Mining for bioactives- the Irish Marine Functional Foods Initiative

Paul Ross, Moorepark Food Research

Drivers

• Extensive National Marineresource

• Under-utilized from a health perspective

• Progressive Dairy Industry with global reach

• Fish and Marine products have a healthy image

Global functional foods market

0

10

20

30

40

50

60

70

1998 2000 2002 2004 2006 2008

Mark

et

siz

e(U

S$

bill

ion)

Functional Foods/ Major Growth Sector

• Japan, US and Europe are the biggestmarkets comprising 86% of globaldemand for functional foods in 2006

• Global market size has been estimatedbetween US$30 billion and US$60 billion

• Sources anticipate that the full potentialof functional foods is 5-10% of the foodand drinks market as a whole

Functional foods are foods which have a positiveeffect on human health over and above basic nutrition.

The Background

Summary of Bioactives Covered

Components with direct health associated bioactivityAntimicrobial peptidesAntihypertensive peptidesImmunoreactive componentsAntiproliferative Fatty Acids

Components which act through gut floraPrebiotics – bioactives which can influence gut flora compositionSubstrates for bioactive production

Mining forBioactives

A1-45A1-54

AA1-45

A1-54A1-45

A1-54

A

Fractionation

Fractionation (size, charge, etc)

Fermentation (with gut microbes)

Hydrolysis (commercial enzymes)

Bioassays

AntimicrobialCell

Proliferation

XHypertension

ImmuneFunction

InfectionControl

BIOASSAYS

Mining forBioactives

A1-45A1-54

AA1-45

A1-54A1-45

A1-54

A

Fractionation

Fractionation (size, charge, etc)

Fermentation (with gut microbes)

Hydrolysis (commercial enzymes)

Antimicrobial

BIOASSAYS

Colin Hill and Paul Ross

a b

Figure 5

IsracidinRPKHPIKHQGLPQEVLNENLLRF αS1(16-38)

Casecidin 15 &17YQEPVLGPVRGPFPI β(193-207)YQEPVLGPVRGPFPIIV β(193-209)

Bovine Colostrum Peptides

MIC-values of 0·5 mg ml-1

Concentration in milk of 0.04 mg ml-1

15-20% growth inhibition of E. coliwith 0.042 mg ml-1

MIC-values of 0·2 mg ml-1

Concentration in milk of 0.035mg ml-1

S1-casein EncryptedAntimicrobial peptides.

Sequence, synthesise and confirm*IKHQGLPQE (9 aa peptide)*VLNENLLR (8 aa peptide)

A1-45A1-54

A1-45A1-54

A1-45A1-54* *

Cecropin P1

Caseicin BCaseicin A

Lactobacillus acidophilus DPC6026

6.7% (w/v)

0.83% (w/v)0.1% (w/v)

0%

Peptide Ingredient can ImproveInfant Formula Safety

Hayes et al., in review

Ingredient kills or stops the growth ofEnterobacter sakazakii

(a pathogen which can be found in infant formula)

Clostridium difficile associated disease (CDAD) becomes anotifiable disease under the Acute InfectiousGastroenteritis Disease (AIG) category, from May 4th 2008

13th May 2008Superbug on the rise in Irish hospitals

Clostridium difficile

Ethanol shocked faecal samples plated on WCAA for 5 days andcolonies ~(30,000) overlaid with C. difficile.

Initial Isolation plateoverlaid with C. difficile

B. thuringiensis DPC 6431

TEMPurified culture

Mining the GIT for Novel Antimicrobials

0.0

0.5

1.0 80

70

60

50

%acetonitrile

Volts

2763 2861

G W V A C V G A C G T V C L A S G G V G T E F A A A S Y F L

G N A A C V I G C I G S C V I S E G I G S L V G T A F T L G

G W V A C V G A C G T V C L A S G G V G X E F A X A S X F L

G N A A C V I G C I G S C V I S E G I G X L V G X A F X L G

-2 -2 -2

-2 -2 -2

Radical SAM proteins ProteaseABC transporter

2769 (-6)

2867 (-6)

A new class ofantimicrobial

peptides?

A new class ofantimicrobial

peptides?

Thuricin operon

Rea et al., unpublished

Thuricin contains unusual post-translational modifications

C. difficile PCR Ribotype 027

Disc

C. difficile PCR ribotype 010

Control Proteinase K Treated

Haliclona simulans

Fergal O’Gara and Alan Dobson

Mining forBioactives

A1-45A1-54

AA1-45

A1-54A1-45

A1-54

A

Fractionation

Fractionation (size, charge, etc)

Fermentation (with gut microbes)

Hydrolysis (commercial enzymes)

InfectionControl

BIOASSAYS

Colin Hill,Cormac Gahan and Paul Ross

GWLTFLKAAG–NH2M-SAA310-mer

Colostrum-derived anti-infective peptide

Human 42-mer:QGWLTFLKAAGQGTKDMWKAYSDMKEANY

Cells+ peptide

Pathogenic E. coli

Gut epithelial cell line

MUCIN

0

20

40

60

80

100

120

140

Na Acetate Scramble Human 10-mer Mouse 10-mer 42-mer LGG

Adh

eren

tEP

EC(C

FU/w

ellX

104 )

P<0.05Human 10-mer, 42-mer reduce EPEC adherence

100%

43%

28%

75%

Milk Peptides

Larson et al. 2003 BBRC, 300, 531-540, Gardiner et al., in press

BIOCLA

Cell Factories which produce fatty acids:

Conjugated Linoleic Acid (CLA)

Linoleic Acid

cis-9, trans-11Most abundant – ruminant fats

Anti-cancerAnti-inflamatory

trans-10, cis-12Anti-obese

Increase lean muscle

trans-9, trans-11Anti-cancer

Catherine StantonRosaleen DeveryGer Fitzgerald

COOH

Linoleic acid

COOHt9, t11 CLA 20ug/ml

Summary of Bioactives Covered

Components with direct health associated bioactivityAntimicrobial peptidesAntihypertensive peptidesImmunoreactive componentsAntiproliferative Fatty Acids

Components which act through gut floraPrebiotics – bioactives which can influence gut flora compositionSubstrates for bioactive production

Catherine Stanton, Fergus ShanahanEamonn Quigley, Liam OMahony, Ger Fitzgerald

Food can programme gut floraand thereby affect health

Gut flora•Gut is home to 1-2 Kg ofbacteria (10 10-1011/g)

•600 different species•30-40% culturable

Vitamin synthesisDigestion andabsorptionImmune stimulationControl pathogens

Function

Control Formula 1 Formula 2 Formula 3

0 7 21 28 0 7 21 28 0 7 21 28 0 7 21 28Days

Faecalbifidobacteria

2.1X 2.3X2.5X* 2.5X*

PFGE21 days

N = 8

6

7

8

Log

cfu

/gfa

eces

0 7 21 28 0 7 21 28 0 7 21 28 0 7 21 28Days

* *

Dia

rrho

easc

ore

(day

7-14

)

11.21.41.61.8

22.22.42.62.8

3

control fucoidan laminarin laminarin/fucoidan

Laminarin *

Mixtures of laminarin and fucoidan reduceETEC associated-diarrhoea

John O Dougherty

B. bre

ve(R

)

B. bre

ve( C

)

B.long

um(E

)

B.lon

gum

(B)

B. bre

ve(H

)

B.long

um(I)

B. bre

ve(G

)

B.bre

ve(P

)

B.long

um(J)

B.lon

gum

(K)

B.lon

gum(L)

B.lon

gum

(D)

B.de

ntium

(M)

B. infan

tis(T

)

B. long

um(O

)

B. brev

e (Q)

B. long

um(F

)

B.lon

gum

(S)

B. cate

nulat

um(N

)

%Conversion

to CLA

Some species of bifidobacteria can make CLA

90

80

70

60

50

49

30

20

10

B. BREVE LINOLEIC ACID SUPERNATANT

0

20

40

60

80

100

120

8 13 18 23 28 33 38 43 48

Retention Time (Min)

Inte

nsity

(mV)

C13:0 Int. Std.

Linoleic Acid

CLA c9t11Oleic Acid

Stearic Acid

Trans Vaccenic

LA

CLA

GLC

Bifidobacterium breve

***

BALB/c mice SCID mice

*

Linoleic acid

Linoleic acid+

Bif. breve

Microbial-induced compositional change in liver fat

n = 9 n = 8

Gut bacteria changing foodcomponents into

Healthy bioactive substances in vivo

Luminal production bygut microbiota

Liver

Bioactive Production

Intestine

Bioacti

vePr

oduc

tion

Adipose

Bioac

tiveP ro

duction

Brain

Bioactive Production

Marine Food Component Pipeline

GutHealth

HeartHealth

HostProtection

WeightManagement

ProbioticFunction

Bioassay Development

Biomarker assessment in models/ human intervention

Product Development/Scale-up and Marketing

The Path to Functional Ingredients

The TeamChair

Professor ofMarine Functional Foods

Chair

Professor ofMarine Bioactives

Conclusions

• ?

1. Food and marine foods in particular contains awide range of bioactive substances which caninfluence human health – scientific basis for functional foods

2. Can have direct activity or signal complexcommunication systems – endocrine,neuronal, genetic

3. Gut flora is a complex bioreactor whosecomposition can affect human health – probiotics atransient bio/immuno reactor

4. Introduced flora can act on dietary (fatty acid)sources to produce bioactive substances

Hippocrates460-377 BC

“Let food be your medicine”

Paradigm shift – from Treatment to Prevention

• Today, our so-called “health systems” arein fact 90% illness systems. They spendalmost all their resources on treating ill-health, and only a small amount onpromoting good health. Imagine thereverse situation

David Byrne, former European

Commissioner for Health and

Consumer Protection

Traditionally the food industryhas been seen as part of theproblem relating to lifestylediseases – in the future theindustry wants to be part of

the solution

Algae Marine Discard Aquaculture Industry

The Raw Material

-Unique sources of health promoting fattyacids, diverse proteins and (prebiotic) complexcarbohydrates

Mining forBioactives

A1-45A1-54

AA1-45

A1-54A1-45

A1-54

A

Fractionation

Fractionation (size, charge, etc)

Fermentation (with gut microbes)

Hydrolysis (commercial enzymes)

XHypertension

BIOASSAYS

Catherine Stanton and Ger Fitzgerald

B-casein structure

Lactobacillus

ACE enzymeSequence, synthesise and confirm*SQSKVLPVPQ (10 aa peptide)**MPFPKYPVEP (10 aa peptide)*IGSENSEKTTMP (12 aa peptide)**EPVLGPVRGPFP (12 aa peptide)*NIPPLTQTPVVVPPFIQ (17 aa peptide)

*43*19*10

Heart Health and ACEACE - Angiotension Converting Enzyme- Key enzyme linked to increased blood pressure

Anti-ACEpeptide

Dick Fitzgerald

Mining forBioactives

A1-45A1-54

AA1-45

A1-54A1-45

A1-54

A

Fractionation

Fractionation (size, charge, etc)

Fermentation (with gut microbes)

Hydrolysis (commercial enzymes)

ImmuneFunction

BIOASSAYS

Liam O’Mahony and Barry Kiely

ECM

Cytoplasm

Nucleus

TNFR

T raf2

IKKα

IKKβ

NE

MO

IB

NFB

OtherTranscription

FactorsNFB-

Binding motif

NFB

•Inflammatory cytokines (IL-1,TNF-αetc)•Immune regulation•Survival•Proliferation

Th1

TNF-α

Th2Treg

Liam O’Mahony, APC

NFB-Downregulation