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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
Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Functional Food Production and ProbioticsProbiotics
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
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
“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.
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
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
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
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)
Potentials of Microorganisms for Potentials of Microorganisms for Functional Food Production and Functional Food Production and ProbioticsProbiotics
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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