.,...,,,.... oncogene carcinogen..

Transcription

1 I. II UR. 2.

2 .,...,,,.... oncogene carcinogen..

3 , %

4 ..,.,,,,.. 10, ( 0.5 )..,. III ,. 2.,,,,.

5 IV. 1.. S. faecalis 40mg/ml, S.cerevisiae.,. ethanol D101 column chromatography HPLC succinic acid, malic acid, acetic acid, oxalic acid, succinic acid. ethanol. pectinase pectin -1.4 polygalacturonse

6 25 /ml 66.7%, 30 /ml 92.6%. pectine ester pectinesterae /ml 15-20%. ethanol,.. JIM Hansenula anomala,,, CHS Torulopsis maris,,,,. CHS JIM ( 5, 1 ). Pseudomonas putida, Pseudomonas putida biotype A, Bacillus megaterium, Enterobacter

7 cloacae Klebsiella pneumoniae, Rhizopus oryzae. Bacillus megaterium. 50,,, Bacillus megaterium. Bacillus megaterium.,,,,.., Aspergillus niger,.,,.. berberine 1H, 13C spectrum berberine...

8 lactobacillus. 2.,,. E.coli O157:H7,.

9 Summary I. Title Development of natural food preservatives from agricultural products II. Significance and purpose of research and development 1. Purpose of research and development This studies are intended to develope the new food preservatives from the natural product including medicinal plants, and agricultural wastes. Almost all the preservatives used for food industry have been imported from other countries rather than national manufacturing. If natural preservatives were developed from domestic agricultural product, a lot of dollars for the imports could be saved. Additionally new industry for the manufacturing of food preservatives could be constructed for this country. Therefore, if natural preservatives were developed through these studies, synthetic preservatives causing in many health problems can be replaced with natural preservatives. Besides patents on this studies could be acquired domestically as well as internationally, and dollars for purchasing synthetic preservatives through trade can

10 be saved. This idea finally might activate domestic industry, even though this country were encountered WTO and UR which make seriously difficult situation to the farmer. 2. Significance of research and development The development of noble preservatives originated from natural product is very important in terms of improving the level of agricultural productivity through the supply and production of raw materials, as well as cultivation of international competition by the product development. It could be said that national competitiveness are dependent on the level of domestic industry. Also industry's competitiveness is depended on the new product developments and endless product improvement. (1) Development of kimchi preservatives There are many reasons korean kimchi is recognized well in the world. The first thing would be a unique taste and flavor from the many fresh raw materials added, as well as Lactobacillus spp resources, which is good to human intestines providing being comfortable and various vitamins. The early studies on Korean Kimchi are quite limited to only on manufacturing, recipe formulations, quality evaluation, micro floral changes during the fermentation, and a little preservation ways of the cabbage kimchi. But radish kimchi was not studied at all on its standard method of making, quality control, and preservation

11 technology so far. Therefore, this study was carried out to investigate characteristics during fermentation in radish kimchi and finally provided the ways of extending self-life. (2) Development of salted soy sauce(ganjang) preservatives Synthetic preservatives like salts of sorbate and benzoic acid derivatives have been used in soy sauce manufacturing for storage at long time circulating process(this period was usually one year). If this synthetic preservatives were dieted to the peoples for a long time, it could be highly exposed to potential carcinogen, as well as it was known to activate various oncogene that known to cause cancer. Therefore, use of synthetic preservatives as food additives must be avoided and limited strictly, also even the health authorities administrate very tightly the amount of use and the place of use. In this study, we have screened food preservatives from natural materials which have been known to nontoxic to our health, and searched antimicrobial activity in soy sauce to inhibit growth of harmful yeast that provide bad taste and flavor of soy sauce. (3) Development of preservatives for ginseng root Ginseng roots are used to be decayed by the various microbes easily so that it is very difficult to the marketing on time. Although Korea is actually a main country of Korea Ginseng, she has

12 no essential high techniques for marketing of raw ginseng (so called Susam, water ginseng). In this study, search and development of natural substances providing antimicrobial activity against causing fresh raw ginseng rottening were investigated. (4) Development of bread preservatives Nowadays, 40% of the world population used to take wheat as their food. As the mainly traditional reasons, the rice has been the staple food of these nation's people for a long history. The food culture of bread in this country, comparatively, is rather short, however it has gradually served as more and more people's breakfast. We know the bread's shelf life is generally very short (about 7 days). The transportation time distributed over the country from bread manufacturer to sale stores usually took 3 5 days, therefore after 2 or 3 days later of distribution, it's self life will be easily expired. In the view of this fact, to develope the techniques for elongating the bread's shelf life is seriously needed. To meet this requirement, we set out to do study on the natural material for elongation shelf life of bread. (5) Development of rice wine preservatives So many kinds of varous wine and spirits have been served for the Korean folks. Among those wine and spirits, Sogokju, Chungmyungju,

13 Smhaeju, Hahyungju, and Nokpaju were counted to the representatives. Sogokju is one of the representative wine is appointed to formless curtural assets. Processing to prepare this rice wine do not force to heat treatment due to classical manufacturing manners. In this main reason, so many bacteria are still alive in its bottle. This cause in easy souring during the time of distribution from market to customer (for the souring, around 10 days in summer, or 30 day in the winter). Unfortunately "the relationships between souring of rice wine and microorganism" are not completely elucidated so far. This studies are intended to identify the souring phenomenon causing organisms, and screen out natural compounds revealing the antimicrobial activity against microorganism responsible for the wind souring. III.Scope and range of research and development This title "Development of natural preservatives from agricultural products" are carried out three years and divided to five subsection as follows: Develoment of Kimchi(fermented vegitable with Chiness cabbage) preservatives. Development of soy source(fermented source with soybean) preservatives. Development of raw ginseng root(so called Susam) preservatives. Development of bread(factory manufactured) preservatives. Development of rice wine(mainly focused work with Sogokju) preservatives Overall scope and range of research and developments could be

14 summarized as follows: 1. By analyzing various classical archives including Bonchogangmok (editor Lee sijeen, Chinese), and Dongwibogam (editor Herjoon, Korean of Yee dynasty), various medicinal plants and natural products are collected from the across the country. 2. Various strains responsible for the putrefying(or souring, or rottening etc.) Kimchi, soy sources, raw ginseng, Factory manufactured bread, and rice wine were screened from its resources, and characterized to understand its putrefying properties. 3. Natural preservatives which show antibiotical activities against each putrefying microorganism selected the each purpose 4. Active fractions showing specific antibiotical activities were further purified using such various techniques as preparative TLC, HPLC, Ion exchange chromatography, elucidated its organic structures for the valuable one. 5. The selected compounds are assayed for the toxicity IV. Results and proposal for the practical use (1) Development of kimchi preservatives These attempts are carried out to find the ways of extending shelf-life of easily soured Radish kimchi in room temperature. The Chloroform fraction of Pugonyoung revealed relatively high antibiotical activities against almost all lactic acid bacteria, and

15 S. faecalis when addid more than 40mg/ml. But S.cerevisiae showed the rather weak inhibition in the this fraction. Schizandra chinensis has been used to chinese medicine of popular remedy for a long time. This study have been carried out to screen of useful components, physiological study, and antimicrobial activity. It has been reported that lignans of Schizandra chinensis have antimicrobial and phygiological activity. This study mainly focused on to investigate the effects of Schizan dra chinensis on the growth of a bacterium, CS6 which was isolated from Kimchi. CS6 was identified to Lactobacillus plantarum that cau ses acidification of Kimchi. The ethanolic extract of Schizandra chinensis(ees) inhibited the growth of L. plantarum. Minimum lethal concentration of EES on L. plantarum was 62.5 mg/ml. In broth culture, 5 /ml of EES completely inhibited the growth of L. plantarum during fermentation. It have been considered that pectinase directly related to the softness of Kimchi`s texture. Addition of EES also inhibited polygalacturonase, and pectineserase. Whereas EES did not significantly suppressed the activity of pectinesterase. In conclusion, the present experiment demonstrated that EES inhibited the growth of L. plantarum, and various enzyme activity. EES-containing Kimchi was sustained the hardness, and initial acidity during fermentation. EES was considered as the possible additive of Kimchi and EES added in Kimchi increase the quality, and storage period of Kimchi. (2) Development of salted soy sauce(ganjang) preservatives We have screened out garlic, Whanggeum, Backgngjam, kilkyung, doragy, etc as food preservatives to inhibit growth

16 of harmful yeast, Hansenula anomala and Torulopsis maris, that provide bad taste and flavor of soy sauce which was isolated from soy sauce. Garlic was finally selected as the best material so that it will provide to extend preservation period for the soy sources. (3) Development of preservatives for ginseng root Ginseng roots used to decayed by the various microbes easily so that it is very difficult to the marketing on time without another trial. In this study, Gaja( ), Omija( ), and Gamgook providing antimicrobial activity against causing fresh raw ginseng rottening, Pseudomonas putida, Pseudomonas putida biotype A, Bacillus megaterium, Enterobacter cloacae, and Klebsiella pneumoniae, were screened. The extract of Gaja and Omija, respectively, showed the most strong antimicrobial activity against Bacillus megaterium, which are considered the most responsible organism to decay (4) Development of bread preservatives The most frequently isolated strain from the rotten bread was the Aspergillus niger, and Aspergillus oryzae, and Penicillum sp. With these two fungi, Natural preservatives to extend shelf life of bread are screened finally to seed of Dongback flower and Whangyeon. The active component was elucidated to berberine by the

17 jointing interpretation of 1H, 13C spectrum from Whangyeun. (5) Development of rice wine preservatives Because many of the rice wine have been encountered the short shelf life especially in the summer season, it is very important to elucidate the souring phenomenons in model system of Sogokju, representative Korean traditional rice wind. It was concluded that Omija showed high growth inhibition to the lactobacillus spp. causing in souring phenomenon. When Omija was added in the rice wine of around 16% alcohol concentration, the shelf life was extended to two time than that of control.

18 CONTENTS Part1. Searching for natural herbs to extend Kacdugi's shelf-life Chapter 1 Introduction 34 Chapter 2 Research method 38 Section 1 Kacdugi's short & long storage properties of herbs and vegetables' extract by water 38 section 2 effect of Kacdugi's ripening by spicy ingridients 40 section 3 effect of antimicrobs and Kacdugi's ripening to Kacdugi's saprogenous bacillus by Omiza & Baekgangzam extracts Kacdugi's manufacturing ph and total acidity total micribs number property of Texture color index antimicrobial test invert sugar content 47 section 4 effect of Kimch Lactobaciilus and inhibition yeast by herbs extract 47

19 1. materials Kacdugi's manufacturing addition manufacturing ph and total acidity hardness used microbs and medium extraction method partition of extracts Soluble silid content antimicrobial test heat stability of antimicrobial material ph stability of antimicrobial material growth rate and ph MIC (minium inhibitary concentration) antimicrobial effect by each chloroform concentrative extracts antimicrobial activity by patition extracts 57 Chapter 3 Results 58 Section 1 Kacdugi's short & long storage properties of herbs and vegetables' extract by water properties of short storage properties of long storage 70 Section 2 effect of Kacdugi's ripening by spicy ingridients red pepper 81

20 2. galic green onion salt water abstract 85 section 3 effect of antimicrobs and Kacdugi's ripening to Kacdugi's saprogenous bacillus by Omiza & Baekgangzam extracts effect of antimicrobs and Kacdugi's ripening to Kacdugi's saprogenous bacillus by& Baekgangzam extracts effect of antimicrobs and Kacdugi's ripening to Kacdugi's saprogenous bacillus by Omiza extracts abstract 88 section 4 effect of Kimch Lactobaciilus and inhibition yeast by herbs extract Change of ph and acidity during Kacdugi's ripening effect of Kacdugi's ripening by natural herbs antimicrobial test of extract by water heat stability of antimicrobial marerials ph stability of antimicrobial material growth rate and ph MIC (minium inhibitary concentration) 100

21 8. antimicrobial effect by each chloroform concent rative extracts antimicrobial activity by patition extracts abstract 106 Chapter 4 Reference 108 Part 2. Development of soy souce preservatives Chapter 1 Introduction 114 Chapter 2 Materials and Methods 117 Section 1. Materials Culture and Identification of strain Natural and others materials 117 Section 2. Methods Isolation of expansive strain Morphological identification by microscope Biochemytrical identification by API kit Extraction of Oleoresine Experiments of anti-microbial 119 Chapter 3. Result 121 Section 1. anti-microbial activity assay of natural materials Biochemystrial identification of soy souce yeast Result of expansion test Extract of Oleoresine 125

22 4. Experiments of anti-microbial 126 Chapter 3. References 130 Part 3. Reserch of ginseng preservatives Chapter 1. Introduction 132 Chapter 2. Material and methods 134 Section 1 Material 134 Section 2 Methods Isolation and identification of rotten wet ginseng microbes Antimicrobial test Inhibition of wet ginseng's rottenness 136 Chapter 3 Results & discussion 138 Section 1 Results rotten wet Genseng microbs Identification of rotten wet Genseng microbs antimicrobial test of rotten wet Genseng microbs Isolation of antimicrobial substances in Gaja extract antimicrobial test of rotten wet Genseng microbs bynatural herbs' essential Inhibitary effect of rotten wet Genseng microbs in Gaja extract 148 Section 2 Abstract 149 Chpter 4 References 150

23 part 4. Developement of bread preservatives. Chapter 1. Introduction 152 Chapter 2. Material and methods 153 Section 1 Material 153 Section 2 Methods Isolation and identification of rotten bread microbes Antifungal test 154 Chapter 3. Results 156 Section 1. Isolation and identification of rotten bread microbes 156 Section 2. Screening of antimicrobial natural preservatives 158 Section 3. Isolation and Identification of Coptis Japonica Makino 164 Chapter 4. References 164 Part 5. Development of alcohol preservation Chapter 1 Introduction 166 Chapter 2 Mathods 168 Section 1 Han-san sogokju 168 Section 2 Screening of natural materials preservitives 169 Chapter 3 Results 171 Section 1 Han-san sogokju 171

24 1. Isolation and identification of microorganisms which exsit in Sogokju Change of ph and acid composition of Sogokju Change of microorganisms distribution in Sogokju as time course Organic acid composition of sogokju and soured sogokju Growth stop temperature of souring microorganism preserving period of sogokju's qality in stop temperature 175 Section 2. natural preservatives Activity screening against sogokju microorganism and acidity observation Change of acidity in sogokju as omisa addition 178 Section 3. Passibility of application as natural preservative of omisa 180 Chapter 4. References 181 APPENDIX 182

25 ph Texture

26 ph Soluble silid ph ph

27 ph ph ph

28 API kit Oleoresine Test Oleoresin Paper disc

29 ,

30 ph

31 1, 1, (1),.,.,.. 2 (24),,,.. (25 26), (25 28), (27 28),

32 ,.,. 3., (radurization) (6-7), (8), (9-11), (12). sorbic acid, sodium dehydroacetate (13-14), ph (15-18), (19-22), (23-24) (13,25-26).., ph,,,.. ph,,., texture

33 (3). Texture. polygalacturonase (4). texture (hardness), (brittleness, crispiness), (chewiness) (5)...,,,, ph, texture. sorbic acid, benzoic acid 13,.,,,,.,,,

34 ,,,,,.(14)

35 2 1 1.,,, , ( ).. NaOH Juncei phenophthalein Sigma GR cm cm 50 g 1.17 g, 1.67 g, 0.84 g, 0.25 g, 1.17 g, 10 ml polyethylene bag 20 1 (Low Temp. Incubator LTI-1000SD, Eyela) 15.

36 . (Food mixer FM-700W, ) (Standard Testing Sieve, Aperture 250 m, No. 60) (Shaking Incubator, VS-8480, SR) (Toyo No.2).. ph ph ph meter (Hanna Instruments 8521) ph.. 0.1% phenophthalein 1.0 N NaOH (Lactic acid, %).. (Hardness) (Hardness) Texture Analyser (XR.RA Dimension, Stable Micro Systems, Version 3.7) TPA (Texture Profile Analysis) TPA Table 1. Texture Analyser 2 force - time curve first bite peak. Table 1. Condition of texture analyzer for texture profile analysis.

37 Sample rate Force threshold Distance threshold Contact area Contact force Pre test speed Post test speed Test speed Strain Time Trigger type 400 pps 20 g 0.5 mm mm2 50 g 10 mm/sec 10 mm/sec 10 mm/sec 75 % 0.5 sec 20 g. 1 ml 100 L MRS (de Man, Rogosa & Sharpe agar). 30 (Incubator, VS-1203 P3, Vision Sci. Co.) 24 colony plate agar. 2 1.,,, , ( ). ( )., ( ), ,,, , ,,, 92343, ,

38 ,,,,, 7. NaOH Juncei phenolphthalein Sigma GR ) 2 2 2cm 50g 1.17g, 1.67g, 0.84g, 0.25g, 1g, 10mL polyethylene bag (Low Temp incubator, LTI-1000SD,Eyela) 20. 2),, +, +, +, g polyethylene bag,, (Low Temp incubator, LTI-1000SD,Eyela).. (Food mixer FM-700W, HANIL) (Standard Testing Sieve, Aperture 250 m, No. 60). 10g ( ) 100ml

39 60 48 shaking (Shaking incubator, VS-8480, SR) 10ml., 95% 1: (EYELA. TOKYO RIKAKIKAI CO., LTD, A-39, Tokyo, Japan) 1/9 membrane filter(whatman 0.2 ).. ph ph ph meter (HANNA Instruments 8521) ph.. 0.1% phenolphthalein 0.1 N NaOH (Lactic acid, %)... 1ml 0.1ml (NA) MRS(de Man, Rogosa & Sharpe agar) (Incubator, VS-1203 P3, Vision Scientific Co.) colony plate agar.. Texture

40 . Texture Texture Analyser (XR.RA Dimension, Stable Micro Systems, Version 3.7) TPA (Texture Profile Analysis) TPA (9). Texture Analyser 2 force - time curve first bite peak.. 3 (Color difference meter, CR-300, JAPAN, Minolta) Hunter L, a, b g(2x2x2) 1g, 1.67g, 0.84g, 0.25g, 1.17g, 10ml. 20 (Incubator LTD-1000SD,EYELA).. 1)

41 (Food mixer FM-700W, Hanil) (Standard Testing Sieve, Aperture 250 m, No. 60). 10% 60 2 (Shaking Incubator, VS-8480, SR) (Toyo No.2). 2) 95% 1: hr (, EYELA, Japan ) 1% 3%. 3).

42 MeOH extract Hexane : MeOH : H2O(10: 1: 9) Hexane layer Evap. under vacuum H2O layer CHCl3 Hexan extract H2O layer CHCl3layer EtOAc Evap. under vacuum CHCl3extract EtOAc layer H2O layer Evap. under vacuum BuOH EtOAc extract BuOH layer H2O layer Evap. under vacumm BuOH extract Evap. under vacuum H2O extract Fig 1. Solvent extraction from methanol extract using various solvent

43 2. ph ph ph meter (HANNA Instruments 8521) ph.. 0.1% phenolphthalein 0.1 N NaOH (Lactic acid, %) ml 0.1ml (NA) MRS(de Man, Rogosa & Sharpe agar) (Incubator, VS-1203 P3, Vision Scientific Co.) colony plate agar. 4. Texture. Texture Texture Analyser (XR.RA Dimension, Stable Micro Systems, Version 3.7) TPA (Texture Profile Analysis) TPA (9). Texture Analyser 2 force - time curve first bite peak. 5.

44 3 (Color difference meter, CR-300, JAPAN, Minolta) Hunter L, a, b. 6. Paper Disc Method. broth 24hr (0.75 % agar) 100 : 1 Pouring 100 disc SDW( ) hr disc clear zone( ). 7. dinitrosalicylic acid(dns),, glucose (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 / ) DNS 2 10 UV spectrophotometer 550. glucose glucose. 4

45 1.,,,, ( ). NaOH Juncei phenophthalein Sigma GR. (Table 2), (Food mixer FM-700W, HANIL) (Standard Testing Sieve, Aperture 250, No. 60) cm cm 50 g 1.17 g, 1.67 g, 0.84 g, 0.25 g, 1.17 g, 10 ml polyethylene bag 20 1 (Low Temp. Incubator LTI-1000SD, Eyela) ((Food mixer FM-700W, ) (Standard Testing Sieve, Aperture 250 m, No. 60) (Shaking Incubator, VS-8480, SR) (Toyo No.2). 4. ph

46 ph ph meter (Hanna Instruments 8521) ph.. 0.1% phenophthalein 1.0 N NaOH (Lactic acid, %). 5. (Hardness) (Hardness) Texture Analyser (XR.RA Dimension, Stable Micro Systems, Version 3.7) TPA (Texture Profile Analysis) TPA Table 2. Texture Analyser 2 force - time curve first bite peak.

47 Table 2. List of natural plants addition on kakdoogi fermentation at 20 1

48 Korean name Scientific name Korean name Scientific name Pueraria thunbergiana Achyranthes japonica Chrysanthemum sinense Rehmannia glutinosa Cassia tora Cinicifuga foetida Chinemys reevesii Manfnolia liliflora Lonicera japonica Hauttuynia cardata Platycodon grandiflorum Ralipanax pictum Camellia sinensis Ganderma lucidum Angelicae gagatis Acanthopanax sessiflorum Rheum undulatum Schizandra chinensis Prunus persia Ginko biloba Eucommia ulmoides Oliver Epimedium koreanum Vitex rotundifolia Aritemisiae capillaris thunb Chaenomeles lagenaria Peaonia lactiflora Saussurea lappa Phyllostachys edulis Phellopterus littoralis Citrus tangerina Imperata cylindrica Plantage asiatica Pachyma hoelen Cnidium officinale Paeonia albiflora Cyathula officinale Angelicae dahuricae Trichosanthes kirilowii Cynanchum silfordii Artemisiae annuae herba Cnidium monnier Zanthoxylum pipertum Sophora subprostrata Thuja orientalis Crataegus pinnatifoda Alismatis plantago Dioscorea batatas Cuscuta australis Zanthoxylum bungeanum Taraxacum mongolicum Morus alba Prunus armeniaca Acorus gramineus Cyperus rotundus Erillae herba Scrophularia buergeriana Pinus rigida Carthamus tinctorius Pinus rigida Scutellaria baicalensis Table 3. Conditions for Texture Profile Analyser

49 Grape type Force vs Time Acquisition rate 200pps Force threshold 20 Force unit Grams Contact force 5.0g Distance format Strain Test speed 10.0 mm/sec Trigger type Auto 20 (Ø cm) (Table 4), brain heart infusion agar(difco), Lactobacilli MRS broth agar(difco), YM broth agar(difco). Table 4. List of used microorganisms Gram positive Streptococcus faecalis KCTC 2011 bacteria Lactic acid Lactobacillus plantarum KCYC 3108 Lactobacillus brevis KCTC 3102 Leuconostoc mesenteroides KCTC 3505 Yeast Saccharomyces cerevisiae KCTC , 1:

50 100% 1:1 3,.(Fig. 1), :90% 1 3 (7.84g), 90% :80% 1 3, 80% :60% % (2.22g).(15) 9. Soluble solid Soluble solid 1ml 105 (mg).(16) 10. (23) 1 Table 5. slant 1 10 broth, Dried leaves of Taraxacum mongolicum (1Kg)

51 MeOH (6L) MeOH extract (63g) CHCl3layer H2O layer Evap. under vacumm CHCl3extract (27g) Hexane layer 90% MeOH Evap. under vacumm Hexane extract (7.9g) CCl4layer 80% MeOH CCl4extarct (6.6g) Evap. under vacumm CH2Cl2layer Evap. under vacumm 60% MeOH Evap. under vacumm CH2Cl2extract(4.1g) 60% MeOH extract(3.4g) Fig 2. Fractionation of the chloloform extract from Taraxacum mongolicum.

52 petridish 15, 5, ( ) filter paper disc(toyo seisakusho, 8 ),, (4 ) 1, 30 incubator disc clear zone ( ). (17) (disc plate method). (17) Table 5. List of herbs used for antimicrobial experiment

53 Korean English name Botanical name plant part name Cassiae torae semen Cassia tora Seeds Lycium Fruit Lycium chinense MILLER Seeds Lonicerae flososs Lonicera japonica Flower Platycodi radix Platycodon grandiflorum Root A. De CANDOLLE Rheum palmatum Imperata cylindrica Paeonia albiflora Angelicae dahuricae Crataegus pinnatifida Dioscorea batatas Ginko biloba Taraxaci herba Taraxacum mongolicum Leaves Moutan radicis Paeonia suffruticosa Bark cortex Citrus tangerina A corus graminens Soland Zanthoxylum pipertum Carthamus tinctorius Root 12. ph ph ph1 13 1, ph7.(17) 13. ph

54 Broth slant 1 10ml broth, 30, 24 2 spectrophotometer(pharmacia Biotech, Ultrospec 3000) 660nm ph. 14. (MIC, minimum inhibitary concentration), membrane filter(0.2 ), , 10, 20, 40, 80mg/ml nm optical density(od) membrane filter(0.2 ), 5, 10, 20, 40, 80mg/ml., 9.8ml 0.1ml 0.1ml 10ml, 30 spectrophotometer(pharmacia Biotech, Ultrospec 3000) 660nm optical density(od). Blank, 0.3.(19) 16.

55 membrane filter(0.2 ), , nm optical density(od).. (18) blank. % inhibitary effect = (control-control blank) (treatment treatment blank) (control-control blank)

56 ph 20 ph Fig 3. ph 6.0, 0.08%(lactic acid) ph (ph 5.5 ), (0.2% ).(19,29 31) (34) 1 kg % (lactic acid). (35) ph ph ph. ph , %(lactic acid).

57 Fig 3. Change in ph, Acidity and Hardness of kakdoogi during fermentation at 20. ph, (hardness) TPA(texture profile analysis). ph ph 4.2, 0.6%(lactic acid) , 2.5,

58 , 2.5, 7, (60 ) ph, Table %(lactic acid). Table (,,,,,,,,,,,,,, ). 2.5 (18-60 ). 15,, (19). (19) ,,,,,,,.,,,,,.

59 ,,,, 1-15.,.(9), (9) %, % 50%,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, % 20, , Texture analyzer,,,,,,,,,,. 30%,,,,,

60 ,,,,,,,, %,,,,,,,,,,,,,,,,,,,,,,, %,,,, 6. 50%(0.3% lactic acid) 30%,,,,,,,, 9.

61 Table 6. Effect of hot water extract of medicinal plants on the fermentation of kadoogi No. Korean Sample Scientific name ph Acidity Time (Lactic acid, (hr) %) Hardness (sample/control) C Control ) Sensory score (-3 +3) 1 Pueraria thunbergiana Chrysanthemum sinense Cassiae tora Sophora angustiflora Chinemys reevesii Lonicera japonica Platycodon grandiflorum Angelicae gagatis Rheum undulatum Prunus persia Eucommia ulmoides Vitex rotundifolia Chaenomeles lagenaria Saussurea lappa Phellopterus littoralis Imperata cylindrica Magmoliakobushimayerbesser Pachyma hoelen Paeonia albiflora Angelicae dahuricae Cynanchum sifordii Cnidium monnier Sophora subprostrata Crataegus pinnatifoda Dioscorea batatas Zanthoxylum bungeanum Saururus chinensis Morus alba Acorus gramineus Sedum sarmentosum Erillae herba Pinus rigida Pinus rigida Achyranthes japonica Rehmannia glutinosa ) Time (hour) to reach the optimum ripeness that shows acidity of 0.6% (lactic acid) 2) Hardness of control is 5870 kg. continued + Acidity below 50% compared with control

62 Table 6. Effect of hot water extract of medicinal plants on the No. Korean fermentation of kakdoogi. Sample Scientific name ph Acidity (Lactic acid, %) Time Hardness (sample/control) C Control ) Sensory score (-3 +3) 36 Cinicifuga foetida Manfnolia liliflora Hauttuynia cardata Ralipanax pictum Forsythia suspensa Ganderma lucidum Acanthopanax sessiflorum Schizandra chinensis Pinus rigida Ginko biloba Epimedium koreanum Artemisiae capillaris thunb Peaonia lactiflora Gleditsia sinensis Phyllostachys edulis Citrus tangerina Plantage asiatica Cnidium officinale Gastorodia elata blume Cyathula oficinalis kuan Trichosanthes kirilowii Artemisiae annuae herba Thuja orientalis Alismatis plantago Cuscuta australis Traxacum plantycarpum Prunus armeniaca Cyperus rotundus Scrophularia buergeriana Corydalis turtsch aninowii Carthamus tinctorius Scutellaria baicalensis Astragalus membranaceus ) Time (hour) to reach the optimum ripeness that shows acidity of 0.6% (lactic acid) 2) Hardness of control is 5870 kg. continued + Acidity below 50% compared with control

63 (60 ) ph, Table %(lactic acid). Table (,,,,,,,,,,,,,,, ). 2.5 (18-60 ). 16,,,, (19). (19)., ph,, (19),., 14. (41-42)..

64 ,,., ,,,,,,,,,,,,,,,,, %,,,,,,,,, 1-15.,,,.(9) %, % 50%,,,,, % 20, , Texture analyzer

65 , %,, % 1. 50%(0.3% lactic acid) 30%,, 3.

66 Table 7. Effect of hot water extract of vegetables and herbs on the No. Korean fermentation of kakdoogi Sample Scientific name ph Acidity (Lactic acid, %) Time (hr) Hardness (sample/control) Sensory score (-3 +3) C Control ) 1 Brassica juncea Brassica cernua Cinnamomum verum Impomoea betatas Capsium annum Camellia sinensis Thymus vulgaris Allium monanathum Daucus carota Aralia elata Perilla fructescens Raphanus sativus Oenanthe japonica Allium tuber Amaranthus patulus Lactuca sativa Apium graveolens Spinacia oleracea Artemisia asiatica Chrysanthemum coronarium Malva verticillata Moringa oleifera Brassica oleracea Allium cepa Nelnumbo nucifera Cucumis sativus Pisum sativum Laurus nobilis Eugenia caryophyllate Brassica oleracea Zanthoxylum pipertum Aster saber Cichorium intybus Brssica oleracea Petroselinum crispum Cucurbita moschata Angelica keiskei ) Time (hour) to reach the optimum ripeness that shows acidity of 0.6% (lactic acid) 2) Hardness of control is 5870 kg. continued + Acidity below 50% compared with control

67 ,, 12 (,,,,,,,,,,, ) 3 Fig cfu/ml,,,,,.,,,,,.. 105,,,,,,,,,,, 12, 30%. Fig. 4

68 2. (13 )>,, (11 )> (10 )>,, (9 )>,, (8 )>, (7 )>,,,,, (6 ). 0.6%(lactic acid) %,,,,,,,,,,,,,, ph,, Table 8, 9, , 15, 7,,,,,,,,, 10., 3. (11 15 ),,,,,,,.

69 (9).,. Lactobacilli sp. (9). 8 Leuconostoc sp. (21,24 27).

70 Table 8. Characteristics of 3-day fermented kakdoogi at 20 Sample Hardness No. Koream Scientific name name Acidity Lactobacilli Bacteria ph (Sample/ (%) ( 105,cfu/g) ( 105,cfu/g) control) c Pueraria thunbergiana Chrysanthemum sinense Brassica cernua Cinnamomum verum Chinemys reevesii Lonicera japonica Platycodon grandiflorum Camellia sinensis Angelicae gagatis Rheum undulatum Prunus persia Chaenomeles lagenaria , Saussurea lappa Phellopterus littoralis Imperata cylindrica , Magmolia kobushi mayer besser Pachyma hoelen Angelicae dahuricae Cynanchum sifordii Sophora subprostrata Crataegus pinnatifoda Dioscorea batatas Zanthoxylum bungeanum Morus alba Acorus gramineus Erillae herba Pinus rigida Pinus rigida Cinicifuga foetida Manfnolia liliflora Moringa oleifera Ganderma lucidum Schizandra chinensis

71 Table 8. Characteristics of 3-day fermented kakdoogi at 20 Sample No. Koream name Scientific name ph Acidity (%) Hardness (Sample/ control) Lactobacilli Bacteria ( 105,cfu/g) ( 105,cfu/g) c Ginko biloba Acanthopanax sessiflorum Epimedium koreanum Artemisiae capillaries thunb Peaonia lactiflora Eugenia caryophyllate Phyllostachys edulis Citrus tangerina Plantage asiatica Cyathula officinalis kuan Trichosanthes kirilowii Artemisiae annuae herba Zanthoxylum pipertum Thuja orientalis Traxacum plantycarpum Alismatis plantago Cuscuta australis Prunus armeniaca Scrophularia buergeriana Carthamus tinctorius

72 Table 9. Characteristics of 7-day fermented kakdoogi at 20 Sample Hardness No. Koream Scientific name Acidity Lactobacilli Bacteria ph (Sample/ name (%) ( 105,cfu/g) ( 105,cfu/g) control) c Pueraria thunbergiana Chrysanthemum sinense Brassica cernua Cinnamomum verum Chinemys reevesii Lonicera japonica Platycodon grandiflorum Camellia sinensis Angelicae gagatis Rheum undulatum Prunus persia Chaenomeles lagenaria Saussurea lappa Phellopterus littoralis Imperata cylindrica Magmolia kobushi mayer besser Pachyma hoelen Angelicae dahuricae Cynanchum sifordii Sophora subprostrata Crataegus pinnatifoda Dioscorea batatas Zanthoxylum bungeanum Morus alba Acorus gramineus Erillae herba Pinus rigida Pinus rigida Cinicifuga foetida Manfnolia liliflora Moringa oleifera Ganderma lucidum Schizandra chinensis

73 Table 9. Characteristics of 7-day fermented kakdoogi at 20 Sample No. Koream Scientific name name ph Acidity (%) Hardness Lactobacilli (Sample/ ( 105,cfu/g) control) c Ginko biloba Acanthopanax sessiflorum Epimedium koreanum Artemisiae capillaries 38 thunb Peaonia lactiflora Eugenia caryophyllate Phyllostachys edulis Citrus tangerina Plantage asiatica Cyathula officinalis kuan Trichosanthes kirilowii Artemisiae annuae herba Zanthoxylum pipertum Thuja orientalis Traxacum plantycarpum Alismatis plantago Cuscuta australis Prunus armeniaca Scrophularia buergeriana Carthamus tinctorius Bacteria ( 105,cfu/g)

74 Table 10. Characteristics of 11-day fermented kakdoogi at 20 Sample Hardness Acidity No. Koream Scientific name ph (Sample/ (%) name control) c Pueraria thunbergiana Chrysanthemum sinense Brassica cernua Cinnamomum verum Chinemys reevesii Lonicera japonica Platycodon grandiflorum Camellia sinensis Angelicae gagatis Rheum undulatum Prunus persia Chaenomeles lagenaria Saussurea lappa Phellopterus littoralis Imperata cylindrica Magmolia kobushi mayer besser Pachyma hoelen Angelicae dahuricae Cynanchum sifordii Sophora subprostrata Crataegus pinnatifoda Dioscorea batatas Zanthoxylum bungeanum Morus alba Acorus gramineus Erillae herba Pinus rigida Pinus rigida Cinicifuga foetida Manfnolia liliflora Moringa oleifera Ganderma lucidum Schizandra chinensis

75 Table 10. Characteristics of 11-day fermented kakdoogi at 20 Sample No. Koream name Scientific name ph Acidity (%) Hardness (Sample/ control) c Ginko biloba Acanthopanax sessiflorum Epimedium koreanum Artemisiae capillaries thunb Peaonia lactiflora Eugenia caryophyllate Phyllostachys edulis Citrus tangerina Plantage asiatica Cyathula officinalis kuan Trichosanthes kirilowii Artemisiae annuae herba Zanthoxylum pipertum Thuja orientalis Traxacum plantycarpum Alismatis plantago Cuscuta australis Prunus armeniaca Scrophularia buergeriana Carthamus tinctorius %, %, % 2

76 26,,,,,,,,,,,,,,,,,,,,,,,, %(lactic acid),,,,,,,,,,,,, (12 13 ),,,,,,,. 3 (10 11 ),,,,,,,,,,. Table 11. Fermentation times to reach the acidity of 1.0% (lactic acid) kakdoogi containg water extract of medical plants or vegetables. Time Medical plants or vegetables (days) 7 control,,,,,,,, 10,,,,,,,,,,, 12,,,,,,,,,,,,,,, 15,,,,,,,,,,,,,,, 3,,,,,,,,,,,,,,,,,,,. 7

77 ,,,,,, 7,,,,,,,.,,,,,, %,,,,,,,, 9.. Table 12 Hardness of kakdoogi containing water extract of medical plants or vegetables during fermentation. Higher than control,,,,,,,,,,,,,, Lower than control,,,,,,,,,,,,,,,,, % (60, 2 ) 20 (ph,

78 ). (Lactic acid, %) 0.6%, % 50% 48, %,,,,,,,,,,, ,, +, +, +, ph 2 ph , ph ph, 5,. ph. > > > 3,,. 0.6%

79 (Lactic acid), 5, 7, 8, 10, 15. TPA,,. 9, 9.,,. 1. ph 12. ph %, 0.6% 5,, ,. 12. A carotene, C. C.

80 capsaicin 0.41%.., Leuconostoc mesenteroides, (10). 2. ph 7 12 ph4,. 0.6% 8., 5,., ph (14) 2 3. (10).,,.,

81 allicin allinase allicin allicin B1.. (11) (0-6%) Leuconostoc mesenteroides.(12) (13) 3%,. ph. 7 ph % ph (14) 3% 25 4,. gingeron shagol citral

82 linalool. L.plantatrum, L.brevis, P.cerevisiae, L.mesenteroides.(14,15), L.mesenteroides ph , 4 (16). (12), ph. ally,.,, A C ph 3,. 0.6% 10..

83 .,.(17) 3% (18,19), (20) 3%, 4%. (21) 3% 6%., (18),,. 5. 5,, %(Lactic acid), 5, 7, 8, 10, 15.,.,,

84 % 3%, 10% 20 ph,,, ( ). ph % 5.24, 1% (Lactic acid, %) 0.6%, 1% 3.2, 3% (cfu/ ) 1% (cfu/ ), 3% (cfu/ ). 3. 1% Paper Disc Method, test,

85 . (ph,, ), ( ),,,. 2. 1%, 3% 10% 20 ph,,, ( ). ph % % (Lactic acid, %) 0.6%, 1% 4.2, 3% (cfu/ ) 1% (cfu/ ), 3% (cfu/ ). 3. 3%, 1% Paper Disc Method, test

86 . test 1.2 clear zone. (ph,, ), ( ),,, % 1: (, EYELA 3%, Japan) membrane filter(whatman 0.2 ) 1% 10% 20 ph,,, ( ). ph % % (Lactic acid, %) 0.6%, 1% 4.2, 3% (cfu/ ) 1% (cfu/ ), 3% (cfu/ ). 3. 3%,

87 1% Paper Disc Method. test, 1.2 clear zone. 95% 1: (, EYELA, Japan) membrane filter(whatman 0.2 ) 1% 3% 10% 20 ph,,, ( ). ph % 5.24, 1% (Lactic acid, %) 0.6%, 1% 3.2, 3% (cfu/ ) 1% (cfu/ ), 3% (cfu/ ). 3. 1% Paper Disc Method. test,.

88 4 1. ph. 20 PH, ph 6.0, 0.08%, ph , %(lactic acid). 20, (60 ) ph Table %(lactic acid). 2.5 (60 ) 0.6% 0.6% 50%,,,,,,,,,,,,,,,,,, % 20,

89 . 40~50%,,,,, 6. 50%(0.3% lactic acid) (30% ),,,,,,, 9. 60,,, 12, 30%.

90 Table 13. Effect of medicinal plants addition on kakdoogi fermentation for 60 hours. Sample name ph Acidity Time Hardness Korean Scientific (Lacticacid,%) (hr) (Sample/ Control) Control ) ) Pueraria thunbergiana * Chrysanthemum sinense * Cassia tora Chinemys reevesii * Lonicera japonica * Platycodon grandiflorum * Camellia sinensis * Angelicae gagatis * Rheum undulatum * Prunus persia Eucommiulmoides Oliver Vitex rotundifolia Chaenomeles lagenaria * Saussurea lappa * Phellopterus littoralis * Imperata cylindrica * Pachyma hoelen * Paeonia albiflora * Angelicae dahuricae * Cynanchum silfordii Cnidium monnier Sophora subprostrata * Crataegus pinnatifoda * Dioscorea batatas * Zanthoxylum bungeanum * Morus alba * Acorus gramineus * Erillae herba * Pinus rigida * Pinus rigida ) Time (hour) to reach the optimum ripeness that shows acidity of 0.6%(lactic acid). 2) Hardness of control is5870kg * acidity below 50% compared with control continued.

91 Table 13. Effect of medicinal plants addition on kakdoogi Korean fermentation for 60 hours Sample name PH Acidity Scientific (Lacticacid,%) Time (hr) Hardness (Sample/ Control) Control Achyranthes japonica Rehmannia glutinosa * Cinicifuga foetida * Manfnolia liliflora Hauttuynia cardata Ralipanax pictum Ganderma lucidum * Acanthopanax sessiflorum * Schizandra chinensis Ginko biloba Epimedium koreanum * Aritemisiae capillaris * thunb Peaonia lactiflora * Phyllostachys edulis * Citrus tangerina * Plantage asiatica Cnidium officinale Cyathula officinale Trichosanthes kirilowii Artemisiae annuae herba * Zanthoxylum pipertum * Thuja orientalis * Alismatis plantago * Cuscuta australis Taraxacum mongolicum Prunus armeniaca * Cyperus rotundus * Scrophularia buergeriana * Carthamus tinctorius * Scutellaria baicalensis

92 Table 14. Antimicrobial activities of chloroform extract on the several microorganisms Plant L.mesent Clear zone on plate (mm) L.plant L.brevis S.faeca S.cere Solublesolid Content (mg/disc) -eroides -arum -Lis -visiae Table 15., L. mesenteroides, L. plantarum L. brevis, S. faecalis,

93 , S. cerevisiae.. (16) 31 B. subtilis L. plantarum. (20)..

94 Table 15. Antimicrobial activities of water extract on the several microorganisms Plant L.mesent Clear zone on plate (mm) L.plant L.brevis S.faeca S.cere Soluble solid Content (mg/disc) -eroides -arum -lis -visiae ~ S. faecalis (Fig. 5), 100 1

95 . Fig 5. Effect of heat treatment on the growth inhibitory activity of chloroform extract for Streptococcus faecalis KCTC ph ph (Fig. 6), ph 1~13.

96 Fig 6. Effect of ph treatment on the growth inhibitory activity of chloroform extract for Streptococcus faecalis KCTC ph ph Fig. 7 Fig. 8.

97 Fig 7. Growth curve of the several microorganisms for 24 hours. Fig 8. ph of the several microorganisms for 24 hours.

98 7. Fig 9. S. faecalis 20mg/ml, L. mesenteroides, L. plantarum, L. brevis 5mg/ml, S. cerevisiae 10mg/ml. Fig 9. Minimum inhibitary concentration(mic) of the chloroform extract against several microorganisms 8. 5~80mg/ml, S. faecalis Fig 13, L. mesenteroides Fig 10, L. plantarum Fig 11, L.brevis Fig 12,

99 S. cerevisiae Fig 14. S. faecalis 20mg/ml 12, 40mg/ml 80mg/ml. L. mesenteroides 10mg/ml 24 20ng/ml, 40mg/ml. L. plantarum 10mg/ml 36 48, 40mg/ml 80mg/ml. L. brevis, 40mg/ml 80mg/ml. S. cerevisiae 10mg/ml, 40mg/ml, 80mg/ml.

100 Fig 10. Effect of growth inhibition by chloroform extract of Taraxacum monglicum on Lenconostoc mesenteroides KCTC Fig 11. Effect of growth inhibition by chloroform extract of Taraxacum monglicum on Lactobacillus plantarum KCTC 3108.

101 Fig 12. Effect of growth inhibition by chloroform extract of Taraxacum monglicum on Lactobacillus brevis KCTC Fig 13. Effect of growth inhibition by chloroform extract of Taraxacum monglicum on Streptococcus faecalis KCTC 2011.

102 Fig 14. Effect of growth inhibition by chloroform extract of Taraxacum monglicum on Saccharomyces cerevisiae KCTC ,, 60% disc plate method Table %, 60%. L. mesenteroides S.faecalis. 60%

103 ., 60%. Fig fraction, 60%..,.(13) Table 16. Antimicrobial activities of fractions from chloroform extract of Taraxacum mongolicum against several Solvent microorganisms. L.mesent Clear zone on plate (mm) L.plant L.brevis S.faeca S.cere Soluble solid content (mg/disc) -eroides -arum -lis -visiae Hexane CCl CH2Cl % MeOH

104 Fig 15. Antimicrobial effect of chloroform fraction in Taraxacum monglicum on the growth of several microorganisms spectrum,,,,, 60%.

105 5 L. mesenteroides S. faecalis 2, 3 5mg/ml. S. faecalis , ph., S. faecalis 40mg/ml, S.cerevisiae.

106 4 1.,,,,,,, :., 5, 217 (1990) 2.,,,, : gamma., 21, 1039 (1989) 3.,,, : polyethylene film., 15(1), 39(1988) 4., :., 16, 443 (1984) 5.,,, :,, 19(6), 515 (1987) 6.,, : -., 5, 5 (1966) 7.,,, :., 23, 188 (1991) 8. :., 4(2), 39 (1988) 9.,,, :., 27(2), 257 (1995) 10.,,, :,, CaCl2.,

107 393, 309 (1988) 11., :., 13, 207 (1970) 12.,, :., 20(1), 102 (1992) 13., : chitosan., 11(3), 97 (1995) 14., :., 8(1), 27 (1992) 15.,,,, :., 21(1), 100 (1989) 16.,, : ph., 15(1), 39 (1982) 17.,, :., 20(4), 483 (1988) 18.,, :., 20(4), 476 (1988) 19.,, :., 20(4), 483 (1988) 20., :., 27(5), 813 (1995) 21., :.

108 , 27(2), 216 (1995) 22.,,,, :,, p105, ,, :,, p75, , : Leuconostoc mesentroides Lactobacillus plantarum,, 10(1), 35 (1994) 25. : ph., 18(3), 327 (1989) 26.,, :., 21(1), 91 (1992) 27., :,, 23(2), 200 (1991) 28. Paul, P.C and Palmer, H.H : Food theory and application. Helen Charly, p 251 (1972) 29., : (1970) 30. :. 150 (1967) 31., :. 152 (1967) 32.,, :. 135 (1967) 33., :. 163 (1968)

109 34.,, :. 35. : Studies on the effect of furyl furamide(af-2) on Korean Kimchi (1969) 36. : ph (1985) 37.,, : (1988) 38. :. (1991) 39.,,,, : (1989) 40.. : (1989) 41.. :, Vitamin C. 42., :., (1988) 43. : mono sodiun glutamate (1990) 44.,, : (1984) 45., : Starter (1988) 46., : (1984)

110 47.,, : Leuconostoc mesenterroides IFO 1260 niacin Escherichiaoh., (1992) 48. : ( 1 ) (1970) 49. : (1970) 50.,, pp 496 (1991) 51.,.,,, 2(2):68 (1986) 52.,,., Calcium acetate potassium sorbate,, 23(1):1 (1991) 53.,.,,, 25(2):224(1997) 54., :., 27(6), 1035(1995) 55.,,,,,, :., 23(6), 1008(1994) 56. Kupchan s partitition scheme, J. Org. Chem.38:178(1973) 57.,,,,,, :., 23(6), 1014(1994) 58. K.J. Klindworth, D.M. Davidson, C.J. Brekke, A.L.Brekke and A.L. Branen:Inhibition of Clostridium perfringens by Butylated Hydroxyanisole. J.Food Sci.,44(2): 564 (1979) 59., :

111 ., 23(2), 205(1991) 60.,,,, : Grapefruit - Grapefruit., 10, 33(1995) 61.,, :.,21(1), 97(1992) 62.,, : Listeria monocytogenes. 29(6), 1236(1997) 63. Wang-GD., Zhang-YM., Xiong-XY : Clinical and experimental study of burns treated locally with Chinese herbs. Chung-His-I-Chieh-Ho-Tsa-Chin, 11(12), 727(1991) 64.,, :., 29(4), 808(1997) 65., :.,13(1),56(1997) 66.,,,,, :., 25(1), 129(1996)

112 2. 1. (1) (2,3). (4).... koji, (5,6)., (7).

113 Saccharomyces rouxii(8) Zygosaccharo-myces salsus, Zygosaccharomyces japonicus, (3,8).... Saccharomyces rouxii Pichia, Hansenula POBA ester(p-hydroxybenzoic acid ester) Na (3)....,,,. 0.3%-0.4% allicin Gram Gram. allicin

114 ..,. garlic oil flavor 200, 900. garlic oleoresin 12, 50. oleoresine.

115 PDA PDB Difco,, tartaric acid PDA. API kit API 20C AUX(BIO MERIEUX, France) Test. Test 160ml 11cm, 13cm % NaCl 9ml

116 1ml 105 PDA Domain, Tartaric acid 10% 2 JIM, CHS Slide glass CHS Yeast JIM Yeast Cover glass 400. CHS JIM Yeast,. 3. API kit API kit API 20C AUX. (9) kit manual. 3 24, 0.85% NaCl 2 kit c medium. ample coupule pasteur pippet., 30 24, 48, plate PDB flask 30 3

117 JIM CHS 5, 20. control, Oleoresine 6 (80strokes/min, 15cm, stroke length) (Toyo No. 5A). 40 oleoresin , 10g methanol, chloroform, H2O, hexane 3, evaporator. petri dish (1.5% agar) 55 flask (0.75% agar) ( : =3 :30 ). 3 disc, 0.85% (75 )

118 pippet disc disc (10,11). disc hr, disc clear zone..

119 API Test kit identification index. CHS O GLU GLY 2KG ARA XYL ADO XLT GAL INO SOR MDG NAG CEL LAC MAL SAC TRE MLZ RAF H/PH Ident.: : Torulopsis maris %id= 99.4 (ADO 16%) (XLT 83%) : Candidas rugosa %id= 68.9 (XLT 79%) (ADO 5%) Torulopsis maris %id= 25.5 (ADO 16%) (XLT 83%) (HYPH 16%) JIM O GLU GLY 2KG ARA XYL ADO XLT GAL INO SOR MDG NAG CEL LAC MAL SAC TRE MLZ RAF H/PH Ident.: : Hansenula anomala 99.8% (Hyphae 86%) : 99.9% CHS Identity 83% Torulopsis maris,

120 JIM Identity 99% Hansenula anomala (Fig.1). Fig. 2. Hansenula, Pichia. ehthyl acetate ester. Pichia.. Hansenula anomala Hansenula. ester.,,., , glucose,. CHS Torulopsis maris, JIM Hansenula anomala. Hansenula anomala. (A) (B) Fig. 1. Application of API 20C AUX for CHS Yeast(A) and JIM Yeast (B)

121 (A) (B) Fig.2. Morphology of CHS Yeast(A) and JIM Yeast(B) isolated soy sauce by using electromicroscope

122 2. Test, JIM CHS control 30 (Fig.1)., CHS JIM,. 7 8 CHS, 10 Fig.2.. (A) (B) (C) Fig.3. Swelling test of Soy sauce added Yeast; Identification from soy sauce (A) Sterilized Soy sauce (B) Sterilized Soy sauce + CHS Yeast (C) Sterilized Soy sauce + JIM Yeast

123 3. Oleoresin 20mesh 20g oleoresin Table. methanol 20.3% 6, ethanol, acetone ethylether, hexane 1%. 64% methanol hyrophilic component oleoresin. methanol. overall flavor 0.2% volatile oil, 60% diallyl disulfide, 20% diallyl trisulfide 6% allyl propyl disulfide. Table.1. Antibiotic effect of solvents on loeoresin extraction from freshy garlic Solvent Yield(%,day basis) Clear zone(cm) CHS Yeast JIM Yeast Methanol Acetone Ethanol Ethyl acetate Ethyl ether Hexane

124 4. Paper disc, JIM CHS test. clear zone petri dish disc. (Table.1). clear zone,,,,,,,. JIM Hansenula anomala,,,, 4.4cm, 3.8cm clear zone. CHS Torulopsis maris 6.8cm,,,,,, 3.2cm clear zone (Table. 2). (A) (B) Fig. 4. Clear zone of media added natural preservatives about JIM Yeast