Microfiltration in whey processing

Desalination . 53 (1985) 143-155 Eleevier Science Publishers B.V ., Amsterdam - Printed in The Netherlands

143

MICROFILTRATION IN WHEY PROCESSING

J .H . HANEHAAIJER Netherlands Institute for Dairy Research (NIZO) P .O . Box 20, 6710 BA EDE (The Netherlands)

ABSTRACT During the past few decades, the processing of whey has developed from mere disposal to transformation into a variety of products with increasing added value . In particular whey protein concentrates (WPC) have attracted considerable attention, primarily because of their excellent nutritional value and their promising functional properties in a range of food products . The applicability - and value - of WPC can be increased by removing certain whey components which unfavourably affect these properties, like lipids, insoluble proteins and micro-organisms . By using microfiltration as a whey pretreatment prior to ultrafiltration, only a portion of the whey lipids is removed, and a considerable amount of valuable protein lost . A modified process is presented, in which by preconcentration of the whey, preferably by reverse osmosis, and subsequent micro- and ultrafiltration in a cascade mode, whey is processed into three fractions : a WPC which is rich in whey lipids ; a highly purified protein concentrate possessing improved functional properties ; and OF permeate . Compared with the processing of whey by ultrafiltration, the process makes a wider range of product applications possible ; however, due to the greater influence of membrane fouling it appears more difficult to control .

INTRODUCTION

During the past twenty to thirty years whey,

a liquid produced when milk is

processed into cheese or casein, has developed from dairy waste into a valuable dairy product . Actual statistics of whey utilization are rare ; for 1978 the world production of whey was estimated at 88 .400 million kg (ref .1) . There is a distinct and growing tendency to transform this pool into a complex of products with a wide range of functional end nutritional properties, for use in a variety of food and feed products . Valuable components of whey are in particular the whey proteins and lactose . It can be seen from Table 1 that whey has a low solids content, and that for many food applications its protein/lactose ratio is rather low, and its salt content high . This is why processes to transform whey into products with added value are necessarily based on separation techniques, such as evaporation/drying,



144

IIANEMAAI .IER

TABLE 1 Average composition of Gouda whey .

whey lipids

0 .05%

proteins

0 .6%

non-protein nitrogen

0 .2%

lactose

4 .5%

salts

0 .6%

lactic acid

0 .1%

total solids

6 .0%

TABLE 2 Some methods of whey processing and resulting products .

technology

products

none (transport)

pig feed

evaporation/drying (EV/D)

whey powder (WP)

demineralization + EV/D

demineralized WP

crystallization + EV/D (+ demineralization)

(demin .) delactosed WP lactose

ultrafiltration • crystallization of permeate • EV/D

whey protein concentrate (UF WPC) lactose delactosed permeate

microfiltration • ultrafiltration • crystallization of OF permeate • EV/D

1

IMF-WPC purified (law-fat) WPC lactose l delactosed permeate

I ,

crystallization, electrodialysis, ion exchange, reverse osmosis and ultrafiltration . Some of the methods used for whey processing are listed in Table 2 . From top to bottom the complexity of the process increases, along with the number of products and their ability to improve the characteristics of various food and feed products . In particular the isolation and purification of the protein fraction in whey is gaining interest . Obviously the production of whey protein concentrates (WPC) of adjustable functionality by flexible processes seems a way to compete successfully with proteins from other sources, such as casein, non-fat dry milk, soy protein and egg albumin .

WANEMAAIJER

145

Flexibility in producing WPC of desired protein content is one of the main advantages of ultrafiltration, and has, together with recent improvements of OF membranes and equipment, contributed to wide acceptance of this technique as a unit operation in dairy processes (refs .2-4) . A recent inventory of the application of OF and RO in the dairy industry showed that the total installed membrane area for OF of whey was about 80 .000 m 2 at the end of 1983, from which it was inferred that the OF WPC production amounted to between 40 .000 and 70 .000 tons (ref .5) . One of the features inherent in the ultrafiltration process is the incorporation in the WPC of certain whey components which are equal in molecular size to - or bigger than - whey proteins, and which may unfavourably affect protein functionality . In particular the presence of whey lipids, insoluble proteins and bacteria hampers the application of WPC in various food products, such as confectionery products and beverages, because of - for example impaired whipping properties, poor solubility at low pH and off-flavours (refs .4,

6-9) .

Improvement of the WPC properties can be achieved by pretreating the whey before ultrafiltration . It is common practice that the whey is centrifugated at the cheese factory, by which most of the curd fines are removed and the fat content is decreased to 0 .4-0 .6 g .l-1 . A number of investigators studied the use of precipitation techniques to remove the remaining substances

(e .g . refs .7, 10,

11) . None of these techniques have so far found acceptance, however, owing to either insufficient purity of the resulting WPC or to unattractiveness of the process technology . The use of microfiltration as a method of pretreating whey was investigated by lee and Merson in 1976 (ref .12) . Their main objective was to increase flux rates during ultrafiltration . After dead-end prefiltration of cottage cheese whey (using 0 .4 pm Nuclepore membranes), a considerable increase of the OF flux was obtained, but heavy deposition of proteins on the microfilters was also observed . No information was made available about operating times before clogging occurred or about amounts of protein lost . A few years later Enka introduced the so-called cross-flow technique for microfiltration (XMF), which makes it possible to treat particle-containing, fouling liquids (such as whey) by microfiltration . Merin et al . (ref .13) and Piot et al . (ref .14) investigated the prefiltration of whey by XMF and measured the permeate flux, the rejection of lipids and bacteria and the protein permeability using a number of MF systems . From their results it is clear that the lipids could not be removed completely : fat/protein ratios in the MF permeate . amounted to > 50 mg .g'1 . For the sake of comparison : the fat/protein ratio typical of whey before centrifugation is about 500 ng .g-1, and that of centrifugated whey 60-100 mg .g -1 ; a decrease to 210 mg .g -1 was obtained by a



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TABLE 3 Fat removal and protein permeability in microfiltration of unconcentrated Gouda whey (rel .17) .

process characteristics system

pore

Enka Gelman

lum) 0 .2 1 .2

I ( ° C) 20 37

pH 6 .6 6 .6

Enka 0 .1 50 5 .2 Enka 0 .2 50 5 .2 Enka 0 .2 50 5 .2 (0 .05% sodium caseinate added) Enka Enka Wafilin Amicon

0 .2 0 .2 0 .1 0 .1

15 50 50 50

3 .7 3 .7 3 .7 3 .7

fat/protein ratio MF permeate ) (mg-g -1

protein permeability (%)

56 > 70

75 > 95

24 61 39

63 81 92

20 9 > 20 2

57 40 9 25

whey pretreatment process developed by De Wit et al . (refs .15, 16), which yielded a high-quality WPC . Also, the protein permeability during preFiltration by XMF was too low for the process to be economically attractive . In our laboratory similar results were obtained, using various types of hF equipment (ref .17) . As is shown in Table 3, in which some of the results are listed, lipid removal could be enhanced by increasing the temperature to 50 ° C and by acidifying the whey to pH = 3 .7, near the isoelectric point of some whey lipoproteins ; probably complex formation of these components with proteins is induced under these conditions . However, sufficient removal of fat could be obtained only at the expense of considerable loss of protein . In this paper a microfiltration process is proposed, in which the removal of lipids has been improved, and the rejected protein is retained in a lipidenriched WPC . LIPID-PROTEIN INTERACTIONS Centrifugated whey contains 0 .4-0 .6 g .1 -1

of "whey lipids", whose composition

is largely unknown . From the lipid composition of milk (e .g . ref .18) it can roughly be derived that the whey lipids are about 50% tri- and diglycerides, present in the - after centrifugation remaining - smallest region (< 2 pm) of the fat globule distribution . Furthermore, less than 10% are free fatty acids, which are capable of freely passing both MF and IF membranes, and therefore will not constitute any significant proportion of the WPC lipids . The remaining fat



HANEMAAIJER 147

consists of lipoproteins, which are present in small particles (about 10 nm), in the fat globule membranes, and as free phospholipids . Microfiltration membranes with pores of 0 .1-0 .4 pm will only reject a part of the fat globules purely by mechanical separation . Further fat removal can only be achieved by physico-chemical processes : lipid-containing aggregates large enough to be rejected have to be formed by inducing solute-solute interactions . Concentration of Gouda whey by evaporation, and especially by reverse osmosis, appears to induce formation of large complexes (> 1 pm, as measured by microscopy), containing protein, fat, calcium, phosphate and citrate . These aggregates develop especially at high concentrations and temperature, and seem to be largely redispersable . A major factor in their formation is probably the precipitation of calcium salts, which become insoluble at increasing concentrations and temperatures ; by decalcifying the whey before concentration aggregate formation was prevented . Concentration by reverse osmosis - as compared with evaporation - resulted in a heavier occurrence of aggregates, which was attributed to the finding that during evaporation of Gouda whey considerable amounts of calcium are retained in the evaporator (ref .19) . It is also suggested that complexes between lipids and proteins can be formed by promoting hydrophobic interactions ; complex formation was induced by lowering the ionic strength or the pH, or by increasing the temperature (refs .16, 20) . This seems to be supported by the results listed in Table 3 (ref .17) .

MATERIALS AND METHODS Whey Centrifugated, pasteurized (68 ° C for 10 a) Gouda whey was obtained from the NIZO cheese factory . The whey was concentrated to 25% total solids by evaporation (Holvrieka two-stage falling-film evaporator) or by reverse osmosis (DDS/Pasilac two-stage pilot unit, equipped with 9 m2 HR-95 membrane) .

Microfiltration equipment Various MF systems were used :

Enka :

tubular membranes, inside diameter di = 5 .5 mm ; mean pore size d p = 0 .2 pm ; membrane surface area S m = 1 m2 .

Enka :

capillary membranes, di = 1 .2 and 1 .8 mm ; dp = 0 .1 and 0 .4 µm ; S m =2m2 .

WafiZin : tubular membranes, di = 15 mm ; dp = approx . 0 .1 pm ; Sm = 2 .5 m 2 . CeZman :

"pleated" membranes ; dp = 1 .2 pm ; Sm = 0 .09 m 2 .

Amicon : capillary membranes, di = 1 .1 mm ; d p = 0 .1 pm ; Sm = 1 .4 m 2 .

148 AANEMAAIIER

Ultrafiltration equipment Alfa-Laval/Remicon pilot unit, equipped with 1 .4 m 2 PM-10 membrane .

Experimental procedure All experiments were performed according to the flow sheet given in Fig . 1 . The concentrated whey was separated by microfiltration, yielding a retentate in which lipids and rejected proteins were concentrated, and an MF permeate which by means of ultrafiltration was processed into a purified protein concentrate and into OF permeate of normal composition . Samples of MF WPC and MF/UF WPC were freeze-dried and stored .

Fig . 1 . Process for combined micro-/ultrafiltration (IF/UF) of concentrated whey .

The micrnfiltration experiments were performed at temperatures of 18, 30 and 50 ° C ; average pressures were 100-150 kPa, depending on the MF system used ; cross-flow velocities were > 1 m .s -1 . All OF runs were performed at 50 ° C and pin/pout 200/100 kPa .

Analytical procedures Total solids (T5) by drying at 105 ° C (ref . 21) . Total protein (TP) by the Kjeldahl procedure, protein factor 6 .38 (ref .22) .

Non-protein-nitrogen ( NPN) as N soluble in 12% TCA filtrate . Fat by the Ruse-Gottlieb extraction procedure (ref .23) ; fat in the NF permeate was measured after concentration by OF to MF/UF WPC . Ash by drying and heating (ref .24) . Calcium by flame emission photometry . Lactose according to Luff-Schoorl (ref .25) . Protein soZubiZity at pH 4 .6 (NSI) (ref .26) .

HANEMAAIJER 149

Protein composition

by 1) high-performance gel permeation chromatography

(HP-GPC) ; detection by UV at 280 na ; buffer 0 .1 mol .l -1 potassium phosphate/0 .15 mol .1 -1 sodium phosphate, pH 6 .0 ; flow rate 1 .0 ml .min -1 (ref .27) and 2) polyacrylamide-gel electrophoresis (PAGE), according to Davies (ref .28) and Hillier (ref .29) .

RESULTS AND DISCUSSION

Process characteristics The influence of MF temperature on Fat removal, protein permeability and permeate flux, standardized for the production of an MF WPC with a protein content of 35% on dry basis, is shown in the upper part of Table 4 . Increase of the temperature to 50'C had a positive effect on both the separation properties and the flux rate of the membrane studied ; the same tendency was found for other membrane types . No experiments were carried out at higher temperatures because of protein denaturation . It was found that the type of equipment used had a considerable effect on separation and flux, as is shown in the lower part of Table 4 . Under the conditions studied the best results were obtained with the Amicon system . The removal of fat is fairly high, in comparison with that obtained with Pf of unconcentrated whey and, for instance, that reached with the clarification process according to De Wit et al . (refs .15, 16) ; the WPC obtained by the latter method can be calculated to have a fat/protein ratio of 8 .4 mg .g 1 .

TABLE 4 Fat removal, protein permeability and flux rate in microfiltration of Gouda whey, concentrated by evaporation (total solids 25%, pH 6 .0 at 20 ° C) . process characteristics

Amicon, Amicon, Amicon,

0 .1 pm, 18 ° C 0 .1 Jim, 30 ° C 0 .1 pm, 50 ° C

Wafilin, 0 .1 pm, 50 ° C Enka, 0 .2 pm, 50 ° C Amicon, 0 .1 pm, 50 ° C Amicon,

0 .1 pm, 50 ° C*)

Fat/protein ratio permeate (mg .g 1 ) (whey :f/p -85) 5 .0 5 .5 < 2 .0 8 .0 6 .5-12 .0 4 .0-7 .5 < 1 .0-2 .5

protein permeability (%)

18 13 19

mean flux for MF WPC 35 (l .m2 .h -1 )

< 10 < 10 -20

4 10-28 9-20

- 10 12-17 20-25

19-34

20-25

*) Experiments in which the whey was concentrated by reverse osmosis .



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RANEMAAIJRR

The difference in behaviour between the various MV systems has to be attributed to differences between the membranes used . Because of the great influence of membrane fouling on separation properties and flux, membrane morphology and surface characteristics will play an important role in the MF process . The Amicon membrane differs from the others by its slightly greater asymmetry, thus providing less opportunity to particles to clog the membrane substructure .

tatlprotein in permute (Mg .

6.0

g)

6.5

pH of the concentrated whey at 20 °C

Fig . 2 . Influence of pH on fat removal and protein permeability during MF of whey, concentrated by RO to 25 % T5 . Amicon MF system, T = 50 ° C .

Furthermore, rather large variations were observed in the amount of fat removed and in protein permeability, as indicated in the lower part of Table 4 . This phenomenon is partly dependent on the whey source : by microfiltration of whey concentrated by reverse osmosis the largest amount of fat was removed and the highest protein permeability obtained (Table 4, last line) . This may be related to the observed difference in aggregate formation between the two concentration techniques (ref .19) . Still, it must be mentioned that the reproducibility of separation is rather poor . Fig . 2 shows the influence of pH on the separation of fat and protein . Slight acidification seems favourable ; no significant effect of the type of acid used (e .g .

citric, phosphoric or hydrochloric) could be observed .

In Fig . 3 the flux during microfiltration is compared with the flux during ultrafiltration, using identical membrane configurations and process conditions, hF shows a higher flux rate which, if measured under the same conditions, is also moderately reproducible . In the right-hand part of Fig . 3 the flux during ultrafiltration of the (50% diluted) hF permeate is given . These flux rates, when related to the protein content, do not differ very much from fluxes during



flux (kg . m' 2. h -1 )

NANEMAAIJER 151

50

T:

50 oC

Romlcon PMIO

Pin :

200 kPa

T:

pout : IOO kPa 40

50 oC

Pin : 200 kPa pout: 100 kPa

MF micon MP0.1

30

20 OF Romicon PM10

10

0 2

5

10

15 20 25

protein in retentate (%

j i

0.5

1 .

5 protein in retentate (%)

10 15

I

L .. 25

35

. . 4550

proteinttotal solids (%

20 35 proteinttotal solids (%)

50 60

Fig . 3 . Permeate flux during micro- and ultrafiltration of concentrated Gouda whey with 25 % T5 (left) and during ultrafiltration of 50 % diluted MF-permeate (right) .

OF of "normal" whey, which points at an only minor influence of lipids and insoluble protein on the flux level . Control of the protein content of both MF and OF retentates by measuring the refraction proved to be not significantly different from common OF operation .

Product characteristics Characterization of the three products resulting from the MF/UF process was performed by determining their composition, by relating this to the composition of products with known characteristics and by evaluating some of the functional properties . In Table 5 the composition of some representative products is given and compared with those of other whey products . The MF WPC differs from "normal" OF WPC by its somewhat higher fat andd ash content ; the latter is to be attributed to the rejection of colloidal salt complexes as a result of the processing of concentrated whey . The purified MF/UF WPC distinguishes itself by the very low fat content . The ash content of the comparable "defatted" OF WPC is lower, because this product is prepared from highly demineralized whey . Apart



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HANEMAAIJER

from a slightly lower content of multivalent salts, the

OF permeate has a normal

composition . Microfiltration alters the protein composition : the smaller proteins, especially a-lactalbumin, pass the membrane preferentially . This is confirmed both by high-performance gel-permeation chromatography (Table 6) and by polyacrylamide-gel electrophoresis (Fig . 4 and Table 7) . The enrichment of a-lactalbumin in the MF/UF WPC could make this product suitable for partial humanization of cows' milk .

TABLE 5 Typical composition of products produced by MF/UF of concentrated whey, compared with those of some other whey products . products

total NPN/ protein/ TS TS (%) (%)

fat/ TS (%)

MF WPC-35 MF WPC-50

35 50

3 .0 2 .5

4 .0 6 .0

MF/UF WPC-35 MF/UF WPC-50 MF/UF WPC-90

35 50 90

4 .0 4 .0 3 .5

0 .04-0 .15 0 .05-0 .25 0 .1 -0 .4

OF permeate

5

4 .5

50% demin . delactosed whey powder OF WPC-35 OF WPC-50 defatted OF WPC-60*)

33 35 50

7 .5 4 .0 3 .0

63

3 .7

lactose/ TS (%)

ash/ TS (%)

Ca/ TS

NSI pH 4 .6

(%)

(%)

50 36

8 .0 7 .0

1 .0 1 .3

85 82

57 42 3 .5

6 .0 5 .0 5 .0

0 .3 0 .4 0 .7

> 98 > 98 > 98

85

8 .5

0 .5

2 .5 3 .0 5 .0

55 50 37

8 .0 6 .0 4 .5

72 88 86

0 .5

29

2 .3

91

*) According to De Wit et al . (ref .15) .

TABLE 6 Composition of the major proteins in whey and MF permeate, estimated by high-performance gel permeation chromatography . (3-lactoglobulin (dimer) MW 36 .700 d (%) whey, 25% TS if permeate (-MF/UF WPC)

67 65

a-lactalbumin MW 14 .200 d (%) 17 30

bovine serum albumin MW 66 .300 d (%) 6 3

imaunoglobulins MW 160 .000-900 .000 d (%) 10 2



I1SNEMAAIJER

B

153

f3-Iactoglobulin

-a-lactalbumin - bovine serum albumin - immunoglobulins

Fig . 4 . Polyacrylamide-gel electrophoresis of WPC powders obtained by MF/UF of concentrated whey, compared with a OF WPC and a casein whey standard . a . OF WPC-65 ; d . W WPC-43 ; b . MF/UF WPC-54 ; e . casein whey . c . MF/UF WPC-60 ;

The solubility of the products at low pH, as determined by the nitrogen solubility index (NSI), is given in the right-hand column in Table 5 . The MF/UF WPC products distinguish themselves by a nearly complete solubility ; even after five months storage NSI values of 99-100% were measured . The high NSI, also in comparison with that of the defatted OF WPC, can be attributed to the diminution of immunoglobulins (Tables 6 and 7), which in undenaturated condition are not fully soluble at pH 4 .6 (ref .16) ; furthermore, the NSI of the defatted OF WPC might be influenced by the lower ionic strength of this product .

TABLE 7 Composition of the major proteins in some whey products estimated by polyacrylamide-gel electrophoresis (Fig . 4) .

(3-lactoglobulin (%) a b c d e

. . . . .

OF WPC-65 MF/UF WPC-54 MF/UF WPC-60 MF WPC-43 casein whey

64 63 60 66 60

a-lactalbumin

bovine serumalbunin

(%)

(%)

21 33 36 17 21

5 2 1 5 6

immunoglobulins (%) 10 2 3 12 13

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HANEMAAIJER

TABLE B Expected comparative functional behaviour of MF WPC and If/UF WPC related to OF WPC .

1 :decreased ; O :not significantly different .

t :improved ;

product characteristics

behaviour, related to OF WPC, of : MF WPC

nutritional quality

0

flavour

application examples (refs .4, 9)

MF/UF WPC

t

baby food

t t

beverages, confectionery beverages

solubility

{

t t

heat setting

t

t

bakery products

emulsification

0

0

salad dressing

whipping

{

t t

confectionery

The high NSI values indicate that no denaturated protein is present in the MF/UF WPC, which was also confirmed with differential scanning calorimetry (DSC) . Whipping properties of the MF/UF WPC in aqueous systems were found to be good, and comparable with those of defatted OF WPC (ref .30) : whipping a 10% protein solution of MF/UF WPC-54, at pH 6 .2 and 20 ° C, showed an overrun of 3150'. and 98% stability . By relating the compositions and measured properties of If WPC and MF/UF WPC with those of, in particular, "normal" OF WPC and "defatted" OF WPC, the functional properties in food systems of both products being investigated extensively (e .g . refs .4, 6, 7, 9,

15), a rough expectation can be made of their

behaviour in food systems . The result of such an attempt is shown in Table 8, in which some properties of both products are related to those of normal OF WPC . It is obvious that the MF/UF process has the potential to widen

the applicability of whey protein

concentrates, by transforming whey into products with adjustable functionality for use in specific food products .

CONCLUSIONS With respect to product aspects, the application of microfiltration and ultrafiltration of concentrated whey in a cascade process seams to be a fairly attractive proposition, since it offers possibilities of producing "tailor-made" whey products which possess specific properties for specific applications . However, from the technological point of view the MF/UF process undoubtedly has some weak points, such as poor reproducibility and susceptibility to changes

RANEMMIJER 155

in the whey source . The feasibility of the process will probably depend on the acquisition of a better understanding of aggregate formation in concentrated whey and of the mechanisms underlying the fouling of membranes .

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to Dr . 3 .N . de Wit for many helpful discussions, to D . Stemerdink, J . Waver and 3 . Klok for performing microfiltration experiments, and to J . Leenders, W . Versluis, C . Brons and J . Kaper for their analytical contributions . Also Enka AG, Gelman Sciences and Wafilin B .V . are acknowledged for the equipment placed at our disposal to perform this work .

REFERENCES 1 D . Allum, J . 5oc . Dairy Technol ., 33 (2) (1980) 59-66 . 2 F .A . Glover, P .J . Skudder, P .H . Stothart and E .W . Evans, J . Dairy Res ., 45 (1978) 291-318 . 3 R . de Boer and J . Hiddink, Desalination, 35 (1980) 169-192 . 4 J .L . Maubois and G . Brul6, Le Lait, 62 (1982) 484-510 . 5 J .H . Hanemaaijer and J . Hiddink, North Eur . Dairy J ., 51 (2) (1985) 33-37 . 6 F .E . McDonough, R .E . Hargrove, W .A . Mattingly, L .P . Posati and J .A . Alford, J . Dairy Sci ., 57 (1974) 1438-1443 . 7 R . de Boer, J .N . de Wit and 3 . Hiddink, J . Soc . Dairy Technol ., 30 (2) (1977) 112-120 . B B .S . Horton, Dairy Record, 83 (12) (1983) 126-142 . 9 3 .N . de Wit, Neth . Milk Dairy J ., 38 (1984) 71-89 . 10 D .A . Grindstaff and W .P . Ahern, Process for pretreating raw cheese whey, US Pat . 3,864,506 (1975) . 11 R .J . Bolzer and 3 . Clanchin, Proc6d6 de traitement d'un produit lactos6rique et produit prot6onique en resultant, Demands de brevet Eur . 0,094 .263 (1983) . 12 D .N . Lee and R .L . Merson, J .Food Sci ., 41 (1976) 403-410 . 13 U . Merin, S . Gordon and G .B . Tanny, New Zealand J . Dairy Sci . and Technol ., 18 (1983) 153-160 . 14 M . Pint, J .L, Maubois, P . Shaegis, R . Veyre and A . Luccioni, Le Lait, 64 (1984) 102-120 . 15 J .N . de Wit, C . Klarenbeek and E . Hontelez-Backx, Neth . Milk Dairy J ., 37 (1983) 37-49 . 16 3 .N . de Wit, G . Klarenbeek and R . de Boer, Proc . 20th int . Dairy Congr ., Paris, 1978, 919 . 17 J .H . Hanemaaijer and D . Stemerdink, unpublished results . 16 P . Walstra and R . Jenness, Dairy Chemistry and Physics, John Wiley and Sons, New York, 1984, 457 pp . 19 3 .H . Hanemaaijer, J . Hiddink, T . Robbertsen and S . Bouman, to be published . 20 K . Yamauchi, M . Shimizu and T . Kamiya, J . Food Sci ., 45 (1980) 1237-1242 . 21 FIL-IDF 15 : 1961 . 22 FIL-IDF 20 : 1962 . 23 C .F . Vaster, Neth . Milk Dairy J ., 16 (1962) 137-144 . 24 FIL-IDF 27 : 1964 . 25 N . Schoorl, Chem . Weekblad, 26 (1929) 130 . 26 AOCS Method Be, 11-65, revised 1969 . 27 G .P . Dimenna and H .J . Segall, J . Liquid Chromatog ., 4 (1981) 639-649 . 28 D .T . Davies, 3 . Dairy Res ., 41 (1974) 217-228 . 29 R .M . Hillier, J . Dairy Res ., 43 (1976) 259-265 . 30 J .N . de Wit, Zuivelzicht, 10 (1975) 228-231 .