production of k-casein concentrate from commercial casein

4
BHUPINDER K. GIRDHAR and POUL M. T. HANSEN Dept of Food Science & Nutrition, Ohio Agricultural Research & Development Center and The Ohio State University, 2121 Fyffe Road, Columbus, OH 43210 PRODUCTION OF K-CASEIN CONCENTRATE FROM COMMERCIAL CASEIN ABSTRACT Differential solubilities of casein fractions in solutions of calcium chloride have been used as the basis for separating calcium-soluble K-casein from calcium-insoluble casein fractions. The amount of protein precipitated increased linearly with increasing calcium concentration up to OSM. The temperature of reacting casein with calcium and the ex- traction temperature for separating the soluble part were found to be significant variables independent of variations in the calcium concentra- tion. Reaction temperatures at 5°C caused impure preparations because of the reformation of casein complexes while reaction temperatures of 60°C caused heavy contamination with p-casein predominatly. In a pilot plant experiment designed from surface response analysis 8 kg of commercial casein were treated with NH, gas in the dry state and dispersed (12%) in water (25°C) and CaCl, was added rapidly to a final concentration of OSM. The mixture was heated to 45°C and filtered. The filtrate was acidified to pH 2.0 to precipitate K-casein which was washed with tap water and steeped overnight in 0.05M ammonium acetate at pH 4.5, dried with acetone, ground and treated with NH, gas to produce the soluble ammonium salt (yield: 8OOg; purity: 75% K-casein; analysis: 0.83% sialic acid, 0.41% P, 0.25% Ca, 14.25% N, Sz ,,w = 15). The preparation exhibited a specific stabilizing capacity of 11.8g of 0~s -casein per g of K-casein as compared with 12.8 for a regular K-caskin. INTRODUCTION K-CASEIN is a fraction of whole casein, insensitive to calcium ions, which is responsible for stabilizing the calciumsensitive as- and /s’-caseins against precipitation in milk. Isolated K-casein fractions stabilize asI -casein in model systems (Waugh and von Hippel, 1956). The role of K-casein in the stability of the casein micelle has been reviewed by Rose (1969), Farrell (1973), Swaisgood (1973) and Slattery (1976). A number of methods are available for isolating K-casein from isoelectric casein made under strict laboratory control and have been reviewed by MacKinlay and Wake (197 1). These methods are generally time consuming, expensive and involve complex techniques. The purpose of this research has been to devise a method for the isolation of K-CaSein from commercial casein which is practical and has potential for use in the food industry. MATERIALS & METHODS Casein The casein used in this study was of French manufacture, supplied by Western Dairy Products, California. A suitable amount of casein was also prepared in the laboratory by isoelectric precipitation (twice) of freshly skimmed milk using hydrochloric acid. Isolation of casein fractions K-Casein was isolated from commercial as well as laboratory manufactured isoelectric casein by the urea-sulfuric acid method of Zittle and Custer (1963) followed by double ethanol purification. 01s -Casein was isolated from laboratory-prepared isoelectric casein by using the method of Zittle et al. (1959). 0022-1147/78/0002-0397$02.25/O 0 1978 Institute of Food Technologists Preparation of K-casein concentrate (Fig. 1) A suitable amount (8 kg) of commercial casein was treated with anhydrous ammonia in a vacuum chamber at slightly reduced pressure (510 mm Hg) for 60 min and was subsequently dissolved in 59.5 L of water. The solution (12%, pH 8.0) was maintained at 25°C and calcium chloride (SM) was added rapidly to a final concentration of 0.5M. After mixing, live steam was used to raise the temperature to 45°C and the precipitate was allowed to settle. The supernatant, containing calcium- soluble K-casein, was decanted and the remaining precipitate reextrac- ted with water at 45°C containing O.lM calcium chloride for 60 min. The supernatants were combined and the temperature raised to 50°C and pH adjusted to 2.0 with dilute sulfuric acid. The precipitated pro- tein was collected over cheesecloth, washed several times with tap water and steeped overnight in 16L of 0.05M ammonium acetate at 10°C (pH 4.5). The curd was removed and dehydrated by successive washings in acetone and dried under vacuum. The dry product was ground in a Waring Blendor and then treated in the dry state with NH, gas at reduced pressure (Holsinger et al., 1977) to produce the soluble ammonium salt which was stored in an airtight container. Stabilization test The method described by Zittle (1961) was followed. In this test the 1 I INJECT LIVE STEAM TO 45°C I RECOVER FOR FURTHER UTILIZATION Fig. l-Manufacture of K-casein concentrate Volume 43 (1978)-JOURNAL OF FOOD SCIENCE- 397

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BHUPINDER K. GIRDHAR and POUL M. T. HANSEN

Dept of Food Science & Nutrition, Ohio Agricultural Research & Development Center and The Ohio State University, 2121 Fyffe Road, Columbus, OH 43210

PRODUCTION OF K-CASEIN CONCENTRATE FROM COMMERCIAL CASEIN

ABSTRACT Differential solubilities of casein fractions in solutions of calcium chloride have been used as the basis for separating calcium-soluble K-casein from calcium-insoluble casein fractions. The amount of protein precipitated increased linearly with increasing calcium concentration up to OSM. The temperature of reacting casein with calcium and the ex- traction temperature for separating the soluble part were found to be significant variables independent of variations in the calcium concentra- tion. Reaction temperatures at 5°C caused impure preparations because of the reformation of casein complexes while reaction temperatures of 60°C caused heavy contamination with p-casein predominatly. In a pilot plant experiment designed from surface response analysis 8 kg of commercial casein were treated with NH, gas in the dry state and dispersed (12%) in water (25°C) and CaCl, was added rapidly to a final concentration of OSM. The mixture was heated to 45°C and filtered. The filtrate was acidified to pH 2.0 to precipitate K-casein which was washed with tap water and steeped overnight in 0.05M ammonium acetate at pH 4.5, dried with acetone, ground and treated with NH, gas to produce the soluble ammonium salt (yield: 8OOg; purity: 75% K-casein; analysis: 0.83% sialic acid, 0.41% P, 0.25% Ca, 14.25% N, S z ,,w = 15). The preparation exhibited a specific stabilizing capacity of 11.8g of 0~s -casein per g of K-casein as compared with 12.8 for a regular K-caskin.

INTRODUCTION ’ K-CASEIN is a fraction of whole casein, insensitive to calcium ions, which is responsible for stabilizing the calciumsensitive as- and /s’-caseins against precipitation in milk. Isolated K-casein fractions stabilize asI -casein in model systems (Waugh and von Hippel, 1956). The role of K-casein in the stability of the casein micelle has been reviewed by Rose (1969), Farrell (1973), Swaisgood (1973) and Slattery (1976).

A number of methods are available for isolating K-casein from isoelectric casein made under strict laboratory control and have been reviewed by MacKinlay and Wake (197 1). These methods are generally time consuming, expensive and involve complex techniques. The purpose of this research has been to devise a method for the isolation of K-CaSein from commercial casein which is practical and has potential for use in the food industry.

MATERIALS & METHODS Casein

The casein used in this study was of French manufacture, supplied by Western Dairy Products, California. A suitable amount of casein was also prepared in the laboratory by isoelectric precipitation (twice) of freshly skimmed milk using hydrochloric acid. Isolation of casein fractions

K-Casein was isolated from commercial as well as laboratory manufactured isoelectric casein by the urea-sulfuric acid method of Zittle and Custer (1963) followed by double ethanol purification. 01s -Casein was isolated from laboratory-prepared isoelectric casein by using the method of Zittle et al. (1959).

0022-1147/78/0002-0397$02.25/O 0 1978 Institute of Food Technologists

Preparation of K-casein concentrate (Fig. 1) A suitable amount (8 kg) of commercial casein was treated with

anhydrous ammonia in a vacuum chamber at slightly reduced pressure (510 m m Hg) for 60 min and was subsequently dissolved in 59.5 L of water. The solution (12%, pH 8.0) was maintained at 25°C and calcium chloride (SM) was added rapidly to a final concentration of 0.5M. After mixing, live steam was used to raise the temperature to 45°C and the precipitate was allowed to settle. The supernatant, containing calcium- soluble K-casein, was decanted and the remaining precipitate reextrac- ted with water at 45°C containing O.lM calcium chloride for 60 min. The supernatants were combined and the temperature raised to 50°C and pH adjusted to 2.0 with dilute sulfuric acid. The precipitated pro- tein was collected over cheesecloth, washed several times with tap water and steeped overnight in 16L of 0.05M ammonium acetate at 10°C (pH 4.5). The curd was removed and dehydrated by successive washings in acetone and dried under vacuum. The dry product was ground in a Waring Blendor and then treated in the dry state with NH, gas at reduced pressure (Holsinger et al., 1977) to produce the soluble ammonium salt which was stored in an airtight container. Stabilization test

’ The method described by Zittle (1961) was followed. In this test the

1 I

INJECT LIVE STEAM TO 45°C I

RECOVER FOR FURTHER UTILIZATION

Fig. l-Manufacture of K-casein concentrate

Volume 43 (1978)-JOURNAL OF FOOD SCIENCE- 397

percent stability is expressed as the relative amount of 01~ -casein stabi- lized in a 10 ml reaction mixture containing 15 mg of &.,-casein and O.OlM calcium and varying amounts of K-casein. For the purpose of comparison between different preparations of K-casein, the term specific stabilization capacity (SSC), was introduced and defined as the grams of as -casein stabilized by lg of K-casein in the reaction mixture described above.

Electrophoresis Urea-starch gel electrophoresis was carried out by the method of

Morr (1974) with 2-mercaptoethanol added to both sample and the gel. Rennet treated samples were exposed to rennet for twice the length of time required to coagulate reconstituted skim milk (usually about 10 min). Ultracentrifugation

The sedimentation coefficient (S, o ,w) was determined at 20°C in a Beckman Model E analytical ultracentrifuge of samples dissolved in phosphate buffer (pH 7.0, 0.1 ionic strength) at 44,000 rpm using an An-D rotor. Chemical analyses

The ester-bound phosphate in casein and K-casein concentrates was determined by the Fiske and Subbarow method (1925). The Warren thiobarbituric acid method (1959) was used for quantitative determina- tion of sialic acid using N-acetyl neuraminic acid (Calbiochem, A grade)

I as a standard. Calcium was determined by using the method of Ntaili- anas and Whitney (1964).

RESULTS PRELIMINARY STUDIES revealed that commercial, dry iso- electric casein may be used as a source for isolating K-casein.

Table l-Analysis of variance of sialic acid content

Treatment D.F. M.S. Significance

Temp of reac. (R) Temp of Ext. (El Calcium (Ca) Ca-Linear Ca-Quadratic Ca-Cubic Ca-Tetragonal RXE RXCa E X Ca RXEXCa

2 0.5116 2 0.1581

(4) 0.1969 1 0.7775 1 0.0047 1 0.0032 1 0.0022 4 0.025 8 0.0059 8 0.0058

16 0.0024

Std. Error of Est. = k 0.049

1.0 I

1% 1% 1% 1% N .S. N .S. N S. 1% N.S. N.S.

E 50 25' 600 5' 25' 60° 5O 25O 60°

- - - R 50 25' 600

E * EI lRRCTION TER R - REACTlON TENP.

Fig. 2-Average variations in sielic acid con tent of K-casein concen- trates with reaction and extraction temperatures (average for five different calcium concentrations).

The preparations obtained by the method of Zittle and Custer (1963) were comparable with those isolated from casein pre- pared under more strict laboratory control in respect to electrophoretic patterns, rennet sensitivity, stabilization properties for (lLsl -casein and in sialic acid and phosphorus content. Optimization of conditions for extraction of K-casein

Attempts were made to improve the yield of food grade K-casein in a scaled-up process and retain stabilizing capacity but to avoid the use of toxic reagents and complex procedures. Experiments were conducted to explore extraction of K-casein in solutions varying in calcium chloride concentration in combination with different temperatures for the addition of calcium chloride (reaction) and centrifugation for the extrac- tion of the soluble K-casein. A total of 45 preparations were made using a three-factorial design with five levels for calcium chloride (O.l-0.5M) and three levels each for reaction and extraction temperatures (5’, 25O and 60’).

Table 1 shows the analysis of variance for the sialic acid content of different fractions which was used as an index of purity since K-CaSein is the only casein fraction which contains sialic acid. Each of the main factors, temperatures of reaction I and extraction and the calcium concentration, were significant 1 at the 1% level with the effect of calcium being linear. The cross product of reaction and extraction temperatures (RXE) was

(

also significant emphasizing the need for identifying a correct combination of these temperatures. Figure 2 shows that the reaction temperature of 25’C in combination with an extrac- tion temperature of 60°C gave a fraction which had the highest amount of sialic acid. This, then, would appear to be the optimum combination to be selected as far as purity is concerned. Figure 3 shows the electrophoretic patterns of K-casein concentrates obtained at 25’C reaction and 60°C ~ extraction in combination with different CaCls concentra- tions. K-Casein in these patterns is represented by a smeared diffused zone overlapping most of the other bands. The K-casein content, however, can be assessed by evaluating the relative intensity of the positively charged protein bands appearing after rennet treatment, corresponding to para- K-CaSein. It was shown by electrophoresis that for a given set of temperature combinations the purity of the fractions im- proved as the calcium concentration was increased from O.l-0.5M with the least contamination by o,i - and /I-casein at a reaction and extraction temperature combination of 25°C and 60°C (Fig, 3).

It was observed (patterns not shown) that the use of low extraction temperature (5’C) in all cases left a considerable amount of /.I-casein in the concentrates which would be expected from the known temperature-dependent disaggre- gation of fl-casein. However, reaction temperatures of 60°C, regardless of extraction temperature, also produced prepara- tions which were heavily contaminated with fl-casein contrary to expectations, The amount of contaminating fl-casein was 1 nearly independent of the calcium concentration as judged by electrophoresis. These observations suggest that a Ca-K-fl-casein complex may have been formed by treating the casein at 60°C which would favor hydrophobic aggregations.

For the purpose of devising a procedure for the K-casein isolation for food use the purity of preparations as judged by electrophoresis and sialic acid content would be less important than the need for selecting conditions which would optimize the yield and ensure a high functional performance. Zittle and ~ Custer (1963) have reported that some of the stabilization performance may be lost when K-casein is highly purified. Sur-

~ ,

face response contours of specific stabilization capacity of the concentrates as affected by varying conditions of preparation are presented in Figure 4 and show that the maximum stabilizing capacity is achieved by using relatively high calcium concentrations (>O.SM) in combination with temperatures of

398-JOURNAL OF FOOD SCIENCE- Volume 43 (1978)

K- CASEIN CONC PRODUCTION FROM COMMERCIAL CASEIN. . .

extraction which favor aggregation of fl-casein (>25’C) in agreement with the findings from the sialic acid analysis and the electrophoretic studies.

Further attempts to optimize the isolation conditions were explored for different levels of CaClz and for casein concentra- tions other than 8%. The results in Table 2 show that no improvement was derived from using CaClz concentrations higher than 0.5M. The results in Table 3 show that purity and relative yield were not affected by using higher casein concentrations. The upper limit on the initial casein concentra- tion would, therefore, be limited only by practical considera- tions such as ease of dispersion and viscosity. Pilot plant production of K-casein

K-Casein was prepared in the pilot plant by using the method outlined in Figure 1. A casein concentration of 12% produced a solution of manageable viscosity. The casein was pretreated with anhydrous ammonia for the ease of solubiliza- tion in water (Girdhar and Hansen, 1974). Holsinger et al. (1977) have reported that ammonia treatment of casein does not produce toxic effects when up to 40% ammoniated casein is incorporated in the diet for 28 days in feeding trials with rats. The extraction temperature was adjusted to 45’C to facilitate the ease of handling of the residual casein which at higher temperatures had a tendency to compact into a gummy mass. The steps for removal of calcium chloride involved an initial precipitation of the K-casien at very low pH with appli- cation of some heat. It was found that in the presence of high amounts of calcium, isoelectric precipitation at pH 4.6 was not possible. Similar difficulties in the precipitation of K-casein at high calcium concentrations have been reported by Morr (1959) who recommended dialysis before precipitation.

CALCIUM (Ml

PARA-K-

START -

Table P-Effect of calcium concentration (reaction 2!? C, extraction 60” Cl on yield and specific stabilization capacity (SSC)

Yield of Stabilization CaCI, K -casei n of 01 S!X Stabilization Yield (a)

(M) 1%) (%? kJ’%lhK) (g as, / lOOg casein)

0.15 9.02 41.13 6.16 55.56 0.30 6.94 64.55 9.68 67.17 0.45 6.94 77.21 11.58 80.36 0.60 5.96 77.21 11.58 69.01 0.75 5.55 75.94 11.39 63.21

a Calculated as the grams of as -casein stabilized by the total quantity of K-casein recovered frbm 1OOg of whole casein (yield X specific stabilization capacity)

Eight hundred grams of K-casein were obtained from 8 kg of commercial casein, representing a yield of lo%, with a purity of about 75% as judge.d by the sialic acid content (Table 4). The phosphorus content of 0.41% was higher than for regular K-casein as would be expected from a preparation con- taminated with fl- and (Ye.-caseins. Alternately, the preparation may possibly contain some h-casein which is a fraction insensi- tive to calcium but with a high (1.18%) phosphorus content (Long et al., 1958). Swaisgood and Brunner (1962) have ob- served material consistent with A-casein, in K-rich isolates ob- tained by the urea-TCA method.

Figure 5 shows that the stabilizing capacity of the K-casein concentrate was comparable to the standard laboratory prod- uct.

U R U RURURU R

Fig. 3-Elecrrophoretic patterns of K-casein concentrates obtained af 25” C reaction and 60” C extraction temperature: R = rennet freated; U = untreated.

SURFACE ESPON __-

SPECIFIC STAB

REACTION TER. - 25’C

200 300 400 500 600

EXTRACTION TEllP ‘C

Fig. 4-Response surface contours for effects of calcium concentration and extraction tempera- ture (25°C) on the specific stabilizing capacity of K-casein concenfrafes.

Volume 43 (19781-JOURNAL OF FOOD SCIENCE- 399

Table d-Effect of different concentrations of ammoniated casein solutions (reaction 25”C, extraction 6@C, CaCI, cone 0.5Ml on yield and specific stabilization capacity &SC)

Yield of Stabilization Casein K-casein of 01

(%?

!SC Stabilization yields

soln (%I (%I (g %, /gJ) (gas3 /lOOg casein)

2 5.5 67.53 10.12 55.66 4 6.0 75.32 11.29 67.74 6 6.2 67.53 10.12 62.74 8 5.7 67.53 10.12 57.68

10 5.9 75.32 11.29 66.61 12 6.0 75.32 11.29 67.74 14 5.8 75.32 11.29 65.48 16 5.9 74.02 11.10 65.49

a Calculated as the grams of 01 s -casein stabilized by the total quantity of K-casein recovered frbm 1OOg of whole casein (yield X specific stabilization capacity)

Table I-Summary of production data for K-casein isolation

Laboratory Pilot methodg plant

Whole casein (g) 100 8000 Yield of K-casein (g) 6 800 Recovery of K-casein (%) 20-40 65 Sialic acid (%I 1 .lO 0.83 Relative purity (%ja 100 75 Phosphorus (%I 0.23 0.41 Calcium (%)b 0.46 0.25 Nitrogen (%I 14.20 14.25 S 2o.w 14-19c 1 sod Time (days) 5-7 l-2 Specific stab. capacitye 12.75 11.81

Stabilization yieldf 76.5 118

a Relative purity based on sialic acid content bThe calcium content for dialyzed samples was invariably higher

than for samples decalcified by acidification precipitation. c Swaisgood (1973) d Extrapolated to zero concentration e Measured as g of as stabilized per g of K f Measured as g of 01s stabilized by K-preps from 1OOg casein g Zittle and Custer (1963)

STABILIZATION TESTS OF K -CASEIN PREPARATIONS -- - 100 .

* l STD.K-CASEIN

- K - CASEIN ISOLATE

I l/15 l/7,5 l/5 113.75 l/3 112.5

RATIO OF K/c&,

DISCUSSION THIS STUDY HAS SHOWN that it is possible to manufacture a K-casein concentrate (75% purity) from commercial casein in approximately 10% yield by a method which utilizes the dif- ferential solubility of casein fractions in CaClz solutions under varying temperature conditions. This product may have potential for incorporation in food products since no toxic chemicals are used. The time required for the isolation process was short (l-2 days) as compared to the lengthy laboratory isolation techniques (5-7 days) making it more acceptable for commercial exploitation.

Many of the procedures published for isolationg K-casein have used the differential solubilities of casein fractions in the presence of high calcium concentration as a basis for separa- tion (Waugh and von Hippel, 1956; Fox, 1958; Hipp et al., 1961; McKenzie and Wade, 1961). In the present study it was found that the temperature of reacting whole casein with calcium and the temperature of extraction were both im- portant variables, independent of the variations in calcium concentration. The significance of this temperature depend- ence was related to a basic problem which must be overcome when using calcium chloride as the separation reagent; namely, the tendency for reuniting of the casein components into stable complexes which remain soluble along with K-casein. There was evidence of such incomplete separation for the preparations obtained at the low reaction temperature which contained both as1- and fl-casein as contaminants. To avoid the reformation of micelles it was found desirable to increase the reaction temperature to the region of P-casein self aggrega- tion (25’C). On the other hand, the application of reaction tem- peratures higher than 25’C were also deleterious to purity, since the preparations reacted at 60°C showed heavy contamination with P-casein suggesting that a heat-induced, soluble complex had been formed between K- and fl-caseins in the presence of calcium. Swaisgood and Brunner (1962) on the basis of free boundary electrophoresis, have also suggested the possible interaction between K- and fl-casein in calcium-free solutions at pH values above and below the isoelectric points of the pro- teins,

Once the calcium reaction had been completed at 25OC the application of higher temperatures (6O’C) was found to be advantageous both for the ease of extraction and for mini- mizing the contamination with @casein. However, because of strong tendencies of the residual casein to form a thermo- plastic, gummy mass at 60°C, difficult for future handling, the pilot plant extraction temperature was adjusted only to 45’C. It should be noted that these conditions were within the optimized parameters established by the surface response analysis.

The high capacity of the K-casein concentrate to stabilize oft 1 -casem makes this product a potential source as an alterna- tive stabilizer to carrageenan for use in evaporated milk and infant milk products. Lin and Hansen (1970) have shown that carrageenan exhibits casein stabilizing properties similar to K-casein and have suggested that the mechanism of stabiliza- tion of processed milk products by carrageenan may be related to this property.

An important aspect for the commercial exploitation of K-CaSein isolation would be the potential use of the residual calcium caseinate which represents about 90% of the starting material. Experimental observations have shown this product to be highly thermoplastic and thus may have application in the manufacture of textured food products. According to a recent report (Anon., 1977) calcium caseinates have shown promise for use in the manufacture of high protein dietary biscuits where they not only serve as a nutritional source but also improve the physico-chemical properties of the dough. Thus, the residual calcium caseinate product would appear to have functional and nutritional proterties to be explored.

Fig. !&Stabilization capacity of K-casein concentrate. -Continued on page 406

400-JOURNAL OF FOOD SCIENCE-Volume 43 (19781