Nestlé Research Center Berries: Industry needs the science Gary Williamson.

Nestlé Research Center

Berries: Industry needs the science

Gary Williamson

Nestlé Research CenterApril 07 COST action on Berries2

Inaugurated in 1987

Nestlé Research Center in Lausanne

Basic knowledge in the food and life sciences

Application in the whole Nestlé Group

Part of the international scientific community


Phytochemicals, Phytonutrients

Flavanols

Hydroxycinnamates (e.g ferulic acid,

caffeic acid) Isoflavones (genistein, daidzein)

Flavonols (e.g. quercetin)

Anthocyanins

Catechins Pro(antho)cyanidins

Polyphenols

Flavanones, flavones

Flavonoids


Modified from Scalbert and Williamson 2000 J Nutr.

•Hertog (1993) 23 mg flavonols per day in the Dutch diet

•NEODIET 350 mg phenolic acids and 20 mg flavanones per day UK women 20-30 y

Food amount (g or ml) phenolic acids flavonols catechins proanthocyanidins flavanones anthocyaninspotato 100 14tomato 50 4 1lettuce 25 2 1onion 20 7apple 100 6 4 10 100cherry 50 37 1 3 17 200

wheat bran 10 50orange juice 100 22

dark chocolate 40 32 172red wine 125 12 2 34 45 4coffee 200 150

black tea 200 8 130total (mg) 275 24 209 334 22 204


0

2000

4000

6000

8000

10000

12000b

lack

be

rry

blu

eb

err

y

cho

keb

err

y

cra

nb

err

y

bla

ckcu

rra

nt

red

gra

pe

rasp

be

rry

stra

wb

err

y

Anthocyanins in berries (mg/kg fresh weight from Clifford, 2000)


Consumer Awareness

85% know the term antioxidant1

54% associate antioxidant with cancer prevention2

1 Mango Logic

2 IFIC 2002 Quantitative Consumer Study

Consumer Awareness


0

10

20

30

40

50

60

blac

kber

ry

redc

urra

nt

rasp

berr

y

stra

wbe

rry

oran

ge

blue

berr

y

red

plum

blac

k gr

ape

tang

erin

e

cher

ry

kiw

i

fig

pear

apric

ot

red

appl

e

whi

te g

rape

bana

na

wat

erm

elon

Antioxidant FRAP values (mol Fe2+/g FW) for fruits (Pellegrini et al 2003 J Nutr 133, 2812)

Nestlé Research CenterApril 07 COST action on Berries

Urine

Dietary polyphenols

Faeces


Cmax for flavonoids in humans(Manach et al 2005 AJCN)

0

0.5

1

1.5

2

2.5

da

idzi

n

ge

nis

tin

he

spe

ridin

na

ring

in

qu

erc

etin

glu

cosi

de

rutin EC

EG

C

EG

CG

an

tho

cya

nid

ins

pro

an

tho

cya

nid

ind

ime

rs

Cm

ax (

M)

isoflavones

catechinsflavonols

flavanones


Summary

Polyphenol composition of berries Antioxidant activity of berries always high in league tables –

this reflects high polyphenol content Industry often use in vitro activity from league tables: this is

not enough! Bioavailability varies for each class of polyphenols, so high

antioxidant actvity in vitro does not necessarily mean effective in vivo


Time

Stress event

Low doseBioactive

Low doseBioactive

Antioxidants are required during stress

Biomarker


Time

Stress event

Optimal doseBioactive

Optimal doseBioactive


Biomarker


Biomarker

Time

Stress event

High dose Bioactive

High doseBioactive



Dias et al 2005 Model of diabetes in rats (streptozotocin-induced)

plasma glucose (mM)

0

5

10

15

20

25

30

35

control quercetin diabetes diabetes plusquercetin

ns

0

2

4

6

8

10

12

14

16

control quercetin diabetes diabetesplus

quercetin

NF-kB activation(relative)

iNOS (relative)

P < 0.05


Some recent studies on cranberry juice in humans

Intervention design subjects main outcomes author date

Cranberry juice (250 ml), single dose at dinner

double-blind, randomized, placebo-controlled cross-over

20 healthy (10 males, 10 female)

E. coli uropathogenic strain showed less adhesion when tested ex vivo, no change in urinary pH Di Martino 2006

Cranberry juice 1 dose per day for 3 months

parallel, placebo controlled, randomized

Children (mean age 4.3 yr), 171 for cranberry, 170 for placebo

no effect on bacterial flora in nasopharynx or bacterial fatty acid composition of stools Kontiokari 2005

cranberry juice parallel 12 normal and 12 calcium oxalate stone formers

urinary calcium and urate increased, urinary pH decreased. Urinary and serum uric acid decreased Gettman 2005

cranberry juicedouble-blind, placebo controlled 376 older patients

Low infection rate than expected (5.6%) seen in all groups. Fewer infections in cranberry group p=0.027 McMurdo 2005

cranberry juice 500 ml for 90 days

double-blind, randomized, placebo-controlled

189 adults with Helicobacter infection aged 48.9 +/- 11.2 yr

Reduction in H. pylori infection in afflicted individuals (p<0.05) Zhang 2005


Summary

Antioxidants have an effect mainly to counteract “stress” We need more human studies on specific effects of berries

and berry components in humans. We already have many on cranberries and bacterial

adhesion, for example Best studies would use berries with high and low polyphenol

contents


Scientific evidence

Supporting Studies– Animal

– In vitro

– Genotype

– Modelling

Intervention Studies–

Observational Studies– Prospective (cohort)

Cross sectional

Case- control-

Supporting Studies–

– In vitro (cellular and molecular)

–

–

–

–

-

Hierarchy of scientific

evidence in support of claims on

foods



foods

Systematic reviews that include meta-analyses of appropriate intervention studies

Intervention StudiesRandomised controlled trials (healthy subjects)

Clinical trials (patients)

Physiological and psychological trials

Strong

Weak

Observational Studies

–

–

–

–

Leve

l of s

cien

tific

evi

denc

e

–

–

–

–

Supporting Studies– Animal

– In vitro

– Genotype

– Modelling

Intervention Studies–

Observational Studies– Prospective (cohort)

Cross sectional

Case- control-

Supporting Studies–

– In vitro (cellular and molecular)

–

–

–

–

-



foods



foods



foods



foods

Systematic reviews that include meta-analyses of appropriate intervention studies

Intervention StudiesRandomised controlled trials (healthy subjects)

Clinical trials (patients)

Physiological and psychological trials

Strong

Weak

Observational Studies

–

–

–

–

Leve

l of s

cien

tific

evi

denc

e

–

–

–

–

Less

strong


The key to a good experiment on flavonoids is the quality of the analytical methods!

20.0 25.0 30.0 35.0

0

200

400

600

800

Retention time (minutes)

Res

pons

e (n

A)

[480 mV]

[400 mV]

[310 mV]

[230 mV]

[150 mV]

[70 mV]

[-10 mV]

[-90 mV]

20.0 25.0 30.0 35.0

0

200

400

600

800


Res

pons

e (n

A)

[480 mV]

[400 mV]

[310 mV]

Ferulic acid 29.2m

[230 mV]

Catechin 18.0m

ECg 32.2m

[150 mV]

EGC 17.1m

EC 21.3m

EGCg 24.5m

[70 mV]

[-10 mV]

[-90 mV]

20.0 25.0 30.0 35.0

0

200

400

600

800


Res

pons

e (n

A)

[480 mV]

[400 mV]

[310 mV]

[230 mV]

[150 mV]

[70 mV]

[-10 mV]

[-90 mV]

20.0 25.0 30.0 35.0

0

200

400

600

800


Res

pons

e (n

A)

[480 mV]

[400 mV]

[310 mV]

[230 mV]

[150 mV]

[70 mV]

[-10 mV]

[-90 mV]

20.0 25.0 30.0 35.0

0

200

400

600

800


Res

pons

e (n

A)

[480 mV]

[400 mV]

[310 mV]

Ferulic acid 29.2m

[230 mV]

Catechin 18.0m

ECg 32.2m

[150 mV]

EGC 17.1m

EC 21.3m

EGCg 24.5m

[70 mV]

[-10 mV]

[-90 mV]

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.5

8618

.908

21.1

77

21.5

10

25.4

07

28.7

99

29.8

93

30.2

83

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.5

7818

.885

21.1

29

21.4

8221

.740

22.0

1922

.300

24.0

80

25.3

63

29.8

29

30.2

13

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.4

9518

.802 21

.051

21.4

1021

.670

21.9

5622

.237

23.8

77

25.2

49

29.6

70

30.0

41

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.5

8618

.908

21.1

77

21.5

10

25.4

07

28.7

99

29.8

93

30.2

83

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.5

7818

.885

21.1

29

21.4

8221

.740

22.0

1922

.300

24.0

80

25.3

63

29.8

29

30.2

13

min16 18 20 22 24 26 28 30 32 34

mAU

0

25

50

75

100

125

150

18.4

9518

.802 21

.051

21.4

1021

.670

21.9

5622

.237

23.8

77

25.2

49

29.6

70

30.0

41

MET “Plasma bioavailability of hesperetin conjugates ”LC-MS analysis of final treatment 2: 0.30 mg hp-7-rut & 3.55 mg Hp-7-glc per g freeze-dried product.Rt 9.64 min: Hesperidin. Rt 13.56min: Hesperetin-7-glucoside.

DAD

Single ion monitoring: 301m/z (hp)

Single ion monitoring: 463m/z (hp-7-glc)


Scan 200-800m/z

Total single ion monitoring: 609, 463, 301m/z

MET “Plasma bioavailability of hesperetin conjugates ”LC-MS analysis of final treatment 2: 0.30 mg hp-7-rut & 3.55 mg Hp-7-glc per g freeze-dried product.Rt 9.64 min: Hesperidin. Rt 13.56min: Hesperetin-7-glucoside.

DAD

Single ion monitoring: 301m/z (hp)



Scan 200-800m/z

Total single ion monitoring: 609, 463, 301m/z


Functional foods

FDA used the Institute of Food Technologists (IFT)'s definition of functional foods as: "foods and food components that provide a health benefit beyond basic nutrition". These include: conventional, fortified, enriched and enhanced foods


Summary

Human studies are needed for regulatory issues within the EU

Future “hard” claims will need high quality human studies on effects of berries

Ultimately, industry like claims – but these must be backed up with human studies

Will new products be “Functional Food”?


Functional foods or not?

Basic: berries or extract with known benefits added to a product and the levels of active compound measured in the product

Advanced: berries or extract with known benefits added to a product and the resulting bioavailability of the active compounds measured in humans

Ideal: berries or extract with known benefits added to a product and the resulting bioefficacy of the product measured in humans


Dose-response – the key!

response

dose

optimum

dose

broadoptimum

Distinct separation between the chemopreventative and toxicdose ranges

Desirable properties of anticancer agents

Adverse response

Best effect on health at optimum levels



response

dose

optimum

dose

broadoptimum



Adverse response


Higher risk of chronic disease at“deficiency level”

Higher risk of chronic disease at“excess intake” – in most cases not

possible from food intake alone



response

dose

optimum

dose

broadoptimum



Adverse response


Normal dietarylevels for most

of the population






response

dose

optimum

dose

broadoptimum



Adverse response





Desired dietarylevels for best health benefit


Nutrient Bioavailability Group at NRC: September 2006

Nestlé Research Center Berries: Industry needs the science Gary Williamson.

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