Effect of High Pressure Treatment on Rheological Characteristics of Egg Components

Abstract

The objective of this study was to evaluate the effect of high pressure treatment on rheological properties of various egg components (i.e., egg white, egg yolk, and whole liquid egg). A five-level central composite design with three independent variables (pressure, 281.8–618.2 MPa; temperature, 8.2–41.8°C; pressure holding time, 1.6–18.4 min) was employed. Samples were packed in plastic pouches, heat sealed, and subjected to pressure treatment in an isostatic press. Shear and time-dependent rheological properties were evaluated using a 3-segment (each 5 min) program with the shear rate initially increasing from zero to 100 s⁻¹ (upward curve), then held at 100 s⁻¹ (hold curve), and finally decreasing from 100 to 0 s⁻¹ (downward curve) using a parallel plate rheometer. Power law model was fitted to the upward (virgin) and downward curves while Weltman time dependency model was fitted for the hold curve (R² > 0.90). Rheological properties were most significantly affected by pressure followed by holding time and temperature for all egg components. In general, they showed thixotropic shear thinning behavior and reduction in time dependency, which was dependent on the level of processing treatment used (combination of pressure, time, and temperature level). The high pressure treatment resulted in a progressive transition of egg components from flowable liquid-like behavior (218–350 MPa) to semi-viscous (350–500 MPa) to highly-viscous (500–618 MPa) behavior. Rheology-based optimal conditions satisfying HP pasteurization needs were identified.

Keywords:

• High pressure processing
• Rheology
• Central composite rotatable design (CCRD)
• Power law
• Egg components

INTRODUCTION

An increase in demand of high quality products with minimal processing, without the use of additives and preservatives, has offered a fertile ground for the nonthermal techniques of food processing to establish its own niche in the global markets. High pressure (HP) processing is one such emerging nonthermal alternative to conventional food processing for producing high quality foods. It works on the principle of application of high pressures to attain shelf stable products and to modify the functional properties of food systems by mildly altering the protein structures, flavor, and nutritional qualities. The isostatic rule which governs HPP states that pressure is instantaneously and uniformly transmitted throughout a sample. Therefore, in contrast to conventional thermal processing, the HP is generally independent of sample size and shape.

HP processing has several advantages over thermal processing, such as better functional properties, no post processing contamination (in container treatment), and better preservation of nutritional quality as it does not require high temperatures.^[1−3] HPP has been shown to inactivate spoilage causing micro-organisms and enzymes resulting in a safe product of desired quality.^[4−8] Its application can contribute towards improvement of the physicochemical properties, like color, gel characteristics, and water-holding capacity of the food systems.^[9] From a safety point of view, HPP has shown to cause a 7-log₁₀ reduction of S. enterica serovar enteritidis in liquid whole eggs.^[10] Denaturation of various proteins, changes in functionality, and structural modifications are some of the outcomes of this technique.^[11,12]

Egg has high amounts of proteins, which undergo denaturation and/or structural changes, such as coagulation, on the application of heat or pressure treatment. Changes in the structure of protein can affect the rheological behavior of the food systems, such as gel and emulsions to a great extent; hence, it would be exciting to explore the flow properties of HP-treated egg.^[13,14] Rheological properties can be used as indices to establish or optimize conditions for transportation of liquid or semi-solid foods throughout the processing line. The consistency, degree of fluidity, and other mechanical properties are important in understanding how long food can be stored, how stable it will remain, and in determining food texture. Knowledge of the rheological properties of food products is essential for the product development, quality control, sensory evaluation, and design and evaluation of the process equipment.

Use of HP processing for denaturation of protein has always been an exciting concept as it can cause modification of functional properties by stimulating protein conformational changes.^[15,16] This protein denaturation can lead to aggregation or gelation depending on the number of factors related to the protein system (nature and composition of proteins), environmental conditions (pH and ionic strength), and HP treatment conditions (pressure level, processing time, and temperature).^[17,18] It can modify the functional properties of food components without affecting other characteristics, thus satisfying increased demand of minimally processed high quality foodstuff. HP processing can affect protein concentration of egg and the impact of HP on aggregation; network formation can also be modulated by pH and its study can be done using rheology.^[19−21] Hence, the detailed study of impact of HP processing on rheology has become an attractive and essential area of research for scientists. The objective of this study was to evaluate the changes in rheological characteristics of various egg components as influenced by HP treatment.

MATERIALS AND METHODS

Sample Preparation

Fresh raw eggs (large A size) were obtained from a local store and were sorted to exclude any cracked or inferior quality eggs. The eggs were randomly picked and some were cracked and the contents mixed to obtain whole liquid egg (WLE). Other eggs were carefully broken at the top and egg white (EW) was allowed to pour out slowly leaving the yolk (EY) behind in the shell. The yolk was cleaned to remove any traces of egg white. EW, EY, and WLE were individually packed into low density 2 oz. polyethylene bags (Whirl Pak^®, USA) and sealed using a heat sealer (QUIK SEAL Type 210B-1, National Instrument Co. Inc., Baltimore, MD, USA) for the processing treatments. Sample preparation was immediately followed by pressure treatment.

High Pressure Treatment

HP treatments were given out in an isostatic press (ACIP 6500/5/12VB; ACB Pressure Systems, Nantes, France) with a cylindrical pressure chamber of 5 L capacity. A high temperature circulating water bath (FP45, Julabo Labortechnik GMBH, Germany) was connected to a HP chamber in order to control the temperature during the processing. The pressurization medium used was water. Test pouches containing whole liquid egg, egg yolk, and egg white were prestabilized at a selected temperature prior to the HP treatment. Since HP treatment was expected to elevate the sample temperature, the initial temperature was kept slightly lower (trial and error) so that the operating conditions were established immediately up on pressurization. The pressure build-up time or releasing time was not considered during the treatment time. Pressurization and depressurization rates were 4.4 and 26 MPa/s, respectively. The pressure-treated pouches containing the sample were immediately transferred to a refrigerator (4°C) followed by rheological analysis. Duplicate measurements were carried out for each run for all of the samples.

Rheological Property Evaluation

A controlled stress rheometer (AR 2000, TA Instruments, New Castle, DE, USA) with attached computer software (Rheology Advantage Data Analysis Program, TA version 2.3 s) was used for all of the rheological measurements for WLE, EW, and EY. Parallel plate geometry (60 mm, 1 mm gap) was used to measure the flow behavior of egg samples with the instrument programmed at 20°C. Zero gap and instrument rotational mapping was efficiently performed before executing the experiments so as to get reliable results. A 2-mL sample of each WLE, EY, or EW was transferred to the flat plate of the rheometer. In order to evaluate the shear and time dependency, a three-cycle shear test was used with the shear rate increasing from 0 to 100 s⁻¹ (upward curve), held at 100 s⁻¹ for 5 min (hold curve), and followed by a decreasing shear rate from 100–0 s⁻¹ in 5 min (downward curve).^[22]

Experimental Design

A central composite design was used to estimate the effect of independent variables, such as pressure (X₁), treatment time (X₂), and temperature (X₃), on the dependent variables, such as flow behavior index (n) and consistency coefficient (m), for upward and downward flow curve, and Weltmann A and B for hold curve of the various egg components (EW, EY, WLE). Twenty combinations of independent variables were selected by the experimental design as shown in Table 1. Quadratic polynomial regression model was used for correlating these values to their coded variables (x_i, i = 1, 2, and 3).

TABLE 1 Minimum and maximum levels of process variables (pressure, temperature, and holding time)

	Pressure (X1)	Temperature (X2)	Holding time (X3)
CCRD levels	(MPa)	(°C)	(min)
−1.68	281.8	8.2	1.6
−1	350.0	15.0	5.0
0	450.0	25.0	10.0
1	550.0	35.0	15.0
1.68	618.2	41.8	18.4

(1)

where b₀ (constant term); b₁, b₂, and b₃ (linear effects); b₁₁, b₂₂, and b₃₃ (quadratic effects); and b₁₂, b₁₃, and b₂₃ (interaction effects) represent the coefficients of the polynomial model. The number of experimental points in the central composite rotatable design (CCRD) was sufficient to test statistical validity of the fitted model and lack-of-fit of the model.^[22] The central point in CCRD was replicated several times to estimate the error due to experimental or random variability. The process was optimized for two independent variables at a time using response surface methodology (RSM).

Rheological Models

Logarithmic plots of shear stress versus shear rate data of treated egg samples were used to calculate n and m values.^[23] Data was tested for various rheological models but power law model (Eq. 2) fitted adequately for upward and downward curves [based on the determination coefficient (R² > 0.90) at all test conditions]:

(2)

where m is the consistency coefficient (Pa.sⁿ), which describes the overall viscosity range of the flow curve. The flow behavior index is denoted as n and it is a dimensionless quantity.

Time Dependent Stress Decay Behavior

In order to investigate the time dependency of shear stress in high-pressure-treated egg products, a steady shear rate of 100 s⁻¹ for 5 min (hold curve) was applied. Stress decay behavior for all conditions was well explained by a modified Weltmann model.^[24] The Weltmann A and B were obtained from the regression of (σ) versus (log t) at a specific shear rate. The Weltman equation is given below:

(3)

where t > t_m, σ = shear stress, t = time (s), t_m = time at maximum observed shear stress, A = intercept when log (t/t_m) = 0 (Pa), and B = slope (time coefficient of thixotropic breakdown) (Pa).

Statistical Analysis

Regression coefficients and the ANOVA table were computed using Design Expert software. Surface graphs were plotted for the predicted values obtained from the models against two different process variables. The models were analyzed for coefficient of determination and standard error.

RESULTS AND DISCUSSIONS

Tables 2–4 show the evaluated powerlaw parameters, consistency coefficient m, and flow behavior index n, under upward and downward ramping as well as the Weltman A and B values during the hold period for WLE, EW, and EY, respectively. An ANOVA was used to fit the m and n values (and Weltman A and B values) to second-order polynomial equations. The sum of squares of a sequential model was evaluated for model fitting. The scope was to test impact of pressure in combination with time and temperature so as to study changes in egg components over a broader range (from completely liquid state to viscous gel form). The suitability of the fitted functions was evaluated by the coefficient of determination (R²). The ANOVA results for WLE are shown in Table 5. Lack of fit was assessed^[25] and found to be insignificant in all cases, thus indicating models employed to be adequate.

TABLE 2 Flow behavior index (n) and consistency coefficient (m) for both upward and downward curves, and Weltman A and B constants for hold curve of whole liquid egg

				Up curve		Hold curve		Down curve
No.	Pressure (MPa)	Time (min)	Temp. (°C)	n	m	A value	B value	n	m
1	350.0	15.0	35.0	0.241	16	2.83	10.3	0.615	5.2
2	450.0	10.0	25.0	0.209	26	2.81	6.25	0.545	2.4
3	450.0	10.0	25.0	0.205	25	2.75	5.81	0.545	2.4
4	350.0	5.0	15.0	0.367	1.0	2.24	3.63	0.705	0.1
5	550.0	5.0	35.0	0.243	22	0.85	3.25	0.386	5.6
6	550.0	15.0	15.0	0.291	30	2.71	51.7	0.331	7.9
7	350.0	5.0	35.0	0.301	2.0	2.82	37.5	0.607	3.3
8	450.0	10.0	25.0	0.206	25	2.68	6.99	0.582	1.0
9	550.0	15.0	35.0	0.156	41	2.16	9.21	0.142	7.8
10	550.0	5.0	15.0	0.341	12	2.22	35.6	0.624	2.3
11	350.0	15.0	15.0	0.312	3.0	2.46	3.99	0.799	0.1
12	450.0	10.0	25.0	0.208	24	2.18	6.55	0.580	1.0
13	450.0	10.0	8.2	0.365	1.0	2.37	5.05	0.630	0.3
14	450.0	10.0	25.0	0.206	24	2.52	6.89	0.550	0.6
15	450.0	18.4	25.0	0.207	28	2.28	55.3	0.386	4.4
16	618.2	10.0	25.0	0.236	37	0.96	5.06	0.313	6.3
17	450.0	10.0	41.8	0.215	22	0.71	0.59	0.333	5.3
18	281.8	10.0	25.0	0.314	2.0	2.53	9.42	0.831	0.1
19	450.0	1.59	25.0	0.311	2.0	2.29	52.5	0.574	0.4
20	450.0	10.0	25.0	0.206	25	2.78	6.32	0.552	0.7

TABLE 3 Flow behavior index (n) and consistency coefficient (m) for upward and downward curve; Weltmann A and B values for hold curve of egg white

				Up curve		Hold curve		Down curve
No.	Pressure (MPa)	Time (min)	Temp. (°C)	n	m	A value	B value	n	m
1	350.0	15.0	35.0	0.316	1.857	1.13	3.37	0.591	0.550
2	450.0	10.0	25.0	0.52	1.03	1.75	15.99	0.545	2.382
3	450.0	10.0	25.0	0.519	1.004	1.75	14.23	0.545	2.382
4	350.0	5.0	15.0	0.216	2.358	0.77	4.89	0.795	0.226
5	550.0	5.0	35.0	0.523	1.165	1.86	22.55	0.386	8.820
6	550.0	15.0	15.0	0.359	3.831	1.92	20.55	0.401	7.437
7	350.0	5.0	35.0	0.32	1.862	0.95	5.82	0.657	0.269
8	450.0	10.0	25.0	0.52	1.00	1.51	11.85	0.551	2.320
9	550.0	15.0	35.0	0.212	4.322	1.79	17.23	0.184	14.60
10	550.0	5.0	15.0	0.524	0.863	1.45	5.08	0.574	1.663
11	350.0	15.0	15.0	0.332	1.469	0.50	5.51	0.799	0.235
12	450.0	10.0	25.0	0.52	1.003	1.76	15.86	0.556	2.336
13	450.0	10.0	8.2	0.481	0.414	0.91	3.89	0.642	0.023
14	450.0	10.0	25.0	0.52	1.005	1.74	15.72	0.482	2.353
15	450.0	18.4	25.0	0.19	2.997	1.86	14.89	0.386	5.190
16	618.2	10.0	25.0	0.31	4.425	2.47	24.57	0.199	13.29
17	450.0	10.0	41.8	0.519	0.862	1.67	7.91	0.333	6.229
18	281.8	10.0	25.0	0.219	4.03	0.83	2.05	0.749	0.096
19	450.0	1.59	25.0	0.336	0.745	1.00	1.36	0.574	0.312
20	450.0	10.0	25.0	0.477	1.157	1.49	16.52	0.480	2.420

TABLE 4 Flow behavior index (n) and consistency coefficient (m) for upward and downward curve and Weltman A and B values for hold curve of egg yolk

				Up curve		Hold curve		Down curve
No.	Pressure (MPa)	Time (min)	Temp. (°C)	n	m	A value	B value	n	m
1	350.0	15.0	35.0	0.113	39	2.87	74.52	0.180	22.6
2	450.0	10.0	25.0	0.108	49	2.95	45.50	0.210	23.3
3	450.0	10.0	25.0	0.105	48	3.07	52.77	0.193	22.8
4	350.0	5.0	15.0	0.107	32	2.24	4.29	0.687	11.1
5	550.0	5.0	35.0	0.101	75	0.85	35.52	0.156	24.2
6	550.0	15.0	15.0	0.102	93	2.72	62.30	0.221	21.4
7	350.0	5.0	35.0	0.112	36	2.81	36.38	0.306	12.7
8	450.0	10.0	25.0	0.108	48	3.38	66.30	0.234	21.9
9	550.0	15.0	35.0	0.104	96	2.72	0.26	0.164	25.6
10	550.0	5.0	15.0	0.099	73	3.13	43.10	0.234	25.7
11	350.0	15.0	15.0	0.113	32	2.47	3.85	0.501	17.0
12	450.0	10.0	25.0	0.109	48	3.18	52.32	0.213	23.4
13	450.0	10.0	8.2	0.101	55	2.37	27.07	0.591	13.9
14	450.0	10.0	25.0	0.106	49	3.01	45.23	0.239	23.6
15	450.0	18.4	25.0	0.106	49	2.90	35.89	0.146	26.0
16	618.2	10.0	25.0	0.097	123	2.47	5.30	0.129	33.5
17	450.0	10.0	41.8	0.104	62	2.68	71.32	0.169	19.9
18	281.8	10.0	25.0	0.113	39	2.50	17.42	0.562	17.9
19	450.0	1.59	25.0	0.101	31	2.92	44.70	0.314	18.7
20	450.0	10.0	25.0	0.104	49	3.18	63.20	0.207	22.3

TABLE 5 ANOVA analysis and regression coefficient of second-order polynomial model for response variables of up upward curve for whole liquid egg

		Flow behavior index			Consistency coefficient
	Source	Sum of squares	DF	Prob > F	Sum of squares	DF	Prob > F
	Block	9.57113E-05	2		24.3	2
	Model	0.0736	9	<0.0001	3031.1	9	<0.0001
Linear	P	7.577 E-003	1	<0.0001	1485.9	1	<0.0001
	T	0.0134	1	<0.0001	682.6	1	<0.0001
	T	0.0283	1	<0.0001	353.9	1	<0.0001
Quadratic	P^2	0.00886	1	<0.0001	35.9	1	0.0058
	t^2	0.00526	1	<0.0001	159.3	1	<0.0001
	T^2	0.013028	1	<0.0001	289.8	1	<0.0001
Interaction	P × t	6.84E-05	1	0.0188	60.8	1	0.0013
	P × T	0.001128	1	<0.0001	9.1	1	0.0968
	T × T	0.000214	1	0.0008	17.6	1	0.0309
	Residual	6.35E-05	8	—	20.57	8
	Lack of fit	5.74E-05	5	0.0926NS	18.29	5	0.1129NS
	R-Squared	0.999					0.993
Hold curve		Constant A				Constant B
	Block	0.55	2		4.67	2
	Model	6.23	9	0.0303	726.16	9	<0.0001
Linear	P	1.859960	1	0.0107	428.06	1	0.0002
	T	0.285091	1	0.2313	83.93	1	0.0228
	T	1.090203	1	0.0351	34.45	1	0.1093
Quadratic	P^2	0.633607	1	0.0895	4.41	1	0.5374
	t^2	0.002640	1	0.9039	40.70	1	0.0859
	T^2	1.118548	1	0.0333	90.11	1	0.0195
Interaction	P × t	0.277513	1	0.2370	10.31	1	0.3533
	P × T	1.058513	1	0.0372	12.60	1	0.3077
	T × T	0.040613	1	0.6380	24.15	1	0.1699
	Residual	1.36	8		84.92	8
	Lack of fit	1.20	5	0.1236NS	78.27	5	0.0693
	R-Squared	0.98			0.999
		Flow behavior			Consistency
Down curve		index			coefficient
	Block	0.00496	2		10.1	2
	Model	0.546503	9	<0.0001	126.6	9	<0.0001
Linear	P	0.327247	1	<0.0001	47.4	1	<0.0001
	T	0.04127	1	<0.0001	19.9	1	<0.0001
	T	0.107051	1	<0.0001	29.2	1	<0.0001
Quadratic	P^2	0.001075	1	<0.0001	11.8	1	<0.0001
	t^2	0.008376	1	<0.0001	5.696	1	<0.0001
	T^2	0.007844	1	<0.0001	8.653	1	<0.0001
Interaction	P × t	0.050944	1	<0.0001	4.517	1	<0.0001
	P × T	0.002614	1	<0.0001	3.266	1	<0.0001
	T × T	0.000169	1	0.0007	0.287	1	<0.0001
	Residual	4.7E-05	8		0.023	8
	Lack of fit	4.05E-05	5	0.1540NS	0.022	5	0.0585NS
	R-Squared	0.999			0.998

NS: Non significant; P: pressure; t: time; T: temperature.

Upward Curve: Effect of HP Treatment on Flow Behavior Index (n Value)

Typical upward, hold, and downward flow curves for egg subjected for selected treatment are shown in Fig. 1 (Tables 2–4). They demonstrate shear thinning behavior both under upward and downward ramps as well as steady shear operating conditions. From the statistical analysis of variance (ANOVA), it was found that all three independent variables—pressure, temperature, and holding time—had a major influence on all rheological parameters of whole liquid egg, egg white, and egg yolk (data shown in Table 5 for WLE). It was found that high pressure treatment caused progressive denaturation of whole liquid egg and they had rheological behavior different from the control sample due to structural breakdown. Typical response surface plots for flow behavior index of the upward curve of WLE are shown in Fig. 2.

FIGURE 1 Typical figure showing up, hold, and down curve for a light, medium, and high intensity treatment (pressure level is high to low from top to bottom).

FIGURE 2 Typical response surface plots for whole liquid egg (WLE) showing the effect of pressure and treatment time on (a) flow behavior index (n) and (b) consistency coefficient (m) for the virgin upward shear curve; and (c) flow behavior index (n) and (d) consistency coefficient (m) for the downward shear curve; (e) Weltman A and (f) Weltman B time dependency parameters during the shear hold.

From rheological analysis, it was found that application of increasing pressure and time caused a small change in n value of whole liquid egg (WLE) (Fig. 2a). The n values were in the range of 0.2 to 0.5 indicating pseudoplastic behavior (n < 1). P values from ANOVA analysis specified that linear and quadratic effect of all process variables, interaction effect of P × t, P × T, and t × T, had a highly significant effect (p < 0.05) on the n value (Table 5).

Flow behavior index (n value) of egg white (EW) was also significantly affected by pressure and time treatments. The interaction due to pressure caused the flow behavior to increase but changes in flow behavior index approached a limiting value of 500 MPa (Table 3). Pressure effect was more pronounced when the treatment times were short (5 min) as compared to longer (15 min) treatment times. It was found that both pressure and temperature increased the n value, but pressure had a more significant impact than temperature. Increase in temperature and time levels caused a small but linear increase in flow behavior index. The flow behavior index values were highest for EW as compared to the other two components, but still remained <1, which indicated pseudo-plastic behavior.

For egg yolk (EY), the n values were far lower than 1.0, demonstrating a more sensitive and deeper pseudoplastic behavior. Increase in pressure level caused a decrease in n value, whereas increasing treatment time and temperature caused an increase in n value but values remained below 1.0 (Table 4). Analysis indicated that pressure (p < 0.05) had the most significant effect on n value followed by time (p < 0.05) and temperature (p < 0.05) for egg yolk. Interactions were less apparent resulting in mostly a linear relationship with the variables. Model fitting and ANOVA were validated by analyzing residuals, including the examination of diagnostic plots and calculation of case statistics. The response surface was generated by keeping one variable at its zero level (center point) and varying the others in their experimental range. In a similar study, it was found that the egg yolk shows slightly pseudoplastic behavior at different total solids content.^[26]

Overall, the flow behavior index relationship between pressure and time for the three egg components varied significantly. As can be expected, the range of values for the WLE laid between those for the other two components. EY appeared to be most influenced by the interactions with n values in the 0.1 region (most pseudoplastic) with EW demonstrating n values at the higher end (still below 1.0). Results indicated a greater sensistivity of EY for the HP treatment resulting possibly from a greater degree of denaturation. EW was least affected and the WLE rheology was moderated by the two.

Upward Curve: Effect of HP Treatment on Consistency Coefficient (m Value)

High pressure was responsible for progressive denaturation of different egg components and had a significant impact on the consistency coefficient. Treated samples had much higher apparent viscosity than the control sample. It was found that pressure, time, and temperature were significant with respect to their influence on m value of WLE, EW, and EY (Fig. 2b, Tables 2–4). Lack of fit was again found to be nonsignificant, suggesting that this model is good for predicting the response variable at different processing conditions (Table 5). For the most part, the interactions were also significant. As with n values, the EY demonstrates the largest change in consistency coefficient and EW the least. Again, the interactions were more significant with EW. WLE appeared to have values around midway between the two in consistence with the proportion of EW and EY in WLE. In a similiar study, it was reported that an increase in pressure level increases the consistency coefficient of egg yolk.^[1] It was found that various phospholipids present in egg yolk are responsible for their viscosity and they also provide stability to egg white by making ovalbumin-phospholipids complex.^[19]

Downward Curve: Effect of HP Treatment on Flow Behavior Index (n Value)

The initial upward curve showed more variability in n and m value as egg samples subjected to upward shear curve are virgin, starting from a well rested system (Tables 2–4). Downward curves are generally more consistent with rheological measurements since the samples are already subjected to shearing (during upward and hold periods) so the system is already in motion. While the up-curve rheology simulates the behavior of virgin samples (not subjected to any shear), the down curve rheological parameters are for stirred sytems and are more useful in flowing liquids. As with the up curves, the ANOVA results for the down curve indicate that the pressure treatment (independent variables and their interactions) influenced flow properties significantly and models relating them were adequate and the lack of fit was not significant (Table 5, Fig. 2c for WLE).

The influence was again more with EY as compared to EW, and the values for WLE were moderated by the two (Tables 2–4). For all egg components, an increase in pressure level, temperature level, and treatment time was accompained with an increase in pseudoplasticity, shown by a decrease in values of flow behavior index (n). Temperature changes caused larger changes in the n value than pressure and time level. Flow behavior was almost unaffected by time. Thus, higher temperature, higher pressure, and higher treatment times seem to work in a synergistic manner to decrease flow behavior index in upward and downward curves. Egg white showed development of an elastic behavior, which showed resistance to the flow. The treatment time had a negligible effect on n value as the latter remained constant with an increase in time, whereas increase in pressure and temperature caused a decrease in the n value. Similar to egg white, all linear effects of all process variables, quadratic effects of temperature, and interaction effect of pressure and time were significant with egg yolk (EY) (p < 0.05).

The low values of flow behavior index (n) signifies that the egg components show flow properties differing from that of the Newtonian behavior, and like many other shear-thinning food products, they have a high viscosity at low shear rates, which decreases dramatically as the shear is increased. It has been reported that a non-Newtonian behavior became important when the flow behavior index is less than 0.6.^[27] It was found that high pressure treatment (400 MPa/25°C/30 min) caused less gelation than cooking in a water bath for 2–10 min.^[15]

Downward Curve: Effect of HP Treatment on Consistency Coefficient (m Value)

For a downward flow curve, an increase in pressure and temperature level caused a major change in m value indicating an increase in the consistency coefficient of WLE while treatment time caused only small changes (Table 2, Fig. 2d). It was also influenced significantly by linear, quadratic, and interaction effects of all process variables (p < 0.05). In EW, model (p < 0.05), linear effect of P, t, and T (p < 0.05), quadratic effect of P, t, and T (p < 0.05), and interaction effect of P × t and P × T were highly significant (p < 0.05) (Table 5). Consistency coefficient (m) of the EW was shifted up with an increase in pressure and temperature level, whereas with increasing treatment time, m value showed a slight increase throughout whole range of treatment time. Pressure and temperature level used had more significant (p < 0.05) effect than treatment time on consistency coefficient (m). For EY, an increase in intensity of process resulted in an increase in consistency causing an increase in m value.

As before, egg yolk had a higher consistency coefficient than EW and WLE, and EW had the lower values. As observed before, egg yolk was more sensitive to pressure treatment and progressively it turned from thin liquid to thicker liquid with enhanced viscosity showing the least flow behavior index and highest consistency coefficient (Table 4). The main reason for the increase in thixotrophy of egg yolk after pressurization could be the relative ease of denaturation and perhaps because it contains low and high density lipoproteins.^[28] Overall, the down curves gave more consistent data that followed more closely with the physical changes that could be observed visually with the samples.

Hold Curve: Effect of HP Treatment on Time Dependent Rheology

In order to evaluate thixotropic time dependent flow behavior of treated egg components, they were subjected to steady shear rheology in which samples were constantly sheared at 100 s⁻¹ for 5 min. In hold curve, Weltman constants A and B, calculated using the Weltman equation, explain about effect of constant shearing on treated samples.

Effect of HP Treatment on Weltman A

Weltman A value has a close correlation with consistency coefficient as it is a measure of initial resistance to shear rate at a time equal to 1 s. A higher value indicates a stronger resistance to start up shear. Tables 2–4 (Fig. 2e for WLE) show that with WLE there was a general, but small, decreasing trend for the Weltman A value, while with EW it was a more steady increasing trend and with EY it was mixed. Also, comparing the magnitude of values for WLE, it appears that the EY effects compensated the EW effects to reverse the Weltman A trend. But the overall ranges for the three components were much closer to each other than with the m and n values observed earlier. Time and time interaction effects were not significant, while the other effects were significant (p < 0.05).

It was proposed that the synergistic interactions between compatible small globular proteins were dependent on the degree of unfolding of the individual proteins in the mixture, which governed optimum exposure of specific groups and thereby optimum interaction.^[29] The protein–protein interactions in the food protein aggregates are dependent on covalent, disulphide bonds. It also affects peptide backbone conformation as well as the microenvironment around the side chains changing interactions of individual and combined egg protein gels.^[19]

Effect of HP Treatment on Weltman B

Weltman B value is a measure of the rate of structure breakdown and is referred to as time coefficient of stress decay, and higher values of Weltman B demontstrate a more pronounced structure loss.^[30] Weltmann B values were also significantly influenced by the process variables indicating greater sensitivity to stress decay at higher treatment severity (Tables 2–4; Fig. 2f for WLE). For all egg components, the model for both parameters was significant (p < 0.05) and the lack of fit was not (p > 0.05). In the case of Weltman B, the trends with WLE and EW were similar, with both demonstrating a progressive linear increase in value with an increase in parametric value while EY showed mixed trends. Hence, the WLE is more dominated by the EW component than EY. The temperature effects and its interactions were largely insignificant (p > 0.05) with Weltmann B. It has been reported that high pressure can cause changes in functional properties like solubility and structural characteristics of ovalbumin, which is a major constituent of egg white. Ovalbumin has been shown to be sensitive to high pressure, which can cause an increase in viscosity due to unfolding of proteins but they retain foaming and other functional properties.^[31]

Rheologically Optimized HP Processing Conditions Meeting Pasteurization Criteria

These were evaluated by using the optimization function of design expert software. The minimal pressure processing conditions were first identified based on available published information for establishing a pressure pasteurization process. Complete inactivation of Salmonella enteritidis in eggs with a two-cycle pressure treatment of 450 MPa and 20°C for 5 min was reported, but single-cycle treatment under the above conditions was considered inadequate.^[32] Since only single-cycle treatments have been used in this study, this information will not be very useful. A detailed study was related to the action of high pressure processing on the inactivation of two food-borne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples in continuous and cyclic treatment conditions.^[33] They found that 15-min treatments at pressure levels ≥400 MPa at room temperature resulted in more than nine log cycle reductions in S. aureus and S. enteritidis inoculated into tryptic soy broth. However, they found it to be less effective when tested in cavier and found that a minimum of 500 MPa for 15 min or multiple cycle treatments were necessary to cause five log reductions. Due to lack of available information, information from these two sources is merged to arrive at minimum HP processing conditions as follows: 500 MPa, 20°C, and 15 min treatment.

TABLE 6 Rheologically optimum processing conditions for WLE (maximize n and minimize m) meeting pasteurization criteria (500 MPa for 15 min at 20°C)

			Lower	Upper	Lower	Upper
Name		Goal	limit	limit	weight	weight	Importance
Pressure		Is in range	500	618	1	1	3
Time		Minimize	15	18	1	1	3
Temp.		Is in range	20	41	1	1	3
n		Maximize	0.142	0.831	1	1	2
m		Minimize	0.052	7.937	1	1	2
Number	Pressure	Time	Temp.	n		m	Desirability
1	500.00	15.00	20.00	0.410		4.437	0.61
2	500.00	15.00	20.17	0.409		4.440	0.60
3	500.00	15.00	20.26	0.408		4.442	0.60
4	500.00	15.00	20.74	0.404		4.455	0.60
5	502.33	15.00	20.46	0.401		4.536	0.59
6	505.86	15.00	20.00	0.397		4.665	0.59
7	500.04	15.15	20.00	0.406		4.528	0.58
8	506.42	15.00	22.30	0.376		4.760	0.57
9	500.00	15.28	20.60	0.397		4.616	0.56
10	500.00	15.39	20.00	0.399		4.670	0.55

Note: n = flow behavior index, m = consistency coefficient.

Based on the results presented in preceeding sections, it is obvious that as the severity of the HPP increases, the rheological properties of WLE, EW, and EY progressively increase resulting in lower values in n, higher values in m (flow curves, both upward and downward), as well as lower values in Weltman A and higher values in Weltman B. Imparting minimal rheological changes means higher n and lower m, higher Weltman A and lower Weltman B. In terms of finding HPP conditions that satisfy minimum pasteurization requirements (550 MPa 15 min at 20°C) and at the same time result in the least changes in rheological properties means one has to look at minimizing changes in rheological property values. This means a condition that would retain maximum n and minimum m values (maximum Weltman A and minimum Weltamn B). Since any imparted change simultaneously decreases n and increases m values (and likewise decreases Weltman A and increases Weltman B values), they result in opposite changes in n versus m and Weltman A versus Weltman B values. Hence, in the search for optimization conditions, one has to maximize retention of n while minimizing the m values (the same with Weltman A versus B values). Hence, one can only get compromising values and, therefore, optimal conditions would be expected to be at intermediate values of n and m values (Weltman A and B values as well). The desirability program generated optimal conditions are listed in Table 6 for WLE that meet the pasteurization criterion and provide intermediate effects on n and m values. The program also prints desirability values with 1 being most desirable and zero being totally undesirable. Since we cannot simultaneously achieve the desired n and m values, the best desirability achieved was about 0.61. Similar results can be computed with other constraints (for using Weltman A and B values), or rheological parameters associated with EW or EY or even considering other pasteurization conditions when they become available (higher pressures with lower treatment times, for example). But the procedure offers an excellent tool for achieving constraint satisfied optimal processing conditions. The desirability method is one of the most commonly used approaches for optimization in industry. This method is based on the theory that quality of a process that has multiple quality parameters is unacceptable when even one of these parameters stays outside of the desired range.

CONCLUSIONS

The effects of high-pressure treatments on rheology of WLE, albumen, and yolk were studied. It was found that all egg samples behaved as thixotropic fluid. The structure breakdown of egg protein enhances with pressure and it completed at 300 MPa for 30 min at 20°C. High pressure affected the protein structural of albumen and WLE; however, electrophoresis results exhibited that the protein coagulation was irreversible. The yolk behaved differently with pressure treatment. It needs further work on pressure–temperature combination and microbiological aspects of post-processed samples before implementing the industrial sector. Rheological properties of egg components (WLE, EW, and EY) were studied as a function of time, temperature, and pressure. All of the egg components demonstrated shear thinning behavior, as well as time dependent thixotrophy. Whole liquid egg containing both the components of egg yolk and egg white behaved differently. At a higher pressure range (550–618 MPa) viscosity of egg yolk increased levels that restricted the free flow of the product. Only limited information was available on pressure pasteurization conditions for egg products and optimal conditions were identified that would meet pasteurization criterion and at the same time impart minimal rheological changes in the treated product.