Potentials of Microorganisms for Potentials of Microorganisms for - PowerPoint PPT Presentation

Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Probiotics Probiotics Functional Food Production and Jun Ogawa Jun Ogawa Research Division of Microbial Sciences, Research Division of Microbial Sciences, Kyoto University, Kyoto, Japan Kyoto University, Kyoto, Japan July 3rd, 2009, Nestle, Lausanne July 3rd, 2009, Nestle, Lausanne

Base of Japan’s Microbial Biotechnology Japan is a country rich in microbial resources. ● We have high affinity to microorganisms, that has been ● obtained traditionally and environmentally. ● There are many active industries using microorganisms. Pe r c e ntage of pate nt applic ants in industr y se c tor 微生物との共存ができます E U Medic al Europe USA Medic al USA Japan F F o o ds & Che mic als e tc o o ds & Che mic als e tc Medic al Japan 豊かな微生物資源が 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% あります バイオ に強い化学工業があ ります

Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Probiotics Probiotics Functional Food Production and Creation of new food function food function based on unique microbial function microbial function : Creation of new based on unique Functional food materials produced by microbial transformation ・ Production of 4-hydroxyisoleucine ・ Production of polyunsaturated fatty acids ・ Production of conjugated fatty acids Food functions based on catalytic activity of microbial enzymes ・ Deodorizing activity derived from laccase Probiotic use of lactic acid bacteria and their metabolisms ・ Probiotics for hyperuricemia prevention Searching unique microbial functions in Japanese microbial Searching unique microbial functions in Japanese microbial diversity and using them for food and chemical industries diversity and using them for food and chemical industries

4-Hydroxyisoleucine (HIL) Target decision: Target decision: by collaboration by collaboration and discussion and discussion with industries. with industries. Fenugreek Fenugreek 4-Hydroxyisoleucine (HIL) is a potential drug candidate ● for the treatment of diabetes and obesity. HIL is contained in fenugreek seeds, but the amount is low. ● Enzymatic processes are promissing for HIL synthesis ● that needs high stereo- and functional-group selectivity.

L-Ile transformation pathway found in Bacillus thuringiensis strain 2e2 O OH NH 2 NH 2 NH 2 COOH COOH COOH L-Ile dioxygenase HIL (IDO) dehydrogenase L-Ile HIL AMKP

HIL production by cell-free extracts of E. coli expressing IDO from B. thuringiensis strain 2e2 NH 2 NH 2 OH Dioxygenase R S S S S COOH COOH Dioxygenase process

Hyper Arachidonic acid producer Mortierella alpina 1S-4 ↓寒天培地上に生育したモルティエレラ・アルピナ ↑高度不飽和脂肪酸の油滴が見られる顕微鏡写真 Searching unique microbial functions in Japanese microbial Searching unique microbial functions in Japanese microbial diversity and using them for food and chemical industries diversity and using them for food and chemical industries 京都大学農学部のキャンパスから分離された“肥満のカビ”モルティエレラ・アルピナ

Fatty acid profile of “Single Cell Oil” produced by M. alpina is quite different from common edible oils and is used as an ingredient for infant formula in the world. “発酵油脂”は 乳幼児用ミルクの 栄養素として 世界中で 使われている 。 見ためは同じでも “発酵油脂”の脂肪酸組成は 植物・動物油脂とは全く違う。

Mutant Screening Mutant isolation, spore cultivation NTG Fatty acid analysis mutation By GLC Retention time (min) Glucose Δ 9 EL1 COOH COOH 18:0 18:1n-9 Δ 6 Δ 5 Δ 12 EL2 COOH COOH COOH COOH 18:2n-6 18:3n-6 20:3n-6, DGLA 20:4n-6, AA ω 3 ω 3 COOH COOH COOH COOH Δ 6 20:5n-3, EPA 20:4n-3 18:3n-3 Δ 5 18:4n-3 EL2

Various PUFAs produced by M. alpina 1S-4 COO COOH COO COOH COOH COO COOH COO COOH COO n-1 n-1 16:3 16 :3n-1 16 16:4 :4n-1 18:4 18 :4n-1 18 18:5 :5n-1 20:5 20 :5n-1 COOH COO COO COOH 20 20:3 :3n-4 18 18:2 :2n-4 Δ 15 EL2 EL2 15 Glucose n-4 n-4 EL EL COO COOH COOH COO COOH COO COOH COO COO COOH 16:2 16 :2n-4 16 16:3 :3n-4 18 18:3 :3n-4 18 18:4 :4n-4 20:4 20 :4n-4 EL2 EL2 EL EL Δ 12 12 COO COOH COOH COO COO COOH COO COOH COO COOH COO COOH 18:2 18 :2n-7 16:2 16 :2n-7 18 18:3 :3n-7 16 16:1 :1n-7 16:0 16 :0 20 20:3 :3n-7 Δ 9 n-7 n-7 EL EL COO COOH COOH COO COO COOH 18 18:1 :1n-7 18:2 18 :2n-7( Δ 5) 5) 24:0 24 :0 EL1 EL1 COOH COO COOH COO 20:1 20 :1n-9 22:0 22 :0 EL EL COO COOH COOH COO COOH COO COO COOH COOH COO COOH COO n-9 n-9 20 20:2 :2n-9 18:1 18 :1n-9 18 18:2 :2n-9 20 20:3 :3n-9, MA MA 20 20:0 :0 18 18:0 :0 Δ 9 Δ 12 12 COO COOH COOH COO COO COOH COOH COO COO COOH COOH COO n-6 n-6 18:3 18 :3n-6 18 18:2 :2n-6 20 20:4 :4n-6, AA AA 20 20:3 :3n-6( Δ 5) 5) 20:2 20 :2n-6 DG DGLA LA Δ 5 Δ 6 EL2 EL2 Δ 5 ω 3 EL2 EL2 COO COOH COO COOH COOH COO COO COOH COO COOH COOH COO n-3 n-3 20:4 20 :4n-3 :4n-6( Δ 5) 20 20:3 :3n-3 18:4 18 :4n-3 20 20:5 :5n-3, EP EPA 20:4 20 5) 18:3 18 :3n-3

Conjugated linoleic acid (CLA) Δ 9 Δ 11 O HO C Δ 10 Δ 12 O HO C inhibits initiation of skin carcinogenesis, and forestomach and mammary tumorigenesis. prevents the catabolic effects of immune stimulation. alters LDL / HDL cholesterol ratio. exhibits anti-arteriosclerosis activity. reduces body fat content and affects body weight gain. Safe and selective CLA production process using lactic acid bacteria!!

Potential strains for CLA production from linoleic acid Fatty acid (mg/ml reaction mixture) Cellular Strain Origin LA Total CLA (CAL1:CLA2) HY1 HY2 FA Enterococcus faecium AKU 1021 0.09 0.72 0.10 (0.04: 0.06) 0.02 0.06 Pediococcus acidilactici AKU 1059 0.14 1.29 1.40 (1.00: 0.40) 0.30 0.43 Propionibacterium shermnjii AKU 1254 0.11 1.42 0.11 (0.09: 0.02) - 0.07 Lactobacillus acidophilus AKU 1137 0.14 0.24 1.50 (0.85: 0.65) 0.11 0.07 Lactobacillus acidophilus IAM 10074 0.25 0.22 0.60 (0.18: 0.42) 0.60 0.18 Lactobacillus acidophilus AKU 1122 0.09 0.91 0.12 (0.02: 0.10) - 0.02 Lactobacillus brevis IAM 1082 0.10 0.16 0.55 (0.23: 0.32) 0.79 - Lactobacillus paracasei IFO12004 0.18 0.83 0.20 (0.05: 0.15) 0.22 0.45 Lactobacillus paracasei JCM 1109 0.17 0.76 0.07 (0.02: 0.05) - 0.57 Lactobacillus paracasei AKU 1142 1.08 0.90 0.07 (0.04: 0.03) 0.05 1.00 Lactobacillus paracasei IFO 3533 0.32 0.93 0.09 (0.05: 0.04) 0.06 0.68 Lactobacillus pentosus AKU 1148 0.10 1.24 0.08 (0.05: 0.03) 0.08 0.05 Lactobacillus pentosus IFO12011 0.09 0.89 0.13 (0.10: 0.03) 0.13 0.74 Lactobacillus plantarum AKU 1138 0.11 0.10 0.45 (0.10: 0.35) 1.21 - Lactobacillus plantarum AKU 1009a 0.07 0.06 3.41 (0.25: 3.16) 0.11 0.16 Lactobacillus plantarum JCM 8341 0.18 0.43 0.19 (0.04: 0.15) 0.27 0.40 Lactobacillus plantarum JCM 1551 0.36 0.02 2.02 (0.10: 1.92) 0.02 0.46 Lactobacillus rhamnosus AKU 1124 0.10 0.22 1.41 (0.69: 0.72) 0.13 0.15 Searching unique microbial functions in Japanese microbial Searching unique microbial functions in Japanese microbial Reactions were carried out in 72 h as described in Materials and methods. diversity and using them for food and chemical industries diversity and using them for food and chemical industries Cellular FA included myristic acid, palmitic acid, palmitoleic acid, oleic acid, trans- vaccenic acid, and 2-hexy-1-cyclopropane-octanoic acid. LA, linoleic acid; HY1, 10-hydroxy- trans -12-octadecaenoic acid; HY2,10-hydroxy- cis -12-octadecaenoic acid; -, not detected.

CLA production by microorganisms Castor oil Lipase Ricinoleic acid Linoleic acid (12-hydroxy- cis- 9-octadecaenoic acid) ( cis -9, cis -12-octadecadienoic acid) O O OH Δ 9 Δ 9 Δ 12 HO C HO C CLA production : 2.5 ~ 7.5 mg/ml CLA production : 20 ~ 40 mg/ml Lactic acid bacteria CLA1 : ~ 50% CLA1 : ~ 75% CLA2 : ~ 82% CLA2 : ~ 97% Free fatty acid Free fatty acid O Δ 9 Δ 11 HO C cis -9, trans -11-octadecadienoic acid O Δ 9 Δ 11 HO C trans -9, trans -11-octadecadienoic acid

Conjugated fatty acids production by lactic acid bacteria CLA production: ～ 40 mg/ml HOOC CLA1 ・ CLA1 selective condition HOOC CLA1 purity: ～ 75% HOOC Linoleic acid CLA2 ・ CLA2 selective condition CLA2 purity: ～ 97% CALA production: ～ 25 mg/ml HOOC ・ CALA1 selective condition CALA1 HOOC CALA1 purity: ～ 85% HOOC α -linolenic acid ・ CALA2 selective condition CALA2 CALA2 purity: ～ 85% CGLA production: ～ 9 mg/ml HOOC CGLA1 ・ CGLA1 selective condition CGLA1 purity: ～ 80% HOOC HOOC ・ CGLA2 selective condition γ -linolenic acid CGLA2 CGLA2 purity: ～ 87%

Non-specific oxidases ・ O R O R ・ O R Peroxidase O R O R H 2 O 2 HO R O R O R O 2 O R Laccase R ・ HO R ・ O O O R R HO R

Deodorization of methylmercaptane by laccase with rosemary extract as a mediator