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Aromatic upgrading of marine by-products
    Sardine (Sardina pilchardus) hydrolysates example


    L. Tripoteau1,2, C. Prost2, M. Cardinal1, JP Bergé1

1 Département  Sciences et Technologie Alimentaires Marines,
     IFREMER centre de Nantes, BP 21105, 44311 Nantes cedex 03,
     France

2   ENITIAA– Equipe de recherche Arôme UMR CNRS 6144 Nantes,
     France


                Members of the SEApro network


                WEFTA 2008, Florence – Italy, 17-19 September 2008   1
CONTEXT

                                                                  Primary
   Biomass                  Processing
                                                                  Products


              40-60%

                         By-products &
  By-catch                  Wastes



                                                              Secondary
             Conversion
                                                                  Products


             WEFTA 2008, Florence – Italy, 17-19 September 2008              2
CONTEXT




                      By-products of
                         sardina



                                                           Secondary
          Conversion
                                                               Products


          WEFTA 2008, Florence – Italy, 17-19 September 2008              3
CONTEXT




                      By-products of
                         sardina



                                                           Secondary
     Enzymatic Hydrolysis
                                                               Products


          WEFTA 2008, Florence – Italy, 17-19 September 2008              4
CONTEXT




                      By-products of
                         sardina




     Enzymatic Hydrolysis                                      Aroma

          WEFTA 2008, Florence – Italy, 17-19 September 2008           5
METHODOLOGY


   Raw material (100)                                        Enzyme (x)
   (sardina heads & viscera)                             (large spectra protease)
                                  Water (50)




        Temperature                                          Time
           (-1 / +1)                                       (-1 / +1)

                                     Reaction




                   WEFTA 2008, Florence – Italy, 17-19 September 2008               6
METHODOLOGY

  Sensory analysis
             Strategy                                     Experimental conditions
          Descriptors
                                               - Trained panel of 13 people
                                               - 5 samples :
   Validation : consensus
                                                     -   T30 t30 A0 S0
            Notation                                 -   T60 t6 A1 S1
                                                     -   T60 t6 A0 S1
                                                     -   T60 t6 A0 S0
  ANOVA                 PCA                          -   Central point

Discriminative     Characterization            - “sniffing” characterization
 descriptors         of samples



                    WEFTA 2008, Florence – Italy, 17-19 September 2008              7
METHODOLOGY

 Aromatic characterization
                Extraction of volatile components :
               Solid phase micro-extraction (SPME)




                 - 20 min equilibrium          Volatile components adsorbed
                                               on SPME CAR/PDMS fiber
                 - 30 min adsorption
                                                Head space

       20 ml of hydrolysate

                                                         Water bath at 30°C




                WEFTA 2008, Florence – Italy, 17-19 September 2008            8
METHODOLOGY

   Aromatic characterization
                          Identification – Semi-quantification


          GC - MS - FID

- Identification by MS and RI

- Internal standard

- Semi-quantification




                      WEFTA 2008, Florence – Italy, 17-19 September 2008   9
PRELIMINARY RESULTS




         WEFTA 2008, Florence – Italy, 17-19 September 2008   10
METHODOLOGY

                                 Maillard reaction


Polysaccharides degradation                                     Carotenoids degradation


                                Aroma from
                                hydrolysates


         PUFA oxydation                                     MUFA oxydation
           (ω3 et ω6)                                                   (ω9)


                   WEFTA 2008, Florence – Italy, 17-19 September 2008                     11
METHODOLOGY

                                 Maillard reaction


Polysaccharides degradation                                     Carotenoids degradation


                                Aroma from
                                hydrolysates


         PUFA oxydation                                     MUFA oxydation
           (ω3 et ω6)                                                   (ω9)


                   WEFTA 2008, Florence – Italy, 17-19 September 2008                     12
METHODOLOGY


    Raw material (100)                                         Enzyme (x)
     (sardina heads & viscera)                             (large spectra protease)
                                    Water (50)
  Antioxydants (-1 / +1)                                     Sugar (-1 / +1)
   (complementary actions)


          Temperature                                          Time
             (-1 / +1)               Reaction                (-1 / +1)
                                           Reaction
                                       Reaction




                     WEFTA 2008, Florence – Italy, 17-19 September 2008               13
METHODOLOGY

 Experimental design


      Parameters                Low                Center         High

    Temperature (°C)              30                  45          60

      Time (min)                  30                 195          360

      Antioxydant                 -1                   0           1

         Sugar                    -1                   0           1




             WEFTA 2008, Florence – Italy, 17-19 September 2008          14
RESULTS

                                                                   Major origin
                                                                  Origine principale
                                         Compound
                                          Composé
                                                                 R             Lipid
                                                                  éactions Oxydation
                                                                 Maillard
                                                                de Maillard Oxydation
                                                                             lipidique
   70 molecules                              2-pentene                          x
                                          2-methyl-furan            x
    identified                          3-methyl-butanal            x
                                            2-ethyl-furan           x
                                         1-penten-3-one                         x
                                        2,3-pentanedione                        x
                                              H exanal                          x
                                           1-Penten-3-ol                        x
                                           (E)-2-hexenal                        x
                                             1-pentanol                         x
                                               octanal                          x
   20 molecules                             2-penten-1-ol
                                            2-nonanone
                                                                                x
                                                                                x
      studied                             2,4-hexadienal
                                    2-furancarboxaldehyde           x
                                                                                x

                                     (5Z )-octa-1,5-dien-3-ol                   x
                                         2,4-heptadienal                        x
                                          benzaldehyde              x           x
                                        2-furan-methanol            x
                                      1,3-cyclo-octadiene                       x



            WEFTA 2008, Florence – Italy, 17-19 September 2008                           15
RESULTS
Processing parameters influence:
       11 molecules were significantly influenced




                   WEFTA 2008, Florence – Italy, 17-19 September 2008   16
RESULTS




          WEFTA 2008, Florence – Italy, 17-19 September 2008   17
RESULTS
 Maillard reaction products




                    WEFTA 2008, Florence – Italy, 17-19 September 2008   18
RESULTS
                                                         Lipid oxydation products




          WEFTA 2008, Florence – Italy, 17-19 September 2008                        19
RESULTS
 Maillard reaction products                                        Lipid oxydation products




                    WEFTA 2008, Florence – Italy, 17-19 September 2008                        20
RESULTS

          Central points




           WEFTA 2008, Florence – Italy, 17-19 September 2008   21
RESULTS




                      DH




          WEFTA 2008, Florence – Italy, 17-19 September 2008   22
RESULTS




          Maillard reaction
                 (++)




               WEFTA 2008, Florence – Italy, 17-19 September 2008   23
RESULTS




                                                           Lipid oxydation
                                                                 (++)




          WEFTA 2008, Florence – Italy, 17-19 September 2008                 24
RESULTS
      Maillard reaction(-)
      Lipid oxydation (-)




             WEFTA 2008, Florence – Italy, 17-19 September 2008   25
RESULTS

          « marine, vegetal »




      « grilled, burnt »

                                                           +
                                                                    « fatty fish, rancid »




               WEFTA 2008, Florence – Italy, 17-19 September 2008                            26
CONCLUSION



Aroma perception of an hydrolysate can be modulated:

   – Aldehyds from lipid oxydation   “fatty and vegetal”
   – Aldehyds from Maillard reaction   “grilled”



Positive correlation between hydrolysis degree (DG) and
  Maillard reaction




                WEFTA 2008, Florence – Italy, 17-19 September 2008   27
PERSPECTIVES



Sensory analysis on all the samples

Identification and quantification of all the molecules

Experimental design interpretation

Feed and Food applications

      Modulate and control the aroma properties


              WEFTA 2008, Florence – Italy, 17-19 September 2008   28
ACKNOWLEDGEMENT



   Many thanks to:
         Charlotte Jacobsen (DTU)
         Sensory analysis panel




           WEFTA 2008, Florence – Italy, 17-19 September 2008   29
THANK YOU FOR YOUR ATTENTION




     WEFTA 2008, Florence – Italy, 17-19 September 2008   30

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Aromatic upgrading of marine by products

  • 1. Aromatic upgrading of marine by-products Sardine (Sardina pilchardus) hydrolysates example L. Tripoteau1,2, C. Prost2, M. Cardinal1, JP Bergé1 1 Département Sciences et Technologie Alimentaires Marines, IFREMER centre de Nantes, BP 21105, 44311 Nantes cedex 03, France 2 ENITIAA– Equipe de recherche Arôme UMR CNRS 6144 Nantes, France Members of the SEApro network WEFTA 2008, Florence – Italy, 17-19 September 2008 1
  • 2. CONTEXT Primary Biomass Processing Products 40-60% By-products & By-catch Wastes Secondary Conversion Products WEFTA 2008, Florence – Italy, 17-19 September 2008 2
  • 3. CONTEXT By-products of sardina Secondary Conversion Products WEFTA 2008, Florence – Italy, 17-19 September 2008 3
  • 4. CONTEXT By-products of sardina Secondary Enzymatic Hydrolysis Products WEFTA 2008, Florence – Italy, 17-19 September 2008 4
  • 5. CONTEXT By-products of sardina Enzymatic Hydrolysis Aroma WEFTA 2008, Florence – Italy, 17-19 September 2008 5
  • 6. METHODOLOGY Raw material (100) Enzyme (x) (sardina heads & viscera) (large spectra protease) Water (50) Temperature Time (-1 / +1) (-1 / +1) Reaction WEFTA 2008, Florence – Italy, 17-19 September 2008 6
  • 7. METHODOLOGY Sensory analysis Strategy Experimental conditions Descriptors - Trained panel of 13 people - 5 samples : Validation : consensus - T30 t30 A0 S0 Notation - T60 t6 A1 S1 - T60 t6 A0 S1 - T60 t6 A0 S0 ANOVA PCA - Central point Discriminative Characterization - “sniffing” characterization descriptors of samples WEFTA 2008, Florence – Italy, 17-19 September 2008 7
  • 8. METHODOLOGY Aromatic characterization Extraction of volatile components : Solid phase micro-extraction (SPME) - 20 min equilibrium Volatile components adsorbed on SPME CAR/PDMS fiber - 30 min adsorption Head space 20 ml of hydrolysate Water bath at 30°C WEFTA 2008, Florence – Italy, 17-19 September 2008 8
  • 9. METHODOLOGY Aromatic characterization Identification – Semi-quantification GC - MS - FID - Identification by MS and RI - Internal standard - Semi-quantification WEFTA 2008, Florence – Italy, 17-19 September 2008 9
  • 10. PRELIMINARY RESULTS WEFTA 2008, Florence – Italy, 17-19 September 2008 10
  • 11. METHODOLOGY Maillard reaction Polysaccharides degradation Carotenoids degradation Aroma from hydrolysates PUFA oxydation MUFA oxydation (ω3 et ω6) (ω9) WEFTA 2008, Florence – Italy, 17-19 September 2008 11
  • 12. METHODOLOGY Maillard reaction Polysaccharides degradation Carotenoids degradation Aroma from hydrolysates PUFA oxydation MUFA oxydation (ω3 et ω6) (ω9) WEFTA 2008, Florence – Italy, 17-19 September 2008 12
  • 13. METHODOLOGY Raw material (100) Enzyme (x) (sardina heads & viscera) (large spectra protease) Water (50) Antioxydants (-1 / +1) Sugar (-1 / +1) (complementary actions) Temperature Time (-1 / +1) Reaction (-1 / +1) Reaction Reaction WEFTA 2008, Florence – Italy, 17-19 September 2008 13
  • 14. METHODOLOGY Experimental design Parameters Low Center High Temperature (°C) 30 45 60 Time (min) 30 195 360 Antioxydant -1 0 1 Sugar -1 0 1 WEFTA 2008, Florence – Italy, 17-19 September 2008 14
  • 15. RESULTS Major origin Origine principale Compound Composé R Lipid éactions Oxydation Maillard de Maillard Oxydation lipidique 70 molecules 2-pentene x 2-methyl-furan x identified 3-methyl-butanal x 2-ethyl-furan x 1-penten-3-one x 2,3-pentanedione x H exanal x 1-Penten-3-ol x (E)-2-hexenal x 1-pentanol x octanal x 20 molecules 2-penten-1-ol 2-nonanone x x studied 2,4-hexadienal 2-furancarboxaldehyde x x (5Z )-octa-1,5-dien-3-ol x 2,4-heptadienal x benzaldehyde x x 2-furan-methanol x 1,3-cyclo-octadiene x WEFTA 2008, Florence – Italy, 17-19 September 2008 15
  • 16. RESULTS Processing parameters influence: 11 molecules were significantly influenced WEFTA 2008, Florence – Italy, 17-19 September 2008 16
  • 17. RESULTS WEFTA 2008, Florence – Italy, 17-19 September 2008 17
  • 18. RESULTS Maillard reaction products WEFTA 2008, Florence – Italy, 17-19 September 2008 18
  • 19. RESULTS Lipid oxydation products WEFTA 2008, Florence – Italy, 17-19 September 2008 19
  • 20. RESULTS Maillard reaction products Lipid oxydation products WEFTA 2008, Florence – Italy, 17-19 September 2008 20
  • 21. RESULTS Central points WEFTA 2008, Florence – Italy, 17-19 September 2008 21
  • 22. RESULTS DH WEFTA 2008, Florence – Italy, 17-19 September 2008 22
  • 23. RESULTS Maillard reaction (++) WEFTA 2008, Florence – Italy, 17-19 September 2008 23
  • 24. RESULTS Lipid oxydation (++) WEFTA 2008, Florence – Italy, 17-19 September 2008 24
  • 25. RESULTS Maillard reaction(-) Lipid oxydation (-) WEFTA 2008, Florence – Italy, 17-19 September 2008 25
  • 26. RESULTS « marine, vegetal » « grilled, burnt » + « fatty fish, rancid » WEFTA 2008, Florence – Italy, 17-19 September 2008 26
  • 27. CONCLUSION Aroma perception of an hydrolysate can be modulated: – Aldehyds from lipid oxydation “fatty and vegetal” – Aldehyds from Maillard reaction “grilled” Positive correlation between hydrolysis degree (DG) and Maillard reaction WEFTA 2008, Florence – Italy, 17-19 September 2008 27
  • 28. PERSPECTIVES Sensory analysis on all the samples Identification and quantification of all the molecules Experimental design interpretation Feed and Food applications Modulate and control the aroma properties WEFTA 2008, Florence – Italy, 17-19 September 2008 28
  • 29. ACKNOWLEDGEMENT Many thanks to: Charlotte Jacobsen (DTU) Sensory analysis panel WEFTA 2008, Florence – Italy, 17-19 September 2008 29
  • 30. THANK YOU FOR YOUR ATTENTION WEFTA 2008, Florence – Italy, 17-19 September 2008 30