Multimodal and multispectral imaging approaches to study the diffusion of pepsin during in vitro digestion of meat Thierry Astruc 1 *, Annie Vénien 1, Olivier Loison 1, Laetitia Théron 1, Christophe Chambon 1, Hua Ding 1, Lara Barotti 1, Michiyo Motoyama 1,2, Delphine Pavon 3, Feng Ming Chian, Mike Boland, Lovedeep Kaur 1 INRA de Theix, UR37 QuaPA, F-63122 St-Genès-Champanelle, France 2 Institute of Livestock and Grassland Science, NARO, Tsukuba, 35-91, Japan 3 Science et Surface, F-6913 Ecully, France Massey University, Riddet Institute, Palmerston North, New Zealand Thierry Astruc, INRA Auvergne-Rhône-Alpes, 63122 Saint-Genès-Champanelle, France Health, Dalian, China, May 26 th - 2 th, 217
Meat Qualities Nutritional quality Health quality Microbiology Contaminants New formed products Composition essential aminoacids Vitamin Fatty acids Organoleptic quality Colour, Texture Jutosity, Tenderness Muscle Fiber Pepsin??? Muscle Fiber (High nutritional value) Fascicle of muscle fibers Technological quality (processing properties)??? Pepsin Cooking yield Cutting yield Emulsifying properties Bioaccessibility of digestive enzymes to the muscle food? Health, Dalian, China, May 26 th - 2 th, 217
In vitro gastric digestion Scientific procedure Cryofixation (-16 C) Cryosections (6-1 µm depth) 2 hours, 37 C Solution of pepsin 125U/mg of protein, ph 1. Effect of the digestion juice on meat structure and pepsin detection Tissues/cells Cells/molecules Molecules/chemistry Element Optical microscopy Histochemistry Optical microscopy immunohistochemistry Microspectroscopies Chemical imaging (IR, UV, Raman, TOF SIMS) Microspectroscopies Chemical imaging Fluorescence X Microanalyse X ToF-SIMS
Pepsin digestion of raw ST muscle Tissue level 25 2 Fiber area 15 1 5 edge center STJ1mcD1 2 mm Formation of a clearer «digestive halo» in the sample periphery.5 mm Astruc T., 21. Muscle structure and digestive enzyme bio-accessibility to intracellular compartment. In: Boland M. J., Golding M. and Singh H editors-in-chief. Food Structure Digestion and Health, Elsevier edition; pp. 193-222.
pepsin digestion of 6 C heated ST muscle Tissue level Pepsin ph 1. STJ1mc1D1 2 mm Swelling of the muscle fibers in the halo. Cumulative effect of low ph and pepsine digestion of extracellular matrix. Clogging disturbs transfer of digestive solution Astruc et al. 212, Food Structure Digestion and health congress, Palmerston North, New Zealand Health, Dalian, China, May 26 th - 2 th, 217
Pepsin digested raw ST muscle Macromolecular level HES staining Immunohistofluorescence of laminin Cy3 Secondary antibody Anti-laminin Laminin Cy3 Secondary antibody Anti-laminin 2 mm Laminin Lost of laminin antigenicity in the halo alteration of ECM by the digestion solution Health, Dalian, China, May 26 th - 2 th, 217
Infrared microspectroscopy on label free section : principle Macromolecular level +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ IN1 Thermo 7 spectra (2 hours acquisition) 3 µm 5 µm MIR cm-1 cm-1
protein macromolecular structure reflected by 165 cm -1 absorbance assigned to a-helices Protein unfolding Macromolecular level Motoyama et al. (in preparation)
Health, Dalian, China, May 26 th - 2 th, 217
MALDI target Maldi-Tof : Matrix Assisted Laser Desorption Ionization - Time of Flight Laser UV START STOP + + + Detector U (kv) G.U MS spectrum Source Acceleration area Analyser (TOF) Free field area Principle of time-of-flight analyzer: The travel time of the ions in the tube depends only on their mass. The lighter ions will reach the detector before the heaviest ones. m/z The measurement of time makes it possible to calculate the m / z and therefore the mass of the ions. Health, Dalian, China, May 26 th - 2 th, 217
11 Mass Spectrometry Imaging: MSI To mass spectrometer (TOF.) + + + + Matrix Analytes Matrix Tissue section Glass slide Health, Dalian, China, May 26 th - 2 th, 217
Pepsin digestion In situ proteolysis characterization 37 C 2 hours Solution of pepsin 125 U/mg of protein. Tissue structure Protein antigenicity 1 µm cryosections Cryo-fixed digested meat sample Proteines peptides maps linear mode from m/z 2, to 3, ; spatial resolution of 1 µm. Normalization and extraction of spectra from regions of interest using FlexImaging software (3., Bruker Daltonics). Health, Dalian, China, May 26 th - 2 th, 217
Maldi-ToF mapping of a digested sample cryosection Pepsin digestion Macromolecular level Health, Dalian, China, May 26 th - 2 th, 217
Molecular imaging of proteins Molecular imaging of peptides More m/z 152 peptides in the digested part. Less m/z 26 proteins in the digested part. Molecular imaging of peptides Co-register image of immuno-labeling of laminin Théron et al. (21). Meat proteolysis by pepsin highlighted by maldi imaging mass spectrometry. In: The 6th International Congress of Meat Science & Technology (ICoMST); 21 August 17-22; Punta Del Este, Uruguay, Archivos Latinoamericanos de Produccion Animal, 22 (5): 535-537. Boland et al. 21. Digestion and digestibility of meat: an investigation of the effects of processing and preparation. NZIFST Conference, 1st - 3rd July, Christchurch, New Zealand Health, Dalian, China, May 26 th - 2 th, 217
Until now, vizualisation of the effects of digestion juice (pepsin + ph) on meat structure Is it possible to detect pepsin directly on digested meat sections? Health, Dalian, China, May 26 th - 2 th, 217
The most useful technique to localize a protein in a biological tissue : immunohistochemistry Anti-pepsin primary antibody Wash to remove excess of anti-pepsin Cy3 fluorescent dye - secondary antibody Observation Image acquisition Wash to remove excess of secondary antibody Health, Dalian, China, May 26 th - 2 th, 217
bright spots only visible in the halo of digestion Cy3 Anti-rabbit rabbit Anti-pepsin pepsin Low pepsin content initially? extraction in the September, washing 23, 215 baths?
Labelling of pepsin with a dye which remain stable at ph 2 : Bodipy dye Pepsin- Pepsin activity? Biochemical analysis Loss of pepsin activity Steric hindrance? Pepsin aggregation BODIPY TR-X NHS ester 63 gmol-1 327 aa, 35 kd (Tang et al. 1973) Health, Dalian, China, May 26 th - 2 th, 217
Label free pepsin detection by ToF-SIMS imaging Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) provides elemental, chemical state, and molecular information from surfaces of solid materials. Digested sample ToF-SIMS V (ION-TOF GmBH, Munster, Germany) 1 µm cryosections Mass spectroscopy mapping Bismuth gun 25 kv (source Bi 3 ) Science et Surface laboratory, Ecully, France Health, Dalian, China, May 26 th - 2 th, 217
15 Protein map 15 Triglyceride map 1 13 5 13 12 1 1 1 3 6 5 2 5 3 1 3 2 mm C15H1NO+ MC: 6; TC:.53e+ 12 mm Sum of: C35H67O+, C37H69O+, C33H63O+, C39H71O+ MC: 1; TC: 1.21e+ 12 Fatty acid map 15 1 15 1 15 1 13 13 13 1 6 1 6 1 6 5 5 5 3 2 3 2 3 2 mm C16H31O2- MC: 1; TC: 5.996e+ 12 mm C1H33O2- MC: 1; TC: 5.52e+ 12 mm C1H35O2- MC: 1; TC: 2.961e+ 12
INORGANIC SPECIES High signal for Na and Cl Negative ions of Nitrogen species CN, CNO, C x N and C x NO Pepsin powder Phosphorus species PO x (PO 3 most represented) ORGANIC SPECIES High signal for (C x H y ) aliphatic and aromatic Positive ions of nitrogen species C x H y N (C H N and C 5 H 12 N most represented) Oxygenated organic species C x H y O z with the C 3 H 3 O + species strongly detected Health, Dalian, China, May 26 th - 2 th, 217
15 15 1 13 1 3 13 1 6 2 5 5 3 1 3 2 mm Sum of CH2N+, CHN+, C2H6N+, C3H6N+, Sum of: CH2N+, CHN+, C2H6N+, C3H6N+, CHN+, CHN+, CH1N+, CH1N+, C5H1N+, C5H1N+, C5H12N+ C5H12N+ MC: MC: 1; TC: 1; TC: 9.31 9.31e+5 e 12 mm Nitrogen species characteristics of pepsin Cl- MC: Chlorine 12 Cl- MC: 1; TC: 2.359 e +5 1; TC: 2.359e+5 15 15 13 12 13 12 1 1 1 1 6 6 5 5 3 2 3 2 mm CN- MC: 1; TC: 5.2 e +5 12 CNO- MC: 13; TC:.57 e mm 12 +5 Astruc et al. 217. CN- Pepsin localization in digested meat: a Tof-SIMS analysis. 5 th International Congress of Food Digestion, Agrocampus Rennes, -6 of april 217. CN-
Conclusions Methods : Diffusion of pepsin in meat can be assed by imaging Indirect methods: Histology, maldi tof imaging, FT-IR microspectroscopy allow to characterize some effects of «digestion juice» entrance in the food including effetcs of pepsin, ph, other hydrolases together. Direct methods of pepsin detection: immunohistofluorescence, SIMS imaging Immunohistofluorescence and pesin labelling show their limits for pepsin detection: High background for immunohistofluorescence, loss of pepsin activity after the dye have been bind. SIMS allows the label free detection of chemicals species specific to pepsin. This approache need confirmation, perhaps by labelling the pepsin with stable isotopes that are easy to evidenced by SIMS. Health, Dalian, China, May 26 th - 2 th, 217
Conclusions Mechanisms of gastric digestion of meat : Pepsin diffuse in meat through a homogeneous front. Pepsin seems also to diffuse along the perimysium sheaths. Perspectives Work in progress: Chewing taken into account Health, Dalian, China, May 26 th - 2 th, 217
Thank you for your attention Any questions? Health, Dalian, China, May 26 th - 2 th, 217