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Funtional Food Ingredients;Fungal Bioproducts for food supplements
Wai Prathumpai, Ph.D.
National Center for Genetic Engineering and Biotechnology
Functional foods ingredients• องคก์ารอาหารและเกษตรแห่งสหประชาชาติ (Food and Agriculture Organization of the United
Nation ; FAO) ไดใ้ห้ความหมาย ของ Functional food วา่ “เป็นอาหารที่บริโภคเหมือนอาหารปกติโดยทัว่ไป โดยประกอบดว้ยสารออกฤทธ์ิทางชีวภาพ (Biological active component) ที่บ ารุงสุขภาพหรือลดความเส่ียงในการเกดิโรคเร้ือรัง (Chronic disease)”
• Probiotics, Proteins & amino acids, Phytochemical & plant extracts, Prebiotics, Fibers & specialty carbohydrates, Omega-3, -6, -9 fatty acids, Carotenoids, Vitamins, Minerals, Dietary fibers, oligosaccharides (Polysaccharides).
Biopolymers
• Homopolysaccharides
Pullulan
Dextran
b-Glucan
Biopolymers• Heteropolysaccharides
Heparin
Algenates
Curdlan
Hyaluronic acid
Fermentation conditionsEnthomopathogenic Fungal Beta-glucan Production
5- liter fermenter (Marubishi Co., Ltd., Pathumthani, Thailand) with a working volume of 4 L
Ophiocordyceps dipterigena
Culture was agitated at 300 rpm and aerated at 1 vvm. pH was not automatically adjusted
glucose 60 gL-1
Yeast extract 2.5 gL-1
Molasses 0.035%
The basal medium
KH2PO40.5 gL-1
K2HPO40.2 gL-1
MgSO47H2O 0.14 gL-1
MnSO4H2O 0.2 gL-1
vitamin solution 1 mLL-1
(Blackmores, NSW, Australia)
Trace element solution contained (per L)
14.3 g of ZnSO4H2O2.5 g of CuSO45H2O0.5 g of NiCl26H2O 13.8 g of FeSO4H2O
About 10% (v/v) seed culture was transferred
Chemical structure of fungal beta-glucan
1,3-b-D-glucan with 1,6-b-d-pyranosyl side chains
Industrial Production of a novel b-glucan oligosaccharide by Ophiocordyceps sp. and its applications
Novel type Beta-glucan, different size glucanoligosaccharide
Raw material Finished products
Human-Functional foodAnimals (Poultry, Swine, Pets)-Feed ingredients-Cosmetic
TECHNOLOGY FEATURES, SPECIFICATIONS AND ADVANTAGES
Size (KDa) Application Competitor
500-900 Obesity,cosmetics
Petroleum
None
70 Cosmetic, food supplement
None
40 Prebiotic Yeast and oat
meal beta-glucan
5-20 Immuno-stimulant
Medical and pharmaceutical
None
TECHNOLOGY OVERVIEW
b-glucan of O.dipterigena
-A Novel type of b-glucan of Ophiocordyceps dipterigena (Pure glucan)-Doesn’t need complicated downstream processes for its purification steps-The size reduction of this b-glucan can be done by gamma-radiation
-Can applied in many applications; Food and Feed, cosmetics, Medical and pharmaceuticals, and petroleum.
Value Chain of Beta-Glucan
Manufacturer/OEMRetailer
Food supplement
Feed supplement
Cosmetics
Poultry Cattles Patients Non-Patients Skin Aging Woundhealing
*1 kDa = 1.66×10-20 grams
*1 kDa = 1.66×10-20 grams
Application in diet supplement of broilers
Bacteria
Cell Numbers (CFU x103/g dried stool)
T1 T2 T3 T4 T5 T6 T7
E. coli 830 80 28 229 35 10.5 27.5
Coliform 1715 170 28 175 150 100 3.5
Lactobacilli 80 870 550 2550 4000 1000 275
Vibrio sp. 75 31 30 365 315 28.5 57
Total bacteria 16850 50700 20250 12950 35850 75500 72500
22PCR- DGGE profiles (40 to 60% denaturing gradient gel) generated from fecal content of 42 days broiler chickens
PCR-DGGE microorganisms identity from fecal samples of treatment 3 (T3) 42 days olds broiler chickens
Band Microorganisms Accession No. Identity(%)
1 Uncultured Lactobacillus sp. DGGE band NSFC2-R8 16S ribosomal RNA gene, partial sequence
GQ280119.1 76
2 Uncultured Lactobacillus sp. clone BV_LV_OTU6424 16S ribosomal RNA gene, partial sequence
JQ962805.2 75
3 Uncultured rumen bacterium clone L206RT-4-F04 16S ribosomal RNA gene, partial sequence
GU303313.1 80
4 Streptomyces sp. 070605-23_L10_11 16S ribosomal RNA gene, partial sequence
FJ626802.1 81
5 Uncultured bacterium clone GDIC2IK01E1CQ4 16S ribosomal RNA gene, partial sequence
JF633402.1 72
6 Uncultured Lactobacillus sp. clone BV_LV_OTU6496 16S ribosomal RNA gene, partial sequence
JQ962830.2 75
7 Uncultured bacterium clone 11LEA01 16S ribosomal RNA gene, partial sequence
FJ163666.1 73
23PCR- DGGE profiles (40 to 60% denaturing gradient gel) generated from fecal content of 42 days broiler chickens
PCR-DGGE microorganisms identity from fecal samples of treatment 4 (T4) 42 days olds broiler chickens
Band Microorganisms Accession No. Identity(%)
1 Uncultured bacterium clone PF1899 16S ribosomal RNA gene, partial sequence
GU619794.1 75
2 Uncultured bacterium clone DZ_G1 16S small subunit ribosomal RNA gene gene, partial sequence
AY702848.1 77
3 Lactobacillus acidophilus clone WWC_C4ALM288 16S ribosomal RNA gene, partial sequence
GU413899.1 84
4 Uncultured rumen bacterium clone P5_D16 16S ribosomal RNA gene, partial sequence
EU382039.1 80
5 Uncultured rumen bacterium clone L206RT-3-F07 16S ribosomal RNA gene, partial sequence
GU303227.1 77
6 Uncultured bacterium clone TS50_a01a09 16S ribosomal RNA gene, partial sequence
FJ369165.1 76
7 Uncultured Escherichia sp. clone BR04AH02 16S ribosomal RNA gene, partial sequence
DQ856911.1 78
8 Clostridium paraputrificum strain ATCC 25780 16S ribosomal RNA, partial sequence >emb|X75907.1| C.paraputrificum 16S rRNA gene
NR_026135.1 73
Molecular weight and IL-8 production
Molecular
weight
50 g/mL 100 g/mL
0
1
2
3
4
5
6
7
8
9
0 50 100 150 200 250 300 350 400 450 500
Cultivation time (h)
MW
x 1
05 (
dalt
on
)
0
500
1000
1500
2000
2500
3000
3500
4000
IL-8
pro
du
cti
on
(p
g/m
L)
Size reduction and IL-8 production
I tems Groups1 SEM P-value
C LS MS HS LL ML HLOrgans at 24 days (%LW)
Pancreas 0.21ab 0.25b 0.24b 0.25b 0.23ab 0.19a 0.25b 0.006 0.03
Spleen 0.074 0.082 0.084 0.090 0.072 0.091 0.085 0.002 0.56
Bursa of Fabricius 0.19a 0.23ab 0.25b 0.25b 0.20a 0.20a 0.18a 0.007 0.02
Thymus 0.13a 0.21ab 0.29b 0.13a 0.16a 0.12a 0.20a 0.015 0.01
HaematologyHaematocrit (%) 26.8 26.8 26.3 27.8 26.7 28.7 27.0 0.33 0.65
Erythrocytes (mm3x106) 4.22 4.21 4.18 4.18 4.22 4.15 4.23 0.01 0.17
Leukocyte (mm3x103) 13.9 14.1 13.8 13.9 13.9 13.6 14.1 0.09 0.87
Blood chemistryPlasma protein (g%) 3.93 4.20 4.16 4.50 3.87 4.20 4.00 0.13 0.95
Blood urea nitrogen (mg/dL) 3.25 2.70 2.98 3.18 3.46 3.70 3.78 0.25 0.94
Creatinine 0.16 0.21 0.20 0.18 0.19 0.21 0.20 0.01 0.35
Alanine aminotransferase 365b 250a 226a 297ab 222a 356b 272a 13.1 0.002
Cholesterol (mg/100ml) 118 129 126 114 115 121 124 3.42 0.92
Triglyceride (mg/100ml) 74.0 107 98.4 78.0 105 104 92.2 4.66 0.30
Phagocytic activity on the abdominal exudative cell (%)Non-opsinized sRBC 26.5a 25.9a 34.9b 35.2b 24.8a 27.6a 25.6a 0.86 0.001
Opsonized sRBC 33.7a 36.0a 44.7b 43.4b 37.0a 35.0a 36.2a 0.76 0.001
Number of sheep red blood cells : phagocytic cellNon-opsinized sRBC 2.41 2.39 2.31 2.43 2.35 2.42 2.42 0.02 0.69
Opsonized sRBC 2.45 2.47 2.42 2.47 2.44 2.39 2.40 0.02 0.85
Antibody titer against ND (log2) 4.00 3.60 4.60 3.20 3.40 3.20 3.40 0.26 0.81
Digestive tract histology (µm)Duodenal villi height 1006 1147 1288 1090 1115 1011 1007 46.2 0.29
Duodenal crypt depth 152 151 173 165 180 169 173 5.78 0.8726
Commercial Beta-glucan
Project by project 2.6-6 MB 20-60 MB
Finished Products• Food
• Food supplements for immune stimulation• Cancer, HIV, Hay fever, cold fever, collagen booster, endurance
• Prebiotic
• Feed• Cattles, swine, poultry-reduce the used of antibiotic and enhance their
immune system.
• House pets-to enhance their immune system
• Cosmetic• Scar reduction
• Moisturizer
• Anti-aging• Mask, day and night cream, serum, acne gel etc..
Application in Food
• Toxicity and Antioxidants test
• Gel and film forming
• Water holding capacity
• Anti-microbial activity
• Solubility properties
• Anti-glycation
• Emulsion property
สายพนัธุ์ถ ัง่เช่าท่ีมีระดบั Cordycepin สูง• Cordyceps sinensis (ถั่งเช่าธิเบต)• Hirsutella sinensis (anamorph)
• Ophiocordyceps sinclairii
• Isaria tenuipes (ถั่งเช่าหิมะ)• Cordycepsmilitaris (ถั่งเช่าสีทอง)
• Isaria cicadae
Estimated living organismsOrganisms Estimated species in
the WorldEstimated Species
in ThailandKnown species in
Thailand
1 Bacteria 40,000 10,000 1,600
2 Fungi 1,500,000 40,000 7,464
3 Microalgae 20,000 5,000 2,300
4 Vascular plant 287,655 10,000 7,000
5 Earth Worm 8,000 100 55
6 Insect 9,600,000 100,000 7,000
7 Millipede 12,000 500 188
8 Reptile & Amphibian 13,900 490 490
9 Bird 9,917 980 980
10 Mammal 5,416 300 300
Groups of fungi in BIOTEC Culture Collection (March,2014)
2275
1036
6573
10822
5822
490
2764
759
1148
1365
2776
4260
3485
Aquatic fungi
Basidiomycetes
Endophytic fungi
Insect Pathogenic fungi
Marine fungi
Lichenized fungi
Mushroom
Palm fungi
Rice blast pathogen
Saprobic fungi
Soil fungi
Wood Decay fungi
Other groups
Number
Other groups, 10.34Wood decay fungi,
12.64
Soil fungi, 8.24
Saprobic fungi, 4.05Rice blast fungi, 3.41
Palm fungi, 2.25Mushroom, 8.20Lichenized fungi, 1.45
Insect fungi, 32.12
Marine fungi, 17.28
>80,000 stains in 2018
Cordyceps militaris• It is a rare combination of a
caterpillar and fungus found
in Sikkim.
• Cordycepins as an active
compounds.
• It improves energy, appetite,
stamina (ความแข็งแรง), libido (อารมณท์างเพศ), endurance (ความแข็งแรง), and sleeping patterns.
Solid state production of fruit bodies
• Take 6-12 months for production.
• Low productivity.
• Time consume
• Large space requirement and labor dependent
Submerged cultivation of mycelium• High productivity
• High quality and contain high active compounds.
• Ease of scale up for industrial production
• Low cost and take shorter time.
Submerged cultivation of mycelium
4 days 7 days
Mannitol and Polysaccharide production by different strains of fungi
Cordycepin production by different strains of fungi
Process development of cordycepin production
BCC 1º screening 2º screening
Large scale
Optimization
Lab scale
สายพนัธ์ุถัง่เช่าท่ีมีสารออกฤทธ์ิ cordycepin สูง
Cordycep millitaris batch
0
10
20
30
40
50
60
70
80
90
0
1
2
3
4
5
6
7
0 20 40 60 80 100 120 140 160
DO
%
pH
Hr
pH and DO profile
pH DO
56.08
41.64
0
10
20
30
40
50
60
0 20 40 60 80 100 120 140 160
Cell mass and Biopolymer
Cell mass Biopolymer
5.648795014
2.5984412392.338035562
0
1
2
3
4
5
6
0 20 40 60 80 100 120 140 160
Active compound
mannitol Cordycepin adenosine
Compound mg/g
Cordycepin 54.23
Adenosine 43.99
Mannitol 111.19
Biopolymer 1000.7
Isaria cicadae batch
0
20
40
60
80
100
120
0
1
2
3
4
5
6
7
0 20 40 60 80 100 120 140 160
DO
%
pH
Hr
pH and DO profile
pH DO
45.22544.08
0
5
10
15
20
25
30
35
40
45
50
0 20 40 60 80 100 120 140 160
Cell mass and Biopolymer
Cell mass Biopolymer
4.580353278
2.99345883.158584
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 20 40 60 80 100 120 140 160
Active compound
mannitol Cordycepin adenosine
Compound mg/g
Cordycepin 83.06
Adenosine 58.33
Mannitol 127.22
Biopolymer 1211.11
Fed-batch fermentation
Fermentation
conditions Aeration 1 vvm
Temperature at
25˚C
400 rpm
Samples were collected every day.
Feeding medium
Using the same
component with batch
medium with high
concentration of
Carbon source
Cordycep militaris fed-batch
0
1
2
3
4
5
6
7
0
20
40
60
80
100
120
0 50 100 150 200
pH
%D
OpH and DO profile
DO pH
66.35
43.9
0
10
20
30
40
50
60
70
0 50 100 150 200 250
Cell mass and Biopolymer
Cell mass Biopolymer
3.1125
2.8522
5.9811
0
1
2
3
4
5
6
7
0 50 100 150 200 250
Active compound
Adenosine Cordycepin Mannitol
Compound mg/g
Cordycepin 53.76
Adenosine 46.83
Mannitol 83.61
Biopolymer 729.53
Isaria cicadae fed-batch
0
1
2
3
4
5
6
7
0
20
40
60
80
100
120
0 50 100 150 200
pH
%D
O
pH and DO profile
DO pH
63.81
38.1
0
10
20
30
40
50
60
70
0 50 100 150 200 250
Cell mass and Biopolymer
Cell mass Biopolymer
Compound mg/g
Cordycepin 64.56
Adenosine 60.96
Mannitol 99.19
Biopolymer 793.38
3.7852
3.5871
6.10025
0
1
2
3
4
5
6
7
0 50 100 150 200 250
Active compound
Adenosine Cordycepin Mannitol
CompoundPB CCD batch Fed-batch
mg/L mg/g mg/L mg/g mg/L mg/g mg/L mg/g
Cordycepin 960.20 37.58 1180 45.54 2590 54.23 3112.5 53.76
Adenosine 145.42 5.69 1270 49.01 2101 43.99 2711.4 46.83
Mannitol 665.18 26.04 1370 52.88 5303 111.19 4841 83.61
Biopolymer 5300 207.52 13,220 510.22 48,003 1000.7 40,200 729.53
Dried cell weight 25.54 g/L/L 25.91 g/L 47.755 g/L 57.04 g/L
Isaria cicadae
Cordyceps militaris
CompoundPB CCD batch Fed-batch
mg/L mg/g mg/L mg/g mg/L mg/g mg/L mg/g
Cordycepin 3920 93.82 4260 93.11 2993 83.06 3587 64.56
Adenosine 2500 59.84 6640 145.13 2071 58.33 3301 60.96
Mannitol 636.8 15.24 1750 38.25 4580 127.22 5512 99.19
Biopolymer 11,340 271.42 42,400 926.77 43,160 1211.11 44,080 793.38
Dried cell weight 41.78 g/L 45.75 g/L 38.88 g/L 55.56 g/L
ถั่งเช่า Adenosine Cordycepin Beta-glucan Mannitol
(mg/g) (mg/L) (mg/g) (mg/L) (mg/g) (mg/L) (mg/g) (mg/L)
China1.30
-3.02
- - - - -
Thailand0.88
-4.10
- - - - -
Cordyceps
militaris46.83 2711.4 53.76 3112.5 729.53 40200 83.61 4841
Isaria
cicadae60.96 3301 64.56 3584 793.38 44080 99.19 5512
ปริมาณ Adenosine. Cordycepin, beta-glucan และ Mannitol ในถ่ังเช่าแต่ละสายพันธ์ุ
Schizochytrium production
Technology
Inoculum
Cultivation in
Bioreactor
Lyophilization
Docosahexanoic acid (Omega-3) production by Schizochytrium sp.
Schizochytrium Arachiconic acid ARA C20:4
Eicosapentaenoic acidEPA C20:5
Yields18-24 g.L-1 DHA
70% TFA
120-200 g.L-1 BIOMASS
Docosahexaenoic acidDHA C22:6
Long chain PUFA production by
Marine microorganisms
Applications:Food Feed
Acknowledgements