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Influence of heating temperature, pressure and pH on recrystallization inhibition activity of antifreeze protein type III
Journal of Food Engineering 187 (2016) 53e61
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Influence of heating temperature, pressure and pH on recrystallization inhibition activity of antifreeze protein type III Andreas Leiter a, *, Stefanie Rau a, Sebastian Winger a, Claudia Muhle-Goll b, c, Burkhard Luy b, c, Volker Gaukel a a
KIT (Karlsruhe Institute of Technology), Institute of Process Engineering in Life Sciences, Section I: Food Process Engineering, Kaiserstraße 12, 76131 Karlsruhe, Germany KIT (Karlsruhe Institute of Technology), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany c KIT (Karlsruhe Institute of Technology), Institute for Biological Interfaces 4, P.O. Box 3640, 76021 Karlsruhe, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Received in revised form 5 April 2016 Accepted 24 April 2016 Available online 29 April 2016
The objective of this work was to investigate the influence of heating temperature, pressure and pH on ice recrystallization inhibition (IRI) activity and on the structure of fish antifreeze protein type III (AFP III). Results showed that temperature treatment up to 80 C for one minute had no influence on IRI activity. 1 H NMR spectroscopy demonstrated a fully reversible denaturation after temperature treatment at 80 C up to 1 h. Adjusting pH with hydrochloric acid or sodium hydroxide increased the IRI activity independent of the pH value, whereas NMR experiments showed no significant structural change. Experiments with sodium chloride showed that the addition of ions is responsible for increased IRI activity and increasing ion concentration led to increased IRI activity up to a limiting value between 0.02 and 0.03 M. Pressure treatment up to 400 MPa for one minute had no influence on IRI activity. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Fish antifreeze protein Ion concentration pH value High pressure Recrystallization inhibition Temperature
1. Introduction Recrystallization is defined as changes in size, number and shape of ice crystals while keeping the total ice volume constant (Cook and Hartel, 2010; Hartel, 2001). These changes may be due to several recrystallization processes like migratory recrystallization, accretion and isomass rounding (Hartel, 2001). During frozen storage recrystallization may affect the quality of frozen food products. For example, recrystallization can lead to high drip loss during thawing and have an important effect on appearance and texture of frozen food (Pham and Mawson, 1997). In ice cream, the ice crystal size has a big impact on mouthfeel. When the ice crystal size increases during storage and exceeds a threshold detection size, the product becomes coarse and unacceptable (Hartel, 1996, 2001). The ice crystal growth due to recrystallization can be influenced by storage temperature (Donhowe and Hartel, 1996; Leiter and Gaukel, 2016) or formulation (Bahramparvar and Mazaheri Tehrani, 2011; Leiter and Gaukel, 2016; Miller-Livney and Hartel,
* Corresponding author. E-mail address: [email protected] (A. Leiter). http://dx.doi.org/10.1016/j.jfoodeng.2016.04.019 0260-8774/© 2016 Elsevier Ltd. All rights reserved.
1997). Traditionally hydrocolloids are added to ice cream to inhibit recrystallization (Adapa et al., 2000; Bahramparvar and Mazaheri Tehrani, 2011), but also ice-binding proteins (IBP) are discussed as very potent agents (Hassas-Roudsari and Goff, 2012; Marshall et al., 2003; Regand and Goff, 2006). Ice-binding proteins were first identified in the blood of fish, living in ice-laden seas (DeVries and Wohlschlag, 1969; Scholander et al., 1953, 1957). Later proteins with similar function were also found in certain insects, plants and microorganisms (Griffith and Ewart, 1995; Venketesh and Dayananda, 2008). IBPs in different organisms can serve different roles, which include thermal hysteresis (TH), ice recrystallization inhibition (IRI), ice structuring, and ice adhesion (Davies, 2014). IBPs could inhibit ice crystal growth in a certain temperature interval by a surface adsorption inhibition (Raymond and DeVries, 1977). By adsorbing to ice crystal surface of IBP, ice growth is only possible between the proteins leading to a microcurvature. According to the Kelvin equation this microcurvature leads to a depression of the temperature of ice growth, called hysteresis freezing point, below the melting point (Kristiansen and Zachariassen, 2005). The melting temperature is also slightly elevated from the melting point by the surface
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adsorption (Celik et al., 2010). The difference between hysteresis freezing point and the melting point has been termed thermal hysteresis (Kristiansen and Zachariassen, 2005). IBPs with high TH, like IBPs of fishes or insects, are also called antifreeze proteins (AFP) (Davies, 2014). Furthermore IBPs are potent agents for ice recrystallization inhibition. In contrast to TH which needs millimolar IBP concentrations, IRI requires only micromolar concentrations (Davies, 2014) and there is no direct correlation between thermal hysteresis and IRI (Gaukel et al., 2014; Yu et al., 2010). Although it is likely that recrystallization inhibition stems from the interaction of IBPs and ice crystals, the exact IRI mechanism of IBPs is not fully understood (Capicciotti et al., 2013; Venketesh and Dayananda, 2008; Yu et al., 2010). Nevertheless the IRI is shown in several systems like sucrose solution (Budke et al., 2009; Gaukel et al., 2014; Regand and Goff, 2005) or ice cream (Crilly et al., 2008; Regand and Goff, 2006). Therefore IBPs have a high potential for industrial application (Venketesh and Dayananda, 2008). For example, commercial ice cream products with IBPs are already available in the market (Crilly et al., 2008). However less is published about the influence of process parameters or pH on IRI activity of IBP. A few studies investigated the influence of temperature on TH (Friis et al., 2014; Kawahara et al., 2007; Li et al., 1998; Marshall et al., 2005; Qiu et al., 2010), but to best of our knowledge only two studies investigated the influence of temperature on IRI activity. Regand and Goff (2005) showed that IRI activity of an IBP from coldacclimated winter wheat grass extract (AWWE) in sucrose solution was reduced significantly after heating to 85 C for 10 min. In contrast pasteurization (74 C, 15 min) of an ice cream mix containing proteins of AWWE improved the IBP activity (Regand and Goff, 2006). Studies on the influence of pH on the TH of AFP type III showed no influence between pH 2 and pH 11. But at pH 1 and 13, the TH decreased by 20% and 27%, respectively (Chao et al., 1994). A few other studies investigated the influence of pH on TH of different IBPs (Gauthier et al., 1998; Kristiansen et al., 2005; Li et al., 1998; Wu et al., 1991), but to what extent the pH value has an influence on the IRI activity of IBPs was not analyzed. To our knowledge there are no studies investigating the influence of high pressure on the activity (TH or IRI) of IBPs. But for example the use of IBPs in combination with high pressure shift freezing (HPSF) has a high potential. This freezing method permits an increase in the ice nucleation rate by creating a high supercooling. As a result, many small ice crystals are formed with a narrow ice crystal size distribution (LeBail et al., 2002). However recrystallization processes during long storage times could diminish the great advantage of a small initial ice crystal size. The use of IBPs in combination with HPSF could be the answer but the effect of pressure on the IRI of IBPs must be known, especially as it is well known that high pressure can affect the structure of proteins (Heremans, 1982; Mozhaev et al., 1996). Therefore in this paper we investigate the influence of temperature, pressure and pH on the IRI activity of AFP type III. In addition we analyzed the impacts of these conditions on the protein structure by NMR spectroscopy. This should provide an insight into a possible correlation between an altered protein structure and their influence on ice recrystallization inhibition. Based on this knowledge production processes can be designed in such a way that the IRI activity of IBPs is kept as large as possible.
was studied in a 49% (w/w) sucrose solution prepared with pure water and AFP III with a total concentration of 2 mg mL1. AFP III, isolated from the fish ocean pout (macrozoarces americanus), was purchased from A/F Protein (Waltham, USA). Composition and structure of the protein were already characterized (Hew et al., 1984, 1988). For the addition of AFP III a parent solution with pure water and a total concentration of 0.2 mg mL1 of AFP was prepared. To get a total concentration of 2 mg mL1 AFP III in the sample solution, 200 ml of the parent solution was added to a 20 mL flask and filled up with the previously prepared sucrose solution. Dilution of sucrose concentration due to the parent solution was adjusted by extra sucrose addition. The influence of pH on the IRI activity was studied in a 49% (w/w) sucrose solution prepared with 0.1 M hydrochloric acid and/or 0.1 M sodium hydroxide. Total AFP III concentration was also 2 mg mL1 and the addition of the protein was performed as described above. The final pH values of the sample solutions were adjusted to 1, 7 and 11 resulting in a final molar ion concentration in all AFP III sample solutions of 0.06 M. In order to investigate the influence of ion concentration on the IRI activity, AFP III sample solution was prepared with 0.005 M, 0.025 M, 0.05 M, or 0.1 M NaCl solution instead of pure water. Thus, the final ion concentration in the AFP III sample solution was 0.003 M, 0.015 M, 0.03 M, or 0.06 M NaCl. 1 H NMR spectroscopy experiments could not be carried out with high sucrose concentration, because sucrose signal would mask the protein signal. In addition the protein concentration was increased to increase the signal-to-noise ratio. Therefore NMR experiments were carried out without sucrose in the solutions and for temperature and pressure experiments the parent solution was used. To investigate the pH influence on protein structure the parent solution and hydrochloric acid and/or sodium hydroxide was mixed (volume ratio 1:1) to get the final pH values 1, 7 and 11. Before 1H NMR spectroscopy measurement, 450 ml of AFP solution was mixed with 50 ml of deuterium. Thus, the final AFP concentration was 0.09 mg mL1 for samples used to measure the effect of pH and 0.18 mg mL1 for samples used to measure the effect of temperature and pressure. 2.2. Heat treatment To investigate the influence of temperature on the IRI activity of AFP III, 800 ml of the sample solution was filled in a 1.5 mL glass vial and sealed. Afterwards the vial was placed in a heated water bath circulator and heated up to 40 C, 60 C or 80 C. When final temperature of the sample solution was reached (heating-up period 3 min for all temperatures), time measurement for heat treatment was started. The treatment times were one minute at 40 C, 60 C, 80 C and 30 or 60 min at 80 C. After heat treatment sample was cooled down immediately to room temperature by placing the vial in ice water. Investigation of temperature on protein structure was done directly in the spectrometer. The solution was heated to 80 C (maximal heating-up period 5 min) and 1H NMR spectrum was acquired after 2 min, 30 min and 60 min. Observation times shorter than 2 min were not feasible, because of the required preparation time for data acquisition. After heat treatment sample was cooled down to room temperature and 1H NMR spectrum was acquired again. 2.3. Pressure treatment
2. Materials and methods 2.1. Sample preparation The influence of temperature and pressure on the IRI activity
Pressure treatment was conducted in a high-pressure multivessel system with five autoclaves (aad GmbH, Frankfurt, Germany) of 20 mL each. The system is fully programmable, allowing reproducible pressure build-up times (SPS, Siemens, Germany). Pressure
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is generated by a multistage pneumatic pressure intensifier. The pressure-transmitting fluid was a mixture of 1/3 water and 2/3 of a glycol based fluid (aad GmbH). For pressurization 600 ml of sample solution was filled into a teflon tube, closed with silicon stoppers and put into the transmitting fluid. Sample solution was pressurized one minute at 30 MPa, 200 MPa and 400 MPa. Time measurement was started when final pressure was reached. 2.4. Sample freezing and storage Sample freezing and storage was executed similar to the method described in Gaukel et al. (2014). For ice recrystallization analysis an amount of 18 ml sample solution was placed between two microscope cover slips stuck on an object slide, then covered with another cover slip and sealed with silicone. After drying of the silicone, samples were subjected to a fast freezing by immersion in liquid nitrogen for a few seconds. This transformed the aqueous solution into a glassy state. This procedure allows the system to crystallize in a uniform way during heating up from the glassy state to the storage temperature. After freezing, the samples were stored at a constant temperature of 12 C, ± 0.1 C, in a small storage chamber. Under these conditions the resulting ice volume fraction is approximately 22% according to the phase diagram by Riedel (1949). To avoid any temperature fluctuations the storage chamber was tempered by a liquid, which was temperature controlled by an external cryostat FP50 (Julabo GmbH, Seelbach, Germany). The storage chamber itself was placed in a cooled glove box with a temperature of 12 C ± 1 C. The temperature inside the storage chamber and glove box was recorded by thermocouples during the storage time of one week. 2.5. Microscopy and image analysis During storage time pictures of ice crystals were taken with a digital camera (Altra SIS20, Olympus, Tokio, Japan) attached to a polarization microscope (BX41, Olympus, Tokio, Japan) installed in
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the glove box. Pictures were taken in regularly time steps of about 3 h or 5 h, 24 h, 48 h, 72 h, 96 h and 168 h or 192 h after freezing. For evaluation of the pictures, the contour of an ice crystal was manually circumscribed on a computer with the software ImagePro Plus 5.0 (Media Cybernetics, Rockville, USA). From the so defined area of each ice crystal the equivalent diameter was calculated as the diameter of a circle with the same area. To get a representative distribution of ice crystal sizes, 300 to 400 ice crystals were analyzed from each object slide and the arithmetic equivalent diameter was calculated. For each experiment two object slides were prepared and each experiment was repeated at least once. Thus, the mean ice crystal diameter x and the standard deviation were determined from the arithmetic equivalent diameters of four object slides. Only the experiments with pure sucrose solution at ph 1 and 11, as well as the AFP III solution with 0.1 M NaCl were executed once and hence mean ice crystal diameter x and the standard deviation were calculated of two object slides. Determination of statistical significance between variances for each sample was carried out using Levene’s test and to determine statistical significance of mean ice crystal diameters one way ANOVA and Tukey’s test or Welch’s t-test was used (alpha ¼ 0.05). 2.6.
1
H NMR spectroscopy
1 H NMR spectra on AFP III were acquired on a 600 MHz Avance I spectrometer (Bruker Biospin, Rheinstetten, Germany) using a triple resonance 5-mm TBI probe equipped with Z gradients. Data acquisition and processing were carried out with Topspin3.2. 1D spectra were acquired with the Watergate W5 pulse sequence with a relaxation delay of 1.5 s. Samples used to measure the effect of pH were acquired with 1024 scans and samples used to measure the effect of pressure were acquired with 256 scans. To investigate the effect of temperature, samples measured after 2 min at 80 C were acquired with 32 scans, after 30 min with 128 scans and after 60 min with 256 scans to reduce the measurement period at shorter heating times. The free induction decays were weighted by
Fig. 1. Influence of temperature treatment on ice recrystallization in a sucrose solution containing AFP III (cAFP III = 2 mg mL-1) during storage at a temperature of -12 C. Time of investigationwas 5 h, 24 h, 48 h, 72 h, 96 h, 168 h, 196 h respectively but to distinguish between thedifferent data points at the same investigation time, data points were shifted along the x-axis.
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Fig. 2. One dimensional 1H NMR spectra of an AFP III solution (cAFP III ¼ 0.18 mg mL1) at 80 C at different heating times compared to the spectrum of an untreated AFP III parent solution.
Fig. 3. One dimensional 1H NMR spectra of cooled AFP III solutions (cAFP untreated AFP III parent solution.
III
¼ 0.18 mg mL1) after different thermal treatment times at 80 C compared to the spectrum of an
an exponential function with a 3 Hz line broadening factor prior to Fourier transformation. 3. Results and discussion 3.1. Influence of temperature on IRI activity and structure of AFP III Ice recrystallization experiments demonstrated a strong IRI activity of AFP III. The mean ice crystal diameter x increased only from
11 mm to 14 mm during storage (Fig. 1). In contrast ice crystals in the frozen sucrose solution increased from 18 mm to 45 mm. The investigation of the influence of temperature on the IRI activity showed that the mean ice crystal diameter x in the one minute heated samples at 40 C, 60 C and 80 C is slightly higher than the mean values of the untreated sample solution. However there is no significant difference between the mean values at the corresponding storage times (P > 0.05). In addition there is no difference between the variances (P > 0.05). Therefore we can conclude that
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Fig. 4. Influence of pH on ice recrystallization in a pure sucrose solution and a sucrose solution containing AFP III (cAFP III ¼ 2 mg mL1) during storage at a temperature of 12 C.
short heating times of one minute up to 80 C have no significant influence on the IRI activity of AFP III. As shown in Fig. 1 longer heating times of 30 and 60 min at 80 C led to higher mean ice crystal diameters compared to the untreated samples, but this difference is statistically not significant (P > 0.05). This is due to the noticeable high standard deviations for the solutions with longer heating times. Statistically there is a significant difference of the variances between short and long heated samples (P < 0.05). According to these results heating times longer than 30 min at 80 C lead obviously to a change or a slight decrease of the IRI activity of AFP III. Fig. 2 shows the one dimensional 1H NMR spectra for the heat treated and untreated samples. A chemical-shift change was observed in the heated samples, indicating a thermal denaturation of the protein. This denaturation was independent of heating time at 80 C because the spectra of all heated samples were similar. The increased noise in the spectrum taken after 2 min heating time was due to a lower number of scans. This was necessary to avoid long exposure to the high temperature. After cooling of the previously heated sample solutions, the original spectrum was restored demonstrating that the protein refolds upon cooling (Fig. 3). Therefore, we can conclude that thermal denaturation of AFP III at 80 C for up to 60 min is fully reversible. This confirms the results of García-Aribas et al. (2007), who showed that thermal denaturation of AFP III is fully reversible after cooling the protein down to room temperature after a heat treatment at 70 C. Also Li et al. (1991) showed a structural transition when AFP III solution was heated from 0 to 60 C. In contrast to García-Aribas et al. (2007) and our research results, Salvay et al. (2007) found that the thermal denaturation is partially irreversible, but the maximal temperature of the thermal treatment was not mentioned. In the case of short heated AFP III sample solution the results of recrystallization experiments could be explained by 1H NMR spectroscopy results. Because thermal denaturation is a fully reversible process, heating doesn’t change the molecular structure and thus not the IRI activity of AFP III. In contrast the slight decrease of IRI activity at longer heating times at 80 C cannot be explained completely by 1H NMR spectroscopy. But one has to keep in mind
that in contrast to the recrystallization experiments the 1H NMR spectroscopy experiments were executed in aqueous solution and not in sucrose solution. It is known that the presence of sucrose increases the stability of proteins and increases the thermal denaturation temperature (Baier and McClements, 2001; Kulmyrzaev et al., 2000). Assuming that the slight decrease of IRI activity at long heating times is based on a change in protein structure, the addition of sucrose and thus an increase in protein stability cannot be the explanation. But a maillard reaction between sucrose and the protein is possible and the reaction speed increases with temperature and time (Ledl and Schleicher, 1990). Therefore it is assumed that at longer heating times at 80 C a small amount of conjugates were produced, which have no or a reduced IRI activity. 3.2. Influence of pH on IRI activity and structure of AFP III To evaluate a possible influence of pH on IRI activity of AFP III, we examined first, as control solution, the influence of pH on the recrystallization in pure sucrose solution. Fig. 4 shows that adjusting pH value to 11 led to a slightly smaller but not significantly different mean ice crystal diameter (P > 0.05), whereas mean ice crystal diameters in pure sucrose solution at pH 1 were significantly increased. As expected Fehling test showed a cleavage of sucrose in glucose and fructose at pH 1. Hereby the molar concentration of sugar molecules increased, resulting in a higher freezing point depression and consequently in a lower ice content. However, the increased recrystallization rate at decreased ice content is contradictory to published results (Budke et al., 2009; Gaukel, 2004; Sutton et al., 1996). At lower ice content the distance between crystals is longer leading to an increased diffusion time between the crystals and less accretion. The only reasonable explanation for the observed results is that the diffusion coefficient of water is higher in the solution of monosaccharides. For example, Sutton et al. (1997) showed that recrystallization rates in sugar solutions decrease with increasing sugar molecular weight. Hagiwara et al. (2006) showed that recrystallization rate in glucose or fructose solution is faster than in sucrose solution at same ice content and storage temperature. In addition the authors
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Fig. 5. One dimensional 1H NMR spectra of AFP III solutions (cAFP III ¼ 0.18 mg mL1) at different pH values adjusted by hydrochloric acid and/or sodium hydroxide compared to the spectrum of an untreated AFP III parent solution.
Fig. 6. Mean ice crystal diameter of a AFP III sucrose solution (cAFP III ¼ 2 mg mL1) with 0.03 M NaCl and of a AFP III sucrose solution (cAFP III ¼ 2 mg mL1) adjusted to pH 7 by adding hydrochloric acid and sodium hydroxide at a temperature of 12 C.
illustrated that there is a correlation between recrystallization rate and water diffusion coefficient in the sugar solutions. This suggests that the increased recystallization rate at pH 1 is due to an increased water diffusion coefficient. In AFP III sample solutions, pH had no clear influence on IRI activity of AFP III. The mean ice crystal diameters of all AFP III sample solutions with hydrochloric acid and/or sodium hydroxide are of the same size and are smaller than the ice crystal diameters in AFP solution with pure water (pH 6.1). These results seem to be
consistent with the research on thermal hysteresis of AFP III by Chao et al. (1994) who showed that TH of AFP III remained unchanged in a pH range from 2 to 11. Interestingly, the cleavage of sucrose in glucose and fructose in the AFP III solution at pH 1 did not affect the IRI activity. By binding to the ice crystal surface and the adsorption inhibition mechanism of AFP III, recrystallization processes are very likely controlled by surface integration process and not by bulk diffusion like in the pure sucrose solution. Thus, an increased water diffusion coefficient in the pH 1 solution would not
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affect the recrystallization rate. But the increased IRI activity of AFP III with hydrochloric acid and/or sodium hydroxide cannot be explained by a dramatic change in molecular structure like complete unfolding. The one dimensional 1H NMR spectra at pH 1, 7 and 11 show a signal dispersion that is comparable to the untreated parent solution (Fig. 5). Around 0 ppm isolated methyl group signals are visible that are indicative of a defined three-dimensional structure and that would disappear if unfolding takes place. However, both at pH 1 and at pH 11 the amide signals, that resonate in the ppm range between 10 and 7 ppm, changed their pattern. This indicates that although the protein is not completely unfolded, partial denaturation may have started. However, this partial denaturation cannot explain the increased IRI activity of AFP III, because at pH 7 there was also an increased IRI but no partial denaturation was visible. Due to our sample preparation the samples at pH 1, 7 and 11 had a higher salt concentration as the sample solution at pH 6.1. This leads to the hypothesis that it is not the pH but probably the ion concentration which affects IRI activity. Evans et al. (2007) have already shown that the addition of different salt types leads to a synergistic enhancement of thermal hysteresis of AFP III. To verify our suggestion that only the ion concentration affects the IRI activity, we investigated the IRI activity of AFP III at pH 7 with an AFP III sucrose solution in pure water (pH 6.1) with the same quantity of ions (NaCl) like in the pH 7 solution. As the results in Fig. 6 show, there is no significant difference between the mean ice crystal diameters between these two samples (P > 0.05). This confirms the suggestion that the addition of ions increased the IRI activity of AFP III and not the pH change. The influence of the slightly increased ion concentration in the pH 7, pH 11 and NaCl solution on the freezing point depression and ice content is negligible as the molar ion concentration was only 0.06 M compared to the molar sucrose concentration of 1.75 M (49%). In addition recrystallization experiments with 49% sucrose and 0.03 M NaCl (0.06 M ionconcentration) showed no difference in recrystallization rate (data not shown). A possible explanation for this salt-induced enhancement of IRI
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activity is given by Kristiansen and Zachariassen (2005). The authors proposed that at the equilibrium melting temperature of an ice crystal, there is a steady-state distribution of AFP molecules in the solution surrounding the ice crystal and in the ice/water interfacial region. Factors that lower the solubility of AFP molecules in the solution are likely to cause a shift in their steady-state distribution in favor of the ice/water interfacial region. Therefore there is an increased surface density of irreversibly adsorbed AFP molecules at the ice surface, when the interfacial region solidifies at a temperature below the equilibrium melting point (Kristiansen et al., 2008; Kristiansen and Zachariassen, 2005). According to Kristiansen et al. (2008) salts reduce the solubility of AFP in the solution and thus TH is increased. Similar results were provided by Hayakari and Hagiwara (2012) in a molecular dynamic analysis of winter flounder antifreeze protein. The analysis showed an enhanced interaction between the AFP molecule and the ice surface due to the presence of ions in the water phase. In accordance with these findings the increased IRI activity in our study is probably due to an increased number of adsorbed AFP molecules on the ice crystal surface. One implication of this presumption is that there should be a limiting ion concentration where the addition of further ions does not increase the IRI activity because a maximum occupancy of AFP molecules on the ice crystal surface is reached. Therefore the influence of NaCl concentration on the IRI activity of AFP III was investigated. Fig. 7 illustrates the mean ice crystal diameter of an AFP III sample solution with different molar NaCl concentrations after different storage times. The results reveal that the mean ice crystal diameter decreases for each storage time with increasing molar NaCl concentration up to a limiting value which is probably between 0.02 M and 0.03 M. An additional increase of salt concentration did not affect the mean ice crystal diameter. Knight et al. (1995) showed that the addition of 0.008 M NaCl does not affect the IRI of AFP I. However, in this research the ice crystal size was analyzed only qualitatively by comparing ice crystal pictures and not quantitatively. Finally, our result supports the presumption that the increased IRI activity of AFP III by the addition of NaCl is due to
Fig. 7. Influence of molar NaCl concentration on ice recrystallization in an AFP III sucrose solution (cAFP
III
¼ 2 mg mL1) during storage at a temperature of 12 C.
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Fig. 8. Influence of hydrostatic pressure on ice recrystallization in an AFP III sucrose solution (cAFP
an increased number of adsorbed AFP III molecules on the ice surface. 3.3. Influence of pressure on IRI activity of AFP III Fig. 8 shows no significant difference between the mean ice crystal diameters at the corresponding storage times of pressure treated and untreated samples (P > 0.05). Also the 1H NMR spectroscopy investigation showed that there is no irreversible denaturation of AFP III at this pressure, because the 1H NMR spectra of the different sample solutions were similar (data not shown). However a reversible denaturation of the protein cannot be ruled out. This result is a first indication of the possibility of the combination of antifreeze proteins with high pressure shift freezing to maintain the small initial ice crystal size for a longer time. 4. Conclusion In this paper we investigated the influence of temperature, pressure, pH, and ion concentration on the ice recrystallization inhibition activity of AFP III. Results showed that thermal treatment at 80 C up to one hour has only a slight effect on IRI activity of AFP III. Furthermore we showed that in pure sucrose solutions the recrystallization rate was increased at pH 1 which can be related to cleavage of sucrose and a diffusion controlled recrystallization, whereas the increased IRI activity at different pH values in AFP solutions can be related to the salt concentration. Up to a limiting value between 0.02 and 0.03 M NaCl the IRI activity increased probably due to an increased AFP concentration on the ice crystals surface. Pressure treatment up to 400 MPa for one minute had no influence on IRI activity. In addition 1H NMR spectroscopy showed high stability of AFP III at pH values between 1 and 11 and pressure up to 400 MPa. Besides 1H NMR spectroscopy demonstrated a fully reversible denaturation of AFP III in aqueous solutions after temperature treatment at 80 C up to 1 h. It is already known that ice-binding proteins have a high potential for different industrial applications, but the findings of this
III ¼ 2
mg mL1) during storage at a temperature of 12 C.
research also shows that the IRI activity of AFP III is not affected by pH or by typical process parameters like temperature or pressure in the range investigated. Thus, these data highlight the high potential of AFP III proteins in industrial applications. Consequently the only barrier for industrial use of AFP III is the high price. That is why more research is required to reduce the costs for protein synthesis or to find alternative substances with similar IRI activity and high process stability like AFP III. Acknowledgement We would like to thank the Max-Buchner-Forschungsstiftung for research fellowship MBFSt 3400 and the financial support. We also thank Max-Rubner Institute for the use of the autoclave and Ralf Lindauer for the support during pressure experiments. Burkhard Luy thanks the DFG (instrumental facility Pro2NMR) and the Helmholtz association (programme BIFTM) for funding. References Adapa, S., Schmidt, K.A., Jeon, I.J., Herald, T.J., Flores, R.A., 2000. Mechanisms of ice crystallization and recrystallization in ice cream: a review. Food Rev. Int. 16 (3), 259e271. Bahramparvar, M., Mazaheri Tehrani, M., 2011. Application and functions of stabilizers in ice cream. Food Rev. Int. 27 (4), 389e407. Baier, S., McClements, D.J., 2001. Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions. J. Agric. Food Chem. 49 (5), 2600e2608. Budke, C., Heggemann, C., Koch, M., Sewald, N., Koop, T., 2009. Ice recrystallization kinetics in the presence of synthetic antifreeze glycoprotein analogues using the framework of LSW theory. J. Phys. Chem. B 113 (9), 2865e2873. Capicciotti, C.J., Doshi, M., Ben, R.N., 2013. Ice recrystallization inhibitors: from biological antifreezes to small molecules. In: Wilson, P. (Ed.), Recent Developments in the Study of Recrystallization. InTech, Rijeka, Croatia, pp. 177e224. Celik, Y., Graham, L.A., Mok, Y.F., Bar, M., Davies, P.L., Braslavsky, I., 2010. Superheating of ice crystals in antifreeze protein solutions. Proc. Natl. Acad. Sci. U. S. A. 107 (12), 5423e5428. € nnichsen, F.D., DeLuca, C.I., Sykes, B.D., Davies, P.L., 1994. StructureChao, H., So function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice. Protein science. A Publ. Protein Soc. 3 (10), 1760e1769. Cook, K., Hartel, R.W., 2010. Mechanisms of ice crystallization in ice cream
A. Leiter et al. / Journal of Food Engineering 187 (2016) 53e61 production. Compr. Rev. Food Sci. Food Saf. 9 (2), 213e222. Crilly, J.F., Russell, A.B., Cox, A.R., Cebula, D.J., 2008. Designing multiscale structures for desired properties of ice cream. Ind. Eng. Chem. Res. 47 (17), 6362e6367. Davies, P.L., 2014. Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. Trends Biochem. Sci. 39 (11), 548e555. DeVries, A.L., Wohlschlag, D.E., 1969. Freezing resistance in some antarctic fishes. Science 163 (3871), 1073e1075. Donhowe, D.P., Hartel, R.W., 1996. Recrystallization of ice in ice cream during controlled accelerated storage. Int. Dairy J. 6 (11e12), 1191e1208. Evans, R.P., Hobbs, R.S., Goddard, S.V., Fletcher, G.L., 2007. The importance of dissolved salts to the in vivo efficacy of antifreeze proteins. Comparative biochemistry and physiology. Part A. Mol. Integr. Physiol. 148 (3), 556e561. Friis, D.S., Johnsen, J.L., Kristiansen, E., Westh, P., Ramløv, H., 2014. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax. Protein science. Publ. Protein Soc. 23 (6), 760e768. García-Aribas, O., Mateo, R., Tomczak, M.M., Davies, P.L., Mateu, M.G., 2007. Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges. Protein Sci. 16 (2), 227e238. Gaukel, V., 2004. Untersuchungen zum Einfluss von Antigefrierproteinen auf die €hrend der Gefrierlagerung, dargestellt an ModRekristallisation von Eis wa €sungen für Eiskrem. GCA-Verlag, Herdecke. elllo Gaukel, V., Leiter, A., Spieß, W.E., 2014. Synergism of different fish antifreeze proteins and hydrocolloids on recrystallization inhibition of ice in sucrose solutions. J. Food Eng. 141, 44e50. Gauthier, S.Y., Kay, C.M., Sykes, B.D., Walker, V.K., Davies, P.L., 1998. Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein. Eur. J. Biochem. 258 (2), 445e453. Griffith, M., Ewart, K.V., 1995. Antifreeze proteins and their potential use in foods. Biotechnol. Adv. 13 (3), 375e402. Hagiwara, T., Hartel, R., Matsukawa, S., 2006. Relationship between recrystallization rate of ice crystals in sugar solutions and water mobility in freeze-concentrated matrix. Food Biophys. 1 (2), 74e82. Hartel, R.W., 1996. Ice crystallization during the manufacture of ice cream. Trends Food Sci. Technol. 7 (10), 315e321. Hartel, R.W., 2001. Crystallization in Foods. Aspen Publishers, Gaithersburg, Md. Hassas-Roudsari, M., Goff, H.D., 2012. Ice structuring proteins from plants: mechanism of action and food application. Food Res. Int. 46 (1), 425e436. Hayakari, K., Hagiwara, Y., 2012. Effects of ions on winter flounder antifreeze protein and water molecules near an ice/water interface. Mol. Simul. 38 (1), 26e37. Heremans, K., 1982. High pressure effects on proteins and other biomolecules. Annu. Rev. Biophys. Bioeng. 11, 1e21. Hew, C.L., Slaughter, D., Joshi, S.B., Fletcher, G.L., Ananthanarayanan, V.S., 1984. Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components. J. Comp. Physiol. B 155 (1), 81e88. Hew, C.L., Wang, N.-C., Joshi, S., Fletcher, G.L., Scott, G.K., Hayes, P.H., Buettner, B., Davies, P.L., 1988. Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus. J. Biol. Chem. 263 (24), 12049e12055. Kawahara, H., Iwanaka, Y., Higa, S., Muryoi, N., Sato, M., Honda, M., Omura, H., Obata, H., 2007. A novel, intracellular antifreeze protein in an antarctic bacterium, Flavobacterium xanthum. Cryoletters 28 (1), 39e49. Knight, C.A., Wen, D., Laursen, R.A., 1995. Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology 32 (1), 23e34. Kristiansen, E., Pedersen, S.A., Zachariassen, K.E., 2008. Salt-induced enhancement of antifreeze protein activity: a salting-out effect. Cryobiology 57 (2), 122e129. Kristiansen, E., Ramløv, H., Hagen, L., Pedersen, S.A., Andersen, R.A., Zachariassen, K.E., 2005. Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.). Comparative biochemistry and physiology. Part B. Biochem. Mol. Biol. 142 (1), 90e97.
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Kristiansen, E., Zachariassen, K.E., 2005. The mechanism by which fish antifreeze proteins cause thermal hysteresis. Cryobiology 51 (3), 262e280. Kulmyrzaev, A., Bryant, C., McClements, D.J., 2000. Influence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins. J. Agric. Food Chem. 48 (5), 1593e1597. LeBail, A., Chevalier, D., Mussa, D., Ghoul, M., 2002. High pressure freezing and thawing of foods: a review. Int. J. Refrig. 25 (5), 504e513. Ledl, F., Schleicher, E., 1990. New aspects of the maillard reaction in foods and in the human body. Angewandte Chemie Int. Ed. Engl. 29 (6), 565e594. Leiter, A., Gaukel, V., 2016. Food freezing: crystal structure and size. In: Smithers, G.W. (Ed.), Reference Module in Food Science. Elsevier, Amsterdam. Li, N., Andorfer, C.A., Duman, J.G., 1998. Enhancement of insect antifreeze protein activity by solutes of low molecular mass. J. Exp. Biol. 201 (15), 2243e2251. Li, X., Trinh, K.Y., Hew, C.L., 1991. Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli improved expression by the modification of the secondary structure of the mRNA. Protein Eng. 4 (8), 995e1002. Marshall, C.B., Chakrabartty, A., Davies, P.L., 2005. Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of alpha-helices. J. Biol. Chem. 280 (18), 17920e17929. Marshall, R.T., Goff, H.D., Hartel, R.W., 2003. Ice Cream, sixth ed. Kluwer Academic/ Plenum Publishers, New York. Miller-Livney, T., Hartel, R.W., 1997. Ice recrystallization in ice cream: interactions between sweeteners and stabilizers. J. Dairy Sci. 80 (3), 447e456. Mozhaev, V.V., Heremans, K., Frank, J., Masson, P., Balny, C., 1996. High pressure effects on protein structure and function. Proteins 24 (1), 81e91. Pham, Q.T., Mawson, F.R., 1997. Moisture migration and ice recrystallization in frozen foods. In: Erickson, M.C., Hung, Y.-C. (Eds.), Quality in Frozen Food. Chapman & Hall, New York, pp. 67e91. Qiu, L.-M., Ma, J., Wang, J., Zhang, F.-C., Wang, Y., 2010. Thermal stability properties of an antifreeze protein from the desert beetle Microdera punctipennis. Cryobiology 60 (2), 192e197. Raymond, J.A., DeVries, A.L., 1977. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc. Natl. Acad. Sci. 74 (6), 2589e2593. Regand, A., Goff, H.D., 2005. Freezing and ice recrystallization properties of sucrose solutions containing ice structuring proteins from cold-acclimated winter wheat grass extract. J. food Sci. 70 (9), E552eE556. Regand, A., Goff, H.D., 2006. Ice recrystallization inhibition in ice cream as affected by ice structuring proteins from winter wheat grass. J. Dairy Sci. 89 (1), 49e57. €gen und Gefriertemperaturen von Fruchts€ Riedel, L., 1949. Brechungsvermo aften in Abh€ angigkeit von der Konzentration. Z. für Leb. eForschung 3, 289e299. Salvay, A.G., Santos, J., Howard, E.I., 2007. Electro-optical properties characterization of fish type III antifreeze protein. J. Biol. Phys. 33 (5e6), 389e397. Scholander, P.F., Dam, L. von, Kanwisher, J.W., Hammel, H.T., Gordon, M.S., 1957. Supercooling and osmoregulation in arctic fish. J. Cell Comp. Physiol. 49, 5e24. Scholander, P.F., Flagg, W., Hock, R.J., Irving, L., 1953. Studies on the physiology of frozen plants and animals in the arctic. J. Cell. Comp. Physiol. 42 (S1), 1e56. Sutton, R.L., Cooke, D., Russell, A., 1997. Recrystallization in sugar/stabilizer solutions as affected by molecular structure. J Food Sci. 62 (6), 1145e1149. Sutton, R.L., Lips, A., Piccirillo, G., Sztehlo, A., 1996. Kinetics of ice recrystallization in aqueous fructose solutions. J. food Sci. 61 (4), 741e745. Venketesh, S., Dayananda, C., 2008. Properties, potentials, and prospects of antifreeze proteins. Crit. Rev. Biotechnol. 28 (1), 57e82. Wu, D.W., Duman, J.G., Cheng, C.-H.C., Castellino, F.J., 1991. Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis. J. Comp. Physiol. B 161 (3), 271e278. Yu, S.O., Brown, A., Middleton, A.J., Tomczak, M.M., Walker, V.K., Davies, P.L., 2010. Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis. Cryobiology 61 (3), 327e334.
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Influence of heating temperature, pressure and pH on recrystallization inhibition activity of antifreeze protein type III Andreas Leiter a, *, Stefanie Rau a, Sebastian Winger a, Claudia Muhle-Goll b, c, Burkhard Luy b, c, Volker Gaukel a a
KIT (Karlsruhe Institute of Technology), Institute of Process Engineering in Life Sciences, Section I: Food Process Engineering, Kaiserstraße 12, 76131 Karlsruhe, Germany KIT (Karlsruhe Institute of Technology), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany c KIT (Karlsruhe Institute of Technology), Institute for Biological Interfaces 4, P.O. Box 3640, 76021 Karlsruhe, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Received in revised form 5 April 2016 Accepted 24 April 2016 Available online 29 April 2016
The objective of this work was to investigate the influence of heating temperature, pressure and pH on ice recrystallization inhibition (IRI) activity and on the structure of fish antifreeze protein type III (AFP III). Results showed that temperature treatment up to 80 C for one minute had no influence on IRI activity. 1 H NMR spectroscopy demonstrated a fully reversible denaturation after temperature treatment at 80 C up to 1 h. Adjusting pH with hydrochloric acid or sodium hydroxide increased the IRI activity independent of the pH value, whereas NMR experiments showed no significant structural change. Experiments with sodium chloride showed that the addition of ions is responsible for increased IRI activity and increasing ion concentration led to increased IRI activity up to a limiting value between 0.02 and 0.03 M. Pressure treatment up to 400 MPa for one minute had no influence on IRI activity. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Fish antifreeze protein Ion concentration pH value High pressure Recrystallization inhibition Temperature
1. Introduction Recrystallization is defined as changes in size, number and shape of ice crystals while keeping the total ice volume constant (Cook and Hartel, 2010; Hartel, 2001). These changes may be due to several recrystallization processes like migratory recrystallization, accretion and isomass rounding (Hartel, 2001). During frozen storage recrystallization may affect the quality of frozen food products. For example, recrystallization can lead to high drip loss during thawing and have an important effect on appearance and texture of frozen food (Pham and Mawson, 1997). In ice cream, the ice crystal size has a big impact on mouthfeel. When the ice crystal size increases during storage and exceeds a threshold detection size, the product becomes coarse and unacceptable (Hartel, 1996, 2001). The ice crystal growth due to recrystallization can be influenced by storage temperature (Donhowe and Hartel, 1996; Leiter and Gaukel, 2016) or formulation (Bahramparvar and Mazaheri Tehrani, 2011; Leiter and Gaukel, 2016; Miller-Livney and Hartel,
* Corresponding author. E-mail address: [email protected] (A. Leiter). http://dx.doi.org/10.1016/j.jfoodeng.2016.04.019 0260-8774/© 2016 Elsevier Ltd. All rights reserved.
1997). Traditionally hydrocolloids are added to ice cream to inhibit recrystallization (Adapa et al., 2000; Bahramparvar and Mazaheri Tehrani, 2011), but also ice-binding proteins (IBP) are discussed as very potent agents (Hassas-Roudsari and Goff, 2012; Marshall et al., 2003; Regand and Goff, 2006). Ice-binding proteins were first identified in the blood of fish, living in ice-laden seas (DeVries and Wohlschlag, 1969; Scholander et al., 1953, 1957). Later proteins with similar function were also found in certain insects, plants and microorganisms (Griffith and Ewart, 1995; Venketesh and Dayananda, 2008). IBPs in different organisms can serve different roles, which include thermal hysteresis (TH), ice recrystallization inhibition (IRI), ice structuring, and ice adhesion (Davies, 2014). IBPs could inhibit ice crystal growth in a certain temperature interval by a surface adsorption inhibition (Raymond and DeVries, 1977). By adsorbing to ice crystal surface of IBP, ice growth is only possible between the proteins leading to a microcurvature. According to the Kelvin equation this microcurvature leads to a depression of the temperature of ice growth, called hysteresis freezing point, below the melting point (Kristiansen and Zachariassen, 2005). The melting temperature is also slightly elevated from the melting point by the surface
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adsorption (Celik et al., 2010). The difference between hysteresis freezing point and the melting point has been termed thermal hysteresis (Kristiansen and Zachariassen, 2005). IBPs with high TH, like IBPs of fishes or insects, are also called antifreeze proteins (AFP) (Davies, 2014). Furthermore IBPs are potent agents for ice recrystallization inhibition. In contrast to TH which needs millimolar IBP concentrations, IRI requires only micromolar concentrations (Davies, 2014) and there is no direct correlation between thermal hysteresis and IRI (Gaukel et al., 2014; Yu et al., 2010). Although it is likely that recrystallization inhibition stems from the interaction of IBPs and ice crystals, the exact IRI mechanism of IBPs is not fully understood (Capicciotti et al., 2013; Venketesh and Dayananda, 2008; Yu et al., 2010). Nevertheless the IRI is shown in several systems like sucrose solution (Budke et al., 2009; Gaukel et al., 2014; Regand and Goff, 2005) or ice cream (Crilly et al., 2008; Regand and Goff, 2006). Therefore IBPs have a high potential for industrial application (Venketesh and Dayananda, 2008). For example, commercial ice cream products with IBPs are already available in the market (Crilly et al., 2008). However less is published about the influence of process parameters or pH on IRI activity of IBP. A few studies investigated the influence of temperature on TH (Friis et al., 2014; Kawahara et al., 2007; Li et al., 1998; Marshall et al., 2005; Qiu et al., 2010), but to best of our knowledge only two studies investigated the influence of temperature on IRI activity. Regand and Goff (2005) showed that IRI activity of an IBP from coldacclimated winter wheat grass extract (AWWE) in sucrose solution was reduced significantly after heating to 85 C for 10 min. In contrast pasteurization (74 C, 15 min) of an ice cream mix containing proteins of AWWE improved the IBP activity (Regand and Goff, 2006). Studies on the influence of pH on the TH of AFP type III showed no influence between pH 2 and pH 11. But at pH 1 and 13, the TH decreased by 20% and 27%, respectively (Chao et al., 1994). A few other studies investigated the influence of pH on TH of different IBPs (Gauthier et al., 1998; Kristiansen et al., 2005; Li et al., 1998; Wu et al., 1991), but to what extent the pH value has an influence on the IRI activity of IBPs was not analyzed. To our knowledge there are no studies investigating the influence of high pressure on the activity (TH or IRI) of IBPs. But for example the use of IBPs in combination with high pressure shift freezing (HPSF) has a high potential. This freezing method permits an increase in the ice nucleation rate by creating a high supercooling. As a result, many small ice crystals are formed with a narrow ice crystal size distribution (LeBail et al., 2002). However recrystallization processes during long storage times could diminish the great advantage of a small initial ice crystal size. The use of IBPs in combination with HPSF could be the answer but the effect of pressure on the IRI of IBPs must be known, especially as it is well known that high pressure can affect the structure of proteins (Heremans, 1982; Mozhaev et al., 1996). Therefore in this paper we investigate the influence of temperature, pressure and pH on the IRI activity of AFP type III. In addition we analyzed the impacts of these conditions on the protein structure by NMR spectroscopy. This should provide an insight into a possible correlation between an altered protein structure and their influence on ice recrystallization inhibition. Based on this knowledge production processes can be designed in such a way that the IRI activity of IBPs is kept as large as possible.
was studied in a 49% (w/w) sucrose solution prepared with pure water and AFP III with a total concentration of 2 mg mL1. AFP III, isolated from the fish ocean pout (macrozoarces americanus), was purchased from A/F Protein (Waltham, USA). Composition and structure of the protein were already characterized (Hew et al., 1984, 1988). For the addition of AFP III a parent solution with pure water and a total concentration of 0.2 mg mL1 of AFP was prepared. To get a total concentration of 2 mg mL1 AFP III in the sample solution, 200 ml of the parent solution was added to a 20 mL flask and filled up with the previously prepared sucrose solution. Dilution of sucrose concentration due to the parent solution was adjusted by extra sucrose addition. The influence of pH on the IRI activity was studied in a 49% (w/w) sucrose solution prepared with 0.1 M hydrochloric acid and/or 0.1 M sodium hydroxide. Total AFP III concentration was also 2 mg mL1 and the addition of the protein was performed as described above. The final pH values of the sample solutions were adjusted to 1, 7 and 11 resulting in a final molar ion concentration in all AFP III sample solutions of 0.06 M. In order to investigate the influence of ion concentration on the IRI activity, AFP III sample solution was prepared with 0.005 M, 0.025 M, 0.05 M, or 0.1 M NaCl solution instead of pure water. Thus, the final ion concentration in the AFP III sample solution was 0.003 M, 0.015 M, 0.03 M, or 0.06 M NaCl. 1 H NMR spectroscopy experiments could not be carried out with high sucrose concentration, because sucrose signal would mask the protein signal. In addition the protein concentration was increased to increase the signal-to-noise ratio. Therefore NMR experiments were carried out without sucrose in the solutions and for temperature and pressure experiments the parent solution was used. To investigate the pH influence on protein structure the parent solution and hydrochloric acid and/or sodium hydroxide was mixed (volume ratio 1:1) to get the final pH values 1, 7 and 11. Before 1H NMR spectroscopy measurement, 450 ml of AFP solution was mixed with 50 ml of deuterium. Thus, the final AFP concentration was 0.09 mg mL1 for samples used to measure the effect of pH and 0.18 mg mL1 for samples used to measure the effect of temperature and pressure. 2.2. Heat treatment To investigate the influence of temperature on the IRI activity of AFP III, 800 ml of the sample solution was filled in a 1.5 mL glass vial and sealed. Afterwards the vial was placed in a heated water bath circulator and heated up to 40 C, 60 C or 80 C. When final temperature of the sample solution was reached (heating-up period 3 min for all temperatures), time measurement for heat treatment was started. The treatment times were one minute at 40 C, 60 C, 80 C and 30 or 60 min at 80 C. After heat treatment sample was cooled down immediately to room temperature by placing the vial in ice water. Investigation of temperature on protein structure was done directly in the spectrometer. The solution was heated to 80 C (maximal heating-up period 5 min) and 1H NMR spectrum was acquired after 2 min, 30 min and 60 min. Observation times shorter than 2 min were not feasible, because of the required preparation time for data acquisition. After heat treatment sample was cooled down to room temperature and 1H NMR spectrum was acquired again. 2.3. Pressure treatment
2. Materials and methods 2.1. Sample preparation The influence of temperature and pressure on the IRI activity
Pressure treatment was conducted in a high-pressure multivessel system with five autoclaves (aad GmbH, Frankfurt, Germany) of 20 mL each. The system is fully programmable, allowing reproducible pressure build-up times (SPS, Siemens, Germany). Pressure
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is generated by a multistage pneumatic pressure intensifier. The pressure-transmitting fluid was a mixture of 1/3 water and 2/3 of a glycol based fluid (aad GmbH). For pressurization 600 ml of sample solution was filled into a teflon tube, closed with silicon stoppers and put into the transmitting fluid. Sample solution was pressurized one minute at 30 MPa, 200 MPa and 400 MPa. Time measurement was started when final pressure was reached. 2.4. Sample freezing and storage Sample freezing and storage was executed similar to the method described in Gaukel et al. (2014). For ice recrystallization analysis an amount of 18 ml sample solution was placed between two microscope cover slips stuck on an object slide, then covered with another cover slip and sealed with silicone. After drying of the silicone, samples were subjected to a fast freezing by immersion in liquid nitrogen for a few seconds. This transformed the aqueous solution into a glassy state. This procedure allows the system to crystallize in a uniform way during heating up from the glassy state to the storage temperature. After freezing, the samples were stored at a constant temperature of 12 C, ± 0.1 C, in a small storage chamber. Under these conditions the resulting ice volume fraction is approximately 22% according to the phase diagram by Riedel (1949). To avoid any temperature fluctuations the storage chamber was tempered by a liquid, which was temperature controlled by an external cryostat FP50 (Julabo GmbH, Seelbach, Germany). The storage chamber itself was placed in a cooled glove box with a temperature of 12 C ± 1 C. The temperature inside the storage chamber and glove box was recorded by thermocouples during the storage time of one week. 2.5. Microscopy and image analysis During storage time pictures of ice crystals were taken with a digital camera (Altra SIS20, Olympus, Tokio, Japan) attached to a polarization microscope (BX41, Olympus, Tokio, Japan) installed in
55
the glove box. Pictures were taken in regularly time steps of about 3 h or 5 h, 24 h, 48 h, 72 h, 96 h and 168 h or 192 h after freezing. For evaluation of the pictures, the contour of an ice crystal was manually circumscribed on a computer with the software ImagePro Plus 5.0 (Media Cybernetics, Rockville, USA). From the so defined area of each ice crystal the equivalent diameter was calculated as the diameter of a circle with the same area. To get a representative distribution of ice crystal sizes, 300 to 400 ice crystals were analyzed from each object slide and the arithmetic equivalent diameter was calculated. For each experiment two object slides were prepared and each experiment was repeated at least once. Thus, the mean ice crystal diameter x and the standard deviation were determined from the arithmetic equivalent diameters of four object slides. Only the experiments with pure sucrose solution at ph 1 and 11, as well as the AFP III solution with 0.1 M NaCl were executed once and hence mean ice crystal diameter x and the standard deviation were calculated of two object slides. Determination of statistical significance between variances for each sample was carried out using Levene’s test and to determine statistical significance of mean ice crystal diameters one way ANOVA and Tukey’s test or Welch’s t-test was used (alpha ¼ 0.05). 2.6.
1
H NMR spectroscopy
1 H NMR spectra on AFP III were acquired on a 600 MHz Avance I spectrometer (Bruker Biospin, Rheinstetten, Germany) using a triple resonance 5-mm TBI probe equipped with Z gradients. Data acquisition and processing were carried out with Topspin3.2. 1D spectra were acquired with the Watergate W5 pulse sequence with a relaxation delay of 1.5 s. Samples used to measure the effect of pH were acquired with 1024 scans and samples used to measure the effect of pressure were acquired with 256 scans. To investigate the effect of temperature, samples measured after 2 min at 80 C were acquired with 32 scans, after 30 min with 128 scans and after 60 min with 256 scans to reduce the measurement period at shorter heating times. The free induction decays were weighted by
Fig. 1. Influence of temperature treatment on ice recrystallization in a sucrose solution containing AFP III (cAFP III = 2 mg mL-1) during storage at a temperature of -12 C. Time of investigationwas 5 h, 24 h, 48 h, 72 h, 96 h, 168 h, 196 h respectively but to distinguish between thedifferent data points at the same investigation time, data points were shifted along the x-axis.
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Fig. 2. One dimensional 1H NMR spectra of an AFP III solution (cAFP III ¼ 0.18 mg mL1) at 80 C at different heating times compared to the spectrum of an untreated AFP III parent solution.
Fig. 3. One dimensional 1H NMR spectra of cooled AFP III solutions (cAFP untreated AFP III parent solution.
III
¼ 0.18 mg mL1) after different thermal treatment times at 80 C compared to the spectrum of an
an exponential function with a 3 Hz line broadening factor prior to Fourier transformation. 3. Results and discussion 3.1. Influence of temperature on IRI activity and structure of AFP III Ice recrystallization experiments demonstrated a strong IRI activity of AFP III. The mean ice crystal diameter x increased only from
11 mm to 14 mm during storage (Fig. 1). In contrast ice crystals in the frozen sucrose solution increased from 18 mm to 45 mm. The investigation of the influence of temperature on the IRI activity showed that the mean ice crystal diameter x in the one minute heated samples at 40 C, 60 C and 80 C is slightly higher than the mean values of the untreated sample solution. However there is no significant difference between the mean values at the corresponding storage times (P > 0.05). In addition there is no difference between the variances (P > 0.05). Therefore we can conclude that
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Fig. 4. Influence of pH on ice recrystallization in a pure sucrose solution and a sucrose solution containing AFP III (cAFP III ¼ 2 mg mL1) during storage at a temperature of 12 C.
short heating times of one minute up to 80 C have no significant influence on the IRI activity of AFP III. As shown in Fig. 1 longer heating times of 30 and 60 min at 80 C led to higher mean ice crystal diameters compared to the untreated samples, but this difference is statistically not significant (P > 0.05). This is due to the noticeable high standard deviations for the solutions with longer heating times. Statistically there is a significant difference of the variances between short and long heated samples (P < 0.05). According to these results heating times longer than 30 min at 80 C lead obviously to a change or a slight decrease of the IRI activity of AFP III. Fig. 2 shows the one dimensional 1H NMR spectra for the heat treated and untreated samples. A chemical-shift change was observed in the heated samples, indicating a thermal denaturation of the protein. This denaturation was independent of heating time at 80 C because the spectra of all heated samples were similar. The increased noise in the spectrum taken after 2 min heating time was due to a lower number of scans. This was necessary to avoid long exposure to the high temperature. After cooling of the previously heated sample solutions, the original spectrum was restored demonstrating that the protein refolds upon cooling (Fig. 3). Therefore, we can conclude that thermal denaturation of AFP III at 80 C for up to 60 min is fully reversible. This confirms the results of García-Aribas et al. (2007), who showed that thermal denaturation of AFP III is fully reversible after cooling the protein down to room temperature after a heat treatment at 70 C. Also Li et al. (1991) showed a structural transition when AFP III solution was heated from 0 to 60 C. In contrast to García-Aribas et al. (2007) and our research results, Salvay et al. (2007) found that the thermal denaturation is partially irreversible, but the maximal temperature of the thermal treatment was not mentioned. In the case of short heated AFP III sample solution the results of recrystallization experiments could be explained by 1H NMR spectroscopy results. Because thermal denaturation is a fully reversible process, heating doesn’t change the molecular structure and thus not the IRI activity of AFP III. In contrast the slight decrease of IRI activity at longer heating times at 80 C cannot be explained completely by 1H NMR spectroscopy. But one has to keep in mind
that in contrast to the recrystallization experiments the 1H NMR spectroscopy experiments were executed in aqueous solution and not in sucrose solution. It is known that the presence of sucrose increases the stability of proteins and increases the thermal denaturation temperature (Baier and McClements, 2001; Kulmyrzaev et al., 2000). Assuming that the slight decrease of IRI activity at long heating times is based on a change in protein structure, the addition of sucrose and thus an increase in protein stability cannot be the explanation. But a maillard reaction between sucrose and the protein is possible and the reaction speed increases with temperature and time (Ledl and Schleicher, 1990). Therefore it is assumed that at longer heating times at 80 C a small amount of conjugates were produced, which have no or a reduced IRI activity. 3.2. Influence of pH on IRI activity and structure of AFP III To evaluate a possible influence of pH on IRI activity of AFP III, we examined first, as control solution, the influence of pH on the recrystallization in pure sucrose solution. Fig. 4 shows that adjusting pH value to 11 led to a slightly smaller but not significantly different mean ice crystal diameter (P > 0.05), whereas mean ice crystal diameters in pure sucrose solution at pH 1 were significantly increased. As expected Fehling test showed a cleavage of sucrose in glucose and fructose at pH 1. Hereby the molar concentration of sugar molecules increased, resulting in a higher freezing point depression and consequently in a lower ice content. However, the increased recrystallization rate at decreased ice content is contradictory to published results (Budke et al., 2009; Gaukel, 2004; Sutton et al., 1996). At lower ice content the distance between crystals is longer leading to an increased diffusion time between the crystals and less accretion. The only reasonable explanation for the observed results is that the diffusion coefficient of water is higher in the solution of monosaccharides. For example, Sutton et al. (1997) showed that recrystallization rates in sugar solutions decrease with increasing sugar molecular weight. Hagiwara et al. (2006) showed that recrystallization rate in glucose or fructose solution is faster than in sucrose solution at same ice content and storage temperature. In addition the authors
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Fig. 5. One dimensional 1H NMR spectra of AFP III solutions (cAFP III ¼ 0.18 mg mL1) at different pH values adjusted by hydrochloric acid and/or sodium hydroxide compared to the spectrum of an untreated AFP III parent solution.
Fig. 6. Mean ice crystal diameter of a AFP III sucrose solution (cAFP III ¼ 2 mg mL1) with 0.03 M NaCl and of a AFP III sucrose solution (cAFP III ¼ 2 mg mL1) adjusted to pH 7 by adding hydrochloric acid and sodium hydroxide at a temperature of 12 C.
illustrated that there is a correlation between recrystallization rate and water diffusion coefficient in the sugar solutions. This suggests that the increased recystallization rate at pH 1 is due to an increased water diffusion coefficient. In AFP III sample solutions, pH had no clear influence on IRI activity of AFP III. The mean ice crystal diameters of all AFP III sample solutions with hydrochloric acid and/or sodium hydroxide are of the same size and are smaller than the ice crystal diameters in AFP solution with pure water (pH 6.1). These results seem to be
consistent with the research on thermal hysteresis of AFP III by Chao et al. (1994) who showed that TH of AFP III remained unchanged in a pH range from 2 to 11. Interestingly, the cleavage of sucrose in glucose and fructose in the AFP III solution at pH 1 did not affect the IRI activity. By binding to the ice crystal surface and the adsorption inhibition mechanism of AFP III, recrystallization processes are very likely controlled by surface integration process and not by bulk diffusion like in the pure sucrose solution. Thus, an increased water diffusion coefficient in the pH 1 solution would not
A. Leiter et al. / Journal of Food Engineering 187 (2016) 53e61
affect the recrystallization rate. But the increased IRI activity of AFP III with hydrochloric acid and/or sodium hydroxide cannot be explained by a dramatic change in molecular structure like complete unfolding. The one dimensional 1H NMR spectra at pH 1, 7 and 11 show a signal dispersion that is comparable to the untreated parent solution (Fig. 5). Around 0 ppm isolated methyl group signals are visible that are indicative of a defined three-dimensional structure and that would disappear if unfolding takes place. However, both at pH 1 and at pH 11 the amide signals, that resonate in the ppm range between 10 and 7 ppm, changed their pattern. This indicates that although the protein is not completely unfolded, partial denaturation may have started. However, this partial denaturation cannot explain the increased IRI activity of AFP III, because at pH 7 there was also an increased IRI but no partial denaturation was visible. Due to our sample preparation the samples at pH 1, 7 and 11 had a higher salt concentration as the sample solution at pH 6.1. This leads to the hypothesis that it is not the pH but probably the ion concentration which affects IRI activity. Evans et al. (2007) have already shown that the addition of different salt types leads to a synergistic enhancement of thermal hysteresis of AFP III. To verify our suggestion that only the ion concentration affects the IRI activity, we investigated the IRI activity of AFP III at pH 7 with an AFP III sucrose solution in pure water (pH 6.1) with the same quantity of ions (NaCl) like in the pH 7 solution. As the results in Fig. 6 show, there is no significant difference between the mean ice crystal diameters between these two samples (P > 0.05). This confirms the suggestion that the addition of ions increased the IRI activity of AFP III and not the pH change. The influence of the slightly increased ion concentration in the pH 7, pH 11 and NaCl solution on the freezing point depression and ice content is negligible as the molar ion concentration was only 0.06 M compared to the molar sucrose concentration of 1.75 M (49%). In addition recrystallization experiments with 49% sucrose and 0.03 M NaCl (0.06 M ionconcentration) showed no difference in recrystallization rate (data not shown). A possible explanation for this salt-induced enhancement of IRI
59
activity is given by Kristiansen and Zachariassen (2005). The authors proposed that at the equilibrium melting temperature of an ice crystal, there is a steady-state distribution of AFP molecules in the solution surrounding the ice crystal and in the ice/water interfacial region. Factors that lower the solubility of AFP molecules in the solution are likely to cause a shift in their steady-state distribution in favor of the ice/water interfacial region. Therefore there is an increased surface density of irreversibly adsorbed AFP molecules at the ice surface, when the interfacial region solidifies at a temperature below the equilibrium melting point (Kristiansen et al., 2008; Kristiansen and Zachariassen, 2005). According to Kristiansen et al. (2008) salts reduce the solubility of AFP in the solution and thus TH is increased. Similar results were provided by Hayakari and Hagiwara (2012) in a molecular dynamic analysis of winter flounder antifreeze protein. The analysis showed an enhanced interaction between the AFP molecule and the ice surface due to the presence of ions in the water phase. In accordance with these findings the increased IRI activity in our study is probably due to an increased number of adsorbed AFP molecules on the ice crystal surface. One implication of this presumption is that there should be a limiting ion concentration where the addition of further ions does not increase the IRI activity because a maximum occupancy of AFP molecules on the ice crystal surface is reached. Therefore the influence of NaCl concentration on the IRI activity of AFP III was investigated. Fig. 7 illustrates the mean ice crystal diameter of an AFP III sample solution with different molar NaCl concentrations after different storage times. The results reveal that the mean ice crystal diameter decreases for each storage time with increasing molar NaCl concentration up to a limiting value which is probably between 0.02 M and 0.03 M. An additional increase of salt concentration did not affect the mean ice crystal diameter. Knight et al. (1995) showed that the addition of 0.008 M NaCl does not affect the IRI of AFP I. However, in this research the ice crystal size was analyzed only qualitatively by comparing ice crystal pictures and not quantitatively. Finally, our result supports the presumption that the increased IRI activity of AFP III by the addition of NaCl is due to
Fig. 7. Influence of molar NaCl concentration on ice recrystallization in an AFP III sucrose solution (cAFP
III
¼ 2 mg mL1) during storage at a temperature of 12 C.
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Fig. 8. Influence of hydrostatic pressure on ice recrystallization in an AFP III sucrose solution (cAFP
an increased number of adsorbed AFP III molecules on the ice surface. 3.3. Influence of pressure on IRI activity of AFP III Fig. 8 shows no significant difference between the mean ice crystal diameters at the corresponding storage times of pressure treated and untreated samples (P > 0.05). Also the 1H NMR spectroscopy investigation showed that there is no irreversible denaturation of AFP III at this pressure, because the 1H NMR spectra of the different sample solutions were similar (data not shown). However a reversible denaturation of the protein cannot be ruled out. This result is a first indication of the possibility of the combination of antifreeze proteins with high pressure shift freezing to maintain the small initial ice crystal size for a longer time. 4. Conclusion In this paper we investigated the influence of temperature, pressure, pH, and ion concentration on the ice recrystallization inhibition activity of AFP III. Results showed that thermal treatment at 80 C up to one hour has only a slight effect on IRI activity of AFP III. Furthermore we showed that in pure sucrose solutions the recrystallization rate was increased at pH 1 which can be related to cleavage of sucrose and a diffusion controlled recrystallization, whereas the increased IRI activity at different pH values in AFP solutions can be related to the salt concentration. Up to a limiting value between 0.02 and 0.03 M NaCl the IRI activity increased probably due to an increased AFP concentration on the ice crystals surface. Pressure treatment up to 400 MPa for one minute had no influence on IRI activity. In addition 1H NMR spectroscopy showed high stability of AFP III at pH values between 1 and 11 and pressure up to 400 MPa. Besides 1H NMR spectroscopy demonstrated a fully reversible denaturation of AFP III in aqueous solutions after temperature treatment at 80 C up to 1 h. It is already known that ice-binding proteins have a high potential for different industrial applications, but the findings of this
III ¼ 2
mg mL1) during storage at a temperature of 12 C.
research also shows that the IRI activity of AFP III is not affected by pH or by typical process parameters like temperature or pressure in the range investigated. Thus, these data highlight the high potential of AFP III proteins in industrial applications. Consequently the only barrier for industrial use of AFP III is the high price. That is why more research is required to reduce the costs for protein synthesis or to find alternative substances with similar IRI activity and high process stability like AFP III. Acknowledgement We would like to thank the Max-Buchner-Forschungsstiftung for research fellowship MBFSt 3400 and the financial support. We also thank Max-Rubner Institute for the use of the autoclave and Ralf Lindauer for the support during pressure experiments. Burkhard Luy thanks the DFG (instrumental facility Pro2NMR) and the Helmholtz association (programme BIFTM) for funding. References Adapa, S., Schmidt, K.A., Jeon, I.J., Herald, T.J., Flores, R.A., 2000. Mechanisms of ice crystallization and recrystallization in ice cream: a review. Food Rev. Int. 16 (3), 259e271. Bahramparvar, M., Mazaheri Tehrani, M., 2011. Application and functions of stabilizers in ice cream. Food Rev. Int. 27 (4), 389e407. Baier, S., McClements, D.J., 2001. Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions. J. Agric. Food Chem. 49 (5), 2600e2608. Budke, C., Heggemann, C., Koch, M., Sewald, N., Koop, T., 2009. Ice recrystallization kinetics in the presence of synthetic antifreeze glycoprotein analogues using the framework of LSW theory. J. Phys. Chem. B 113 (9), 2865e2873. Capicciotti, C.J., Doshi, M., Ben, R.N., 2013. Ice recrystallization inhibitors: from biological antifreezes to small molecules. In: Wilson, P. (Ed.), Recent Developments in the Study of Recrystallization. InTech, Rijeka, Croatia, pp. 177e224. Celik, Y., Graham, L.A., Mok, Y.F., Bar, M., Davies, P.L., Braslavsky, I., 2010. Superheating of ice crystals in antifreeze protein solutions. Proc. Natl. Acad. Sci. U. S. A. 107 (12), 5423e5428. € nnichsen, F.D., DeLuca, C.I., Sykes, B.D., Davies, P.L., 1994. StructureChao, H., So function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice. Protein science. A Publ. Protein Soc. 3 (10), 1760e1769. Cook, K., Hartel, R.W., 2010. Mechanisms of ice crystallization in ice cream
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