보안과제( ), 일반과제( o ) S120910L120100 임업기술연구개발, 보조포함 고부가가치갈매보리수나무육종및기능성소재개발연구 Study on improvement and development of valuable materials with Vitamin trees(hippophae rhamnoides) 동국대학교산학협력단 산림청
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Table 1. Morphological characteristics of 5 seed sources of Hippophae rhamnolides Seed sources M1 M2 R1 R2 C Width (mm) 2.3±0.47 2.03±0.18 2.27±0.45 2.2±0.41 2.23±0.43 Length (mm) 5.3±0.53 2.47±0.57 4.97±0.76 4.93±0.83 5.73±0.45 No. of Seed (10g) 779 1252 661 834 566 Seed coat color brown, luster light brown, luster brown dark brown dark brown Seed shape medium small round long oval long oval long & narrow - 16 -
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Fig 1-2. Effects of GA 3 and Filterpaper for Germination on White media. - 18 -
Table 1-2. Effects of treatments of Filterpaper and Carbon sources for germination on White media in H. rhamnoides. Carbon sources Filterpaper Germination(%) Contamination(%) stem Length(cm) root 3% sucrose 3% sugar O 90.57 0.94 1.9±0.72 2.27±1.25 X 86.76 1.47 1.62±0.55 2.18±1.24 O 87.25 0.98 1.57±0.76 2.00±1.24 X 86.58 2.44 1.55±0.74 2.07±1.39 Mean±standard deviation - 19 -
A B C D Fig 1-3. Seed germination of H. rhamnoides in in vitro. A. excluding filterpaper, B. including filterpaper, C. 3% sucrose, D. 3% sugar - 20 -
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Table 1-3. Effect of carbon source for germination from seed of different clone on WPM media in H. rhamnoides. Clone Carbon source Germination(%) Contamination(%) Mongol-1 Mongol-2 China Russia-1 Russia-2 Sucrose 82.4±4.2 5.8±0.8 Sugar 95.0±2.6 8.3±0.5 Sucrose 52.7±6.7 21.8±9.6 Sugar 67.2±6.2 21.6±1.1 Sucrose 61.8±5.0 26.0±3.0 Sugar 73.3±7.5 25.0±3.4 Sucrose 71.1±9.5 67.8±7.4 Sugar 78.2±7.8 45.8±7.02 Sucrose 26.7±6.7 100.0 Sugar 50.0±17.3 90.0±10.0-22 -
Table 1-4. Effects of PGRs for the formation of somatic embryo and germination in SH media in H. rhamnoides. PGRs (mg/l) Kin 1.0 + IAA 0.5 Kin 2.0 + IAA 1.0 BA 1.0 + IAA 0.5 BA 2.0 + IAA 1.0 Somatic embryo formation(%) 11.11 20.59 34.78 77.77 Somatic embryo germination(%) 5.98 8.82 8.69 0.00-23 -
Table 1-5. Effects of media and concentrations of GA3 on somatic embryo germination in H. rhamnoides. Media SH WPM Concentrations of GA 3 (mg/l) 1.0 3.0 1.0 3.0 Germination of Somatic embryo(%) 4.00 6.15 6.00 4.00-24 -
A B C D Fig 1-4. Somatic embryo formation and germination in H. rhamnoides. (A) explant transfer to the media for somatic embryo formation (B) somatic embryo formation from explant after 3 weeks (C) initial stage of somatic embryo germination (D) elongation from germinated somatic embryo - 25 -
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Table 1-6. Effects of PGRs for organogenesis from cotyledon(from in vitro) and leaf(from ex vitro) on WPM media in H. rhamnoides. Clone PGRs(mg/L) Induction of organogenesis(%) Germination rates BA Kin IAA cotyledon leaf cotyledon leaf 1.0 5.0 63.9±21.93 93.1±4.2-36.7±7.0 Mongol-1 1.0 5.0 36.1±19.9 73.4±12.5-11.1±4.5 1.0 1.0 5.0 69.13±8.44 81.1±10.2-66.7±9.0 Mongol-2 China-1 1.0 5.0 40.3±10.2 25.9±11.1 - - 1.0 5.0 38.8±7.5 22.2±3.7 - - 1.0 1.0 5.0 45.5±3.2 77.8±11.1 - - 1.0 5.0-90.0±4.8-14.8±3.7 1.0 5.0 63.0±14.8 77.8±5.2 - - 1.0 1.0 5.0-96.8±3.2-33.3±11.1 1.0 5.0-75.5±15.1 - - Russia-1 1.0 5.0 38.9±5.5 66.7±33.3 - - 1.0 1.0 5.0-95.6±4.4-33.3±10.5 1.0 5.0-60.1±15.1 - - Russia-2 1.0 5.0-55.7±3.3 - - 1.0 1.0 5.0-85.6±13.4 - - - 27 -
A B C D E F Fig 1-5. Organogenesis from leaf(from ex vitro) supplemented with 1.0 mg/l BA, 1.0 mg/l Kin and 5.0 mg/l IAA on WPM medium (A) and (B) Proliferation of adventitious bud with 1.0 mg/l BA, Kin and 5.0 mg/l IAA, (C) and (D) Early proliferation for shoot, (E) Root developed, and (F) Shoot development. - 28 -
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Table 1-7. Germination rates on storage duration in UR media. Storage period 0 6 month ago 1 year ago Weeks 4 8 4 8 4 8 Germination(%) 66.9 ± 3.9 73.9 ± 2.5 59.3 ± 5.5 65.3 ± 3.1 43.5 ± 2.7 62.2 ± 4.5 Mean ± standard deviation - 30 -
A B C Fig 1-6. Effects of storage period for seed germination of H. rhamnoides in ex vitro. (A) 1 year, (B) 6 month, and (C) directly after harvest. - 31 -
A B C D Fig 1-7. Seed germination in ex vitro and acclimation. (A) germination on sand plastic pot, (B) germination on UR media, (C) and (D) transplant in field. - 32 -
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Fig 1-8. Effect of pot germination rates(%) on clone (M1) of H. rhamnoides. - 34 -
Fig 1-9. Effect of pot for length of shoot on clone (M1) of H. rhamnoides. - 35 -
Table 1-8. Different of weeks number of seedling from different 6 clones on H. rhamnoides. weeks M1 M2 M3 R1 R2 C1 2 0 148 224 25 16 10 4 4 150 340 160 24 140 6 6 44 280 210 9 170 8 4 10 75 350 17 131-36 -
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Fig 1-10. Seedling growth of H. rhamnoides in first year and second year in open ground. - 38 -
Fig 1-11. Seedling growth reproduced by cutting with 1 year branch from 5 years stock trees. - 39 -
μ μ μ μ μ - 40 -
Fig 1-12. Different clones of chlorophyll contents on H. rhamnoides from leaf of grown the ex vitro condition. - 41 -
Fig 1-13. Photosynthetic of 6 different clones of H. rhamonides. - 42 -
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Fig 1-14. Antioxidative enzyme activities of H. rhamnoides on different clones - 44 -
A B C D Fig 1-15. Histological examination of the adventitious bud differentiation from leaf segment. (A) early stage cell division on the portion of adventitious bud formation, (B) meristem for adventitious bud in central-leaf, (C) cross-section from induction shoot, and (D) expansion of meristem - 45 -
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Dry powder Hexane extraction Hexane extracts cake Ethanol extraction Ethanol extracts cake Hot water extraction Hot water extracts Methanol extraction cake waste Methanol extracts Water solubles Fig 2-1. Fractionation procedure of Sea buckthorn components - 49 -
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Table 2-1. HPLC analysis conditions for the separation of SE components Standard compounds Sample preparation method Conditions of analysis EGCG & Epigallocatechin & Catechin & Gallocatechin Gallate & Epicatechin & Epicatechin Gallate - sample in 10 ml Volumetric flask - 1) Sonication after added 10ml MeOH 2) Sonication after added 10ml MeOH and 0.1% HCl - Centrifuge (3500 rpm, 10 min) & Syringe filter(0.45 μm) - Analysis 1. Equipment Model Agilent 1200 HPLC 2. Column ZORBAX Eclipse XDB-C 18 250x4.0mmI.D.,5μm 3. Guard column ZORBAX Eclipse XDB-C 18 12.5x4.6mmI.D.,5μm 4. Mobile phase 5. Detector UV 280 nm 6. Column Temp. 30 7. Flow rate 8. Injection volume 5 ~ 10 μl Conditions 1 : 0.1% TFA in water : Acetonitrile = 85 : 15 Conditions 2 : 0.1% TFA in water : Acetonitrile = 95 : 05 Conditions 1 : 0.35 ml/min Conditions 2 : 0.2 ml/min - 54 -
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Fig 2-2. Fractionation procedure of Sea buckthorn fruits - 60 -
Fig 2-3. Oil fractionation by column chromatography - 61 -
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Fig 2-4. Solvent fractionation of Sea buckthorn materials - 64 -
Fig 2-5. Bleaching of hexane solution of Sea buckthorn oil by adsorbents - 65 -
Fig 2-6. Bleaching of hexane solution of Sea buckthorn oil by active carbon - 66 -
Fig 2-7. Absorption spectra of the hexane solution of Sea buckthorn oil bleached by active carbon (190nm 800nm) - 67 -
Fig 2-8. Effect of active carbon on the bleaching of Sea buckthorn oil - 68 -
Fig 2-9. Absorption spectra of pigments removed by active carbon from the Sea buckthorn oil - 69 -
Fig 2-10. Bleaching of hexane solution of Sea buckthorn oil by acid clay - 70 -
Fig 2-11. Absorption spectra of the hexane solution of Sea buckthorn oil bleached by acid clay (190nm 800nm) - 71 -
Fig 2-12. Effect of acid clay on the bleaching of Sea buckthorn oil - 72 -
Fig 2-13. Absorption spectra of pigments removed by acid clay from the Sea buckthorn oil - 73 -
Fig 2-14. Absorption spectra of pigments recovered from the spent earth by ethanol - 74 -
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Fig 2-15. Fatty acid compositions of oil fractions recovered from the Sea buckthorn fruits (2008) - 76 -
Table 2-2. Fatty acid compositions of various oil fractions recovered from Seabuckthorn fruits (2009) ID JH JHT JHP DH DHT DHP SH SHT SHP C14:0 0.30 0.25 0.45 0.53 0.46 0.85 0.11 0.09 0.17 C15:0 0.04 0.05 0.06 0.06 0.06 0.09 0.09 0.10 C16:0 35.99 36.03 35.67 34.06 34.10 34.51 6.02 5.95 6.44 C16:1 41.65 41.03 40.87 38.51 38.83 38.60 0.46 0.41 0.62 C16:1 0.13 0.14 0.14 0.15 0.14 0.14 C16:2 1.81 1.72 2.01 2.16 2.10 2.35 C17:0 0.04 0.04 0.04 0.05 C18:0 0.71 0.78 0.73 0.88 0.87 0.89 2.63 2.57 2.42 C18:1 2.68 2.84 2.59 2.71 2.74 2.57 15.64 16.06 14.36 C18:1 6.02 6.31 5.80 6.88 6.86 6.44 1.63 1.65 1.69 C18:2 9.69 9.86 10.43 11.81 11.50 11.73 38.84 39.25 38.86 C18:3 0.67 0.71 0.92 1.17 1.00 1.49 32.01 32.02 34.52 C20:0 0.18 0.20 0.17 0.25 0.24 0.21 0.39 0.40 0.36 C20:1 0.29 0.28 0.22 sample ID: JH: hexane extract of juice, JHT: neutral oil of JH, JHP: coloring compounds of JH DH: hexane extract of debris, DHT: neutral oil of DH, DHP: coloring compounds of DH SH: hexane extract of seed, SHT: neutral oil of SH, SHP: coloring compounds of SH - 77 -
Fig 2-16. Radical scavenging activity of Sea buckthorn components - 78 -
Fig 2-17. Protection effects of the extracts on cell damage induced by UVB (+UV, HaCaT, A; L-ascorbic acid 25µM) - 79 -
Fig 2-18. Protection effects of the extracts on cell damage induced by UVB (+UV, Fibroblast, A; L-ascorbic acid 25µM) - 80 -
Fig 2-19. Anti-inflammatory activity of extracts on UVB-induced TNF-α D; Dexamethasone 1µM - 81 -
Fig 2-20. Effect of extracts on the production of MMP-1. AS : Ascorbic acid 25μM - 82 -
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Fig 2-21. Protection effects of the SE extracts on cell damage induced by (+UV, Fibroblast, A; L-ascorbic acid 25µM) - 84 -
Fig 2-22. Collagen synthesis effect by SE extracts after UVB irradiation (AS : Ascorbic acid 25μM) - 85 -
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Table 2-3. DPPH free radical scavenging activity of Sea buckthorn components Concentration (mg/ml) 0.5 0.25 0.125 0.0625 0.03125 0.015625 JH 12.6 14.2 3.3 0.0 4.0 0.0 JE 95.9 72.4 40.2 22.5 14.4 8.3 JW 46.8 30.5 27.6 21.7 17.9 11.0 DH 29.0 14.4 7.3 3.7 0.0 0.0 DE 93.8 77.5 54.9 34.1 23.8 14.3 DW 66.8 57.8 53.6 45.2 32.3 23.7 SH 26.0 16.3 3.5 0.9 1.0 0.0 SE 87.9 91.9 93.3 93.9 94.3 94.4 SW 77.5 82.1 80.7 73.0 55.5 40.5 LH 70.8 84.5 88.1 66.7 40.6 21.5 LE 51.2 75.0 65.5 40.7 25.1 14.2 LW 73.1 82.6 81.8 81.7 78.7 64.5-87 -
Fig 2-23. Composition of Sea buckthorn fractions - 88 -
Fig 2-24. Heat Stability of SE, LW fractions - 89 -
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Fig 2-25. TLC Separation of phenolic compounds 1-92 -
Fig 2-26. TLC Separation of phenolic compounds 2-93 -
Table 2-4. The color developments of phenolic compounds by different visualization techniques Visualization No Vanillin AlCl 3 DPPH Light source 254 366 white 254 366 white 254 366 white 254 366 white t-cinnamic bk - - bk pgy - bk - - bk - - quercetin sbk br br bl sskybl br bk swt br bk br pk salicylic pbl skybl - bk pgy - bl skybl - pbl skybl - p-coumaric bk bl - bk gy ppp bk - - bk bl ppk ferulic bk skybl - pbl skybl ppp bk pbl pbr bk skybl pk gallic bk - bk bk bk pbr bk pbl gy bk gy pk catethin pbk - pbk sbk dpp pp bk pbl pbr pbk gy pk bk:black, bl:blue, gy:gray, pp:purple, wt:white, pk:pink, s:strong, p:pale, d:dark, sky:sky - 94 -
Fig 2-27. TLC Separation of SE fraction - 95 -
Fig 2-28. TLC Separation of ethanol fractions from Sea buckthorn 1-96 -
Fig 2-29. TLC Separation of ethanol fractions from Sea buckthorn 2-97 -
Fig 2-30. TLC Separation of fractionated SE 1-98 -
Fig 2-31. TLC Separation of fractionated SE 2-99 -
Fig 2-32. HPLC separation of SE fraction 1-100 -
Fig 2-33. HPLC separation of SE fraction 2-101 -
a. 6.4 minute peak of condition 1 b. 54.0 minute peak of condition 2 c. 71.6 minute peak of condition 2 Fig 2-34. UV absorption spectra of major peaks - 102 -
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Fig 2-35. ESI-MS of SE fraction - 106 -
Fig 2-36. Polyphenol contents of different ethanol extracts of Sea buckthorn seeds - 107 -
Fig 2-37. DPPH free radical scavenging activity of BHA and ascorbic acid - 108 -
Fig 2-38. DPPH free radical scavenging activity of the different ethanol extracts of Sea buckthorn seeds - 109 -
Fig 2-39. Effect of SHE and SEO on the cell viability in human fibroblasts by the MTT assay. - 110 -
Fig 2-40. Cell counting using the BrdU assay - 111 -
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Fig 2-41. Effect of SHE and SEO on the collagen synthesis in human fibroblasts by the collagen assay. - 114 -
Fig 2-42. Effect of SHE and SEO on the glycosaminoglycanollagen synthesis in human fibroblasts by the GAG assay. - 115 -
Table 2-5. DPPH free radical scavenging activity and total phenolic content (TPC) of the solvent-dependent extracts of Sea buckthorn seeds Compound Solvent Antioxidant activity TPC IC 50 /DPPH (μg/ml) a) (μg GAE/mg) b) SBSH Hexane 1288.21 ± 176.28a 6.70 ± 2.13c SBSE Ethanol 8.33 ± 0.39d 126.96 ± 6.76a SBSW Water 163.72 ± 3.93b 41.63 ± 0.96b Ascorbic acid - 11.37 ± 0.29c - a) The antioxidant activity was evaluated as the content of the test sample required to decrease the b) absorbance at 517 nm by 50% in comparison to the control; Total reducing capacity of Sea buckthorn seed as determined by Folin-Cioculteu assay. TPC values are expressed as gallic acid equivalent (GAE)/mg in dry weight. Values are expressed as mean of triplicate determinations ± standard deviation; Different letters in the same column show significant differences from each other at P<0.05 level. - 116 -
Fig 2-43. Effect of SBSE concentrations on the cytotoxicity in human fibroblasts by the MTT assay. Data are expressed as percentage of live cells compared to untreated controls. *P < 0.01 vs exposed controls and **P < 0.001 vs exposed controls. - 117 -
Fig 2-44. Effect of SBSE on the cell viability of skin fibroblasts exposed to UVB radiation at 30 mj/cm2,determined using the MTT assay. Data are expressed as percentage of live cells compared to unexposed controls. *P < 0.01 vs exposed controls and **P < 0.001 vs exposed controls. - 118 -
β β β α α α - 119 -
Fig 2-45. SBSE blocked the UVB-induced increase of IL-1β expression in cultured fibroblasts. - 120 -
Fig 2-46. SBSE inhibited UVB-induced increase in IL-6 and COX-2 expression in human dermal fibroblasts. - 121 -
Fig 2-47. SBSE inhibited UVB-induced increase in TNF-α expression in human Keratinocytes. - 122 -
Fig 2-48. SBSE inhibited UVB-induced expression of MMP-1 in human dermal fibroblasts. - 123 -
Fig 2-49. SBSE stimulated the synthesis of type I procollagen in human dermal fibroblasts. - 124 -
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Crushing to 20-50 mesh for sea buckthorn seed ò Mixing of ethanol and crushed sea buckthorn seed to a concentration of 5%(w/v) in reactor ò Extraction for 18 hr at 40-60 ò Separation of supernatant ò Vacuum filtering after celite addition ò Vacuum evaporation to a dry weight of 2.5-3.5% at 60 ò Storage at -20 5 for 0.5 20 hr after addition of celite in evaporated extract ò Vacuum filtering ò Packaging Fig 2-50. The extraction process of Sea buckthorn seeds - 126 -
Table 2-6. Comparison of the extraction yield, antioxidant activity (IC 50 ),and stability at the different steps of the separation process used for ethanol extraction of Sea buckthorn seeds CR CE CE-CS Extraction yield(%) 14.0 13.1 12.6 IC 50 ( μg /ml) 5.5 7.5 8.0 Phase separation in stability test during 70 days under room temperature CR : the crude ethanol extract without separation CE : the extract separated by the use of celite CE-CS : the extract filtering CR, which contains celite, after the storage of 16 h in refrigerator of 4 IC 50 : 50% inhibition concentration - 127 -
100 90 Antioxidant activity (%) 80 70 60 50 40 30 20 CR CE CE-CS 10 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Concentration of SBS extract (ug/ml) Fig 2-51. Antioxidant activity at different steps of ethanol extraction from Sea buckthorn seeds (CR : the crude ethanol extract without separation, CE : the extract separated by the use of celite, CE-CS : the extract filtering CR, which contains celite, after the storage of 16 h in refrigerator of 4 ) - 128 -
Antioxidant activity (%) Antioxidant activity (%) 100 100 90 80 80 70 60 60 50 40 40 30 20 20 SBS extract BHA 0 day (SBS Vitamic extract) C 70 days (SBS extract) 0 day (BHA) 70 days (BHA) 0 day (Vitamic C) 70 days (Vitamin C) 10 0 0 0 20 40 60 80 100 120 0 10 20 30 40 50 60 70 80 Concentration (ug/ml) Concentration (ug/ml) Fig 2-53. Effect of storage time on the antioxidant activity Fig 2-52. Comparison of the SBS extract with other antioxidant agents - 129 -
100 SBS extract 90 Antioxidant activity (%) 80 70 60 50 40 30 20 Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C 10 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Concentrations of SBS extract (ug/ml) 100 90 BHA Antioxidant activity (%) 80 70 60 50 40 30 20 Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C 10 0 100 90 0 10 20 30 40 50 60 Concentrations of BHA (ug/ml) Vitamin C Antioxidant activity (%) 80 70 60 50 40 30 20 Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C 10 0 0 10 20 30 40 50 60 Concentrations of vitamin C (ug/ml) Fig 2-54. Effect of temperature on the antioxidant activity - 130 -
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150 100 Bactericidal ratio (%) 50 0-50 -100-150 -200 Control SBS extract Vitamin C BHA -250-300 0 1 2 3 4 5 6 7 8 Time (hr) Fig 2-55. Comparisons of the antibacterial activities of the SBS extract, vitamin C and BHA - 133 -
100 90 Bactericidal ratio (%) 80 70 60 50 40 30 20 37 O C 25 O C 10 0 0 1 2 3 4 5 6 7 8 Time (hr) Fig 2-56. Effect of culture temperature on the antibacterial activities (E.coli 200 rpm) - 134 -
100 90 Bactericidal ratio (%) 80 70 60 50 40 30 20 250 ug/ml 500 ug/ml 1000 ug/ml 2000 ug/ml 10 0 0 1 2 3 4 5 6 7 8 Time (hr) Fig 2-57. Effect of SES extract concentration on the antibacterial activities (E.coli 200 rpm) - 135 -
100 90 Bactericidal ratio (%) 80 70 60 50 40 30 20 E. coli P. aerusinosa S. aureus B. subtilis 10 0 0 1 2 3 4 5 6 7 8 Time (hr) Fig 2-58. Antibacterial activity against other bacteria - 136 -
Fig 2-59. Prototype status of cosmetics adding ethanol extract from Sea buckthorn seeds - 137 -
Fig 2-60. Comparison of heat stability in the each LW fraction - 138 -
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시약과 - 140 -
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Fig 3-1. Physical composition of Sea buckthorn fruit - 144 -
Fig 3-2. Condition of GC-MS analysis - 145 -
Fig 3-3. SAFE (Solvent Assisted Flavour Evaporation) - 146 -
Fig 3-4. SPME (Solid Phase Micro Extraction) - 147 -
Table 3-1. Method of making Home-brewed beer by adding Sea buckthorn fruit 맥주제조기계 맥주제조방법 맥주제조방법 단계 발효및숙성 생수를발효조에 을채운후 를모두혼합함 생수 를넣고효모를넣음 상온에서일주간숙성시킴 단계 차숙성 단계가끝나면 에서 일간숙성시킴 단계 차숙성 맥주 에설탕 넣고흔든후상온에서 주일간숙성시킴 갈매보리수나무열매착즙액첨가 단계 발효및숙성 발효조에생수 를채움 뜨거운물에맥주원액캔을담아둠 생수 에부스터를넣어녹인후끓임 끓인부스터에맥주원액캔을부어혼합후발효조에넣음 발효조에 리터의생수를넣은후 가되면효모를넣고상온에서 주간발효시킴 단계 차숙성 맥주 에설탕 넣고 주간숙성시킴 갈매보리수나무열매착즙액첨가 단계 차숙성 에서 주간숙성시킴 - 148 -
μ - 149 -
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Fig 3-5. H. rhamnoides berry juice - 151 -
Table 3-2. The questionnaire of functional jellies contained H. rhamnoides juice sensory test. Sensory test methods Sensory characteristics Appearance Interest : The higher samples get grades, the better assessors feel characteristics. Suitability : 4 points mean the most suitable intensity of samples' characteristics, while higher points than 4 mean intensity is strong and lower points than 4 mean intensity is weak. Grades Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Taste Too bad Bad Little bad Not good not bad Little good Good So good 1 2 3 4 5 6 7 Flavor Too bad Bad Little bad Not good not bad Little good Good So good 1 2 3 4 5 6 7 Overall Color Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Too Little Little Too Weak Suitable Strong weak weak strong strong 1 2 3 4 5 6 7 Sweet-ness Too weak Weak Little weak Suitable Little strong Strong Too strong 1 2 3 4 5 6 7-152 -
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Table 3-3. The questionnaire of functional beverages containing H. rhamnoides juice sensory test. Sensory test methods Sensory characteristics Appearance Taste Flavor Overall Color Sweet-ness Sourness Interest : The higher samples get grades, the better assessors feel characteristics. Suitability : 4 points mean the most suitable intensity of samples' characteristics, while higher points than 4 mean intensity is strong and lower points than 4 mean intensity is weak. Grades Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Little Not good So Too bad Bad Little good Good bad not bad good 1 2 3 4 5 6 7 Too Little Little Too Weak Suitable Strong weak weak strong strong 1 2 3 4 5 6 7 Too Little Little Too Weak Suitable Strong weak weak strong strong 1 2 3 4 5 6 7 Too Little Little Too Weak Suitable Strong weak weak strong strong 1 2 3 4 5 6 7-154 -
μ μ μ μ μ μ μ μ - 155 -
μ μ μ - 156 -
μ φ μ μ μ - 157 -
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Fig 3-6. Physical composition of Sea buckthorn fruit - 160 -
Fig. 3-7. Total flavonoid content of the each samples - 161 -
Table 3-4. Comparision of total flavonoid content of the each samples Type Catechin equivelnet (mg/l) Standard deviation Sea buckthorn 941.11 26.43 Fermented Enzyme 1050.00 24.19 (Enzyme+Lactic acid bacteria) Fermentation 1132.22 36.55 Natural Fermentation 974.44 19.31 Lactic acid bacteria fermentation 836.67 43.20 Laurel fruit extract 616.50 11.42-162 -
Table 3-5. Chemical composition of Sea buckthorn fruit Chemical composition Average(%) Standard deviation Moisture 78.19 9.46 Crude Fat 4.28 1.17 Crude Protein - - Reducing Sugar 14.84 3.77 Crude Fiber 0.41 0.21-163 -
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Fig 3-8. GC chromatogram for volatile compounds of Sea buckthorn fruit with SAFE - 165 -
- 166 -
Table 3-6. volatile compounds of Sea buckthorn fruit by analyzing SAFE peak Possible compound no. 1) I Retention % of total Time (min) 1 ethyl butylrate 1029 7.25 1.82 2 ethyl 2-methylbutyrate 1046 7.62 4.47 3 ethyl 3-methylbutyrate 1062 7.97 2.75 4 pentanol 1246 12.09 7.54 5 ethyl hexanoate 1229 13.18 13.55 6 trans-beta-ocimene 1245 14.15 1.07 8 3-methybutyl 3-methylbutyrate 1292 15.30 7.82 12 propylhexnoate 1316 16.15 1.53 13 ethyl heptanoate 1330 16.72 0.75 14 methylpenta 3-ol,2 1151 17.25 1.54 17 methyl octanoate 1381 18.92 2.56 18 ethyl octanoate 1432 20.98 9.86 19 butyric acid 1432 21.37 1.77 20 methylbutyl hexanoate 3-1650 21.79 0.57 21 6-hepten-1-ol,2 methyl 1457 21.98 0.37 33 methyl 3-hydroxybutyrate 1515 24.44 0.52 38 methyl benzoate 1616 28.14 1.28 42 methylbutyric acid,3-1680 29.86 3.38 51 benzyl alcohol 1874 37.53 0.47 52 ethyl hexanoate 1229 37.73 1.28 54 methylbutyl benzoate 1907 38.59 2.56 55 phenyl ethanol 2-1910 38.84 0.62 67 pentacosane 2400 56.38 3.35 68 heptasosane * 65.87 5.17 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 167 -
- 168 -
(a) polydimetysiloxane (PDMS) SPME fiber chromatogram Fig 3-9. Amount of adsorption according to fiber type (b) carbowax/divinylbenzene (DVB/CAR/PDMS) SPME fiber chromatogram Fig 3-9. Amount of adsorption according to fiber type - 169 -
Fig 3-10. GC chromatogram for volatile compounds of Sea buckthorn fruit with SPME - 170 -
- 171 -
Table 3-7. Volatile compounds of Sea buckthorn fruit with SPME peak No. 1) 2) Possible compound 1) I Retention Time (min) % of total 1 ethyl2-methylbuturate 1046 7.66 5.92 2 ethyl hexanoate 1229 8.39 16.23 3 isoamylbutyrate 2) * 10.49 1.61 4 3-methylbutyl 2-methylbutyrate 1292 10.92 8.99 5 methylbutyl 3-methylbutylate 3, 1361 11.31 2.51 6 propylhexanoate 1245 12.98 0.47 7 6-methyl 5-hepten 2-one * 13.18 0.21 8 ethyl hex-2-enoate 1341 15.27 0.15 9 isobutyl hexanoate * 15.33 0.19 10 1-hexanol 1381 15.44 0.18 11 dodecamethylcyclohexasiloxane 1477 15.51 0.21 12 methyl octanoate 1386 16.13 1.72 13 ethyl octanoate 1432 17.17 37.77 14 butanoic acid * 17.32 0.25 15 methylbutyl octanoate 3-1658 18.74 1.05 16 methly but-2-enoate * 19.19 0.29 17 ethyl oct-4-enoate 1462 19.45 2.75 18 ethyl sorbate 1503 20.11 0.29 19 ethyl oct-3-enoate 1690 20.42 0.08 20 propyl octanoate 1518 20.49 0.08 21 ethyl 4,7-octadienoate (Z) 1515 21.01 0.49 22 ethyl nonanoate 1534 21.39 0.08 23 ethyl oct-2-enoate (E) 1551 21.92 0.14 24 methylbenzoate 1616 23.29 0.29 25 ethyl decanoic acid 1658 25.18 12.75 26 butanoic acid 1636 25.83 0.09 27 ethyl E-4-decenoate 1662 26.06 1.22 28 ethyl benzoate 1680 26.68 2.06 29 ethyl dec-4-noate 1663 26.82 0.07 30 methyl diethyl borinic acid * 28.87 0.11 31 methyl decadienoate * 30.06 0.07 32 ethylphenyl aceate 1783 32.69 0.10 33 phenylethyl acetate 2-1813 32.97 0.31 34 ethyl dodecanoate 1843 33.72 0.10 35 hexanoicacid 1921 34.05 0.21 36 decahydro naphthalene * 34.17 0.39 37 methyl 1-butanol, benzoate * 34.33 0.58 I mean Kovats retention index on DB-WAX * mean no identified in Kovats index - 172 -
총산 초산으로서 N 소비량 N 의 검체량 ml - 173 -
- 174 -
- 175 -
Table 3-8. Using Sea buckthorn fruit for 15 home-brewed beers and control GA GA5g GAS5% GAS3% GAS1% GA5% GA3% GA1% AA AAS5% AAS3% GL GLS3% PA PAS3% Con Control(Con), Golden Ale(GA), Golden Ale + sugar5g(ga5g), Golden Ale (GAS) Amber Ale(AA), Amber Ale (AAS), Golden Larger(GL), Golden Larger (GLS), Pale Ale(PA), Pale Ale (PAS) - 176 -
- 177 -
Table 3-9. Total acid content of 16 kinds of beers Beer Total acid Beer Total acid GA 0.174 AA 0.244 GA5g 0.192 AAS5% 0.394 GA5% 0.27 AAS3% 0.308 GA3% 0.192 GL 0.154 GA1% 0.18 GLA3% 0.18 GAS5% 0.258 PA 0.14 GAS3% 0.204 PAS3% 0.174 GAS1% 0.174 Con 0.12-178 -
- 179 -
Table 3-10. Total sugar content of 16 kinds of beers Beer Total sugar(%) Beer Total sugar(%) GA 2.99±0.32 1) AA 1.60±0.02 GA5g 2.82±0.37 AAS5% 1.78±0.07 GA5% 2.81±0.18 AAS3% 1.47±0.06 GA3% 3.15±0.19 GL 2.67±0.12 GA1% 2.82±0.43 GLS3% 2.67±0.01 GAS5% 1.97±0.73 PA 2.92±0.06 GAS3% 2.83±0.57 PAS3% 2.35±0.12 GAS1% 3.02±0.49 Con 2.55±0.06-180 -
Table 3-11. Total reducing sugar content of 16 kinds of beers Beer Reducing sugar(%) Beer Reducing sugar(%) GA 1.28±0.34 1) AA 1.22±0.06 GA5g 1.39±0.28 AAS5% 1.05±0.01 GA5% 1.13±0.36 AAS3% 1.14±0.07 GA3% 1.31±0.34 GL 0.89±0.02 GA1% 1.28±0.31 GLS3% 0.91±0.02 GAS5% 0.93±0.35 PA 1.00±0.02 GAS3% 1.26±0.30 PAS3% 0.88±0.03 GAS1% 1.20±0.34 Con 0.74±0.03-181 -
Table 3-12. Ethanol content of 16 kinds of beers - 182 -
Fig 3-12. a) Sensory evaluation b) Sensory evaluation paper - 183 -
Table 3-13. Sensory evaluation of golden larger and pale ale(40 panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.38 b 2.78 b 3.58 b 3.15 b 6.03 a Color 4.79 bc 3.98 c 5.88 a 4.18 bc 4.98 ba Flavor 3.62 b 3.90 b 4.38 b 3.65 b 6.20 a Taste 3.13 b 2.90 b 3.23 b 3.30 b 5.88 a Sourness 3.38 cb 3.10 c 4.05 b 3.38 cb 5.73 a Sweetness 3.56 b 3.53 b 3.48 b 3.60 b 5.49 a Bitterness 3.77 b 3.90 b 3.85 b 4.09 b 5.65 a Sparkling 3.00 cb 2.73 c 3.15 cb 3.75 b 5.46 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 184 -
Table 3-14. Sensory evaluation of golden larger and pale ale(16men panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.75 b 2.69 b 3.50 b 2.88 b 6.06 a Color 4.69 a 4.00 a 5.56 a 3.81 a 4.88 ba Flavor 4.00 a 4.13 b 4.63 b 3.31 b 6.38 a Taste 3.38 b 2.69 b 2.94 b 2.75 b 5.94 a Sourness 3.38 b 3.31 b 4.06 b 3.00 b 6.00 a Sweetness 3.31 b 3.38 b 3.00 b 3.13 b 5.81 a Bitterness 3.44 b 3.44 b 3.63 b 3.13 b 5.81 a Sparkling 2.81 b 2.50 b 3.44 b 2.28 b 5.69 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 185 -
Table 3-15. Sensory evaluation of golden larger and pale ale(24 women panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.13 b 2.83 b 3.63 b 3.33 b 6.00 a Color 4.87 b 3.96 b 6.08 a 4.42 b 5.04 a Flavor 3.35 b 3.75 b 4.21 b 3.88 b 6.08 a Taste 2.96 b 3.04 b 3.34 b 3.67 b 5.83 a Sourness 3.39 b 2.96 b 4.04 b 3.63 b 5.54 a Sweetness 3.74 b 3.63 b 3.79 b 3.92 b 5.27 a Bitterness 4.00 b 4.21 b 4.00 b 4.73 ba 5.54 a Sparkling 3.13 b 2.88 b 2.96 b 4.00 b 5.31 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 186 -
Table 3-16. Sensory evaluation of Amber ale(41 panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.39 b 2.12 b 2.27 b 5.71 a Color 4.20 b 3.20 c 3.04 c 6.71 a Flavor 2.76 b 3.37 b 3.17 b 6.00 a Taste 2.37 b 2.05 b 2.34 b 5.46 a Sourness 3.98a 4.24 a 4.37 a 4.87 a Sweetness 3.44 b 3.26 b 3.44 b 5.07 a Bitterness 4.37 a 4.29 a 3.89 a 4.63 a Sparkling 3.02 b 2.78 b 2.24 b 4.98 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 187 -
Table 3-17. Sensory evaluation of Amber ale(15 men panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.27 b 2.33 b 2.27 b 5.33 a Color 3.53 b 3.27 b 3.13 b 6.27 a Flavor 2.38 b 3.47 b 3.00 b 6.12 a Taste 2.33 b 2.40 b 2.33 b 5.33 a Sourness 4.53 a 4.73 a 4.73 a 4.67 a Sweetness 2.80 b 2.93 ba 3.53 ba 4.53 a Bitterness 4.67 a 4.20 a 3.80 a 4.07 a Sparkling 3.67 ba 2.73 b 2.33 b 5.13 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 188 -
Table 3-18. Sensory evaluation of Amber ale(26 women panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.46 b 2.00 b 2.27 b 5.92 a Color 4.58 b 3.15 c 3.00 a 6.96 a Flavor 2.38 b 3.31 b 3.00 b 6.12 a Taste 2.38 b 1.85 b 2.35 b 5.54 a Sourness 3.65 a 3.96 a 4.15 a 5.00 a Sweetness 3.81 b 3.46 b 3.38 b 5.38 a Bitterness 4.19a 4.35a 3.92a 4.96 a Sparkling 2.65 b 3.81 b 2.19 b 4.88 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test). - 189 -
- 190 -
Fig 3-13. GC Chromatogram for volatile compounds of Control beer by SAFE - 191 -
- 192 -
Table 3-19. volatile compounds of Control beer with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol 1083 4.17 1.04 2 isoamyl acetate 1119 4.90 1.80 3 isoamyl alcohol 1216 7.23 10.68 4 ethyl hexanoate 1231 7.69 0.25 5 3-methylbut-2-en-1-ol 1332 10.53 0.01 6 ethyl heptanoate 1332 10.91 0.03 7 acetic acid 1448 15.13 0.03 8 furfural 1456 15.36 0.03 9 1-heptanol 1459 15.56 0.01 10 ethyl octanoate 1475 16.12 0.02 11 linalool 1550 18.98 0.04 12 octanol 1563 19.45 0.02 13 propanoic acid 1568 19.64 0.04 14 5,5-dimethyl 2(5H)furanone 1589 20.45 0.03 15 butyrolactone 1602 20.98 0.04 16 ethanol * 21.47 0.02 17 butyric acid 1626 21.84 0.10 18 ethyl decanoate 1637 22.24 0.07 19 2-furanmethanol 1659 23.06 0.23 20 butanoic acid 1668 23.38 0.28 21 alpha-terpineol 1692 24.26 0.02 22 3-methlythiopropanal 1710 24.90 0.18 23 delta-cadinene 1752 25.93 0.02 24 citronellol 1772 27.09 0.01 25 phenyl ethylacetate 1804 28.22 3.10 26 hexyl but-2-enoate 1854 29.94 0.07 27 hexanoic acid 1845 29.61 1.52 28 benzyl alcohol 1866 30.34 0.05 29 phenyl ethylalcohol 1910 31.81 32.89 30 2-acetylpyrrole 1957 33.33 0.13 31 octanoic acid 2109 36.66 3.67 32 pyrrole-2-carboxaldehyde 2120 37.38 0.03 33 cis-ethyl cinnamate 2138 38.14 0.01 34 2-phenoxyethanol 2150 36.68 0.04 35 decanoic acid 2269 43.03 0.28 36 phenol 2307 44.19 0.01 37 dodecanoic acid 2491 48.98 0.05 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 193 -
Fig 3-14. GC Chromatogram for volatile compounds of Pale ale(3%) by SAFE - 194 -
Table 3-20. Volatile compounds of Pale ale(3%) with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol 1083 4.17 2.04 2 isoamyl acetate 1115 4.79 1.84 3 isoamyl alcohol 1214 7.19 35.85 4 ethyl hexanoate 1231 7.68 0.32 5 4-methylpentanol 1317 10.36 0.14 6 3-methylbut-2-en-1-ol 1323 10.59 0.09 7 1-hexanol 1355 11.76 0.27 8 ethyl octanoate 1433 14.61 1.09 9 furfural 1454 15.36 0.05 10 1-heptanol 1460 15.57 0.08 11 ethyl octanoate 1472 16.00 0.02 12 Ethanol 1522 17.89 0.58 13 linalool 1552 19.04 0.27 14 octanol 1563 19.45 0.09 15 propanoic acid 1567 19.62 0.31 16 trans-caryophyllene 1577 20.02 0.08 17 5,5-dimethyl 2(5H)furanone 1589 20.46 0.08 18 butyrolactone 1602 20.98 0.15 19 butyric acid 1627 21.89 0.11 20 ethyl decanoate 1636 22.22 0.30 21 alpha-humulene 1647 22.61 0.24 22 3-methylbutyl octanoate 1655 22.91 0.04 23 ethyl benzoate 1651 22.75 0.12 24 2-furanmethanol 1659 23.05 0.70 25 ethyl succinate 1673 23.58 0.10 26 alpha-terpineol 1692 24.27 0.07 27 3-methlythiopropanol 1710 24.90 0.39 28 delta-cadinene 1741 26.02 0.05 29 citronellol 1770 27.05 0.03 30 phenyl ethylacetate 1801 28.13 0.68 31 hexanoic acid 1844 29.58 1.73 32 phenyl ethylalcohol 1903 31.60 42.38 33 malto 1947 33.01 0.54 34 2-acetylpyrrole 1957 33.32 0.60 35 octanoic acid 2105 36.61 3.15 36 pyrrole-2-carboxaldehyde 2120 37.36 0.06 37 cis-ethyl cinnamate 2138 38.14 0.07 38 benzoic acid 2180 39.95 0.15 39 2-methoxy-4-vinylphenol 2195 40.23 0.06 40 beta-eudesmol 2206 41.02 0.05 41 phenol 2307 44.19 0.24 42 decanoic acid 2332 44.81 0.46 43 dodecanoic acid 2492 49.01 0.09 44 benzoic acid 2613 52.06 0.06 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 195 -
- 196 -
Fig. 3-15 GC Chromatogram for volatile compounds of Pale ale by SAFE - 197 -
Table 3-21. Volatile compounds of Pale ale with SAFE 1) peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol 1083 4.17 2.26 2 isoamyl acetate 1115 4.79 3.16 3 isoamyl alcohol 1213 7.14 34.92 4 ethyl hexanoate 1231 7.68 0.28 5 4-methylpentanol 1317 10.36 0.1 6 3-methylbut-2-en-1-ol 1323 10.59 0.01 7 1-hexanol 1350 11.76 0.05 8 ethyl octanoate 1433 14.61 0.93 9 furfural 1454 15.36 0.07 10 1-heptanol 1460 15.57 0.04 11 Ethanol 1522 17.89 0.01 12 linalool 1552 19.04 0.42 13 octanol 1563 19.45 0.09 14 propanoic acid 1567 19.62 0.37 15 trans-caryophyllene 1577 20.02 0.10 16 5,5-dimethyl 2(5H)furanone 1589 20.46 0.10 17 butyrolactone 1602 20.98 0.14 18 butyric acid 1627 21.89 0.10 19 ethyl decanoate 1636 22.22 0.18 20 alpha-humulene 1647 22.61 0.53 21 3-methylbutyl octanoate 1655 22.91 0.03 22 ethyl benzoate 1651 22.75 0.05 23 2-furanmethanol 1659 23.05 0.86 24 ethyl succinate 1673 23.58 0.10 25 alpha-terpineol 1692 24.27 0.07 26 3-methlythiopropanol 1710 24.90 0.42 27 delta-cadinene 1741 26.02 0.06 28 phenyl ethylacetate 1801 28.13 1.28 29 hexanoic acid 1844 29.58 0.09 30 phenyl ethylalcohol 1903 31.60 48.78 31 malto 1947 33.01 0.18 32 2-acetylpyrrole 1957 33.32 0.50 33 octanoic acid 2105 36.61 2.87 34 pyrrole-2-carboxaldehyde 2120 37.36 0.06 35 cis-ethyl cinnamate 2138 38.14 0.05 36 benzoic acid 2180 39.95 0.21 37 2-methoxy-4-vinylphenol 2195 40.23 0.04 38 beta-eudesmol 2206 41.02 0.11 39 phenol 2307 44.19 0.04 40 decanoic acid 2332 44.81 0.14 41 dodecanoic acid 2492 49.01 0.02 42 benzoic acid 2613 52.06 0.22 I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 198 -
Fig. 3-16 GC Chromatogram for volatile compounds of Golden ale(3%) by SAFE - 199 -
Table 3-22. Volatile compounds of Golden ale(3%) with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol 1083 4.19 1.87 2 isoamyl acetate 1115 4.80 1.02 3 isoamyl alcohol 1214 7.19 49.24 4 ethyl hexanoate 1233 7.73 0.19 5 4-methylpentanol 1318 10.39 0.08 6 3-methylbut-2-en-1-ol 1323 10.59 0.02 7 ethyl heptanoate 1333 10.93 0.04 8 ethyl octanoate 1435 14.66 1.02 9 furfural 1455 15.39 0.04 10 1-heptanol 1460 15.58 0.07 11 ethyl octanoate 1472 16.00 0.02 12 Ethanol 1522 17.89 0.26 13 linalool 1553 19.04 0.19 14 octanol 1558 19.48 0.07 15 propanoic acid 1566 19.58 0.17 16 trans-caryophyllene 1590 20.05 0.10 17 5,5-dimethyl 2(5H)furanone 1590 20.48 0.20 18 butyrolactone 1603 21.01 0.08 19 butyric acid 1627 21.87 0.09 20 ethyl decanoate 1637 22.25 0.42 21 alpha-humulene 1648 22.64 0.30 22 2-furanmethanol 1659 23.07 1.24 23 ethyl benzoate 1651 22.75 0.12 24 ethyl succinate 1673 23.58 0.10 25 alpha-terpineol 1692 24.27 0.07 26 3-methlythiopropanol 1710 24.90 0.39 27 delta-cadinene 1741 26.02 0.05 28 citronellol 1770 27.05 0.03 29 phenyl ethylacetate 1801 28.13 0.68 30 hexanoic acid 1844 29.58 1.73 31 phenyl ethylalcohol 1903 31.60 42.38 32 malto 1947 33.01 0.54 33 2-acetylpyrrole 1957 33.32 0.60 34 octanoic acid 2105 36.61 3.15 35 pyrrole-2-carboxaldehyde 2120 37.36 0.06 36 cis-ethyl cinnamate 2138 38.14 0.07 37 benzoic acid 2180 39.95 0.15 38 2-methoxy-4-vinylphenol 2195 40.23 0.06 39 beta-eudesmol 2206 41.02 0.05 40 phenol 2307 44.19 0.24 41 decanoic acid 2332 44.81 0.46 42 dodecanoic acid 2492 49.01 0.09 43 benzoic acid 2613 52.06 0.06 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 200 -
Fig 3-17. GC Chromatogram for volatile compounds of Golden ale by SAFE - 201 -
Table 3-23. Volatile compounds of Golden ale with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol 1083 4.19 1.87 2 isoamyl acetate 1115 4.80 1.02 3 isoamyl alcohol 1214 7.19 49.24 4 ethyl hexanoate 1233 7.73 0.19 5 4-methylpentanol 1318 10.39 0.08 6 3-methylbut-2-en-1-ol 1323 10.59 0.02 7 ethyl heptanoate 1333 10.93 0.04 8 ethyl octanoate 1435 14.66 1.02 9 furfural 1455 15.39 0.04 10 1-heptanol 1460 15.58 0.07 11 ethyl octanoate 1472 16.00 0.02 12 Ethanol 1522 17.89 0.26 13 linalool 1553 19.04 0.19 14 octanol 1558 19.48 0.07 15 propanoic acid 1566 19.58 0.17 16 trans-caryophyllene 1590 20.05 0.10 17 5,5-dimethyl 2(5H)furanone 1590 20.48 0.20 18 butyrolactone 1603 21.01 0.08 19 butyric acid 1627 21.87 0.09 20 ethyl decanoate 1637 22.25 0.42 21 alpha-humulene 1648 22.64 0.30 22 2-furanmethanol 1659 23.07 1.24 23 ethyl benzoate 1651 22.75 0.12 24 ethyl succinate 1673 23.58 0.10 25 alpha-terpineol 1692 24.27 0.07 26 3-methlythiopropanol 1710 24.90 0.39 27 delta-cadinene 1741 26.02 0.05 28 citronellol 1770 27.05 0.03 29 phenyl ethylacetate 1801 28.13 0.68 30 hexanoic acid 1844 29.58 1.73 31 phenyl ethylalcohol 1903 31.60 42.38 32 malto 1947 33.01 0.54 33 2-acetylpyrrole 1957 33.32 0.60 34 octanoic acid 2105 36.61 3.15 35 pyrrole-2-carboxaldehyde 2120 37.36 0.06 36 cis-ethyl cinnamate 2138 38.14 0.07 37 benzoic acid 2180 39.95 0.15 38 2-methoxy-4-vinylphenol 2195 40.23 0.06 39 beta-eudesmol 2206 41.02 0.05 40 phenol 2307 44.19 0.24 41 decanoic acid 2332 44.81 0.46 42 dodecanoic acid 2492 49.01 0.09 43 benzoic acid 2613 52.06 0.06 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 202 -
- 203 -
Fig 3-18. GC Chromatogram for volatile compounds of control beer by SPME - 204 -
Table 3-24. Volatile compounds of Control beer with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 2-methylpropyl acetate 1012 2.95 0.17 2 ethyl butyrate 1030 3.25 0.37 3 isobutyl alchol 1082 4.14 0.11 4 isoamyl acetate 1112 4.74 14.23 5 D-limonene 1188 6.47 0.08 6 isoamyl alcohol 1206 6.93 7.63 7 ethyl hexanoate 1228 7.60 2.82 8 octan-3-one 1246 8.14 0.13 9 benzaldehyde 1281 9.17 0.36 10 ethyl undecanoate 1386 12.88 0.34 11 ethyl octanoate 1435 14.66 6.77 12 acetic acid 1448 15.13 0.04 13 ethyl octanoate 1435 15.62 1.04 14 1-heptanol 1435 15.39 0.50 15 alpha-cubebene 1475 16.11 0.15 16 ethanol 1524 17.98 0.35 17 ethyl nonanoate 1536 18.42 0.28 18 linalool 1551 19.01 0.14 19 octanol 1564 19.49 0.40 20 propanoic acid 1570 19.75 0.09 21 trans-caryophyllene 1578 20.02 0.07 22 benzaldehyde 1582 20.19 0.06 23 5,5-dimethyl 2(5H)furanone 1589 20.48 0.07 24 undecan-2-one 1596 20.74 0.04 25 pyrrole 1606 21.12 0.03 26 ethyl decanoate 1636 22.21 0.25 27 alpha-humulene 1652 22.78 0.20 28 3-methylbutyl octanoate 1657 23.00 0.39 29 alpha-terpineol 1696 24.43 0.78 30 delta-cadinene 1745 26.16 0.52 31 citronellol 1769 27.00 0.39 32 ethyl dodecanoate 1840 29.47 1.08 33 hexanoic acid 1853 29.88 0.14 34 benzyl alcohol 1865 30.30 0.16 35 phenylethyl alcohol 1898 31.41 4.85 36 2-acetylpyrrole 1960 33.43 0.16 37 octanoic acid 2060 36.74 0.25 38 3-methyldiphenyl 2223 41.54 0.40 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 205 -
Fig 3-19. GC Chromatogram for volatile compounds of Pale ale(3%) by SPME - 206 -
Table 3-25. Volatile compounds of Pale ale(3%) with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 ethyl 2-methylbutyrate 1030 3.26 0.67 2 ethyl butyrate 1045 3.51 1.02 3 isobutyl alchol 1068 3.91 0.77 4 isoamyl acetate 1112 4.71 9.69 5 isoamyl alcohol 1206 6.93 22.50 6 ethyl hexanoate 1228 7.59 2.49 7 ethyl 3-methylbutyrate 1289 9.42 1.09 8 methyl hex-2-enoate 1327 10.75 0.59 9 ethyl undecanoate 1383 12.78 0.19 10 ethyl octanoate 1433 14.60 6.27 11 1-heptanol 1459 15.56 0.21 12 (E)-ethyl oct-4-enoate 1470 15.96 0.16 13 ethyl nonanoate 1535 18.42 0.07 14 linalool 1551 19.01 1.11 15 trans-caryophyllene 1578 20.02 0.33 16 acetic acid 1606 21.11 0.04 17 ethyl decanoate 1636 22.20 0.66 18 alpha-humulene 1647 22.60 1.12 19 ethyl benzoate 1650 22.74 0.30 20 3-methylbutyl octanoate 1655 22.90 0.10 21 2-furanmethanol 1659 23.06 0.23 22 butanoic acid 1671 23.51 0.30 23 (Z)-ethyl dec-4-enoate 1687 24.09 0.40 24 alpha-terpineol 1691 24.24 0.12 25 heptadec-1-ene 1725 25.43 0.02 26 delta-cadinene 1745 26.16 0.07 27 1-decanol 1765 26.88 0.02 28 citronellol 1769 27.00 0.05 29 2-phenylethyl acetate 1801 28.14 2.01 30 hexanoic acid 1848 29.71 0.33 31 ethyl dodecanoate 1840 29.46 0.06 32 butyl benzoate 1894 31.28 0.12 33 phenylethyl alcohol 1898 31.41 15.53 34 2-acetylpyrrole 1957 33.32 0.07 35 octanoic acid 2058 36.68 1.00 36 cis-ethyl cinnamate 2108 38.18 0.02 37 decanoic acid 2274 43.20 0.03 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 207 -
Fig 3-20. GC Chromatogram for volatile compounds of Pale ale by SPME - 208 -
Table 3-26. Volatile compounds of Pale ale with SPME peak no. Possible compound 1) I Retention % of total Time (min) 1 2-methylpropyl acetate 1010 2.91 0.65 2 ethyl butyrate 1027 3.21 1.02 3 isobutyl alchol 1080 4.11 1.11 4 isoamyl acetate 1112 4.71 15.44 5 isoamyl alcohol 1206 6.93 32.25 6 ethyl hexanoate 1228 7.59 2.41 7 1-hexyl acetate 1268 8.78 0.11 8 methyl hex-2-enoate 1327 10.75 1.46 9 benzaldehyde 1281 9.17 0.40 10 ethyl undecanoate 1383 12.78 0.26 11 ethyl octanoate 1433 14.60 5.23 12 1-heptanol 1459 15.56 0.13 13 propanoic acid 1496 16.89 0.25 14 ethyl nonanoate 1535 18.42 0.08 15 linalool 1551 19.01 1.70 16 octanol 1564 19.49 0.20 17 trans-caryophyllene 1578 20.02 0.34 18 ethyl decanoate 1636 22.20 0.36 19 3-methylbutyl octanoate 1655 22.90 0.05 20 2-furanmethanol 1659 23.06 0.20 21 tetradecanol 1725 25.43 0.05 22 delta-cadinene 1745 26.16 0.10 23 1-decanol 1765 26.88 0.03 24 citronellol 1769 27.00 0.08 25 2-phenylethyl acetate 1801 28.14 1.66 26 hexanoic acid 1848 29.71 0.25 27 benzyl alcohol 1865 30.30 0.16 28 phenylethyl alcohol 1898 31.41 10.16 29 2-acetylpyrrole 1957 33.32 0.04 30 octanoic acid 2058 36.68 0.59 31 decanoic acid 2274 43.20 0.03 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 209 -
Fig 3-21. GC Chromatogram for volatile compounds of Golden ale(3%) by SPME - 210 -
Table 3-27. Volatile compounds of Golden ale(3%) with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 ethyl 2-methylbutyrate 1030 3.26 0.67 2 ethyl butyrate 1045 3.51 1.02 3 isobutyl alchol 1068 3.91 0.77 4 isoamyl acetate 1112 4.71 9.69 5 isoamyl alcohol 1206 6.93 22.50 6 ethyl hexanoate 1228 7.59 2.49 7 ethyl 3-methylbutyrate 1289 9.42 1.09 8 methyl hex-2-enoate 1327 10.75 0.59 9 ethyl undecanoate 1383 12.78 0.19 10 ethyl octanoate 1433 14.60 6.27 11 1-heptanol 1459 15.56 0.21 12 (E)-ethyl oct-4-enoate 1470 15.96 0.16 13 ethyl nonanoate 1535 18.42 0.07 14 linalool 1551 19.01 1.11 15 trans-caryophyllene 1578 20.02 0.33 16 acetic acid 1606 21.11 0.04 17 ethyl decanoate 1636 22.20 0.66 18 alpha-humulene 1647 22.60 1.12 19 ethyl benzoate 1650 22.74 0.30 20 3-methylbutyl octanoate 1655 22.90 0.10 21 2-furanmethanol 1659 23.06 0.23 22 butanoic acid 1671 23.51 0.30 23 (Z)-ethyl dec-4-enoate 1687 24.09 0.40 24 alpha-terpineol 1691 24.24 0.12 25 heptadec-1-ene 1725 25.43 0.02 26 delta-cadinene 1745 26.16 0.07 27 1-decanol 1765 26.88 0.02 28 citronellol 1769 27.00 0.05 29 2-phenylethyl acetate 1801 28.14 2.01 30 hexanoic acid 1848 29.71 0.33 31 ethyl dodecanoate 1840 29.46 0.06 32 butyl benzoate 1894 31.28 0.12 33 phenylethyl alcohol 1898 31.41 15.53 34 2-acetylpyrrole 1957 33.32 0.07 35 octanoic acid 2058 36.68 1.00 36 cis-ethyl cinnamate 2108 38.18 0.02 37 decanoic acid 2274 43.20 0.03 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 211 -
Fig 3-22. GC Chromatogram for volatile compounds of Golden aleby SPME - 212 -
Table 3-28. Volatile compounds of Golden ale with SPME peak no. Possible compound 1) I Retention % of total Time (min) 1 2-methylpropyl acetate 1011 2.92 0.64 2 ethyl butyrate 1045 3.51 0.73 3 isobutyl alcohol 1081 4.12 0.96 4 isoamyl acetate 1112 4.71 0.96 5 D-limonene 1188 6.47 0.80 6 isoamyl alcohol 1206 6.93 23.51 7 ethyl hexanoate 1228 7.59 1.99 8 octan-3-one 1246 8.14 0.04 9 benzaldehyde 1281 9.17 0.70 10 3-methylbutyl butyrate 1293 9.53 0.13 11 ethyl heptanoate 1330 10.85 0.58 12 ethyl undecanoate 1383 12.78 0.29 13 ethyl octanoate 1433 14.60 6.86 14 1-heptanol 1459 15.56 0.09 15 alpha-cubebene 1479 16.27 0.17 16 ethyl nonanoate 1490 16.66 0.09 17 linalool 1552 19.03 0.96 18 trans-caryophyllene 1578 20.02 0.75 19 ethyl decanoate 1636 22.21 0.42 20 alpha-humulene 1647 22.60 2.52 21 3-methylbutyl octanoate 1655 22.90 0.06 22 2-furanmethanol 1659 23.06 0.15 23 delta-cadinene 1745 26.16 0.22 24 citronellol 1769 27.00 0.06 25 phenylethyl acetate 1774 27.16 1.46 26 ethyl dodecanoate 1840 29.46 0.07 27 hexanoic acid 1848 29.72 0.11 28 benzyl alcohol 1867 30.35 0.02 29 phenylethyl alcohol 1898 31.41 0.58 30 2-acetylpyrrole 1957 33.32 0.05 31 octanoic acid 2059 36.71 0.15 32 decanoic acid 2274 43.19 0.03 33 benzoic acid 2683 53.81 0.13 1) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index - 213 -
Fig 3-23. Total flavonoid content of each beer types - 214 -
- 215 -
- 216 -
Fig 3-24. DPPH free radical scavenging activity of Sea buckthorn fruit - 217 -
Fig 3-25. ABTS radical cation activity of the home-brewed beers - 218 -
Fig 3-26. Functional jellies containing H. rhamnoides berry juices (sample A : containing 10% juices, sample B : containing 20% juices, sample C : containg 30% juices) - 219 -
Table 3-29. Interest sensory test of functional jellies containing H. rhamnoides juice. Sensory characteristics Sample A Sample B Sample C Appearance 4.58±0.75 1) 5.26±0.78 5.11±0.72 Taste 5.11±0.97 b 3.74±0.96 a 4.58±1.04 b Flavor 5.05±1.05 b 3.79±0.77 a 3.42±1.31 a Overall 5.26±0.71 b 3.84±0.93 a 4.11±1.12 a 1) All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test. - 220 -
Table 3-30. Suitability sensory test of functional jellies contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Color 2.74±0.85a 1) 4.16±0.49b 4.47±0.68b 1) Sweetness 3.89±0.55 3.79±0.89 3.68±0.57 All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test. - 221 -
Fig 3-27. Functional beverages containing H. rhamnoides berry juices (sample A : containing 0% juices, sample B : containing 5% juices, sample C : containg 10% juices, sample D : containing 15% juices) - 222 -
Table 3-31. Interest sensory test of functional beverages contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Sample D Appearance 4.45±1.32 1) 4.39±1.21 4.32±1.06 4.35±1.33 Taste 4.65±1.26 c 3.81±1.57 ab 4.13±1.36 bc 3.23±1.29 a Flavor 4.55±1.10 b 3.81±1.26 a 3.58±1.10 a 3.45±1.43 a 1) Overall 4.61±1.10 c 4.32±1.30 bc 3.87±1.18 ab 3.35±1.36 a All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test. - 223 -
Table 3-32. Suitability sensory test of functional beverages contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Sample D Color 4.74±1.14 b 1) 4.32±1.25 ab 3.90±1.25 a 3.84±1.30 a Sweetness 4.48±1.34 c 4.06±1.13 bc 3.77±1.18 ab 3.26±1.41 a 1) Sourness 3.55±1.16 a 4.48±1.10 b 4.55±1.10 b 5.23±1.21 c All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test. - 224 -
μ μ - 225 -
μ μ - 226 -
Fig 3-28. DPPH radical scavenging activity of H. rhamnoides L fruit extract. - 227 -
Fig 3-29. Concentration-dependent anti-oxidative effect of H. rhamnoides L fruit extract in MEF cells. - 228 -
Fig 3-30. Morphological changes in MEF cells exposed to H 2 O 2 and H. rhamnoides L fruit extract. - 229 -
Fig 3-31. Effect of H. rhamnoides L fruit extract treatment on cell cycle progression in oxidative damaged cells. - 230 -
Fig 3-32. Effect of H. rhamnoides fruit extract treatment on cell cycle progression in oxidative damaged cells. - 231 -
- 232 -
Fig 3-33. Effects of H. rhamnoides fruit extract treatment on apoptotic cell death assessing by flow cytometry. - 233 -
Fig 3-34. Effects of the H. rhamnoides L. fruit extract in dysregulation cell cycle of the oxidative damaged cell (1; control group, 2; H 2 O 2, 3; H. rhamnoides, 4; Co-Treat) - 234 -
- 235 -
Fig 3-35. Protective effect of H. rhamnoides fruit extract on DNA damaged cells induced by DNA alkylating agent (MMS). - 236 -
Fig 3-36. Effect of H. rhamnoides fruit extract on cytotoxicity in three different human cancer cell lines (A; AGS, B; HepG2, C; HeLa). - 237 -
Fig 3-37. Apoptotic cell death increases according to H. rhamnoides fruit extract treatment times. - 238 -
Fig 3-38. Expression profiles of characteristic intrinsic apoptotic proteins (Bax, Bcl-2) after exposure to H. rhamnoides fruit extract in AGS cell for various times (24, 48, 72h). - 239 -
- 240 -
Fig 3-39. H. rhamnoides fruit extract inhibits AGS cell proliferation through the cell cycle arrest. - 241 -
Fig 3-40. Anti-proliferative effect through G2/M cell cycle arrest by 72 h administration times. - 242 -
Fig 3-41. Effect of H. rhamnoides fruit extract on related to G2/M cell cycle checkpoint regulating proteins. - 243 -
Fig 3-42. Expression of caspase-dependent apoptotic proteins exposure to H. rhamnoides extract in AGS cells after 72h administration times. - 244 -
- 245 -
Fig 3-43. Bacteria for examination of antibacterial performance. (A ; E. coli, B ; B. cereus, C ; S. aureus) - 246 -
Table 3-33. Selection of bacteria and cultivation conditions. Bacteria Culture media time of culture temp. Gram negative bacteria Escherichia coli (ATCC 25922) 18 h Trypticase Gram positive bacteria Bacillus cereus (ATCC 14579) Soy 16 h Broth 37 Gram positive bacteria Staphylococcus aureus (ATCC 25923) 15 h - 247 -
Fig 3-44. Antibacterial effect of H. rhamnoides extract against E. coli. - 248 -
Control H. rahmnoides treatment Fig 3-45. Clear zone of H. rhamnoides extract against E. coli. - 249 -
Table 3-34. Examination of forming clear zone against E. coli. Clear zone (mm) Kanamycin H. rhamnoides treatment 17.3 ± 1.5 - - 250 -
Fig 3-46. Antibacterial effect of H. rhamnoides L. extract against B. cereus. - 251 -
Control H. rhamnoides treatment Fig 3-47. Clear zone of H. rhamnoides extract against B. cereus. - 252 -
Table 3-35. H. rhamnoides fruit extract form clear zone against B. cereus. Clear zone(mm) Kanamycin H. rhamnoides treatment 20.3 ± 1.1 10.3 ± 0.71-253 -
μ - 254 -
Fig 3-47. Antibacterial effect of H. rhamnoides extract against S. aureus. - 255 -
Control H. rhamnoides treatment Fig 3-48. Clear zone of H. rhamnoides extract against S. aureus. - 256 -
Table 3-36. Examination of forming clear zone against S. aureus. Clear zone (mm) Kanamycin H. rhamnoides treatment 17.3 ± 1.5 - - 257 -
Fig 3-49. Set the effective minimal concentration of inhibition bacterial performance. - 258 -
Table 3-37. Minimal concentration of antibacterial effectiveness. Minimum antibacterial concentration (μg/ml) H. rhamnoides treatment E. coli B. cereus S. aureus - > 100 - - 259 -
Table 3-38. Examination of antibacterial performance against gram positive and gram negative bacteria. Clear zone (mm) H. rhamnoides treatment E. coli B. cereus S. aureus - ++ - - : No clear zone, + : 7 9 mm Clear zone, ++ : 9 12 mm Clear zone, +++ : > 12 mm Clear zone - 260 -
Fig 3-50. Large gel 2-DE separation of total protein lysate of MEF cell. - 261 -
Table 3-39. Comparative proteomic analysis of oxidative stress and H. rhamnoides extract using 2-DE. Spo t Accession number Protein name PI M. W (kda) Sequence covered Score ratio Reported function 1 gi 7948999 peroxiredoxin-4 6.67 31261 29% 86 1.66 up Prx4 plays a role in inhibition of NF-kB (nuclear factor 'kb) function as a cytosolic factor Arp2/3 complex is 2 gi 23956222 Actin-related protein 3 5.61 47783 35% 150 1.81 down major role in the regulation of the actin cytoskeleton Ratio: ratio of H. rhamnoides L. / H 2 O 2-262 -
Fig 3-51. The peptide mass fingerprinting of peroxiredoxin-4. - 263 -
- 264 -
Fig 3-52. Large gel 2-DE separation of total protein lysate of AGS cells. - 265 -
- 266 -
- 267 -
- 268 -
- 269 -
Fig 4-1. Climate information of investigate site. - 270 -
Table 4-1. Germination properties of H. rhamnoides seeds at different temperatures. Temperature ( ) GP (%) MGT (day) 10 32.7 24.1 0.72 1.4 15 40.7 12.1 1.85 3.4 20 40.7 7.8 3.04 5.3 25 32.7 4.0 5.17 8.1 30 30.0 5.7 2.61 5.4 35 3.3 6.7 0.26 0.5 GR GPI - 271 -
Table 4-2. Quadratic regression equations, r2 value and cardinal temperatures (Tb, Tm and To) for influence of temperature on H. rhamnoides seed germination. Characteristic Regression equations r 2 T b a T m T o a GP y = -0.131x 2 + 4.847x - 2.952 0.88 * 0.6 36.4 18.5 Base(T b ), maximum(t m ), and optimum(t o ) temperatures ( ), respectively. * : p<0.05. - 272 -
Table 4-3. Regression equations, r2 and cardinal temperatures (Tb, Tm and To) by linear sub- and supra-optimal models for germination rate in H. rhamnoides seed as a function of temperature response. a Characteristic Function type Model r 2 T b a GR Sub-optimal Supra-optimal y = 0.290x - 2.393 y = -0.491x + 17.4 0.93 * 8.3 0.93 * - Base(T b ), maximum(t m ), and optimum(t o ) temperatures ( ), respectively. * : p<0.05. T m - 35.4 T o 25.3-273 -
Fig. 4-2. Regression analysis models of GP and GR in H. rhamnoides seed as a function of temperature response. - 274 -
- 275 -
- 276 -
Table 4-4. Growth characteristics of H. rhamnoides seedling according to light conditions. Light condition Stem length(cm) Root collar diameter(mm) Survival(%) full light 49.4 9.88 86 50% light 50.3 8.39 82 25% light 43.1 7.05 68 5% light 37.4 6.95 68-277 -
Fig 4-3. Growth characteristics of H. rhamnoides seedling according to light quality conditions. - 278 -
Fig 4-4. Electrical conductivity in leachate of H. rhamnoides seed by seed age. - 279 -
Fig 4-5. Inorganic compounds in leachate of H. rhamnoides seed by seed age. - 280 -
Table 4-5. Change of soil properties before and after H. rhamnoides seedling planting Classification Rep. ph Before After OM (%) T-N (%) EP (mg/kg) CEC (cmol + /kg) EC (Ms/m) Salinity (%) 1 6.49 2.13 0.08 138.00 11.45 0.29 0.02 2 6.46 2.03 0.08 138.70 11.32 0.43 0.03 3 6.34 2.11 0.09 142.10 12.09 0.35 0.02 4 6.35 1.93 0.08 153.30 11.87 0.42 0.03 5 6.68 1.64 0.08 129.40 12.31 0.31 0.02 6 6.69 1.71 0.08 128.70 12.01 0.45 0.03 7 6.64 2.15 0.07 129.10 13.03 0.44 0.03 8 6.61 2.22 0.08 130.20 12.43 0.45 0.03 9 6.26 1.21 0.06 136.90 11.77 0.31 0.02 10 6.23 1.56 0.07 133.80 11.27 0.42 0.03 11 6.24 1.53 0.08 146.00 11.83 0.32 0.02 12 6.23 1.22 0.06 149.10 10.80 0.43 0.03 Mean 6.44 1.78 0.080 137.94 11.85 0.38 0.02 1 6.93 2.13 0.09 83.70 9.27 0.31 0.020 2 6.91 2.03 0.09 82.60 9.39 0.31 0.020 3 6.95 2.11 0.09 112.80 9.72 0.36 0.023 4 6.99 2.33 0.10 117.40 10.16 0.36 0.023 5 7.30 1.64 0.08 100.50 10.84 0.35 0.022 6 7.42 1.71 0.08 101.90 11.62 0.34 0.022 7 7.18 2.15 0.09 140.10 11.83 0.34 0.022 8 7.12 2.22 0.10 143.60 11.95 0.34 0.022 9 7.10 2.21 0.09 134.50 11.28 0.41 0.026 10 7.09 1.56 0.07 136.10 11.07 0.40 0.026 11 7.31 2.53 0.10 121.10 12.65 0.40 0.026 12 7.35 2.22 0.10 121.60 12.42 0.40 0.026 13 7.73 2.13 0.09 73.10 11.53 0.40 0.025 14 7.76 2.03 0.09 78.90 11.65 0.39 0.025 15 7.57 2.11 0.09 73.40 11.07 0.36 0.023 16 7.48 1.93 0.09 69.80 10.99 0.36 0.023 17 7.13 1.64 0.08 33.30 10.03 0.32 0.020 18 7.10 1.71 0.08 37.70 9.73 0.31 0.020 19 7.32 2.15 0.07 74.40 10.41 0.40 0.026 20 7.37 2.22 0.09 76.80 10.62 0.40 0.026 21 7.40 2.21 0.11 109.20 12.20 0.39 0.025 22 7.42 1.96 0.07 110.90 11.76 0.39 0.025 23 7.79 1.53 0.10 115.10 12.57 0.54 0.035 24 7.89 2.22 0.10 119.20 12.50 0.54 0.035 Mean 7.32 2.03 0.088 98.65 11.14 0.38 0.02-281 -
- 282 -
Fig 4-6. Germination of H. rhamnoides seeds with different irrigation intervals - 283 -
Fig 4-7. Height and survival of H. rhamnoides seedlings with different irrigation intervals - 284 -
Fig 4-8. Growth of H. rhamnoides seedlings with different irrigation intervals. - 285 -
Fig 4-9. Height and root collar diameter of H. rhamnoides seedlings by various fertilization condition. - 286 -
Fig 4-10. Chlorophyll contents of H. rhamnoides seedlings by various fertilization condition. - 287 -