Transcript Raman Spectroscopic Studies on Meat Quality

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.
Monday, 06th November, 2000.
•Introduction to Raman spectroscopy
•Comparison with NIR
•Previous work on research area
•Current results from research:
Initial work on lipids – model systems
Meat lipids – adipose and intramuscular fat
Aspects of meat quality – cooking and ageing
•Future plans
•Potential for Raman
•Introduction to resonance Raman spectroscopy
Raman Spectroscopy
• Irradiate sample with monochromatic radiation
• Collect inelastically scattered light
• Frequency difference gives vibrational spectrum
Rayleigh
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Intensity
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Advantages
Disadvantages
•Minimal sample prep.
•Weak effect
•Very general
•Expensive
•Rich in information
•Experimentally difficult
•Aqueous samples
•Fluorescence interferes
•“Special” techniques
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.
Schematic layout diagram for the CCD system
Diffraction
Grating
Spectrograph
C.C.D.
l=785nm
Ti-Saph
Lasers
Ar+
Telescope
Depolariser
Holographic
Notch Filter
Sample
Comparison of NIR & Raman Spectroscopy:principles of measurement
Near Infrared Reflectance
Non Destructive
Spectroscopic
Molecular Vibrations +
Electronic Configuration
Difficult to assign peaks
Particle size
Large water effect
Raman Spectroscopy
Non Destructive
Spectroscopic
Molecular Vibrations
Assignable peaks
Physical State
Low water interference
Comparison of NIR & Raman Spectroscopy:Practical Aspects
Near Infrared Reflectance
Raman Spectroscopy
Large area of measurement
Small area of measurement
Fibre optic system
Fibre optic system
Compact systems
Compact systems under
development
Cost £30k upwards*
Cost £30k upwards*
User friendly
Considerable Training needed
* This price is for a general purpose bench-top instrument, rather than smaller
task orientated devices
Foodstuffs
NIR
Raman
•Sample preparation frequently required
•No sample preparation
•Sensitive to water
•Insensitive to water
•Main food groups all give spectra
•Main food groups all give detailed spectra
Raman Intensity
Absorbance
Carbohydrate
Protein
Fat
1100
1300 1500
1700
1900
2100 2300
Wavelength (nm)
2500
1800
1400
1000
Wavenumber (cm-1)
600
Absorbance
2.5
NIR Spectra of
Water
water
2
1.5
1
1100
1300
1500
1700
1900
Wavelength
2100
2300
2500
Comparison of the effect of water on the spectra of
sugars.
NIR
Raman
Absorbance
Raman Intensity
Honey
Granular
Fructose
1100
1300 1500
1700
1900
2100 2300
Wavelength (nm)
2500
1800
1400
1000
Wavenumber (cm-1)
600
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 0C.
Intact single fibers:
• 70% a-helical structure (78% for myosin).
• Contraction did not significantly change the secondary structure of the fiber.
• Amino acid residue peaks changed upon interaction with Ca2+ 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.
O
OH
Triglycerides
O
OH
HO
CH2
HO
CH
HO
CH2
O
OH
Previous work suggests•cis/trans isomer ratios
•iodine values
but may be fluorescence problems with unpurified samples
unless FT Raman is used
Raman Spectrum of a Triglyceride
Scattering Intensity
H-C-H
H-C-H
C-C
=C-H
C=C
C1-C2
C=O
800
1000
1200
Raman Shift/cm-1
1400
1600
EFFECT OF INCREASING CHAIN LENGTH
Model Fats : FAMEs
CH2
5
C8
C7
C6
C5
Relative Band Intensity
4.5
C=O
R2 = 0.991
4
3.5
3
2.5
2
1.5
1
0.5
0
1000
1200
1400
1600
Wavenumber
1800
(cm-1)
0
5
10
Chain length
15
20
EFFECT OF INCREASING UNSATURATION
Model Fats : FAMEs
Commercial Fats and Oils
n(C=C)
d(CH2)
0.5
n(C=O)
18:4cis
0.45
Relative Peak Area
d(=C-H)
0.4
R2 = 0.982
0.35
18:2cis
0.3
0.25
18:1cis
18:0
0.2
0.15
0.1
20
800
1000 1200 1400 1600 1800
Wavenumber (cm-1)
30
40
50
60
70
Iodine Value
80
90
100
Raman Intensity
Comparison of the Raman spectrum of butter fat in
different physical states
80oC
21oC
-10oC
-176oC
700
1200
Raman Shift / cm-1
1700
Raman Intensity
Spectra of Various Animal Fats
Chicken
Pork
Lamb
Beef
800
1000
1200
1400
Raman Shift/cm-1
1600
Unsaturation Level vs. Depth through a cross
section of lamb adipose tissue
Unsaturation Level
0.14
0.13
0.12
0.11
0.1
0.09
0
1000
2000
3000
Depth
4000
5000
6000
Raman spectra of intramuscular fat before and after cooking.
Raman signal
Chicken
raw
Chicken
60 min
Beef
raw
Beef
60 min
700
900
1100
1300
1500
Raman Shift / cm-1
1700
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 C1-C2 stretching mode as an internal standard.
Fat Content:
•Ratio the C1-C2 stretch or the C-C stretch at 1060 cm-1 to the phenylalanine
peak (internal standard for meat protein).
600
800
n(C-C,N)
1000
1200
Raman Shift cm-1
Amide III
1400
Tryptophan
n(COO- )
d(CH2) sc
d(CH2) tw
Tyrosine + Phenylalanine
n(C-N)
Phenylalanine
n(C-C):a-helix
Tyrosine
Methionine
Cysteine
Peak Identity in Raman Spectrum of Meat
Amide I
1600
Raman spectra of various types of meat
a-helix mode
Phenylalanine
Amide III
Amide I
Raman Intensity
Chicken
Pork
Beef
650
1000
1400
Raman Shift / cm-1
1750
Difference spectra showing changes in protein secondary structure
upon cooking of meat samples.
(Sample after 60 minutes cooking - sample after10 minutes cooking time)
a-helix
b-sheet
Relative Intensity
Pork
600
Beef
1000
1500
Raman Shift / cm-1
Effect of Proteolysis on the Raman Spectrum of Meat
1 Day
14 Days
Cys Met
Tyr Skeletal n(C-N) Amide III CH2sc Amide I
a-helix b-sheet
Difference
Projected
Residual
600
800
1000
1200
1400
Raman Shift cm-1
1600
Principal Component Analysis of the Raman spectra of Pork
as it is aged
100
80
60
Day 1
Day 4
Day 7
Day 10
49Bc
94Ac
93Ac
47Ab
40
34Ac 50Bc
94Ab
47Bc
50Bb
94Bb
93Ab
34Bb
49Ab
20
t[3]
49Bb
47Ac
34Bc
49Ac
34Ab
50Ab
47Bb
93Bb
49Ba 34Aa
49Aa
93Ba
94Ba
93Aa
50Ba
47Aa
47Ba
50Aa
93Bd
0
-20
-40
93Bc
50Ac
94Bc
94Bd
47Ad
94Ad
49Ad
50Ad 49Bd
93Ad
-60
47Bd
34Ad
34Bd
-80
50Bd
-100
-100
0
t[2]
100
Loadings for PCA analysis of Pork ageing
Cys
Met
Tyr
Peptide Bond bands
0.080
Skeletal Amide III
Amide I
p[2]
0.040
0.000
-0.040
2nd component: amide
hydrolysis and residue effects
-0.080
0.100
p[3]
0.050
0.000
-0.050
3rd component: secondary
structure and residue shifts
-0.100
0
100
200
300
400
Pixel Num
500
600
700
800
Conclusions
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.
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
The future of Raman
Meat Quality Attributes
Instrumental/Rapid Method
Appearance
Flavour
Texture
Nutritional Quality
Proximate Analysis
Characterisation:Lipid
Protein/Amino Acids
Carbohydrates
Reflectance
Electronic nose +Raman?
NIR? Raman?
NIR?
Raman?
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.
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 Fearon
Mr. Griff Kirkpatrick
Mr. Alan Beattie
Mr. Colum Connelly
Resonance Raman Spectroscopy
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Chromophore
lmax
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Excitation hn
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•Irradiate sample with
monochromatic radiation
corresponding to adsorption
band in UV-Vis spectrum
• 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.
Non-Resonance
Raman
Intensity
• Bands associated with this
adsorption are enhanced by a
factor of ~103 to 104 relative to
the ground state Raman and
Rayleigh.
Rayleigh
Resonance
Raman
0
n-n’
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.
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 mm laser to give a 3 ns
temperature jump (~60 0C). 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.