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High-resolution solid-state NMR of food materials
Review Many foods and ingredients are best described as solids. High-resolution NMR is conventionally used to obtain detailed chemical information on liquid-state systems; examples are discussed in other articles in this issue. Recent advances in techniques and hardware now allow the routine acquisition of high-resolution 1JC NMR spectra from solid materials. Commercial hardware has been available for more than a decade and many applications to solids in a wide variety of areas have been described. Although 1H is the most abundant NMR nucleus in solid food materials, there are technical difficulties in high-resolution solid-state 1H NMR that make 1JC the nucleus of choice. This article outlines some of the information of relevance to food science that has been obtained recently using 1JC NMR.
There are many differences in NMR parameters between solids and liquids. One of the most well known is the variation in the 'H T2 value, as this underlies the ability to determine non-destructively the solid/liquid ratio in fats and oils by NMR. In addition to relaxation effects, there are two other major influences on the ability to obtain high-resolution J3C NMR signals from solids. One is dipolar broadening of signals due to heteronuclear interactions between the abundant 'H nuclei and the dilute i3C nuclei. This interaction depends on molecular orientations, but has an average value over all orientations of zero. In liquids and solutions, molecular motion is fast enough that orientations average out over the timescale of the experiment, and no significant dipolar broadening is usually observed. This is not the case for solids, where residual dipolar broadening is of the order of at least 10 kHz (at least 100 ppm in a i3C spectrum on a 400 MHz spectrometer). A second factor that differs between NMR in solids and NMR in solutionslliquids is chemical shift anisotropy. As chemical shifts are determined by electrons shielding nuclei from the applied magnetic field, they will have a definite directional component because electron densities are not arranged spherically around nuclei. Local molecular motions average this anisotropy to zero in solution, whereas this mechanism is not available to solids. The practical solution to these two difficulties in obtaining solid-state NMR spectra is to use 'magicangle' spinning to simulate motional averaging, and high-power decoupling to minimize dipolar interactions. The so-called 'magic' angle is -54 0 44', and is that angle for which 3cos 28 = I. This is appropriate as the equations describing the angular dependence of both dipolar decoupling and chemical shift anisotropy factors contain the term (3cos 28 - I). Spinning at the 'magic' angle needs to be at frequencies greater than the anisotropy in chemical shifts; for most solids, -3 kHz is Michael J. Gidley is with Unilever Research, Colworth House, Sharnbrook, UK MK441LQ.
Trends in Food Science & Technology August/September 1992 [Vol. 3J
High-resolution solid-state NMR of food materials Michael J. Gidley considered to be sufficient. Residual dipolar interactions are removed by high-power 'H decoupling. Whereas conventional solution-state NMR uses decoupling powers of 5 W or less, removal of dipolar interactions in solids requires typically 50-100 W. The requirement for rapid spinning at a defined angle to the direction of the magnetic field, together with the need to use high decoupling powers, means that nonstandard NMR equipment is needed. However, manufacturers offer the required equipment as accessories to all modem spectrometers. A magic-angle spinning probe and a typical sample holder are shown in Fig. 1. Having minimized broadening effects due to dipolar coupling and chemical shift anisotropy, a choice of spectral acquisition strategies is available. In practice, one technique, cross-polarization, has come to dominate high-resolution 1JC NMR in solids. In this method, magnetization is transferred from 'H nuclei by dipolar coupling to 1JC nuclei. This results in an increase in inherent sensitivity and, because relaxation is via the rapid 'H pathway rather than the slower 1JC route, experiments can be repeated more rapidly, with consequent increases in signal-to-noise ratio. The cross-polarization method is most effective for the least molecularly mobile systems. Where molecular Plobility is greater, a conventional single-pulse acquisition can be used. This forms the most common route to mobility-resolved spectroscopy in solids and is discussed further below. More detail on the principles of high-resolution solid-state NMR and broad descriptions of application areas are given elsewhere 1- s.
Lipids, proteins and carbohydrates Lipids, proteins and carbohydrates form the bulk of carbon-containing components in food systems. In many applications, a solid form is important and, hence, their study by high-resolution solid-state NMR has been investigated. Typical J3C spectra are shown in Fig. 2. The application of NMR methods to food lipids has recently been reviewed6 • High-resolution solid-state NMR spectroscopy has been used to study abundant lipids in seeds 7•8 and crystal polymorphism in triglycerides9 • Oilseed composition has been established using magic-angle spinning and dipolar decoupling with observation either by single 7 or cross-polarization8 pulse strategies. Sufficient molecular information is obtained to suggest that solid-state NMR methods may be useful in the non-invasive analysis of composition in single
©1992, Elsevier Science Publishers ltd, (UKI
231
oilseeds. As NMR is sensitive to chemical effects on the atomic distance scale, it is a powerful adjunct to X-ray diffraction in determining crystalline structures in, for example, triglycerides, which are of great importance in many fat-structured foods. Bociek et al. 9 showed how distinct spectral features can be observed for crystalline
Fig. 1 (a) Magic-angle spinning assembly in a commercial (Bruker) probe; the field in the NMR magnet is in the vertical direction. (b) Magic-angle spinning rotor (Bruker), constructed of ceramic with a polymeric fluorocarbon cap.
232
polymorphs of tripalmitin and tristearin. In addition to permitting the interpretation of chemical shift effects in terms of local molecular structure, the technique provided significant information on the relative mobility of individual sites within polymorphs that was consistent with thermodynamic analyses 10. There appears to be considerable further potential for the fruitful application of high-resolution solid-state NMR to molecularly ordered lipid systems of relevance to foods. The wealth of resonances observed in NMR spectra of proteins (due to the diversity of amino acid compositions and sequences) has been exploited in many outstanding solution studies that have led to complete resonance assignments to a known sequence and detailed three-dimensional structure proposals. Unfortunately the same approach cannot be applied to solid proteins, with the result that spectra are rich in information (Fig. 2) but difficult to interpret other than in broad functional group terms. Due to its importance in foods, gluten has been the subject of high-resolution solid-state NMR studies by a number of groups 11-14. A general conclusion from these reports is that reproducible chemical shift differences that reflect baking quality have not been found. However, information on molecular mobility in powders and hydrates has been obtained that aids the development of appropriate molecular descriptions of gluten functionality. Food carbohydrates have been the subject of several solid-state NMR studies. For low molecular weight materials, crystallinity leads to sharp signals in a crosspolarization solid-state spectrum, whereas noncrystalline forms give broader signals. This is likely to be important in studies of amorphous-to-crystalline transitions, which are a subject of considerable current research interest. Crystalline polymorphism (e.g. in lactosel 5) can be probed by solid-state NMR with the 'rule of thumb' being that a separate resonance should be observed for each inequivalent atomic site within the crystalline unit cell l6 . This can mean multiple resonances for single molecular sites due to asymmetries of molecular packing within the crystal. Such effects are also found in polysaccharides, which, although rarely crystalline, are often present in defined molecularly ordered confonnations (e.g. helices). Examples of multiple resonances include ordered forms of starch l6.17 (Fig. 3) and celluloseI 8 .1 9 • Due to the tendency of polysaccharides to form molecularly ordered but noncrystalline structures, high-resolution solid-state NMR is being applied in many studies, some of which are described below. In general, solid-state NMR studies are most informative in systems with relatively simple major molecular structure features (e.g. triglycerides, simple sugars and many polysaccharides). For more complicated spectral types (e.g. proteins), residual line widths, potential multiple resonances from single sites and the lack of detailed assignment strategies make interpretation very difficult. It is interesting that this is the converse of the situation for solution-state NMR, where the simplicity of spectra for 'simple' molecules reduces information Trends in Food Science & Technology August/September 1992 [Vol. 31
(d)
100
(c)
90
80
70
60
Chemical shift (ppm) 180
160 140
120 100
80
60
40
20
Chemical shift (ppm)
Fig. 3 Ordered and amorphous model starch materials observed by cross-polarization and magic-angle spinning NMR. A-type (a) and
Fig. 2
B-type (b) crystalline forms show clear multiplicity effects at least for
1lC
the anomeric carbon signal (-100 ppm), and all three crystalline
cross-polarization and magic-angle spinning spectra of typical
food lipid, protein and carbohydrate ingredients: (a), tripalmitin;
forms, including the V-type form (c), have chemical shift patterns
(b), ovalbumin; (c), waxy maize starch.
that are distinct from that of amorphous material (d).
content, but complicated spectra (e.g. from small proteins) can be completely assigned and the consequent enormous information content used to derive threedimensional structures.
estimates of crystallinity obtained from X-ray diffraction studies I7 .2o • Spectra of native cellulose have similarly been interpreted as a composite of crystalline and amorphous features l8 , although detailed interpretation is still controversial 19. Although such use of solid-state NMR for the identification and quantification of biopolymer conformations is gaining in importance, it would clearly be beneficial if the relationships between chemical shift and conformation were understood. There is a significant amount of experimental data now available for defined ordered forms of polysaccharides and proteins with simple repeat structures 21 • Increasingly it is becoming clear that probably the major feature contributing to the range of chemical shifts encountered (e.g. 10 ppm for the C-l site in a(l~4)-linked glucans; Fig. 3) is a diversity of local conformations. Effort has therefore turned to examining the effect of conformation-defining torsion angles on chemical shifts 21 - 23 • For proteins, systematic effects of a-helix and ~-sheet local conformations on chemical shifts have been reported 21 . In polysaccharides, a(l ~4) linked glucans have been most studied due to their conformational diversity. Correlations between chemical shift and glycosidic bond conformation have been
Biopolymer conformation As illustrated in Fig. 3, amorphous or differently ordered conformations of the same biopolymer give very different solid-state DC NMR spectra. These provide excellent 'fingerprint' spectra for the identification of ordered states in other environments (e.g. as described below for gels). A general strategy would be to obtain solid-state ]lC NMR spectra for crystalline samples exhibiting X-ray diffraction effects, and use these to identify and quantify molecularly ordered but non-crystalline forms. One example is in the analysis of granular starches, whose solid-state ]lC NMR spectra (Fig. 2) can be modelled by a combination of appropriate ordered and amorphous spectral features (Fig. 3), which not only allow the identification of molecular conformations, but also permits quantification of their relative abundances 17 • This information provides the ratio of doublehelical (ordered) to single-chain (amorphous) populations, which can be compared with quantitative Trends in Food Science & Technology August/September 1992 [Vol. 3J
233
sequences. In particular, single-pulse and crosspolarization experiments often give complementary spectral information 1, with less mobile sites being detected by cross-polarization excitation and more mobile sites by single-pulse excitation. This approach has been used to resolve mobility populations for a range of gluten fractions '2 , and has been particularly informative Mobility-resolved spectroscopy Once signals have been separated in a high-resolution in studies of molecular structure in polysaccharide spectrum, there are two approaches to gaining infor- gels 25 ,26. Figure 4 shows 13C spectra for K-carrageenan in mation on the relative mobility of individual structural the solution form (single-pulse experiment) and the gel features. One involves measurement of relaxation form (using cross-polarization). Highly significant chemicharacteristics of isolated signals, which gives infor- cal shift differences are seen, consistent with the tranmation concerning both local molecular motions, and sition from coil to double helix that is proposed to be rewhether different signals belong in the same relaxation quired for gel formation. A single-pulse experiment on domain - potentially a very powerful means of studying the gel (with magic-angle spinning and high-power demUlti-phase food systems. It is much used in deter- coupling) showed very low intensity, suggesting a single mining the compatibilities of synthetic polymer blends, dominant conformation (presumably the double helix) and has recently been used to show that protein domains in the gel (Gidley, M.J., Darke, A.H. and Ablett, S., unwithin wheat flours are at least 50 A in diameter24 . published). For amylose gels, a different response was Molecular motion within crystalline triglycerides has observed, as shown in Fig. 5. In this case, significant been probed using the interrupted decoupling technique, intensity was observed in a single-pulse experiment without magic-angle spinning. Residual breadth in these which gives spectra enriched in mobile species9 • The second general approach to mobility-resolved signals was shown to be due to chemical shift spectroscopy is to use different excitation pulse anisotropy, as magic-angle spinning reduced signal widths dramatically. The resultant spectrum had identical chemical shifts to those of amylose in solution. The cross-polarization experiment (with magic~angle spinning) gave a totally different spectrum, which was identified by fingerprint matching with a reference spectrum as being due to double helices of the B-type crystalline form (Fig. 5). Together with confirmatory relaxation measurements, this led to a molecular model for amylose gels 25 , involving 'junction zones' of aggregated double helices interconnected by segments of mobile chains that are prevented from undergoing full reorientation by being connected to the network. A third category of polysaccharide gels are those that show intensity in both single-pulse and cross-polarization experiments, with essentially identical chemical shifts but different relative intensity ratios for resonances in the two spectra. Examples of this third category are locust bean gum and konjac mannan gels, where the less mobile regions (as detected by cross-polarization) are enriched in mannose and glucose features, respectively26. The combination of defined conformations and a range of segmental mobility in polysaccharide gels makes high-resolution solid-state NMR a method of choice for the study of underlying molecular mechanisms. reported and have been successfully used to model the C-I resonance for amorphous materiaF3. All of these correlations, however, are empirical; a major challenge is to rationalize these observed effects in terms of chemical shift theory.
70
Examples of application areas
Chemical shift (ppm)
Fig. 4 1le
NMR spectra of lC-carrageenan obtained for 4% solution and gel forms at various temperatures, and using single-pulse and cross-
polarization excitation in combination with magic-angle spinning:
Many applications of high-resolution solid-state 13C NMR have been described in preceding sections. A miscellany of further applications are now discussed, with examples drawn from areas where recent progress has been made as well as from areas of potentially greater application in food science.
6Soe solution with single-pulse excitation; (b), soe gel with soe gel with Starch cross-polarization excitation and magic-angle spinning; (d), -20 oe In addition (al,
single-pulse excitation and magic-angle spinning; (c),
gel with cross-polarization excitation and magic-angle spinning.
234
to identifying ordered forms (Fig. 3) and quantifying the ratio of double helical to amorphous
Trends in Food Science & Technology August/September 1992 [Vol.
31
residues in granular starch 17 , it has now been shown in a multi-technique study that individual double helices are the key structural stabilizer in starch granules, and that the enthalpy of gelatinization is directly related to the content of double helices 20 . In light of this result, solidstate NMR studies of starches and starch-containing foods are predicted to become more widely exploited. Many cereal starches contain a small (typically less than 1% dry weight) amount of lipid within the granules. It has long been debated whether this lipid is present as a complex with amylose or in a free state. Recent solidstate 13C NMR spectra strongly suggest that lipids are present predominantly as amylose complexes, at least in barley starches 27 •
(a)
(c)
Flours and doughs A recent study of wheat flours and doughs24 has opened up several lines of enquiry into the mechanisms involved in flour and dough hydration, and in particular into the difference between hard and soft wheat flours. Much greater mobilization/plasticization of protein is observed in soft wheat flours than in hard wheat flours, with correspondingly greater mobilization/plasticization of starch in hard than in soft flours 24 . Such mapping of water relationships could potentially be extended to more molecular detail and is clearly important in understanding the effects of varietal flours on baking characteristics.
(d)
110
100
90 80 70 Chemical shift (ppm)
60
Fig. 5 llC spectra for a 10% (w/v) amylose gel using single-pulse excitation without (a) and with (b) magic-angle spinning, or cross-polarization excitation with magic-angle spinning (c). Spectrum (d) was obtained using cross-polarization excitation and magic-angle spinning on solid B-type crystalline amylose.
Plant cell walls Cell walls have a major impact on the texture and processing characteristics of edible plants. As they are composed primarily of polysaccharides, augmented in some cases by structural proteins and phenolics, plant cell walls should be amenable to study by high-resolution solid-state 13C NMR. This possibility has been experimentally realized, with spectra being obtained from celery strands 28 and from intact and fractionated cell walls of bean sprouts 29 . There is significant potential in this area, as earlier relaxation studies identified domains within cell walls 30 that could conceivably be probed by mobility-resolved spectroscopy - as could ripening-related changes in, for example, apples 31 .
Frozen systems An early practical demonstration of high-resolution solid-state 13C NMR involved low-temperature observation of room-temperature liquids 32 . Variable temperature operation is possible on standard spectrometers with 'solids' accessories and, hence, there is no practical barrier to obtaining spectra. It is perhaps surprising, therefore, that little work has been reported in this field. Two relevant applications of 'frozen' 13C NMR are in assessing the range (rather than the motional average as observed at room temperature) of conformations in solutions of oligo- and polysaccharides33 , and in immobilizing chain segments within polysaccharide gels so that all of the polymer is detected in a cross-polarization experiment 25 . Given the commercial importance of Trends in Food Science & Technology August/September 1992 [Vol. 3J
frozen food systems, it is anticipated that this will be a growth area for high-resolution solid-state 13C NMR spectroscopy.
References 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Harris, R.K. (1983) Nuclear Magnetic Resonance Spectroscopy: A Physicochemical View, Pitman Sanders, J.K.M. and Hunter, B.K. (1987) Modern NMR Spectroscopy, Oxford University Press Yannoni, CS. (1982) Acc. Chern. Res. 15, 201-208 Maciel, G.E. (1984) Science 226,282-288 Chmelka, B.F. and Pines, A. (1989) Science 246, 71-77 Eads, T.M. and Croasmun, W.R. (1988) J. Am. Oil Chern. Soc. 65, 78-83 Rutar, V. (1989) J. Agric. Food Chern. 37, 67-70 Haw, J.F. and Maciel, G.E. (1983) Anal. Chern. 55, 1262-1267 Bociek, S.M., Ablett, S. and Norton, LT. (1985) J. Am. Oil Chern. Soc. 62,1261-1266 Norton, LT., Lee-Tullnell, CD., Ablett, S. and Bociek, S.M. (1985) J. Am. Oil Chern. Soc. 62, 1237-1244 Schofield, J.D. and Baianu, I.C (1982) Cereal Chern. 59, 240-245 Belton, p.s., Shewry, P.R. and Tatham, AS. (1985) J. Cereal Sci. 3, 305-317 Moonen, J.H.E., Hemminga, M.A. and Graveland, A. (1985) J. Cereal Sci. 3, 319-327 Ablett, S., Barnes, D.J., Davies, AP., Ingman, S.J. and Patient, D.W. (1988)]. Cereal Sci. 7,11-20 Earl, w.L. and Parrish, FW. (1983) Carbohydr. Res. 115,23-32 Marchessault, R.H., Taylor, M.G., Fyfe, CA and Veregin, R.P. (1985) Carbohydr. Res. 144, C1 -C5 Gidley, M.J. and Bociek, S.M. (1985) J. Am. Chern. Soc. 107, 7040-7044
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1B 19 20 21 22 23 24 25
Atalla, R.H. and VanderHart, D.L.11984) Science 223, 283-285 Horii, F., Hirai, A. and Kitamaru, R. 11987) Macromolecules 20, 2117-2120 Cooke, D. and Gidley, MJ (1992) Carbohydr. Res. 227, 103-112 Saito, H. (1986) Magn. Reson. Chem. 24, 835-852 Veregin, R.P., Fyie, e.A., Marchessault, R.H. ano Taylor, M.G.119S7) Carbohydr. Res. 160, 41-56 Gidley, MJ and Bociek, S.M. (1988) J. Am. Chem. Soc. 110, 3820-3829 Garbow, J.R. and Schaeier, J. (1991) J. Awic. Food Chem. 39, 877-880 Gidley, M.J. (1989) Macromolecules 22, 351-358
26
Gidley, I'v\.I., McArthur, A.I. dnd Underwood, D.R.119911 Food Hydrocolloids 5, 129-140
27 2B 29 30
Morrison, W.R., Tester, RJ. and Gidley, M.I. em'al Cherll. lin press) larvis, M.c. and Apperley, D.e. (19901 Plant Physiol. 92. 61-65 Jarvis. M.e. 119901 Carbohydr. Res. 201. 327-333 Mackay, A.L., Wallace, I.e.. Sasaki. K. and Taylor, I.E.P. 119881 Biochemistry 27. 1467-1473 Irwin, P.L., Pietier. P.E., Gerasimowicz. W.V .• Pressey. R. and Sams. e.E. 119841 Phvtochemistry 23.2239-2242 Fyie. e.A., Lyerla, I.R. and Yannoni, e.S. (19781/. Am. Chem. Soc. 100, 5635-5636 Gidley, M.I. and Bociek, S.M. 119881 Carbohvdr. Res. 183, 126-130
31 32 33
Review
Applications of
NMR in •
sensory sCience
sensory science IS In its infancy. New applications are just waiting to be explored.
Advantages of NMR
Terri Robertson, Shelly Schmidt and Barbara Klein NMR spectroscopy is already of great value in determining the relationships between a molecule's structure and its odor, flavor, color and textural characteristics. Newer applications of NMR techniques in sensory science include studies of the effects of ion and water binding on taste perception; for example, 2lNa NMR has been used to examine the effects on perceived saltiness of Na+ binding to ionic and non-ionic gums. The range of useful applications is expanding as both NMR and sensory science techniques continue to develop,
Sensory science uses formal experimental designs and statistical methods to evaluate the perception of foods and materials by sight, smell, taste, touch and hearing'. An important aspect of sensory science is the study of the relationships between the physicochemical properties of a molecule and its flavor, texture and/or odor perception. NMR spectroscopy and imaging are powerful tools that have been used to examine a variety of physicochemical features (e.g. molecular structure, conformation and dynamics) of diverse systems (e.g. solutions, suspensions and solids) at the molecular level, by probing NMR-active nuclei such as 'H, 11C or 21Na. As in many fields, the application of NMR techniques in Terri Robertson, Shelly Schmidt and Barbara Klein are with the Division oi Foods & ,"utrition, University oi Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801, USA.
236
NMR spectroscopy and imaging are non-invasive, nondestructive techniques. Thus, NMR provides the opportunity to view a system at the molecular level and then use the same sample for other physicochemical measurements and sensory testing by subjects. Most foods may be analysed by NMR techniques, which can be applied to solid, semisolid or liquid samples. Additionally, numerous isotopes can be studied; 'H and "C are the most frequently studied. To date, molecular chemists and food scientists have used NMR to identify the structures and conformations of molecules with known tastes. The tastes that have received the greatest attention are bitterness, sweetness and saltiness. Traditionally NMR spectroscopy is complemented by ultraviolet (UV) or infrared (IR) spectroscopy, mass spectrometry, X-ray crystallography and/or conformational energy calculations in the identification process. More recently, the dynamics of a food system, such as molecular mobility, competitive ion binding and water binding, as determined by NMR, have been related to taste and texture perception as analysed by sensory analysis techniques.
Bitterness The structures and conformations of bitter-tasting compounds found in citrus fruits2-'), zucchinis and beer!> have been determined by NMR. In citrus fruits, naringin is a bitter flavanone glycoside that includes the disaccharide rhamnose. To determine the link between rhamnose and bitterness, Kamiya et al. 2 synthesized six flavanone glycosides with (I ~2)-linked disaccharides. The structures and conformations of these compounds were confirmed by 'H NMR, UV and IR spectroscopy. A taste panel composed of five young women matched the bitterness of the naringin control to concentrations of the synthetic compounds. The authors reported that when the a-L-rhamnosyl residue in the naringin molecule was replaced by either a-D-glucosyl or Trends in Food Science & Technology August/September 1992 IVol. 31
There are many differences in NMR parameters between solids and liquids. One of the most well known is the variation in the 'H T2 value, as this underlies the ability to determine non-destructively the solid/liquid ratio in fats and oils by NMR. In addition to relaxation effects, there are two other major influences on the ability to obtain high-resolution J3C NMR signals from solids. One is dipolar broadening of signals due to heteronuclear interactions between the abundant 'H nuclei and the dilute i3C nuclei. This interaction depends on molecular orientations, but has an average value over all orientations of zero. In liquids and solutions, molecular motion is fast enough that orientations average out over the timescale of the experiment, and no significant dipolar broadening is usually observed. This is not the case for solids, where residual dipolar broadening is of the order of at least 10 kHz (at least 100 ppm in a i3C spectrum on a 400 MHz spectrometer). A second factor that differs between NMR in solids and NMR in solutionslliquids is chemical shift anisotropy. As chemical shifts are determined by electrons shielding nuclei from the applied magnetic field, they will have a definite directional component because electron densities are not arranged spherically around nuclei. Local molecular motions average this anisotropy to zero in solution, whereas this mechanism is not available to solids. The practical solution to these two difficulties in obtaining solid-state NMR spectra is to use 'magicangle' spinning to simulate motional averaging, and high-power decoupling to minimize dipolar interactions. The so-called 'magic' angle is -54 0 44', and is that angle for which 3cos 28 = I. This is appropriate as the equations describing the angular dependence of both dipolar decoupling and chemical shift anisotropy factors contain the term (3cos 28 - I). Spinning at the 'magic' angle needs to be at frequencies greater than the anisotropy in chemical shifts; for most solids, -3 kHz is Michael J. Gidley is with Unilever Research, Colworth House, Sharnbrook, UK MK441LQ.
Trends in Food Science & Technology August/September 1992 [Vol. 3J
High-resolution solid-state NMR of food materials Michael J. Gidley considered to be sufficient. Residual dipolar interactions are removed by high-power 'H decoupling. Whereas conventional solution-state NMR uses decoupling powers of 5 W or less, removal of dipolar interactions in solids requires typically 50-100 W. The requirement for rapid spinning at a defined angle to the direction of the magnetic field, together with the need to use high decoupling powers, means that nonstandard NMR equipment is needed. However, manufacturers offer the required equipment as accessories to all modem spectrometers. A magic-angle spinning probe and a typical sample holder are shown in Fig. 1. Having minimized broadening effects due to dipolar coupling and chemical shift anisotropy, a choice of spectral acquisition strategies is available. In practice, one technique, cross-polarization, has come to dominate high-resolution 1JC NMR in solids. In this method, magnetization is transferred from 'H nuclei by dipolar coupling to 1JC nuclei. This results in an increase in inherent sensitivity and, because relaxation is via the rapid 'H pathway rather than the slower 1JC route, experiments can be repeated more rapidly, with consequent increases in signal-to-noise ratio. The cross-polarization method is most effective for the least molecularly mobile systems. Where molecular Plobility is greater, a conventional single-pulse acquisition can be used. This forms the most common route to mobility-resolved spectroscopy in solids and is discussed further below. More detail on the principles of high-resolution solid-state NMR and broad descriptions of application areas are given elsewhere 1- s.
Lipids, proteins and carbohydrates Lipids, proteins and carbohydrates form the bulk of carbon-containing components in food systems. In many applications, a solid form is important and, hence, their study by high-resolution solid-state NMR has been investigated. Typical J3C spectra are shown in Fig. 2. The application of NMR methods to food lipids has recently been reviewed6 • High-resolution solid-state NMR spectroscopy has been used to study abundant lipids in seeds 7•8 and crystal polymorphism in triglycerides9 • Oilseed composition has been established using magic-angle spinning and dipolar decoupling with observation either by single 7 or cross-polarization8 pulse strategies. Sufficient molecular information is obtained to suggest that solid-state NMR methods may be useful in the non-invasive analysis of composition in single
©1992, Elsevier Science Publishers ltd, (UKI
231
oilseeds. As NMR is sensitive to chemical effects on the atomic distance scale, it is a powerful adjunct to X-ray diffraction in determining crystalline structures in, for example, triglycerides, which are of great importance in many fat-structured foods. Bociek et al. 9 showed how distinct spectral features can be observed for crystalline
Fig. 1 (a) Magic-angle spinning assembly in a commercial (Bruker) probe; the field in the NMR magnet is in the vertical direction. (b) Magic-angle spinning rotor (Bruker), constructed of ceramic with a polymeric fluorocarbon cap.
232
polymorphs of tripalmitin and tristearin. In addition to permitting the interpretation of chemical shift effects in terms of local molecular structure, the technique provided significant information on the relative mobility of individual sites within polymorphs that was consistent with thermodynamic analyses 10. There appears to be considerable further potential for the fruitful application of high-resolution solid-state NMR to molecularly ordered lipid systems of relevance to foods. The wealth of resonances observed in NMR spectra of proteins (due to the diversity of amino acid compositions and sequences) has been exploited in many outstanding solution studies that have led to complete resonance assignments to a known sequence and detailed three-dimensional structure proposals. Unfortunately the same approach cannot be applied to solid proteins, with the result that spectra are rich in information (Fig. 2) but difficult to interpret other than in broad functional group terms. Due to its importance in foods, gluten has been the subject of high-resolution solid-state NMR studies by a number of groups 11-14. A general conclusion from these reports is that reproducible chemical shift differences that reflect baking quality have not been found. However, information on molecular mobility in powders and hydrates has been obtained that aids the development of appropriate molecular descriptions of gluten functionality. Food carbohydrates have been the subject of several solid-state NMR studies. For low molecular weight materials, crystallinity leads to sharp signals in a crosspolarization solid-state spectrum, whereas noncrystalline forms give broader signals. This is likely to be important in studies of amorphous-to-crystalline transitions, which are a subject of considerable current research interest. Crystalline polymorphism (e.g. in lactosel 5) can be probed by solid-state NMR with the 'rule of thumb' being that a separate resonance should be observed for each inequivalent atomic site within the crystalline unit cell l6 . This can mean multiple resonances for single molecular sites due to asymmetries of molecular packing within the crystal. Such effects are also found in polysaccharides, which, although rarely crystalline, are often present in defined molecularly ordered confonnations (e.g. helices). Examples of multiple resonances include ordered forms of starch l6.17 (Fig. 3) and celluloseI 8 .1 9 • Due to the tendency of polysaccharides to form molecularly ordered but noncrystalline structures, high-resolution solid-state NMR is being applied in many studies, some of which are described below. In general, solid-state NMR studies are most informative in systems with relatively simple major molecular structure features (e.g. triglycerides, simple sugars and many polysaccharides). For more complicated spectral types (e.g. proteins), residual line widths, potential multiple resonances from single sites and the lack of detailed assignment strategies make interpretation very difficult. It is interesting that this is the converse of the situation for solution-state NMR, where the simplicity of spectra for 'simple' molecules reduces information Trends in Food Science & Technology August/September 1992 [Vol. 31
(d)
100
(c)
90
80
70
60
Chemical shift (ppm) 180
160 140
120 100
80
60
40
20
Chemical shift (ppm)
Fig. 3 Ordered and amorphous model starch materials observed by cross-polarization and magic-angle spinning NMR. A-type (a) and
Fig. 2
B-type (b) crystalline forms show clear multiplicity effects at least for
1lC
the anomeric carbon signal (-100 ppm), and all three crystalline
cross-polarization and magic-angle spinning spectra of typical
food lipid, protein and carbohydrate ingredients: (a), tripalmitin;
forms, including the V-type form (c), have chemical shift patterns
(b), ovalbumin; (c), waxy maize starch.
that are distinct from that of amorphous material (d).
content, but complicated spectra (e.g. from small proteins) can be completely assigned and the consequent enormous information content used to derive threedimensional structures.
estimates of crystallinity obtained from X-ray diffraction studies I7 .2o • Spectra of native cellulose have similarly been interpreted as a composite of crystalline and amorphous features l8 , although detailed interpretation is still controversial 19. Although such use of solid-state NMR for the identification and quantification of biopolymer conformations is gaining in importance, it would clearly be beneficial if the relationships between chemical shift and conformation were understood. There is a significant amount of experimental data now available for defined ordered forms of polysaccharides and proteins with simple repeat structures 21 • Increasingly it is becoming clear that probably the major feature contributing to the range of chemical shifts encountered (e.g. 10 ppm for the C-l site in a(l~4)-linked glucans; Fig. 3) is a diversity of local conformations. Effort has therefore turned to examining the effect of conformation-defining torsion angles on chemical shifts 21 - 23 • For proteins, systematic effects of a-helix and ~-sheet local conformations on chemical shifts have been reported 21 . In polysaccharides, a(l ~4) linked glucans have been most studied due to their conformational diversity. Correlations between chemical shift and glycosidic bond conformation have been
Biopolymer conformation As illustrated in Fig. 3, amorphous or differently ordered conformations of the same biopolymer give very different solid-state DC NMR spectra. These provide excellent 'fingerprint' spectra for the identification of ordered states in other environments (e.g. as described below for gels). A general strategy would be to obtain solid-state ]lC NMR spectra for crystalline samples exhibiting X-ray diffraction effects, and use these to identify and quantify molecularly ordered but non-crystalline forms. One example is in the analysis of granular starches, whose solid-state ]lC NMR spectra (Fig. 2) can be modelled by a combination of appropriate ordered and amorphous spectral features (Fig. 3), which not only allow the identification of molecular conformations, but also permits quantification of their relative abundances 17 • This information provides the ratio of doublehelical (ordered) to single-chain (amorphous) populations, which can be compared with quantitative Trends in Food Science & Technology August/September 1992 [Vol. 3J
233
sequences. In particular, single-pulse and crosspolarization experiments often give complementary spectral information 1, with less mobile sites being detected by cross-polarization excitation and more mobile sites by single-pulse excitation. This approach has been used to resolve mobility populations for a range of gluten fractions '2 , and has been particularly informative Mobility-resolved spectroscopy Once signals have been separated in a high-resolution in studies of molecular structure in polysaccharide spectrum, there are two approaches to gaining infor- gels 25 ,26. Figure 4 shows 13C spectra for K-carrageenan in mation on the relative mobility of individual structural the solution form (single-pulse experiment) and the gel features. One involves measurement of relaxation form (using cross-polarization). Highly significant chemicharacteristics of isolated signals, which gives infor- cal shift differences are seen, consistent with the tranmation concerning both local molecular motions, and sition from coil to double helix that is proposed to be rewhether different signals belong in the same relaxation quired for gel formation. A single-pulse experiment on domain - potentially a very powerful means of studying the gel (with magic-angle spinning and high-power demUlti-phase food systems. It is much used in deter- coupling) showed very low intensity, suggesting a single mining the compatibilities of synthetic polymer blends, dominant conformation (presumably the double helix) and has recently been used to show that protein domains in the gel (Gidley, M.J., Darke, A.H. and Ablett, S., unwithin wheat flours are at least 50 A in diameter24 . published). For amylose gels, a different response was Molecular motion within crystalline triglycerides has observed, as shown in Fig. 5. In this case, significant been probed using the interrupted decoupling technique, intensity was observed in a single-pulse experiment without magic-angle spinning. Residual breadth in these which gives spectra enriched in mobile species9 • The second general approach to mobility-resolved signals was shown to be due to chemical shift spectroscopy is to use different excitation pulse anisotropy, as magic-angle spinning reduced signal widths dramatically. The resultant spectrum had identical chemical shifts to those of amylose in solution. The cross-polarization experiment (with magic~angle spinning) gave a totally different spectrum, which was identified by fingerprint matching with a reference spectrum as being due to double helices of the B-type crystalline form (Fig. 5). Together with confirmatory relaxation measurements, this led to a molecular model for amylose gels 25 , involving 'junction zones' of aggregated double helices interconnected by segments of mobile chains that are prevented from undergoing full reorientation by being connected to the network. A third category of polysaccharide gels are those that show intensity in both single-pulse and cross-polarization experiments, with essentially identical chemical shifts but different relative intensity ratios for resonances in the two spectra. Examples of this third category are locust bean gum and konjac mannan gels, where the less mobile regions (as detected by cross-polarization) are enriched in mannose and glucose features, respectively26. The combination of defined conformations and a range of segmental mobility in polysaccharide gels makes high-resolution solid-state NMR a method of choice for the study of underlying molecular mechanisms. reported and have been successfully used to model the C-I resonance for amorphous materiaF3. All of these correlations, however, are empirical; a major challenge is to rationalize these observed effects in terms of chemical shift theory.
70
Examples of application areas
Chemical shift (ppm)
Fig. 4 1le
NMR spectra of lC-carrageenan obtained for 4% solution and gel forms at various temperatures, and using single-pulse and cross-
polarization excitation in combination with magic-angle spinning:
Many applications of high-resolution solid-state 13C NMR have been described in preceding sections. A miscellany of further applications are now discussed, with examples drawn from areas where recent progress has been made as well as from areas of potentially greater application in food science.
6Soe solution with single-pulse excitation; (b), soe gel with soe gel with Starch cross-polarization excitation and magic-angle spinning; (d), -20 oe In addition (al,
single-pulse excitation and magic-angle spinning; (c),
gel with cross-polarization excitation and magic-angle spinning.
234
to identifying ordered forms (Fig. 3) and quantifying the ratio of double helical to amorphous
Trends in Food Science & Technology August/September 1992 [Vol.
31
residues in granular starch 17 , it has now been shown in a multi-technique study that individual double helices are the key structural stabilizer in starch granules, and that the enthalpy of gelatinization is directly related to the content of double helices 20 . In light of this result, solidstate NMR studies of starches and starch-containing foods are predicted to become more widely exploited. Many cereal starches contain a small (typically less than 1% dry weight) amount of lipid within the granules. It has long been debated whether this lipid is present as a complex with amylose or in a free state. Recent solidstate 13C NMR spectra strongly suggest that lipids are present predominantly as amylose complexes, at least in barley starches 27 •
(a)
(c)
Flours and doughs A recent study of wheat flours and doughs24 has opened up several lines of enquiry into the mechanisms involved in flour and dough hydration, and in particular into the difference between hard and soft wheat flours. Much greater mobilization/plasticization of protein is observed in soft wheat flours than in hard wheat flours, with correspondingly greater mobilization/plasticization of starch in hard than in soft flours 24 . Such mapping of water relationships could potentially be extended to more molecular detail and is clearly important in understanding the effects of varietal flours on baking characteristics.
(d)
110
100
90 80 70 Chemical shift (ppm)
60
Fig. 5 llC spectra for a 10% (w/v) amylose gel using single-pulse excitation without (a) and with (b) magic-angle spinning, or cross-polarization excitation with magic-angle spinning (c). Spectrum (d) was obtained using cross-polarization excitation and magic-angle spinning on solid B-type crystalline amylose.
Plant cell walls Cell walls have a major impact on the texture and processing characteristics of edible plants. As they are composed primarily of polysaccharides, augmented in some cases by structural proteins and phenolics, plant cell walls should be amenable to study by high-resolution solid-state 13C NMR. This possibility has been experimentally realized, with spectra being obtained from celery strands 28 and from intact and fractionated cell walls of bean sprouts 29 . There is significant potential in this area, as earlier relaxation studies identified domains within cell walls 30 that could conceivably be probed by mobility-resolved spectroscopy - as could ripening-related changes in, for example, apples 31 .
Frozen systems An early practical demonstration of high-resolution solid-state 13C NMR involved low-temperature observation of room-temperature liquids 32 . Variable temperature operation is possible on standard spectrometers with 'solids' accessories and, hence, there is no practical barrier to obtaining spectra. It is perhaps surprising, therefore, that little work has been reported in this field. Two relevant applications of 'frozen' 13C NMR are in assessing the range (rather than the motional average as observed at room temperature) of conformations in solutions of oligo- and polysaccharides33 , and in immobilizing chain segments within polysaccharide gels so that all of the polymer is detected in a cross-polarization experiment 25 . Given the commercial importance of Trends in Food Science & Technology August/September 1992 [Vol. 3J
frozen food systems, it is anticipated that this will be a growth area for high-resolution solid-state 13C NMR spectroscopy.
References 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Harris, R.K. (1983) Nuclear Magnetic Resonance Spectroscopy: A Physicochemical View, Pitman Sanders, J.K.M. and Hunter, B.K. (1987) Modern NMR Spectroscopy, Oxford University Press Yannoni, CS. (1982) Acc. Chern. Res. 15, 201-208 Maciel, G.E. (1984) Science 226,282-288 Chmelka, B.F. and Pines, A. (1989) Science 246, 71-77 Eads, T.M. and Croasmun, W.R. (1988) J. Am. Oil Chern. Soc. 65, 78-83 Rutar, V. (1989) J. Agric. Food Chern. 37, 67-70 Haw, J.F. and Maciel, G.E. (1983) Anal. Chern. 55, 1262-1267 Bociek, S.M., Ablett, S. and Norton, LT. (1985) J. Am. Oil Chern. Soc. 62,1261-1266 Norton, LT., Lee-Tullnell, CD., Ablett, S. and Bociek, S.M. (1985) J. Am. Oil Chern. Soc. 62, 1237-1244 Schofield, J.D. and Baianu, I.C (1982) Cereal Chern. 59, 240-245 Belton, p.s., Shewry, P.R. and Tatham, AS. (1985) J. Cereal Sci. 3, 305-317 Moonen, J.H.E., Hemminga, M.A. and Graveland, A. (1985) J. Cereal Sci. 3, 319-327 Ablett, S., Barnes, D.J., Davies, AP., Ingman, S.J. and Patient, D.W. (1988)]. Cereal Sci. 7,11-20 Earl, w.L. and Parrish, FW. (1983) Carbohydr. Res. 115,23-32 Marchessault, R.H., Taylor, M.G., Fyfe, CA and Veregin, R.P. (1985) Carbohydr. Res. 144, C1 -C5 Gidley, M.J. and Bociek, S.M. (1985) J. Am. Chern. Soc. 107, 7040-7044
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1B 19 20 21 22 23 24 25
Atalla, R.H. and VanderHart, D.L.11984) Science 223, 283-285 Horii, F., Hirai, A. and Kitamaru, R. 11987) Macromolecules 20, 2117-2120 Cooke, D. and Gidley, MJ (1992) Carbohydr. Res. 227, 103-112 Saito, H. (1986) Magn. Reson. Chem. 24, 835-852 Veregin, R.P., Fyie, e.A., Marchessault, R.H. ano Taylor, M.G.119S7) Carbohydr. Res. 160, 41-56 Gidley, MJ and Bociek, S.M. (1988) J. Am. Chem. Soc. 110, 3820-3829 Garbow, J.R. and Schaeier, J. (1991) J. Awic. Food Chem. 39, 877-880 Gidley, M.J. (1989) Macromolecules 22, 351-358
26
Gidley, I'v\.I., McArthur, A.I. dnd Underwood, D.R.119911 Food Hydrocolloids 5, 129-140
27 2B 29 30
Morrison, W.R., Tester, RJ. and Gidley, M.I. em'al Cherll. lin press) larvis, M.c. and Apperley, D.e. (19901 Plant Physiol. 92. 61-65 Jarvis. M.e. 119901 Carbohydr. Res. 201. 327-333 Mackay, A.L., Wallace, I.e.. Sasaki. K. and Taylor, I.E.P. 119881 Biochemistry 27. 1467-1473 Irwin, P.L., Pietier. P.E., Gerasimowicz. W.V .• Pressey. R. and Sams. e.E. 119841 Phvtochemistry 23.2239-2242 Fyie. e.A., Lyerla, I.R. and Yannoni, e.S. (19781/. Am. Chem. Soc. 100, 5635-5636 Gidley, M.I. and Bociek, S.M. 119881 Carbohvdr. Res. 183, 126-130
31 32 33
Review
Applications of
NMR in •
sensory sCience
sensory science IS In its infancy. New applications are just waiting to be explored.
Advantages of NMR
Terri Robertson, Shelly Schmidt and Barbara Klein NMR spectroscopy is already of great value in determining the relationships between a molecule's structure and its odor, flavor, color and textural characteristics. Newer applications of NMR techniques in sensory science include studies of the effects of ion and water binding on taste perception; for example, 2lNa NMR has been used to examine the effects on perceived saltiness of Na+ binding to ionic and non-ionic gums. The range of useful applications is expanding as both NMR and sensory science techniques continue to develop,
Sensory science uses formal experimental designs and statistical methods to evaluate the perception of foods and materials by sight, smell, taste, touch and hearing'. An important aspect of sensory science is the study of the relationships between the physicochemical properties of a molecule and its flavor, texture and/or odor perception. NMR spectroscopy and imaging are powerful tools that have been used to examine a variety of physicochemical features (e.g. molecular structure, conformation and dynamics) of diverse systems (e.g. solutions, suspensions and solids) at the molecular level, by probing NMR-active nuclei such as 'H, 11C or 21Na. As in many fields, the application of NMR techniques in Terri Robertson, Shelly Schmidt and Barbara Klein are with the Division oi Foods & ,"utrition, University oi Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801, USA.
236
NMR spectroscopy and imaging are non-invasive, nondestructive techniques. Thus, NMR provides the opportunity to view a system at the molecular level and then use the same sample for other physicochemical measurements and sensory testing by subjects. Most foods may be analysed by NMR techniques, which can be applied to solid, semisolid or liquid samples. Additionally, numerous isotopes can be studied; 'H and "C are the most frequently studied. To date, molecular chemists and food scientists have used NMR to identify the structures and conformations of molecules with known tastes. The tastes that have received the greatest attention are bitterness, sweetness and saltiness. Traditionally NMR spectroscopy is complemented by ultraviolet (UV) or infrared (IR) spectroscopy, mass spectrometry, X-ray crystallography and/or conformational energy calculations in the identification process. More recently, the dynamics of a food system, such as molecular mobility, competitive ion binding and water binding, as determined by NMR, have been related to taste and texture perception as analysed by sensory analysis techniques.
Bitterness The structures and conformations of bitter-tasting compounds found in citrus fruits2-'), zucchinis and beer!> have been determined by NMR. In citrus fruits, naringin is a bitter flavanone glycoside that includes the disaccharide rhamnose. To determine the link between rhamnose and bitterness, Kamiya et al. 2 synthesized six flavanone glycosides with (I ~2)-linked disaccharides. The structures and conformations of these compounds were confirmed by 'H NMR, UV and IR spectroscopy. A taste panel composed of five young women matched the bitterness of the naringin control to concentrations of the synthetic compounds. The authors reported that when the a-L-rhamnosyl residue in the naringin molecule was replaced by either a-D-glucosyl or Trends in Food Science & Technology August/September 1992 IVol. 31