Coagulation Kinetics of High Pressure Treated Acidified Milk Gel for Preparation Chhana (An Indian Soft Cottage Cheese)

Abstract

Chhana, an Indian cottage cheese, is one of the most popular heat-acid coagulated milk products. It forms the base material for a variety of milk-based sweets. Application of high pressure treatment for denaturation of milk protein has successfully been carried out. In the present study, high pressure in the range of 200–400 MPa for 0 to 100 min at coagulation temperature of 30–70°C was applied at constant acidity of milk-acid mixture in order to study the coagulated milk gel properties for preparation of chhana. The experiments were conducted using a central composite rotatable experimental design. Linear and non-linear regression equations, using the coded values of the independent variables were developed to study the effect of process variables on coagulated gel properties viz., lag time, mean coagulation rate and inflexion time. The optimum values of independent variables corresponding to minimum lag time, maximum coagulation rate and minimum inflexion time was obtained were high pressure - 280 MPa; pressurization time - 47 min; and coagulation time - 52°C. At this optimum condition of independent variables the lag time, mean coagulation time and inflexion time were 0.0028 min, 3.87 min, and 5.19 min, respectively.

Keywords:

• High pressure treatment
• Chhana
• Milk coagulation
• Lag time
• Mean coagulation time
• Inflexion time

INTRODUCTION

Chhana, an Indian counter part of soft cottage cheese is a heat-acid coagulated product of milk. It forms the base material for production of a variety of milk products like paneer, rasogolla, sandesh, and gulabjaman in Asian countries. Pattern of milk consumption indicates that about 6% of milk produced in India is coagulated to produce chhana.[1]

Chhana is a rich source of fat and protein. It also contains fat-soluble vitamins A and D. With high protein and low sugar content, chhana is highly recommended for diabetic patients.[2] According to the definition of the Bureau of Indian Standard (BIS), chhana should not contain more than 70% moisture and milk fat should not be less than 50% of the dry matter.[3] According to the Prevention of Food Adulteration Rules, chhana is defined as a milk product obtained by precipitating a part of milk solid by boiling whole milk of cow and or buffalo or a combination thereof by addition of lactic acid, citric acid or any other suitable coagulating agent and subsequent drainage of whey.[4]

For preparation of chhana, whole milk of cow and/or buffalo is heated to its boiling point. The heated milk is allowed to cool to 70°C and a coagulating solution of lactic acid, citric acid or calcium lactate is added to coagulate the milk. Coagulation of milk involves destabilization of casein micelles. Acid affects the stability of casein directly by disturbing the charges carried by particles and indirectly by releasing the calcium ion from colloidal calcium-caseinate-phosphate complex. The destabilization results in formation of large, firm, and cohesive structural aggregates of casein micelles in which milk fat, other colloidal and soluble solids are entrained along with deproteinated whey. The whey is drained out and the dewheyed product is known as chhana.

Quality of chhana is affected by the heat treatment given to milk prior to its acidification, acidity of milk-acid mixture at the time of coagulation and residence time of the coagulum prior to separation of milk solids, besides the type of milk and its initial composition.[5] The heat treatment of milk before acidification is characterized by the temperature to which milk is heated, the rate of heating, the temperature to which the milk is cooled and the rate of cooling.[6] Heating causes denaturation of whey proteins and they get associated with casein micelles. The degree of denatured whey proteins depends on the time-temperature combination given to milk and is mainly determined by the maximum temperature to which milk is heated.[7–11]

High pressure processing is one of the most promising non-thermal processes for food processing and of great concern in food industry because of its potential to achieve interesting functional, structural, chemical, and sensory attributes in the final product. High pressure processing have been found to improve the cheese making properties such as coagulation time, cheese yield, gel strength and effect on syneresis.[12–17] Some authors reported milk curdling by renneting under pressure.[12,18] Many authors have reported that the coagulation properties of milk improved after pressure treatment,[15,18,19] however, there is little information available in the literature characterizing the influence of pressure processing conditions (pressure, pressurization time, and coagulation temperature) on acidified milk gel properties.

The specific objective of the present research was to study the effect of high pressure treatments viz., applied pressure and pressurization time, and coagulation temperature on acidified gel kinetics viz., lag time, mean coagulation rate, and inflexion time of milk, and to optimize the applied pressure, and pressurization time, and coagulation temperature based on minimum lag time, minimum coagulation time and minimum inflexion time for preparation of chhana.

MATERIALS AND METHODS

Milk Sample Preparation

Cow milk was used to study the acid coagulation of milk. The raw milk obtained from a particular cow from the nearby cow yard at the University of Reading, UK was dried into powder using a buchi spray dryer (Model–456/R, USA). The milk was dried in order to maintain the composition of its constituents same, which was further reconstituted immediate after drying. This spray dried milk powder was reconstituted by adding distilled water. The fat content in the reconstitute milk was balanced at 4% making a mass balance over fat and solid-not-fat content. The reconstituted milk samples were stirred at the ambient temperature for at least 10 h before further treatment to ensure complete equilibration.

Moisture content of reconstituted milk and chhana was determined as per IS: 5162.[20] Fat content of the milk was determined by Gerber method.[21] Lactose content (% actic acid) and titratable acidity (% lactic acid) of the milk were determined by using Lane-Eynon method as described in BIS.[22] Ash content of milk was determined according to ISI: 5162.[20] Protein content was determined by Kjeldahl method using 6.38 as nitrogen to protein conversion factor.[22] The proximate compositions of milk, as obtained from the experiments were: moisture 0.861 ± 0.015, protein 0.047 ± 0.008, fat 0.041 ± 0.007, lactose 0.0361 ± 0.002, and ash 0.011 ± 0.001 kg per kg milk. The average titratable acidity of milk was 0.168 ± 0.005% equivalent lactic acid.

High Pressure Treatment

A high pressure unit (Model S-FL-085-9-W, Stansted Fluid Power, UK) was used in this study to subject milk to various pressure conditions. The high pressure unit consists of a pressure chamber, with an internal diameter of 10 cm and height of 55 cm with an enclosed metal jacket. The metal jacket allows temperature control in the pressure chamber by circulating hot/cold water at the desired coagulation temperature. Water containing 2% water-soluble oil (part no 5019, ABB Autoclave Engineers, UK) was used as the hydrostatic fluid in the pressure chamber. The milk filled in test pouches were submerged in water in the pressure chamber and subjected to various pressure and time combinations as mentioned in Table 1. Adiabatic heating results in temperature rise in test samples and pressure medium (approx. 3°C/100 MPa). In order to achieve and maintain an approximate average coagulation temperature during test run, milk samples were equilibrated to lower initial temperatures and the temperature of circulating water was kept slightly below the coagulation temperature. Average temperature indicated during the run was within ±2°C of the set point. Following treatment, test pouches were removed from the chamber and processed for analysis.

Table 1 Experimental design of independent variables

	Real values of independent variables
Coded values of independent variables	Pressure X1 (MPa)	Pressurisation time X2 (min)	Coagulation temperature X3 (°C)
+1.682	400	100	70
+1	330	85.5	60
0	300	50	50
−1	270	14.5	40
−1.682	200	0	30
	Experimental plan with coded form of independent variables
Sl No	x1	x2	x3
1	−1	−1	−1
2	−1	−1	1
3	−1	1	−1
4	−1	1	1
5	1	−1	−1
6	1	−1	1
7	1	1	−1
8	1	1	1
9	−1.682	0	0
10	1.682	0	0
11	0	−1.682	0
12	0	1.682	0
13	0	0	−1.682
14	0	0	1.682
15	0	0	0
16	0	0	0
17	0	0	0
18	0	0	0
19	0	0	0
20	0	0	0
21	0	0	0
x1: Coded value of pressure; x2: Coded value of pressurization time; x3: Coded value of coagulation temperature; LT: Lag time; MCT: Mean coagulation time; and IT: Inflexion time.

A central composite rotatable design (CCRD) was employed to carry out the experiments at various combinations of independent variables, e.g., applied pressure, pressurization time and coagulation temperature. The levels chosen for pressure X ₁, time X ₂, and coagulation temperature X ₃ were; X ₁: 200, 270, 300, 330, and 400 MPa; X ₂: 0, 14.5, 50, 85.5, and 100 min; and X ₃: 30, 40, 50, 60, and 70°C. These values were considered based on literature and preliminary trails. In the design (Table 1), the real values of independent variables viz., pressure, pressurization time and coagulation temperature were coded as x_{1 ,} x₂, and x₃ . The coded values were set to lie in the range +1.682 and −1.682.[23–25] The relationship between the real and coded variables is given by the following equations:

(1)

(2)

(3)

(4)

where X_max is the maximum value of X, and +1.682 is the coded value associated with X_max , X_min is the minimum value of X and −1.682 is its coded value; andX_m is the arithmetic mean of X_max and X_min . A total number of 21 experiments were carried out (Table 1). The experiments were conducted in random order. Six repeated experiments were conducted at the central points of the coded variables. This was necessary for calculating the error sum of square and the lack of fit of the developed regression equations.[25]

To find the effect of independent variables x (viz., applied pressure, pressurization time and coagulation temperature) on the dependent variables y (viz., lag time (LT), mean coagulation time (MCT) and inflexion time (IT)), following linear and non-linear regression forms of equations were fitted.

(4)

(5)

where, a ₀, a ₁, a ₂, a ₁₁,a ₂₂, a ₃₃, a ₁₂, a ₁₃, and a ₂₃ are regression coefficients; and x ₁, x ₂, and x ₂ are the coded values of the independent variables X, viz., pressure (X ₁), time (X ₂), and coagulation temperature (X ₃), respectively.

Milk Coagulation and Rheological Parameters Measurement

Citric acid solution was used as the coagulating agent for coagulation of milk. Strength of the acid solution required for the coagulation was calculated based on acidity and density of milk, acidity of milk-acid mixture, and ratio of milk and acid solution at the time of coagulation.[26] The ratio of weight of milk to citric acid solution was fixed at 5:1.[6] The acidity of milk-acid mixture at the time of coagulation was fixed at 0.52% lactic acid.[6] Using the procedure, the strength of the citric acid solution used was 1.62 gm per 100 gm of citric acid solution.

The reconstituted milk was treated with high pressure as per the experimental design given in Table 1. The citric acid solution, previously heated to different coagulation temperatures as mentioned in Table 1 was immediately added to the high pressure treated samples. Immediately after addition of citric acid solution to milk samples, 1 ml sample of acid treated mass were poured into three disposable 10 mm polythene micro-cuvettes and transferred to a Beckman DU 65 spectrophotometer (Beckman Instrument, Inc., Fullerton, CA) equipped with a Beckman auto 6-sampler accessory, a six-sample cell holder. Three of the six cells were used to simultaneously follow coagulation in three replicate samples. The coagulation temperature was maintained with circulation hot/cold water. Optical density (OD) was recorded at 700 mm at 12 s intervals for 50 min by using a Beckman DU Data Leader software.

Properties of High Pressure Treated Acid Coagulated Milk Gel

Optical density ratio was used as an index to quantify the gel quality. The optical density ratio (ODR) was calculated by dividing the measured optical density value by the average of initial optical density values for the first 5 data points (1 min) after acidification. Since the optical density increased with time, the ODR vs. time represented a normalized transient optical density curve (Fig. 1), the coagulation process can be modeled as a sigmoid equation[27–28] as shown in Eq. (6):

(6)

where ODR: optical density ratio; a: minimum ODR; b: slope coefficient; c: inflexion point coefficient; d: maximum ODR at infinite time; e: a symmetry parameter; and t: time (min). In Eq. (6), b is the indicative of the rate of coagulation. The values of a, b, c, d, and e were obtained by fitting the coagulation curve to the Eq. (6) using software Origin 6.0. Eq. (6) and its first and second derivatives allow the determination of lag time (t _lag) which is defined as the time when ODR indicates an increase by 0.001 units from the initial value of 1.0, the inflexion time (t _max), which is the time when the rate of coagulation is maximum, and D _max, which is the coagulation rate (slope) at the inflexion point which is the maximum. The values are calculated from the sigmoid model as shown below[28]:

(7)

(8)

(9)

Figure 1 Typical coagulation curve of optical density ratio vs. time.

RESULTS AND DISCUSSION

Table 2 shows the different parameters of the coagulation curve at various levels of coagulation variables. The coagulation curves thus obtained are appeared to be good fit for the ODR and time data. The model fitted had R ² values in the range of 0.929–0.997. As shown in the Fig. 1, the coagulation started at approximately 5 min after acid addition and reached the peak value at the inflexion point approximately 18 min. An average coagulation rate was computed from observed rates around the inflexion point to be 0.0038 min⁻¹, while the peak value was 0.0043 min⁻¹. The inflexion times for various levels of the treatments were obtained from the first derivative of the Fig. 1.

Table 2 Estimated values of quality of acid gel

	Independent variables			Dependent variables
Sl No	X 1	X 2	X 3	LT	MCT	IT
1	270	14.5	40	0.0018	4.18	5.76
2	270	14.5	60	0.0022	3.87	6.28
3	270	85.5	40	0.0026	4.23	4.78
4	270	85.5	60	0.0027	4.02	6.43
5	330	14.5	40	0.0035	3.03	7.34
6	330	14.5	60	0.0027	4.12	5.81
7	330	85.5	40	0.0023	3.76	3.79
8	330	85.5	60	0.0015	4.17	3.12
9	200	50	50	0.0016	4.01	3.98
10	400	50	50	0.0022	3.12	4.23
11	300	0	50	0.0025	2.86	5.34
12	300	100	50	0.0018	3.12	3.34
13	300	50	40	0.0018	2.98	3.54
14	300	50	70	0.0027	3.02	3.01
15	300	50	50	0.0024	2.67	3.54
16	300	50	50	0.0023	2.76	3.67
17	300	50	50	0.0023	2.71	3.55
18	300	50	50	0.0025	2.77	3.49
19	300	50	50	0.0024	2.76	3.55
20	300	50	50	0.0025	2.72	3.51
	300	50	50	0.0025	2.72	3.51
X 1: Pressure (MPa); X 2: Pressurization time (min); X 3: Coagulation temperature (°C); LT: Lag time (min); MCT: Mean coagulation time (min); and IT Inflexion time (min).

Effect of Independent Variables on High-Pressure Treated Acid Coagulated Gel Properties

The values of the coagulation process responses obtained for different combinations of pressure treatment and coagulation temperature are mentioned in Table 3. Linear regression Eqs. (10), (11), and (12), using the coded values (x ₁, x ₂, and x ₃) of the independent variables (X ₁, X ₂, and X ₃) have been developed to find out the effect of high pressure (x ₁), pressurization time (x ₂) and coagulation temperature (x ₃) on the acidified milk gel properties (viz., lag time (LT), mean coagulation rate (MCT), and inflexion time (IT).

Table 3 Estimated values of quality parameters of liquid honey powder obtained from the optimized combinations of the ingredients

Sl. No	Quality parameters	Estimated values
1	Lag time (min)	0.0028
2	Mean coagulation time (min)	3.87
3	Inflexion time (min)	5.19

(10)

(11)

(12)

The relationship between the real (X) and the coded (x) values are:

(13)

(14)

(15)

By comparing the coefficients and sign of the coefficients of Eqs. (10), (11), and (12), and the surface plots drawn with coded values of independent variables for various responses, we may arrive at the following conclusions.

Equation (10)

It is observed that lag time will increase with increase in pressurization time, applied pressure, and coagulation temperature. The high pressure has the significant effect on lag time compared to coagulation temperature. Surface plots demonstrating the effect of coded values of different process variables on lag time are shown in Fig. 2. The lag time increased with an increase in applied pressure and pressurization time (Fig. 2a). Moreover, the effect of coagulation temperature was more significant followed by pressurization time and applied high pressure. Both increased value of pressure and coagulation time increased the lag time equally as observed from Fig. 2b. The effect of pressurization time and coagulation temperature (Fig. 2c) on lag time increased, but the effect of coagulation temperature was more prominent than the pressurization time.

Figure 2 Surface plots showing the effect of coded values of high pressure variables on lag time (a) pressure and pressurization time; (b) pressure and coagulation temperature; and (c) pressurisation time and coagulation temperature.

Equation (11)

The mean coagulation time will decrease with increase in applied high pressure, but it increases with increases in pressurization time and coagulation temperature. Pressurization time has the significant effect on the mean coagulation time compared to applied pressure. The effect of various coded values of process variables on the time at inflexion point is shown in Fig. 3. With coagulation temperature fixed, a decrease in applied pressure and increases in pressurization time resulted in an increase in the inflexion time (Fig. 3a). Longer inflexion times were apparent at longer pressures and pressurization time. There was a reduction in the inflexion time as the pressure levels and coagulation temperature increased. The treatment effect on inflexion time appears to be more sensitive at the higher coagulation temperature (Eq. 11).

Figure 3 Surface plots showing the effect of coded values of highpressure variables on mean coagulation time (a) pressure and pressurisation time; (b) pressure and coagulation temperature; and (c) pressurisation time and coagulation temperature.

Equation (12)

The equation shows that inflexion time increases with increase in applied pressure and pressurization time, but it decreases with coagulation temperature. Pressurization time has the most significant effect on inflexion time than applied pressure. Figure 4 shows the response surface plots of the combined effect of coded values of different processing variables on inflexion time.

Figure 4 Surface plots showing the effect of coded values of high pressure variables on inflexion time (a) pressure and pressurisation time; (b) pressure and coagulation temperature; and (c) pressurisation time and coagulation temperature.

Optimum Coagulation Conditions

In order to find out the level of applied pressure (X ₁), pressurization time (X ₂), and coagulation temperature (X ₃) required to attain the minimum lag time, maximum coagulation rate and minimum inflexion time, second order regression equations were fitted with coded values of X ₁, X ₂, and X ₃ by using the experimental values of the response given in Table 2.

(16)

(17)

(18)

The range of optimum values of x ₁, x ₂, and x ₃ at the desired values, i.e., minimum lag time, minimum coagulation time and minimum inflexion time were found out by solving the above equations using Micro Soft Excel Solver. The values of x ₁, x ₂, and x ₃ thus obtained are: applied pressure (x ₁): 0.8; pressurization time (x ₂): 0.627; and coagulation temperature (x ₃): 0.867. The real value (viz., X ₁, X ₂, and X ₃) of these process variables was obtained by using Eqs. (17)—(18) and they are: applied high pressure (X ₁): 280 MPa, pressurization time (X ₂): 47 min and coagulation temperature (X ₃): 52°C. Using optimum ranges of the independent variables, x ₁, x ₂ and x ₃ as obtained above, the values of lag time, mean coagulation time and inflexion time of high pressure treated coagulated acidified milk gel were estimated using Eqs. (16), (17), and (18). The estimated values of the above parameters are given in Table 3.

CONCLUSIONS

High pressure processing significantly affected the coagulation properties of acid cogulated milk. The coefficients obtained from ODR vs. time curve showed that higher-pressure levels and higher temperatures significantly decreased the lag, inflexion time, and coagulation time. A multiple correlation model was developed for each coagulation parameter to relate them to the process variables viz. pressure, pressurization time and coagulation temperature. The optimum values of independent variables corresponding to minimum lag time, maximum coagulation rate and minimum inflexion time was obtained were: high pressure - 280 MPa; pressurization time - 47 min; and coagulation time - 52°C. At this optimum condition of independent variables the lag time, mean coagulation time and inflexion time were 0.0028 min, 3.87 min, and 5.19 min, respectively.

ACKNOWLEDGEMENT

The author is thankful to the Society of Chemical Industry (SCI), UK, for felicitating as Seligman APV Fellow for the year 2007, and for providing financial support to get in hand experience of high pressure machine at The University of Reading, UK.