Food Chemistry 141 (2013) 1328–1334

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Food Chemistry

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Effect of high pressure processing on rheological and structuralproperties of milk–gelatin mixtures

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.074

⇑ Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7422.E-mail address: y.hemar@auckland.ac.nz (Y. Hemar).

Anastasia Fitria Devi a,b, Li Hui Liu b, Yacine Hemar c,⇑, Roman Buckow b, Stefan Kasapis a

a School of Applied Sciences, RMIT University, City Campus, Melbourne, Victoria 3001, Australiab CSIRO Animal, Food and Health Sciences, Werribee, Victoria 3030, Australiac School of Chemical Sciences, The University of Auckland, Auckland Central, Auckland 1142, New Zealand

a r t i c l e i n f o

Article history:Received 4 January 2013Received in revised form 20 March 2013Accepted 21 March 2013Available online 3 April 2013

Keywords:Skim milkGelatinHigh pressure processingViscosityPhase separation

a b s t r a c t

There is an increasing demand to tailor the functional properties of mixed biopolymer systems that findapplication in dairy food products. The effect of static high pressure processing (HPP), up to 600 MPa for15 min at room temperature, on milk–gelatin mixtures with different solid concentrations (5%, 10%, 15%and 20% w/w milk solid and 0.6% w/w gelatin) was investigated. The viscosity remarkably increased inmixtures prepared with high milk solid concentration (15% and 20% w/w) following HPP at 300 MPa,whereas HPP at 600 MPa caused a decline in viscosity. This was due to ruptured aggregates and phaseseparation as confirmed by confocal laser scanning microscopy. Molecular bonding of the milk–gelatinmixtures due to HPP was shown by Fourier-transform infrared spectra, particularly within the regionsof 1610–1690 and 1480–1575 cm�1, which reflect the vibrational bands of amide I and amide II,respectively.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

One of the first applications of static high pressure processing(HPP) on a food product was reported by Hite (1899) on milk forextending its shelf-life. Since then HPP has been utilised not onlyfor extending shelf life of perishable food, while ensuring its safetyand nutrients, but also for modifying food macromolecule func-tionality (Knorr, Heinz, & Buckow, 2006). The extent of macromol-ecule rearrangements affected by high pressure is dependent on itsintrinsic properties, physicochemical environment, appliedpressure and temperature, holding time and decompression rate(Michel & Autio, 2002).

The structure–function properties of foods are mainly governedby two groups of macromolecules: proteins and polysaccharides,consequently appointing them as the essential constructionalmaterials and suitable components to model foods, which are com-plex systems (Morris, 2007). When two biopolymers are mixed,commonly the destabilisation force is bigger than the stabilisationforce. As a result, unstable solutions are often encountered, whichlead to either segregative or associative phase separation. The typeof phase separation taking place in a mixed gel is determined bythe procedure of gel formation that guides final application(Walkenstrom & Hermansson, 1997a). Less likely, synergetic inter-actions between biopolymers may occur, being recognised by a

substantial increase in yield stress and elastic modulus in mixtures(Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998).

Gel fabrication by HPP may create textures with unique proper-ties that cannot be achieved by its thermal counterpart. The latterfavours gel formation through disulfide bonding, whereas pressuretreatment leads to structure development mainly via hydrogenbonds, although disulfide bonding can occur if the HPP is per-formed at sufficiently high pressure (e.g. 500 MPa) or at relativelyhigh temperatures (Totosaus, Montejano, Salazar, & Guerrero,2002). Weak bonds, such as hydrogen, electrostatic, van der Waalsand hydrophobic forces can be destabilised by HPP, due to theirlimited energy levels. However, covalent associations of biopoly-mers are usually not affected under high pressure (Boonyaratanak-ornkit, Park, & Clark, 2002).

Gelatin, a proteinaceous material derived from collagen, is animportant macromolecule able to impart a variety of textures(Poppe, 1992). Interaction between gelatin and other food macro-molecules is an attractive research field, since the resultant knowl-edge is used to model novel food product development (Djagny,Wang, & Xu, 2001). In addition, there is an increasing demand toexplore the functional properties of mixed biopolymer systemsthrough HPP in an effort to add value to formulations. Clearly, mix-tures of gelatin and milk proteins, which find application in thefood industry, can be of interest in this regard. However, publica-tions on the phase behaviour and structural properties of thesebinary mixtures involving high pressure processing are scarce(Hemar, Liu, Meunier, & Woonton, 2010; Walkenstrom &Hermansson, 1997a, 1997b).

A.F. Devi et al. / Food Chemistry 141 (2013) 1328–1334 1329

This present paper aims to investigate the effect of HPP on therheological and structural properties of milk–gelatin mixtures, pre-pared with selected solid concentrations, which relate to a range ofcurrent dairy product formulations including yoghurts. Character-isation of rheological, broader structural properties, and biopoly-mer interactions will provide a valuable guide on thefunctionality of these materials subjected to static pressure.

2. Materials and methods

2.1. Materials

Gelatin, produced through alkaline hydrolysis of beef skin (typeB), was purchased from Melbourne Food Ingredient Depot, Austra-lia. This material had an isoelectric point (pI) of 4.5 and Bloom va-lue of 220.

The milk protein utilised in this study was skim milk powder(SMP) produced with low heat to minimise denaturation of thewhey protein component. It was obtained from Tatura, Victoria,Australia, and contained 33.6% protein, 0.7% fat, 2.8% ash, and0.44% lactose. A solution of 10% w/w in deionised water gave apH value of 6.7.

2.2. Methods

2.2.1. Sample preparationAqueous gelatin solution (0.6% w/w) was prepared by soaking

gelatin granules in deionised water overnight at 5 �C then heatedand stirred for 40 min at 65 �C. Gelatin solutions were cooled toroom temperature prior to pH adjustment to 6.7 with 0.1 M NaOH.Reconstituted skim milk with concentrations of 5%, 10%, 15% and20% w/w were prepared by dispersing SMP in deionised water un-der stirring for 2 h, followed by overnight storage at 5 �C to allowcomplete hydration. Milk was then warmed at 40 �C for 10 min un-der stirring. The adjustment of milk to pH 6.7 was conducted atroom temperature using dropwise 1 M NaOH or 1 M HCl. Mixturesof milk–gelatin with final concentrations of 5%, 10%, 15% and 20%w/w of milk solids and 0.6% w/w of gelatin were prepared sepa-rately from reconstituted skim milk (25% w/w) and gelatin solu-tions (1%, 2%, 3% and 4% w/w). Each parental solution wasprepared following the above instructions and pH adjustmentwas performed after mixing both solutions under stirring at40 �C for 10 min and cooling to room temperature.

2.2.2. High pressure processingSamples were transferred into 50-mL bottles (3.5 cm in diame-

ter and 8 cm in height; PB packaging, Mordialloc, Australia) withno headspace, which then were sealed in polyethylene bags (VenusPackaging, Melbourne, Australia) filled with water. The sampleswere treated at pressures of 150, 300, 450 and 600 MPa for15 min in an HPP system (QFP 35 L-600-S Food Press, Avure Tech-nologies AB, Västerås, Sweden) at room temperature. Recordedtemperature profile during HPP showed that at the highest pres-sure used (600 MPa) the temperature increased to approximately40 �C. This temperature is not high enough to induce thermal dena-turation of whey proteins. Following the holding time, the pressurewas released instantaneously to atmospheric pressure, and sam-ples were stored overnight at 5 �C prior to measurements.

2.2.3. Rheological measurementsApparent viscosity and dynamic oscillation measurements were

performed using a stress-controlled rheometer (Paar – PhysicaMCR 301, Anton Paar GmbH, Ostfildern, Germany). Measurementswere conducted at 5 and 35 �C. Viscosity measurement at 5 �C fol-lowed the following protocol: Step 1 – where samples were condi-tioned at 5 �C for 5 min, continued with Step 2 � where the shear

rate was applied from 0.01 to 1000 s�1, and finished with Step 3 �where the shear rate was applied from 1000 to 0.01 s�1.Corresponding measurements at 35 �C were performed in a similarmanner, except that the sample was conditioned at 35 �C for30 min in a water bath and for 5 min in the rheometer prior toexperimentation. For milk-only systems (low viscosity samples),the double gap (TEZ-DG26.7/Ti 3524) was used, and for gelatin,cup and bob (TEZ-CC.27 6325) was the geometry of choice. Dy-namic oscillation was performed at 5 �C, following a pre-shear stepat 10 s�1 for 2 min to remove trapped air, with the samples beingallowed to rest for the next 2 min. A frequency sweep from 0.01to 10 Hz at a constant strain of 0.5% was conducted followed bya strain sweep from 0.1% to 1000% at a constant frequency of1 Hz. All rheological measurements were performed in duplicateand average values of effectively overlapping traces are reported.

2.2.4. Confocal laser scanning microscopy (CLSM)Images were taken using a Leica SP5 confocal laser scanning

microscope (Leica Microsystems, Wetzlar, Germany). A drop of FastGreen (0.4% w/v in deionised water) was gently mixed by handwith approximately 10 mL of a prepared sample prior to cold stor-age at 5 �C. For pressure-treated samples, the dye was addedimmediately after HPP. A small amount of dyed sample was placedonto a glass cavity slide and covered with a glass cover slip. Thesamples were observed using an HCX PL APO 63.0 � 1.30 GLYC37 �C UV objective lens, immediately after being taken from thecold room. Fast Green was excited by HeNe 633 nm laser and theemitted light was recorded at 643–737 nm.

2.2.5. Fourier transform infrared (FTIR) spectroscopyInfrared spectroscopy (Spectrum 100 FTIR spectrometer; Perkin

Elmer, Waltham, MA) was carried out from 4000 to 650 cm�1 at aresolution of 4 cm�1 taking eight scans and averages are reported.Spectra were transferred into absorbance to examine the nature ofmolecular interactions, specifically secondary protein structure,between gelatin and milk protein in atmospheric and pressurisedpreparations. All FTIR measurements were performed in duplicate.

2.2.6. Thermal analysisThis was performed using a Setaram VII Micro DSC (Setaram

Instrumentation, Caluire, France). Approximately 800 mg of sam-ple were transferred into the sample pan and the same amountof deionised water was used in the reference pan. Weighing andsample loading were done at room temperature. Samples werescanned at a heating/cooling rate of 1 �C/min, as follows: (1) milkwas conditioned at 25 �C for 10 min then heated to 95 �C; (2) gel-atin was conditioned at 25 �C for 10 min, heated to 60 �C, cooled to5 �C, held for 2 h at 5 �C and reheated to 60 �C; (3) milk–gelatinmixture was conditioned at 25 �C for 10 min, heated to 40 �C,cooled to 5 �C, held for 2 h at 5 �C and heated to 95 �C.

3. Results and discussion

3.1. Rheology of the pressure treated gelatin gel

Gelatin gels 0.6% w/w were analysed first to build up a baselineof behaviour for subsequent characterisation of the mixed counter-parts. Gelatin gels became weaker following HPP, particularly afterpressurisation above 300 MPa (Fig. 1). There was no notable differ-ence in the values of apparent viscosity or storage modulus (G0)upon HPP at 450 and 600 MPa. This result is contrary to earlierstudies, which claimed that HPP substantially increased the G0 ofvariable-origin gelatin gels, i.e., cod gelatin at 6.67% w/w (Montero,Fernandez-Diaz, & Gomez-Guillen, 2002), bovine skin gelatin up to10% w/w (Kulisiewicz & Delgado, 2009), and pigskin gelatin at 3%

(a)

(b)

Fig. 1. Apparent viscosity (a) and storage modulus (b) of gelatin gels 0.6% w/wprepared without HPP (j) and with HPP at 150 MPa (h), 300 MPa (d), 450 MPa (s)and 600 MPa (�). All pressure treatments were conducted for 15 min at roomtemperature and measurements were taken at 5 �C.

Fig. 2. Apparent viscosity (a) and storage modulus (b) of gelatin gels 0.6% w/w (j),and milk–gelatin mixtures prepared with 0.6% w/w gelatin and various milk–solidconcentrations: 5% w/w (h), 10% w/w (d), 15% w/w (s) and 20% w/w (�). Allmeasurements were conducted at 5 �C.

1330 A.F. Devi et al. / Food Chemistry 141 (2013) 1328–1334

w/w (Walkenstrom & Hermansson, 1997a). However, Hemar et al.(2010) observed no effect of HPP, up to an application of 600 MPafor 15 min at room temperature, on G0 of unbuffered gelatin solu-tions with varying concentrations from 0.2% to 1.0% w/w. Clearly,gelatin origin, concentration, and pH of the analysed samples affectheavily the material’s response to experimental pressure (Devi,Buckow, Hemar, & Kasapis, 2013).

Storage modulus reduction of gelatin gels after HPP in thisstudy was almost tenfold compared to that of the unpressurisedcounterpart. To date, only Montero et al. (2002) mentioned a de-crease in G0 values after HPP, for megrim gelatin gels prepared at6.67% w/v. This was explained on the basis of incomplete unfoldingof the gelatin chain, which was dissolved in water at 60 �C for20 min followed by cold setting overnight at 7 �C and pressurisa-tion at 200 MPa for 12 min at 7 �C. Since the gelatin solution in thiswork was prepared by soaking overnight and heating under stir-ring at 65 �C for 40 min to allow complete dissolution, the reasonpostulated by Montero et al. (2002) cannot be the reason behindthe weakening of gelatin gels in our observations.

Guo, Colby, Lusignan, and Howe (2003) have offered an expla-nation for our observation based on thermodynamic consider-ations. Weakening of gelatin gels upon HPP should be theoutcome of hindered triple helix formation. Accordingly, the kinet-ics of triple helix formation is controlled by a two-stranded nu-

cleus, which requires a positive change in enthalpy and volume(+DH and +DV). Recalling that �DH and �DV are often the fa-voured reaction conditions at high pressures (e.g., 600 MPa), thetwo-stranded nucleation of gelatin chain in solution is then re-tarded (Guo, Colby, Lusignan, & Whitesides, 2003b). Experimentalwork by Kasapis (2007) is congruent to this, having found a reduc-tion of �0.23 �C in the temperature of the coil-to-helix transitionfor every 100 MPa of pressure increase in 15% gelatin gels in thepresence of 63% w/w small molecule co-solute.

3.2. Rheology of untreated milk–gelatin mixtures

Incorporation of milk solids as low as 5% w/w into the 0.6%(w/w) gelatin solution caused a remarkable viscosity enhancement(Fig. 2a). The viscosity of a binary biopolymer solution is largelyinfluenced by the concentration and interactions of the two con-stituents (Schmitt et al., 1998). Qualitatively, the viscosity ofmilk–gelatin mixtures shows a similar pattern of shear-rate depen-dence to that of the gelatin solution shown in Fig. 1a. This outcome,in combination with the considerable increase in viscosity values,can be considered as an indication of phase separation phenomenain the system. Commonly, when phase separation is taking place,structural properties of the different phases are often enhancedcompared to those of the pure constituents. However, the

A.F. Devi et al. / Food Chemistry 141 (2013) 1328–1334 1331

rheological behaviour of the phase separated systems is qualita-tively similar to that of the continous phase (Walkenstrom & Her-mansson, 1997b).

The proposed morphology of phase separation is further sup-ported by the fact that, at the pH (�6.7) used in this investigation,both dairy proteins and gelatin are negatively charged. Accordingto Polyakov, Grinberg, and Tolstoguzov (1997), increase in thenet charge of proteins in the mixtures enhances their incompatibil-ity, which is the driving force of macromolecular phase separation.As shown in Fig. 2b, addition of milk solids contributes to highervalues of G0 in the mixture, compared to the aqueous gelatin solu-tion. This is due to the dispersed casein molecules that add resis-tance to flow and the high capacity of casein particles to holdwater at approximately four gram per gram of protein (Damoda-ran, 2008). The phase morphology of the mixture is based on gela-tin forming a continuous phase, which is interrupted by theabundance of milk solids.

3.3. Rheology of pressure-treated milk–gelatin mixtures

The influence of HPP to steady shear viscosity varies dependingon the composition of the formulation and applied static pressure.HPP at 300 MPa reduced the viscosity of mixtures with low

(a)

(c)

Fig. 3. Apparent viscosity of milk–gelatin gels prepared with 0.6% w/w gelatin and variouwithout the influence of HPP (j) and after HPP at 300 MPa (h) and 600 MPa (d). All presswere taken at 5 �C.

milk–solid content (5% and 10% w/w) in Fig. 3a and b, but tendedto increase the system viscosity at higher milk concentrations(15% and 20% w/w) in Fig. 3c and d. Applying HPP at 600 MPa re-duced the viscosity of all milk–gelatin samples (Fig. 3a–c), exceptthe one prepared with 20% w/w milk solids (Fig. 3d).

Although gelatin exhibits a decrease in both viscosity and stor-age modulus upon HPP (Fig. 1), the presence of relatively highamounts of milk solids appear to assist in the protein’s structureformation. Recalling that caseins at the natural pH of milk possiblybreak down once subjected to intermediate pressure (250–300 MPa according to Orlien, Boserup, and Olsen (2010)), the smal-ler fragments of casein should stabilise the overall structure of themixture. As highlighted by Tolstoguzov (2000), the dissociation ofcomplex proteins (such as caseins) into simpler forms increases theinterfacial contact between the two polymeric constituents leadingto enhanced stabilisation of the gelling component, i.e., gelatin’striple helix, in the present mixture. In addition, the mixture pre-pared with 20% w/w milk solids could resist HPP at 600 MPa, withnearly unchanged viscosity, as opposed to the viscosity reductionobserved at more dilute preparations (Fig. 3). At this level of ap-plied pressure, casein submicelles cannot contribute to viscosityenhancement, since gelatin’s gelling ability should have been com-promised to a significant extent.

(b)

(d)

s milk–solid concentrations: 5% w/w (a), 10% w/w (b), 15% w/w (c) and 20% w/w (d),ure treatments were conducted for 15 min at room temperature and measurements

Table 1Melting temperature of gelatin gels and in milk–gelatin mixtures, prepared fromgelatin and various milk–solid concentrations as shown, with and without highpressure treatment.

Samples Melting temperature (Tm) (�C)

0.1 MPa 300 MPa 600 MPa

Gelatin gel (0.6% w/w) 27.5 27.8 27.7Milk 5% w/w + gelatin 0.6% w/w 27.7 29.0 29.5Milk 10% w/w + gelatin 0.6% w/w 27.7 27.8 28.0Milk 15% w/w + gelatin 0.6% w/w 28.3 28.3 28.7Milk 20% w/w + gelatin 0.6% w/w 29.3 27.5 28.3

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Finally, a rheological measurement was carried out at 35 �C, i.e.,at the vicinity of the gelatin network melting temperature, to as-sess the nature of the composite’s morphology following HPP. Forboth untreated and pressurised mixtures, steady shear viscositydrops dramatically, due to the collapse of the gelatin network, tovalues well below 10 mPa s (results not shown). Thermal analysisof the samples using micro-DSC corroborates this observation by

Fig. 4. Confocal images of gelatin gels 0.6% w/w (a–c) and milk–gelatin mixtures preparuntreated samples being on the left-hand side (a, d, and g), samples treated at 300 MPa bright-hand side (c, f, and i). All pressure treatments were conducted for 15 min at room

showing the expected melting temperature of the protein gel withor without dairy solids (<30 �C in Table 1). This outcome argues forthe absence of direct associative interactions, in the form of elec-trostatic attraction or via conformational compatibility, betweenthe two polymeric components in atmospheric and HPP mixtures.The importance of a phase separated gelatin network for structureformation is, hence, further supported. Note that the rheologicalmeasurements performed on milk samples alone are not reportedin the previous figures because the samples did not form a gel and,thus, yield very small values (G0 < 0.1 Pa) for the complex modulus.

3.4. Tangible evidence of phase morphology in pressure treated milk–gelatin mixtures

The microstructure of milk–gelatin mixtures was investigatedwith CLSM (Fig. 4). Milk proteins, stained with Fast Green, appeargreen whilst the gelatin phase remains black. The latter shows thinstrands and small pores prior to HPP (Fig. 4a), reflecting the trans-parent nature of its network. It becomes unevenly dense following

ed from gelatin 0.6% w/w and milk solids at 10% w/w (d–f) or 20% w/w (g–i), witheing placed in the middle (b, e, and h) and those being treated at 600 MPa are on thetemperature. The length of the scale bar is 75 lm of images is 1024 � 1024 lm.

Fig. 5. FTIR spectra of aqueous milk–gelatin mixtures, prepared from gelatin 0.6%w/w and milk solids at 20% w/w, without the influence of HPP (black curve) andafter HPP at 300 MPa (blue curve) and 600 MPa (red curve). All pressure treatmentswere conducted for 15 min at room temperature. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web versionof this article.)

A.F. Devi et al. / Food Chemistry 141 (2013) 1328–1334 1333

pressurisation, with coarse aggregates surrounding bigger waterholding voids (Fig. 4b and c), an outcome which is congruent withthe declining G0 values shown in Fig. 1b.

The phase morphology of the mixture exhibits inclusions ofmilk–protein rich regions within a gelatin-rich continuous phasefor atmospheric samples (Fig. 4d and g). This is in agreement withthe concept of phase separation postulated presently and also re-ported for similar milk solid (2–18% w/w) and higher gelatin(1.5–3% w/w) concentrations (Hermansson, Altskär, & Jordansson,1998). For all experimental samples, gelatin forms the continuousphase and the discontinuous milk protein inclusions grow biggeras the quantity of the latter is increased in preparations; this find-ing is in agreement with an earlier observation by Hermanssonet al. (1998) with 1.5% w/w gelatin and 2–14% w/w of SMP.

Cloudy confocal images are captured in milk–gelatin mixturesfollowing HPP at 300 MPa shown in Fig. 4e. These do not closelyresemble the three dimensional arrangement of the unpressurisedsamples, an outcome which reflects the destruction of casein mi-celles with increasing applied pressure bringing the two polymericsystems into proximity. Protein dissociation in electrostatically at-tracted i-carrageenan and casein under HPP raise the opportunityfor direct interaction of the two macromolecules (Abbasi & Dickin-son, 2004). However, ‘coacervation’ leading to coprecipitation andreduced viscosity is likely to occur in these cases (Barth, 2007),which is not the observation in our systems. Instead, highly rup-tured colloidal casein produces dense aggregates (Fig. 4h), whichfurther increase the stability of the gelatin helix, leading to an in-creased viscosity in the composite with 20% w/w milk protein(Fig. 3d).

Finally, application of 600 MPa leads to considerable disruptionof the colloidal casein network (Fig. 4f) and corresponding reduc-tion in viscosity (Fig. 3b). Mixtures prepared with 20% w/w milksolids form dense aggregates through the intense pressurisationof 600 MPa (Fig. 4i) and exhibit high viscosities (Fig. 3d).

3.5. FTIR spectra of pressure treated milk–gelatin mixtures

Changes in protein structure due to HPP can be traced throughFTIR spectra within the range of amide vibrations. Amide I band is

the primary band to consider for protein secondary structure and islocated between 1600 and 1690 cm�1. It reflects the C@O stretch-ing, which is interfered with by protein unfolding or interactionswith the physicochemical environment. In general, FTIR spectraof high-pressure treated proteins show decreasing frequency ofamide I band, due to reforming of hydrogen bonds under high pres-sure and consequent disturbance of the C@O bond (Carbonaro &Nucara, 2010). In the present work, milk–gelatin mixtures showeda small decrease in absorbance intensity between 1600 and1700 cm�1 and a limited increase in absorbance intensity between3000 and 3500 cm�1 following HPP of our systems (Fig. 5).

Altered arrangement of hydrogen bonds can be recognised fromamide II band (1480–1575 cm�1), since it influences the NH bend-ing vibration (Barth, 2007; Carbonaro & Nucara, 2010). Spectra inFig. 5 show a small decrease in amide II band but, thus far, thisband is rarely used to give further information on high-pressure-treated proteins (Dzwolak, Kato, & Taniguchi, 2002). There is alsothe amide III band (1229–1301 cm�1), which reflects CN stretchingand NH bending. Samples containing milk solids give amide IIIvibrations typical of native micellar casein (Hussain, Gaiani, Aber-kane, & Scher, 2011). There is also a limited decrease in absorbancein this band due to HPP in our materials (Fig. 5), but no supportingscientific publication has been found in relation to the effect ofpressure on the secondary structure of proteins.

4. Conclusions

The present work showed that the rheology and microstructureof milk–gelatin mixtures can be tailored by controlling the degreeof aggregation. This depends on composition, applied pressure and,of course, temperature, in the case of gelatin. HPP can increase theviscosity of milk–gelatin mixtures, due to segregative phase sepa-ration between the two polymeric components. Application ofintermediate to high pressure (300–600 MPa) results in changingmicrostructure in the mixture depending on the milk–solid con-tent. It appears that in the presence of milk solids at levels ofindustrial interest (<20% w/w), gelatin structure formation atrefrigeration temperature is enhanced following application ofhigh pressure. Results are of interest, since high pressure isincreasingly seen as a substitute for conventional thermal treat-ment, with possible application in yoghurt manufacture where gel-atin is used as a stabiliser.

Acknowledgement

Author A.F. Devi would like to acknowledge the support for herPh.D. study provided by the Endeavour International PostgraduateResearch Scholarship (IPRS).

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