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

▶
potentials of microorganisms for potentials of
SMART_READER_LITE
LIVE PREVIEW

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

Nov 24, 2022 193 likes •404 views

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

slide-1
SLIDE 1

Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Functional Food Production and Probiotics Probiotics 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

slide-2
SLIDE 2

Base of Japan’s Microbial Biotechnology

  • Japan is a country rich in microbial resources.
  • We have high affinity to microorganisms, that has been
  • btained traditionally and environmentally.
  • There are many active industries using microorganisms.

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Japan USA Europe

Pe r c e ntage of pate nt applic ants in industr y se c tor

USA E U Japan

Medic al Medic al Medic al

F

  • o ds & Che mic als e tc

F

  • o ds & Che mic als e tc

豊かな微生物資源が あります 微生物との共存ができます バイオに強い化学工業があ

ります

slide-3
SLIDE 3

Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Functional Food Production and Probiotics Probiotics

Creation of new Creation of new food function food function based on unique based on unique microbial function microbial function: 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

slide-4
SLIDE 4

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.

4-Hydroxyisoleucine (HIL)

Target decision: Target decision: by collaboration by collaboration and discussion and discussion with industries. with industries.

slide-5
SLIDE 5

COOH

HIL

NH2 OH

L-Ile AMKP L-Ile dioxygenase (IDO) HIL dehydrogenase

COOH NH2 COOH O NH2

L-Ile transformation pathway found in Bacillus thuringiensis strain 2e2

slide-6
SLIDE 6

HIL production by cell-free extracts of E. coli expressing IDO from B. thuringiensis strain 2e2

S S COOH NH2 S COOH NH2 OH S R

Dioxygenase Dioxygenase process

slide-7
SLIDE 7

↑高度不飽和脂肪酸の油滴が見られる顕微鏡写真 ↓寒天培地上に生育したモルティエレラ・アルピナ

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

Hyper Arachidonic acid producer Mortierella alpina 1S-4 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

slide-8
SLIDE 8

“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。

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.

slide-9
SLIDE 9

Mutant Screening

spore Mutant isolation, cultivation

NTG mutation Retention time (min) Fatty acid analysis By GLC

COOH COOH COOH COOH COOH COOH COOH COOH COOH COOH

18:0 18:1n-9 18:2n-6 18:3n-6 20:3n-6, DGLA 20:4n-6, AA 18:3n-3 18:4n-3 20:4n-3 20:5n-3, EPA Glucose

Δ9 EL1 Δ5 ω3 Δ6 Δ12 EL2 Δ6 Δ5 ω3 EL2

slide-10
SLIDE 10

Various PUFAs produced by

  • M. alpina 1S-4

Δ12 12

COO COOH

20 20:3 :3n-9, MA MA

COO COOH

20 20:2 :2n-9

COO COOH

18 18:2 :2n-9

COO COOH

18 18:0 :0

COO COOH

16 16:0 :0

COO COOH

18 18:1 :1n-9

COO COOH

20 20:4 :4n-6, AA AA

COO COOH

DG DGLA LA

COO COOH

18 18:3 :3n-6

COO COOH

18 18:2 :2n-6

COO COOH

18 18:3 :3n-3 18 18:4 :4n-3

EL2 EL2 Δ6

COO COOH

16 16:1 :1n-7

COO COOH

16 16:2 :2n-7

COO COOH

18 18:2 :2n-7

COO COOH

18 18:3 :3n-7

COO COOH

20 20:3 :3n-7

COO COOH

18 18:1 :1n-7

COO COOH

18 18:2 :2n-7(Δ5) 5) 20 20:4 :4n-4

COO COOH

18 18:4 :4n-4

COO COOH

18 18:3 :3n-4

COO COOH

20 20:3 :3n-4

COO COOH

16 16:3 :3n-4

COO COOH

16 16:2 :2n-4

COO COOH

18 18:2 :2n-4

COO COOH COO COOH

20 20:2 :2n-6

COO COOH

20 20:3 :3n-3

COO COOH

20 20:3 :3n-6(Δ5) 5)

COO COOH

20 20:4 :4n-6(Δ5) 5)

n-4 n-4 n-7 n-7 n-9 n-9 n-6 n-6

COO COOH

20 20:5 :5n-3, EP EPA

COO COOH COO COOH

20 20:4 :4n-3

ω3 Δ5

n-3 n-3

Glucose

Δ15 15

20 20:5 :5n-1

COO COOH

18 18:5 :5n-1

COO COOH

18 18:4 :4n-1

COO COOH

16 16:4 :4n-1

COO COOH

16 16:3 :3n-1

COO COOH

n-1 n-1

Δ9 Δ5 EL2 EL2 EL1 EL1 EL2 EL2 EL2 EL2 EL EL EL EL EL EL Δ9

COO COOH

20 20:1 :1n-9

EL EL

COO COOH

20 20:0 :0

COO COOH

22 22:0 :0

COO COOH

24 24:0 :0

Δ12 12

slide-11
SLIDE 11

C O HO

Δ9 Δ11

C O HO

Δ10 Δ12

Conjugated linoleic acid (CLA)

inhibits initiation of skin carcinogenesis, and forestomach and mammary tumorigenesis. prevents the catabolic effects of immune stimulation. alters LDL / HDL cholesterol ratio. reduces body fat content and affects body weight gain. exhibits anti-arteriosclerosis activity.

Safe and selective CLA production process using lactic acid bacteria!!

slide-12
SLIDE 12

Potential strains for CLA production from linoleic acid

Fatty acid (mg/ml reaction mixture) Strain (CAL1:CLA2) HY1 HY2 Enterococcus faecium (0.04: 0.06) 0.02 0.06 Pediococcus acidilactici (1.00: 0.40) 0.30 0.43 Propionibacterium shermnjii (0.09: 0.02)

  • 0.07

Lactobacillus acidophilus (0.85: 0.65) 0.11 0.07 Lactobacillus acidophilus (0.18: 0.42) 0.60 0.18 Lactobacillus acidophilus (0.02: 0.10)

  • 0.02

Lactobacillus brevis (0.23: 0.32) 0.79

  • Lactobacillus paracasei

(0.05: 0.15) 0.22 0.45 Lactobacillus paracasei (0.02: 0.05)

  • 0.57

Lactobacillus paracasei (0.04: 0.03) 0.05 1.00 Lactobacillus paracasei (0.05: 0.04) 0.06 0.68 Lactobacillus pentosus (0.05: 0.03) 0.08 0.05 Lactobacillus pentosus (0.10: 0.03) 0.13 0.74 Lactobacillus plantarum (0.10: 0.35) 1.21

  • Lactobacillus plantarum

(0.25: 3.16) 0.11 0.16 Lactobacillus plantarum (0.04: 0.15) 0.27 0.40 Lactobacillus plantarum (0.10: 1.92) 0.02 0.46 Lactobacillus rhamnosus Origin AKU 1021 AKU 1059 AKU 1254 AKU 1137 IAM 10074 AKU 1122 IAM 1082 IFO12004 JCM 1109 AKU 1142 IFO 3533 AKU 1148 IFO12011 AKU 1138 AKU 1009a JCM 8341 JCM 1551 AKU 1124 Cellular FA LA Total CLA 0.09 0.72 0.10 0.14 1.29 1.40 0.11 1.42 0.11 0.14 0.24 1.50 0.25 0.22 0.60 0.09 0.91 0.12 0.10 0.16 0.55 0.18 0.83 0.20 0.17 0.76 0.07 1.08 0.90 0.07 0.32 0.93 0.09 0.10 1.24 0.08 0.09 0.89 0.13 0.11 0.10 0.45 0.07 0.06 3.41 0.18 0.43 0.19 0.36 0.02 2.02 0.10 0.22 1.41 (0.69: 0.72) 0.13 0.15

Reactions were carried out in 72 h as described in Materials and methods. 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.

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

slide-13
SLIDE 13

Δ9 Δ12 C O HO

Linoleic acid

(cis-9,cis-12-octadecadienoic acid) CLA production : 20 ~ 40 mg/ml CLA1 : ~ 75% CLA2 : ~ 97% Free fatty acid

CLA production by microorganisms

Δ9 Δ11 C O HO cis-9,trans-11-octadecadienoic acid Δ9 Δ11 C O HO trans-9,trans-11-octadecadienoic acid Δ9 C O HO

Ricinoleic acid

(12-hydroxy-cis-9-octadecaenoic acid) OH

Lactic acid bacteria

CLA production : 2.5 ~ 7.5 mg/ml CLA1 : ~ 50% CLA2 : ~ 82% Free fatty acid

Castor oil

Lipase

slide-14
SLIDE 14

HOOC HOOC HOOC HOOC HOOC HOOC HOOC HOOC HOOC

Linoleic acid α-linolenic acid γ-linolenic acid CLA1 CLA2 CALA1 CALA2 CGLA1 CGLA2

Conjugated fatty acids production by lactic acid bacteria

CLA production: ～40 mg/ml ・CLA1 selective condition CLA1 purity: ～75% ・CLA2 selective condition CLA2 purity: ～97% CGLA production: ～9 mg/ml ・CGLA1 selective condition CGLA1 purity: ～80% ・CGLA2 selective condition CGLA2 purity: ～87% CALA production: ～25 mg/ml ・CALA1 selective condition CALA1 purity: ～85% ・CALA2 selective condition CALA2 purity: ～85%

slide-15
SLIDE 15

R HO R O

・

R O

・

R O

・

R O

・

R O R O R O R O R HO R O R O R HO R O

Peroxidase Laccase

H2O2 O2

Non-specific oxidases

slide-16
SLIDE 16

Deodorization of methylmercaptane by laccase with rosemary extract as a mediator

slide-17
SLIDE 17

Background ( Hyperuricemia & Purine)

  • Hyperuricemia

is a disease, which results from upper serum uric acid level. Some hyperuricemic individuals develop gout .

  • Over 20% of male adults (in Japan) develop

hyperuricemia.

  • Hyperuricemia

is influenced by a high dietary intake of purine (meat, seafood and alcoholic beverages).

  • Low-purine

diets are used for hyperuricemia clinic, but difficult for patients to adhere.

slide-18
SLIDE 18

Mammalian purine metabolism

O HN N H H N N H O O O HN N H N N H O O HN N N N H HN N N N O Rib HN N N N H O H2 N HN N N N O H2 N Rib

Inosine Xanthine Guanosine Guanine Uric acid IMP GMP AMP Adenosine Adenine

HN N N N H N HN N N N N Rib HN N N N NH2 Rib P P HN N N N O H2 N Rib HN N N N O Rib P

nucleotide nucleotide nucleoside nucleoside base base

Hypoxanthine

slide-19
SLIDE 19

Result of screening

Lactobacillus (187), Bifidobacterium (29), Leuconostoc (15), Enterococcus (13), Pediococcus (9), Clostridium (1), Bacillus (12), Saccharomyces (1) 267 strains Lactobacillus (58), Leuconostoc (4), Pediococcus (2), Clostridium (1), Saccharomyces (1) 66 strains

  • L. fermentum

(7) , L. brevis (2) , L. mali (1)

  • L. vaccinostercus (1) , L. homohiochi (1) , L. pentosus

(1) 13 strains First screening; reaction time 2 h Second screening; reaction time 30 min

Third screening; Rat model experiment

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

slide-20
SLIDE 20

Results of rat experiment

Days （day）

Serum uric acid concentration (mg/dl)

*

0.0 1.0 2.0 3.0 4.0

  • 2

2 5 8

Untreated group Control group

  • L. fermentum

Days （day）

Serum uric acid concentration (mg/dl)

#

0.0 1.0 2.0 3.0 4.0

  • 2

2 5 8

Untreated group Control group

  • L. fermentum

0.0 1.0 2.0 3.0 4.0

Days （day） #

  • 2

2 5 8

Untreated group Control group

  • L. pentosus

Serum uric acid concentration (mg/dl)

slide-21
SLIDE 21

Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Functional Food Production and Probiotics Probiotics

Creation of new Creation of new food function food function based on unique based on unique microbial function microbial function: 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

These microbial functions might be useful by themselves and to endow agricultural products with extra-qualities increasing added values of primary agro-products.