1672 JOUI~NAL OF DAIRY SCIENCE

sensitive (as) casein by kappa-casein. Effect of chymotrypsin and heat on kappa-casein. J. Dairy Sci., 44: 2101.

(9) Zittle, C. A., and J. It. Custer. 1963. Purifi- cation and some of the properties of as-Ca- sein and K-casein. J. Dairy Sei., 46: 1183.

Calcium Ion Concentration in Milk, Whey, and ~-Lactoglobulin as Influenced by Ionic Strength, Added Calcium, Rennet Concentration, and Heat

Abstract

Employing a calcium-sensitive electrode to estimate the calcium activity, additions of up to 2.5 mmoles of CaCle/liter of milk resulted in a linear response curve with about one-third of the added calcium remaining in the ionic state. The addition of rennet to milk resulted in a decrease in calcium ion concentration until coagulation occurred, at which time the calcium ion concentration in the curd-whey mixture gradually rose. fl-Lactoglobulin heated at 65 or 75 C for 30 rain did not bind more calcium than an unheated specimen. The 75 C treatment caused an increase in opti- cal density which was greater at higher levels of added calcium. Samples heated at 65 C showed no such increase in optical density.

The calcium-sensitive electrode has been used to determine the calcium ion concentration in milk (2, 4) and appears to have promise in quality control in the manufacture of dairy products. The importance of an adequate cal- cium concentration to rennet action is well recognized and has been extensively reviewed by Ernstrom (3).

Yamauchi (8) was unable to find a signifi- cant difference between the calcium-binding power of a-casein and rennet coagulated casein by a membrane equilibrium method. Casein binds calcium at the ratio of about 0.016 g per gram of casein (7). With the exception of coagulability, casein and paracasein have almost identical physical and chemical prop- erties (5). Thus, one gram of paracasein will not combine with more than 0.016 g calcium or about 0.4 g calcium per liter of milk. This 0.4 g is about five times as much calcium as is present in ionic form. I f ionic calcium reacts with paracasein to form curd, there is a rapid shift of calcium from the colloidal calcium phosphate to ionic calcium at the time of coagulation. Pyne (5) rationalized that the ionic calcium being in equilibrium with the

J. DAIRY SCIENCE ¥OL. 52, NO. I0

colloidal calcium phosphate might determine to a large extent the amount of calcimu phos- phate in the paracaseinate complex. Calcium phosphate serves as a source of calcium ions and as a sensitizer of rennin-altered casein to calcium ions, both factors contributing to rennin eoagulability of the system.

The present report deals with factors related to the determination of ionic calcium and of changes in the calcium ion concentration when milk and fi-lactoglobulin are heated and when milk undergoes rennet action.

Experimental Procedure

An Orion calcium-sensitive electrode assembly as described earlier (2) was used to determine calcium ion concentration. The effect of ionic strength in the range of 0.06 to 0.10 on the electrode response was determined on 18 trials using aqueous solutions of potassium chloride and calcium chloride containing 1 or 3 mmoles calcium ion per liter. Statistical analysis was done as described by Steel and Torrie (6) for split plot designs using the calcium ion con- centration as the main treatment and the ionic strength as a secondary treatment.

Varying amounts of 0.1 ~ CaCI 2 were added to milk and the mixtures analyzed for electrode response. To convert the electrometer readings to calcium ion concentrations, a standard curve was constructed each day by plotting the readings from the expanded scale p t I meter on solutions of constant ionic strength of 0.10 on the abscissa and the calcium ion concentra- tions on the ordinate of semi-log paper. Raw and homogenized milk were held for one hour at room temperature to determine the influence of such aging upon the calcium ion concentration.

For the experiments on rennet coagulation, calcium standards ranging from 1 to 3 mmoles per liter and at constant ionic strengths of 0.07 were made up from calcium chloride and potas- sium chloride. Calcium ion concentration was determined on 100 ml of raw milk at 35 C, then 2 or 5 ml of 5% rennet were added to the milk. The calcium-sensitive electrode was

T E C H N I C A L N O T E S 1673

~735 t-

uJ c~" 7.16 Z

~: 7.10

UA

I MILL IMOLE CALCIUM PER LITER

:3 MILLIMOLES CALCIUM ~ - - - - - ~ 9 _ PER LITER ~ / - - - - ~ - - ~ ~ b=0 .93

H d ~ . , , ~ , , o ~ 0.06 0.07 0.08 0.09 0.10

IONIC STRENGTH FIG. 1o Influence of ionic strength upon calcium

ion determination in water.

immersed in the milk-rennet mixture and readings taken each 15 see until coagulation occurred. Readings were taken after coagula- tion at 3-min intervals up to 30 rain after addition of rennet to the milk. Coagulation time was noted by the presence of curd on a glass rod alternately immersed in and with- drawn from the milk.

fl-Laetoglobulin was isolated from raw milk by the method of Aschaffenburg and Drewry

5.8

z 3.6 £

= 5 ~ - 3.4 W,-I

g~. 3.2 --O H~

2~ 3.0 _J

O

2.8

0 I I I I | 0 0.5 1.0 1.5 2.0 2,5

MILLIMOLES CeCl 2 ADDED PER LITER OF MILK

FIG. 2. Relationship between added calcium to milk and the calcium ion concentration.

(1). The protein preparation was dialyzed against distilled water at 4 C until the water was free of sulfate. To 10 ml of this protein preparation 0.005 ~ calcium chloride solution was added in quantities ranging up to 1 ml, and

205

z 200 0

n.- ~- ~- 1.95 z - - i iJ_l

O n ~ 1.90 o3 z w og

~ 1.85

r j 1.80

o 0

r

,oo

COAGULATION / /

~,'~ / / ~ ~2ML~0F 5°1° RENNET ~ o - W S PER I00 ML MILK

q COAGULATION

I I 1 I I I I I I 200 400 600 800 I000 1200 1400 1600 1800

TIME (SECONDS) FIG. 3. Calcium ion concentration of milk undergoing rennet action.

3. DAIRY SCIENCE VOL. 52, NO. 10

1674 J O U R N A L OF D A I R Y SCIENCE

water added to make a total volume of 11 ml. The protein-calcium preparations were heated at 65 or 75 C for 30 rain, the calcium ion concentration determined, and the turbidity measured on a colorimeter at 425 t~ wavelength.

Results and Discussion

The amount of calcium in potassium chloride-calcium chloride aqueous sohltions, as well as the ionic strengths of the solutions, had significant effects (P < 0.01) upon the elec- trometer readings. The linear regression lines are shown in Figure 1.

Electrometer readings taken after the solu- tions set for 30 min were not different (P>0.05) from the original readings. Average calcium ion concentration of 32 samples of raw milk was 2.24-+-0.64 mmoles per liter, and after one hour at room temperature the concentration was 2.03 ± 0.57. Likewise, fresh and aged homogenized milk had concentrations of 2.29 ± 0.52 and 2.16 ± 0.56, respectively. The in- fluence of aging was not significant (P>0.05) .

Results of 17 trials in which various amounts of 0.1 ~ calcium chloride were added to milk indicated that about one-third of the added calcium remained in the ionic state (Fig. 2). However, as the quantity of added calcium was increased beyond 5 mmoles per liter by adding 1.0 ~I CaC12 solution, essentially the entire added amount remained in the ionic state.

Milk used in 19 trials with 5 ml of 5% rennet per 100 ml had an average calcium ion con- centration of 1.73 mmoles per liter and milk used in the 20 trials when 2 ml of 5% rennet was added contained an average of 1.76 mmoles per liter. The first determination of the cal- cinm ion concentration in the milk-rennet mix- ture containing 2 and 5 ml rennet, respectively, was 0.10 and 0.12 mmoles per liter higher than in the milk. This could have been due to calcium contained in the rennet solution, as addition of 2 or 5 mt of 5% rennet to 100 mI of 0.002 calcium chloride increased calcium ion con- centration an average of 0.18 mmole per liter. Data in Figure 3, adjusted for the difference in calcium ion concentration in the two sets of milk, show a slight decline in calcium ion con- centration before coagulation, followed by a gradual increase. Milk containing the greater amount of rennet coagulated faster, showed less of a decrease in calcium ion concentration before coagulation and a faster increase fol- lowing coagulation. At about 1,000 sec after addition of the rennet, the two mixtures had almost the same calcium ion concentration. At the end of the 30-min period those samples

• ]', DAII~Y SEIEITCE VOL. 52, NO. 10

0.8

0.6

z Llul t"-, . j 0.4

__o I.- 0. o Q2

z 0,8

,,z, ~ o.6 ca

8 ~ oo.4

9 0.2

6 5 ° C

] I I I

I I I I I 0 , 0 9 0.18 0 .27 0 . 3 6 0 ,45

C A L C I U M ADDED (MICROMOLES PER ML PROTEIN SUSPENSION)

FIG. 4. Relationship of added calcium to calcium ion concentration and optical density in heated ~t-lactoglobulin suspensions.

having only 2 ml of 5% rennet added had a higher calcium ion concentration than those samples containing 5 ml of the same rennet solution. Zittle et al. (9) also found a greater binding of calcium by casein than by paracasein.

Calcium ion concentrations in the fl-lactoglo- bulin preparations are shown in Figure 4. Six trials, each using eight different concentrations of calcium, were conducted. The heat treat- ments (none, 65 for 30 rain, or 75 for 30 rain) did not influence the calcium ion concentration; thus, the three treatment effects were combined into a single line. The low concentration of calcium was approaching the limits of reliable detection by the electrode, which may explain increases in calcium ion concentration in the protein suspensions exceeding that from cal- cium added. The 75-C heal treatment, however, did influence the optical density as shown in Figure 4, indicating a calcium-influenced aggre- gation of fl-lactoglobulin at 75 C which did not occur at 65 C. Zittle et al. (10) found that heating fl-laetoglobulin at 90 C for 30 min in the presence of calcium ions resulted in aggre-

TECHNICAL NOTES 1 6 7 5

gation but did not affect the amount of calcium bound.

B. J. DEMOTT, Dairy Department, The Uni- versity of Tennessee, Knoxville 37901

References

(1) Aschaffenburg, R., and J. Drewry. 1957. Improved method for the preparatio~ of fl-lactoglobulin and a-lactalbumin from cow's milk. Biochem. J., 65: 273.

(2) Demott, B. J. 1968. Ionic calcium in milk and whey. J. Dairy Sci., 51: 1008.

(3) Ernstrom, C. A. 1965. Rennin action and cheese chemistry. Part I. Rennin and other enzyme actions, pp. 590-623. In Byron It. Webb and Arnold It. Johnson, Fundamen- tals of Dairy Chemistry. The AVI Pub- lishing Co., Westport, Connecticut.

(4) Muldoon, P. J., and B. J. Liska. 1969. Com- parison of a resin ion-exchange method mad a liquid ion-exchange method for determina- tion of ionized calcium in skimmilk. J. Dairy Sci., 52: 460.

(5) Pyne, G. T. 1955. The chemistry of casein. Dairy Sci. Abstr., 17: 532.

(6) Steel, 1~. G. D., and J. It. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York. 481 pp.

(7) Tessier, tI., and D. Rose. 1961. IIeat sta- bility of casein in the presence of calcium and other salts. J. Dairy Sci., 44: 1238.

(8) Yamauchi, K. 1959. Studies on milk coagu- lating enzymes. XV. Comparison of alpha casein and para alpha casein. J. Agr. Chem. Soe. Japan, 33: (13), 1134. [Dairy Sci. Abstr., 22: (8), no. 2358. 1960.]

(9) Zittle, C. A., E. S. DellaMonica, and L. Pepper. 1959. Casein and paracasein.: Neu- tralization with alkali, precipitation by cal- cium chloride and binding of calcium. Arch. Biochem. Biophys., 81: 187.

(10) Zitt]e, C. A., E. S. DellaMonica, R. K. Rudd, and J. It. Custer. 1957. The binding of calcium ions by beta lactoglobulia both be- fore and after aggregation by heating in the presence of calcium ions. J. Amer. Chem. Soc., 79: 4661.

Electrophoretic Proteolytic Patterns in Cheddar Cheese by Rennet and Fungal Rennets: Their Significance to International Classification of Cheese Varieties

Abstract

Polyacrylamide gel electrophoretie pat- terns of rennet and fungal rennet Cheddar cheeses proved useful in determining the type of milk coagulant for cheese, and in screen-testing poteutial rennet substitutes. The electrophoretic pattern of cheese varied widely with type of milk coagulant. I t is emphasized that the many casein compo- nent variations in cheese may complicate attempts to classify cheese varieties inter- nationally.

Supply shortages and quality variations of rennet from young calves have spum'ed efforts to discover suitable alternative rennet substi- tutes. The activities of such enzyme prepara- tions, clotting, proteolysis, and lipolysis, can shift by changing source, production conditions, and extraction procedures. Rennet substitutes have the potential for being modified to hn- prove over-all cheese quality and lower cost of cheesemaking. Their availability minimizes supply fluctuations, and those of microbial or higher plant origins remove religious dietary objections.

Rennet substitutes for commercial cheese dis-

tributed interstate in this countl T must be ap- proved for safety and suitability by the United States Food and Drug Administration. Thus, sensitive and effective methods for determining the type of milk coagulant used in a given cheese and for ascertaining the coagulant's desirable qualities are required. This paper presents results of the proteolytic effects in cheese of rennet and fungal rennets and their implications in cheese classification.

Experimental Procedure

Cheddar cheeses were made as described by Kosikowski (6), with rennet obtained from a commercial supply. Three lots of milk co- agulant from Endothi~ pc~rasitica and two lots of coagulants from Mucor sources were used. For convenience the coagulants from the source indicated are labeled endothia (Endothia para- sitica ; Charles Pfizer & Company, Inc.), Mucor 1 (Mucor michel; WalleTstein Company), and Mucor 2 (Mucor pusillus ; Dairyland Food Lab- oratories, Inc.). One per cent commercial lac- tic starter (Marlac) was added to ripen cheese milk of Lot 1, and 1% each of lactic and entcrococci (Flav-O-Lac Flakes D-K) starters to that of Lot 2. Curing of the cheese occurred

J. D~II~Y SCIENCE VOL. 52, I~O. 10