0 1.5 1.0 0.5 0.0-0.5 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 number Fracration Fig. 12. Sephadex G-75 gel filtration peak - 130 -
80000 75000 70000 Albumin 65000 60000 55000 50000 45000 40000 35000 ES-2 Carbonic anhydrase 30000 25000 20000 15000 Cytochrome C 10000 Aprotinin ES-3 5000 Somatostatin 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 number Fraction Fig. 13. molecular weight * ES-2: enzyme hydrolysis sample from Dioscorea batatas (no. 56) * ES-3: enzyme hydrolysis sample from Dioscorea batatas (no. 95) 4 Ⅱ Ⅲ. Ⅱ 56, Ⅲ 95 HMG-CoA reductase peak 56 29 kda Carbonic Anhydrase, 95 1.6 kda Somatostatin. - 131 -
80000 75000 Albumin 70000 FS-1 65000 60000 55000 50000 45000 40000 35000 30000 Carbonic anhydrase 25000 20000 15000 Cytochrome C 10000 Aprotinin 5000 Somatostatin 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 number Fraction Fig. 14. molecular weight * FS-1: fermented sample from Dioscorea batatas (no. 22) M.W (Fig. 14). Ⅰ HMG-CoA reductase Ⅱ 22. Fig. 12 peak 22 66 kda albumin.. 1) ethyl acetate ethanol. maillard reaction. - 132 -
Fig. 15. Fig. 16. protamex - 133 -
. 100,000 dalton 50,000 dalton, 30,000 dalton, 10,000 dalton. Fig. 17. Fig. 18. - 134 -
2) n-butanol ethanol, ethyl acetate. maillard reaction. Fig. 19. Fig. 20. - 135 -
, 100,000 dalton.. Fig. 21. Fig. 22. - 136 -
6.. 1) ph 5 ph, 100:0, 80:20, 50:50, 20:80 ph 6.2~6.5, ph 4.3, 0:100 ph 6.89, ph 5.21 4. Table 86. ph : 100:0 80:20 50:50 20:80 0:100 0 6.25 6.21 6.22 6.49 6.89 1 5.83 6.02 6.03 6.12 6.71 2 5.72 5.81 5.83 5.61 5.85 3 4.79 4.56 4.95 5.33 5.41 4 4.52 4.42 4.45 5.25 5.25 5 4.41 4.34 4.41 4.59 5.13 6 4.35 4.33 4.36 4.35 4.99 7 4.32 4.33 4.35 4.34 5.06 8 4.28 4.32 4.35 4.32 5.21 2) 5, 100:0, 80:20, 50:50, 20:80, 0:100. - 137 -
Fig. 23. 3),..,.,. 20:80. - 138 -
Table 87. : 100:0 80:20 50:50 20:80 0:100 6.4 6.4 5.4 4.6 3.4 7.5 7.5 6.2 4.5 3.5 4.0 3.2 3.0 2.4 1.6 1.8 2.0 2.5 3.8 4.2 5.0 5.3 6.0 7.2 7.4 3.2 3.0 4.2 6.0 5.2. 1) ph,, ph 4.. ph 4.3, 0.33, 15%. Table 88. ph,, : 0:0 0:1 1:0 1:1 ph 4.4 4.3 4.3 4.4 (%) 0.34 0.33 0.32 0.33 (%) 14.7 14.9 14.9 15.0 2),,,,.. - 139 -
Table 89. : 0:0 0:1 1:0 1:1 7 7 8 9 8 7 7 6 8 6 5 4 6 7 7 8 6 6 7 7 5 7 6 8. 1) Table 90, 5 %, 20 %, 10 %. Table 90. 5% 10% 15% 20% 25% 30% 7.63 6.34 5.23 4.98 3.87 2.46 1.12 2.54 4.65 5.43 6.54 8.03 ( ) 6.74 6.28 5.15 2.84 1.95 1.21 5.43 6.53 4.51 4.72 3.37 2.62 2) Table 91,.,, 15%. - 140 -
Table 91. 5% 10% 15% 20% 25% 30% 6.87 7.88 8.43 8.67 8.85 8.97 2.80 3.27 4.21 4.56 4.64 4.72 ( ) 6.91 5.85 5.02 4.27 4.07 3.78 7.05 7.31 7.95 6.19 6.33 6.24 3) Table 92,. 3%. Table 92. 1% 3% 5% 7% 10% 7.64 6.53 5.42 5.44 4.76 3.79 4.57 5.88 6.98 7.02 ( ) 8.25 7.89 7.21 6.87 3.64 4.59 4.68 3.25 3.11 2.15-141 -
Fig. 24.. 1), 2%. 1.5%, 2.5%. Table 93. 0.5% 7.23 5.23 2.01 7.41 2.35 1.0% 7.34 5.30 4.41 7.50 5.61 1.5% 7.19 5.26 4.76 7.43 6.28 2.0% 7.20 5.37 5.25 7.56 8.47 2.5% 7.31 5.34 7.68 7.39 4.65 3.0% 7.28 5.31 7.92 7.51 2.31 2) - 142 -
, 50%,. Table 94. 30% 6.59 4.10 4.08 6.71 6.65 35% 6.52 4.28 4.16 6.63 6.94 40% 6.34 5.34 4.35 7.21 7.23 45% 6.12 5.86 5.27 7.18 7.58 50% 5.97 6.45 5.65 7.34 8.14 3), 10% 20% 30%. Table 95. 10% 6.09 6.87 5.32 8.51 8.12 20% 6.34 6.63 5.25 8.39 7.68 30% 6.62 5.89 5.33 8.27 7.59 4) 20%, 30%. - 143 -
Table 96. 10% 1.13 6.15 5.39 6.86 7.98 20% 1.07 6.16 5.43 6.73 8.35 30% 1.18 6.21 5.50 6.69 8.07 5) 30%. Table 97. 10% 1.23 7.31 5.47 7.42 6.32 20% 1.11 7.29 5.58 7.58 6.54 30% 1.26 7.24 5.69 7.72 6.81 6) 5%,. Table 98. 3% 1.61 4.87 5.15 5.97 3.57 5% 1.64 4.78 5.24 5.68 3.83 7% 1.78 4.66 5.39 5.34 3.19 7),,,,,,,. - 144 -
. 2. 2 %, 50 % 10 %, 20 %, 5 %.,,. Table 99. 10% 20% 30% 5% 6.09 1.07 1.26 1.64 6.87 6.16 7.24 4.78 5.32 5.43 5.69 5.24 8.51 6.73 7.72 5.68 8.12 8.35 6.81 3.83 Fig. 25. - 145 -