Dynamic Confocal Scanning Laser Microscopy Methods for Studying Milk Protein Gelation and Cheese Melting

Transcription

1 SCANNING VOL. 21, (1999) FAMS, Inc. Dynamic Confocal Scanning Laser Microscopy Methods for Studying Milk Protein Gelation and Cheese Melting M.A.E. AUTY, M.A. FENELON, T.P. GUINEE, C. MULLINS, D.M. MULVIHILL* Dairy Products Research Centre, Moorepark, Fermoy; *Food Chemistry, Department of Food Science and Technology, University College, Cork, Ireland Summary: Dynamic confocal scanning laser microscopy (CSLM) methods were developed to enable observation of milk protein gelation and cheese melting. Protein aggregation and the formation of gel networks in renneted fullfat and low-fat milks and glucono-δ-lactone (GDL)-acidified skim milks were observed by CSLM and observations correlated with increases in shear modulus (G ) and dynamic viscosity (η*) as determined by dynamic amplitude oscillatory rheology. Confocal scanning laser microscopy observation of low-fat and full-fat cheeses showed changes in fat distribution and an increase in staining intensity during cheese melting. Key words: confocal scanning laser microscopy, dynamic studies, protein gelation, microstructure, cheese melting PACS: Introduction Tt; 82.7.Gg Food texture is largely influenced by the microstructure of the individual components and their relationship to each other. Proteins and fats are important structural elements in a wide variety of foods, influencing texture and flavour release. The ability to form gels is a fundamental functional property of many proteins, particularly in products such as yoghurt, cheese, and processed meat products. Electron microscopy (EM) techniques have been used extensively to elucidate the mechanisms of gel formation in milk products (Emmons et al. 198; Harwalkar and Kalab 198, 1981; Schellhaase and Morris 1985). However, EM techniques, in particular transmission electron microscopy (TEM), often involve considerable sample preparation including chemical fixation, dehydration, resin embedding, sectioning, and staining. Each of these treatments may Address for reprints: M.A.E. Auty Dairy Products Research Centre Moorepark, Fermoy Co. Cork, Ireland influence the microstructure of the sample under investigation, increasing the likelihood of artefacts (Kalab 1984). Electron microscopy studies also have the limitation that sampling has to take place at fixed time points, making it difficult to follow rapid time- or temperature-related morphologic changes. Confocal scanning laser microscopy (CSLM) is being used increasingly in food microstructure studies. Advantages of CSLM are as follows: Fixation and/or dehydration is usually unnecessary, thereby minimising sample preparation time and artefacts; (2) optical sectioning below the surface enables threedimensional (3-D) reconstruction of the undisturbed microstructure; (3) fluorescent probes can be used to identify specific food components (Heertje et al. 1987); and (4) food microstructure can be continuously monitored. Despite the advantages, CSLM has some limitations; resolution of individual proteins and casein micelles is restricted by the diffraction-limited.2 µm spatial resolution of the confocal microscope, although CSLM detection of subresolution particles is possible given an efficient signal-to-noise ratio (Pawley 1995). A further limitation is that CSLM observations are currently restricted to quiescent, nonsheared food systems although the possibility of examining foods during shearing is being investigated (Hermannsson 1998) In recent years, improved image acquisition rates and high-efficiency detectors have facilitated the study of realtime dynamic events by CSLM (Nie et al. 1995). Hassan et al. (1995) used CSLM in reflectance (backscattered) mode to study skim milk gels acidified by both gluconoδ-lactone (GDL) and microbial cultures. The GDL-induced casein gels aged for approximately 17 h have been studied by CSLM (Bremer et al. 1991, Lucey et al. 1997). Other than these studies, which concentrated on gels once they had formed, there are few published data showing the gelation process. Auty et al. (1997) reported on the use of hotstage CSLM together with a protein and fat stain for the continuous monitoring of rennet-induced milk gelation. There appear to be no published data on the use of CSLM to study cheese melting. In this study, dynamic CSLM methods were developed for the direct observation of rennet- and acid-induced gelation of milk and for melting of full- and low-fat cheeses.

2 3 Scanning Vol. 21, 5 (1999) Materials Raw, full-fat, and low-fat cheesemilks were obtained from the Dairy Products Research Centre (DPRC) processing hall. Low-heat skim milk powder (SMP) was produced in the DPRC processing hall. Full-fat and low-fat cheddar cheeses were produced as described by Fenelon and Guinee (1998). One-week-old cheese was used for CSLM observations while 2- day-old cheese was used for low-amplitude oscillatory rheology (LAOR). Young cheeses were chosen to study the effect of fat content per se, thus eliminating textural effects due to proteolysis. Gross compositions of the milks and cheeses used are shown in Table I. Nile blue (sulphate salt, C.I.5118), Rhodamine B (C.I. 4517), GDL, silicone melting bath oil, and probe clips were obtained from Sigma-Aldrich Ireland Ltd, Dublin, Ireland. Methods All microscopy experiments were carried out using a Zeiss LSM 31 CLSM (Carl Zeiss Ltd., Herts., UK) fitted with a Kr/Ar laser giving excitation wavelengths of 488 nm and 568 nm, and a He/Ne laser giving an excitation wavelength of 633 nm. A long pass 58 nm barrier (emittance) filter was used. A Linkam C12 warm stage (Linkam Scientific Instruments Ltd., Surrey, UK) was used to study protein gelation and cheese melting. Time lapse animations of rennet- and GDL-induced milk gelation were produced at a frame rate of 15 frames/s using Videotrope (Ver. 1.) animation software (Digital Workshop, Banbury, Oxon, UK). Monitoring Milk Protein Gelation by Confocal Scanning Laser Microscopy and Low-Amplitude Oscillatory Rheology Rennet-induced gelation of full-fat and low-fat milks: Full-fat (3.5,%,w/v) and low-fat (.5,%,w/v) milks were treated with chymosin (Double Strength Chy-max, 5, TABLE I Compositions of standardised milks and cheeses used in the study Milk Full-fat, %, w/v Low-fat, %, w/v Protein Fat Lactose Cheese Full-fat, %, w/w Low-fat, %,w/w Moisture Protein Fat FDM Abbreviation: FDM = Fat-in-dry-matter. MCU/ml, Pfizer Inc., Milwaukee, Wisc., USA), diluted 1:1 with deionized water, at a level of 7.3 ml/kg. Immediately following rennet addition, Nile blue (.1,%, w/v, aq.) was added to a final concentration of 1 mg/ml. Approximately 75 µl of renneted milk containing the stain was then placed into a well 13 mm in diameter and 1 mm deep (volume ~13 µl) placed on the warm stage of the CSLM using a silicone rubber spacer gasket, taken from a probe clip, and placing a coverslip on top (Fig. 1). The edge of the coverslip was sealed with silicone oil to prevent moisture loss. Confocal scanning laser microscopy images were acquired at 2 min intervals over a period of 45 min. Samples were analysed using confocal mode and images acquired at or pixel resolution at scan rates 1 or 4 s/frame. Images were scanned approximately 2 3 µm below the level of the coverslip using the 633 nm laser to detect Nile blue stained protein; a 514 nm excitation filter was used in conjunction with the 488 nm laser to image fat droplets. Dynamic low-amplitude oscillation rheometry was carried out during milk gelation using a controlled strain Bohlin CVO Rheometer (Bohlin Instruments, Cirencester, UK) operating at an angular frequency (ω) of 1 Hz, as described by Guinee et al. (1997). Changes in the storage modulus (G ) and dynamic viscosity (η*) of the renneted milks were recorded at 31 o C. GDL-induced gelation of skim milk: Reconstituted skim milk was prepared by dissolving 1 g of low-heat skim milk powder in 9 ml distilled water at 4 o C. After 2 h stirring, the reconstituted milk was heat treated at 85 o C for 15 min, then cooled in an ice bath to approximately 1 o C. Glucono- lactone (GDL,1,%,w/v) was added to the cooled milk and the temperature was raised to 4 o C over 3 min. Three aliquots of the GDL-containing milk were taken, one for ph measurement, one for CSLM, and one for rheological analysis by LAOR. The ph was monitored continuously at Sample + Sample stain stain Probe clip Probe clip silicone silicone spacer spacer gasket CSLM objective CSLM objective Coverslip Coverslip Warm stage plate held at 31 C, or 4 C, or ramped at 3 C/min from 2 to 8 C 1 mm 1mm FIG. 1 Diagram showing hot-stage modified for dynamic confocal scanning laser microscopy (CSLM) studies of rennet-induced milk gelation and melting of cheese.

3 M.A.E. Auty et al.: SSLM for milk protein gelation and cheese melting study 31 4 o C over 3 h using a glass electrode. To study GDLinduced skim milk gelation by CSLM, Rhodamine B (1,%, aq.) was added to the milk to a final concentration of 1 µg/ml. The GDL-containing stained milk was then incubated at 4 o C using the same warm stage procedure as described above for the renneted milk. Confocal scanning laser microscopy images ( pixels) were acquired at 3 s intervals over a period of 3 h. The 568 nm laser was used to image protein stained with Rhodamine B. Rheological parameters, G and η*, of the GDL-treated milk were measured by LAOR in the Bohlin CVO rheometer, as described above for the renneted milk, during incubation at 4 o C for 3 h. The rheological characteristics (G and η*) of GDL-containing milk were unaffected by the added Rhodamine B. Monitoring Cheddar Cheese Melting by Confocal Scanning Laser Microscopy and Low-Amplitude Oscillatory Rheology Dynamic changes in the microstructure of low-fat and full-fat cheddar cheese on heating were studied using a warm-stage attachment to the confocal microscope. Small sections of cheese approximately mm thick, were placed on the warm stage. Rhodamine B, ~5 µl of.1% aq., was then applied to the cheese surface and a coverslip was placed on top. The edges of the cheese and coverslip were sealed with silicone oil to prevent moisture loss. The cheese was then heated from 2 to 8 o C at a controlled heating rate of 3 o C/min. The CSLM images were acquired at 1 o C intervals using the 568 nm laser. Images were scanned approximately 1 µm below the level of the coverslip. Changes in the rheological characteristics of the low-fat and full-fat cheddar cheeses during heating were measured using low-amplitude oscillation in a controlled strain Bohlin CVO rheometer, essentially according to the method of Horne et al. (1993). Slices, 2 mm thick, were cut from cheeses using a Berkel slicer (Model 3G; Avery Berkel Group, Smethwick, Warley, West Midlands, UK); discs (15 mm diam.) were then cut from the slices using a stainless steel borer. The discs were tempered at 2 C for 15 min, then placed between the two parallel serrated plates (to prevent slippage on melting) of the rheometer cell. To prevent sample dehydration during subsequent heating, the exposed cheese surface was coated with high melting point lithiumbased LM grease (Castrol, UK. Limited, Swindon, UK). The sample was equilibrated at 2 C for 3 min and subjected to a harmonic low-amplitude shear strain (γ) of.5, at an angular frequency (ω) of 1 Hz. These conditions ensured that the samples were within the linear viscoelastic range during testing. In preliminary experiments, the yield strain for all the cheeses tested was found to be ~.15 at 2 C and >>.15 at 8 C. The temperature was raised from 2 to 8 C at a rate of 3 C/min. The storage modulus, G, the loss modulus, G, and the phase angle, δ, were monitored from the initiation of heating. The relationship between the moduli and δ is given by: tan δ = G /G. (1) δ is o for an ideal elastic solid and 9 o for a Newtonian liquid and is between these values for a viscoelastic material such as cheese. Results and Discussion Monitoring Milk Protein Gelation by Confocal Scanning Laser Microscopy and Low-Amplitude Oscillatory Rheology Rennet-induced gelation of full-fat and low-fat milks: A sequence of CSLM images, taken from a time lapse animation during rennet-induced gelation of full-fat milk, is shown in Figure 2. Initially, the protein appeared as a fine dispersion of small particles <.5 µm in diameter, presumed to be large individual casein micelles or small micelle aggregates. After ~ 15 min the casein particles began to aggregate, eventually forming a three-dimensional (3-D) network at the onset of gelation. Fat droplets appeared as dark circular regions bounded by a protein-rich coating of variable thickness. Excitation with the 488 nm laser (514 nm excitation filter) confirmed that the dark circles were fat (Brooker 1995). The CSLM images of low-fat milk gelation were generally similar to those of full-fat, except that fat droplets were far fewer and the low-fat milk gelled more slowly. Following the onset of gelation (~ 15 min), there was a gradual increase in protein aggregation together with a corresponding decrease in stained material in the surrounding aqueous phase. After 45 min, the renneted milk system appeared as a continuous 3-D protein network with occluded fat droplets and moisture. Changes in microstructure were accompanied by increases in η*and G up to the end of the experiment, 45 min after rennet addition (Fig. 3). For both samples, aggregation and gel formation, as observed by CSLM, coincided with an increase in η* and G. In agreement with previous results (Chapman 1974, Guinee et al. 1997), increasing fat level in the milk resulted in higher η* and G values. The slower gelation of the low-fat milk sample (as determined by both CSLM and LAOR) may be due to the fat exerting a volume exclusion effect, increasing the volume fraction of protein in the aqueous phase of the full-fat milk, leading to more rapid gelation. Further work is necessary to clarify these observations. GDL-induced gelation of skim milk: Figure 4 shows a sequence of CSLM images taken from a time-lapse animation during GDL-induced milk gelation. In agreement with previous electron microscopic observations (Heertje et al. 1985), CSLM analysis of skim milk showed that slow, quiescent acidification using GDL

4 32 Scanning Vol. 21, 5 (1999) FIG. 2 Sequence of confocal scanning laser microscopy (CSLM) images during rennet- induced gelation of full-fat milk containing.1% w/v Nile blue. Bright areas are protein, dark circles are fat droplets. Numbers refer to minutes after rennet addition. Images were acquired using 633 nm wavelength He/Ne laser excitation. Bar = 25 µm. resulted in (1) initiation of aggregation of casein micelles at ph ~5.58, (2) the onset of gelation as reflected by a reduction in protein mobility at ph 5.48, and (3) gradual formation of a 3-D gel network as the ph decreased below From 1 to 2 min (ph ) after GDL addition, small particles of protein, <1µm in diameter, were observed to move rapidly in random directions, presumably due to Brownian motion and/or small, localised convection currents. After 3 min, the ph had dropped to 5.58 and aggregation of milk proteins into small (<2 µm) particles was evident (results not shown). The onset of gelation and the formation of a visible protein network occurred at ph ~5.4, at which point much of the stained protein became immobilized (Fig. 4c). A 3-D reconstruction of a CSLM image stack of the GDL-induced gel confirmed the 3-D nature of the protein network. Time-lapse animation of the CSLM image sequence of GDL-induced gelation enabled dynamic visualisation of the gelation process. Further reduction in ph to 5. was accompanied by an increase in the staining intensity of the network (Fig. 4 g j). Simultaneously, the aqueous phase became darker indicating a decrease in staining intensity. Whilst most of the protein appeared to be immobile at ph <5.4, time-lapse animation revealed that some protein strands attached to the network appeared to flex. The onset of gelation at ph 5.48 coincided with large increases in G and η (Fig. 5); both rheological parameters continued to increase as the ph decreased to 4.9. The GDL-induced protein network was more homogeneous than the rennet-induced gel networks described above. Storage modulus (Pa) Time (min) FIG. 3 Low-amplitude oscillatory rheometry (LAOR) data for rennet coagulated low-fat and full-fat milk showing apparent dynamic viscosity (η*) and storage modulus (G ). Key: full-fat η* (+); lowfat η* (*); full-fat G ( ); low-fat G ( ). Monitoring Cheddar Cheese Melting by Confocal Scanning Laser Microscopy and Low-Amplitude Oscillatory Rheology Figures 6 and 7 show a sequence of CSLM images confocal images of protein-stained, full-fat and low-fat cheddar cheeses, respectively, taken during heating from 2 to 8 o C. The protein phase appeared as a homogeneous and continuous bright region containing irregularly shaped, dark unstained fat inclusions. Heating from 2 to 45 o C resulted in little visible change in microstructure. There was an increase in staining intensity of the protein for both fullfat and low-fat cheeses as the temperature approached 5 o C. Above 5 o C, a gradual collapse of the cheese sam- Dynamic viscosity (Pa.S)

5 M.A.E. Auty et al.: SSLM for milk protein gelation and cheese melting study 33 1min, ph6.51 2min, ph5.64 4min, ph5.43 6min, ph5.33 8min, ph5.25 a b c d e 1min, ph min, ph min, ph5.8 16min, ph5.3 18min, ph4.98 f g h i j FIG. 4 Sequence of confocal scanning laser microscopy (CSLM) images of glucono-δ-lactone (GDL)-induced gelation of skim milk containing 1 µg/ml Rhodamine B. Bright areas are protein. Numbers refer to minutes after rennet addition. Images were acquired using 568 nm wavelength Kr/Ar laser excitation. Bar = 25 µm. ph Storage modulus, G, (Pa) Time (min) FIG. 5 Low-amplitude oscillatory rheometry (LAOR) data for glucono- lactone (GDL)-treated skim milk showing ph ( ), dynamic viscosity (-), and storage modulus ( ). Dynamic viscosity, η * (Pa.S) ple occurred making it difficult to maintain the same field of view. Fat droplets in the full-fat cheese began to coalesce at temperatures above 6 o C, resulting in the formation of spherical fat pools as the temperature approached 8 o C. There was very little fat coalescence in the low-fat cheese compared with the full-fat cheese, a trend expected because of the lower volume fraction of the fat phase and the more widely dispersed fat globules. Figure 8 shows the changes in storage modulus (G ) and phase angle (δ) of low-fat and full-fat cheddar cheeses during heating from 2 to 8 o C. The low-fat cheddar cheese had a higher initial storage modulus than the full-fat cheese, reflecting the higher concentration of the protein-structuring element in the former cheese. The storage moduli of the two cheeses appeared to converge as the temperature approached 8 o C. The increase in phase angle of both full-fat and low-fat cheeses at > 6 o C indicated a reduction in elasticity and an increase in the viscous nature of the sample (Horne et al. 1993). FIG. 6 Sequence of confocal scanning laser microscopy images taken during heating of full-fat cheddar cheese stained with.1% aq., w/v, Rhodamine B. Bright areas are protein, dark regions are fat. Heating rate 3 o C/min. Images were acquired using 568 nm Kr/Ar laser excitation. Bar = 25 µm.

6 34 Scanning Vol. 21, 5 (1999) FIG. 7 Sequence of confocal scanning laser microscopy images taken during heating of low-fat cheddar cheese stained with.1% aq., w/v, Rhodamine B. Note increase in staining intensity at 5 o C. Heating rate 3 o C/min. Images were acquired using 568 Kr/Ar nm laser excitation. Bar = 25 µm. Phase angle ( ) FIG. 8 Low-amplitude oscillatory rheometry (LAOR) data showing changes in phase angle (δ) and storage modulus (G ) of full-fat and low-fat cheddar cheeses during heating from 2 to 8 o C. Key: fullfat δ ( ),low-fat δ ( ), full-fat G ( ), low-fat G ( ). Conclusions These results indicate that CSLM is a valuable technique for studying dynamic events in relation to physical measurements. It can thus be a predictive tool for studying changes in food texture, such as gelation and melting, and can be used to identify key processing effects in product development. Acknowledgment The authors would like to thank David Oldfield for providing the skim milk powder. This work was partly funded by European Union Structural Funds (European Regional Development Fund). References Temperature ( C) 25, 2, 15, 1, 5, Auty MAE, Fenelon MA, Guinee TP: Observation of rennet-induced gelation using confocal scanning laser microscopy (abstr). Irish J Ag Food Res 36, 263 (1997) Bremer LGB, van Vliet T, Walstra P: Confocal scanning laser microscopy as a tool to study the fractal nature of casein gels. In Past, Present and Future of Electron Microscopy in Agricultural Research (Ed. A. Boekestein). Dienst Landbouwk undig Onderzoek-Technische en Fysische Dienst voor de Landbouw. Wageningen, The Netherlands (1991) Storage modulus (Pa) Brooker BE: Imaging food systems by confocal scanning laser microscopy. In New Physico-Chemical Techniques for the Characterisation of Complex Food Systems (Ed. E. Dickenson). Blackie Academic and Professional, London (1995) Chapman HR: The effect the chemical quality of milk has on cheese quality. Dairy Ind 39, (1974) Emmons DB, Kalab M, Larmond E, Lowrie, RJ: Milk gel structure. X. Texture and microstructure in cheddar cheese made from whole milk and from homogenised low-fat milk. J Texture Stud 11, (198) Fenelon MA, Guinee TP: The effect of milk fat on yield in Cheddar cheese. J Dairy Sci (in press) (1998) Guinee TP, Gorry CB, O Callaghan J, O Kennedy BT, O Brian N, Fenelon MA: The effects of composition and some processing treatments on the rennet coagulation properties of milk. Int J Dairy Technol 5, (1997) Harwalkar VR, Kalab M: Milk gel structure. XI. Electron microscopy of glucono-δ lactone-induced skim milk gels. J Texture Stud 11, (198) Harwalkar VR, Kalab M: Effect of acidulants and temperature on microstructure, firmness and susceptibility to syneresis of skim milk gels. Scan Electr Microsc III, (1981) Hassan AN, Frank JF, Farmer MA, Schmidt KA, Shalabi SI: Formation of yoghurt microstructure and three-dimensional visualisation as determined by confocal scanning laser microscopy. J Dairy Sci 78, (1995) Heertje I, Visser J, Smits P: Structure formation in acid gels. Food Microstruct 4, (1985) Heertje I, van der Vlist P, Blonk JCG, Hendrickx HAC, Brackenhof GJ: Confocal scanning laser microscopy in food research: Some observations. Food Microstruct 6, (1987) Hermannsson A-M: Microscopy the art of food technology. Food Microstructure conference, Leatherhead Food RA, Surrey, UK (September 1998) Horne DS, Banks JM, Leaver J, Law AJR: Dynamic mechanical spectroscopy of cheddar cheese. In Cheese Yield & Factors Affecting Its Control. IDF Seminar, Brussels (1993) Kalab M: Artefacts in conventional scanning electron microscopy of some milk products. Food Microstruct 3, (1984) Lucey JA, van Vliet T, Grolle K, Geurts T, Walstra P: Properties of acid casein gels made by acidification with glucono-δ lactone. 2. Syneresis, permeability and microstructure properties. Int Dairy J 7, (1997) Nie S, Chiu DT, Zare RN: Real-time detection of single molecules in solution by confocal fluorescence microscopy. Anal Chem 67, (1995) Pawley J: Fundamental limits in confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd Edition (Ed. Pawley JB). Plenum Press, New York (1995) Schellhaase SM, Morris HA: Rheological and scanning microscopic examination of skim milk gels obtained by fermenting with ropy and non-ropy strains of lactic acid bacteria. Food Microstruct 4, (1985)