High-pressure small-angle neutron scattering for food studies

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Colloid & Interface Science

High-pressure small-angle neutron scattering for food studies Susana C. M. Teixeira1,2 Abstract

This article highlights contributions and the tremendous potential of high pressure (HP) small-angle neutron scattering for our understanding of biopolymer stability and phase behavior, in the context of nutrition and food properties. The use of HP processing as a nonthermal sterilization technique is well established in the food industry, and many other applications are emerging in recent years. Green chemistry, consumer preferences, and nutritional trends push for further developments, which require a database of experimental data. Unbiased studies on pressure-induced effects on colloids and amphiphiles are paramount for the development of new food molecules and methodologies. Biopolymer phase diagrams are described, with an emphasis on proteins. HP small-angle neutron scattering research capabilities and future directions are discussed. Addresses 1 Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA 2 NIST Center for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, USA Corresponding author: Teixeira, Susana C.M. ([email protected])

Current Opinion in Colloid & Interface Science 2019, 42:99–109 This review comes from a themed issue on X-Ray and Neutron Scattering Edited by Jeff Penfold and Norman J. Wagner For a complete overview see the Issue and the Editorial https://doi.org/10.1016/j.cocis.2019.05.001 1359-0294/Published by Elsevier Ltd.

Keywords Small-angle neutron scattering, High pressure, Food processing, Cold denaturation.

Use of high pressure in food science and technology Sterilization: safer, better tasting food

D. Papin was first reported to deliberately use pressure cooking in 1679, when he invented the steam digester. In the late 19th century, Hite [1] investigated the use of high pressure (HP) for the preservation of milk, fruits, and meat. Hite was seeking alternatives to chemical and thermal sterilization, which alters food

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and its organoleptic properties. The seminal work by Bridgman [2] on the coagulation of albumin then brought the hen egg to center stage. To this day, hen eggs are recognized as a food matrix with applications as an emulsifier and foaming agent, as well as in gelation. The pursuit of additive-free food excellence has only intensified, fueled by modern consumer preferences that drive the consumer market like contemporaries of Hite and Bridgman could not. For example, HP is an equally viable intervention for prominent food-borne pathogens associated with raw bivalve shellfish and crustacea, where it is also used to assist shucking [3]. The future costs and sustainability of food processing technology are beyond the scope of this review. The present reality is a booming market for HP products across the globe. Pressures typically under 1 GPa increase the shelf life of liquid and solid foods with a high content of moisture. Toward ‘greener’ food

In the early 2000s, not long after ionic lipids were first suggested as a ‘greener’ substitute for conventional solvents, high hydrostatic pressure-assisted extraction entered the food industry arena as an environmentfriendly technology. High hydrostatic pressureassisted extraction is energy efficient and improves yields of extraction, by enhancing the mass transfer of solvents into materials without heat degradation. Polyphenols, ascorbic acid, carotenoids, and other natural ingredients are cold extracted from herbs, dietary plants, and fruits at 100e1000 MPa [4]. Thermodynamically stable supercritical fluids (SCFs), such as SCF H2O or CO2, are alternatives to damaging solvents in industrial processes [5]. SCFs have low viscosity and dielectric constants that can be tuned by the operating pressure and temperature for higher selectivity. SCFs penetrate the pores of heterogeneous matrices more efficiently because of the absence of surface tension, at pressures typically below 500 MPa. In 2015, when the state laws in the US (and elsewhere) were taking the first steps toward banning single-use plastics, Geyer et al. [6] reported that only 9% of plastics are recycled. SCF extraction can be used to remove contaminants from post-consumer polyolefin plastics, making them suitable for direct food contact applications such as packaging [7].

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The good and bad sides of high pressure

Despite no direct effect on covalent bonds at the range typical of food processing, by altering the enzymatic activity, HP may result in chemical changes. Predictably, because it inactivates pathogenic cells, yeasts, spores, or viruses, pressure causes changes on all food biopolymer structures and dynamics. Above w200 MPa, phase transitions, interface separation, and membrane protein unfolding collapse the cell membrane structure. Lipids are largely the most HPsensitive biopolymers, undergoing changes in density, crystallization kinetics, and melting temperature shifts of þ10  C to þ20  C per increase of 100 MPa [8]. HP processing of dietary fiber improves cholesterol sorbent capacity, digestibility, and stability [9]. In contrast, processing of meats accelerates lipid oxidation at pressures of 300e500 MPa, which induce liberation of radicals and oxidation-prone lipids upon membrane breakdown [10]. Triacylglycerols and cholesterol-derivative lipids are particularly susceptible to oxidation. Such issues can however be circumvented by combinations of temperature and HP cycles, shown to improve meat texture while prolonging the shelf life. Proteins and polysaccharides are also pressure sensitive, which makes them amenable to HP processing. Starch is a known example that is composed of amylose and branched amylopectin; starch granules are concentric shells of alternating semicrystalline and soft amorphous layers. Starch gelatinization is a major process in the food industry, providing textural quality and a longer shelf life. Compared with thermal treatments, HP causes limited granule swelling and poorer leaching of amylose, ensuing more homogeneous gelatinization [11] and emulsions. Food emulsions can incorporate bioactive ingredients from natural sources, which are popular in the functional food market. Starch and other carbohydrates are used in many emulsions, interacting with proteins such as whey proteins, b-lactoglobulin, and bovine serum albumin [12]. HP processing acts on the structure and deformation of protein/polysaccharide complexes and consequently the rheological, texture, and stability properties. Pressure promotes the ionization of carboxyl groups in both proteins and polysaccharides, and it causes pH shifts that drive the interactions toward negatively or positively charged polysaccharides, depending on the protein isoelectric point (pI). In general, HP favors the formation of charged species, where increased electrostriction by newly formed charges decreases the total molar volume of water. The impact of pH on food cannot be overemphasized as it can dictate color and taste, and it influences HP processes such as SCF extraction, chemical reactions (e.g. Maillard browning), and other applications to biopolymers [13]. Even in the presence of natural or pH Current Opinion in Colloid & Interface Science 2019, 42:99–109

regulators, acid dissociation constants (Ka) for all food components, not in the least water, depend on both pressure (P) and temperature (T), as approximately described by Plank’s equation: 

d ln Ka dP

 ¼ T

DV RT

(1)

where R is the gas constant (8.3145 J K1 mol1), and the reaction volume, DV (mL/mol), is the difference in the partial molar volumes of the acid and the ionized products in equilibrium. Acidebase equilibria are shifted toward negative DV, according to Le Chatelier’s principle, to counteract the effect of pressure (see Table 1). Anionic buffers tend to have negative and relatively large reaction volumes: phosphate undergoes a decrease of w0.4 pH units for every 100-MPa pressure rise [14]. On the other hand, cationic and zwitterionic buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES) or 2-amino2-hydroxymethyl-propane-1,3-diol (TRIS), are able to maintain their buffering abilities relatively well under pressure. Considering the typically complex nature of food matrices, the choice of buffer to be used under HP requires careful consideration on the parameters of interest. Considering the typically complex nature of food matrices, the choice of buffer to use under HP requires careful consideration on the parameters of interest. Quinlan and Reinhart [15] have designed baroresistant buffer mixtures that can be used to minimize both HP- and temperatureinduced pKa shifts, particularly suitable for pH-sensitive and cold denaturation studies. The percentage of D2O in the bufferdoften used for nuclear magnetic resonance (NMR) or small-angle neutron scattering (SANS) techniquesdshould also be taken into consideration when setting up the pH/pD of a solution for HP (for a discussion see Rubinson [16]). Table 1 Effects of pressure and temperature on common biological pH buffers. Common Useful pH range buffer name (25  C)

Acetate MES Imidazole Phosphate (dibasic) HEPES TRIS

DV (mL/mol)

pKaa 4  C 25  C 37  C

3.6–5.6 5.5–6.7 6.2–7.8 5.8–8.0

4.77 6.33 7.37 7.26

4.76 6.10 6.95 7.20

4.76 5.98 6.71 7.16

−11.2 ([35]) +3.9 ([94]) −2.0 ([35]) −23.8 ([35])

6.8–8.2 7.5–9.0

7.77 8.80

7.48 8.06

7.31 7.70

+9.4 ([94]) +1.0 ([35])

HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MES, 2-(Nmorpholino)ethanesulfonic acid; TRIS: 2-amino-2-hydroxymethylpropane-1,3-diol . Note that common food additives, known in Europe for their ‘E’ number, include acidity regulators: for example, E260-E269 (acetates) and E340349 (phosphates). a

At 100 mM (Biological Buffers, URL: http://www.applichem.com/ fileadmin/Broschueren/BioBuffer). www.sciencedirect.com

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HP studies of food biopolymers The energy landscapes

HP is increasingly used to investigate the architecture and energetics of biopolymers (typically 0.5e 500 MPa). Pressure triggers a reduction in volume, achieved at various levels through changes in morphology, packing, and shortening of interatomic distances. For oligomeric proteins, dissociation occurs at relatively lower pressures (usually < 200 MPa), favored by extended hydration of interfaces. The mechanisms behind HP-induced effects have been controversial, for protein unfolding in particular, but it is now clear that rationalizing unfolding of biopolymers requires model compounds with higher levels of complexity [17]. Protein unfolding equilibrium can be given using a two-state thermodynamic description first used by Hawley [18], whereby a second order expansion of the free energy, DG, is defined around a reference point T0, P0 (usually 295 K, 0.1 MPa): DG0 þ DV0 ðP  P0 Þ  DS0 ðT  T0 Þ þ DaP ðP  P0 Þ DCP DbT ðT  T0 Þ  ðT  To Þ2 þ ðP  P0 Þ2 ¼ 0 2T0 2 (2) where DV0 is the reaction volume and CP is the heat capacity. fP is the expansion coefficient, and the compressibility coefficient, bT, quantifies the ability of a protein to undergo an HP-induced change in bulk volume. Eq. (2) may describe a phase diagram of an elliptical, parabolic, or hyperbolic shape when projected onto the P-T plane. At present, most experimental data on protein phase diagrams are consistent with elliptical-shaped equilibrium boundaries, for which the mathematical constraint is as follows:

D a2P T0  DCP DbT >0

(3)

DCP is positive and relatively large for proteins, so Eq. (3) imposes a decrease in compressibility upon unfolding. Figure 1 shows P-T diagrams for proteins, nucleic acids, and a polysaccharide. Disruption of microbial lipidic membranes during HP sterilization of food leads to the loss of components from the cell, including nucleic acids. No significant health risk has been associated with ingestion of nucleic acids, but interest in pressure effects on their structure does not end there. How do microorganisms rapidly acquire resistance to pressures in the GPa range [19]? Why are interactions between nucleic acids and proteins disturbed at pressures that match the upper pressure limit for microbial growth [20]? There is much yet to learn. Phase diagrams of nucleic acids are arguably the most understudied of all biopolymers. The Gibbs free energy at equilibrium for nucleic acids can also be approximated by a quadratic equation, in reference to the helixecoil transition [21]. HP effects vary significantly with the melting temperature TM at which the helical structure unwinds. The literature commonly reports on nucleic acids with TM > 50  C, in the range of 263e393 K, where the thermal stability of duplexes increases up to 200 MPa, and decreases above 200 MPa (Figure 1). The double helix dissociation into a single-stranded structure is characterized by a small positive DV0 of 1e3 ml/mol (cf. for globular proteins, DV0 -20 to 200 ml/mol [22]). The single-strand form can however be stabilized by base-stacking, hydration [23], and entropic contributions of w23 cal mol1 K1 per nucleotide [24]. The elasticity of nucleic acidsdreflected in HP-induced A-

Figure 1

Nonmonotonic transition phase diagrams for biopolymers present in food. Proteins: generic illustration of equations (2) and (3), within typical P-T ranges, where Df determines the orientation of the ellipse. DS = 0 at the maximum pressure for stability, when the slope of the tangent to the ellipse is zero. At the highest temperature for stability, DV = 0 where the slope of the tangent is infinite. Nucleic acids (adapted by permission from [21], Copyright 2001 American Chemical Society): dependence of the phase diagram on Tm, with a turning point for all slopes ~200 MPa, across the temperature range. Starch: (adapted by permission from Springer Nature: J. Verbraucher. Lebensmittel. [102], Copyright 2010) phase transition lines between the gelatinized and liquid polysaccharide, for starch-water suspensions after ~15 min of isothermal/isobaric treatment. www.sciencedirect.com

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DNA to -Z-DNA [25] and B-DNA to -Z-DNA [26] transitionsdis doubtless central to the microorganism’s survival at HP. As shown in Figure 1 for starch, a phase diagram can also be drawn for polysaccharides by considering the process of gelatinization. For wheat in particular, HP treatment decreases the gelatinization temperature of starch granules. Once more, experimentally observed P-T effects have been interpreted using a second-order model very similar to that described by Eq. (2) [27]. A discussion on HP gelatinization mechanisms, still not clearly understood, can be found in [28]. Despite widespread use of the elliptical description for phase diagrams of various biopolymers, it is based on approximations shown to fail for various studies. For myoglobin, an enzyme involved in lipid oxidation in meat, the phase boundary shows distortions from the elliptical shape [29]. Eq. (2) is a truncation of thirdorder derivatives of DfP, DbT, and DCP, which introduce P-T dependence and is particularly fallible at high T, P [30]. The assumption of the two-state equilibrium also does not hold for reports of irreversible changes under pressure, such as the denaturation of ovalbumin [31] or the misfolding of b-lactoglobulin. Furthermore, a hyperbolic phase diagram was recently described by Klamt et al. [32], from 1He15N NMR measurements on a recombinant Zn finger protein, apoKti11. The study reports significantly populated folded/unfolded states and favorable slow exchange for the NMR chemical shift timescale. HP-induced pH changes are a potential source of unaccounted destabilization, but the authors limit shifts to a maximum of w1 pH unit at the highest pressure used (240 MPa). In their conclusions, Klamt et al. [32] suggest that above 200 MPa: “[.] the volume of the native state drops below the unfolded state and the equilibrium shifts back toward the native state with increasing pressure.” These findings are however compatible with the presence of a dry molten globule intermediate, a more compact ‘native-like’ structure with an HP-stabilized secondary structure, often reported in HP denaturation [33]. Despite measurement of the thermodynamic parameters in Eq. (2) at numerous P-T points, contributions from various molecular determinants to observed changes in volume remain difficult to evaluate (even for a relatively small protein such as the aforementioned apoKti11). Both the solvent-excluded volume VSE and the hydration volume Vhyd contribute toward the total volume occupied by a protein. Vtotal ¼ VSE þ Vhyd ¼ ðVcavities þ VVDW Þ þ Vhyd

(4)

where VSE accounts for solvent-excluded cavities, Vcavities, and the total van der Waals volume VVDW of the polymer. At atmospheric pressure, cavities are stabilized because the Current Opinion in Colloid & Interface Science 2019, 42:99–109

formation of 4-H-bonded water clusters at apolar surfaces would decrease entropy [34]. Up to z 300 MPa, pressure destabilizes bound water clusters, removing the entropic penalty and making exposure of apolar residues more favorable. In contrast, above z300 MPa, hydrophobic interactions are enhanced by the higher compressibility of free water compared with that of bound water [35,36]. Vcavities can amount up to 30% of Vtotal, yet volume changes under HP can be positive, and typically only 4% to þ0.5% relative to Vtotal [37]. Chen and Makhatadze [17] have shown that, for the native ensemble in particular, solventexcluded volume scales linearly with protein size. The balance between VSE and Vhyd in Eq. (4) determines the polymer-specific effects of HP. Filling of cavities with protein side-chains ensuing HP-induced structural relaxation has been shown to play a role in volume changes [38]. It is currently accepted that the dominant drive for unfolding is the filling of solvent-excluded cavities [39], irrespective of the size of the polypeptide chain [40]. SANS and the kinetics of HP-induced transitions

Protein folding spans a broad range of time regimes. Near unfolding conditions, kinetic effects are known to cause equilibration to take hours [41]. Slow kinetics is not an issue for SANS techniques, being nondestructive and extremely sensitive to the formation/dissociation of oligomers or aggregates. This is a significant advantage when working, for example, with radiation-sensitive heme iron proteins (e.g. HP-SANS study on a form of myoglobin, a protein present in meat [42]). In situ HP small-angle x-ray scattering (SAXS) studies are at a disadvantage from this viewpoint because of the rapid onset of radiation damage [43] and the technical difficulties associated with a flow cell under the pressure ranges of interest. Combined, HP-SAXS and HP-SANS are however able to cover a broad range of kinetics and provide information on a paramount aspect of biopolymer stability: the hydration layer [44] and proteinesolvent interactions. Small-angle scattering techniques measure radii of gyration (Rg)doften insufficient to assess volume differences within an ensemble [45]dbut also pair distribution functions, using no prior structure information and irrespective of the size of the molecule. For food studies, it is important to recall that contrast matching is a critical advantage of SANS. At the relatively low resolution at which SANS techniques operate, the difference in average atomic composition of each biopolymer is reflected by a characteristic neutron scattering length density (SLD). Contrast is provided by the difference between the neutron SLD of a component of interest and that of the solvent [46]. Using isotope labeling, or simply variations of the percentage of D2O present in the solution, contrast of some components can be matched to the solvent to minimize its contribution. By deconvoluting contributions in multicomponent phase diagrams, contrast aids assignment of the data, for example, in proteinelipid interactions at the oilewater interface in food and foam emulsions [47]. www.sciencedirect.com

HP small angle neutron scattering for food studies Teixeira

Early in this century, the first measurements of DV and DbT for HP-induced protein unfolding were measured by densitometry [48]. At present, most state-of-the-art biophysical techniques adopted pressure instrumentation. HP circular dichroism (CD) studies were enabled by compatible materials for pressure-resistant lenses [49], while Fourier-transform infrared (FTIR) [50], fluorescence [51], and light scattering studies [52] under pressure are carried out on a regular basis. Pressure perturbation calorimetry is now also providing key results on protein and phospholipid phase transitions, as well as interactions of biomolecular systems [53]. Typically requiring smaller amounts of sample and being available at in-house facilities, spectroscopic techniques are poised for screening of HP-induced kinetics, complementary to NMR and scattering data. HP effects on kinetics are described by reaction rates k and activation volume DVs: 

 d ln k DV s ¼ dP T RT

(5)

Reactions characterized by negative DVs will be accelerated by pressure, for example, to assist enzymatic activity in the food industry [54]. Those with positive DVs will be slowed down, enabling, for example, HPextended shelf life of fruit [55]. For protein folding/ unfolding, pressure jump [56,57] and HP stopped-flow experiments [58] have shown that pressure decreases kfolding and increases kunfolding, by lowering the reconfiguration diffusion coefficient. Diffusion of the polypeptide chain, as it adopts its final structure, is slowed down, effectively extending the lifetime of metastable transition state ensembles and enabling their study [59]. Understanding the formation of such transition states is central to processes on prion diseases such as bovine spongiform encephalopathy, attributed to misfolding followed by ordered aggregation. Ingestion of contaminated meat from cattle with bovine spongiform encephalopathy can cause CreutzfeldtJakob disease in humans [60]. Prion proteins have been shown to become prone to aggregation when their reconfiguration rate is slow enough to expose hydrophobic residues on the same timescale that bimolecular association occurs [61]. Cold denaturation

Processing, storage, and transport require stability of foodstuff. It is no small deed, given that protein stability is only marginal, with typical DGunfolding w5% of the interaction energy of a single CeC bond [62]. As drawn in Figure 1, for proteins, the two dashed lines (DS = 0 and DV = 0) divide the elliptical diagramin 4 regions, where 3 are highlighted with arrows. When DS>0 and DV<0, pressure denaturing can occur at high P and high T. Because it is more Because they are more difficult to www.sciencedirect.com

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access experimentally, such aggressive conditions are equally avoided in food industry. Heat denaturationda.k.a. thermal denaturation or high temperature meltingdis well known in cooking and food processing and occurs in the phase region where DS>0 and DV>0. The denaturation temperature Tm of biopolymers is typically under boiling conditions and tends to promote unfolded states that are key intermediates in aggregation processes, often irreversible. As mentioned in Section The energy landscapes, high T and P introduce pressure and temperature dependencies of thermodynamic parameters. Upon heat denaturation, the simultaneous change of enthalpy of unfolding and heat capacity break the linearity between changes observed for Tm and changes in free energy [63], making experimental results difficult to fit accurately. In contrast, cold denaturation happens at low temperatures, rationalized by weaker hydrophobic interactions together with solvation effects [62]. Cold denaturation is of practical interest for the food industry. Protein destabilization and aggregation can be triggered during frozen storage or freeze thawing, as well as the cryoconcentration of fruit juices [64], in the context of lipid oxidation of frozen meats and changes underlying storage or transport at low temperatures [65]. The Gibbse Helmholtz equation describes unfolding at constant pressure.   T þ DCP ðT  Tm Þ DGðT Þ ¼ DHm 1  Tm   T  T DCP ln Tm

(6)

where DG(Tm) = 0. Cold denaturation is a consequence of the large positive DCP for unfolding of proteins, which introduces a strong curvature in DG(T). Cold denaturation typically occurs at subzero temperatures: few proteins have cold and heat denaturation temperatures above freezing (such as the apoKti11 example introduced in Section The energy landscapes). At freezing temperatures, the presence of ice alters samples and limits the techniques that can be used. To artificially shift Tm toward temperaturesabove 273 K, different perturbation techniques have been deployed, and a comparison of the corresponding results offers comprehensive insight [40]: the addition of chemical denaturants, pH shifts, reverse micelle encapsulation [66], or pressure. HP-assisted cold denaturation takes advantage of the phase diagram of water, which remains liquid at w200 MPa and down to 20  C. Furthermore, recent studies detected cold-mediated aggregation, even in the absence of freezing stresses, suggesting a useful alternative to heat stress for extrapolating predictions of protein shelf life at refrigerated conditions [67]. Data on cold denaturation, still scarce at present, will support our understanding of the mechanisms involved and the development of unifying models for pressure and thermal denaturation [68]. Experimentally, the importance of temperature control Current Opinion in Colloid & Interface Science 2019, 42:99–109

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must be underlined when carrying out HP measurements. Thermal equilibration is slow, and volumetric properties are sensitive to temperature. Here too, the non-destructive nature of SANS techniques offers the advantage of probing for changes until equilibration is reached. HP-SANS instrumentation

HP-SANS sample environments are by no means new at neutron centers [69]. Albeit there are numerous contributions from coherent and incoherent scattering studies of biopolymers (e.g. see [70e74]), these are still scarce compared with reports from NMR, SAXS, or spectroscopic techniques. SANS studies require allocation of beamtime at the reactor or spallation neutron centers, where HP cells were typically not appropriate for weak scatterers with limited sample availability, often the case for biopolymers. Combined with a significant lack of supporting information to guide the design of neutron experiments, notably on kinetics, HPSANS studies were hampered by large sample amounts (hundreds of mg), unknown equilibration times, and difficult or ambiguous data interpretation. The two last decades have seen a significant change in this scenario. HP capabilities were adopted by complementary techniques, as mentioned in the Section SANS and the kinetics of HP-induced transitions, providing a holistic view by monitoring different structural parameters and the validity of the two-state equilibrium, such as the local environment observable through tryptophan fluorescence, the secondary structure content observable by FTIR or CD, or the tertiary/quaternary structures observable by small angle scattering. Developments in biological SANS in the last 10 years [75] transformed the technique intoa natural tool for food sciences [76]. Extended pressure capabilities at synchrotron facilities, for both SAXS [77] and crystallography studies [78], have equally contributed to a renewed awareness of the ability of pressure to assist

structural and dynamic studies. In the context of the boom of HP technology in food and pharmaceuticals, HP-SANS is now ideally placed to contribute essential information. Both academic groups and in-house teams at neutron facilities have contributed toward the buildup of sample environment suites around the world. Table 2 lists HP-SANS instrumentation suitable for the study of food biopolymers, which, albeit incomplete, highlights the experimental parameters currently accessible. It should be noted at this point that the success of HP techniques is highly reliant on both careful control of P-T conditions, and on giving samples enough time to equilibrate once a new P-T environment is applied. Neutron scattering allows for probing of equilibration, so that studies of hysteresis in reversibility cannot be biased. HP cell design for SANS takes into consideration the balance between the resolution needed and the highest pressure achievable. The smallest real space distance measurable (z2p/q) is related to the magnitude of the momentum transfer q: q ¼ ð4p=lÞsin q

(7)

where l is the neutron wavelength, and 2q is the scattering angle. SANS techniques can deploy wavelengths, or wavelength bands for time-of-flight approaches, that cover a very broad q range. Sample volumes are a critical parameter for both SAXS and SANS. With X-ray radiation, the exposed volume of a stopped-flow HP cell plays into the impact of radiation damage [43], while for neutrons, the relatively weak brightness of the beam imposes minimum illuminated volumes and/or concentrations. The strongly penetrating power of neutrons affords the use of a broad range of materials for HP cells. Pressures up to 300 MPa are easily achievable using pressure cells where neutrons illuminated and scattered by the sample go through parallel high-transmission windowsdthe so-called

Table 2 Examples of HP-SANS cells for solution studies of biopolymers.

JRR-3M, Japan LLB, France LLB, France LLB, France PSI, Switz. FRM-II, Germany Quokka, Australia NIST, USA HFIR, USA

qmax (Å−1)

Pmax (MPa)

T (K)

Particular features and comments

0.03 0.4 0.26 0.2 0.35 0.23 0.3 0.3 0.15

200 300 530 600 500 500 350 350 200

273–523 RTa RTa 277–363 277–353 277–353 RTa 253–338 283–473

Low or high viscosity solutions [95] Sample volume 6 mL [79] Sample volume 240 mL [96] Metallic windows [80] In-situ light scattering, P-jumps [97] PSI-based cell design [98] Sample volume 50 mL [99] Subzero T, sample volume 3.5 mL [100] High T corrosion resistant [101]

HP-SANS, high-pressure small-angle neutron scattering; RT, room temperature; NIST, National Institute of Standards and Technology, PSI, Paul Scherrer Institute, HFIR, high-flux isotope reactor. a

Typically controlled with a thermostatic bath to prevent heating. It should be noted that the q-max values reported are relative to the highest reported experimental data and may differ slightly for different experimental settings.

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HP small angle neutron scattering for food studies Teixeira

flat geometrydwhich are typically made of sapphire. Cylindrical designs for metallic alloy cells also exist, with broader measurable 2q, but this geometry proved to be less well adapted for HP-SANS [79]. A potential alternative was recently proposed for a flat geometry with metallic windows, reaching 600 MPa [80]. Probing of more HP-resistant protein phases will be enabled with higher pressures. Glucose isomerase, for example, a potential secondary standard for HP-SANS studies [81] with a large market in the food industry, is known to be relatively resistant to pressure. HP-SANS cells with the ability for simultaneous spectroscopic measurements, such as the Paul Scherrer Institute (PSI) cell in Table 2, will provide powerful sensitivity at local and quaternary structure levels alike, while ensuring that measurements are obtained from the same sample.

Highlighted applications of HP-SANS to food Milk is the archetypical food emulsion, both from the nutrition and technology viewpoints. Its constituent biopolymers have functionalities and several commercial applications in HP processing [82]. Casein micelles (CMs) illustrate well the challenges faced by research in milk and dairy products. Various models have been proposed and refined for CMs, to provide a mechanistic understanding of renneted milk gels or the rheological behavior of concentrated micellar suspensions. CMs are stabilized by intermolecular protein hydrophobic interactions and ionic interactions between phosphorylated residues of f-caseins and b-caseins with calcium phosphate. The latter arrests amorphous calcium phosphate before it can mature into higher ordered structures. The resulting colloidal nanoclusters are distributed in a highly hydrated network within the micelle, which is coated with a k-caseineenriched surface. Jackson and McGillivray [83] carried out HP-SANS studies on CMs, up to 350 MPa. Contrast variation was used to assign scattering measured in a q-range covering real space distances of z3 nm to 6 mm. Scattering intensities of dilute CM solutions with different percentages of D2O were measured [84]: IðqÞ ¼ f Vp Dr2 PðqÞ SðqÞ þ B

(8)

where Dr is the difference between the SLDs of the CM and the solvent (effectively, the contrast above the background B), f is the volume fraction of CM, VP is the volume of CM particles, and P(q) is the normalized form factor of the CM. The interparticle structure factor of the solution was taken as S(q)z1 for all the contrasts measured, given the low volume fraction (1%) and broad size distribution of the micelles, where the polydispersity also models the diffuseness of the k-casein surface layer. Jackson and McGillivray modeled I(q) as the sum of contributions from colloidal nanoclusters and the hydrated internal CM structure. The National Institute of Standards and Technology (NIST) HP-SANS cell was used for this study (see www.sciencedirect.com

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Table 2; this cell now has the capability for cold denaturation studies at subzero T). Key findings include the partial irreversibility of HP effects; assignment of a feature of ˚ -1 to contributions from hydrated protein w3.5  102 A and not colloidal calcium phosphate; and observations that, above 100 MPa, the micelles release particles of ca. 20 nm into the solution, which grow with increasing pressures. Above 250 MPa, the CM particle size increases at the cost of a decrease in CM population. It was also noted that CMs are more sensitive to pressure when no serum proteins are present in the buffer. In 2015, Tromp et al. [85] used SANS to study effects of the rate and history of HP processing on CMs, using the PSI HP cell (Table 2). At 300 MPa, CM disruption was found to be incomplete, regardless of the rate at which it was applied. Pressure history affected a w30-nm scattering entity emerging above 100 MPa, presumably released from CMs, while a w10-nm feature appears as pressure is taken up to 300 MPa (irrespective of the HP rate): the molecular underpinnings for this bimodal distribution are unclear. The characteristic timescale of both disruption at 300 MPa and reassembling after pressure release was in the order of 10 min. In 2016, Ingham et al. [86] have revisited the interpretation of SAXS data for ˚ -1 CMs, including the assignment of the w3.5  102 A feature (typically not observable from SAXS data). Using Ca-edge resonant soft X-ray scattering, it was attributed to a correlation peak from colloidal calcium phosphate. Beyond natural size distributions for different milk samples, it seems likely that apparent inconsistencies in experimental data come from the use of different buffers and concentrations of samples. CMs are a perfect example of the need to address complex food matrices with complementary techniques, over a range of conditions. Future SANS studies on CMs at subzero temperatures are still largely unexplored, as well as studies of milk protein concentrates, which are of significant interest for commercially available products [87,88].

Studies on other components of milk have also highlighted the ability of HP-SANS techniques to contribute thermodynamic parameters. For b-lactoglobulin, present in dairy gels and emulsions, and an allergen in cow milk, unfolding, dissociation, and aggregationegelation behavior are of particular interest. Using the LLB 300MPa HP cell (Table 2), Loupiac et al. [89] studied blactoglobulin. Using an expansion of the Guinier approximation to take into account concentration effects, which introduce the second virial coefficient, the evolution of Rg with pressure was monitored to establish that w300 MPa irreversible aggregates begin to form between dimeric units of the protein. Ortore et al. [90] monitored aggregation of b-lactoglobulin in D2O and D2O/ethylene glycol solutions using HP-SANS. A Kratky plot highlighted the partial unfolding of b-lactoglobulin at 280 MPa. pH, concentration, and ionic strength were set to establish a reference thermodynamic condition of near equimolar amounts of monomers and dimers. I(q) was described as in Eq. (8), where S(q) z1. P(q) was described by the sum of the monomer and dimer form Current Opinion in Colloid & Interface Science 2019, 42:99–109

106 X-Ray and Neutron Scattering

factors, weighted by the respective fractions as fitting parameters. Applying Eqs. (1) and (2) in the context of the equilibrium constant and free Gibbs energy of the dimer dissociation of b-lactoglobulin, Ortore et al. used a global fit to analyze the SANS data at different pressures and constant temperature. The data were constrained to have the same DG0, DV0, and DbT, and the calculated DG0 determined for the two solvent conditions was very similar. b-lactoglobulin volume and compressibility changes at dissociation were comparable with the values found for pH-induced dissociation in aqueous buffers. Changes in compressibility at dissociation are larger in water than in ethylene glycolecontaining buffers, showing the importance of the buffer to the behavior of the molecule under study.

Standards and Technology nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Conflict of interest statement Nothing declared.

Acknowledgements The author is very grateful for the general support and useful discussions with the staff at the NIST Centre for Neutron Research (NCNR), the University of Maryland Institute for Bioscience and Biotechnology Research, and the Centre for Neutron Science and the C. Roberts group at the Chemical and Biomolecular Engineering department of the University of Delaware. This work was supported with funding from CNS NIST Cooperative Agreement 70NANB12H239.

References Conclusions HP processing is nowadays the leading nonthermal technology in terms of consumer and regulatory acceptance, with industrial applications on the global market [91]. Understanding how HP drives phase transitions, and making use of its ability to stabilize uncharacterized intermediates, is paramount. The limitations of the theoretical framework for phase diagrams of biopolymers are well known. When it comes to biopolymers, seemingly weak effects drive phase transitions through additive or cooperative mechanisms spanning large distances in the molecular topology. Albeit still based on relatively coarse experimental mapping of the energy landscape and stability of biopolymers, improved biopolymer modeling and fits of stability and free energy are emerging. For food biopolymers in particular, a finer sampling is needed to support the design of new HP techniques and molecules and further our understanding of factors such as the conditioning of the structure and kinetic trapping [92]. HP-SANS cells are expanding accessible experimental P-T ranges. Cold denaturation studies are largely underexplored, despite tremendous potential in trapping reaction intermediates that are of interest from the food and health viewpoints. HP-SANS is now in an ideal context to contribute, supported by complementary techniques for which pressure cells became available commercially. In this respect, the NIST HP-SANS cell stands out among those currently available to neutron users. The examples given for HP-SANS studies of milk highlight how neutron scattering can go far beyond qualitative assessments of size changes under pressure, in an area where it can continue supporting the use of HP in dairy processing [93].

Disclaimer Certain commercial equipment, instruments, suppliers, and software are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Current Opinion in Colloid & Interface Science 2019, 42:99–109

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