Funtional Food Ingredients

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

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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

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80

90

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7

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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

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Active compound

mannitol Cordycepin adenosine

Compound mg/g

Cordycepin 54.23

Adenosine 43.99

Mannitol 111.19

Biopolymer 1000.7

Isaria cicadae batch

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120

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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

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1.5

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2.5

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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

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pH

%D

OpH and DO profile

DO pH

66.35

43.9

0

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20

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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

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3

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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

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60

80

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120

0 50 100 150 200

pH

%D

O

pH and DO profile

DO pH

63.81

38.1

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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

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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