Comparative studies on thermal stability of poultry ovotransferrins

Abstract

A comparative study was carried out on the thermal stability of ovotransferrins (OTrfs) from chicken, duck, guinea fowl, Japanese quail, ostrich and turkey by differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF) and turbidimetry. The average temperature at the denaturation midpoint in DSC (Tm_DSC) was 42.8 °C at pH 4.0, 63.8 °C at pH 7.0 and 59.4 °C at pH 9.0, and the differences in the Tm_DSC values between chicken and the other poultry, except ostrich, were less than 3.5 °C. The results of DSF were almost comparable to those of DSC. In an analysis of poultry OTrf solution turbidities, ostrich OTrf solution remained clear up to 95.0 °C at pH 4.0 and pH 8.0, while the others became turbid at around 40 °C at pH 4.0 and 60 °C at pH 8.0. Turkey, Japanese quail and duck OTrfs remained soluble at pH 9.0 up to 73.0 °C, 85.0 °C and 95.0 °C, respectively, though turbidity appeared up to 60 °C in the others. Such findings on the similarities and differences among poultry OTrfs should be significant for the utilization of poultry eggs other than chicken eggs since the thermal processes required for chicken egg products often depends on the behavior of heat-susceptible chicken OTrf.

Introduction

Chicken (Chi) eggs are widely utilized for food products because of their functionality, nutritional values and palatability. Thermal treatments during food processing are almost always used for pasteurization to avoid salmonella contamination and for gel formation in some products. In such processes, the thermal effects on the properties of ovotransferrin (OTrf) are often examined because Chi OTrf is a major heat-susceptible component of egg white proteins, comprising about 12% of all egg white proteins, and because OTrf is rich in beneficial functional properties, such as solubility, gelling properties, foaming ability and metal binding. Therefore, considerable efforts have been made to develop thermal conditions for pasteurization and manufacturing of Chi egg products in order to minimize the deterioration of the useful properties of Chi OTrf. Pasteurization temperatures for Chi egg white have been studied extensively (Cunningham and Lineweaver, 1965; Mizutani et al., 2006), and the effects of heat on the structure (Donovan and Ross, 1975; Nakamura et al., 1979; Watanabe et al., 1985; Matsudomi et al., 1991, 2002), gelling properties (Koyama et al., 2021), foaming power (Nakamura et al., 1979) and metal binding of Chi OTrf (Price and Gibson, 1972; Donovan and Ross, 1975) have been reported.

Other than the eggs of Chi, OTrfs are also found in eggs of poultry such as duck (Duk), guinea fowl (Gui), Japanese quail (Jpq), ostrich (Ost) and turkey (Tky), though their contents range from 2% to 12% of egg white proteins depending on the species (Feeney et al., 1960). Such OTrfs in the transferrin (Trf) family have sequences containing 682 to 686 amino acids (Reference Sequenceⁱ⁾). The sequences are well aligned with that of Chi OTrf, which has a sequence similarity of 90% with Jpq and Tky OTrfs, 86% with Gui OTrf, 80% with Duk OTrf and 73% with Ost OTrf. As for Chi and Duk OTrfs, the secondary and tertiary structures and the sites of disulfide bridges have been reported (Kurokawa et al., 1999). The molecules consist of two homologous domains, each capable of binding iron similar to other proteins in the transferrin family (Li-Chan and Kim, 2007).

It is therefore suggested that Gui, Jpq, Ost and Tky OTrfs have similar tertiary structures to Chi and Duk OTrfs. Such similarity between Chi OTrf and other poultry OTrfs might reflect their thermal characteristics and, if so, the thermal conditions used for Chi egg products, often determined in favor of Chi OTrf, might be applicable to other poultry eggs and facilitate the utilization of poultry eggs other than Chi eggs. However, a detailed comparative study on the thermal properties of poultry OTrfs has not been conducted to date. It is therefore essential to obtain basic knowledge about the denaturation of poultry OTrfs and the thermal characteristics of their solubility and aggregation in order to utilize poultry eggs other than those from Chi.

In the present study, the thermal stability of five poultry OTrfs, i.e., Duk, Gui, Jpq, Ost and Tky OTrfs, was compared with reference to that of Chi OTrf in terms of protein denaturation and aggregation by differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF) and turbidimetry.

Materials and Methods

Poultry eggs    Chicken (Chi, Gallus gallus domesticus, White Leghorn) eggs were obtained from the Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (Tsukuba, Japan); duck (Duk, Anas platyrhynchos domesticus) eggs from the Research Institute of Environment, Agriculture and Fisheries, Osaka Prefecture (Habikino, Japan); guineafowl (Gui, Numida meleagris) eggs from Jafra Trading (Kasumigaura, Japan); Japanese quail (Jpq, Coturnix japonica) eggs from Motoki Co., Ltd. (Tokorozawa, Japan); ostrich eggs (Ost, Struthio camelus, African Black) from Jonan Green System Co., Ltd. (Ishioka, Japan); and turkey (Tky, Meleagris gallopavo) eggs from the Association for the Production of Turkey (Tosa, Japan). All eggs were obtained one to two days after laying.

Separation and purification of poultry ovotransferrin (OTrf)    The egg white without chalaza was isolated, homogenized at 5 000 rpm for 5 min with a homogenizer (Polytron PT 10–35 GT, Kinematica AG, Luzern, Switzerland) fitted with a generator shaft (SE-30, Kinematica AG), and used immediately for the preparation of OTrf, or stored frozen at −20 ° until use. Crude OTrf fractions of poultry egg white were prepared by ethanol precipitation, essentially according to Ko and Ahn (2008). Briefly, egg white was diluted with an equal volume of purified water and NaHCO₃. NaCl and FeCl₃ were added to the concentrations of 0.05, 0.15 and 0.02 mol/L, respectively, to form OTrf-Fe (III). The solution was adjusted to pH 9.0 with 1 mol/L hydrochloric acid and stirred at room temperature for 30 min. Then, cold ethanol was added to the solution up to 40% for Duk and 43% for the other poultry. The mixture was allowed to stand at 5 °C for 1 h and was then centrifuged at 9 500 × g at 5 °C for 20 min. The supernatant was collected and the ethanol concentration was gradually increased up to 58% for Duk and 60% for the other poultry at 5 °C. The ethanol concentrations described above were used to minimize contaminants, ovalbumin or ovomucoid. After 1 h of ethanol precipitation, the mixture was centrifuged at 9 500 × g at 5 °C for 15 min. The precipitate was dissolved with purified water and the solution was adjusted to pH 4.7 with 0.2 mol/L citric acid to dissociate the iron ions from OTrf, and was then mixed and stirred with ion exchange resin (AG 1-X2, Bio-Rad Laboratories, California, U.S.A.) for 1 h. The resin was removed by filtration (No. 5A, Advantech, Tokyo, Japan). The filtrate containing the crude OTrf fraction was dialyzed against purified water at 5 °C and lyophilized. The poultry OTrfs were purified at 25 °C by anion exchange chromatography using FPLC (ÄKTA Purifier, GE Healthcare, Tokyo, Japan) connected to a fraction collector. The column (HiTrap DEAE FF, 5 mL, GE Healthcare) was equilibrated with 0.2 mol/L Bis-Tris HCl buffer (pH 6.4) and the bound proteins were eluted by a linear gradient using the same buffer containing 1 mol/L NaCl at a flow rate of 5 mL/min. The protein was monitored at 280 nm. The fractions containing OTrf almost free of ovalbumin and ovomucoid were collected, repeatedly dialyzed against purified water, then lyophilized and stored frozen at −20 °C until use. The purity of the poultry OTrf was more than 99%, as confirmed by SDS-PAGE and densitometry.

OTrf solutions    The purified poultry OTrfs were dissolved at a concentration of 1 mg/mL in 0.01 mol/L hydrochloric acid at pH 2.0, 0.05 mol/L citrate buffer at pH 4.0, 5.0 and 6.0, 0.05 mol/L phosphate buffer at pH 7.0 and 8.0, or 0.05 mol/L glycine-NaOH buffers at pH 9.0.

Differential scanning calorimetry    DSC was carried out with a Nano DSC (TA Instruments-Waters, Delaware, U.S.A.). Aliquots of 300 µL of poultry OTrf solutions (1 mg/mL at pH 4.0, 7.0 or 9.0) were scanned from 20 °C to 95 °C at a heating rate of 1 °C/min. The DSC data were processed by analysis software (Nano Analyze Data Analysis ver. 3.10.0, TA Instruments) and the changes in the molar heat capacity of poultry OTrfs related to temperature were recorded. The molecular weights of poultry OTrfs estimated based on their amino acid composition were: Chi OTrf; 75 823 Da (NCBI accession number: NM_205304), Duk OTrf; 75 065 Da (XM_005028068), Gui OTrf; 75 838 Da (XM_021393807), Jpq OTrf; 75 735 Da (XM_015871233), Ost OTrf; 75 731 Da (XM_009666861), Tky OTrf; 75 745 Da (NM_001303207). These molecular weights were used in the calculation of the molar heat capacity. The temperature at the maximum molar heat capacity showing the temperature at the denaturation midpoint (Tm) in DSC (Johnson, 2013) was designated as Tm_DSC. The enthalpy of denaturation, ΔH, for each OTrf was estimated by the peak area in the DSC thermogram.

Differential scanning fluorimetry    DSF was carried out with a Light Cycler 480 System II (Roche Diagnostics, Basel, Switzerland), essentially according to Huynh and Partch (2016). OTrf solutions (1 mg/mL at pH 4.0, 7.0 or 9.0) and SYPRO Orange (#S6650, Thermo Fisher Scientific, Massachusetts, U.S.A.) were added to the wells of 96-well PCR plate (#04729692001, Roche Diagnostics) in triplicate. Plates were sealed with transparent film (#04729757001, Roche Diagnostics) and scanned at a rate of 1 °C/min from 30 °C to 95 °C. The fluorescence intensity (F) data, which were dependent on the heating temperature (T), were processed by QtiPlot software (IonDev SRL, Bucuresti, Romania) and the differential fluorescence intensity (dF/dT) versus temperature was obtained. The temperature at the maximum dF/dT showing Tm in DSF (Huynh and Partch, 2016) was designated as Tm_DSF. The significance of the difference among the Tm_DSF values for poultry OTrfs was calculated by multiple comparison tests in SPSS software and the significance level was set at 5%.

Turbidimetry    One-hundred µL aliquots of poultry OTrf solutions (1 mg/mL) at different pH were dispensed in an 8-unit PCR strip tube (AS ONE, Osaka, Japan). A thermal cycler (T100 Thermal Cycler, Bio-Rad) equipped with a temperature-gradient heating function was used for heat treatment of the samples. With this device, a temperature gradient in the specified range is automatically formed between the tubes at both ends of the 8-unit PCR strip tube. The samples in the 8-unit PCR strip tube were isothermally heated for 40 min in the specified range of temperature depending on the poultry OTrfs between 30 °C and 95 °C, then immediately cooled to 5 °C and allowed to stand overnight in order to ensure irreversibility. The heating time of 40 min was chosen since it was long enough to allow irreversible aggregation of the six poultry OTrf solutions at pH 7.0, and then a duration of 40 min was applied to the experiments at the other pH values. Duplicate samples subjected to the same heat treatment were well mixed and 170 µL aliquots were transferred to a microplate (#439454, Thermo Fisher Scientific). The experiments were carried out in triplicate and the turbidity at 655 nm was measured with a microplate reader (iMark, Bio-Rad). The lowest temperature where the turbidity was detected (≥ 0.03, absorbance at 655 nm) was defined as T_trb.

Other methods    Protein was determined by bicinchoninic acid assay (#23225, Thermo Fisher Scientific) using bovine serum albumin as a standard. SDS-PAGE was carried out using a 12.5% polyacrylamide gel plate (PAGEL, ATTO, Tokyo, Japan) according to Laemmli (1970).

Results

DSC analysis of poultry ovotransferrins    The molar heat capacity for the poultry OTrfs for different temperatures at pH 4.0, pH 7.0 and pH 9.0 are shown in Fig. 1, except that for Ost OTrf at pH 4.0, as Ost OTrf did not show any endothermic peak in DSC under such conditions. At pH 4.0, thermal denaturation of OTrfs occurred between 30 °C and 50 °C, though the ranges for Gui and Chi OTrf shifted slightly lower. The six poultry OTrfs exhibited similar curves between 57 °C and 72 °C at pH 7.0 and between 51 °C and 68 °C at pH 9.0. Based on the DSC thermograms, the Tm_DSC for poultry OTrfs was estimated at pH 4.0, pH 7.0 and pH 9.0, and the results are summarized in Table 1. The Tm_DSC for Chi OTrf was 42.1 °C at pH 4.0, 64.7 °C at pH 7.0 and 60.1 °C at pH 9.0. At pH 4.0, Duk, Jpq and Tky OTrfs showed 1.8 °C to 3.1 °C higher Tm_DSCs than Chi OTrf, while Tm_DSC for Gui OTrf was 3.5 °C lower than that for Chi OTrf. At pH 7.0, Duk, Gui, Ost and Tky OTrfs showed 1.0 °C to 2.3 °C lower Tm_DSC values than Chi OTrf, while the Tm_DSC for Jpq OTrf was 0.9 °C higher than that for Chi OTrf. At pH 9.0, Duk, Gui, Ost and Tky OTrfs showed 0.2 °C to 2.6 °C lower Tm_DSC values than Chi OTrf, while the Tm_DSC for Jpq OTrf was 1.1 °C higher than that for Chi OTrf. The ΔH values for the poultry OTrfs are shown in Table 2. The ΔH for Chi OTrf was 1 768 kJ/mol at pH 7.0, which was about 1 000 kJ/mol lower than the ΔH at pH 4.0 and pH 9.0. The ΔHs for the poultry OTrfs showed the following order: Gui < Chi < Duk < Jpq < Tky at pH 4.0, Gui < Ost < Duk < Jpq < Chi < Tky at pH 7.0 and Gui < Duk < Ost < Jpq < Chi < Tky at pH 9.0.

Fig. 1.

Molar heat capacity (Cp) for poultry OTrfs related to temperature.

DSC analysis was carried out on poultry OTrf solutions (1 mg/mL at pH 4.0, pH 7.0 or pH 9.0) scanning at a heating rate of 1 °C/min from 20 °C to 90 °C. Representative results for Otrfs are shown. Most samples were scanned in duplicate. DSC thermograms were processed by Nano DSC software including baseline correction. (a) pH 4.0; (b) pH 7.0; (c) pH 9.0.

Table 1. Summary of TmDSC values (°C) for poultry OTrfs.

pH	OTrf
	Chi	Duk	Gui	Jpq	Ost	Tky
pH 4.0	42.1	43.9	38.6	45.2	ND	44.4
pH 7.0	64.7	62.9	63.7	65.6	62.4	63.4
pH 9.0	60.1	57.5	59.9	61.2	58.8	58.8

TmDSC values, the temperatures at the maximum molar heat capacity were obtained by DSC analysis on poultry OTrf solutions at pH 4.0, pH 7.0 or pH 9.0. ND represents “not determined”.

Table 2. Summary of ΔH values (kJ/mol) for poultry OTrfs.

pH	OTrf
	Chi	Duk	Gui	Jpq	Ost	Tky
pH 4.0	706	792	569	945	ND	1 058
pH 7.0	1 768	1 622	1 440	1 691	1 505	1 987
pH 9.0	634	458	452	562	471	698

The enthalpies of denaturation, ΔH values were obtained by DSC analysis on poultry OTrf solutions at pH 4.0, pH 7.0 or pH 9.0. ND represents ‘not determined’.

DSF analysis of poultry ovotransferrins    The profiles for the differential fluorescence intensity (dF/dT) related to temperature (T) for the poultry OTrfs at pH 4.0, pH 7.0 and pH 9.0 are shown in Fig. 2. Details of Tm_DSF, the temperature at the peak for the poultry OTrfs shown in Fig. 2, are summarized in Table 3. The Tm_DSF values for Chi OTrf were 37.0 °C at pH 4.0, 63.0 °C at pH 7.0 and 57.9 °C at pH 9.0. The average Tm_DSF for the poultry OTrfs was 39.5 °C at pH 4.0, 62 °C at pH 7.0 and 57.3 °C at pH 9.0. At pH 4.0, Gui and Ost OTrfs showed similar Tm_DSF values to Chi OTrf, while the Tm_DSF values for Duk, Jpq and Tky OTrfs were significantly higher than that for Chi OTrf by 4.0 to 5.0 °C. At pH 7.0, the Tm_DSF values for Duk, Gui, Ost and Tky OTrfs were 1.0 °C to 3.1 °C lower than that for Chi OTrf, while Jpq OTrf showed a 0.9 °C higher Tm_DSF than Chi OTrf. At pH 9.0, the Tm_DSF for Gui OTrf was the same as that for Chi OTrf, while the Duk, Ost, and Tky OTrfs showed 0.9 °C to 1.9 °C lower Tm_DSF values than Chi OTrf. The Tm_DSF for the Jpq OTrf was higher than that for Chi OTrf by 1.1 °C.

Fig. 2.

Differential fluorescence intensity (dF/dT) for poultry OTrfs related to temperature.

DSF analysis of poultry OTrf solutions was carried out with a qPCR system using SYPRO Orange as a fluorescent probe, scanning at a rate of 1 °C/min from 30 °C to 95 °C. The fluorescence intensity (F) data, which were dependent on the heating temperature (T), were processed by QtiPlot software and the average dF/dT in triplicate were plotted versus temperature. (a) pH 4.0; (b) pH 7.0; (c) pH 9.0.

Table 3. Summary of Tm_DSF values (°C) for poultry OTrfs.

pH	OTrf
	Chi	Duk	Gui	Jpq	Ost	Tky
pH 4.0	37.0 ± 0.00a	42.0 ± 0.47b	37.9 ± 0.43a	42.0 ± 0.48b	37.0 ± 0.00a	41.0 ± 0.47b
pH 7.0	63.0 ± 0.00d	60.9 ± 0.00b	62.0 ± 0.00c	63.9 ± 0.00e	59.9 ± 0.00a	62.0 ± 0.00c
pH 9.0	57.9 ± 0.00c	56.0 ± 0.00a	57.9 ± 0.00c	59.0 ± 0.00d	57.0 ± 0.00b	56.0 ± 0.00a

Tm_DSF values, the temperatures at the maximum dF/dT were obtained by DSF analysis of poultry OTrf solutions at pH 4.0, pH 7.0 or pH 9.0. Tm_DSF values for poultry OTrf were compared using multiple comparisons in SPSS software. Values shown with the different superscript in the same raw were significantly different. p < 0.05, n = 3

Turbidimetric analysis of poultry OTrf solutions isothermally heated at different temperatures for 40 min    The results of turbidimetric analysis at pH 4.0, pH 7.0, pH 8.0 and pH 9.0 are shown in Fig. 3. At pH 4.0, all of the poultry OTrf solutions except Ost OTrf became turbid by heating between 40 °C and 45 °C, while Ost OTrf remained soluble up to 95 °C (Fig. 3a). At 59 °C and pH 7.0, the poultry OTrf solutions, except Jpq and Ost OTrf, became turbid, while the Jpq and Ost OTrfs remained soluble; however, all of the poultry OTrf solutions developed turbidity above 62 °C (Fig. 3b). At pH 8.0, all of the poultry OTrf solutions, except Ost, developed turbidity up to 64 °C, while Ost OTrfs remained soluble up to 95 °C (Fig. 3c). At pH 9.0, Chi, Gui and Ost OTrf solutions developed turbidity up to 60 °C, while Tky, Jpq and Duk OTrfs remained soluble up to 73 °C, 85 °C and 95 °C, respectively (Fig. 3d).

Fig. 3.

The turbidity for poultry OTrf solutions isothermally heated at specified temperatures.

The poultry OTrf solutions were isothermally heated for 40 min at a specified temperature, then immediately cooled to 5 °C and allowed to stand overnight. The average turbidity measured at 655 nm with a microplate reader in triplicate were plotted versus heating temperature with error bars representing SD. (a) pH 4.0; (b) pH 7.0; (c) pH 8.0; (d) pH 9.0.

Based on the turbidimetric data, T_trb, the lowest temperature at which turbidity was detected in poultry OTrf solutions at various pH were determined and are compared in Fig. 4. Actually, T_trb was not determined at pH 2.0, since Chi OTrf as well as the other poultry OTrfs remained soluble by heating to 95.0 °C. At pH 4.0, the T_trb values for Chi and Duk were about 45 °C and higher than those for Gui, Jpq and Tky by 5 °C. The T_trb for Ost could not be determined because of the solubility up to 95.0 °C (Fig. 3a). At pH 5.0, the T_trb values for all poultry were similar at around 50 °C. The T_trb values for all of poultry were similar at around 60 °C at pH 6.0 and pH 7.0. At pH 8.0, the T_trb values for Chi and other poultry, except Ost, were similar at around 63 °C, and the T_trb for Ost could not be determined because of the solubility up to 95.0 °C (Fig. 3c). At pH 9.0, the T_trb values for Gui and Ost were about 55 °C and lower than that of Chi by 5 °C. The T_trb values for Tky and Jpq were 76 °C and 90 °C, respectively, and the T_trb for Duk could not be determined because of the solubility up to 95.0 °C (Fig. 3d).

Fig. 4.

Summary of T_trb val e (?) for poultry OTrf solutions.

T_trbvalues, the lowest temperatures where the turbidity (≥ 0.03, absorbance at 655 nm) was detected, were estimated by turbidimetry for poultry OTrf solutions at pH 2.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0 or pH 9.0. ‘*’ represents that the turbidity was not detected by heating up to 95.0 °C.

Discussion

DSC is used to study structural changes in proteins in response to heating (Johnson, 2013). The results show the denaturation temperature ranges, Tm_DSC and thermodynamic parameters for proteins during denaturation. In this study, we focused on the temperature ranges of denaturation, Tm_DSC, and the enthalpy of denaturation, °CH, for poultry OTrfs from Duk, Gui, Jpq, Ost and Tky and compared them to that for Chi. The ranges of denaturation temperature for Chi and other poultry OTrfs, except Ost OTrf, were similar between 30 °C and 50 °C at pH 4.0, 57 °C and 72 °C at pH 7.0 and 51 °C and 68 °C at pH 9.0 (Fig. 1). This clearly shows that OTrfs are more stable under neutral conditions than under acidic and alkaline conditions. Ost OTrfs did not show distinct endothermic peaks, probably because the energy required for thermal denaturation after acid denaturation might be below detectable levels, as described previously for Chi OTrf (Watanabe et al., 1985). The Tm_DSC values for Chi OTrf were 42.1 °C at pH 4.0, 64.7 °C at pH 7.0, and 60.1 °C at pH 9.0 (Table 1). Under acidic conditions, it has been reported that the thermal denaturation temperatures for Chi OTrf in egg white are 48.5 °C at pH 4.5 and 58.0 °C at pH 5.5, while no endothermic peak corresponding to OTrf was detected at pH 2.5 and pH 3.5 by DSC analysis (Watanabe et al., 1985). Under neutral and alkaline conditions, the denaturation temperatures for Chi OTrf have been reported to be 63.0 °C at pH 7.5 and 64.0 °C at pH 8.3 (Donovan and Ross, 1975), and the Tm for Chi OTrf was between 60 °C and 63 °C depending on the buffer at pH 7.5 (Mizutani et al., 2006). The denaturation temperature for Chi OTrf in egg white determined by DSC has been reported to be between 65 °C and 70 °C depending on pH (Donovan et al., 1975; Mineki et al., 2005; Van der Plancken et al., 2006). Our results are similar comparable to those in the literature but it should be taken into consideration that the Tm_DSC of isolated Chi OTrf was lower than that in egg white and that Tm_DSC was greatly affected by pH condition, ionic strength and the rate of heating in DSC (Donovan et al., 1975). The differences between Chi and other poultry OTrfs in Tm_DSC were less than 3.5 °C at pH 4.0, 2.3 °C at pH 7.0 and 2.6 °C at pH 9.0 (Table 1). These DSC findings suggest that the denaturation temperatures for the six poultry OTrfs are similar, though slight differences were observed at acidic pH. Despite these similarities, in the comparison of °CH, the energy required for denaturation was lower for the Gui OTrf and higher in the Tky OTrf compared to the Chi OTrf under any of the pH conditions tested.

Recently, DSF using a qPCR instrument has been used to analyze the thermal denaturation of proteins and protein-ligand interactions (Huynh and Partch, 2016). The principles of the method depend on the exposure of hydrophobic regions of a tested protein due to structural changes in response to thermal denaturation and the binding of fluorescent probes to the regions. In the present study, DSF was carried out under the same conditions as DSC in terms of the protein concentration (1 mg/mL) and the temperature scanning rate (1 °C/min). The Tm_DSF values for Chi OTrf were 37.0 °C, 63.0 °C and 57.9 °C at pH 4.0, pH 7.0 and pH 9.0, respectively (Table 3). The average Tm_DSF values for the six poultry breeds were 39.5 °C at pH 4.0, 62.0 °C at pH 7.0 and 57.3 °C at pH 9.0, and the difference in the Tm_DSF values among poultry OTrfs was less than 5 °C at any pH tested. The results of the DSF analysis were comparable to those of the DSC analysis, as shown above, though the DSF analysis consistently showed Tm values that were approximately 2 °C lower than those obtained by DSC analysis for equivalent samples. DSF has not always been successfully applied to proteins, but these findings showed that it can be used to monitor the denaturation of OTrfs and can be a powerful tool to study the conformational changes of OTrfs under various environments owing to its high-throughput characteristics.

In summary, the DSC and DSF results clearly show that the six poultry OTrfs were not markedly different in terms of their denaturation temperatures. This suggests that the pasteurization temperatures used for Chi eggs may be applicable to products produced with other poultry eggs, since the original methods for processing Chi eggs were developed in order to avoid salmonella contamination and to minimize the deterioration of the useful properties of Chi OTrf (Cunningham and Lineweaver 1965; Mizutani et al., 2006). However, optimization of the thermal process for each poultry egg white should be undertaken.

The thermal aggregation resulting from the association of denatured OTrf molecules was investigated by turbidimetry using 1 mg/mL OTrf solutions, as in the DSC and DSF analyses. However, the turbidimetric measurements were carried out by isothermal heating at a specified temperature for 40 min, which was different to the DSC and DSF analyses which used temperature scanning. Numerous studies have been conducted on the changes in solubility or turbidity of Chi OTrf on heating (Cunningham and Lineweaver, 1965; Nakamura et al., 1979; Matsuda et al., 1981; Matsudomi et al., 1991; Matsudomi et al., 2002, Mizutani et al., 2006). Although the thermal conditions such as heating temperature and heating time were quite different among the experiments, the current results for Chi OTrf were almost comparable to those published in the literature. Ost OTrf remained soluble even after heating to 95 °C, while Chi and the other poultry OTrfs aggregated at 40 °C to 45 °C at pH 4.0 or 61 °C to 64 °C at pH 8.0. Duk, Jpq and Tky OTrfs remained soluble even after heating to 95 °C, 85 °C and 73 °C, respectively, at pH 9.0, while Chi and the other poultry OTrfs aggregated between 54 °C and 60 °C (Fig. 4). As for the other pH values, the thermal properties of Chi and the other poultry OTrfs were similar; all the poultry OTrfs were soluble up to 95 °C at pH 2.0 and aggregated at approximately 51 °C at pH 5.0 and approximately 60 °C at pH 6.0 and pH 7.0. In contrast to the similarity in the denaturation temperatures for the six poultry OTrfs across the wide range of pH tested, marked differences in the aggregation temperatures were observed among OTrfs under some pH conditions (Fig. 3, Fig. 4). The results of the turbidimetric analysis suggest that the thermal process used for Chi eggs might not always be applicable to other poultry eggs, and that appropriate thermal process should be developed to take advantage of the properties that are unique to the OTrfs of each poultry breed.

The tertiary structures of the OTrfs of the different poultry are considered to be similar to those of Chi. In the denaturation and aggregation process for OTrfs, the conformational changes of the molecules and their intermolecular interactions should occur. In such changes, newly formed hydrophobic and hydrogen bonds, electrostatic interactions and van der Waal's bonds might be involved. Some disulfide interactions and/or sulfhydryl group formation may also occur. However, only limited information is available on such changes at present. In order to clarify the mechanisms that affect similarities and differences in denaturation temperatures and aggregation properties, comparative studies on changes in disulfide bonds and secondary structures of OTrfs during thermal processing should be undertaken. In addition, comparative analyses of thermal aggregates and heated solutions should also be performed.

Conclusion

1. Duk, Gui, Jpq, Ost and Tky OTrfs showed similar denaturation temperatures to Chi OTrf in the DSC analysis. This suggests the potential applicability of the pasteurization procedures developed for processing chicken egg white to the egg whites of other poultry in order to avoid contamination by salmonella and deterioration of the functionality of poultry OTrfs.

2. The results obtained by DSF were comparable to those obtained by DSC, showing that DSF is applicable to studies on the thermal transition of OTrfs.

3. In contrast to the similarities in denaturation temperatures observed among different poultry eggs, marked differences were observed in the thermal aggregation properties of Chi OTrf and the OTrfs of Duk, Jpq and Ost under some pH conditions. The results suggest that appropriate thermal processing of egg products should be developed to take advantage of the properties that are unique to some poultry OTrfs.

Acknowledgements    The authors would like to thank Ms. Minako Maeda of TA Instruments Japan Inc. for useful advice and technical support with the DSC analysis.

Conflict of interest    There are no conflicts of interest to declare.

Abbreviations

Chi

chicken

DSC

differential scanning colorimetry

DSF

differential scanning fluorimetry

Duk

duck

Gui

guinea fowl

Jpq

Japanese quail

Ost

ostrich

OTrf

ovotransferrin

Tky

turkey

References

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• 1) https://www.ncbi.nlm.nih.gov/nuccore/2157111428 (Jun. 8, 2022)