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Effect of pH, Calcium, and Heat Treatment on Curd Tension of Casein Fraction Fortified Skim Milk1
Effed of pH, Calcium, and Heat Treatment on Curd Tension of Casein Fraction Fortified Skim Milk 1 D. L. SCHULTZ and U. S. ASHWORTH Department of Food Science and Technology Washington State University, Pullman 99163 Abstract
tors appear responsible for curd tension. However, pH, calcium concentration, and casein content are major factors affecting curd strength. Casein was considered a pure protein until electrophoretic patterns revealed three main components: a-, fl-, and y-casein in order of decreasing mobilities (10). Electrophoretie analysis of whole casein indicates that about 55% is a-casein, 30% fl-casein, and 15% minor components (9, 11). Because curd tension is dependent upon casein content, the question of the influence of individual casein fractions on curd character arises. This study investigated the effect of fortifying skim milk with either a- or fl-casein and measured the effect on rennet curd tension. Heat treatment effects on the casein fraction fortified skim milk were also studied by the curd tension method.
The effect of a- or/3-casein fortification (12 to 28% above control at 3.3% total protein) on skim milk curd strength was studied. After addition of 1 N hydroehloric acid to pH 6.0, a-casein fortified skim milk produced a weaker curd than /~-casein fortification. With added calcium chloride (.01 M), both fractions formed a firmer curd with a-casein slightly stronger than fl-easein. Alpha and fl-easein fortified skim milk cultured with Streptococcus lac~is had similar curd tension within a pH range of 5.20 to 5.85. Calcium addition (.01 M) increased a-casein fortified skim milk curd tension but had a depressing effect on fl-casein as compared to their respective curd strengths without added calcium. However, as pH approached 5.20, E-casein curd strength increased faster per pH unit than that of the a-casein fortified samples. Heat treatments within an effective heat range of .05 to .42 (holding time 10 min) allowed a-casein fortified skim milk to form a stronger curd than that fortified with E-casein. When compared to unfortified skim milk, the greatest difference in curd tension between the a- and fl-casein fortifications was at effective heat .42. Alpha casein curd tension was reduced nearly three times as fast as E-casein when skim milk fortified samples were heated at .42 in the presence of .005 M calcium chloride addition.
Experimental Procedure
Introduction
Factors influencing rennet curd strength have long been investigated. Because of the complex physico-chemical systems, many facReceived January 16, 1974. 1 Scientifie Paper No. 4178, Washington Agricultural Experiment Station, Pullman, Project No. 1809.
Casein preparation. Pooled pasteurized skim milk was diluted with two volumes of distilled water and by slowly adiusting the pH to 4.6 with 1 N HC], isoelectric casein was obtained. The urea method, Hipp et al. (5), was used to prepare whole a- and/3-casein; all fractions were precipitated once and washed five times with distilled water to remove any traces of urea. Sample preparation. Skim milk or 9% solidsnot-fat (SNF) reconstituted milk powder was fortified with sufficient casein fraction to increase the protein content 12 to 28% above a base of 3.3% total protein. To hasten casein fraction dispersion, the fortified sample was placed in a Waring blender and blended 2 rain. After dispersion the sample was divided into two 170-ml lots; one received no added CaCI2 while the other received 1.7 ml of 1 M CaCI2 (.01 M addition). After allowing about 3 h for calcium equilibration, the 170-m] lots were divided into three 50-ml samples; all samples were adiusted to pH 6.0 by the addition of 1 N HC1. Unfortified skim milk with and without added GaClz served as controls. Curd tension procedure. Curd strength was
992
993
CURD TENSION OF CASEIN FRACTIONS
measured by the method of Ashworth and Nebe (2); however, 50-ml samples were used to facilitate additional test trials. After sample tempering (33.3 C), 1 ml of 2% rennet ( 2 / 100 dilution of commercial rennet) was added. To ensure adequate mixing, the sample was poured back and forth twice into an empty beaker, covered and placed in a 33.3 C water bath for 30 min. Curd tension was read with a Cherry-Burrell Curd Tension Meter. Curd tension values were computed from the average of three sample readings. The same procedure was followed to determine the effect of heat treatment on curd tension of skim milk fortified with t~- or fl-casein, Samples were heated in an open water bath with close surveillance of come-up, holding, and cooling time. All heat treatments consisted of 10 rain holding and rapid cooling to below 62,8 C. Upon cooling to 33.3 C, rennet was added, and curd tension values were determined after 30 min. Because come-up and cooling time varied, effective heat values (E) were calculated for comparing time-temperature treatment of milk as described by Burton (3), and modified by Ashworth and Nebe (2). The ~z- and/3-casein preparations were electrophoreseed in Veronal Buffer (pI-I 8.6) containing 2.5 M urea to measure fraction purity (9, 12). Only trace amounts of fl-casein were in the s-fraction and trace amounts of tz-casein in the fl-fraction. Protein determination. Acid Orange-12 dye binding was used to determine percent protein in the milk samples (1). The amount of unbound dye was measured with a Beckman B Spectrophotometer containing a flow-through euvette (light path .75 mm) at 475 ran. The percentage of protein was read from a standard curve based o n Kjeldahl analysis of sim-.'lar samples. Calcium determination. After measurement of curd tension, the whey was separated from the eoagulum by filtration through Whatman No. 1 filter paper. A 5-ml aliquot was removed ~rom the filtrate and titrated with Versene (7). By determining the molar concentration of calcium in the whey sample, the amount of ca]eium retained in the curd was calculated by difference. Results and Discussion
Effect of whole casein [ortiI~eation on skim milk curd tension. Before the urea fractionation of whole acid precipitated casein (APC), fresh skim milk was fortified with APC and varying CaC12 added. Increased concentrations of casein and CaClz favor strong curd charae-
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Fic. 1. Effect of protein concentration and calcium addition on rennet curd tension (pH 6.0, 33.3 C for 30 min). × = skim milk; (~) = skim milk + whole casein; • --- skim milk + whole casein + Ca(.01 M); A = skim milk + whole casein + Ca(.02 M). ter (2, 4, 13); Fig. 1 supports these relationships up to 4.2% protein. A 4 to 5% increase in total milk protein content (without additional calcium) increased curd tension 25 to 30%. However, as more protein was added to the system, a corresponding increase in curd tension was not observed; therefore, calcium is the limiting factor. (APC is devoid of calcium salts due to the isoelectrie method of preparation. ) The addition of CaC12 to the casein fortified milk system had a pronounced effect on rennet curd tension. Increased protein content of 18 to 20% raised curd tension almost 50%. However, the results show that too much calcium may actually decrease curd tension. There is an optimal percent of calcium which will yield maximum curd tension values. 34 /% /%
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FIc. 2. Molar concentration of calcium in rennet whey. X = skim milk; O -----skim milk + whole casein; • ---- skim milk + whole casein .t_ Ca(.01 M); A = skim milk + whole casein + Ca( .02 M ). JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 9
994
SCHULTZ AND A S H W O R T H
Versene titration of the whey sample sup-
ports the fact that curd strength is not a function of any single factor (2). Fig. 1 shows calcium addition increased curd tension while Fig. 2 reveals that titratable whey clacium is relatively constant over the range of percent protein fortification, with or without added calcium. Therefore, within the given protein range, it appears that constant calcium was retained in the curd regardless of its protein concentration while the excess was soluble in the whey.
Effect of alpha or beta casein on curd strength o~ skim milk. Reconstituted milk powder (9% SNF) was fortified with a- or fi-casein isolated by urea fractionation of APC; this treatment increased total milk protein content 12 to 28%. Each set of fortified samples received sufficient CaCle to increase the milk calcium concentration by .01 M. After rennet curd tension, the whey samples were titrated with Versene, and the molar concentration of calcium was calculated. Fig. 3 and 4 show the results of this experiment. Ordinate values are expressed as Curd Tension/% Protein; plotting curd tension values on a unit protein basis tends to eliminate the effect of variable protein content. We define this as specific curd tension
(SpCT). Without additional CaC12 (Fig. 3), increasing isoelectric casein fractions produced a decrease in SpCT; the a-casein fortit~cation yielded a softer curd than t-casein throughout the protein range. The t-casein curve results are comparable to the skim milk control until 4.0% protein is reached. With the addition of 40 35 =
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Fro. 3. Effect of ~- or p-casein fortification on rennet curd tension of reconstituted skim milk (pH 6.0, 33.3 C for 30 rain). × = skim milk; O = skim milk q- ~-casein; • = skim mi:k -b a-casein q- Ca(.01 M); /~ = skim milk -t- pcasein; ,A, = skim milk q- p-casein q- Ca(.01 M). JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 9
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Fro. 4. Molar concentration of calcium in rennet whey. × = skim milk; O = skim milk + acasein; • : skim milk + a-casein + Ca(.01 M); A = skim milk + p-casein; ~ = skim milk + g-casein + Ca(.01 M). CaClz, curd tension of both the a- and t-casein fortifications increased; the a-casein produced a slightly stronger curd than the t-casein. Filtered rennet whey contained .015 to .017 M calcium when none was added and .020 to .023 M with .01 M added calcium (Fig. 4). This suggests that about 50% or 5 mM of the added calcium was retained in the curd whether a- or t-casein was used for fortification.
Effect of culture activity on curd strength of skim milk fortified with alpha or beta casein. Jen and Ashworth (6) found rennet curd tension of reconstituted milk (9% SNF) increased until pH 5.8 with the addition of an acid or a lactic starter, but lowering pH by means of the starter gave more uniform curd tensions below pH 6.0. The present experiment was designed to test the effect of culture activity on the curd tension of a- or t-casein fortified milk. Reconstituted milk powder (9% SNF) was fortil~ed with a- or /3-casein (total protein 3.9%) plus sufficient 1 M CaC12 to increase milk calcium .01 M. Lactic starter (3%) was added to each sample and incubated at 33.3 C for 2 h. Following incubation, rennet was added (final dilution of 1:9_500) and curd tension measured 30 rain later; the final pH value was obtained immediately after curd tension determination. Variable pH between sample sets was a hmction of culture activity. Whey samples were collected and titrated for calcium content. Fig. 5 shows SpCT plotted against final pH values. With a decrease in pH due to starter activity (5.9 to 5.2), all curves show progressively firmer curd production. The skim milk cgntrol curd tension levels off at pH 5.5, then
CURD T E N S I O N OF CASEIN FRACTIONS
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Fro. 5. Effect of culture activity on rennet curd tension of a- or p-casein fortified reconstituted skim milk (33.3 C for 30 rain). × -- skim milk; C) = skim milk + a-casein; • = skim milk + a-casein + Ca(.01 M); A = skim milk + /~easein; , ~ = skim milk + /8-casein + Ca(.01 M). drops slightly. The a - and t-casein fortified skim milks show lower SpCT values than the control skim milk until pH 5.6 to 5.7; below this pH, the a- and t-casein fortifications had similar curd tensions but higher than control. Calcium addition decreased fi-casein curd tension and increased a-casein curd tension with respect to skim milk and corresponding fractions Without added calcium. However, with decreasing pH, t-casein p'.us calcium produced a more rapid increase in curd tension per pH unit than ~casein plus added calcium, although
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Fro. 6. Molar concentration of calcium in rennet whey. X = skim milk; C) = skim milk + acasein; • = skim milk + a-casein + Ca(.01 M); A = skim milk + B-casein; , ~ = skim milk + B-casein + Ca(.01 M).
995
t-casein fortified wheys revealed similar amounts of whey calcium (Fig. 6). This was expected because calcium left the curd as the isoelectrie point of casein was approached. Within the pH range 5.6 to 5.2, the three curves are nearly linear with 1.5 to 2.0 mM soluble calcium being released from the caseinate complex per .1 unit of p H change. The a- and t-casein calcium curves appear similar between pI-I 5.6 to 5.2 with each casein fraction fortified skim milk binding approximately 4.5 to 5.0 mM or 50% of the added calcium. Effect of heat treatment on curd strength of skim milk fortified with alpha or beta casein. During cheese ripening, casein undergoes proteolysis fielding nitrogenous compounds which contribute to its flavor. With Swiss and Port du Salut cheeses, a-casein degradation precedes that of t-casein. Ledford et a]. (8) found asl-casein proteolyzed in most ripening cheeses while t-casein remained intact in some types of cheese and not others; this variation in casein fraction degradation may account for major differences among commercial cheese. Therefore, it appears that by fortifying milk with either a- or t-casein, any number of cheese textures or flavors may be produced. However, because most milk is heat treated prior to manufacturing, the following experiment was designed to measure the effect of heat on the curd tension of a- or t-casein fortified skim milk. The casein fraction fortified skim milk samples (4.10% total protein) and skim milk controis were heated over a 68.3 to 76.7 C range with a holding time of 10 min at pH 6.0. Come-up and cooling times were carefully recorded; 62.8 C served as the base temperature; below this temperature, heat denaturation of milk protein was negligible. Following heat treatment, all samples were immediately cooled in tap water to 33.3 C; rennet was added, and curd tension measured after 30 rnin. Effective heat value calculations (E) were based on ]3urton's method (3) as modified by Ashworth and Nebe (2). As shown in Fig. 7, SpCT values of skim milk fortified with a-casein are higher over the entire heat treatment range. At E ----- .05 (68.3 C) to .16 (72.8 C). fl-fortil~'ed curd tension closely paralleled that of the skim milk control. Curve slope appeared to be a function of temperature with E ---- .42 (76.7 C) producing the most difference in treatments and E = .16 the least. Because initial calcium content of all samples was virtually identie-~, limited serum protein denaturation may ac]OURNAL OF DAIRY SCIENCB VOL. 57, NO. 9
996
SCHULTZ AND ASHWORTH
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count for some of the differences in curd tension values. However, the greater heat resistance of the a-caseinate system was apparent. Less denatured whey protein l~inds to the casein complex, thereby making it more susceptible to rennin action which results in higher curd tension. Calcium chloride addition (.005 M) produced little effect on skim milk curd tension; a-fortified curd strength was reduced nearly 23 SpCT units as compared to 8 units for fl-easein at E ---- .42 (Fig. 8). Heat denatured serum protein aggregation or precipitation is enhanced in the presence of added calcium. This may account for some of the similarity between the three curves; the denatured serum proteins precipitate on the caseinate complex thus hindering rennin action. By doubling calcium addition (.01 M), the e-casein fortification yielded a much stronger curd than the fl-samples. Excess calcium (.01 M) tended to destabil~Te tho milk system which was exhibited by floe formation or precipitation during the heating period prior to rennin activity. All samples heated above E = .21 (73.9 C) in the presence of .01 M CaCI2 precipitated. However, the a-casein fortified samples w e r e more resistant to heat treatment than the fl-casein fortified samples or skim milk controls at the higher calcium addition (.01 M). At JOURNAL OF DAIRY SCIENCE VOL. 57. NO. 9
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Fxc. 8. Effect of heat treatment on rennet curd tension of a- or fl-casein fortified skim milk with added calcium (pH 6.0, 33.3 C for 30 min). [2] ---- skim milk + Ca(.005 M); • = skim milk + Ca(.01 M); O ---- skim milk + a-casein + Ca(.00~ M); • = skim milk + a-casein + Ca(0.01 M); /k = skim milk + fl-casein + Ca(.005 M); ~h. = skim milk + fl-casein jr Ca(.01 M). E = .11 (71.1 C), a-casein yielded about 25 SpCT units as compared to zero for fl-easein and skim milk. Between E = .05 to .11, SpCT values of fl-easein and the skim milk control were measurable. Conclusion
The curd strength of pasteurized skim milk could be increased by the addition of whole casein (APC). Insufficient milk calcium appeared to limit maximum rennet curd tension. A combination of added casein plus extra ealciurn produced a much stronger curd than casein alone. However, excessive calcium aetually decreased curd tension (Fig. 1). Alpha casein forttfed skim milk with no added calcium produced a softer curd than fl-casein at p H 6.0. The addition of calcium caused a considerable increase in curd tension of both ~ractions with the a-casein fortification slightly firmer (Fig. 3). As shown by Versene titration, both casein fraction systems had approximately the same affim'ty for the added calcium, binding about 50%. Within a lactic culture environment (pH 5.20 to 5.85), a- or fl-casein fortified skim milk yielded similar curd tension values. The addi-
CURD TENSION OF CASEIN FRACTIONS
t.ion of CaC12 (.01 M) had two effects: (1) slightly decreased fl-casein curd tension, and (2) increased a-casein curd tension. However, as the pH approached 5.20, fl-casein with added calcium rapidly increased in curd strength, but the a-casein plus calcium had a firmer curd over the entire pH range. Skim milk forti~ed with a-casein produced a stronger curd than the fl-casein fortification after heat treatment in the range of 68.3 to 76.7 C (holding time 10 min at pH 6.0). This difference was greatest at 76.7 C. Therefore, a-casein fortified skim milk without added calcium appears more heat stable than the fl-easein fortification. When compared to the skim milk control (Fig. 7), CaCl2 addition (.005 M) reduced a-casein curd strength n e a r l y 23 SpCT units while only 8 for fl-casein at 76.7 C (Fig. 8). Between 71.1 to 76.7 C, the effect of added calcium (.005 M) also appeared significant; both casein fractions produced lower curd tension values than their respective fraction without added calcium. Under these conditions (Fig. 8), a- and fl-casein curd strength are similar. However, as calcium content increased .01 M, the curd strength produced by either fraction dropped sharply; a-casein gave considerably higher curd tension values than either fl-casein or the unfortified skim milk control (68.3 to 73.9 C). Above 73.9 C, all samples (a-casein, fl-easein, and t h e skim milk control) precipitated during heating in the presence of .01 M calcium.
(3)
(4)
(5) (6)
(7)
(8)
(9)
(10) (11)
(12) References
(1) Ashworth, U. S. 1966. Determination of protein in dairy products by dye-binding. J. Dairy Sei. 49:133. (2) Ashworth, U. S., and J. L. Nebe. 1970.
(13)
997
Comparison of rennet curd tension with undenatured whey protein as a measure of heat treatment. J. Dairy Sei. 53:415. Burton, H. 1951. The calculation of an equivalent pasteurization time for any time-temperature treatment of milk. Dairy Industries 16:823. Dill, G. W., and W. M. Roberts. 1959. Relationships of heat treatment, solids-notfat, and calcium chloride to the curd tension of skim milk. J. Dairy Sci. 42:1792. Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. McMeekin. 1952. Separation of a-, fl-, and 3,-casein. J. Dairy Sci. 35:272. Jen, j. j., and U. S. Ashworth. 1970. Factors influencing the curd tension of rennet coagulated milk. Salt balance. J. Dairy Sci. 53:1201. Jenness, R. 1953. Titration of calcium and magnesium in milk and milk fractions with ethylenediamino tetraaeetate. Anal. Chem. 25:966. Ledford, R. A., A. C. O'Sullivan, and K. R. Nath. 1066. Residual casein fractions in ripened cheese determined by polyacrylamide-gel electrophoresis. J. Dairy Sci. 49: 1098. Libbey, L. M., and U. S. Ashworth. 1961. Paper electrophoresis of casein. I. The use of buffersi containing urea. J. Dairy Sci. 44:1016. Mellander, O. 1939. Elektrophoretishe untersuchung yon casein. Biochem. Z. 300: 250. Mercier, J. C., F. Grosclaude, and B. R. Dumas. 1972. Primary structure of bovine caseins. A review. Milehwissensehaft 27: 402. Rapid Electrophoresis Manual No. 70176 B. 1966. Gelrnan Instrument Company, Ann Arbor, Michigan. Sanders, G. P., K . J . Matheson, and L. A. Burkey. 1936. Gm:d tension of milk and its relationship to firmness of curd in cheesemaking. J. Dairy SoL 19:395.
JOURNAL OF DAIRY SCIENCE VOL. 57, N O . 9
tors appear responsible for curd tension. However, pH, calcium concentration, and casein content are major factors affecting curd strength. Casein was considered a pure protein until electrophoretic patterns revealed three main components: a-, fl-, and y-casein in order of decreasing mobilities (10). Electrophoretie analysis of whole casein indicates that about 55% is a-casein, 30% fl-casein, and 15% minor components (9, 11). Because curd tension is dependent upon casein content, the question of the influence of individual casein fractions on curd character arises. This study investigated the effect of fortifying skim milk with either a- or fl-casein and measured the effect on rennet curd tension. Heat treatment effects on the casein fraction fortified skim milk were also studied by the curd tension method.
The effect of a- or/3-casein fortification (12 to 28% above control at 3.3% total protein) on skim milk curd strength was studied. After addition of 1 N hydroehloric acid to pH 6.0, a-casein fortified skim milk produced a weaker curd than /~-casein fortification. With added calcium chloride (.01 M), both fractions formed a firmer curd with a-casein slightly stronger than fl-easein. Alpha and fl-easein fortified skim milk cultured with Streptococcus lac~is had similar curd tension within a pH range of 5.20 to 5.85. Calcium addition (.01 M) increased a-casein fortified skim milk curd tension but had a depressing effect on fl-casein as compared to their respective curd strengths without added calcium. However, as pH approached 5.20, E-casein curd strength increased faster per pH unit than that of the a-casein fortified samples. Heat treatments within an effective heat range of .05 to .42 (holding time 10 min) allowed a-casein fortified skim milk to form a stronger curd than that fortified with E-casein. When compared to unfortified skim milk, the greatest difference in curd tension between the a- and fl-casein fortifications was at effective heat .42. Alpha casein curd tension was reduced nearly three times as fast as E-casein when skim milk fortified samples were heated at .42 in the presence of .005 M calcium chloride addition.
Experimental Procedure
Introduction
Factors influencing rennet curd strength have long been investigated. Because of the complex physico-chemical systems, many facReceived January 16, 1974. 1 Scientifie Paper No. 4178, Washington Agricultural Experiment Station, Pullman, Project No. 1809.
Casein preparation. Pooled pasteurized skim milk was diluted with two volumes of distilled water and by slowly adiusting the pH to 4.6 with 1 N HC], isoelectric casein was obtained. The urea method, Hipp et al. (5), was used to prepare whole a- and/3-casein; all fractions were precipitated once and washed five times with distilled water to remove any traces of urea. Sample preparation. Skim milk or 9% solidsnot-fat (SNF) reconstituted milk powder was fortified with sufficient casein fraction to increase the protein content 12 to 28% above a base of 3.3% total protein. To hasten casein fraction dispersion, the fortified sample was placed in a Waring blender and blended 2 rain. After dispersion the sample was divided into two 170-ml lots; one received no added CaCI2 while the other received 1.7 ml of 1 M CaCI2 (.01 M addition). After allowing about 3 h for calcium equilibration, the 170-m] lots were divided into three 50-ml samples; all samples were adiusted to pH 6.0 by the addition of 1 N HC1. Unfortified skim milk with and without added GaClz served as controls. Curd tension procedure. Curd strength was
992
993
CURD TENSION OF CASEIN FRACTIONS
measured by the method of Ashworth and Nebe (2); however, 50-ml samples were used to facilitate additional test trials. After sample tempering (33.3 C), 1 ml of 2% rennet ( 2 / 100 dilution of commercial rennet) was added. To ensure adequate mixing, the sample was poured back and forth twice into an empty beaker, covered and placed in a 33.3 C water bath for 30 min. Curd tension was read with a Cherry-Burrell Curd Tension Meter. Curd tension values were computed from the average of three sample readings. The same procedure was followed to determine the effect of heat treatment on curd tension of skim milk fortified with t~- or fl-casein, Samples were heated in an open water bath with close surveillance of come-up, holding, and cooling time. All heat treatments consisted of 10 rain holding and rapid cooling to below 62,8 C. Upon cooling to 33.3 C, rennet was added, and curd tension values were determined after 30 min. Because come-up and cooling time varied, effective heat values (E) were calculated for comparing time-temperature treatment of milk as described by Burton (3), and modified by Ashworth and Nebe (2). The ~z- and/3-casein preparations were electrophoreseed in Veronal Buffer (pI-I 8.6) containing 2.5 M urea to measure fraction purity (9, 12). Only trace amounts of fl-casein were in the s-fraction and trace amounts of tz-casein in the fl-fraction. Protein determination. Acid Orange-12 dye binding was used to determine percent protein in the milk samples (1). The amount of unbound dye was measured with a Beckman B Spectrophotometer containing a flow-through euvette (light path .75 mm) at 475 ran. The percentage of protein was read from a standard curve based o n Kjeldahl analysis of sim-.'lar samples. Calcium determination. After measurement of curd tension, the whey was separated from the eoagulum by filtration through Whatman No. 1 filter paper. A 5-ml aliquot was removed ~rom the filtrate and titrated with Versene (7). By determining the molar concentration of calcium in the whey sample, the amount of ca]eium retained in the curd was calculated by difference. Results and Discussion
Effect of whole casein [ortiI~eation on skim milk curd tension. Before the urea fractionation of whole acid precipitated casein (APC), fresh skim milk was fortified with APC and varying CaC12 added. Increased concentrations of casein and CaClz favor strong curd charae-
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g,
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Fic. 1. Effect of protein concentration and calcium addition on rennet curd tension (pH 6.0, 33.3 C for 30 min). × = skim milk; (~) = skim milk + whole casein; • --- skim milk + whole casein + Ca(.01 M); A = skim milk + whole casein + Ca(.02 M). ter (2, 4, 13); Fig. 1 supports these relationships up to 4.2% protein. A 4 to 5% increase in total milk protein content (without additional calcium) increased curd tension 25 to 30%. However, as more protein was added to the system, a corresponding increase in curd tension was not observed; therefore, calcium is the limiting factor. (APC is devoid of calcium salts due to the isoelectrie method of preparation. ) The addition of CaC12 to the casein fortified milk system had a pronounced effect on rennet curd tension. Increased protein content of 18 to 20% raised curd tension almost 50%. However, the results show that too much calcium may actually decrease curd tension. There is an optimal percent of calcium which will yield maximum curd tension values. 34 /% /%
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I 4.3
% Protein
FIc. 2. Molar concentration of calcium in rennet whey. X = skim milk; O -----skim milk + whole casein; • ---- skim milk + whole casein .t_ Ca(.01 M); A = skim milk + whole casein + Ca( .02 M ). JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 9
994
SCHULTZ AND A S H W O R T H
Versene titration of the whey sample sup-
ports the fact that curd strength is not a function of any single factor (2). Fig. 1 shows calcium addition increased curd tension while Fig. 2 reveals that titratable whey clacium is relatively constant over the range of percent protein fortification, with or without added calcium. Therefore, within the given protein range, it appears that constant calcium was retained in the curd regardless of its protein concentration while the excess was soluble in the whey.
Effect of alpha or beta casein on curd strength o~ skim milk. Reconstituted milk powder (9% SNF) was fortified with a- or fi-casein isolated by urea fractionation of APC; this treatment increased total milk protein content 12 to 28%. Each set of fortified samples received sufficient CaCle to increase the milk calcium concentration by .01 M. After rennet curd tension, the whey samples were titrated with Versene, and the molar concentration of calcium was calculated. Fig. 3 and 4 show the results of this experiment. Ordinate values are expressed as Curd Tension/% Protein; plotting curd tension values on a unit protein basis tends to eliminate the effect of variable protein content. We define this as specific curd tension
(SpCT). Without additional CaC12 (Fig. 3), increasing isoelectric casein fractions produced a decrease in SpCT; the a-casein fortit~cation yielded a softer curd than t-casein throughout the protein range. The t-casein curve results are comparable to the skim milk control until 4.0% protein is reached. With the addition of 40 35 =
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Fro. 3. Effect of ~- or p-casein fortification on rennet curd tension of reconstituted skim milk (pH 6.0, 33.3 C for 30 rain). × = skim milk; O = skim milk q- ~-casein; • = skim mi:k -b a-casein q- Ca(.01 M); /~ = skim milk -t- pcasein; ,A, = skim milk q- p-casein q- Ca(.01 M). JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 9
35
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Fro. 4. Molar concentration of calcium in rennet whey. × = skim milk; O = skim milk + acasein; • : skim milk + a-casein + Ca(.01 M); A = skim milk + p-casein; ~ = skim milk + g-casein + Ca(.01 M). CaClz, curd tension of both the a- and t-casein fortifications increased; the a-casein produced a slightly stronger curd than the t-casein. Filtered rennet whey contained .015 to .017 M calcium when none was added and .020 to .023 M with .01 M added calcium (Fig. 4). This suggests that about 50% or 5 mM of the added calcium was retained in the curd whether a- or t-casein was used for fortification.
Effect of culture activity on curd strength of skim milk fortified with alpha or beta casein. Jen and Ashworth (6) found rennet curd tension of reconstituted milk (9% SNF) increased until pH 5.8 with the addition of an acid or a lactic starter, but lowering pH by means of the starter gave more uniform curd tensions below pH 6.0. The present experiment was designed to test the effect of culture activity on the curd tension of a- or t-casein fortified milk. Reconstituted milk powder (9% SNF) was fortil~ed with a- or /3-casein (total protein 3.9%) plus sufficient 1 M CaC12 to increase milk calcium .01 M. Lactic starter (3%) was added to each sample and incubated at 33.3 C for 2 h. Following incubation, rennet was added (final dilution of 1:9_500) and curd tension measured 30 rain later; the final pH value was obtained immediately after curd tension determination. Variable pH between sample sets was a hmction of culture activity. Whey samples were collected and titrated for calcium content. Fig. 5 shows SpCT plotted against final pH values. With a decrease in pH due to starter activity (5.9 to 5.2), all curves show progressively firmer curd production. The skim milk cgntrol curd tension levels off at pH 5.5, then
CURD T E N S I O N OF CASEIN FRACTIONS
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Fro. 5. Effect of culture activity on rennet curd tension of a- or p-casein fortified reconstituted skim milk (33.3 C for 30 rain). × -- skim milk; C) = skim milk + a-casein; • = skim milk + a-casein + Ca(.01 M); A = skim milk + /~easein; , ~ = skim milk + /8-casein + Ca(.01 M). drops slightly. The a - and t-casein fortified skim milks show lower SpCT values than the control skim milk until pH 5.6 to 5.7; below this pH, the a- and t-casein fortifications had similar curd tensions but higher than control. Calcium addition decreased fi-casein curd tension and increased a-casein curd tension with respect to skim milk and corresponding fractions Without added calcium. However, with decreasing pH, t-casein p'.us calcium produced a more rapid increase in curd tension per pH unit than ~casein plus added calcium, although
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Fro. 6. Molar concentration of calcium in rennet whey. X = skim milk; C) = skim milk + acasein; • = skim milk + a-casein + Ca(.01 M); A = skim milk + B-casein; , ~ = skim milk + B-casein + Ca(.01 M).
995
t-casein fortified wheys revealed similar amounts of whey calcium (Fig. 6). This was expected because calcium left the curd as the isoelectrie point of casein was approached. Within the pH range 5.6 to 5.2, the three curves are nearly linear with 1.5 to 2.0 mM soluble calcium being released from the caseinate complex per .1 unit of p H change. The a- and t-casein calcium curves appear similar between pI-I 5.6 to 5.2 with each casein fraction fortified skim milk binding approximately 4.5 to 5.0 mM or 50% of the added calcium. Effect of heat treatment on curd strength of skim milk fortified with alpha or beta casein. During cheese ripening, casein undergoes proteolysis fielding nitrogenous compounds which contribute to its flavor. With Swiss and Port du Salut cheeses, a-casein degradation precedes that of t-casein. Ledford et a]. (8) found asl-casein proteolyzed in most ripening cheeses while t-casein remained intact in some types of cheese and not others; this variation in casein fraction degradation may account for major differences among commercial cheese. Therefore, it appears that by fortifying milk with either a- or t-casein, any number of cheese textures or flavors may be produced. However, because most milk is heat treated prior to manufacturing, the following experiment was designed to measure the effect of heat on the curd tension of a- or t-casein fortified skim milk. The casein fraction fortified skim milk samples (4.10% total protein) and skim milk controis were heated over a 68.3 to 76.7 C range with a holding time of 10 min at pH 6.0. Come-up and cooling times were carefully recorded; 62.8 C served as the base temperature; below this temperature, heat denaturation of milk protein was negligible. Following heat treatment, all samples were immediately cooled in tap water to 33.3 C; rennet was added, and curd tension measured after 30 rnin. Effective heat value calculations (E) were based on ]3urton's method (3) as modified by Ashworth and Nebe (2). As shown in Fig. 7, SpCT values of skim milk fortified with a-casein are higher over the entire heat treatment range. At E ----- .05 (68.3 C) to .16 (72.8 C). fl-fortil~'ed curd tension closely paralleled that of the skim milk control. Curve slope appeared to be a function of temperature with E ---- .42 (76.7 C) producing the most difference in treatments and E = .16 the least. Because initial calcium content of all samples was virtually identie-~, limited serum protein denaturation may ac]OURNAL OF DAIRY SCIENCB VOL. 57, NO. 9
996
SCHULTZ AND ASHWORTH
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Fic. 7. Effect of heat treatment on rennet curd tension of a- or fl-casein fortified skim milk (pH 6.0, 33.3 C for 30 min). X ---- skim milk; C) = skim milk + a-casein; /k _- skim milk -b flcasein.
count for some of the differences in curd tension values. However, the greater heat resistance of the a-caseinate system was apparent. Less denatured whey protein l~inds to the casein complex, thereby making it more susceptible to rennin action which results in higher curd tension. Calcium chloride addition (.005 M) produced little effect on skim milk curd tension; a-fortified curd strength was reduced nearly 23 SpCT units as compared to 8 units for fl-easein at E ---- .42 (Fig. 8). Heat denatured serum protein aggregation or precipitation is enhanced in the presence of added calcium. This may account for some of the similarity between the three curves; the denatured serum proteins precipitate on the caseinate complex thus hindering rennin action. By doubling calcium addition (.01 M), the e-casein fortification yielded a much stronger curd than the fl-samples. Excess calcium (.01 M) tended to destabil~Te tho milk system which was exhibited by floe formation or precipitation during the heating period prior to rennin activity. All samples heated above E = .21 (73.9 C) in the presence of .01 M CaCI2 precipitated. However, the a-casein fortified samples w e r e more resistant to heat treatment than the fl-casein fortified samples or skim milk controls at the higher calcium addition (.01 M). At JOURNAL OF DAIRY SCIENCE VOL. 57. NO. 9
0
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Curd Tension (Groms) / % Protein
Fxc. 8. Effect of heat treatment on rennet curd tension of a- or fl-casein fortified skim milk with added calcium (pH 6.0, 33.3 C for 30 min). [2] ---- skim milk + Ca(.005 M); • = skim milk + Ca(.01 M); O ---- skim milk + a-casein + Ca(.00~ M); • = skim milk + a-casein + Ca(0.01 M); /k = skim milk + fl-casein + Ca(.005 M); ~h. = skim milk + fl-casein jr Ca(.01 M). E = .11 (71.1 C), a-casein yielded about 25 SpCT units as compared to zero for fl-easein and skim milk. Between E = .05 to .11, SpCT values of fl-easein and the skim milk control were measurable. Conclusion
The curd strength of pasteurized skim milk could be increased by the addition of whole casein (APC). Insufficient milk calcium appeared to limit maximum rennet curd tension. A combination of added casein plus extra ealciurn produced a much stronger curd than casein alone. However, excessive calcium aetually decreased curd tension (Fig. 1). Alpha casein forttfed skim milk with no added calcium produced a softer curd than fl-casein at p H 6.0. The addition of calcium caused a considerable increase in curd tension of both ~ractions with the a-casein fortification slightly firmer (Fig. 3). As shown by Versene titration, both casein fraction systems had approximately the same affim'ty for the added calcium, binding about 50%. Within a lactic culture environment (pH 5.20 to 5.85), a- or fl-casein fortified skim milk yielded similar curd tension values. The addi-
CURD TENSION OF CASEIN FRACTIONS
t.ion of CaC12 (.01 M) had two effects: (1) slightly decreased fl-casein curd tension, and (2) increased a-casein curd tension. However, as the pH approached 5.20, fl-casein with added calcium rapidly increased in curd strength, but the a-casein plus calcium had a firmer curd over the entire pH range. Skim milk forti~ed with a-casein produced a stronger curd than the fl-casein fortification after heat treatment in the range of 68.3 to 76.7 C (holding time 10 min at pH 6.0). This difference was greatest at 76.7 C. Therefore, a-casein fortified skim milk without added calcium appears more heat stable than the fl-easein fortification. When compared to the skim milk control (Fig. 7), CaCl2 addition (.005 M) reduced a-casein curd strength n e a r l y 23 SpCT units while only 8 for fl-casein at 76.7 C (Fig. 8). Between 71.1 to 76.7 C, the effect of added calcium (.005 M) also appeared significant; both casein fractions produced lower curd tension values than their respective fraction without added calcium. Under these conditions (Fig. 8), a- and fl-casein curd strength are similar. However, as calcium content increased .01 M, the curd strength produced by either fraction dropped sharply; a-casein gave considerably higher curd tension values than either fl-casein or the unfortified skim milk control (68.3 to 73.9 C). Above 73.9 C, all samples (a-casein, fl-easein, and t h e skim milk control) precipitated during heating in the presence of .01 M calcium.
(3)
(4)
(5) (6)
(7)
(8)
(9)
(10) (11)
(12) References
(1) Ashworth, U. S. 1966. Determination of protein in dairy products by dye-binding. J. Dairy Sei. 49:133. (2) Ashworth, U. S., and J. L. Nebe. 1970.
(13)
997
Comparison of rennet curd tension with undenatured whey protein as a measure of heat treatment. J. Dairy Sei. 53:415. Burton, H. 1951. The calculation of an equivalent pasteurization time for any time-temperature treatment of milk. Dairy Industries 16:823. Dill, G. W., and W. M. Roberts. 1959. Relationships of heat treatment, solids-notfat, and calcium chloride to the curd tension of skim milk. J. Dairy Sci. 42:1792. Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. McMeekin. 1952. Separation of a-, fl-, and 3,-casein. J. Dairy Sci. 35:272. Jen, j. j., and U. S. Ashworth. 1970. Factors influencing the curd tension of rennet coagulated milk. Salt balance. J. Dairy Sci. 53:1201. Jenness, R. 1953. Titration of calcium and magnesium in milk and milk fractions with ethylenediamino tetraaeetate. Anal. Chem. 25:966. Ledford, R. A., A. C. O'Sullivan, and K. R. Nath. 1066. Residual casein fractions in ripened cheese determined by polyacrylamide-gel electrophoresis. J. Dairy Sci. 49: 1098. Libbey, L. M., and U. S. Ashworth. 1961. Paper electrophoresis of casein. I. The use of buffersi containing urea. J. Dairy Sci. 44:1016. Mellander, O. 1939. Elektrophoretishe untersuchung yon casein. Biochem. Z. 300: 250. Mercier, J. C., F. Grosclaude, and B. R. Dumas. 1972. Primary structure of bovine caseins. A review. Milehwissensehaft 27: 402. Rapid Electrophoresis Manual No. 70176 B. 1966. Gelrnan Instrument Company, Ann Arbor, Michigan. Sanders, G. P., K . J . Matheson, and L. A. Burkey. 1936. Gm:d tension of milk and its relationship to firmness of curd in cheesemaking. J. Dairy SoL 19:395.
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