Preparation of “Native” Phosphocaseinate by Combining Membrane Ultrafiltration and Ultracentrifugation

Preparat ion o f " N a t i v e " Phosphocaseinate by Combin ing

Membrane Ul t ra f i l t ra t ion and Ul t racentr i fugat ion

G. BRULE, J. FAUQUANT, and J.-L. MAUBOIS /aboratoire de Recherches Laitidres

Institut National de la Recherche Agronomique 65, rue de Saint Brieuc

35042 Rennes Cedex, France

ABSTRACT

Combining membrane ultrafiltration and ultracentrifugation is proposed for developing a physical process leading to industrial production of "native" skim milk phosphocaseinate. Effect of ultracentrifugation characteristics on the composition of the separated products was studied.

At 50 C, the higher the protein content of the centrifuged retentate, the higher the sedimentation yield. At lower temperatures, efficiency of ultracentrifugation decreased because of the large increase in the viscosity of ultrafiltration retentates. Protein content of sediments was not related to the protein content of the retentates but to the intensity of the centrifugal field and the centrifugation temperature. Washing of the sediment or ultracentrifugation of diafiltrated retentates led to purified phosphocaseinate.

The sediment exhibited a pseudoplas- tic rheological behavior, and its viscosity varied strongly with the temperature.

INTRODUCTION

Membrane uhrafiltration has emerged as a pragmatic means to concentrate milk and whey selectively for cheese and other dairy product manufacture (7, 8, 10, 11). Thanks to decisive progress in recent years in making high mechan- ically resistant steels, ultracentrifugation in large and open bowls rotating at speeds be- tween 50,000 and 100,000 rpm has become an industrial reality especially for isotopic separa- tion from uranium hexafluorure (2).

The manufacturing processes in the caseinate

Received October 10, 1978.

1979 J Dairy Sci 62:869-875

industry involve formation of a curd by added mineral acids, washing the curd, and preparation of an aqueous colloidal solution by addi- tion of an alkali (12). Such processes lead to handling of dangerous products, production of acid whey, a low value by-product, and losses of casein as fine particles in the whey.

Ultracentrifugation has been used as a physical tool for studying structure of casein micelles for a long time (3 to 13). Industrial extrapolation of the method used in dairy research laboratories for preparing native calcium phosphocaseinate from skim milk did not appear realistic because of the large amounts of liquid to be ultracentrifuged. Combining membrane ultrafiltration and ultracentrifugation seemed a more advantageous (less volume to be ultracentrifuged) possibility to obtain a wide choice of products, not only as sediments but also as supernatants, having interesting compositions and properties for the dairy industry (9).

In view of this new process, it was desirable to determine the effects of ultracentrifugation characteristics not only on the composition of the separated products, sediment and supernatant, but also on the efficiency of this step, i.e. the ultracentrifugation yields. This report deals with the effect of the composition of ultracentrifuged retentates (protein and total solids contents), intensity of the centrifugal field, temperature, and time of the ultracentrifugation step. The rheological behavior of the sediments is also reported. Its knowledge was required for studying and building the contin- uous extracting device for ultracentrifuged sediments.

MATERIAL AND METHODS

Milk

Milk was mostly raw skim milk (separated at 32 C) produced on the experimental dairy farm

869

e"

TABLE 1. Composition of products after ultracentrifugation of skim milk and its retentates at 44 K, 78 K, and 105 K × g.a

00

< O

O~ t J

Z

Centrifugal field

44 K Not Products centrifuged Sediment Supernatant Sediment

78 K 105 K

Supernatant Sediment Supernatant

Milk Protein 3.07 21.80 1.06 23.58 .98 25.60 .84 Total solids 8.53 27.90 6,40 29.42 6.30 30.76 6.14 Protein/total solids .36 .78 .16 .80 .15 .83 .13

Retentate 2 Protein 7.56 20.49 3.82 22.04 2.84 23.38 2.38 Total solids 13.36 27.70 9,39 28.77 8.43 29.46 7.87 Protein/total solids .56 .74 .40 .76 .33 .79 .30

Retentate 3 Protein 9,60 21.47 5.98 21.95 4.39 23.31 3.04 Total solids 15.58 27.92 11.70 28.47 10.10 29.80 8.62 Protein/total solids .61 .76 .51 .77 .43 .78 .35

Retentate 4 Protein 13.00 20.78 10.69 22.18 8.97 22.60 5.63 Total solids 19.40 28.15 17.02 28.87 15.30 29.00 11.75 Protein/total solids .67 .73 .62 .76 .58 .77 .47

Retentate 5 Protein 17.53 21.04 16.62 22.66 15.47 22.56 10.17 Total solids 25.56 28.10 24.48 30.08 22.81 29.94 17.52 Protein/total solids .68 .74 .67 .75 .67 .75 .58

aAll results are in g/100 g.

"NATIVE" PHOSPHOCASEINATE

TABLE 2. Ultracentrifugation yields.

871

Centrifugal field 44 K 78 K 105 K 150 K

Milk Yw 9.6 9.2 9.0 9.0 Protein %: 3.0 Yp 68.8 71.9 75.1 76.4

Ym 31.7 31.9 32.5 32.3 Ypl 2.1 2.1 2.3 2.3 Yml 2.7 2.7 2.7 3.0

Retentate 2 Yw 22.1 24.3 24.4 24.9 Protein %:7.5 Yp 60.1 70.9 75.6 80.4

Ym 46.0 52.4 53.9 52.0 Ypl 4.5 5.3 5.7 6.0 Yml 6.1 7.0 7.2 7.9


Ym 41.9 54.2 61.9 61.2 Yp1 5.0 6.5 7.5 7.7 Yml 6.5 8.4 9.6 10.2


Ym 33.2 45.4 64.9 71.2 Ypl 4.7 6.7 9.8 10.7 Yml 6.4 8.8 12.5 13.9


Ym 22.6 33.7 69.6 75.3 Ypl 4.3 6.4 13.4 14.7 Yml 5.7 8.6 17.7 19.3

of the I.N.R.A. research Center. Some experi- ments utilized industrial high temperature short time (HTST) pasteurized skim milk.

Membrane Ultrafiltrations

Milk retentates were prepared with Rhone- Poulenc equipped with 4 m z of IRIS 3042 membranes and Romicon equipped with 2.8 m z of hollow fiber XM 50 membranes.

Uhrafiltrations were according to Maubois and Mocquot (10).

Ultracentrifugations

Ultracentrifugations were with a preparative Beckman SPINCO ultracentrifuge and rotors 21, 30, 40, and 50 used at their maximum speeds, i.e. 21,000, 30,000, 40,000, and 50,000 rpm corresponding to average centrifuge fields of 44,373, 78,480, 105,651, and 150,925 x g.

Analytical Determinations

Analytical methods were those described by

Brule and al. (4) and included total solids, ash, pH, and Kjeldahl protein (N x 6. 38). Noncasein nitrogen (NCN) was determined according to Aschaffenburg and Drewry (1). Determinations of viscosity were with a Rheotest II co-axial cylinder viscosimeter which included facilities for control of sample temperatures (-+ .2 C) by a thermostatic water bath. Density determinations were by direct weighing of 100 ml of samples at 20 + .1 C.

Ultracentrifugation Y ields

Ultracentrifugation yield can be expressed as 1)Sediment weight in g for 100 g for liquid

centrifuged (Yw) 2) Protein proportion collected in the

sediment in g/100 g of protein centrifuged (Yp) 3) Dry matter collected in the sediment in

g/100 g of dry matter centrifuged (Ym) 4) Protein weight collected in the sediment

in g proteins/100 g liquid centrifuged (Ypl) 5) Dry matter collected in the sediment in g

dry matter /100 g of liquid centrifuged (Yml)

Journal of Dairy Science Vol. 62, No. 6, 1979

872 BRULE ET AL.

v m

is~oog

o~ooo g

o ~ ~sooog

, ,ooog

~OrEIN N . ~ a a ~

D ; ,'o ,; ;o

Figure 1. Effect of protein concentration on protein weight ultracentrifugation yield at different centrifugal fields.

Y p l ~o

~o m.

4Ore.

eeoTerN o

Figure 2. Effect of protein concentration and centrifugation time on protein weight uhracentrifugation yield.

RESULTS

Effect of the Centrifugal field

Typical compos i t ion of p roduc t s af ter uhracen t r i fuga t ion during 1 h at 20 C is in Table 1 for centr ifugal fields varying f rom

44,000 x g to 105,000 x g and pro te in c o n t e n t o f milk and re ten ta tes varying f rom 3.0% to 17.5%. It appears tha t for a given centr ifugal field, a large variat ion in compos i t i on of the u l t racent r i fuged liquid has little e f fec t on the s ed imen t compos i t ion .

TABLE 3. Composition of the products after uhracentrifugation at 50 C and 78,000 X g.

Protein Total (%) solids Ash NCN YW Ypl

Milk 2.94 Sediment milk 29.42 Supernatant milk .65

Retentate 2 7, 50 Sediment R 2 30.88 Supernatant R 2 1.57

Retentate 3 11.77 Sediment R 3 30.77 Supernatant R 3 2.93

Retentate 4 14.14 Sediment R 4 30.54 Supernatant R 4 3.92

8.65 .78 .70 38.00 4.29

6.14 .49 .61

13.76 1.21 1.56 39.47 3.97

7.28 .55 1.50

18.40 1.61 2.32 38.49 3.91 8.89 .58 2.48

21.10 1.85 2.70 38.29 3.81 10.08 .63 3.17

(%)

7.9 2.3

20.2 6.2

31.7 9.8

38.9 11.7

Retentate 5 17.95 25.05 2.20 3.31 Sediment R 5 28.69 37.27 3.68 Supernatant R 5 5.22 11.97 .72 4.21

54.2 15.6


"NATIVE" PHOSPHOCASEINATE 873

The different yields of ultracentrifugation expressed as above are in Table 2. The higher the protein content of the ultracentrifuged liquid, the greater is the effect of the increase of centrifugal field on yields. For a centrifugal field of 150,000 × g, when the protein content of the liquid ultracentrifuged is increased by a factor of 5.7, the weight yield (Yw) increases from 9 (milk) to more than 63% (retentate 5).

Figure 1 represents the variation of the yield of protein weight (Ypl) as a function of the protein content of the ultracentrifuged liquid. Above a protein content of 9% in the retentate, only a part of the casein micelles were sedi- mented at centrifugal fields lower than 78,000 × g. When the centrifugation of the casein micelles was complete (centrifugal field equal to or higher than 105,000 × g), Ypl varied linearly with the protein content of the retentate; the slope of the regression line was .86.

TABLE 4. Purification of the sediment a by washing with water.

Protein Total total

Protein solids solids

(%)

Retentate 12.1 19.1 63 Sediment 19.4 27.0 72 Sediment W b 20.3 25.0 81

Retentate D c 11.3 15.5 73 Sediment D 19.9 23.8 84

aultracentrifugation 60 min; 20 C; 78,480 × g. bWashing, 2 volumes of distilled water added to 1

volume of sediment. CDiafiltration, 1 volume of distilled water added

continuously to 1 volume of retentate.

Effect of the Ultracentrifugation Time

Effect of time ultracentrifugation was studied at 105,000 × g and at 20 C. Variation of Ypl as a function of the protein content of the retentates is in Figure 2. When the protein content of the retentate was lower than 8%, the sedimentation of casein micelles was maximum after 20 min of centrifugation. Above 8% of protein, there was a sharp decline of the 20- min yield, and the maximum was reached only after 60 rain.

diafiltration of the UF retentate or by washing the sediment which requires a second ultracentrifugation. Results by both procedures are in Table 4. The washing of the sediment increased the protein/TS ratio from 72 to 81%. A similar purification was obtainable by ultracentrifugation of a diafiltrated retentate. Increasing the diafiltration rate or repeated washings of the sediment led to a protein/TS ratio near of 90% which represents the theoritical maximum value.

Effect of the Ultracentrifugation Temperature

In Table 3 are typical results at 50 C during 60 min with a centrifugal field of 78,000 × g. A comparison of these data with those of Table 1 shows, first, an increase in the total solids (TS) and protein contents of the sediment (TS increased from 29% to 38%; proteins increased from 22% to 30%); second, as at 20 C, gross sediment composition was not affected by the composition of the centrifuged liquid; third, protein yields of centrifugation (Ypl) were maximum for even higher protein retentates, and finally, the supernatant composition varied exactly in the same way as that of the composition of the centrifuged liquid.

Effect of the Washing of the Sediment

Increasing the ratio of protein to total solids of the sediment can be attained either by a

Rheology of the UF Retentate and Ultracentrifugation Sediment

Respective variations of the retentate density and viscosity at 20 C as a function of the protein content are in Figure 3. Density of retentate varied linearly from 1.03 (milk, 3% protein) to 1.09 (retentate, 19% protein). Retentate viscosity determined at a shear rate of 437 s. -1 varied logarithmically.

Raising the temperature from 20 C to 60 C reduced the viscosity of a 20% protein sediment by a factor of 13 (Table 5). Under similar testing conditions, the sediment viscosity was 5 times higher titan the viscosity of an UF retentate (5). Washing the sediment increased its viscosity by a factor varying from 2 at 20 C to 1.5 at 60 C.

The rheological behavior of these products was non-Newtonian (Figure 4). Viscosity


874 BRULE ET AL.

VI~O~IT y C~ DENSITY

N, 6.38

v l s COSITY

l 00

00,4

S ] 2 0 ° c

s] 30 °c

, i 2;0 500 75° b VE LOC I S - T Y I G RAEIIENT

Figure 3. Effect of protein concentration on viscosity and density of U. F. retentates.

Figure 4. Effect of velocity gradient on sediment viscosity (S) and on washed sediment viscosity (WS) at 20 C and 30 C.

decreased when the veloci ty gradient increased over the veloci ty gradient range. Such a result is characterist ic of a s t ructure viscosity or a

TABLE 5. Variation of the sediment viscosity as a function of temperature.

Viscosity a (poises)

Temperature Washed (C) Sediment b sediment c

10 12.23 20 4.40 8196 30 2.24 3.46 43 .84 1.28 50 .57 .75 60 .34 .48

aviscosity determined at a velocity gradient of 437.4 s -1 .

bSediment composition:28.3% total solids; 20.85 % protein.

Cwashed sediment composition (1 v sediment + 2 v water) : 23.94% total solids; 19.51% protein.

pseudoplast ic behavior (5).

DISCUSSION

Combining membrane ul t raf i l t ra t ion and ul t racentr i fugat ion can lead to a process for manufac tur ing "na t ive" calcium phosphocaseinate f rom skim milk by only physical separa- t ion techniques. A yield of u l t racent r i fugat ion near the theori t ical m a x i m u m is ob ta ined by using a centrifugal field higher than 75,000 × g and a tempera ture of 50 C. The vo lume of l iquid to be centr i fuged is reduced by a factor of 5 by in t roducing UF re tenta te with a prote in con ten t o f 17%. Two by-products are ob ta ined with the proposed process, the UF permeate and the u l t racentr i fugat ion supernatant . But both are more suitable for animal feeding than acid casein whey. Permeate of UF can be used for manufac tur ing molasses or lick blocks (6 to 9). Ul t racentr i fugat ion supernatant has the same pH as skim milk, and Table 3 shows that its gross compos i t ion varies f rom that of a sweet whey to that of a whey prote in concen-


"NATIVE" PHOSPHOCASEINATE 875

trate, including that o f a milk (supernatant f rom ul t racent r i fugat ion of a 12% prote in retentate) .

Viscosity appears to be the mos t impor t an t fac tor which must be control led for obtaining no t only the best ef f ic iency of the ul tracen- t r i fugat ion step (sedimenta t ion rate, prote in yield) but also the mos t concent ra ted sediment . If our results (nearly 40% TS and 31% protein) are conf i rmed by the industrial ul t racentr i fuga- t ion equ ipmen t buil t by Bertin Society, an impor tan t savings in the energy cost required for drying calcium caseinate can be expected . As shown, viscosity is affected strongly by t empera tu re and ve loc i ty gradient, but more s tudy is required to de te rmine effects of re ten ta te pH, mineral compos i t ion of the re tentate , and previous heat t reatments .

Increasing the prote in con ten t of the l iquid u l t racentr i fuged increased the amount of whey proteins in the sediment . This can improve the nutr i t ional value of the p roduc t and also affect its funct ional properties. Washing of the sedi- men t reduces its whey prote in con ten t bu t also reduces the eff ic iency of the u l t racent r i fugat ion step owing to the increase in viscosity o f the washed sediment mos t l ikely due to an increase in the water binding propert ies of the casein micelles. However, when the presence of few whey proteins is no t undesirable, diaf i l t ra t ion o f UF re tenta te is more suitable for preparing low lactose calcium phosphocaseinate .

Data in Table 3 show an increase in mineral salts of the u l t racent r i fugat ion supernatants when prote in of the centr i fuged l iquid rises. Apparent ly , a small por t ion o f caseins, com- plexed with mineral salts, is not sed imented whatever protein was in the UF retentate . This soluble or serum casein represents about 2 to 3% of the total proteins of the ul t racentr i fuged liquid.

ACKNOWLEDGMENTS

This work was suppor ted by grants f rom D61~gation ~ ]a Recherche Scient i f ique et Techn ique (Contra t D.G.R.S.T. N ° 75 70 385). The authors are indebted to F. V. Kosikowski and G. Mocquo t for their critical and helpful suggestions and advice.

REFERENCES

1 Aschaffenburg, R., and J. Drewry. 1959. New procedure for the routine determination of the various non-casein proteins of milk. XVth Int. Dairy Congr. London. 3:1651.

2 Bertin and Co. 1974. Document NO. 03 74 41. 3 Bloomfield, V. A., and C. V. Morr. 1973. Structure

of casein micelles: physical methods. Netherlands Milk Dairy J. 103:

4 Brule, G., E. Real Del Sol, J. Fauquant, and C. Fiaud. 1978. Mineral salts stability in the aqueous phase of milk: influence of heat treatments. J. Dairy Sci. 61:1225.

5 Culioli J., J. P. Bon, and J. L. Maubois. 1974. Etude de la viscositff des "r~tentats" et des "pr& fromages" obtenus apr~s traitement du lair par ultrafiltration sur membrane. Lait 54:481.

6 Hargrove, R. E., F. E. McDonough, and J. A. Alford. 1974. A whey fraction converted into animal feed without drying. Food Eng. 46:77.

7 Lang F., and A. Lang. 1976. Ultrafiltration in the dairy industry. Milk Ind. 78:16.

8 Maubois, J.-L. and G. Mocquot. 1971. Preparation of cheese from liquid precheese obtained by ultrafiltration of milk. Lait 51:495.

9 Maubois, J. -L., J. Fauquant, and G. Brule, 1974. Procede de traitement de matieres contenant des proteines telles que le lair. French Patent No. 74 39311.

10 Maubois, J. -L., and G. Mocquot. 1975. Applica- tion of membrane ultrafiltration to preparation of various types of cheese. J. Dairy Sci. 58: 1001.

11 Maubois, J. -L. 1978. Application of membrane techniques in the cheese industry. 20th Dairy Congr., Paris.

12 Muller, L. L. 1971. Manufacture and uses of casein and co-precipate. DSA 33:659.

13 Waugh, D. F., and P. H. yon Hippel. 1956. K-casein and the stabilization of casein micelles. J. Amer. Chem. Soc. 78:4576.


Documents

Preparation of “Native” Phosphocaseinate by Combining Membrane Ultrafiltration and Ultracentrifugation