Effect of type and concentration of cryoprotectant on glass transition temperature (
1. Introduction
Inulin is a generic term applied to heterogeneous blends of fructo-oligosaccharides [1] which are reserve carbohydrate sources present in many plant foods such as bananas, onions, garlic, leeks, artichokes and chicory, which represents the main commercial source. This polysaccharide has a wide range of both, nutritional and technological applications. Nutritionally, inulin is regarded as a soluble fiber which promotes the growth of intestinal bacteria, acting as a prebiotic. Also, is a non-digestible carbohydrate with minimal impact on blood sugar and unlike fructose, it is not insulemic and does not raise triglycerides being generally considered suitable for diabetics and potentially helpful in managing blood sugar-related illnesses [2-4]. Among the technological benefits, inulin is used as fat and sugar replacement, low caloric bulking agent, texturing and water-binding agent [5,6]. One general property of the saccharides is the stabilization of proteins by their incorporation into carbohydrate solutions before freeze-drying being this a known preservation procedure [7-10]. The previous incorporation of saccharide promotes the formation of amorphous, glassy systems, inhibits crystallization and influences the kinetics of deteriorative reactions upon storage by which its structured integrity is maintained [8,9,11,12]. To act successfully as a protectant, the saccharides should have a high glass transition temperature (
The protein preserved by freeze-drying simplifies aseptic handling and enhances stability of protein products, with limited shelf lives in solution, by obtaining a dry powder without excessive heating. However, during the freeze-drying process the protein may lose its activity and must be protected from conformational changes or denaturation [11,15]. The stabilization of proteins conferred by saccharides during freeze- drying has been explained by several mechanisms. First, replacing the hydrogen bonding between water and protein stabilizes the protein during drying processes, and second, the formation of a glass matrix where the protein is encapsulated avoiding its unfolding and thus preserving its conformation during freeze-drying [8,12,16-18]. Therefore, through the correct selection of the saccharide it is possible to improve the stability of proteins through their encapsulation in a glassy matrix, where molecular mobility is quite limited so that the rates of diffusion-controlled reactions, like protein unfolding or protein aggregation, are reduced [16,19,20].
Information about the energy of a protein can be obtained by means of thermal denaturation studies, allowing the characterization of their behavior during freeze-drying cycle. Differential scanning calorimetry (DSC) is one of the most useful methods for assessing protein thermal behavior and to obtain thermodynamic parameters of folding-unfolding transitions [21].
During the freeze-drying of a protein solution with or without saccharides to protect the structure, the primary drying is the most time consuming stage of the process. It should be carry out at the maximum allowable temperature usually associated to the glass transition temperature of the maximally freeze concentrate solution (
A diagram of phases for the water-saccharide system is shown in Figure 1. The curve of the freezing temperature separates the zones corresponding to the liquid and the solid (ice) solution phases. In fact, this procedure is aimed at obtaining a glassy system at room temperature as indicated in D. To get to this state, the freeze-dried process indicated by the curve A-B-C-D-E is carried out. The curve for the glass transition temperature (
Therefore, the determination of the freeze-drying cycle is important because of physical changes that occur in the solution during the process, its study can be applied to improve processability, quality, and stability of the product during storage [29].
Although many authors reported the use of saccharides as cryoprotectants of proteins and inulin as a good protector agent of some compounds, the present study is an attempt to evaluate inulin as cryoprotector of food proteins such as bovine plasma proteins, taking profit of the nutritional and technological benefit of the polysaccharide. Also there is a limited amount of data on glass transition temperatures for multicomponent mixtures and on the comparison of experimental and predicted values for such mixtures [28]. Then, the purposes of this study were
The glass transition temperatures of the maximally concentrated frozen solutions (
2. Materials and methods
2.1. Raw materials
The inulin used as cryoprotectant is mainly constituted by linear chains of fructose, with a glucose terminal unit, and has a molecular weight of 2400 g/mol. The commercial product was provided by Orafti Chile S.A. and was obtained from chicory. The other saccharides employed to compare their performance were:
The protein used in the study was spray dried bovine plasma (Yerubá S.A. Argentine). The molecular weights of the proteins were in the range of 15.000 to 80.000 Da. The composition was 76±5% proteins, <0.1% fat, 10% ash, 4% water, 1% low molecular weight compounds.
2.2. Preparation of Protein/carbohydrate samples: Concentration of bovine plasma proteins through ultrafiltration and freeze-drying treatments
The protein concentrate was obtained by means of a membrane process, which allowed protein concentration, eliminating insoluble macroscopic components, reducing the saline content [18]. The steps of the process were: i) the bovine plasma was dissolved in de-ionized water to a concentration of 3% w/v using a mixer at a low speed to avoid the formation of vortex and to minimize the appearance of foam; ii) the solution was passed through a porous support (Viledon FO 2431D, Germany) to remove macroscopic aggregates and reduce the saline content; iii) the feed solution (3 L) was thermostatized in a water bath and impelled with a centrifugal pump, first through a frontal flow stainless steel filter, with a pore size of 60 μm (Gora, Argentine) (this procedure of microfiltration (MF) reduces the amount of bacteria and spores and acts as cold pasteurization, moreover this stage protects the ultrafiltration (UF) membrane from fouling); and finally, iv) the UF was performed using Pellicon cassette module (Millipore, Bedford, MA, USA), containing modified polyethersulfone membranes with a molecular weight cut-off (MWCO) of 10 kDa, with a membrane area of 0.5 m2. The concentration of proteins by UF was carried out by continuously removing the permeate stream until the desired concentration of 4% (w/v), was achieved. The experimental runs were performed at a transmembrane pressure (
The UF membrane undergoes a fouling process during protein permeation so a cleaning protocol may be applied. It was performed by applying a "Cleaning in Place" (CIP) procedure according to the manufacturer's instructions. At the end of each run, a cycle of water/ alkali (NaOH, pH=12.5 ± 0.5)/ water wash was applied to the membrane at (40 ± 2) °C and at a transmembrane pressure of 1 bar. Furthermore, a cleaning step using NaClO (commercial grade) 300 ppm was carried out at the same temperature and pressure to ensure sanitation and cleaning. Measurements of normalized water permeability were performed in order to verify recovery of flow through the membrane which ensures the recuperation of membrane permeability.
The bovine plasma protein (BPP) concentrate obtained by UF (concentration: 4 % w/v) was fractioned: A fraction as witness sample was reserved and the protective agent (glucose, sucrose, inulin) was added to the rest, in concentrations of 5%, 10% and 15% (w/v). A part of these solutions was reserved for DSC analysis to determine
2.3. Differential Scanning Calorimetry (DSC) measurements
Determination ofThe solutions containing plasma proteins–saccharides mixture were analyzed to determine
Heat induced conformational changes on freeze-dried bovine plasma protein concentrate (BPP concentrate) in the amorphous carbohydrate matrix. The freeze-dried solids were analyzed to determine
Freeze–dried solids were equilibrated at 0 °C, held for 1 min and then warmed up to 200 °C at heating rate of 2 °C/min. To check the irreversibility of the reaction of heat-induced conformational changes, the samples after the end of the first heating stage described before, were re-scanned. For this, the protein-saccharide samples were cooled to 20 °C and stabilized during 5 min, and then warmed up to 200°C. Samples of freeze dried bovine plasma protein concentrate (BPP concentrates) in the amorphous carbohydrate matrix at pH 8, 6 and 4, at different heating rates of 2 and 5 °C/min in the temperature range 20–200 °C were analyzed. The pH was adjusted using 0.1 N of NaOH and HCl. Measurements were carried out on three separate samples (replicates). The following parameters were calculated at least in triplicate:
In the freeze dried samples, at temperatures above
2.4. Determination of native protein content
The native protein content is a measure of protein functionality preservation. It was determined after isoelectric precipitation of denatured/aggregated protein [18,35]. Dispersions of protein concentrate at 1% (w/v) were adjusted to pH value inferior of the pI of plasma proteins (~ 4.8) using 0.1 N of NaOH and HCl. An aliquot of the solution was centrifuged in a refrigerated ultracentrifuge (Beckman J2-HS) at 20,000 rpm 30 min at 5 ºC. Protein concentration in the supernatants was diluted in a dissociating buffer (EDTA 50 mM, urea 8 M, pH= 10) and determined by molecular absorptiometry at 280 nm. The results were reported as percentage of the total protein concentration [36]. The percentage of native protein content of suspensions at pH 4.8 was obtained as the ratio between soluble protein (
2.5. Scanning electron microscopy
The microstructure of freeze-dried plasma concentrates with and without saccharides was analyzed by scanning electron microscopy (SEM) using an LEO1450VP equipment (Zeiss, Germany). Powder samples were mounted on double-sided carbon adhesive tape on aluminum stubs and gold-coated and processed in a standard sputter. The micrographs were obtained in high vacuum at 10 KeV.
2.6. Statistical analysis
The experimental data were statistically analyzed by the Tukey-Kramer multiple comparison test, in the cases where 2 or more comparisons were considered, assuming that a
3. Theoretical considerations
3.1. Equations for T´ g prediction
The Miller/Fox equation can be used for the determination of
where
The Gordon and Taylor equation [40] predicts the plasticizing effect of water on the
where
Eqs. (2) and (3) were used for the determination of
3.2. Theory of protein unfolding
Unfolding of protein is suggested to involve at least two steps according to Lumry and Eyring model (1954). The first step is a reversible unfolding of the native protein (
A special case was when
where the first-order rate constant
Experimentally, the irreversibility of unfolding was verified in a rescan. For an irreversible process, in the DSC rescanned thermograms no transition could be observed.
4. Results and discussion
4.1. Effect of saccharides on glass transition of the freeze concentrated matrix
As was previously mentioned, to avoid collapse of the products during the freeze-dried process, a temperature below the glass transition temperature of the frozen concentrated solutions, must be attained. Inulin as protein protective agent was comparatively studied, employing mono and disaccharides. The thermograms of Figure 2 show the transition temperatures of the frozen solutions of bovine plasma with inulin compared to the other saccharides, obtained in a single scan.
The result indicated that at each saccharide concentration,
Thermograms of bovine plasma solutions revealed the existence of two glass transitions (
Saccharide | Concentration (%, w/v) | |||
Glucose | 5 | -62.50 ± 0.58a | -39.24 ± 0.75a | 16.31 ± 0.38a |
10 | -61.06 ± 0.45a,b | -39.91 ± 0.83a | 41.52 ± 0.29b | |
15 | -59.82 ± 0.68b | -44.96 ± 0.49b | 60.31 ± 0.48c | |
Sucrose | 5 | -51.48 ± 1.05c | -31.15 ± 0.40c | 48.01 ± 0.56d |
10 | -50.12 ± 1.03c,d | -31.86 ± 0.60c | 52.48 ± 0.52e | |
15 | -48.42 ± 0.98d | -33.72 ± 0.45d | 64.28 ± 0.46f | |
Inulin | 5 | -26.96 ± 0.68e | - | 48.85 ± 0.35d |
10 | -23.67 ± 0.55f | - | 66.18 ± 0.69g | |
15 | -22.40 ± 0.45f | - | 69.25 ± 0.45h |
The effect of water as a plasticizer of the mixture protein-saccharide was predicted by the Miller/Fox and Gordon–Taylor equations, the results, were compared with experimental values (Table 1). The data of
The densities of bovine plasma proteins, glucose, sucrose and inulin (at room temperature) were determined with a digital densimeter, and the results were: 0.4 ± 0.08 g/cm3, 0.6 ± 0.05 g/cm3, 0.8 ± 0.04 g/cm3 and 0.3 ± 0.05 g/cm3, respectively.
From literature the
Saccharide | |||
5 % (w/v) | 10% (w/v) | 15 % (w/v) | |
Glucose | -85 | -79 | -72 |
Sucrose | -59 | -53 | -46 |
Inulin | -17 | -15 | -13 |
Glucose %(w/v) | Sucrose %(w/v) | Inulin %(w/v) | |||||||
15 | 10 | ||||||||
1.039 | 1.042 | 1.059 | 1.033 | 1.041 | 1.056 | 1.032 | 1.039 | 1.049 | |
-63.99 | -60.46 | -56.1 | -54.07 | -51.24 | -47.04 | -30.43 | -23.64 | -19.51 | |
Difference (%) | 2.32 | 0.99 | 6.63 | 4.79 | 2.18 | 2.95 | 11.40 | 0.12 | 14.8 |
Taylor modified) | -62.69 | -61.26 | -60.03 | -51.38 | -50.58 | -48.47 | -26.70 | -24.23 | -22.11 |
Difference (%) | 0.30 | 0.33 | 0.35 | 0.19 | 0.91 | 0.10 | 0.97 | 2.31 | 1.31 |
3.5 | 4.1 | 4.5 |
4.2. Effect of saccharides on glass transition of the freeze-dried samples
The storage temperature of frozen or freeze-dried foods should be below the glass transition temperature as previously established [22,27,42,50]. Figure 3 shows the thermograms of the freeze-dried samples containing inulin compared with glucose and sucrose at different concentrations. The existence of these transitions evidenced the glassy state of the freeze–dried plasma protein/saccharides mixtures. Besides, Table 1 shows that
4.3. Effect of saccharides on crystallization temperature of the freeze-dried samples
It is important to determine the crystallization temperature (
Fig 4 shows the crystallization temperature (
4.4. Thermal denaturation of BPP in a matrix of saccharide
4.4.1. Effect of saccharide type and concentration
The thermal stability of BPP in a matrix of inulin compared with other saccharides was investigated using DSC. Table 4 shows the values of
Saccharide | Concentration (%, w/v) | ||
Glucose | 5 | 110.07 ± 1.22a | 0.84 ± 0.32a |
Sucrose | 132.78 ± 2.12b | 5.08 ± 0.98b,c | |
Inulin | 143.81 ± 0.89c | 2.97 ± 0.55a,d,c | |
Glucose | 10 | 107.27 ± 0.85a,d | 12.26 ± 0.92e |
Sucrose | 144.95 ± 2.34c | 22.40 ± 1.23 | |
Inulin | 156.21 ± 1.12e | 12.22 ± 1.43e | |
Glucose | 15 | 104.91 ± 0.89d | 3.77 ± 0.98d,f |
Sucrose | 126.66 ± 1.54f | 7.01 ± 1.22b | |
Inulin | 132.57 ± 1.34b | 5.78 ± 0.76b,f,c |
The functional structure of a protein in solution is determined by electrostatic forces, hydrogen bonds, Van der Waals interactions and hydrophobic interactions. All these interactions are influenced by water, becoming essential for the functional unfolding of most of the proteins. As water is eliminated during freeze-drying, peptide-peptide interactions prevail causing an alteration in the secondary, tertiary or quaternary structure of the protein, i.e. a conformational change of it. However, the presence of sugar displaces and supplants water forming hydrogen bonds with the dry protein which maintains its structured integrity into the glass matrix. In the case that the formation of the glass structure did not occur, the sugar would be excluded and it would not be available for the formation of hydrogen bonds to protect the dry protein from its unfolding or loss of conformation [13,14].
The protective effect of saccharides depends on its concentration, since as the concentration increases there are more possibilities of forming hydrogen bonds with the protein [11,18]. However, when concentrations were higher than 10 % (w/v), a lower protection was obtained. This result can be explained taking into account that at high concentrations, the saccharide starts to crystallize during freeze-drying, being prevented the formation of hydrogen bonds with the dry protein [12]. This behavior was confirmed by determination of the native proteins in the protein-saccharide matrixes employing eq. (1). The results are presented in Figure 5, which shows that there is a maximum at a concentration of 10% (w/v) for the different saccharides analyzed, indicating higher protein protection and stability.
4.4.2. Effect of pH
To determine the application of these formulations is important to know the variation of
Saccharide | pH | ||
Glucose | 8 | 107.27 ± 0.85a | 12.26 ± 0.82a |
Sucrose | 144.95 ± 1.34b | 22.40 ± 0.97b | |
Inulin | 156.21 ± 1.12c | 12.22 ± 0.55a | |
Glucose | 6 | 102.94 ± 1.33d | 34.74 ± 0.92c |
Sucrose | 134.56 ± 2.16e | 43.15 ± 1.23d | |
Inulin | 152.98 ± 1.52c,f | 42.95 ± 1.45d | |
Glucose | 4 | 101.74 ± 1.27d | 9.58 ± 0.98a,e |
Sucrose | 107.67 ± 1.56a | 9.32 ± 0.72e | |
Inulin | 151.84 ± 1.89f | 9.35 ± 0.96e |
With increasing alkalinity of the medium there is an increase in the values of
4.4.3. Effect of scanning rate
The protein-saccharide mixtures were studied at different scanning rates (2 °C/min and 5 °C/min). As an example Figure 6 shows the transition temperature and enthalpy for sucrose at 10 % (w/v).
It was found for all the saccharides that
4.4.4. Study of Irreversibility of the Thermal Denaturation of BPP
The irreversibility of BPP denaturation was investigated by a multiple reheating experiment, according to the method proposed by by Idakieva and Michnik [45,60]. From the initial DSC scan, we have determined the values of the transition temperatures at 107°C, 145 °C and 156 °C for glucose, sucrose and inulin at 10% w/v, respectively (Table 5). DSC tests were carried out as successive scans, where the heating was carried out up to different final temperatures, with a cooling up to 20°C between scans (Figure 7).
For glucose, sucrose and inulin, the first heating was carried out up to 75°C, and 85°C (temperatures below the
with the addition of protective agents the activation energy increased; besides with increasing molecular weight, the activation energy also increased. Therefore, the addition of saccharides, especially of inulin caused a decrease in the rate of degradation reactions, obtaining a higher stabilization upon storage [8,14,18].
4.4.5. Study of the blends morphology through SEM
Figure 8 sowed the SEM micrographs of blends of protein-saccharides.
It was observed phases homogeneously distributed, indicating miscibility of the component in the matrix. The shapes were uniform, which was an attribute, linked with thermodynamic compatibility [62]. Based on the data previously obtained, comparing the transitions of the blends with respect to the value of the individual components, showed an increase in the value
5. Conclusions
The thermodynamic properties of the solution and the freeze–dried bovine plasma proteins–saccharides mixtures were investigated in this study. The DSC thermograms demonstrated that the bovine plasma proteins– inulin mixtures have the highest glass transition temperature for the protein solution and also the highest glass transition and denaturation temperature for the freeze–dried powder, optimizing the freeze–drying process and also stabilizing and protecting the proteins during storage in conditions below the collapse temperature of the material. Thermograms revealed the existence of two glass transitions in solutions (
Therefore, the results showed highest values of
The findings regarding the protective effect of inulin on bovine plasma proteins, suggest that may be interesting the study of the behavior of formulated foods elaborated with the analyzed matrices (protein-saccharide-water) exposed to treatments such as cooling and freeze-drying.
References
- 1.
Niness K R 1999 Inulin and Oligofructose: What Are They? J. nutr.129 7 1402 1406 - 2.
Abrams S Griffin I Hawthorne K Liang L Gunn S Darlington G Ellis K A 2005 Combination of Prebiotic Short- and Long-chain Inulin-type Fructans Enhances Calcium Absorption and Bone Mineralization in Young Adolescents. Am. j. clin. nutr.82 471 476 - 3.
Hempel S Jacob A Rohm H 2007 Influence of Inulin Modification and Flour Type on the Sensory Quality of Prebiotic Wafer Crackers Eur. food res. technol.224 335 341 - 4.
Nazzaro F Fratianni F Coppola R Sada A Pierangelo O 2009 Fermentative Ability of Alginate-prebiotic Encapsulated Lactobacillus Acidophilus and Survival under Simulated Gastrointestinal Conditions J. funct. food1 3 319 323 - 5.
Kip P Meyer D Jellema R H 2006 Inulins Improve Sensory and Textural Properties of Low-Fat Yoghurts. Int. dairy j.16 1098 1103 - 6.
Ronkart S N Paquot M Fougnies C Deroanne C Blecker C S 2009 Effect of Water Uptake on Amorphous Inulin Properties Food hydrocolloid23 922 927 - 7.
M R (Baeza R I Pilosof A 2002 Calorimetric Studies of Thermal Denaturation of b-Lactoglobulin in the Presence of Polysaccharides. Lebensm.-wiss. technol.35 393 399 - 8.
Buera P Schebor C Elizalde B 2005 Effects of Carbohydrate Crystallization on Stability of Dehydrated Foods and Ingredient Formulations J. food eng.67 157 165 - 9.
Claude J Ubbink J 2006 Thermal Degradation of Carbohydrate Polymers in Amorphous States: A Physical Study Including Colorimetry Food chem.96 402 410 - 10.
Santivarangkna C Higl B Foerst P 2008 Protection Mechanisms of Sugars During Different Stages of Preparation Process of Dried Lactic Acid Starter Cultures Food microbiol.25 429 441 - 11.
Allison S D Chang B Randolph T W Carpenter J F 1999 Hydrogen Bonding Between Sugar and Protein is Responsible for Inhibition of Dehydration-Induced Protein Unfolding Biochem. biophys.365 289 298 - 12.
Carpenter J F Crowe L M Crowe J H 1987 Stabilization of Phosphofructokinase with Sugars during Freeze-drying: Characterization of Enhanced Protection in the Presence of Divalent Cations. Biochim. biophys. acta923 1 109 115 - 13.
H W ( LHinrichs W J Prinsen M G Frijlink 2001 Inulin Glasses for the Stabilization of Therapeutic Proteins. Int. j. pharmaceut.215 163 174 - 14.
H W ( LHinrichs W J Sanders N N De Smedt S C Demeester J Frijlink 2005 Inulin is a Promising Cryo- and Lyoprotectant for PEGylated Lipoplexes. J. control. release103 465 479 - 15.
Rey Cabinet L d’Etudes L, Switzerland J C (2004 Freeze Drying/lyophilization of Pharmaceutical and Biological Products. Maryland, U.S.A: Center for Biologics Evaluation and Research Food and Drug Administration. - 16.
Liao Y H Brown M B Martin G P 2004 Investigation of the Stabilization of Freeze-dried Lysozyme and the Physical Properties of the Formulations. Eur j. pharm. Biopharm.58 15 24 - 17.
Minson E I Fennema O Amundson C H 2006 Efficacy of Various Carbohydrates as Cryoprotectants for Casein in Skim Milk J. food sci.46 5 1597 1602 - 18.
Rodriguez Furlán L T Pérez Padilla A, Campderrós M (2010 Inulin Like Lyoprotectant of Bovine Plasma Proteins Concentrated by Ultrafiltration Food res. int.43 788 796 - 19.
Costantino H R Curley J G Wu S Hsu C C 1998 Water Sorption Behavior of Lyophilized Protein-sugar Systems and Implications for Solid-state Interactions. Int. j. pharm.166 211 221 - 20.
Passot S Fonseca F Alarcon-lorca M Rolland D Marin M 2005 Physical Characterization of Formulations for the Development of Two Stable Freeze-dried Proteins During Both Dried and Liquid Storage. Eur. j. pharm. biopharm.60 335 348 - 21.
Guzzi R Sportelli L Sato K Cannistraro S Dennison C 2008 Thermal Unfolding Studies of a Phytocyanin. Biochim. biophys. acta1784 1997 2003 - 22.
Chen T Oakley D M 1995 Thermal Analysis of Proteins of Pharmaceutical Interest Thermochim. acta248 229 244 - 23.
Schenz T W 1995 Glass Transitions and Product Stability-an Overview. F ood hydrocolloid9 4 307 315 - 24.
Rheological, Calorimetric and Dielectric Behavior of Selected Indian Honey. J. food eng.Ahmed J Prabhu S T SRaghavan G V Ngadi M 2007 Physico-chemical 79 1207 1213 - 25.
Gallegos Infante J A Ochoa Martínez L A, Ortiz Corral C (2005 Glass Transition Temperature Behavior of a Model Blend of Carbohydrates Cien. Tecnolog. Alimen.5 6 10 - 26.
Noel T R Parker R Ring S G Tatham A S 1995 The Glass-transition Behaviour of Wheat Gluten Proteins Int j. boil. macromol.17 2 81 85 - 27.
Roos Y 1995 Characterization of Food Polymers using State Diagrams J. food eng.24 339 360 - 28.
Shah B N Schall C A 2006 Measurement and Modeling of the Glass Transition Temperatures of Multi-component Solutions Thermochim. acta443 78 86 - 29.
Katkov I I Levine F 2004 Prediction of the Glass Transition Temperature of Water Solutions: Comparison of Different Models 49 62 82 - 30.
Tattini Jr V Parra D F, Polakiewicz B, Pitombo R N M (2005 Effect of Lyophilization on the Structure and Phase Changes of PEGylated-bovine Serum Albumin. Int. j. pharm.304 124 134 - 31.
Sunooj K V Radhakrishna K George J Bawa A S 2009 Factors Influencing the Calorimetric Determination of Glass Transition Temperature in Foods: a Case Study Using Chicken and Mutton J. food eng.91 347 352 - 32.
Akköse A Aktas N 2008 Determination of Glass Transition Temperature of Beef and Effects of Various Cryoprotective Agents on Some Chemical Changes Meat sci.80 875 878 - 33.
Cao X Li J Yang X Duan Y Liu Y Wang C 2008 Nonisothermal Kinetic Analysis of the Effect of Protein Concentration on BSA Aggregation at High Concentration by DSC Thermochim. acta467 99 106 - 34.
Dàvila E Parés D Cuvelier G Relkin P 2007 Heat-induced Gelation of Porcine Blood Plasma Proteins as Affected by pH Meat Sci.76 216 225 - 35.
De Wit J N 1990 Thermal Stability and Functionality of Whey Proteins J. dairy Sci.73 3602 3612 - 36.
Giroux H J Britten M 2004 Heat Treatment of Whey Proteins in the Presence of Anionic Surfactants Food hydrocolloid18 685 692 - 37.
SAS USER GUIDE: Statistic Versión (1989 SAS Inst. Inc., Cary, NC, USA. - 38.
Fox T G 1956 Influence of Diluent and Copolymer Composition on the Glass Temperature of a Polymer System. B. am. phys. soc. 2(1): 123. - 39.
Miller D P De Pablo J J Corti H 1997 Thermophysical Properties of Trehalose and its Concentrated Aqueous Solutions. Pharm res-dord14 5 578 590 - 40.
Gordon M Taylor J S 1952 Ideal Copolymers and the Second-order Transitions of Synthetic Rubbers. I. Non-crystalline Copolymer s. J. appl. chem.2 493 500 - 41.
K W ( MGeorget D R Smith A C Waldron 1999 Thermal Transitions in Freeze- dried Carrot and its Cell Wall Component s. Thermochim. acta332 203 210 - 42.
Maitani Y Aso Y Yamada A Yoshioka S 2008 Effect of Sugars on Storage Stability of Lyophilized Liposome/DNA Complexes with High Transfection Efficiency. Int. j. pharm.356 69 75 - 43.
Rodriguez Furlán L T Lecot J, Pérez Padilla A, Campderrós M, Zaritzky N (2011 Effect of Saccharides on Glass Transition Temperatures of Frozen and Freeze-dried Bovine Plasma Protein J. food eng.106 74 79 - 44.
H A M (Creveld L D Meijberg W JBerendsen H C Pepermans 2001 DSC Studies of Fusarium Solani Pisi Cutinase: Consequences for Stability in the Presence of Surfactants. Biophys. Chem.92 65 75 - 45.
Idakieva K Parvanova K Todinova S 2005 Differential Scanning Calorimetry of the Irreversible Denaturation of Rapana Thomasiana (Marine Snail, Gastropod) Hemocyanin Biochim. biophys. acta1748 50 56 - 46.
Ramprakash J Doseeva V Galkin A Krajewski W Muthukumar L Pullalarevu S Demirkan E Herzberg O Moult J Schwarz F P 2008 Comparison of the Chemical and Thermal Denaturation of Proteins by a Two-state Transition Model. Anal. biochem.374 221 230 - 47.
Ohkuma C Kawaib K Viriyarattanasaka C Mahawanichc T Tantratianc S Takaia R Suzuki T 2008 Glass Transition Properties of Frozen and Freeze-dried Surimi Products: Effects of Sugar and Moisture on the Glass Transition. Food hydro colloid22 255 262 - 48.
P J A ( RTelis V N Sobral 2002 Glass Transitions for Freeze-dried and Air-dried Tomato Food res. int.35 435 443 - 49.
S O (Sun W Q Davidson P Chan H 1998 Protein Stability in the Amorphous Carbohydrate Matrix: Relevance to Anhydrobiosis. Biochim. biophys. acta1425 245 254 - 50.
In: Sal, A. (Ed.), Handbook of Powder Technology,Salman A D Hounslow M J PSeville J K 2006 Granulation 11 España: Elsevier. - 51.
Gabbott P 2008 Principles and Applications of Thermal Analysis Blackwell Publishing. Chapter 9. - 52.
Dilworth S E Buckton G Gaisford S Ramos R 2004 Approaches to Determine the Enthalpy of Crystallization, and Amorphous Content, of Lactose from Isothermal Calorimetric Data. Int. j. pharm.284 83 94 - 53.
[53] Relkin P 1996 Thermal Unfolding of β-Lactoglobulin, α-Lactalbumin and Bovine Serum Albumin. A Thermodynamic Approach. Crit. rev. food sci.36 6 565 601 - 54.
Yamul D K Lupano C E 2003 Properties of Gels from Whey Protein Concentrate and Honey at Different pHs Food res. int.36 25 33 - 55.
Landro L. (Penco M Sartore L Bignotti F D Antone S Di 2000 Thermal Properties of a New Class of Block Copolymers Based on Segments of Poly(D,L-lacticglycolic Acid) and Poly(e-caprolactone). Eur. polym. j.36 901 908 - 56.
Kavitha M Bobbili K B Swamy M J 2010 Differential Scanning Calorimetric and Spectroscopic Studies on the Unfolding of Momordica Charantia Lectin. Similar Modes of Thermal and Chemical Denaturation 92 58 64 - 57.
Schubring R 1999 DSC Studies on Deep Frozen Fishery Products Thermochim. acta337 89 95 - 58.
Yu Sakharov I, Jadan A P, van Huystee, R B, Villar E, Shnyrov V L (Zamorano L S Pina D G Gavilanes F Roig M G 2004 Two-state Irreversible Thermal Denaturation of Anionic Peanut (Arachis Hypogaea L.) Peroxidase. Thermochim. acta417 67 73 - 59.
W ( WVermeer A P Norde 2000 The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein. Biophys. j.78 394 404 - 60.
Michnik A Drzazga Z Kluczewska A Michalik K 2005 Differential Scanning Microcalorimetry Study of the Thermal Denaturation of Haemoglobin. Biophys. chem.118 93 101 - 61.
Rodriguez Furlán L T Lecot J, Pérez Padilla A, Campderrós M E, Zaritzky N (2012 Stabilizing Effect of Saccharides on Bovine Plasma Protein: A Calorimetric Study Meat sci. In press. - 62.
Gallego K López B L Gartner C 2006 Estudio de Mezclas de Polímeros Reciclados para el Mejoramiento de sus Propiedades. Rev. Fac. Ing.37 59 70 - 63.
G (Mousavioun P ODoherty W S George 2010 Thermal Stability and Miscibility of Poly(hydroxybutyrate) and Soda Lignin Blends. Ind. crop. prod.32 3 656 661 - 64.
Rodriguez Furlán L T Pérez Padilla A, Campderrós M E (2010 Functional and Physical Properties of Bovine Plasma Proteins as a Function of Processing and pH, Application in a Food Formulation Adv. j. food sci. tech.2 5 256 267 - 65.
Rodriguez Furlán L T Rinaldoni A N, Padilla A P, Campderrós M E (2011 Assessment of Functional Properties of Bovine Plasma Proteins Compared with other Proteins Concentrates, Application in a Hamburger Formulatio n. Am. j. food tech.6 9 717 729