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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
Nestlé Research CenterApril 07 COST action on Berries3
Nestlé Research CenterApril 07 COST action on Berries4
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
Nestlé Research CenterApril 07 COST action on Berries5
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
Nestlé Research CenterApril 07 COST action on Berries6
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)
Nestlé Research CenterApril 07 COST action on Berries7
Consumer Awareness
85% know the term antioxidant1
54% associate antioxidant with cancer prevention2
1 Mango Logic
2 IFIC 2002 Quantitative Consumer Study
Consumer Awareness
Nestlé Research CenterApril 07 COST action on Berries8
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
Nestlé Research CenterApril 07 COST action on Berries10
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
Nestlé Research CenterApril 07 COST action on Berries11
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
Nestlé Research CenterApril 07 COST action on Berries12
Time
Stress event
Low doseBioactive
Low doseBioactive
Antioxidants are required during stress
Biomarker
Nestlé Research CenterApril 07 COST action on Berries13
Time
Stress event
Optimal doseBioactive
Optimal doseBioactive
Antioxidants are required during stress
Biomarker
Nestlé Research CenterApril 07 COST action on Berries14
Biomarker
Time
Stress event
High dose Bioactive
High doseBioactive
Antioxidants are required during stress
Nestlé Research CenterApril 07 COST action on Berries15
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
Nestlé Research CenterApril 07 COST action on Berries16
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
Nestlé Research CenterApril 07 COST action on Berries17
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
Nestlé Research CenterApril 07 COST action on Berries18
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
Hierarchy of scientific
evidence in support of claims on
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)
–
–
–
–
-
Hierarchy of scientific
evidence in support of claims on
foods
Hierarchy of scientific
evidence in support of claims on
foods
Hierarchy of scientific
evidence in support of claims on
foods
Hierarchy of scientific
evidence in support of claims on
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
Nestlé Research CenterApril 07 COST action on Berries19
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
Retention time (minutes)
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
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
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
Retention time (minutes)
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)
Single ion monitoring: 609m/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)
Single ion monitoring: 463m/z (hp-7-glc)
Single ion monitoring: 609m/z (hp-7-glc)
Scan 200-800m/z
Total single ion monitoring: 609, 463, 301m/z
Nestlé Research CenterApril 07 COST action on Berries20
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
Nestlé Research CenterApril 07 COST action on Berries21
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”?
Nestlé Research CenterApril 07 COST action on Berries22
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
Nestlé Research CenterApril 07 COST action on Berries
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
Nestlé Research CenterApril 07 COST action on Berries
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
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
Nestlé Research CenterApril 07 COST action on Berries
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
Normal dietarylevels for most
of the population
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
Nestlé Research CenterApril 07 COST action on Berries
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
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
Desired dietarylevels for best health benefit
Nestlé Research CenterApril 07 COST action on Berries27
Nutrient Bioavailability Group at NRC: September 2006