THE INFLUENCE OF VARIOUS FACTORS ON MILK CLOTTING TIME

The influence of pH (6.5 and 5.8), amount of added CaCl2 (0, 200 and 400 mg/l)), coagulation temperature (30oC and 35oC) and heat treatment of milk (65oC/30 min and 87oC/10 min) on the rate of rennet induced milk coagulation (s) were investigated. The time (s) from rennet addition to onset of gelation (as indicated by the first visible floccules) was measured. The milk samples heat-treated at 87oC/10 min, with 400 mg/l added CaCl2, which were coagulated at 35oC and pH 5.8, coagulated 23.28-fold faster than the same samples without added CaCl2, which were coagulated at 30oC and pH 6.5. The results of investigations related to the influence of particular coagulation factors on the coagulation rate of heat-treated milk showed that at pH 6.5 the most pronounced influence was demonstrated by the amount of Ca and temperature of coagulation. At pH 5.8, different amounts of Ca and used temperatures of coagulation did not influence coagulation rate regardless of the used heat treatment of milk. The influence of used heat treatment of milk was particularly pronounced during coagulation of samples without added CaCl2 that coagulated at 30oC and pH 6.5. The used heat treatment of milk practically did not influence the milk coagulation rate at pH 5.8. The greatest influence on milk coagulation rate was showed by pH. This influence was the most marked in coagulation of samples in which the coaggregates were formed, regardless of the amount of added Ca and used coagulation temperatures.


I n t r o d u c t i o n
Milk coagulation and formation of rennet-induced gel (coagulum) is the most important and the most sensitive process in the production of the rennet curd cheese varieties.It was principally manifested through the action of numerous factors, which, on the other hand, control both biochemical and physico-chemical processes during coagulation and directly influence rheological characteristics of rennet-induced casein gel.
Milk coagulation induced by proteolytic enzymes was the oldest technological process in the cheesemaking ( It is well known that milk coagulates with the extract of the stomach of young milk-fed calves, kids or lambs, which is usually called rennet. Milk coagulation could be presented as two-stage process that involves primary phase, i.e. limited hydrolysis of κ-casein, and secondary phase when hydrolyzed casein micelles cross-link in the presence of Ca 2+ to form gel (D a l g l e i s h , 1983, 1986, 1993, D j o r d j e v i ć , 1987, G a v a r i ć , 1988, S c o t t , 1986).
Numerous factors influence primary and secondary coagulation phase as well as rheological properties of formed gels.The most essential factors are: casein concentration, milk pH value, type and concentration of enzymes, concentration of Ca 2+  Nevertheless, milk coagulation rate, activity of enzymes used for coagulation in cheesemaking as well as rheological properties of rennet-induced casein gel greatly depend on previously applied heat treatment of milk (D a l g l e i s h , 1990, G u i n e e , et al., 1997, M a ć e j et al., 1996, M a r s h a l l , 1986, S i n g h and F o x , 1988).
Investigations of numerous authors showed that longer milk heating above 70 o C leads to the formation of chemical complex between κ-casein and whey proteins (milk proteins coaggregates) ( It was also concluded that coaggregates formation leads to a lesser sensitivity of casein to proteolytic enzymes even if the concentration of Ca 2+ is greater than in raw milk.
This problem requires additional investigations, which could solve numerous problems associated with coagulation of milk in which coaggregates were formed, with the aim to create optimum coagulation conditions for the use of heat treated milk in cheese production.Also, this is a way to increase cheese yield due to a greater utilization of whey proteins which are lost with whey in traditional cheese production.
The aims of this investigation were to investigate the influence of pH, amount of added CaCl 2 , coagulation temperature and applied heat treatment on the clotting time, as well as to maintain optimum conditions for the production of gel with good rheological properties from milk in which coaggregates were formed.

Material and Method
Raw milk gained from Dairy Beograd "AD IMLEK" Padinska Skela was used for investigations.The amount of 200 ml per sample was used.The experiments were performed at the Department of Dairy Technology -Faculty of Agriculture, Belgrade.
Clotting time (s) indicated as time from rennet addition to the formation of the first visible floccules were measured visually.The influence of the following factors were investigated: -Milk pH value (6.5 and 5.8) -Amount of added CaCl 2 (0, 200 and 400 mg/l) -Applied heat treatment (65ºC/30 min and 87ºC/10 min) -Coagulation temperature (30ºC and 35ºC) Milk heat treated at 65ºC/30 min was designated as Control sample, while milk heat treated at 87ºC/10 min was designated as Experimental sample.
The 10% lactic acid was used to modulate milk pH value, while 20% solution of CaCl 2 was used for modulation of CaCl 2 amount.Liquid rennet "Biopak", declared activity 1:5000, was used for coagulation.
The following analyses of raw milk were performed: a) Determination of total solids by standard drying method at 102±2ºC, (I D  All experiments were repeated 5 times.Statistical analysis was performed.All data for the investigated parameters are shown as mean values.Also, analyses of variance for all data were performed (Standard deviation and coefficient of variation).

Results and Discussion
Results of investigation are shown in Tables 1 According to the results presented in Table 2. it can be seen that control samples, coagulated at 30ºC and pH 6.5, without added CaCl 2 had clotting time 2393 s.Control samples with added 200 and 400 mg/l of CaCl 2 , respectively, coagulated 1.59-fold and 2.19-fold faster, which indicate that clotting times were reduced by 891.20 s and 1299 s, respectively.
Conversely, experimental samples had lengthened clotting time.Experimental samples without added CaCl 2 had clotting time 5205 s that was 2.17-fold longer than in control samples.It can also be concluded that addition of CaCl 2 decreased clotting time, but even then clotting times were longer than clotting time of control samples.
Clotting time of experimental samples with added 200 mg/l of CaCl 2 was 2537.60 s and was 2.05-fold shorter than for samples without added CaCl 2 .On the other hand, clotting time was 1.69-fold and 1.06-fold longer than for control samples with added 200 mg/l of CaCl 2 and without added CaCl 2 , respectively.
Clotting time of experimental samples with added 400 mg/l of CaCl 2 was 1567.20 s and was 2.05-fold and 1.62-fold shorter, respectively, than for samples without added CaCl 2 and samples with added 200 mg/l of CaCl 2 .
It can be concluded that milk in which coaggregates were formed when 400 mg/l CaCl 2 was added coagulate 1.53-fold faster than control samples without added CaCl 2 .In contrast, clotting time was 1.04-fold and 1.43-fold longer compared with control samples with added 200 and 400 mg/l of CaCl 2 , respectively.
At the pH 6.5 clotting times of both control and experimental samples decreased by raising coagulation temperature to 35ºC.
Control samples without added CaCl 2 had clotting time 2245.20 s.Control samples with added 200 mg/l of CaCl 2 had clotting time 1324.40s that was 1.69fold shorter.Control samples with added 400 mg/l of CaCl 2 had clotting time 927.20 s which was 2.42-fold and 1.43-fold shorter, respectively, than for samples without and with added 200 mg/l of CaCl 2 .
The influence of coagulation temperature was very pronounced, because the samples without added CaCl 2 that coagulated at 30ºC had 1.06-fold longer clotting time than samples that coagulated at 35ºC.Control samples with added 200 and 400 mg/l of CaCl 2 that coagulated at 30ºC had 1.13-fold and 1.18-fold longer clotting time, respectively, than the same samples that coagulated at 35ºC.
The increase of coagulation temperature to 35ºC influenced decreasing of clotting time of experimental samples, which was still longer than in control samples, regardless of the concentration of Ca 2+ .
Clotting time of experimental samples without added CaCl 2 was 3599.60 s.Clotting time of samples with added 200 and 400 mg/l CaCl 2 was 1829 s and 1142.80 s, respectively, and was 1.97-fold and 3.15-fold shorter than for samples without added CaCl 2 .
Clotting time of experimental samples without and with added 200 and 400 mg/l CaCl 2 , respectively, was 1.60-fold, 1.38-fold and 1.23-fold longer than for control samples with the same concentration of Ca 2+ .Decreasing of pH value from 6.5 to 5.8 had important influence on milk clotting time.The influence of pH value and other factors on milk clotting time is shown in Fig. 1.Clotting time of control samples without and with added 200 and 400 mg/l of CaCl 2 that coagulated at 30ºC was 426 s, 354.20 s and 314.60 s, respectively.
At pH 5.8 and 30ºC control samples without and with added 200 and 400 mg/l of CaCl 2 , respectively, coagulated 5.62-fold, 4.24-fold and 3.48-fold faster than the same samples at pH 6.5.
Unexpectedly, experimental samples had shorter clotting time than control samples, regardless of the concentration of Ca 2+ .Clotting time of experimental samples without added CaCl 2 was 416 s and was 1.02-fold shorter than clotting time of control samples.Samples with added 200 and 400 mg/l of CaCl 2 had 1.09fold and 1.04-fold shorter clotting time.
Similar trend was observed when coagulation temperature was increased to 35ºC.Milk samples in which coaggregates were formed showed greater sensibility to rennet than control samples, apart from the amount of added CaCl 2 .
Experimental samples without added CaCl 2 coagulated 1.08-fold faster than control samples, while samples with added 200 and 400 mg/l of CaCl 2 , respectively, coagulated 1.12-fold and 1.07-fold faster than control samples with the same amount of added CaCl 2 .
All aforementioned showed that clotting time was more or less influenced by the investigated coagulation factors.The influence of these factors was more pronounced for samples of milk in which coaggregates were formed.This is confirmed by the fact that clotting time of milk samples heat-treated at 87ºC/10 min, with added 400 mg/l of CaCl 2 that coagulated at pH 5.8 and 35ºC, was 23.28-fold shorter compared with the same samples without added CaCl 2 that coagulated at pH 6.5 and 30ºC.Clotting time of control samples, on the other hand, with added 400 mg/l of CaCl 2 that coagulated at 35ºC and pH 5.8 was 10.04-fold shorter compared with control samples without added CaCl 2 , which coagulated at 30ºC and pH 6.5.
The above discussed facts opened new questions, which could be classified as follows: 1) Which of the investigated factors have the greatest influence on clotting time; 2) To which extent is interaction among factors significant; 3) Interaction between which factors provides the most pronounced effect on clotting time; 4) How to explain lesser susceptibility of casein from heat-treated milk to rennet; 5) Is it possible to find out an optimal combination of factors to form casein gel with good rheological characteristics?Tables 3., 4., 5., 6. and 7. give analysis of results with drawn conclusions that answer the above posed questions.
Results shown in Table 2. and Fig. 1. categorically indicate cumulative effect of pH, concentration of Ca 2+ , coagulation temperature and applied heat treatment on clotting time.
The influence of investigated parameters on index of milk clotting time (IMCT) is shown in Table 3 Clotting time of samples heat-treated at 87ºC/10 min without added CaCl 2 that coagulated at 30ºC and pH 6.5 was designated by index 100%.
From the results shown in Table 3. we can see cumulative effect and interaction among investigated parameters on IMCT.IMCT of control samples was smaller by 54.03% than IMCT of experimental samples that coagulated under the same conditions.
The differences were less pronounced for both milk in which coaggregates were formed and control milk when concentration of Ca 2+ was increased.IMCT of experimental samples with added 400 mg/l of CaCl 2 that coagulated at 30ºC and pH 6.5 was 30.11%, while control samples under the same coagulation conditions had IMCT 21.02%.Also, it is clearly evident from results that temperature increase induced IMCT decrease.Experimental samples with 400 mg/l added CaCl 2 that coagulated at 35ºC and pH 6.5 had IMCT 21.96%, while control samples under the same conditions had IMCT 17.81%.
As can be seen from Table 3., the decrease of pH value from 6.5 to 5.8 leads to the more pronounced decrease of IMCT.Also, experimental samples, regardless of the amount of added CaCl 2 and applied coagulation temperature, had smaller IMCT than control samples.It can be concluded that casein from heattreated milk showed increased susceptibility to rennet under these coagulation conditions.At pH 5.8, the influence of Ca 2+ and coagulation temperature on the IMCT was smaller than at pH 6.5.As can be seen in table 4., the influence of Ca 2+ was more pronounced at pH 6.5 and coagulation temperature of 30ºC for milk samples in which coaggregates were formed, therefore IMCT decreased to 48.75% and 30.11%, respectively, when 200 and 400 mg/l of CaCl 2 was added.Also, the influence of added Ca 2+ is evident when milk samples in which coaggregates were formed coagulated at 35ºC, so IMCT was 50.81% and 31.75%,respectively, when 200 and 400 mg/l of CaCl 2 was added.
The influence of Ca 2+ was less pronounced for control samples than for experimental samples, regardless of the used coagulation temperature.Compared with control samples without added CaCl 2 , IMCT of control samples with 200 and 400 mg/l of added CaCl 2 was 62.76% and 45.72%, respectively.However, at pH 5.8, Ca 2+ had only insignificant influence.To be precise, at pH 5.8, clotting time was the shortest, while concentration of Ca 2+ , coagulation temperature and applied heat treatment play insignificant role.This is confirmed by the results shown in Tables 2. and 3., which show that clotting time and IMCT are manifold shorter at pH 5.8.
The influence of coagulation temperature on IMCT is shown in Table 5.
T a b .As can be seen from the results, at pH 6.5 the influence of coagulation temperature was noticeably less pronounced on control than on experimental samples.At 35ºC, IMCT of control samples without added CaCl 2 was only by 6.18% smaller than IMCT of control samples that coagulated at 30ºC.Under the same conditions, IMCT of experimental samples was even by 30.84% smaller.The influence of coagulation temperature on the IMCT of control samples increased with increase of Ca 2+ .For example, IMCT decreased by 11.81% and 15.25%, respectively, with the addition of 200 and 400 mg/l of CaCl 2 .The influence of coagulation temperature, on the other hand, was less obvious for samples heat-treated at 87ºC/10 min with added 400 mg/l of CaCl 2 .Experimental samples without added CaCl 2 had smaller IMCT by 30.84%, while at 35ºC IMCT of experimental samples with added 200 and 400 mg/l of CaCl 2 , respectively, was by 27.93% and 27.08% smaller.It can be concluded that coagulation temperature has insignificant influence on IMCT of control samples with added 200 and 400 mg/l of CaCl 2 , while coagulation temperature has more pronounced influence on IMCT of milk samples heat-treated at 87ºC/10 min.
Also, the influence of coagulation temperature on control samples was increased when pH decreased from 6.5 to 5.8 in spite of added CaCl 2 .At 35ºC, control samples without added CaCl 2 had smaller IMCT by 19.16% than the same samples that coagulated at 30ºC.Control samples with added 200 and 400 mg/l of CaCl 2 had smaller IMCT by 20.33% and 24.22%, respectively, which indicates more pronounced decrease of IMCT at pH 5.8 than at 6.5.
The influence of coagulation temperature on IMCT was greater for milk samples in which coaggregates were formed, while different concentrations of Ca 2+ practically did not have any influence on IMCT.
The results show that for milk samples heat-treated at 87ºC/10 min, the influence of coagulation temperature was less pronounced at pH 5.8 than at pH 6.5.
The results in Table 6.show the influence of heat treatment on IMCT.
T a b .The results demonstrate that at pH 6.5, IMCT was greatly influenced by the applied heat treatment.IMCT of control samples without added CaCl 2 that coagulated at 30ºC was by 54.03% smaller than that of experimental samples under the same conditions.The influence of heat treatment was less pronounced when 200 and 400 mg/l of CaCl 2 was added, although differences were still significant, so IMCT of control samples was by 40.82% and 30.19% smaller.
The differences among IMCT of control and experimental samples were significantly reduced when coagulation temperature was increased from 30ºC to 35ºC.Control samples without added CaCl 2 had smaller IMCT by 37.63% than experimental samples under the same coagulation conditions.Smaller differences are (27.59%and 18.87%, respectively) when 200 and 400 mg/l of CaCl 2 was added.
The pH reduction from 6.5 to 5.8 did not influence IMCT of control and experimental samples at 30º.The difference between IMCT of control and experimental samples was 2.40%, 8.72% and 4.31%, respectively, for samples without and with added 200 and 400 mg/l of CaCl 2 .Under these conditions, heattreated milk samples had smaller IMCT than pasteurized samples.The differences are greater when coagulation temperature was increased from 30ºC to 35ºC.
All aforementioned indicate that under these coagulation conditions susceptibility of casein from heat-treated milk to rennet was regenerated.
Table 7. and Figures 2a. and 2b.show the influence of pH on the alteration of IMCT.
The results show that decrease of pH from 6.5 to 5.8 leads to manifold reduction of IMCT, but this influence is more significant for the heat-treated milk samples.At pH 5.8 control samples without added CaCl 2 that coagulated at 30ºC had smaller IMCT by 82.20% than at 6.5.The reduction of IMCT was 76.42% and 71.24%, respectively, for control samples with added 200 and 400 mg/l of CaCl 2 (Fig. 2a).The influence of pH on IMCT of samples in which coaggregates were formed is more pronounced as Fig. 2b.shows.At pH 5.8, IMCT of experimental samples without added CaCl 2 that coagulated at 30ºC was by 92.01%smaller than at 6.5.The reduction of IMCT was 87.16% and 80.76%, respectively, when 200 and 400 mg/l of CaCl 2 was added.However, when milk samples coagulated at 35ºC, decrease of pH from 6.5 to 5.8 induced faster coagulation, i.e.IMCT of control samples decreased in spite of Ca 2+ concentration.
According to the less pronounced differences of experimental samples, it can be concluded that higher coagulation temperature did not influence the change of IMCT.
The results of our investigation agree well with the results of other authors.G u i n e e et al., (1997) assumed that milk heat treatment impaired rennet coagulation properties of milk, increased clotting time and decreased curd-firming rate.M a ć e j (1989) concluded that heat treatment at 87ºC/10 min multiplies coagulation time.M a ć e j et al., (1997) investigated the influence of heat treatment on the milk coagulation rate and detected lesser sensitivity of casein to rennet even when 200 and 400 mg/l CaCl 2 was added to heat-treated milk, which, on the other hand, increased Ca 2+ concentration to the level greater than in raw milk.When milk heat-treated at 95ºC/20 min and raw milk are mixed in the ratio of 1:1, susceptibility of casein to rennet was regenerated and clotting time of this mixture was the same as clotting time of milk pasteurized at 65ºC/30 min.M a r s h a l l (1986) and S i n g h and F o x (1988) considered that severe heat treatments of milk have more influence on the secondary than on the primary phase of coagulation.In contrast, F o x (1986) assumed that both the primary and secondary phases of coagulation are retarded in the milk heat-treated to the temperatures that influence whey protein denaturation and coaggregate formation via thiol-disulphide interchange, which makes Phe-Met bond (rennet-susceptible bond) less susceptible to rennet.The same opinion has D a l g l e i s h (1990) who found out a linear increase of milk clotting time with heating of milk to 85ºC and 90ºC.
M a ć e j et al., (1989) investigated the influence of various factors on milk coagulation rate at different coagulation temperatures and concluded that pH had the greatest influence on the coagulation rate and after that concentration of Ca 2+ , while coagulation temperature had the smallest influence.P u d j a (1992) and G u i n e e et al. (1992) found that protein concentration and pH had the greatest influence on the coagulation rate of milk in which coaggregates were formed.P u d j a (1992) found that milk heat-treated at 100ºC/2 min did not coagulate during 2 hours, but when UF milk (concentrated to the level 5) was added to increase protein concentration, coagulation completed in 20 min, which agree well with the clotting time of a control sample.
According to v a n H o o y d o n k et al. (1987) lowering of pH of heat-treated milk to 6.0 decreased clotting time.By the addition of 4mM CaCl 2 at constant pH value, clotting time of heat-treated milk was decreased but coagulation rate of untreated milk is unattainable.B r i n g e and K i n s e l l a (1986) regarded concentration of Ca 2+ as a significant factor of coagulation rate and found out that coagulation begins at a lower level of κ-casein hydrolysis (30% instead of 90%) if concentration of Ca 2+ was increased from 3 mM to 60 mM.S i n g h et al. (1988) concluded that heat-treated milk, after acidification and subsequent neutralization to pH 6.6, had shorter clotting time than untreated milk.

C o n c l u s i o n
According to the aforementioned, it could be concluded that: Cumulative effects of investigated parameters had the greatest influence on milk clotting time.Experimental samples with added 400 mg/l of CaCl 2 that coagulated at 35ºC and pH 5.8 had 23.28-fold shorter clotting time or had lower index of milk clotting time by 95.70% than the same samples without added CaCl 2 that coagulated at 30ºC and pH 6.5.This indicates a significant cumulative interaction among investigated parameters.
The results of investigations related to the influence of tested factors on clotting time showed that at pH 6.5 both Ca 2+ and coagulation temperature had particular influence on clotting time of milk in which coaggregates were formed.At pH 5.8 different concentrations of Ca 2+ and applied coagulation temperatures did not have great influence on the reduction of IMCT, regardless of the used heat treatment of milk.
The influence of applied heat treatment of milk was particularly marked when samples without added CaCl 2 coagulated at 30ºC and pH 6.5.The variations were lesser, but still obvious, when concentration of Ca 2+ and coagulation temperature were increased.However, at pH 5.8, applied heat treatment did not have any influence on IMCT.According to this fact, it could be concluded that casein became more sensitive to rennet under these coagulation conditions.
A b d E l -S a l a m et al., 1993, D a l g l e i s h , 1979, 1986, 1990, D j o r d j e v i ć , 1987, D j o r d j e v i ć and C a r i ć , 1970, 1974, E r n s t r o m and W o n g , 1974, G r e e n and M o r a n t , 1981, H i l l et al., 1974, M a ć e j et al., 1998, M e h a i a and C h e r y a n , 1983, M u l v i h i l l and F o x , 1979, P e j i ć , 1956, P u d j a et al., 1995, S h a l a b i and F o x , 1982).
and coagulation temperature (B a n k s and M u i r , 1984, D a l g l e i s h , 1983, 1993, F o x , 1987, G a v a r i ć et al., 1989, L o p e z et al., 1998, P u d j a et al., 1996).
D j o r d j e v i ć et al., 1987, E l f a g m and W h e e l o c k , 1978a,b, E u b e r and B r u n n e r , 1982, H a q u e and K i n s e l l a , 1988, H a r t m a n and S w a n s o n , 1965, J a n g and S w a i s g o o d , 1990, K i r c h m e i e r et al., 1985, L o n g et al., 1963, M a ć e j , 1983, 1989, M a ć e j and J o v a n o v i ć , 1998, M c K e n z i e et al., 1971, M o r r et al., 1962, P u d j a et al., 1995, P u r k a y a s t h a et al., 1967, S i n g h and F o x , 1987, S m i t s and van B r o u w e r s h a v e n , 1980).
F S t a n d a r d 4A:1982) b) Determination of total nitrogen matter by Kjeldahl method, (IDF s t a n d a r d 20A:1986) c) Titratable acidity determination according to Soxlet-Henkel, (C a r i ć et al., 2000) d) Determination of pH with pH-meter Sentron 1001 e) For density determination lactodensimeter was used, (C a r i ć et al., 2000)

Fig. 2 .
Fig. 2. -Index of milk clotting time (IMCT) as influenced by pH value 1.A b d E l -S a l a m , M.H., A l i c h a n i d i s , E., Z e r f i r i d i s , G.K. (1993): Domiati and Feta Type cheeses in Cheese: chemistry, physics and microbiology.Volume 2. Major cheese groups.Second edition.Chapter 11, 301-355.Ed. by Fox, P. F., Chapman &Hall, London and New York.2. B a n k s , J.M., M u i r , D.D. (1984): Coagulum strength and cheese yield.Dairy Ind. Internat.49 (9), 17-19, cont.on pages 21, 36.3. B r i n g e , N.A., K i n s e l l a , J.E. (1986): Influence of calcium chloride on chymosin-initiated coagulation of casein micelles.J. Dairy Res.53 (3), 371-379.4. C a r i ć , M ., M i l a n o v i ć , S ., V u c e l j a , D .( 2000): Standardne metode analize mleka i mlečnih proizvoda.Prometej, Novi Sad.
. and 2. The quality parameters of milk used for investigations are shown in Table1.
., where the longest clotting time is designated as 100%.

Table 4 .
shows the influence of Ca 2+ on IMCT.
5. -Index of milk clotting time as influenced by coagulation temperature 6. -Index of milk clotting time as influenced by heat treatment of milk