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Raman Spectroscopic Studies on Meat Quality
Ph.D. Research Degree by Renwick BeattieSupervisors: 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 systemsMeat lipids – adipose and intramuscular fatAspects 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
hn
hn
hn
hn
hnhn’
hn’
Rayleigh
Intensity
-n n’0
• Weak effect
• Expensive
• Experimentally difficult
• Fluorescence interferes
Advantages
• Minimal sample prep.
• Very general
• Rich in information
• Aqueous samples • “Special” techniques
Disadvantages
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
DiffractionGrating
HolographicNotch Filter
Sp
ectr
ogra
ph
C.C
.D.
Ar+ Ti-SaphLasers
Telescope
Sample
Depolariser
l=785nm
Comparison of NIR & Raman Spectroscopy:- principles of measurement
Near Infrared Reflectance Raman SpectroscopyNon Destructive Non Destructive
Spectroscopic Spectroscopic
Molecular Vibrations + Molecular Vibrations
Electronic Configuration
Difficult to assign peaks Assignable peaks
Particle size Physical State
Large water effect Low water interference
Near Infrared Reflectance Raman Spectroscopy
Large area of measurement Small area of measurement
Fibre optic system Fibre optic system
Compact systems Compact systems underdevelopment
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
Comparison of NIR & Raman Spectroscopy:- Practical Aspects
Foodstuffs• Sample preparation frequently required
• Sensitive to water
• Main food groups all give spectra
Wavenumber (cm-1)
1800 1400 1000 6001100 1300 1500 1700 1900 2100 2300 2500
Wavelength (nm)
Carbohydrate
Protein
Fat
RamanNIR• No sample preparation
• Insensitive to water
• Main food groups all give detailed spectra
Ab
sorb
ance
Ram
an In
tens
ity
1
1.5
2
2.5
1100 1300 1500 1700 1900 2100 2300 2500
Wavelength
Ab
sorb
ance
water
NIR Spectra of Water
Wavenumber (cm-1)
1800 1400 1000 6001100 1300 1500 1700 1900 2100 2300 2500
Wavelength (nm)
Honey
Granular Fructose
Ab
sorb
ance
Ram
an In
tens
ity
RamanNIR
Comparison of the effect of water on the spectra of sugars.
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.
Previous work suggests-
• cis/trans isomer ratios
• iodine values
but may be fluorescence problems with unpurified samples unless FT Raman is used
Triglycerides
OH
OOH
O
OH
O
HO
HO
HO
CH2
CH2
CH
Sc
atte
rin
g In
ten
sit
yRaman Spectrum of a Triglyceride
C=O
C=C
H-C-H
=C-H
H-C-H
C-C
C1-C2
800 1000 1200 1400 1600
Raman Shift/cm-1
EFFECT OF INCREASING CHAIN LENGTH
C5
C6
C7
C8
1200 1400 1600 18001000
Wavenumber (cm-1)
CH2
C=O
Chain length
Re
lati
ve
Ba
nd
In
ten
sit
y0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
R2 = 0.991
0 5 10 15 20
Model Fats : FAMEs
EFFECT OF INCREASING UNSATURATION
Wavenumber (cm-1)
1200 1400 1600 18001000800
18:4cis
18:2cis
18:1cis
18:0
n(C=C)
n(C=O)d(CH2)d(=C-H)
Model Fats : FAMEs
R2 = 0.982
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
20 30 40 50 60 70 80 90 100
Iodine Value
Rel
ativ
e P
eak
Are
a
Commercial Fats and Oils
700 1200 1700Raman Shift / cm-1
Ram
an I
nten
sity
80oC
21oC
-10oC
-176oC
Comparison of the Raman spectrum of butter fat in different physical states
Pork
Spectra of Various Animal Fats
800 1000 1200 1400 1600Raman Shift/cm-1
Ram
an I
nten
sity
Lamb
Beef
Chicken
Unsaturation Level vs. Depth through a cross section of lamb adipose tissue
0.09
0.1
0.11
0.12
0.13
0.14
0 1000 2000 3000 4000 5000 6000
Depth
Un
sat
ura
tio
n L
ev
el
Raman spectra of intramuscular fat before and after cooking. R
aman
sig
nal
Chickenraw
Chicken 60 min
Beefraw
Beef60 min
700 900 1100 1300 1500 1700
Raman Shift / cm-1
Fat Composition and Content DeterminationFat 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).
Peak Identity in Raman Spectrum of Meat
600 140012001000800 1600Raman Shift cm-1
Ph
enyl
alan
ine
Tyr
osin
e
Cys
tein
e
Met
hio
nin
e
n(C-C,N) Amide III Amide I
n(C
-C):a
-hel
ix
n(C
-N)
d(C
H2)
sc
d(C
H2)
tw
n(C
OO
- )
Try
pto
ph
an
Tyr
osin
e +
Ph
enyl
alan
ine
Chicken
Pork
Beef
Raman Shift / cm-1
Ram
an I
nte
nsi
ty
650 1000 1400
Raman spectra of various types of meat
1750
Amide Ia-helix mode Phenylalanine Amide III
Difference spectra showing changes in protein secondary structure upon cooking of meat samples.
(Sample after 60 minutes cooking - sample after10 minutes cooking time)
Rel
ativ
e In
ten
sity
Raman Shift / cm-1
Pork
Beef
a-helix
b-sheet
600 15001000
600 140012001000800 1600Raman Shift cm-1
1 Day
Effect of Proteolysis on the Raman Spectrum of Meat
Projected Residual
14 Days
Difference
Tyr SkeletalMetCys CH2scAmide IIIn(C-N) Amide I-a helix b-sheet
-100
-80
-60
-40
-20
0
20
40
60
80
100
-100 0 100
t[3]
34Aa
34Ab
34Ac
34Ad
34Bb
34Bc
34Bd
47Aa
47Ab 47Ac
47Ad47Ba
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
94Bb94Bc
94Bd
Principal Component Analysis of the Raman spectra of Pork as it is aged
t[2]
Day 1Day 4
Day 10Day 7
0.100
0
p[3
]
Pixel Num
0.050
-0.100
0.000
-0.050
100 400300200 700600500 800
p[2
]
0.080
-0.080
-0.040
0.000
0.040
Loadings for PCA analysis of Pork ageing
Peptide Bond bands
2nd component: amide hydrolysis and residue effects
3rd component: secondary structure and residue shifts
Tyr
Skeletal
MetCys
Amide III Amide I
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
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 RamanMeat Quality Attributes
AppearanceFlavourTexture
Nutritional Quality
Proximate AnalysisCharacterisation:-LipidProtein/Amino AcidsCarbohydrates
Instrumental/Rapid Method
ReflectanceElectronic nose +Raman?NIR? Raman?
NIR? Raman?
RamanRaman?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. Alan BeattieMr. Griff Kirkpatrick Mr. Colum Connelly
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 ~103 to 104 relative to the ground state Raman and Rayleigh.
hn
hn’
hn
hn’
hn’
hn
Excitation
lmax
hnChromophore
RayleighIn
ten
sity
-n n’0
Non-Resonance Raman
Resonance Raman
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.