Modifications structurales des viandes au cours des transformations et conséquences sur la bioaccessibilité des solutions digestives au sein de l'aliment

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Modifications structurales des viandes au cours des transformations et conséquences sur la bioaccessibilité

des solutions digestives au sein de l’aliment

Thierry Astruc

To cite this version:

Thierry Astruc. Modifications structurales des viandes au cours des transformations et conséquences sur la bioaccessibilité des solutions digestives au sein de l’aliment. Metaseminaire CEPIA-modaltub- 2013, Mar 2013, Paris, France. 24 p. �hal-01601299�

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Modifications structurales des viandes au cours des

transformations et conséquences sur la bioaccessibilité des solutions digestives au sein de l'aliment.

Thierry Astruc

Unité « Qualité des Produits Animaux » INRA de Clermont-Ferrand-Theix

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Séminaire Modaltub 21&22 Mars 2013

Yellow: Muscle fibre Red: connective tissue

Intracellular proteins (90 %)

45 % essential aminoacids

Connective tissue (Extracellular Matrix ;10 %) Poor Nutritional value

Muscle (meat) composition 75% water 19% proteins 3% lipids

& glucides, µnutriments …

(4)

What is the effect of the structural organization on the bioaccessibility of digestive enzymes to myofibrillar protein of raw and processed meat ???

Muscle Fiber

(High nutritional value)

Fascicle of muscle fibers

Pepsin Pepsin

Muscle Fiber ???

???

(5)

Material and Methods

(6)

Semi-tendinosus (ST)

D1 postmortem

Control No processed

Control (no digestion)

Pepsin 2 H

Pepsin + Trypsin+

Chymotrypsin 2 H Marinating

pH 4.5 24 H

Heating 60°C 45 min

Heating 90°C 45 min

pH 4.5 + 60°C 45 min

pH 4.5 + 90°C 45 min D12 postmortem

(controlateral)

Infraspinatus (IS)

Experimental Design

Charolais cow, 7 years old

Bloc (1x1x1.5) cm

ST

IS

Pepsin 125 U/mg proteins in pH 1.8 glycine buffer, trypsin (150 U/mg proteins) and chymotrypsin (0.1 U/mg proteins) in a pH 8 glycine buffer

(7)

Quantification of Peptides

from meat digestion

Morphology quantification

Fluorochrome

Antigen

Histochemistry Muscle fibers collagen stainings

Immunohistofluorescence

First antibody Second antibody

Protein localisation

Antigen degradation

Biochemistry

(8)

Results

Effects of process on meat structure

(Meat storage & cooking)

Effects of process on digestive solution bio-accessibility

(9)

Postmortem changes during meat storage

1 day postmortem (D1)

For each condition, measurement on 30 images, about 3000 cells

100 µm

50 µm Fi

Fi

ECS

50 µm Fi Fi Fi

ECS

**

Extracellular spaces

0 20 40 60 80 100 120

IS ST

D1 D12

1012days postmortem (D12)³µm²

0 5 10 15 20

IS ST

D1 D12

0 5 10 15 20

IS ST

D1 D12

**

fiber area 10³µm²

0 20 40 60 80 100 120

IS ST

D1 D12

0 20 40 60 80 100 120

IS ST

D1 D12 Extracellular

spaces area 10³µm²

**

Extracellular spaces

0 20 40 60 80 100 120

IS ST

D1 D12

10³µm²

1 µm Fi Fi

100 µm

Fi

(10)

45 min heating 60 °C

Cooking (ST muscle)

100 µm 100 µm

0 2 4 6 8 10 12 14

raw 60°C 90°C

Fibers area

0 50 100 150 200 250 300 350

raw 60°C 90°C

Extracellular spaces

10³µm² 10³µm²

Heating shrinks the muscle fibers and increase the extracellular spaces

(11)

Séminaire Modaltub 21&22 Mars 2013 Cooking effect on connective tissue (ST D1, 60°C)

200 µm 200 µm

collagen laminin

exposure time X 4 /raw muscle

Partial heat denaturation of proteins of the extracellular matrix that remain visible and well localized

Immunohistofluorescence Sirius red staining Muscle fibers : yellow Collagen : red

(12)

Technological treatments affect meat microstructure - Extracellular matrix desorganisation

(ageing, marination, cooking) - Increase in extracellular spaces (cooking)

Which consequences on

- digestive enzyme accessibility ? - meat digestion ?

Pepsin Trypsin Chymotrypsin

(13)

STJ1m0c0D1 2 mm

Pepsin digestion of raw ST muscle

0.5 mm

Formation of a clearer « digestive halo » in the sample periphery

0 5 10 15 20 25

edge center

Fiber area

(14)

Effect of enzymatic digestion on muscle fibers structure and ultrastructure

Control Pepsin Pepsin+Trypsin+Chymotrypsin

50 µm Fi

Fi

Fi Fi

50 µm

Enlargement of postmortem proteolysis break

Cooperative phenomenon?

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

OD 280 nm Fi

Fi Fi

50 µm

(15)

Séminaire Modaltub 21&22 Mars 2013 pepsin digestion of 60°C heated ST muscle

STJ1m0c1D1 2 mm

Swelling of the muscle fibers in the halo. Cumulative effect of low pH and pepsine digestion of extracellular matrix

(16)

2 mm

Peptides from digestion Migration of digestion juices

pepsin pepsin +trypsin +chymotrypsin

raw 60°C 90°C (OD 280 nm)

0 0.5 1

90°C raw 60°C

(mm)

0 1 2

(17)

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

raw marinated 60°C marinated

& 60°C

90°C marinated

& 90°C Process

Peptides in the digestion solution

more aggressive is the process, lower is the digestion

OD 280 nm

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

ST IS Muscle

OD 280 nm

(18)

MALDI-TOF mass spectra of ST muscle after pepsin digestion (same profile for IS muscle)

Raw Marinated

90°C Marinated

& heated 90°C

1433.7 1433.7

1828 1828

1018.4 1018.4

(19)

Séminaire Modaltub 21&22 Mars 2013 HES staining

2 mm

Lost of laminin antigenicity in the halo

 alteration of ECM by the pepsin solution Pepsin digested raw & marinated ST muscle

Immunohistofluorescence of laminin

(20)

200 µm

1 mm 20 µm

Pepsin digested raw ST muscle

Double labelling of collagen (green) and myosin (red)

Decrease of myosin antigenicity in cells of the halo, suggesting a pepsin penetration in the intracellular compartment.

(21)

Séminaire Modaltub 21&22 Mars 2013 Pepsin digested heated 60°C ST muscle

Double lebelling of collagen (green) and myosin (red)

Large swelling of cells and decrease of myosin antigenicity in the halo

Board Middle

100 µm 100 µm

(22)

HES Sirius red laminin pepsin

2 mm

Pepsin is detected in the halo but seems to be more located in the connective tissue Pepsin digested sample of marinated ST muscle

(23)

Low pepsin content initially ?

extraction in the washing baths?

bright spots only visible in the halo of digestion

Cy3

pepsin

Anti-pepsin From rabbit Anti-rabbit

(24)

Conclusions

Postmortem time

D12>D1 : endogen proteolysis increased the digestive enzyme accessibility and digestion process

Process

change the tissue structure.

But decrease

- digestive accessibility of enzymes (halo) - level of digestion (OD 280 nm)

Swelling of cells in the halo Access more difficult ?

Changes in molecular structure of proteins Recognition of cleavage sites of enzymes ?

(25)

Perspectives

Improvement of the pepsin labelling

Identification of the peptides found in the digestion solution Chewing effect ?

Fundings ANR project

« pronutrial »

Labellisation of trypsin & chymotrypsin

Peristaltic contractions ?

Conférence FSDH (New Zealand)

Échanges avec M. Boland et L. Kaur (Riddet Institute)

Chapitre d’ouvrage FSDH (soumis)

Programme Hubert Curien (2013-2014)

Astruc, Santé-Loutellier, Kaur, Boland