Determination of Rennet Clotting Time by Texture Analysis Method

In this study, texture analysis method was used for the determination of rennet flocculation time (tfloc) and rennet clotting time (tclot) of rennet-induced reconstitued milk samples with different CaCl2 concentrations. The rennet flocculation time (RFT) and rennet clotting time (RCT) were also determined by using the Berridge test and sensory evaluation. The hardness value versus renneting time curves derived from texture analysis gave a good modified exponential relationship for each CaCl2 concentration and the curves were used to calculate flocculation time and clotting time parameters. It was found that the parameters (tfloc and tclot) appeared strongly correlated with RFT and RCT, respectively. Texture analysis was proved as a suitable method to control the rennet-induced coagulation and determine the rennet clotting time. It was also determined that enrichment of milk with CaCl2 leaded to a decrease in flocculation and clotting times and an increase in rate of clotting and gel hardness.


Introduction
The first stage of cheese manufacture is the conversion of liquid milk to cheese curd. After the addition of chymosin to the milk, there is little apparent reaction for some time and then the milk coagulates rapidly. During this lag phase, the enzyme hydrolyses the κ-casein which stabilizes the casein micelles. This phenomenon, which is the first step of cheesemaking, results from two stages. The enzymatic proteolysis forms the first or primary phase. In this phase, milk-clotting enzymes spli κ-casein at the junction between the para-κ-casein and macropeptide moieties, i.e. in bovine κ-casein, at the Phe 105 -Met 106 bond. When sufficient amount of κ-casein has been hydrolyzed, the destabilized micelles begin to aggregate and this eventually leads to a three-dimensional cheese curd [1,2]. In casein micelles, proteolysis of a small number of κ-casein molecules will have much less effect on the aggregation properties, since the micelles contain many hundreds or even thousands of such molecules. The para-κ-casein produced in the micelles by renneting can only aggregate when the whole micelle is capable of aggregating, and it is this that causes the lag phase before aggregation is observed. Renneted micelles appear to be incapable of aggregating until about 60% -80% of their κ-casein has been destroyed, after which the concentration of micelles capable of aggregating increases rapidly. This behavior can be explained either by the loss of surface charge during renneting or by loss of steric stabilization [1].
The aggregation rate of renneted micelles is unaffected by the concentration of rennet or by the size of the micelles. However, it is very sensitive to the concentration of ionic calcium. It is thought that the ionic calcium does not directly affect the enzymatic phase, although addition of CaCl 2 does reduce milk pH, which accelerates the hydrolysis reaction. However, addition of calcium reduces the rennet coagulation time, even at constant milk pH, and flocculation occurs at a lower degree of κ-casein hydrolysis. Addition of calcium also increases the rate of firming of rennet-induced milk gels and firmness of the gel. This effect of ionic calcium could be explained by the masking of charged groups and the hydrophobicity increase [3].
The most easily detected outcome of chymosin proteolysis and rennet clotting is the visible observation of the presence of flocs in a milk sample in a rotating tube. The time taken for their appearance is defined as the rennet coagulation time, and for the cheese producers interested in the activity of an enzyme preparation, this may be the only quantity of interest [1]. Numerous devices have been developed to study and control of phenomena occurred during the rennet-induced coagulation due to the importance of the curd cutting time on the final cheese quality [4]. Various laboratory techniques have also been described for measuring visco-elastic properties of milk gels as a reference for cheese making; the most widely used are Formagraph and low-amplitude dynamic shear measurements [5]. Payne et al. [6] used a diffuse reflectance technique to predict optimal cutting time as measured by Formagraph on composite milks prepared from varying proportions of cream, skim milk and condensed skim milk. They found that the inflection point of the sigmoidal phase of the diffuse reflactance curve was well correlated with the Formagraph measure of the rennet clotting time. It was compared that the performance of a NIR transmission probe with the Formagraph used skimmed and whole milk and it has found that there was a good correlation between the time to inflection point of the transmission signal and the rennet coagulation time by the Formagraph.
Sharma et al. [7] evaluated on-line measurements of coagulation time and coagulation firmness of renneted milk using a torsional vibration technique, namely a Nametre viscometer. It was defined as the point where complex viscosity became higher than the initial value at rennet addition using a coagulation time parameter.
Turbidity or light scattering methods were used for following early aggregation phase while the gel formation and development are most monitored by rheometer. Each technique suffers from limitations. Light scattering requires a dilute dispersion of particles so that only singly scaretted photons are collected at the detector. Studies using light scattering are thus limited to initial stage of aggregation, where growth of molecular weight or degree of polimerization is obtained as function of reaction time. Rheological measurements suffer from the opposite failing. There, the limitation is instrument-sensitivity and a detectable signal is realized only after the reaction has progressed to a significant extent [1].
Some of the techniques mentioned above have also been used to determine the influence of various factors such as temperature, pH, milk composition and CaCl 2 concentration on the rennet coagulation process.
In most cases, studies were carried out with using techniques mentioned above. There is no research on determination of both of the rennet flocculation and clotting times with using the texture analysis. The objective of this study is to predict both of the flocculation and clotting times with using results of hardness measurements at different CaCl 2 concentrations during the ren-netting.

Rennet Coagulation Experiment
Maxiren 600 ® (DSM Foods, Delft, The Netherlands) with a declared activity of 160 IMCUmL −1 was used for renneting of the reconstitued milk. A solution of rennet was prepared daily by diluting 1 mL of Maxiren 600 ® in 100 mL of deionized water. Rennet solution was added to the reconstitued milk at 32˚C to a final concentration of 0.01% (v/v). Samples were divided into several assays (200 ml each) for preparing renneting time-series (0 -70 min).

Measurement of Gel Hardness
The hardness (N) of viscoelastic rennet gels was determined by means of texture analysis. A texturometer model TA-PLUS (Lloyd Instruments, Fareham, UK) with a 10 N cross-head was used to measure the hardness of rennet gels. The measuring probe consisted of an acrylic cone (diameter 4 cm and 60˚ angle) was thrust into gels in cylindrical containers (200 ml in volume) by the 80% compression depth from surface. A single compression method (one cycle) was applied at a constant crosshead velocity of 2 mm·s −1 . The hardness value of the gels was calculated on line by using the software, Nexygen ® 2.0 software (Lloyd Instruments, Fareham, UK). Exponential relationships between rennetting time (t, min) and hardness value (H, N) have been determined in the rennet gels at different CaCl 2 concentrations. It was found that the curves fitted "modified exponential model" (correlation coefficient, r ≥ 0.98; standard error, SE < 0.006 were found). According to this model, equation derived from the curves was given as such; where H is hardness value (N), t is the time after the addition of rennet (min), H o is the initial hardness value (N), a is the regression coefficient. Four different parameters were extracted from the hardness changes according to renneting time; rennet clotting time (t clot ) corresponding to the time when the Open Access FNS pH values of the reconstitued milk samples were determined with using a pH meter (Sartorius PB-20, Germany).

Statistical Analysis
Each experiment was carried out in triplicate. The data were analyzed statistically by using SPSS for Windows ® software (SPSS Inc., Chicago, IL, USA).

Results
The rennet floculation time (RFT), the time from addition of the rennet to the first formation of visible floccules, was determined according to the method by Berridge [8]. The rennet clotting time (RCT) was determined with sensory evaluation by using a spatula by means of traditionally detection of curd-cutting time.
The hardness values of the samples measured during 70 min after the addition of rennet were shown from Figure  1. It was observed that the hardness was increased to a maximum and it was then kept almost constant for all CaCl 2 concentration during the renneting. There was also a lag phase observed at the samples added CaCl 2 at below of 0.10%. The differences between slopes of the curves and length of the lag phase obtained by different CaCl 2 concentrations, allowed to the determination of the rennet flocculation and clotting times and the explanation of the effect of CaCl 2 on the renneting with using texture analysis method.

Total Protein Content
The total protein content of reconstituted milk was determined by Bradford method [9].

Calcium Ion Activity
Plotting the hardness value as a function of the renneting time gave a good modified exponential relationship (correlation coefficient ≥ 0.98; standard error < 0.006) for each CaCl 2 concentrations. Rennet clotting Calcium ion activity of the reconstitued milk samples was determined with an Ion Analyzer (Orion, Model 407 A, USA) and an ion selective electrode (Sartek, UK).
Rate of clotting values (r clot , N/min) were calculated from the slope of the plot of hardness versus renneting time. r clot values corresponding to the rate of hardness increase as a function of renneting time were presented in Table 1.
It was also shown from Table 2 the rennet flocculation time (RFT) and rennet clotting time (RCT) determined by Berridge method and sensory evaluation, respectively. It was found that the time parameters (t floc and t clot ) derived from the hardness changes according to the renneting time which were in correlation with the RFT and RCT (determination coefficients, R 2 = 0.993 for t floc -RFT line and R 2 = 0.999 for t clot -RCT line). The relationship was validated on the samples at different CaCl 2 concentrations (0.02% -0.12%) (Figure 2).    Table 2. pH of the samples decreased with increasing CaCl 2 concentration of the samples because of the "Lewis acid role of the calcium". The addition of CaCl 2 not only increased the calcium concentration but also reduced the pH of milk, resulting in an increased aggregation rate [10]. It was also reported that the percentage of κ-casein proteolyzed was dependent on milk pH and ionic calcium content [1].
As it is shown from in Figure 1, decreasing of CaCl 2 concentration leads to prolongation of the lag phase of the renneting that represents the preliminary of flocculation stage of para-κ-casein micelles. Both of t floc -t clot and RFT-RCT decreased with increasing CaCl 2 concentration (Table 1 and 2). CaCl 2 has also leaded to an increase in the gel hardness during the coagulation process. The hardness value at 70 min after the addition of rennet (H 70 ) increased with increasing CaCl 2 concentration. Rate of clotting (r clot ) also increased with increasing CaCl 2 concentration ( Table 1).

Discussion
In this study, the milk samples were characterized by different rennet coagulation properties due to CaCl 2 added at different concentration to the reconstituted milk. Different CaCl 2 concentration of rennet gels allowed evaluation of the method on the samples characterized by different rennet flocculation and clotting times. Two different methods were used and compared to determine rennet flocculation and clotting times of the rennet gels. It was showed that the textural analysis allowed determining both of the rennet flocculation and clotting times. There are many techniques in the literature provided to determine or predict individually early aggregation phase and gel formation-development. Initial aggregation phase of rennetting was generally followed by some methods such as using turbidity or light scaretting whereas the gel formation and development was monitored by another method such as rheometer [1]. However, in this study it was found that the hardness measurement (texture analysis) allowed monitoring the both of early phase and curding cutting step during the rennetting via evaluation of the time parameters (t floc and t clot ).
Castillo et al. [11] studied on prediction of both gelation time and cutting time in Cottage cheese-type gels and monitored the coagulation process simultaneously using a light backscatter sensor, a rheometer and a pHmeter. It was developed the models for the prediction of the rheologically defined gelation time using light backscatter and for the prediction of cutting time using light backscatter. It was determined gelation time by using an equation and cutting time by using another equation.
However, in this study one method based on texture analysis during the renneting was provided determination of rennet flocculation and clotting times as a function of hardness. This is confirmed that the texture analysis method was sensitive to all stages of renneting. It was showed that determination of rennet flocculation and clotting times with this method is possible for all given CaCl 2 concentrations. It was exposed that flocculation and clotting times can be determined by using the same equation Besides the determination of the time parameters, this method contributed to the observation of the hardness changes during the renneting and evaluation of influence of CaCl 2 on rennet flocculation and coagulation phases. κ-casein molecules hydrolyse to para-κ-casein and macropeptide during the lag phase of renneting and aggregation phase starts after the sufficient amount of κ-casein hydrolyses [1]. The length of lag phase decreases with increasing calcium ion activity. Above of the 0.08% addition of CaCl 2 , there was no lag phase observed. This result indicates that the CaCl 2 concentration affects the primary phase of renneting. It possibly means that flocculation may occur at a lower degree of κ-casein hydrolysis at higher ionic calcium concentrations.
It is known that the rate of formation and final strength of rennet gels are influenced greatly by calcium [12]. While not completely understood, it has been shown that ionic calcium influence coagulation and gel formation [13,14]. In this study, it was observed that the maximum hardness value of the gels increased and the time needed to reach to the maximum hardness decreased with increasing CaCl 2 concentration (Figure 1 and Table 2). Furthermore, an increase of rate of clotting with increasing CaCl 2 concentration could be explained mainly by charge neutralization of the negatively charged groups on the micelle surface and possibly by the formation of calcium bridges ( Table 1). It is reported that enrichment of milk with calcium leads to an increase in casein aggregation rate during the coagulation process, consequently decreased the flocculation time, and an increase in firmness of rennet gel [4]. It was shown from Table 1 and Table 2, rennet flocculation and clotting times decreased with increasing CaCl 2 concentration. Sample F (0.12% CaCl 2 ) has reached maximum H 70 values while sample A (0.02% CaCl 2 ) has minimum H 70 value during the renneting ( Table 1). In this study, it was shown that the CaCl 2 affects on the both of the primary and secondary phases of renneting with using the texture analysis method.

Conclusion
In this study, it was indicated that the texture analysis could be used accurately for determination of rennet clotting time during the renneting of milk. The obtained hardness values were plotted against the renneting time and evaluated for calculation of rennet flocculation and clotting times. It was found to be important of texture analysis method (hardness measurement) at the rennet-induced samples with different CaCl 2 concentrations in terms of prediction both of the flocculation time and clotting time. Furthermore, it was shown that CaCl 2 concentration which is the one of the main parameters in the renneting process was affected on the primary and secondary phases of renneting with using the texture analysis method.

Acknowledgements
The author expresses her gratitude to Prof. Dr. Yaşar Kemal Erdem for his permition for using the texturometer and to Miss Elif Avcı for her helpings.