Potentials of Microorganisms for Functional Food Production ......Potentials of Microorganisms for...

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

Jun OgawaJun OgawaResearch Division of Microbial Sciences, Research Division of Microbial Sciences,

Kyoto University, Kyoto, JapanKyoto University, Kyoto, JapanJuly 3rd, 2009, Nestle, LausanneJuly 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 beenobtained traditionally and environmentally.

●There are many active industries using microorganisms.

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

Japan

USA

Europe

Percentage of patent applicants in industry sector

USA

EU

Japan

Medical

Medical

Medical

Foods & Chemicals etcFoods & Chemicals etc

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


Creation of new Creation of new food functionfood function

based on unique based on unique microbial functionmicrobial 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 industriesdiversity and using them for food and chemical industries

FenugreekFenugreek

●

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.

COOH

HIL

NH2OH

L-Ile AMKP

L-Ile dioxygenase(IDO)

HILdehydrogenase

COOH

NH2

COOH

O NH2

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

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

S S COOH

NH2

S COOH

NH2OH

SR

Dioxygenase

Dioxygenase process

↑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 producerMortierella alpina 1S-4


“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.

Mutant Screening

spore Mutant isolation,cultivationNTG

mutation

Retention time (min)

Fatty acid analysisBy 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

Δ9EL1

Δ5

ω3

Δ6Δ12

EL2Δ6 Δ5

ω3

EL2

Various PUFAs

produced by

M. alpina 1S-4

Δ12

COOH20:3n-9, MA

COOH20:2n-9

COOH18:2n-9

COOH18:0

COOH16:0

COOH18:1n-9

COOH20:4n-6, AA

COOHDGLA

COOH18:3n-6

COOH18:2n-6

COOH18:3n-3 18:4n-3

EL2Δ6

COOH16:1n-7

COOH16:2n-7COOH18:2n-7 COOH

18:3n-7 COOH20:3n-7

COOH18:1n-7

COOH18:2n-7(Δ5)

20:4n-4COOH

18:4n-4COOH

18:3n-4COOH

20:3n-4COOH

16:3n-4COOH

16:2n-4COOH

18:2n-4COOH

COOH20:2n-6

COOH20:3n-3

COOH20:3n-6(Δ5)

COOH20:4n-6(Δ5)

n-4

n-7

n-9

n-6

COOH20:5n-3, EPA

COOH COOH20:4n-3

ω3Δ5

n-3

Glucose Δ15

20:5n-1COOH

18:5n-1COOH

18:4n-1COOH

16:4n-1COOH

16:3n-1COOH n-1

Δ9

Δ5 EL2

EL1

EL2

EL2EL

EL

ELΔ9

COOH20:1n-9

ELCOOH

20:0

COOH22:0

COOH24:0

Δ12

C

O

HOΔ9 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!!

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 0.06Pediococcus acidilactici (1.00: 0.40) 0.30 0.43Propionibacterium shermnjii (0.09: 0.02) - 0.07Lactobacillus acidophilus (0.85: 0.65) 0.11 0.07Lactobacillus acidophilus (0.18: 0.42) 0.60 0.18Lactobacillus acidophilus (0.02: 0.10) - 0.02Lactobacillus brevis (0.23: 0.32) 0.79 -Lactobacillus paracasei (0.05: 0.15) 0.22 0.45Lactobacillus paracasei (0.02: 0.05) - 0.57Lactobacillus paracasei (0.04: 0.03) 0.05 1.00Lactobacillus paracasei (0.05: 0.04) 0.06 0.68Lactobacillus pentosus (0.05: 0.03) 0.08 0.05Lactobacillus pentosus (0.10: 0.03) 0.13 0.74Lactobacillus plantarum (0.10: 0.35) 1.21 -Lactobacillus plantarum (0.25: 3.16) 0.11 0.16Lactobacillus plantarum (0.04: 0.15) 0.27 0.40Lactobacillus plantarum (0.10: 1.92) 0.02 0.46Lactobacillus rhamnosus

Origin

AKU 1021AKU 1059AKU 1254AKU 1137IAM10074

AKU 1122IAM 1082IFO12004

JCM 1109AKU 1142IFO 3533

AKU 1148IFO12011

AKU 1138AKU1009aJCM 8341JCM 1551AKU 1124

Cellular FA LA Total CLA

0.09 0.72 0.100.14 1.29 1.400.11 1.42 0.110.14 0.24 1.500.25 0.22 0.600.09 0.91 0.120.10 0.16 0.550.18 0.83 0.200.17 0.76 0.071.08 0.90 0.070.32 0.93 0.090.10 1.24 0.080.09 0.89 0.130.11 0.10 0.450.07 0.06 3.410.18 0.43 0.190.36 0.02 2.020.10 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.


Δ9 Δ12CO

HO

Linoleic acid(cis-9,cis-12-octadecadienoic acid)

CLA production : 20 ~ 40 mg/mlCLA1 : ~ 75%CLA2 : ~ 97%Free fatty acid

CLA production by microorganisms

Δ9 Δ11CO

HOcis-9,trans-11-octadecadienoic acid

Δ9 Δ11CO

HO

trans-9,trans-11-octadecadienoic acid

Δ9C

O

HO

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

OH

Lactic acid bacteriaCLA production : 2.5 ~ 7.5 mg/mlCLA1 : ~ 50%CLA2 : ~ 82%Free fatty acid

Castor oilLipase

HOOC

HOOC

HOOC

HOOC

HOOC

HOOC

HOOC

HOOCHOOC

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 conditionCLA1 purity: ～75%

・CLA2 selective conditionCLA2 purity: ～97%

CGLA production: ～9 mg/ml

・CGLA1 selective conditionCGLA1 purity: ～80%

・CGLA2 selective conditionCGLA2 purity: ～87%

CALA production: ～25 mg/ml

・CALA1 selective conditionCALA1 purity: ～85%

・CALA2 selective conditionCALA2 purity: ～85%

RHO

RO・

RO

・

RO

・

RO ・

RO

RO

RO

RO

RHO

RO

RO

RHO

R

O

Peroxidase

Laccase

H2O2

O2

Non-specific oxidases

Deodorization of methylmercaptane

by laccase with rosemary extract as a mediator

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.

Mammalian purine

metabolism

O

HN

NH

HN

NH

O

O

O

HN

NH

N

NH

O

O

HN

N

N

NH

HN

N

N

N

O

Rib

HN

N

N

NH

O

H2

N

HN

N

N

N

O

H2

N

Rib

Inosine

Xanthine

Guanosine Guanine

Uric acid

IMP

GMP

AMPAdenosine Adenine

HN

N

N

NH

NHN

N

N

N

N

Rib

HN

N

N

N

NH2

RibP

P

HN

N

N

N

O

H2

N

Rib

HN

N

N

N

O

RibP

nucleotidenucleotide nucleosidenucleoside basebase

Hypoxanthine

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


Results of rat experiment

Days （day）Seru

m u

ric

acid

con

cent

ration

(mg/

dl)

*

0.0

1.0

2.0

3.0

4.0

-2 2 5 8

Untreated groupControl groupL. fermentum

Days （day）

Seru

m u

ric

acid

con

cent

ration

(mg/

dl)

#

0.0

1.0

2.0

3.0

4.0

-2 2 5 8

Untreated groupControl groupL. fermentum

0.0

1.0

2.0

3.0

4.0

Days （day）

#

-2 2 5 8

Untreated groupControl group

L. pentosus

Seru

m u

ric

acid

con

cent

ration

(mg/

dl)


Creation of new Creation of new food functionfood function

based on unique based on unique microbial functionmicrobial 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.

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