No.3....

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2 2005 Contents

3 2005 Contents ext F 16 1 TEL: GPN: ISSN: FAO GMO GM Israeli Jaffa Sweetie 70 4% 7~10 30

4 IFT (Institute of Food Technologists) functional safety nutritional nutraceutical functional foods nutraceuticals 2002~2005 IFT Functional Safety Nutritional Antioxidant Weight Nutraceutical Soy Bioactive Aging Soybean Cardiovascular Peptide Tea Obesity Genetic Gene Diabetes Polyphenol Isoflavone

5 Functional Foods Nutraceuticals EPA DHA HealthFocus 2005 ITIS %

6 ( ) What an ingredient does -- How an ingredient works Ordinary foods ( ) Nutrition labeling and Education Act (1990) ILSI-Europe opinion (CODEX Alimentarius) Food for Specific Health Use Act (1991) Dietary supplements ( ) Dietary Supplement Health and Education Act (1994) Health food Manufactures Association (bar) (Cereal) Nutrition business Journal, 2005 Food Technology, Vol.59, No.5, May 2005 ITIS (functional foods) Nutrition Business Journal % 32%25% % 21% 5,000 Market standard Healthy for you Natural/Organic Functional food Market standard Healthy for you Natural/Organic Functional food E 2003 Market standard 82% Healthy for you 11% Natural/Organic 3% Functional food 4% Mark K. Schmidl (Fresh produce) (Healthy Snacks and Meal Replacements) / ( Herbs) (Medical Foods) , Market standard 76% Healthy for you 13% Natural/Organic 5% Functional food 6% Datamonitor 6-7%

7 sterol sterol esters Danone sterol esters Minute Maid Tesco Lactobacillus acidophilus inulin Semper (novel probiotic delivery system) Paul Yamaguchi and Associates Inc % FOSHU % FOSHU FOSHU FOSHU 63% 50 FOSHU 29% 110 corroborative project (biomarker of fatigue) (alternative medications) (dietary supplements) R (blueberry) (collagen) (hyaluronic acid) (ceramide) (lotus germ extract)(rose petal extract) (conjugated linoleic acid CLA) (L-carnitine) CoQ10 18 CoQ α- 21 β

8 (estrogen-like) (probiotics) (Morinaga) (lactoferrin yogurt) (Koiwai)Lactobacillus yogurt (Meiji) LG 21 Pylori yogurt (pylori bacteria) R (Calpis) R Daizu Peptide Nysankin 4,000 R Powerade Toraku 8,000 R The Peptide Tokiwa R (sardine peptide) Lapis Support (Kao) Healthya Green Tea 36% (therapeutic) (liquid) / FOSHU a Mize-Mizu-Shia Suntory ( b ) Resistant starch Blenry Coffee with Oligosaccharide Ajinomoto General Foods Coffee (manno-) oligosaccharide c Van Hou Ten Milk Cocoa Fiber Plus Kataoka Trading Cocoa mix Mine-zu Mitsukan Prety-o Gamma amino butyric acid Healthia Oolong Tea Re-setta Soft Margarine MCT a:from Asahi Shimbun(Japanese daily newspaper), March 26, b: c: Bifidobacterium Food Technology, Vol.59, No.5, May 2005 ITIS

9 , AB ) 35.3% 24 / % 22 7 β-glucan HMG- CoA EPA DHA 10.3% 7 4 G G Daidzein nystose 8.8% 6 4 ab N 2.9% 2 2.9% 2 (Healthy Resetta Diet Oil) AB 2001 ABC / LGG (Econa Cooking Oil) OilliO (Healthy Resetta Diet Oil) Econa OilliO

10 10 AMIRU-S Econa Health ,600 Econa Health 1998 (Banso-reicya) AMIRU-S FOSHU 100 FOSHU 1999 Survival food Convenience food Engineered food % 89% 10 Functional food Nutraceutical Nutrigenetic food 18th 19th 20th 21th Century Fi Asia -China Conference, ITIS (Nutrigenetic Food Age)

11 Nutrigenetic (Survival food) (Convenience food) (Functional food) (Nutrigenetic food)

12

13 dietary supplement (Food for Specific Health Use, FOSHU) dietary supplement dietary supplement (This statement has not been evaluated by the FDA. This product is not intended to diagnose, treat, cure or prevent any disease.) (medical claim) (1)

14 (2) (3) (structure/function claim) (health claim) (medical claim)

15 ( ( gov.tw)

16 68 (94) 8 28 Flavonoids allyl sulfides carotenoids monoterpenes isothiocyanates phytosterols ,, 28, 90, (Genotoxicity study) (1) (2)

17 (Ames test) S. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA1535 S. typhimurium TA1537 TA97 TA97a S. typhimurium TA102 E. coli WP2 uvr A E. coli WP2 uvr A (pkm101) (1)(In vitro Chromosomal aberration test with mammalian cells in culture) : (2) tk (In vitro mouse lymphoma tk assay) tk cell cell (1) (Chromosomal aberrations in bone marrow cells of rodents) : (2) (Micronuclei in bone marrow cells of rodents) : (3) (Micronuclei in peripheral blood of rodents) : Acridine orange (1000X) (1000X) 5/ / (5g/kg 10ml/kg) SD (no-observed-adverse-effect level, NOAEL) 28 5 (w/w) 1000 mg/kg

18 28 28 (NOAEL) NOAEL (acceptable daily intake, ADI) 10 5 WORLDNUTRA 2004 (1) Oligonol Polyphenols Chitosan Soy protein Fish oil Sea algae Anthocyanins Probiotics Phytoestrogens Lutein CoQ10 Lycopene Blueberries CLnA (Isomers of Conjugated Linolenic Acid) Galactooligosacchrides Vitamin Ginseng Noni Sterols Dietary Supplements (2) (3) (4) (5) (6) (7)(8) (9) 250g 250g Cy3xGG Cy3xSGG 500g Cy3xGG Cy3xSGG (1) GMO (2)(3) (4) (5) (1) (2) / / Dr. Ruckmany GRAS DHEA Novel Foods Food Supplements directive (1) Ames test metaphase analysis mammalian cell mutation assay Gastrointestinal study(2)single dose oral study 4/8-week oral repeat dose study 90-day oral repeat dose study St. John's Wort (Hypericum perforatum L.) MAO COMT Cortisol 5-HTGABA-A Beta-adrenoceptors 5-HT /3 Cadmium

19 3 FDA Dr. Blumenthal 6 (1) EphedraFDA (2)Bitter orange Synephrine ephedrine (3)black cohosh CSPI FDA 32 (4) Kava FDA (5)St. John's wort P450 (6)Comfrey CO2 (7)scullcapgermander (8)Asian ginseng 1979 JAMA caffeine (9) Magnolia bark 1990 Aristolochic acid Tubocurarine Magnocurarine10 Aristolochia Magnolia bark Aristolochic acid Magnolia bark Magnocurarine Magnolia bark Tubocurarine lobelia 4 (1) 1 (2) 2 (3) 3 (4) Dr. Stohs ( Citrus Aurantium) synephedra (CNS) Dr. Pujari PCB(EPA 8082 SDIRaPID Assay ) Dioxin(EPA 625) Pb Cd Hg As (EPA 6020/200.8) (AOAC991.39) Dr. Tauscher

20 (EWG-no. 209/91) Dr. Kumii mg Isoflavone (IF)1 HPLC-ECD 644 ng/ml Daidzein 295 ng/ml Equol 287 ng/ml Genistein 7 4 EquolIF WBC RBC Hb Ht Thyroid steroid IF 1 Dr. Kim (Ca) (Pb) 6.7 mg/g 1.1 mg/g 2.3 mg/g FAO/WHO : (1) (2) (3) : (ADME) 6. Flavonoids allyl sulfides carotenoids monoterpenes isothiocyanates phytosterols

21 10 4 II I (Naringin) anti-cd71-fitc III IV Propidium lodide I - II III IV 5000 in vitro Caco-2 cell line 3R

22 Probiotics Prebiotics prebiotics synbiotics B (1) (2) ph (3) 3. K 4. Obradovic

23 200 ml mmol/l 20 L. para. subsp. paracasei NTU A synbiotic 3 (prebiotic) 1 ( )

24 synbiotics Lactobacillus sporogenes (Bacillus coagulans) AB (prebiotic) Bifidobacterium longum Lactobacillus acidophilus ( ) Lactobacillus bulgaricus Bifidobacterium bifidum Bifidobacterium longum Streptococcus thermophilus ABC / (Daidzein nystose) TCELL-1 LGG (synbiotic) (synbiotic) (synbiotic) 4 3 ABC / AB TCELL-1 LGG

25 11 LGG ABC / AB TCELL-1 (Lactobacillus) (Bifidobacterium) Lactobacillus acidophilus Lactobacillus casei Lactobacillus delbrueckii Lactobacillus paracasei Lactobacillus casei sp. Rhamnosus Lactobacillus rhamnosus Lactobacillus bulgaricus Lactobacillus fermentum Bifidobacte-rium longum Bifidobacterium lactis Bb-12 Bifidobacterium bifidum Enterococcus faecium Streptococcus thermophilus AB 2 2 Lactobacillus acidophilus La-5 Bifidobacterium lactis Bb Bifidobacterium longum BB536 + Bifidus 2 2 Bifidobacterium longum Lactobacillus acidophilus 1 1 Bifidobacterium lactis HN Bifidobacterium longum Lactobacillus acidophilus Bifidobacterium lactis Bb-12 Lactobacillus casei Enterococcus faecium 2 2 Streptococcus thermophillus Lactobacillus delbrueckii 3 3 DR10TM Bifidobacterium longum Streptococcus thermophillus Lactobacillus delbrueckii

26 / ABC 3 5 Streptococcus thermophillus Lactobacillus delbrueckii Lactobacillus acidophilus Bifidobacterium longum Lactobacillus paracasei 1 1 Bifidobacterium longum 2 3 Bifidobacterium lactis Lactobacillus acidophilus Lactobacillus casei 3 4 Bifidobacterium lactis Lactobacillus acidophilus Enterococcus faecium Lactobacillus paracasei 1 1 Lactobacillus casei sp. rhamnosus GG 2 3 Bifidobacterium lactis Lactobacillus acidophilus Lactobacillus casei 1 1 Lactobacillus casei Shirota 2 2 Bifidobacterium longum Lactobacillus acidophilus 2 3 Bifidobacterium lactis Lactobacillus acidophilus Lactobacillus casei Bifidus TCELL Lactobacillus rhamnosus Tcell Lactobacillus sporogenes (Bacillus coagulans) Bifidobacterium longum powder 3 5 Lactobacillus acidophilus Lactobacillus bulgaricus Bifidobacterium bifidum Bifidobacterium longum Streptococcus thermophilus LGG 3 7 Lactobacillus bulgaricus Streptococcus thermophilus Lactobacillus rhamnosus GG Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus Lactobacillus casei (Daidzein 1 1 Lactobacillus fermentum nystose)

27 46 52 T-cell 38 Lactobacillus rhamnosus Tcell (GOT GPT) (RBC HGB HCT MCV MCH MCHC PLT WBC) 28 (28-day feeding toxicity study)spf L. rhamnosus Tcell-1 28 Wistar 20 1 g/kg (7x10 11 CFU) 0.5 g/kg (3.5x10 11 ) 0.1 g/kg (7x10 10 CFU) L. rhamnosus Tcell-1 L. rhamnosus Tcell-1 HPRT (S9) 6(S9) 3.125, 6.25, 12.5, 25, 50, 100 µl/ml 0.1 µl/ml methanesulfonic acid, ethyl ester 18~24 (S9) , 3.125, 6.25, 12.5, 25, 50 µl/ ml 100 µg/ml N- nitroso-dimethylamine 18~24 HPRT ICR ml/kg mitomycin C 1 mg/kg 36~48 ICR S9 3 S S9, 3 +S9, 3 -S9,

28 5 (µl/ml) 6.25, 12.5, 25, 50, 100-S9 mitomycin C 1 µm +S9 cyclophosphamide 40 µm SD ml/kg (20 ml/kg) 14 SD SD ml/kg/day (15 ml/kg/day) (1) Streptococcus type Lactobacillus type (2) Lactobacillus acidophilus 2004 (3) (L. reuteri) Co-culture/overlayer (RPLR) L. GG Caco Clostridium difficile Caco-2 (4) L. acidophilus L. bulga

29 ricus Streptococcus lactis SK2 1993L. plantarum L. plantarum Escherichia coli, Pseudomonas aeruginosa Bacillus cereus (5) 1995 Bifidobacterium -D- 2~3 B. longum B. bifidum (6) 1995 Bacillus coagulans Leuconostoc mensenteroides Pediococcus cerevisiae L. brevis L. plantarum (7) L. plantarum Pediococcus acidilacici Pediococcus pentosaceus (8) 1995 L. bulgaricus L. acidophilus Streptococcus thermophilus Lactococcus lactis -18 C -3l C 5 7 log CFU/mL (9) 1995 glutathione Lactobacillus paracasei subsp. paracasei NTU 101 Bifidobacterium longum BCRC : C CFU/mL L. para. subsp. paracasei NTU 101L. plantarum NTU 102 L. para. subsp. paracasei NTU 101 L. plantarum NTU 102 L. para. subsp. paracasei NTU L. plantarum NTU LDL HDL L. plantarum NTU 102 L. para. subsp. paracasei NTU 101 (10)

30 1994 (probiotics) (11) C 3 30 ppm 0 ppm ethyl carbamate 7 ppb 35 ppb 130 ppb (12) 1995 L. acidophilus B. bifidum (13) 1989 Streptococcus lactis Bacillus subtilis nisin 1986 Leuconostoc sp. TMB Ca-alginate gel pnlp1 p (14) 1995 Streptococcus faecium (1) (Helicobacter pylori) (rotavirus diarrhoea) (gastritis) (2)

31 (Monascus)van Tieghem 1884 (septa) (ascocarp) (Fungi) (Ascomycota) (Ascomycetes) (Eurotiales) (Monascaceae) 1908 Monascus purpureus 1930 Monascus anka Anka 30 C (monascidin) 3. (monacolins) 4. (GABA) 5. (flavonoids)

32 (monacolins) 51 (19.2 ) , Monascus ruber monacolin K HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) mevalonic acid (low density lipoprotein cholesterol, LDL-C) Monacolin K (isoprene) Monacolin K ~ mmhg 180 mmhg γ- (γaminobutyric acid, GABA) (glucosamine) 1987 Kohama (spontaneously hypertensive rats SHR) 1992 Tsuji (Monascus pilosus IFO 4520) γ

33 300 µg 27 g (monascidin) M. purpureus 1977 Wong Bau monascidin A Monascidin A Bacillus Streptococcus Pseudomonas monascidin ABlanc 1995 GC-MS NMR IR monascidin A citrinin monascorubrin rubropunctatin (ammoniemia) Yasukawa 12-o-tetradecanoyl-phorbol-13-acetate (TPA) (inflammatory) monascorubrin ~ 0.3% 23~33% 19~29% (ergosterol) D (Monascus) 1999 Aniya α α -diphenyl-β - picrylhydrazyl (DPPH) Aniya dimerumic acid Juzlova 1996 C 14 ~C 24 GC-MS iso anteiso 14 (monoenoic acid) 2 (dienoic acid) 1 α- (α-linolenic acid) (superoxide dismutase SOD) (ribonuclease) α- N (α-galactosidase) (pectinase) citrinin (monascidin A) (hepatonephrotoxic mycotoxin) Citrinin Citrinin Penicillium citrinum Aspergillium spp. Monascus spp. Citrinin C 13 H 14 O 5 alcohol dioxane dilute alkali nm LD mg/kg (mouse) 67 mg/kg (rat) O OH HOOC O O CH 3 CH 3 CH 3 Citrinin

34 Citrinin gram-positive Bacillus Streptococcus Pseudomonas citrinin (nephrotoxin and hepatotoxin)monascus ruber citrinin citrinin citrinin Blanc 1995 Monasus purpureus Monascus ruber citrinin 100~400 mg/l citrinin citrinin citrinin citrinin citrinin citrinin 1999 Monica citrinin 0.2~17.1 µg/kg Ames Salmonella-microsome assay Salmonella-hepatocyte assay citrinincitrinin monacolin K/- 6. monacolin K/ - monacolin K (II)

35 , azaphilones pravastatin (HMG-CoA reductase inhibitor) M citrinin

36 3. HPLC CE citrinin 4. citrinin 5. citrinin 6. citrinin 1. citrinin monacolin K citrinin monacolin K citrinin HPLC C18 citrininmonacolin K monacolin K 1 g 100 ethanol (10 ml) 60 C 30 min citrinin monacolin K monacolin K HPLC acetonitrile-water-trifluoacetate (550:450:0.5) citrinin monacolin K monacolin K LC-MS J. AOAC International 2. -γ- monacolin K Monascus γ- (γ-aminobutyric acid, GABA) monacolin K M. purpureus BCRC31615 NaNO 3 GABA monacolin K monacolin K 378 mg/ kg GABA 368 mm GABA K 2 HPO mm (J. Industrial Microbiology and Biotechnology) 3. monacolin K GABA citrinin M. purpureus 0.5 ethanol citrinin ppb ppb monacolin K mg/kg mg/kg GABA 1.06 mg/g 7.45 mg/g 30 C 0.3 ethanol 145 ml monacolin K mg/kg citriinin ppb GABA 5.00 mg/g citrinin 0.25 M NaHCO 3 30 citrinin monacolin K (J. Industrial Microbiology and Biotechnology) 4. citrinin monacolin K GABA citrinin monacolin K GABA Monascus purpureus NTU

37 Monascus citrinin Bacillus subtilis Monascus Bacillus subtilis citrinin monacolin K citrinin 301 citrinin ppb wild type 50 monacolin K mg/kg 90 GABA 5.25 mg/g wild type monacolin K9500 mg/kg monacolin K (J. Agriculture and Food Chemistry) 5. - monacolin K monascin Monascus purpureus NTU mg/kg monacolin K 5.37Monascin monacolin K monascin (J. Industrial Microbiology and Biotechnology) 6. α α-diphenyl-βpicrylhydrazyl (DPPH) Monascus sp. NiA M. anka M M. purpureus BCRC31615 M. anka M monacolin K (J. Agriculture and Food Chemistry) 7. Monascus purpureus NTU 568 (total cholesterol) (triglyceride) (LDL-C) HDL-C LDL-C/HDL-C citrinin GOT (glutamic-oxaloacetic transaminase) GPT (glutamic-pyruvic transaminase) (Applied Microbiology and Biotechnology) Wistar Monascus purpureus NTU g/kg 1 g/kg

38 mg/dl mg/dl mg/dl (p 0.05) mg/dl mg/ dl mg/dl (Applied Microbiology and Biotechnology) 9. Monacolin K 48(Hy-line) HDL LDL (0 : , 2 : , 5 : , 8 : mg/egg; p 0.05) (0 : , 2 : , 5 : , 8 : mg/dl; p 0.05) LDL (0 : , 2 : , 5 : , 8 : mg/dl; p 0.05) HDL (0 : , 2 : , 5 : , 8 : mg/dl; p 0.05) MDA (0 : , 2 : , 5 : , 8 : µm; p 0.05) HPLC monacolin K (J. Agriculture and Food Chemistry) 100 Science News LDL-C5.0 LDL-C (p 0.05) % (Applied Microbiology and Biotechnology) 11. (mechanical attrition method) 26 µm ml (10 ) 600 g

39 100 ml 3000 rpm 3 hr(dynamic light scattering, DLS) nm nm (Scanning electron microscope SEM) monacolin K citrinin mg/ kg mg/kg monacolin K citrinin (1) citrinin citrinin citrinin citrinin polyketide citrinin citrinin NTG citrinin citrinin (2) (intron) (gene cluster) polyketide

40 (Chang, 2005) Sasaki(G. applanatum) (Sasaki et al, 1971) -2(IL-2) T (Lai and Lin, 1992) T α DNAT (Lai and Lin, 1991) IL-1,IL-2,IL-6,INF-α,INF-γ (Lieu et al, 1992)

41 (AIDS) β (1-3) β (1-6) G. applanatum G. lucidum G. tsugae (G. applanatum) G. lucidum G. tsugae (Wang et al,1993) T TNF-α IFN-γ (Wang et al, 1997) F3, GLPS, PS-G -1(IL-1) TLR4 (Toll-like receptor-4) (Hsu et al, 2004, Lin et al, 2005) Kubota G. lucidum A.B. (ganoderic acid A. B.) (Kobota et al,1982) 1999 Kim HIV (Kim and Kim,1999) 1989 Kino G. lucidum LZ- (Ling Zhi-8) LZ ,420 Da (Tanaka, 1989) LZ-8 (homodimmer) (systemic anaphylaxis reaction) (Arthus reaction) LZ-8 (Kino, 1989) LZ-8 β-d β-b α-a β-a α-b β-f β-g β-e β-c t-a SDTALIFRLAWDVKKLSFDYTPNWGRGNPN NFIDTVTFPKVLTDKAYTYRVAVSGRNLGV KPSYAVESDGSQKVNFLEYNSGYGIADTNT IQVFVVDPDTNNDFIIAQWN LZ-8 (Lin, 1997)

42 FIP-gts FIP-fve FIP-vvo (Lin et al. 1997) 1989 Kino LZ-8 LZ-8 (nonobese diabetic, NOD) (Kino, 1990) LZ-8 (immunomodulatory drug) : CsA (cyclosporin A) R FK506 tacrolimus LZ-8 (ven der Hem, 1995) LZ-8 (G. tsugae) 13 kd FIP-gts (fungal immunomodulatory protein-gts) LZ-8 LZ-8 (human peripheral lymphocytes) 3 H-thymidine 5 µg/ml RT-PCR LZ-8 (IL-2, IL-4) (IFN-γ)(TNF-α) 1997 FIP-gts LZ kd FIP-fve (Ko et al. 1995) FIP-vvo (Hsu et al. 1997) LZ-8 FIP-fve FIP-vvo G. applanatum G. boninense G. formosamun G. fornicatum G. lucidum G. microsporum G. neojaponicum G. oerstedii G. resinaceum G. sinense G. tropicum G. tsugae G. valesiacum G. weberianum LZ-8 lz-8 LZ-8 genome walking G. microsporum G. fornicatum gmi gfo-1 gfo-2 lz-8 gmi gfo-1 Pichia pastoris KM71 replz regmi regfo-1 MALDI- TOF

43 300 mg/l BALB/c (dendritic cells, DCs) IL-12 J774A.1 TNF-α T Jurkat cells IL-2reGMI 5 µg/ml DCs IL-12 replz 2005 Pichia pastoris (laccase, ) (manganese peroxidase, ) (lignin peroxidase, ) (Servili et al., 2000) (Leonowicz et al., 2001) (Abadulla et al., 2000) (Mougin et al., 2000) S. cerevisiae S. cerevisiae (Larsson et al., 2001) Li Steffens (Li and Steffens, 2002) 1990 Ko (G. lucidum ) (isozymes) Galc3 ph3-ph10 (Ko et al., 2001) D'souza LZ-8 ( 2005)

44 Veratryl alcohol 0.2U/ml (D'souza et al., 1999) 26 (intron) G. lucidum G. tsugae G. fornicatum RZ.lac lac lac bp 2019 bp 2110bp 9 intron signal peptide Cysteine Phenylalanine AOX lac1 Pichia pastoris KM71 ph C BMMHY 30 C AY (Pleurotus ostreatus ) AY (Polyporous ciliatus lcc3) lac lac1 AY lac lac2 947 RZ.lac RZ-2.lac1 RZ-3.lac lac1 997 RZ.lac1 RZ-34a.lac lac1 1.5K lac lac AY (G.sp.BS-1 lac2) 0109.lac lac1 AF lac2 987 RZ.lac3 RZ.lac4 RS.lac lac1 999 AY (T. pubescens lap2) AY (T. Versicolr lcc3) AY (G.sp.BS-1 lac1) 0109.lac lac3 1.5K 0705.lac lac lac2 RS.lac

45 6.6U/ml 81.7U/mg re1109.lac1 55 C Global Industry Analysis % β 2010 (Molecular pharming, biopharming pharming) 80(Aspergillus spp.) (chymosin) B (virus-like particle) (Burns et al, 2005, Chen et al, 2000) (green fluorescent protein) (Hirano et al, 2000) pan7-1 hygromycin Bhph - hygromycin B(1998) 2001 GPD promoter (2001 Sun et al,) 2004 restriction enzyme-mediated integration (2004 Kim et al,)

46 GMI GFO-1 Pichia pastoris Abadulla E, Tzanov T, Costa S, Robra K.H., Cavaco-Paulo A, Gubitz G.M Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta. Appl Environ Microbiol 66(8): Burns, C., K. E. Gregory, M. Kirby, M. K. Cheung, M. Riquelme, T. J. Elliott, M. P. Challen, A. Bailey, Foster G. D Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet Biol 42: Chang S.T Ganoderma lucidum A prominent source for the healthcare market in the 21th century. Proceedings of the first Symposium on development of China's medicinal fungi industry Chen, X., M. Stone, C. Schlagnhaufer, Romaine C. P A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66: D'Souza T.M., Merritt C.S., Reddy C.A Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum. Appl Environ Microbiol 65(12): Frendscho M.H., Kino, Sone T. and Jardieu P Ling Zhi-8:A novel T cell mitogen includes cytokine production and upregulation of ICAM-1 expression. Cell immunol.150, Hirano, T., T. Sato, K. Yaegashi, Enei H Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance. Mol Gen Genet 263: Hsu H.C., Hsu C.I., Lin R.H., Kao C.L., Lin J.Y Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem J 323 (Pt 2): Hsu H.Y., HUA K.F., Lin C.C., Hsu J., Wong C.H Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways. J Immunol. 173(10): Janusz M.J., Austen K.F. and Czop J.K Isolation of a yeast heptaglucoside that inhibets monocyte phagocytosis of zymosan particles. J. Immunol. 142: Kim S., Song J., Choi H.T Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzymemediated integration. FEMS Microbiology letters 233: Kim H.W. and Kim B. K Biomedicinal triterpenoids of Ganoderma lucidum (Curt.:Fr.)P Karst. (Aphyllophoromycetideae). Intl. J. Med. Mushrooms 1: Ko E.M., Leem Y.E., Choi H.T Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum. Appl Microbiol Biotechnol 57(1-2):

47 22. Kino K., Yamsshita A., Yamaoka K., Watanabe J., Tanaka S., Ko K. and Tsunoo H Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8(LZ-8),form Ganiderma lucidum. J.Biol. Chem. 264, Kino K., Mizumoto K., Sone T., Yamaoka J., Watanabe A., Yamashita K., Yamaoka K., Ko K., Tsunoo H An immunomodulatory protein, Ling Zhi-8, prevents insulitis in non-obese diabetic mice. Diabetologia. 33, Ko J.L., Hsu C.I., Lin R.H., Kao C.L., Lin J.Y A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem 228(2): Kubota T., Asaka Y., Miura I. and Mori H Structures of ganoderic acids A and B, two new lanostane type bitter triterpenes from Ganoderma lucidum(fr.)karst. Helv Chim Acta, 65, Larsson S, Cassland P, Jonsson L.J Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol 67(3): Leonowicz A, Cho N.S., Luterek J, Wilkolazka A, Wojtas-Wasilewska M, Matuszewska A, Hofrichter M, Wesenberg D, Rogalski J Fungal laccase: properties and activity on lignin. J Basic Microbiol 41(3-4): Lei L.S. and Lin Z.B Effect of Ganoderma polysaccharides on T cell subpopulations and production and production of interleukin2 in mixed lymphocyte response. Acta Pharmaceuica Sinica 27(5): Lei L.S. and Lin Z.B Effect of Ganoderma polysaccharides on the activity of DNA polymerase in -spleen cells stimulated by alloantigens in mice in vitro. T. Beijing Medical University 23(4): Li L, Steffens J.C Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215(2): Lieu C.W., Lee S.S. and Wang S.Y The effect of Ganoderma lucidum on induction of differentiation in leulsemic U937 cells Anticancer Research 12: Lin Y.L., Liang Y.C., Lee S.S., Chiang B.L Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways. J Leukoe Biol.78(2): Miyasaka N., Inoue H., Sone T. and Jardieu P Ling Zhi-8:facilitates cellular interaction through modulation of adhesion molecules. Biochem. Biophys. Res. Commun. 186, Mizuno T., Sakai T. and Chihara G Health foods and medicinal usages of mushrooms. Food Review International 11 (1) Mougin C, Boyer F.D., Caminade E, Rama R Cleavage of the diketonitrile derivative of the herbicide isoxaflutole by extracellular fungal oxidases. J Agric Food Chem 48(10): Servili M, DeStefano G, Piacquadio P, Sciancalepore V A novel method for removing phenols from grape must. Am. J. Enol. Vitic. 51: Sun L., Cai H.Q., Xu W.H., Hu Y.L., Gao Y., Lin Z.P Efficient transformation of the medicinal mushroom Ganoderma lucidum. Plant Molecular Biology Reporter 19: 383a-383j. 38. van der Hem L.G., van der Vliet J.A., Bocken C.F., Kino K, Hoitsma A.J., Tax W.J Ling Zhi-8: studies of a new immunomodulating agent. Transplantation 60(5): Wang G., Zhang J., Mizuno T., Zhuang C., Ito H., Mayuzumi H., Okamoto H. and Li J Antitumor active polysaccharides from the Chinese mushroom Song Shan Lingzhi, the fruiting body of Ganoderma tsugae. Biosci.Biotech. Biochem. 57 (6):

48 (Bacillus subtilis) (Bacillus natto spore) 2000 B 1 B 2 E K B1 B2 K2 kcal g g g g mg mg mg mg mg mg mg U/g

49 E 1995 Angiotension K K SOD K 2 Catalase 1967 KMD B (LDL) : 1986 K 2 O-157 (Helicobacter pylori)

50 angiotensin converting enzyme (ACE) K NHK 4,5 (nattokinase) DNA ph4 ~12 ph7~9 Nattokinase Enzyme Ralph E. Holsworth: Nattokinase and Cardiovascular Health, (urokinase) (FU) (thiol reductant) 6,7 FDATulane , ~60 B 6 E K

51 (reactive oxygen species, ROS) 9 KMD1126 KMD1126 Ehrlich ascites carcinoma (interferon) 30 (edamame therapy) (purine) K 2 (osteoclast calcium) Gla 10 K 2 (coagulation) K 2 K 2 K 2 K 2 11,

52 13 (probiotics) (endospore) (γ-poly-glutamic acid, γ- PGA) 14,15 - (urokinase) (tissue plasminogen activator, t-pa) (ACE inhibitor) K 2 17,18,19 (100 C) (vegetative cells) D

53 ( ) ( ) (2005) (nutraceuticals) Nutrition Pharmaceutical Nutraceutical Science Safety LDL

54 R(peptides)(glycopeptides) 6 -O-succinyl 19 PPQ (pyrroloquinoline quinone) (Bifidus) (natto micronutrients) (amylase) (Protease) (natural antioxidative factors) 20 Euromonitor ( ) 1. Zuber, P., M. M. Nakano, and M. A. Marahiel Peptide antibiotics, p In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C. 2. T. Yamashita, E. Oda a, J.C. Giddings, J. Yamamoto: The effect of dietary Bacillus natto productive protein on in vivo endogenous thrombolysis, Pathophysiology of Haemostasis and Thrombosis 2003; 33: Kudva, I. T., P. S. Evans, N. T. Perna, T. J. Barrett, F. M. Ausubel, F. R. Blattner, and S. B. Calderwood. Strains of Escherichia coli O157:H7 differ primarily by insertions or deletions, not single-nucleotide polymorphisms. J. Bacteriol. 2002; 184: Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimuro S: Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bull 1995; 18: Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S: Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl Environ Microbiol 1996; 62:

55 6. Yamamoto, K. et al. Plasminogen activator inhibitor-1 is a major stress-regulated gene: implications for stress-induced thrombosis in aged individuals. Proceedings of the National Academy of Sciences. 2002; 99(2): Urano T, Ihara H, Umemura K, Suzuki Y, Oike M, Akita S, Tsukamoto Y, Suzuki I, Takada A: The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1. J Biol. Chem. 2001; 276: Pesce, Noel ; Eyster, Kathleen M.; Williams, John L. ; Wixon, Regina ; Wang, Chunyang ; Martin, Doug S.: Effect of Genistein on Cardiovascular Responses to Angiotensin II in Conscious Unrestrained Rats, Journal of Cardiovascular Pharmacology. 2000; 36(6): Iwai K, Nakaya N, Kawasaki Y, Matsue H: Antioxidative functions of natto, a kind of fermented soybeans: Effect on LDL oxidation and lipid metabolism in cholesterol-fed rats. J Agric Food Chem 2002; 50: Kaneki M, Hedges SJ, Hosoi T, et al. Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk. Nutrition 2001 Apr; 17(4): Tsukamoto Y, Ichise H, Kakuda H, Yamaguchi M: Intake of fermented soybean (natto) increases circulating vitamin K 2 (menaquinone-7) and gamma-carboxylated osteocalcin concentration in normal individuals. J Bone Miner Metab 2000; 18/4: Katsuyama H, Ideguchi S, Fukunaga M, Saijoh K, Sunami S: Usual dietary intake of fermented soybeans (Natto) is associated with bone mineral density in premenopausal women. J Nutr Sci Vitaminol (Tokyo) 2002; 48/3: Tomohiro Hosoi, Akio Ametani, Kan Kiuchi, and Shuichi Kaminogawa: Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin, 2000: Can. J. Microbiol./Rev. can. Microbiol. 46(10): Ashiuchi, M., C. Nawa, T. Kamei, J. J. Song, S. P. Hong, M. H. Sung, K. Soda, and H. Misono Physiological and biochemical characteristics of poly-γ-glutamate synthetase complex of Bacillus subtilis. Eur. J. Biochem. 268: Ashiuchi, M., and H. Misono. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl. Microbiol. Biotechnol, 2002; 59: Liu BY, Song HY: Molecular cloning and expression of nattokinase gene in Bacillus subtilis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002; 34: Chang CT, Fan MH, Kuo FC, Sung HY: Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem 2000; 48: Yamaguchi M, Ma Z J. Inhibitory effect of menaquinone-7 (vitamin K 2 ) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro. Mol Cell Biochem 2001; 228(1-2): Toda T, Uesugi T, Hirai K, Nukaya H, Tsuji K, Ishida H. New 6-O-acyl isoflavone glycosides from soybeans fermented with Bacillus subtilis (natto). I. 6-O-succinylated isoflavone glycosides and their preventive effects on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol Pharm Bull 1999; 22(11): Mentor-Marcel R, Lamartiniere CA, Eltoum IA, Greenberg NM, Elgavish A. Dietary genistein improves survival and reduces expression of osteopontin in the prostate of transgenic mice with prostatic adenocarcinoma (TRAMP). 2005: J Nutr; 135(5):

56 (Original Design Manufactures ODM) 2005 cgmp (Small Business Innovation Research SBIR) LP33Aller Relief(Nu Skin) ODM... Q1 A1 Pediatric Allergy and Immunology

57 - Q2 A DNA PCR DNA API CH50 Q3 A3 600 cgmp LP33 (Lactobacillus paracasei33)

58 396 (ph2.5) (0.3%) CTL Th NK B (helper T) Th1 γ (IFN-γ) B G IgG Th2 IL-4 IL-5 E(IgE) (humoral immunity) Older sibs: many infections (Th1 stimuli) LP33 Th1 No allergies Birth Th2 Allergen exposure GOT Only child: few infections Still Th2 Allergies GTP T Th2 IgE Th1 Th2 LP33 IgE IFN γ (antihistamine) Q4 LP33 A LP33LP33 IgE LP LP33 LP33 IgE IFN γ

59 LP33 Q5 A5 Q6 A Q7 A7 SBIR cgmp

60 % G.M.P Q1 A

61 1.55 % 1.55 % 5.43 % A00043 T Q2 A copy Q3 A3 A (A) (B) (C) (D) (E) (B/A) 3.3% (D/C) 22.3%

62 Q4 A Antrodia Q5 A5 14% 3% 24% ,284 31,823 93, , ,475 59%

63 /2 93 2/3 Q6 A WTO Q7 A ODM OEM 8 Shaker L 3 200L 2 300L 1 500L

64 2 Ton Ton 1 5 Ton 1 20 Ton 1 40 Ton 1 50 Ton

65 Bacillus subtilis natto

66 Q1 A1(B. subtilis Natto)40 C (Natto- Kinase) (C) (A) (B) (C) (A) (B) Pyrazines Q2 A2 K

67 PUB Q3 A3 Q4 A4 know-how Q5 A5 Q6 A6 60 Q7 A7 70% Amgen (Epogen)Amgen Amgen

68 Amgen Q8 A8 25% SBIR Q9 25% A9 Q10 A10 Q11 A11 1 Q12 A12 20 Nutrition Business Journal % 32%25%

69 Bt 1000 Gerard Barry Bt AGBIOS.COM 2005/04/ % ISNA Tehran Times 2005/05/

70 FAO GMO (FAO) (GMOs) Monsanto 90% 650 FAO GM GMOs (Consumers International)2000 GM GM (WFP) GM GMOs GM FAO GMOs Tehran Times 2005/05/17 GM (GM) T25 Syngenta Bt176 Monsanto ]MON810, Topas 19/2 MS1xRF1 20 GM AGBIOS BBC News 2005/06/

71 Frost & Sullivan GI GI (pure glucose)100 2 (GI =100) 2 Frost & Sullivan AGBIOS 2005/09/ (luciferrin) (luciferase)(adenosine triphosphate ATP) (bio-luminescence testing method) AGBIOS 2005/09/

72 Israeli Jaffa Sweetie pomelit Israeli Jaffa Sweetie pomelit pomelit (bypass surgery) pomelit 200 pomelit pomelit (low-density lipoprotein, LDL) (blood albumin) (blood fibrinogen) pomelit pomelit Science Daily AGBIOS

73 4% ITIS 4.6% 2% 2.6% 3% 4% 5.9% WTO ITIS IT ERP CRM SCM KM GMP CAS HACCP ISO /02/ zerumbone humulene α-curcumene gingerol /03/

74 94/05/ (1 3) -β-d-glucan /06/

75 10,256 WTO 2005/06/ /06/

76 /07/ B 94/07/

77 60 genistin daidzin 94/08/