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Raman Spectroscopic Studies on Meat Quality Ph.D. Research Degree by Renwick Beattie Supervisors: Drs B. Moss and S. Bell Faculty of Agriculture and Science,

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Presentation on theme: "Raman Spectroscopic Studies on Meat Quality Ph.D. Research Degree by Renwick Beattie Supervisors: Drs B. Moss and S. Bell Faculty of Agriculture and Science,"— Presentation transcript:

1 Raman Spectroscopic Studies on Meat Quality Ph.D. Research Degree by Renwick Beattie Supervisors: Drs B. Moss and S. Bell Faculty of Agriculture and Science, The Queen’s University of Belfast Funded by: Department of Agriculture and Rural Development, N.I. Department of Agriculture and Rural Development, N.I.

2 Monday, 06 th November, 2000. Introduction to Raman spectroscopyIntroduction to Raman spectroscopy Comparison with NIRComparison with NIR Previous work on research areaPrevious work on research area Current results from research:Current results from research:  Initial work on lipids – model systems  Meat lipids – adipose and intramuscular fat  Aspects of meat quality – cooking and ageing Future plansFuture plans Potential for RamanPotential for Raman Introduction to resonance Raman spectroscopyIntroduction to resonance Raman spectroscopy

3 Raman Spectroscopy Irradiate sample with monochromatic radiation Collect inelastically scattered light Frequency difference gives vibrational spectrum h h h h h h ’ Rayleigh Intensity ’’ 0

4 Weak effect Expensive Experimentally difficult Fluorescence interferes Advantages Minimal sample prep. Very general Rich in information Aqueous samples “Special” techniques Disadvantages

5 Low-Cost, Compact Raman Spectrometers Enabling Technologies Diode lasers: Wide range of wavelengths and also tunable lasers to allow increased flexibility. Notch filter: Eliminate the strong laser line, preventing detector saturation. CCDs (Charge Coupled Detectors): Ultra high quantum efficiency detectors for detection of very low levels of light.

6 Schematic layout diagram for the CCD system Diffraction Grating Holographic Notch Filter Spectrograph C.C.D. Ar + Ti-Saph Lasers Telescope Sample Depolariser = 785nm

7 Comparison of NIR & Raman Spectroscopy:- principles of measurement Near Infrared ReflectanceRaman Spectroscopy Non Destructive Spectroscopic Molecular Vibrations +Molecular Vibrations Electronic Configuration Difficult to assign peaksAssignable peaks Particle sizePhysical State Large water effectLow water interference

8 Near Infrared ReflectanceRaman Spectroscopy Large area of measurementSmall area of measurement Fibre optic system Compact systemsCompact systems under development Cost £30k upwards* User friendlyConsiderable Training needed * This price is for a general purpose bench-top instrument, rather than smaller task orientated devices Comparison of NIR & Raman Spectroscopy:- Practical Aspects

9 Foodstuffs Sample preparation frequently requiredSample preparation frequently required Sensitive to waterSensitive to water Main food groups all give spectraMain food groups all give spectra Wavenumber (cm -1 ) 180014001000600 11001300150017001900210023002500 Wavelength (nm) Carbohydrate Protein Fat Raman NIR No sample preparationNo sample preparation Insensitive to waterInsensitive to water Main food groups all give detailed spectraMain food groups all give detailed spectra Absorbance Raman Intensity

10 NIR Spectra of Water

11 Wavenumber (cm -1 ) 180014001000600 11001300150017001900210023002500 Wavelength (nm) Honey Granular Fructose Absorbance Raman Intensity RamanNIR Comparison of the effect of water on the spectra of sugars.

12 Previous Work Whole Muscle: Observed spectra very similar to myosin spectrum (~50% of the total muscle protein). Intact Muscle contains ~20% bound water, remaining supercooled at –10 0 C. Intact single fibers: 70%  -helical structure (78% for myosin). Contraction did not significantly change the secondary structure of the fiber. Amino acid residue peaks changed upon interaction with Ca 2+ and ATP (which affect the effective charge density of the fiber). Isolated proteins: Myosin, Actin, Acto-myosin, Tropomyosin, Troponin and sub-fragments. Effect of different conditions (pH, salts and temperature) on protein secondary structure.

13 Previous work suggests- cis/trans isomer ratios iodine values but may be fluorescence problems with unpurified samples unless FT Raman is used Triglycerides OH O O O HO CH 2 CH

14 Scattering Intensity Raman Spectrum of a Triglyceride C=O C=C H-C-H =C-H H-C-H C-C C 1 -C 2 8001000120014001600 Raman Shift/cm -1

15 EFFECT OF INCREASING CHAIN LENGTH C5C5 C6C6 C7C7 C8C8 1200 1400 1600 18001000 Wavenumber (cm -1 ) CH 2 C=O Chain length Relative Band Intensity 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 R 2 = 0.991 05101520 Model Fats : FAMEs

16 EFFECT OF INCREASING UNSATURATION Wavenumber (cm -1 ) 12001400160018001000800 18:4cis 18:2cis 18:1cis 18:0 (C=C) (C=C) (C=O) (C=O)  (CH 2 )  (=C-H) Model Fats : FAMEs R 2 = 0.982 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 2030405060708090100 Iodine Value Relative Peak Area Commercial Fats and Oils

17 700 12001700 Raman Shift / cm -1 Raman Intensity 80 o C 21 o C -10 o C -176 o C Comparison of the Raman spectrum of butter fat in different physical states

18 Pork Spectra of Various Animal Fats 8001000120014001600 Raman Shift/cm -1 Raman Intensity Lamb Beef Chicken

19 Unsaturation Level vs. Depth through a cross section of lamb adipose tissue 0.09 0.1 0.11 0.12 0.13 0.14 0100020003000400050006000 Depth Unsaturation Level

20 Raman spectra of intramuscular fat before and after cooking. Raman signal Chicken raw Chicken 60 min Beef raw Beef 60 min 7009001100130015001700 Raman Shift / cm -1

21 Fat Composition and Content Determination Fat Composition: Similar to determination for free fat except:- Problems: Fat peaks mixed in with protein peaks Carbonyl stretch, the usual internal standard, is unsuitable as the protein matrix shifts the peak below the amide I band. Solutions: Isolate Fat peaks by taking baseline at set points each side of each peak. Use the C 1 -C 2 stretching mode as an internal standard. Fat Content: Ratio the C 1 -C 2 stretch or the C-C stretch at 1060 cm -1 to the phenylalanine peak (internal standard for meat protein).

22 Peak Identity in Raman Spectrum of Meat 6001400120010008001600 Raman Shift cm -1 Phenylalanine Tyrosine Cysteine Methionine (C-C,N) Amide III Amide I (C-C):  -helix (C-N)  (CH 2 ) sc  (CH 2 ) tw (COO - ) Tryptophan Tyrosine + Phenylalanine

23 Chicken Pork Beef Raman Shift / cm -1 Raman Intensity 65010001400 Raman spectra of various types of meat 1750 Amide I  -helix mode PhenylalanineAmide III

24 Difference spectra showing changes in protein secondary structure upon cooking of meat samples. (Sample after 60 minutes cooking - sample after10 minutes cooking time) Relative Intensity Raman Shift / cm -1 Pork Beef  -helix  -sheet 60015001000

25 6001400120010008001600 Raman Shift cm -1 1 Day Effect of Proteolysis on the Raman Spectrum of Meat Projected Residual 14 Days Difference TyrSkeletalMetCysCH 2 scAmide III (C-N) Amide I  helix  -sheet

26 -100 -80 -60 -40 -20 0 20 40 60 80 100 -1000100 t[3] 34Aa 34Ab 34Ac 34Ad 34Bb 34Bc 34Bd 47Aa 47Ab 47Ac 47Ad 47Ba 47Bb 47Bc 47Bd 49Aa 49Ab 49Ac 49Ad 49Ba 49Bb 49Bc 49Bd 50Aa 50Ab 50Ac 50Ad 50Ba 50Bb 50Bc 50Bd 93Aa 93Ab 93Ac 93Ad 93Ba 93Bb 93Bc 93Bd 94Ab 94Ac 94Ad 94Ba 94Bb 94Bc 94Bd Principal Component Analysis of the Raman spectra of Pork as it is aged t[2] Day 1 Day 4 Day 10 Day 7

27 0.100 0 p[3] Pixel Num 0.050 -0.100 0.000 -0.050 100400300200700600500800 p[2] 0.080 -0.080 -0.040 0.000 0.040 Loadings for PCA analysis of Pork ageing Peptide Bond bands 2 nd component: amide hydrolysis and residue effects 3 rd component: secondary structure and residue shifts Tyr Skeletal MetCys Amide IIIAmide I

28 The results so far have indicated dispersive Raman Spectroscopy can be applied to:- Quantitative analysis of Fatty acid parameters: chain length, unsaturation level, solid fat. Understanding some of the mechanisms of biochemical change in proteins during cooking and formation of meat. Correlations currently under investigation include:- Quantitative analysis of fat composition in butters, adipose tissue and meat. Quantitation of total fat content in meat. Speciation using fat and/or meat. Level of proteolysis in muscle/meat. Conclusions

29  Plans for Research:  Speciation of meat (by muscle and/or fat). Cold shortening – contraction of meat. Tenderness – state of contraction, hydrolysis of proteins etc. Taste – can Raman predict which pieces of meat taste good? Final internal temperature of cooked meats. Leanness/ Total fat content. Fatty Acid composition – incorporate work on lipids. Raman spectra will be compared to standard tests and to taste tests

30 The future of Raman Meat Quality Attributes Appearance Flavour Texture Nutritional Quality Proximate Analysis Characterisation:- Lipid Protein/Amino Acids Carbohydrates Instrumental/Rapid Method Reflectance Electronic nose +Raman? NIR? Raman? Raman Raman? Dispersive Raman spectroscopy has long been neglected for food analysis, largely due to the problem of fluorescence and expense. However, our research has shown that by using a laser on the boundary of visible and near-infrared radiation, one can easily determine many nutritional and qualitative parameters using the cheaper dispersive Raman instruments rather than expensive FT-NIR Raman instruments.

31 Acknowledgements DARD – for the award of a postgraduate studentship, enabling me to carry out this research. Drs Bruce Moss and Steven Bell, for their supervision and help Dr Ann FearonMr. Alan Beattie Mr. Griff KirkpatrickMr. Colum Connelly

32 Resonance Raman Spectroscopy Excite the particular bond involved in the adsorption to give longer lived excited state. Increases the probability of change in vibrational state before energy is released. Irradiate sample with monochromatic radiation corresponding to adsorption band in UV-Vis spectrum Bands associated with this adsorption are enhanced by a factor of ~10 3 to 10 4 relative to the ground state Raman and Rayleigh. h h ’ h h Excitation max h Chromophore Rayleigh Intensity ’’ 0 Non-Resonance Raman Resonance Raman

33 Applications of Resonance Raman Spectroscopy Resonance Raman spectroscopy (RRS) probes particular bonds (chromophores) resulting in: Very precise information about specific bonds. Detection of very low concentrations of the chromophore (less than 10 -6 M). Detection of small changes in the chromophore. This is useful for meat analysis because: The amide bond of meat is a chromophore and has a well established relationship with the secondary and tertiary structure of the protein. RRS can improve analysis of changes in amide bonding hence structure of the protein or level of proteolysis.

34 Resonance Raman Spectroscopy of Proteins. The amide bonds of proteins has a strong adsorption band in the UV and 204 nm lasers can be used to provide RR spectra with the bands due to the amide bonds enhanced. RRS has recently been used to probe the dynamic changes involved in protein folding and unfolding. The peptide (penta-alanine) was probed with a 1.9  m laser to give a 3 ns temperature jump (~60 0 C). The peptide was then probed with the 204 nm laser at a pulse rate of 3 ns to follow peptide folding from a few ns up to a few ms. Initial increase due to temperature is observed before actual unfolding begins at around 50 ns. After 95 ns the peptide is ~30% unfolded. Kinetic calculations from the results indicate it is not a simple transition between two states, but involves intermediate conformations.

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