A Research Note Application of Reflection Photometry to the

Measurement of Milk Coagulation

JOEL HARDY and JACQUES FANNI

ABSTRACT The kinetics of milk coagulation can be observed by reflection pho- tometry using a color difference meter. The lightness increases after rennet addition. Visible clotting appears when lightening starts to slacken. Slope of the lightness curves plotted versus time ‘after renneting depends on temperature, nature and concentration of enzyme. When calcium chloride is added to the milk, a shoulder can be observed on the curve. This shoulder depends on the salt concen- tration. Attempts are made to explain the mechanisms of light dif- fusion by milk under coagulation.

INTRODUCTION THE MEASUREMENT of color is a widely used technique to control the quality of food products (Clydesdale, 1975). In recent years, it has been also applied to milk and dairy products (Langsrud and Solberg, 1976; Basset et al, 1977; Basset and Blanc, 1978). A test of measurement of the milk color during the coagulation has never been made. Only measures of turbidity by means of spectrophotom- eters (Payens, 1978) or absorption of light (Butkus et al., 1978) have been carried out during the coagulation.

In this paper variations of milk lightness in different conditions of coagulation have been studied with a Hunter- lab tristimulus Colormeter. A measuring device has been specially developed .to control the evolution of the “L” parameter of the skim milk, according to the nature and the proportion of enzyme, the temperature and the amounts of calcium chloride.

EXPERIMENTAL Determination of milk lightness during coagulation

Color deviations have been measured using a Hunterlab D 25 - 2 Colormeter, equipped with a D 25 - L optical head. This meter has the following criteria: optical geometry 45’/0” C.I.E. (Commis- sion Internationale de l’Eclairage) with circumferential lighting (360”) of the sample, C.I.E. standard illuminant C, aperture diam- eter: 5 cm.

The values were read in the L1, al, bI system according to Hunter (1958). Only values of L (lightness or whiteness) have been retained in the data. During the experiments, lightness variations (AL) of the renneted milk have been calculated and the plots repre- sent AL versus time after renneting. The nonrecording Hunterlab meter used shows values of L with one figure after the decimal.

Milk coagulation experiments were performed in a 6-cm diameter flat bottomed cuvette made of transparent glass and thermostated at f O.l”C at the periphery. A case, which is black inside, was placed over the cuvette in order to cover it completely. A measure of a blank gives L = 0.0. Under these conditions, settling of a milk-bed entails and increase of the L-value.

Milk lightening during coagulation was measured as follows: after the temperatures of cuvette and milk were stabilized at the same value, 5 ml milk are poured out and immediately renneted with 0.5 ml of an enzyme dilution. A chronometer is set in motion at the end of the rennet addition and the contents of the cuvette

Authors Hardy and Fanni are affiliated with Laboratoire de Science et Technologie des Alinienis, lnstitut National Polytechnique de Lorraine, 32, rue Sainte Catherine, 54000 Nancy, France.

1956-Volume 46 (1981)-JOURNAL OF FOOD SCIENCE

is stirred rapidly. The cuvette is put on the colormeter aperture and covered with the black case. The L-value is noted every 30 seconds and A L calculated from the initial value of renneted milk.

Clotting studies Each experiment was performed with log skim milk powder

reconstituted under standard conditions in 90g of calcium chloride solutions or distilled water. After stirring for 5 min, the milk was stored at room temperature for 30 min.

By this procedure, the action of the following clotting enzymes was studied: rennet of animal origin, a mixture in equal parts of pepsin and rennet and a microbial enzyme from Mucor mieihei. All these commercial enzymes had an activity of 10,000 lnterna- tional Units (I.U.).

In addition, three other clotting parameters were studied: enzyme dilution (from 50 I.U. to 1,000 I.U.), concentration (from 0-0.02M) of the calcium chloride solution used to dilute the milk powder, and temperature in the range 25-32°C.

RESULTS&DISCUSSION IN EVERY EXPERIMENT, a variation (AL) of the light- ness of renneted milk can be observed a short time after rennet addition. This variation increases with increasing time and reaches, after a few moments depending on the experiments, an approximative value of 2.0 (AL max = 2.0, Fig. 1).

The beginning of AL increase appears before visible coagulation time determined according to Berridge’s method (1952b); arrows on the curves show the moment when the first signs of granulation appear in a test tube containing the same milk renneted in the same conditions.

If the activity of the enzyme is increased, the first varia- tion of L appears .earlier and the curve, AL versus time, shows a more abrupt slope. However, maximum deviation (AL max) seems to be the same in each experiment. Thus, at 27’C, with milk prepared with distilled water and clotted with rennet, the following results were obtained: with rennet activity at 1,000 I.U. the time, toeI, required to reach AL = 0.1 is 1 min; with 200 I.U., t0.1 is 4 min and with 80 I.U., to.l increases to 8 min.

~7~-~0-~~-30 35 40 TIME AFTER RENNETING lrmn i

Fig. l-Lightness deviation (AL) of Sk/m milk versus t/me after renneting at different temperatures. Amount of calcium chloride = 0. Enzyme: rennet, 50 I. U. (Arrows show visible clotting time).

MILK LIGHTNESS DURING COAGULATION.. .

When varying temperature, alterations of the curves can be observed (Fig. 1). With other clotting enzymes, the time tc.r always depends on enzyme dilution and on coagu- lation temperature.

When studying the action of the different enzymes, AL max depends on the nature of the enzyme. A mixture of pepsin and rennet, which is known to be more proteolytic than rennet alone, or Mucor mieihi enzyme (Tam and Whitaker, 1972) gives AL max equal to 1.7 instead of 2.0 for pure rennet or microbial enzyme.

If milk is reconstituted in calcium chloride solutions, a shoulder appears on the curve (Fig. 2). This shoulder moves according to the concentration of calcium salt. In addition, its shape (height and width) varies according to the calcium chloride concentration. By reconstituting milk in distilled water, the shoulder appears late or simply does not appear.

The results obtained in experiments performed with dis- tilled water and the observations made by Payens (1978, 1979) can be compared. Payens used turbidimetry to study enzymic clotting of casein micelles in milk. He showed that, in the initial stages of coagulation, turbidity of the system passes through a shallow minimum followed by an explosive increase. He explained the minimum as cleavage of the macropeptide from x-casein by the clotting enzyme, and the turbidity increase as aggregation of the resulting particles.

In the same way, it is possible that lightening of renneted milk corresponds to an increase of the average dimension of particles which are aggregating. When this aggregation be- comes complete, a continuous network occurs and lightness reaches its maximum value. In experiments in which milk is reconstituted with calcium chloride, the shoulder can be seen as a result of several physical interactions.

Generally, light diffusion caused by suspended particles

-t 25

-:CaCI, 0.002 M

;I I -----.:CoCh 0.004 M 82.

5 ___.__ :CoCI, O.OlM

I= t :CaCI> 0.02M

,' ,' 0 5 1015-~0-- RENNETN?hn 30 35 40

TIME AF-fEi3 1

Fig. Z-Effect of amounts of calcium chloride on lightness devia- tion (AL) versus t ime after renneting. Temperature = 25.3 f O.PC. Enzyme: rennet, 50 I.U. (Arrows show visible clotting time).

in a liquid medium follows Rayleigh’s law for particles smaller than about % the wavelength of light. For these particles diffusion is proportional to the cube of their diameter. As the particle diameter increases, diffusion decreases inversely with the square of the particle diameter. According to these laws, diffusion reaches a maximum level when particle size becomes about % the wavelength of light. The Hunter colormeter measures whole diffused light in the visible spectrum (0.4-0.8 microns). Since the average size of native casein micelles is about 0.09 microns, depend- ing on the amount of calcium ions (Munya and Larsson- Raznikiewicz, 1980), it is quite possible that appearance of the shoulder could be connected with a rate of the par- ticles aggregation in the presence of Ca++. At the top of the shoulder, average size of aggregating particles could be estimated to be 0.1-0.2 microns. This aggregation occurs before any visible appearance of coagulation. When visible coagulation occurs, aggregation of particles is followed by a rearrangement resulting in a continuous network which probably scatters or reflects light according to more complex laws.

Diffused reflection measurement of milk during coagula- tion can constitute a significant means of investigation of clotting mechanisms. Furthermore, it should be possible to use reflection photometry to study and compare clotting enzymes. On the basis of the data gathered in this investi- gation, further studies to explain lightening of milk under coagulation and the effect of calcium salts are underway.

REFERENCES Berridge, N.J. 1952% Some observations on the determination of

the activity of rennet. Analyst. 77: 57. Berridge, N.J. 195213. An improved method of observing the clotting

of milk containing rennin. J. Dairy Res. 19: 328. Boss&, J.O., Ruegg, M. and Blanc, B. 1977. La couleur du fromage

et sa mesure: essai de determination par photome&? de reflexion. Schweiz. Milchw. Forschung. 6: 1.

Basset, J.O. and Blanc, B. 1978. La mesure de la couleur du Iait et des produits laitiers par photomdtrie de reflexion. 20th. Inter. Dairy Congr. F: 433.

Butkus, K., Bernatoms, G.V. and Butkuus. R.C. 1978. Appareil oour determiner les auahtes d’un coarmlum lactiaue. 20th. Inter. b&y Cons. F: 416. -

Clydesdale. F.M. 1975. Methods and measurements of food colour. In “Theory, Determination and Control of Physical Properties of Food Materials.” p. 275. D. Reidel Publ. Co., Dordrecht, The Netherlands.

Hunter, R.S. 1958. Photoelectric color difference meter. J. Opt. Sot. Am; 48: 985.

Langsrud, T. and Solberg. P. 1976. Effect of dairy processes on SUP face colour of milk and milk Droducts. MeieriDosten. 65: 873.

Mun a J.K. and Larsson-Raznikiewicz. M. 1980. The infhrence of Ca I+* on the size and light scattering properties of casin miceIIes. l-Ca++ removal. Milchwiss. 35: 604.

Payens, T.A. 1978. On different modes of casein clotting: the kine- tics of enzymatic and nonenzymatic coagulation compared. Neth. Milk Dairy J. 32: 170.

Pawns, T.A. 1979. Casein micelles: the colloid-chemical approach. J. Dairy Res. 46: 291.

Tam, J.J. and Whitaker. J.R. 1972. Rates and extents of hydrolysis of several caseins by pepsin, rennin, Endothia parasitica protease and Mucor pusiBus protease. J. Dairy Sci. 55: 1523.

M S received 2/20/81;revised 5121181; accepted 5/30/81.

Volume 46 (1981)-JOURNAL OF FOOD SCIENCE-1957