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Investigating in vitro digestion of food models to explainin vivo micronutrient bioavailability

Manon Hiolle, Beatrice Gleize, N. Meunier, B. Pereira, R. Richard, IsabelleSavary-Auzeloux, Marie-Agnès Peyron, Catherine Veyrat, Olivia Ménard,

Caroline Buffière, et al.

To cite this version:Manon Hiolle, Beatrice Gleize, N. Meunier, B. Pereira, R. Richard, et al.. Investigating in vitrodigestion of food models to explain in vivo micronutrient bioavailability. 6.International Conferenceon Food Digestion, Apr 2019, Grenade, Spain. 2019. �hal-02100429�

Investigating in vitro digestion of food modelsto explain in vivo micronutrient bioavailability

Speaker : Manon Hiolle

Nutritional quality : beyond food composition

2

FOOD

Blo

od

stream

DIGESTION :Complex processing of

food disintegrationfor delivery of nutrients

Interactions

A composition A structure

« Food, not nutrients, is the fundamentalunit in nutrition »

Jacobs & Tapsell (2007)

3

Nutritional quality : beyond food composition

Food structure

Metabolic fate of

nutrients

Models micronutrients : - vitamins B9 and B12- vitamin D and Lutein

Bioaccessibility

Metabolic responses

Kinetics

Bioavailaibility

Macronutrients : ☑

Micronutrients : composition effectversus structure effect ?

4

Strategy : clinical study conduct

Four productswithidenticalcomposition

Custard

Pudding

Sponge cake

Biscuit

Water content

+

-

Clinical study

• 12 healthy male subjects• 20 to 30 years old• Cross-over experimental design

Enriched with the fourmicronutrients studied

• 8 hours postprandial follow-up

• Hourly blood samples and plasma separation by centrifugation

Bioavailability

• Absorption kinetics: quantitative analysis of micronutrients in plasma or chylomicrons

Liquid

Soft gel

Foamy solid

Hard solid Results for B9 : poster n°378/214, session 2

Products characterization• Microstructure : confocal microscopy• Macrostructure : texture analysis

5

Strategy : development of food models

Matrices Biscuit Sponge cake Pudding Custard

Photo

Water content 6 % 38 % 48 % 68 %

Heatingtreatment

180 °C/18 min 180 °C/30 min 180 °C/20 min110 °C/20 min

80 °C/7 min

Portion size 135 g 206 g 242 g 512 g

Same composition on dry matter :

- 17 % proteins- 52 % carbohydrates- 30 % lipids

: lipids (Red Nile): proteins (Fast Green)

Enriched in (per portion) : - Lutein : 20 mg- Vitamin D : 1,25 mg- B9 : 1 mg- B12 : 12 µg

6

Results - clinical study

0

5

10

15

20

25

30

35

40

45

50

0 60 120 180 240 300 360 420 480

Dco

nte

nt

of

lute

in (

nm

ol/

L)

Time after meal ingestion (min)

Mean ± SEM

0

2

4

6

8

10

12

0 60 120 180 240 300 360 420 480

Dco

nte

nt

of

vita

min

D3

(nm

ol/

L)

Time after meal ingestion (min)

Mean ± SEM

→ Extraction of 2 parameters from the curves : - TAmax : Time of

maximal absorption

- AUC : Area under the curve

Lutein Vitamin D

7

Results - clinical study

Later TAmax for harder texture

0

50

100

150

200

250

300

350

TAm

ax (

min

)

Time of maximal concentration

a

b b b

a

b bb

0

200

400

600

800

1000

1200

1400

1600

Biscuit Spongecake

Pudding Custard

AU

C

Area under the curve

Hardness

+-

aa,b

a,b

b

a,b

a b

a

Hardness+

-

Vitamin D Lutein

Tendency to higher total bioavailability for softer matrices

8

Research question and strategy

Food structure impacts kinetics of matrix disintegration during digestion

Can we explain in vivo digestion mechanismsby using an in vitro digestion model ?

Following INFOGEST in vitro protocol with special conditions : Use of human saliva in oral phase (mix 1:1) Constant water content between matrices

(difference adjustment with water)

Focus point : Matrix disintegration

ProteolysisLipolysisAmylolysis

Release of micronutrients (lutein and vitamin B9)

(Minekus, 2014)

Digestion kinetics

(S0, G0, G30, G120, I0, I30, I120)

9

Principal component analysis – graph of variables

-1.0 -0.5 0.0 0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

ACP n°1 : Graphe des variables

Dim 1 (59.98%)

Dim

2 (

12

.29

%)

GlcLip

LutFA

Brad

AireTot

MKJ

OPA

Classe1

Classe2 Classe3

Classe4

Classe5

Classe6

Classe7Glyc

TAG

AGL

CHOL

DAG

MAG

PL

LipTot

Temps

Axis n°2 : Solubilization of macronutrients

in the supernatant

Axis n°1 : Hydrolysis of

macronutrients

Axis n°1 : - Low molecular weight peptides (< 25 kDa) and free

amino group content- FFA, MAG and DAG- Carbohydrates less than 10 glucose units equivalent

→ Hydrolysis of macronutrients

Axis n°2 : - High molecular weight peptides (> 100 kDa)- Triglycerides and total lipids

→ Solubilization of macronutrients in the supernatant

PCA = Principal Component Analysis

10

Principal component analysis – map of individualsAxis n°2 :

Solubilization of macronutrients in the supernatant

Axis n°1 : Hydrolysis of

macronutrients

-6 -4 -2 0 2 4 6 8

-50

51

0

ACP n°1 : Graphe des individus

Dim 1 (59.98%)

Dim

2 (

12

.29

%)

22

36

57

23

3758

24

38

59

25

3960

26

4061

27

41

62 28

42

63

1

50

712

51

72

352

73

4

5374 554

75 6

5576 7

56

77

15

43

64

16

4465

17

4566

1846

67

19

47 68

2048

69

2149 70

8

29

78

9

30

79

10

31

801132

81

12

33

82

133483 14

3584

BCA

F

G

G0

G120

G30 I0

I120

I30S0

BCA

FG

1°) Grouped by matrix : no matrix effect highlighted

2°) Grouped by digestion time : evidence of a strong "digestion time" effect on individuals→ Samples split according to digestion time

Oral phase

Gastric phase

Intestinal phase

11

-4 -2 0 2 4 6

-4-2

02

4

Dim 1 (49.30%)

Dim

2 (

28

.44

%)

B.S0

B.G0

B.G30

B.G120B.I0

B.I30

B.I120

CA.S0

CA.G0CA.G30

CA.G120

CA.I0CA.I30

CA.I120

F.S0

F.G0

F.G30

F.G120

F.I0

F.I30

F.I120

G.S0

G.G0

G.G30G.G120

G.I0G.I30

G.I120

B

CA

F

G

S0G0

G30G120

I0I30I120

Multiple factor analysis – map of individuals

Axis n°2 : Solubilization of macronutrients

in the supernatant

Axis n°1 : Hydrolysis of

macronutrients

Matrix effect highlighted by this analysis- Axis n°1 isolates biscuit- The 3 softer matrices are separated along the axis n°2

Focus on custard behavior during in vitro digestion :

- Oral phase : macronutrients solubilized in the supernatant

- Gastric phase : drop in pH leads to macronutrients aggregation

- Intestinal phase : re-solubilization and hydrolysis of macronutrients

Water content

Heat treatment

-4 -2 0 2 4 6

-4-2

02

4

Dim 1 (49.30%)

Dim

2 (

28

.44

%)

B.S0

B.G0

B.G30

B.G120B.I0

B.I30

B.I120

CA.S0

CA.G0CA.G30

CA.G120

CA.I0CA.I30

CA.I120

F.S0

F.G0

F.G30

F.G120

F.I0

F.I30

F.I120

G.S0

G.G0

G.G30G.G120

G.I0G.I30

G.I120

B

CA

F

G

S0G0

G30G120

I0I30I120

12

AGL > 100 kDa 50 kDa 25 kDa 10 kDa 5 kDa 1 kDa < 1 kDa DAG B9 Glc Glyc Lip Lut MAG MKJ OPA TAG

AGL 0.1752 0.0505 0.6405 <.0001 <.0001 <.0001 <.0001 <.0001 0.0131 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

> 100 kDa 0.1752 0.7055 0.4193 0.2575 0.1624 0.2762 0.5820 0.1630 0.0032 0.0627 0.4111 0.3951 0.0758 0.2104 0.5493 0.1655 0.0103

50 kDa 0.0505 0.7055 <.0001 0.2164 0.6387 <.0001 <.0001 0.0923 0.6511 0.1283 0.0338 0.0200 0.1880 0.1097 0.0260 0.0166 0.0162

25 kDa 0.6405 0.4193 <.0001 0.4850 0.0662 0.0197 0.0117 0.9289 0.5243 0.9186 0.7120 0.2819 0.6995 0.8595 0.6308 0.4731 0.4055

10 kDa <.0001 0.2575 0.2164 0.4850 <.0001 <.0001 <.0001 <.0001 0.1846 <.0001 <.0001 0.0002 <.0001 0.0007 <.0001 <.0001 0.0057

5 kDa <.0001 0.1624 0.6387 0.0662 <.0001 <.0001 <.0001 <.0001 0.2743 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0002

1 kDa <.0001 0.2762 <.0001 0.0197 <.0001 <.0001 <.0001 <.0001 0.3762 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

< 1 kDa <.0001 0.5820 <.0001 0.0117 <.0001 <.0001 <.0001 <.0001 0.0172 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

DAG <.0001 0.1630 0.0923 0.9289 <.0001 <.0001 <.0001 <.0001 0.0074 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

B9 0.0131 0.0032 0.6511 0.5243 0.1846 0.2743 0.3762 0.0172 0.0074 0.0278 0.0240 0.0325 0.1914 0.0385 0.0308 0.0034 0.0017

Glc <.0001 0.0627 0.1283 0.9186 <.0001 <.0001 <.0001 <.0001 <.0001 0.0278 <.0001 <.0001 0.0001 <.0001 <.0001 <.0001 <.0001

Glyc <.0001 0.4111 0.0338 0.7120 <.0001 <.0001 <.0001 <.0001 <.0001 0.0240 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

Lip <.0001 0.3951 0.0200 0.2819 0.0002 <.0001 <.0001 <.0001 <.0001 0.0325 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

Lut <.0001 0.0758 0.1880 0.6995 <.0001 <.0001 <.0001 <.0001 <.0001 0.1914 0.0001 <.0001 <.0001 0.0005 <.0001 <.0001 0.1704

MAG <.0001 0.2104 0.1097 0.8595 0.0007 <.0001 <.0001 <.0001 <.0001 0.0385 <.0001 <.0001 <.0001 0.0005 <.0001 <.0001 <.0001

MKJ <.0001 0.5493 0.0260 0.6308 <.0001 <.0001 <.0001 <.0001 <.0001 0.0308 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

OPA <.0001 0.1655 0.0166 0.4731 <.0001 <.0001 <.0001 <.0001 <.0001 0.0034 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

TAG <.0001 0.0103 0.0162 0.4055 0.0057 0.0002 <.0001 <.0001 <.0001 0.0017 <.0001 <.0001 <.0001 0.1704 <.0001 <.0001 <.0001

Matrix of Pearson correlations – p-values

13

Matrix of Pearson correlations – p-values

Vitamin B9 correlates with : - Solubilization of macronutrients (TAG, total lipids,

high weight class of proteins)- Markers of matrix disintegration (lipolysis,

proteolysis, amylolysis)

Lutein correlates with : - Markers of matrix disintegration

(lipolysis, proteolysis, amylolysis)

Hydrolysis of all macronutrient classes has an impact on micronutrients release from the matrix

→ The key factor of micronutrients bioaccessibility seems to be matrix disintegration

TAGTotallipids

FFA DAG MAGFFA/

tot lipTotal

nitrogen> 100 kDa

50 kDa 25 kDa 10 kDa 5 kDa 1 kDa < 1 kDaFree

aminoGlc B9 Lut

B9 0.0017 0.0240 0.0131 0.0074 0.0385 0.0325 0.0308 0.0032 0.6511 0.5243 0.1846 0.2743 0.3762 0.0172 0.0034 0.0278 0.1914

Lut 0.1704 <.0001 <.0001 <.0001 0.0005 <.0001 <.0001 0.0758 0.1880 0.6995 <.0001 <.0001 <.0001 <.0001 <.0001 0.0001 0.1914

Lipolysis Proteolysis Amylolysis

Lipids in supernatant

Proteins in supernatant

14

Lutein is released continuously during digestion → driven by proteolysis ?

Release of B9 mainly during gastric step: impacted by solubilization of macronutrient

Drop in B9 bioaccessibility in intestinal phase : low solubility at neutral pH

0

50

100

150

200

250

0

20

40

60

80

100

120

140

B9

bio

acce

ssib

ility

(µ

g)

Lute

in b

ioac

cess

ibili

ty (

%)

LuteinB9

S0 G0 G30 G120 I0 I30 I120

Oral phasepH 6,5

amylase

Gastric phasepH 3

protease

Intestinal phasepH 7

proteaselipase

amylase

15

Conclusion and perspectives

Is the structure solely involved in bioavailability differences observed in vivo?

→ In vitro dynamic study using the DIDGI to better mimic physiological responses during digestion (gastric emptying, progressive addition of enzymes, etc.)

Food structure impacts kinetics of matrix disintegration during digestion

Food structure has an impact on matrix behaviour during in vitro digestion

Béatrice Gleize

Catherine Caris-VeyratEmmanuelle Reboul

Isabelle Savary-Auzeloux

Caroline Buffière

Nathalie Meunier

Ruddy Richard

Bruno Pereira

Françoise Nau

Didier Dupont

Olivia Menard

Nathalie Boulier-Monthean

Jordane Ossemond

Thank you for your

attention

If you want more information :

manon.hiolle@inra.fr

Poster n°378/214, session 2