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Effect of lecithin on the reconstitutability of whole milk powder. Toma, Sadiq Jawad 1971

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FFSCT OF LECITHIN ON THE RECONSTITUTABILI 1 OF WHOLE MILK P O W D E R hy SADIQ JAWAD TOMA 5.Sc. U n i v e r s i t y of Baghdad 1965 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE . i n the Department of FOOD SCIENCE F a c u l t y of A g r i c u l t u r a l Sciences We accept t h i s t h e s i s as conforming to th required standard. THE UNIVERSITY OF BRITISH COLUMBIA May 1971 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Depa-rtment or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Food Science The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date May 26, 1971 ABSTRACT In the l a s t few decades, considerable emphasis has been placed on the problem of manufacturing very rea d i l y soluble whole milk powder. A s a t i s f a c t o r y instant whole milk powder is d i f f i c u l t to produce l a r g e l y because of the presence of fat in the system. In the present study, certain substances were added to the whole milk powder to help the dispersion of i t s constituents. Attempt has been made to investigate the effect of l e c i t h i n from d i f f e r e n t sources on the i n s t a n t i z a t i o n of whole milk powder and. to find out whether this effect i s due to a chemical interaction between the casein and l e c i t h i n or due to a change in the physical properties of the powder. I t was found that the powder p a r t i c l e s agglomerated p a r t i a l l y and as a re s u l t , the r e c o n s t l t u t a b i l i t y in cold water, 4°C, was improved, remarkably. Different degrees of improvement i n the s i n k a b i l i t y were observed when the powder was treated with ?.% l e c i t h i n from d i f f e r e n t sources. There was no evidence f o r casein l e c i t h i n interaction but the results show that a l t e r a t i o n in the i n t e r f a c i a l tension of butter o i l and the surface tension of water by l e c i t h i n s would play an important role in the sink-a b i l i t y improvement of the powder. - I I I -TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 4 METHODS AND MATERIALS 12 A d d i t i o n o f L e c i t h i n and Commerc ia l E m u l s i f i e r 12 D i s p e r s i b i l i t y T e s t l2 E x t r a c t - a b i l i t y o f L e c i t h i n f rom a M i x t u r e o f A c i d C a s e i n and. L e i t h i n 12 U l t r a c e n t r i f u p - a t i o n 14 P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 14-P r e p a r a t i o n o f Sample 1 5 S i n k a b i l i t y T e s t 15 . F r e e - f l o w a b i l i t y T e s t 17 I n t e r f a c i a l T e n s i o n o f B u t t e r O i l 17 I o d i n e V a l u e o f L e c i t h i n 17 RESULTS ' 18 E f f e c t o f L e c i t h i n on the D i s p e r s i b i l i t y o f D r i e d Whole M i l k Powder J S E f f e c t o f Commerc ia l E m u l s i f i e r s on t he D i s p e r s i -b i l i t y o f Dr ied. Whole M i l k 18 E f f e c t of D i f f e r e n t L e c i t h i n s and E m u l s i f i e r s on t h e . I n t e r f a c i a l T e n s i o n o f B u t t e r O i l and the Surfa.ce Ten s i on o f Water 20 I o d i n e V a l u e o f L e c i t h i n and i t s E f f e c t on the F r e e -f l o w a b i l i t y o f Whole M i l k Powder 2 5 H y d r o p h y l i c - l i p o p h y l i c P r o p e r t y o f M i l k C o n s t i t u e n t s 2 5 E f f e c t o f P a r t i c l e S i z e on t he S i n k a b i l i t y o f t he L e c i t h i n T r ea ted Powder 2 8 - i v -Page Concentration of L e c i t h i n and the S i n k a b i l i t y of 28 the Powder DISCUSSION 3 5 SUMMARY 4-3 APPENDIX 4 5 BIBLIOGRAPHY cyx LIST OF TABLES Table I Iodine Value of Dif f e r e n t Lecithins and Their Eff e c t on the I n t e r f a c i a l Tension (Oil-Water) of Butter O i l , the Surface Tension of Water and the S i n k a b i l i t y of Dried Whole Milk Page 2| Table II Ef f e c t of Span and Tween Type Emulsifiers on the I n t e r f a c i a l Tension of Butter O i l and. the Surface Tension of Water and the Disper-s i b i l i t y of Whole Milk Powder 24-Table III Angle of Repose of Whole Milk Powder Treated with 2% Different L e c i t h i n 2'? - v i -Figure 1 Figure 2 Figure 3 Figure k Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 LIST OF FIGURES D i s p e r s i b i l i t y Hates f o r Whole Milk Powder, Instant Skim Milk Powder, and Whole Milk Powder Containing 2% Lecithins of Di f f e r e n t Origins D i s p e r s i b i l i t y Rates of Whole Milk Powder Containing 2% D i f f e r e n t Span Type Emulsiflers D i s p e r s i b i l i t y Rates of Whole Milk Powder Containing 2% D i f f e r e n t Tween Type Emulsifiers Page 1 9 21 22 Iodine Value of L e c i t h i n and i t s E f f e c t on the Free-flowability (Angle of Repose) of Whole Milk Powder Containing 2% L e c i t h i n . 26 E x t r a c t a b i l i t y of L e c i t h i n from Casein-Lecithin Mixtures of Di f f e r e n t Weight Ratio 2 9 E f f e c t of P a r t i c l e Size on the S i n k a b i l i t y (15 Sees. Test Time) of Whole Milk Powder Contain-ing Different Additives JO E f f e c t of Concentration of L e c i t h i n (Vegetable Lecithin) on the S i n k a b i l i t y (30 Sees. Test Time) and the D i s p e r s i b i l i t y of Whole Milk Powder ? d D i s t r i b u t i o n of L e c i t h i n as Related to P a r t i c l e Size of Dired Whole Milk Powder Treated with 2% Vegetable L e c i t h i n D i s t r i b u t i o n of ©article Size of the Dried Whole Milk Powder Before and A f t e r Treating With 2% Vegetable L e c i t h i n . 32-34 - v i i -ACKNOWLBDGEMENTS I wish to express my gratitude to Dr. S" . Eakai f o r h i s constant advise and encouragement. Thanks are also expressed to the members of the Department of Food Science, p a r t i c u l a r l y Dr. W . D . Powrie, Dr. J.P. Richards and Dr. N.R. Bulley. Suggestions from senior graduate students, Reginald Clarke and Thomas Beveridge are appreciated. - 1 -I H T R O D U C T I O N Dry milk production has become an Increasingly important segment of the dairy industry. The production of dry whole milk i n North America i s roughly about 100 m i l l i o n pounds yearly. The ultimate aim of the industry Is to obtain dry products which recombine with water e a s i l y giving very l i t t l e or no sign of detrimental change compared to the o r i g i n a l l i q u i d product. A dried milk, to be e a s i l y d i s p e r s i b l e and reconstitu-table, required certain physical-chemical properties, e.g., good w e t t a b i l i t y and s i n k a b i l i t y , absence of any lumping when placed into water, rapid and. complete dispersion of p a r t i c l e s , no tendency to form a water-repellent f i l m on the walls of the container or fat clumps on the surface of the reconstituted product and absence of floc c u l a t e d protein on the walls of the container. Regular milk powder i s not however, r e a d i l y accepted by the housewives for beverage purposes due to the d i f f i c u l t y with which i t reconstitutes i n cold water and i t s poor flow c h a r a c t e r i s t i c s . Ease of reconstitution of dried milk i s impor-tant also l n the ice cream industry where large amounts of milk powder are being used. The drawbacks of regular powder can be overcome by subjecting the powder to the so-called i n s t a n t i z i n g process by one of the following three general methods: 1. The re-wet method or the agglomeration method i n which agglomeration i s accomplished by wetting the p a r t i c l e s , using steam or water, s u f f i c i e n t l y to cause the surface to be tacky - 2 -whereby the p a r t i c l e s w i l l agglomerate. Re-drying to normal moisture content i s usually done by hot a i r . During the process, a s u b s t a n t i a l part of the lactose changes to c r y s t a l l i n e form, mainly c<-iactose hydrate. 2. The s t r a i g h t through or single-pass method which gives a powder generally termed semi-instant. This method consists of adjusting the spray drying conditions to give a coarse powder. Such large p a r t i c l e s are obtained when the v i s c o s i t y of the feed to the atomizer i s high. High t o t a l s o l i d s and low feed tempera-ture or a combination of both w i l l give a viscous concentrate. The powder produced i n t h i s way w i l l not be as coarse as powder from a re-wet i n s t a n t i z e r , and w i l l therefore not possess the same instant properties. 3. Fake-instantization, which i s based on the use of additives to give powders with w e t t a b i l i t y and d i s p e r s i b i l i t y c h a r a c t e r i s t i c s s i m i l a r to i n s t a n t i z e d milko E f f e c t of sodium c i t r a t e , disodium phosphate, sugar, Span, Tween, Myverol type emulsifiers and l e c i t h i n on the r e c o n s t i -t u t a b i l i t y of dried milk powder has been studied,, L e c i t h i n has been used recently i n many patents f o r i n s t a n t i z a t i o n of dried whole milk powder <> In the present study attempts have been made to in v e s t i g a t e the e f f e c t of l e c i t h i n from d i f f e r e n t sources on the i n s t a n t i -zation of dried whole milk and to determine whether t h i s e f f e c t i s due to chemical i n t e r a c t i o n between the casein and l e c i t h i n or due to a change i n the phys i c a l properties of the powder• - 3 -- D i s p e r s i b i l i t y and s i n k a b i l i t y tests were used as a measure of r e c o n s t i t u t a b i l i t y of the powder. In order to study the mechanism involved i n the improvement of the r e c o n s t i t u t a b i l i t y of the powder by l e c i t h i n , i n t e r f a c i a l tension between water and butter o i l containing l e c i t h i n ; the surface tension of water containing l e c i t h i n were measured a Casein l e c i t h i n i n t e r a c t i o n was investigated by gel electrophoresis, u l t r a c e n t r i f u g a t i o n and the e x t r a c t a b i l i t y of l e c i t h i n from casein l e c i t h i n mixture© LITERATURE REVIEW When the agglomeration method was applied to whole milk powder, i t was found ( 1 8 ) that the resulting milk powders contained approximately four times as much free fat as the original powder. This substantial increase i n free fat con-tent was due to the formation of lactose crystals of the <<-form. When lactose crystalizes, fat bound to lactose w i l l separate, and further, lactose crystals w i l l rupture fat globule membranes. Flavour deterioration of the. powder deve-loped during re-drying and storage due to large amount of free f a t . Those results indicate that the re-wet method i s unsuitable for whole milk powder. The application of the second method to whole milk powder offers advantages that crystaliLzation of lactose can be avoided but powder prod-uced i n this way may not be as coarse as powder from a re-wet instantizer, and w i l l therefore not possess the same instant properties (18).Since the f i r s t and second methods are not applicable for the "instantization of whole milk powder ,surface active agents such as l e c i t h i n have been used i n many of the instantization processess for whole milk powder. Mclntire and Loo ( 2 0 , 2 1 ) developed two l e c i t h i n processess assigned to dairy foods. The principle of Mclntire process entails the dry blending with a predetermined amount of l e c i t h i n which can be accomplished mechanically by any suitable blending apparatus or by heating the l e c i t h i n to an extent sufficient to reduce i t s viscosity and then spraying or pumping the l e c i --5-t h i n through a nozzle upon tiie s t a r t i n g powder. The r e s u l t i n g mixture i s agitated or blended s u f f i c i e n t l y to e f f e c t a thorough Intermixture. Another l e c i t h i n process was developed by Sjollema (39). Spray dried milk powder was heated to 70°C i n a steam jacketed rotary mixer. Soy l e c i t h i n was heated to 70°C also i n a separate vessel and mixed with the milk powder to give a mixture of 2 per cent by weight. The mixture was then cooled under evacuated drum, and the product revealed excellent "instant" property i n cold water 0 Shields, et a l , (36) used l e c i t h i n i n a p a r t i c u l a r manner to produce a fat-containing milk product having instant properties„ During the f i n a l drying operation of whol6 milk and while the material was s t i l l i n a moist condition, i t was Intermixed with l e c i t h i n (0.25-1$). These authors claimed that when a quantity of t h i s powder was deposited upon the surface of cold water, the water quickly penetrated the mass and the Individual porous aggregates without blocking, whereby the entire mass quickly wetted and sank. Nova, s i aX (28) modified the above method by atomization of dispersed l e c i t h i n i n steam. Nova _et a l ( 29) described a method fo r the production of r e a d i l y reconstitutable f a t containing milk powder. L e c i t h i n was incorporated onto the surface of the whole milk powder by atomizing steam, containing l e c i t h i n drop-l e t s , onto moistened whole milk powder at a temperature between 5^-71°Co This l e d to an agglomerated powder. Another process was developed by Tumerman, et a l ( 3^ i n - 6 -i 1966. Spray dried whole milk powder, 26$ fat, was injected by means of high velocity a i r stresun, into an agglomeration chamber. An emulsion of 4$ soya bean l e c i t h i n in water was introduced into the chamber by an independently operated spray nozzle. The wetted particles, moisture content 15$, were graded for size, and dried to a moisture level of 3$« The f i n a l product contained 0.06$ l e c i t h i n , mainly on the surface of the particles, and was instantly soluble in water. William j i t a l (44) produced instant fat-containing milk powder, by dissolving l e c i t h i n ( 1.1$) in the milk fat at a temperature sufficient to maintain the fat in a l i q u i d state. The milk f a t - l e c i t h i n mixture was subsequently added to concen-trated skim milk (in sufficient quantity to give a fat content of 6-48$ and an active l e c i t h i n ranging from 0.05-4.0$ in the f i n a l powder). The mixture was homogenized and spray dried by a process designed to increase the bulk density of the powder. Another method which produced quickly soluble milk powders with a high fat content involved spray drying of a precondensed whole milk to which l e c i t h i n was added to the milk fat portion in addi-tion to some suitable thickners l i k e alginate (30). Nonfat dry milk solids of improved d i s p e r s i b i l i t y and s o l u b i l i t y were pre-pared by incorporating 0.033-0.066$ active l e c i t h i n followed by moistening and agglomerating the powder to increase bulk density (39). The l e c i t h i n was mixed with vegetable o i l (1:3) at 60°C and then added at a rate of 1 drop/5 sec. to the freshly spray dried milk powder at 37-50°C while mixing in a ribbon blender for 15 minutes. A German patent issued to Shields et a l (3?) - 7 -covers the production of dried milk and cream having instant properties. The milk powder particles were treated with 0.1-1% f i n e l y dispersed l e c i t h i n either during or after agglomera-tion of the powder. A, readily dispersible or "instant" whole milk powder with good free flowing characteristics has been produced by the New Zealand Dairy Research Institute (34). The milk powder was sprayed with a mixture of anhydrous milk fat and l e c i t h i n to give a level of 0.2$ l e c i t h i n i n the f i n a l powder which was heat tempered and rapidly cooled. Lecithin has been used also for manufacturing of instant soluble cocoa powder (8) by mixing the cocoa powder with 0.5-5% l e c i t h i n i n a rotating drum. Nelson et a l (25) observed that Tween 80, Tween 60 or l e c i t h i n when added to the whole milk or milk powder precon-densate at a level of 1% by weight of the f a t , improved s i g n i f i -cantly the s i n k a b i l i t y of powder i n water at 25°C. The sink-a b i l i t y increased through a range of 1-4% of the additives. However, at concentrations of 2,3» and 4% p a r t i a l churning of the fat took place during reconstitution. The greatest increase in s i n k a b i l i t y , with almost no occurrance of churning was attained with a mixture of Tween 80 and Atmos 300 (hydro-p h i l i c - l i p o p h i l i c balance of 8.0), added to the precondensate or mixed dry with the powder. However, the effect of commercial surface active agents had been investigated before publication of Nelson's and Winder's work. Hibbs and Ashworth (13) used surface active agents (Span 62, Tween 60, Myverol 1800, and Myverol 1885) to improve the recon-s t i t u t a b i l i t y of whole milk powder. These authors found that the incorporation of the above surface active agents to the precondensate at the rate of 0.05$ i n a reconstituted bases resulted i n churning. Samples stored at 7.2°C churned consider-ably a f t e r one month. . This churning could be remarkably ; reduced by storing at a higher temperature, i . e . , 29°C. Gibson and Raithby ( 12) observed a marked Increase i n w e t t a b i l i t y on adding polyoxyethylene sorbitan monolaurate (Tween 20 and Tween 21) to the precondensate (0.5-1$ of the t o t a l s o l i d s ) . However, the sorbitan monolaurate (Span 20) was almost i n e f f e c t i v e . The authors ascribed the e f f e c t of surface active agents either to a lowering of the surface tension of water, thus f a c i l i t a t i n g i t s penetration into the i n t e r p a r t i c l e spaces of the powder, or to an orien t a t i o n of these substances into a l a y e r on the powder p a r t i c l e surface, a t t r a c t i n g the water into the powder. Hollender ( 1^ ) and Mather and Hollender (19) Investigated the e f f e c t of surface active agents upon the s e l f ^ d i s p e r s i o n and churnability of dried whole milk i n more detail„ Tween 81 at a concentration of 0„1$ (added to the precondensate on a f l u i d milk bases) produced a r e a d i l y d i s p e r s i b l e powder0 However, an undesirable churning occurred during re c o n s t l t u t i o n by mechanical ag i t a t i o n . Both improved s e l f - d l s p e r s i b l l l t y and absence of churning during reconstltution were obtained at a proper hydro-p h i l i c - l i p o p h i l i c balance of surface active agents., This balance was achieved using a combination of two substances, e«.g., 0»05$ polyoxyethylene sorbitan monostearate (Tween 60) and 0.05$ sorbitan monostearate (Span 62). The e f f e c t of the i n t e r f a c i a l tension of butter o i l on the we t t a b i l i t y of the powder was studied by Baker et a l , ( 7 ). Their results showed that a l t e r a t i o n s i n the i n t e r f a c i a l tension of the f a t component of a milk powder by Span and Tween type emulsifiers did not i n i t s e l f exert a marked e f f e c t on wettabil-i t y . The f a t content of dried milk and i t s degree of unsaturation affects the w e t t a b i l i t y as well as the s e l f - d i s p e r s i o n of the powder. Self-dispersion of dry whole milk was 4.559, 1.448, 0.955 and 0.52?g of f a t contents of 1.4, 9.8, 27.0 and 34.9$ respectively (41). Reinke et a l , ( 3 5 ) found that reducing the f a t content of milk increased the s e l f - d i s p e r s i b i l i t y of the powder. Ashworth ( 3 ) found that i n freshly made fat-containing powders the effect of the fat content on the w e t t a b i l i t y was only small; the low, medium and high f a t contents (12, 25, and 38$) giving s i m i l a r high values f o r w e t t a b i l i t y . At low storage temperature (1.7 - 7.2°C) these powders maintained t h e i r high w e t t a b i l i t y even up to 2 years storage. However, storage at 29.4°C caused considerable de t e r i o r a t i o n of w e t t a b i l i t y l n medium and high fat powders. The dried skim milk showed a w e t t a b i l i t y of the same value i n both low and high storage temperatures which was not affected by the time of storage. A temperature-conditioning was developed by Bullock and Winder ( 9) which allows the recovery of the i n i t i a l reconstituta--Mo-b i l i t y possessed immediately after drying. This phenomenon was attributed to the physical rearrangement of the glycerides of the surface fat on the powder particles. Pyne, ( 31) has measured the contact angles of water drop-lets on the f l a t surface of highly compressed tablets of dry milk products. The non-fat dry milk surface was readily wettable as shown by a cosine of. 0.86. However, the wettability (cosine 0) decreased with an increase in the fat content. Pyne also showed that the surfaces of particles containing fat in the li q u i d state were more wettable (higher cos 0) than those containing fat in thessolid state 0 A process was devised for manufacturing Instant whole milk powder by coating the powder particles with the fraction of the milk fat which is li q u i d at a low temperature (l6-18°C) (24,2? ). The coating material may also contain skim milk solids, l e c i t h i n and/or a glyceride of vegetable origin.. I t was shown by Baker et a l ( 6) that a milk powder con-taining a low-melting (19-21°C) butter o i l fraction had approxi-mately the same wettability and d i s p e r s i b i l i t y at 24°Q as did a superior grade of instant skim milk powder. The interfacial tension (oil-water) of this low melting butter o i l fraction was lower than that of higher-melting fractions which yielded much less wettable milk powders. However, Stone et a l , (41) ooncluded from their studies on the self-dispersion of milk powders, in which the butter fat was replaced by corn o i l , that the superior d i s p e r s i b i l i t y of the powder was due to the fact -11-that the f a t was i n the l i q u i d state. Nelson and Winder (25) observed that the highest s i n k a b i l -i t y i n water at 25°C was always associated with the highest l i q u i d f a t content of spray dried whole milk. The temperature of water had l i t t l e e f f e c t on the s e l f -dispersion of spray-dried whole milk between 1.7°C to 32.2°C (41). However, between 35 and 37°5°C near the melting range of milk f a t , a steep increase i n s e l f - d i s p e r s i o n took place, followed by gradual Increase between 37°5 and 60°C. Similar s i g n i f i c a n t increases i n w e t t a b i l i t y of dried whole milk at water temperature of about 35°G was observed by others i Mol and de Vries , (22), Radema and van Dijk, (32) and Pavstova (10). -12-METHODS AND MATERIALS A l l measurements were performed i n d u p l i c a t e . Addition of L e c i t h i n and CommecEcial E m u l s i f i e r to the Powder An appropriate amount of l e c i t h i n * was weighed and d i s s o l v e d i n 10 ml. pet. ether, then t h i s s o l u t i o n was added to an appro-pr i a t e amount of commercial spray d r i e d whole milk powder** to give a f i n a l concentration of 2% l e c i t h i n (w/w) with continuous s t i r r i n g f o r about one minute. The powder was exposed to the a i r f o r 3-4 hours i n f l a t container at room temperature f o r pet. ether:evaporation. The same solvent was used f o r a d d i t i o n of Span type e m u l s i f i e r s and acetone f o r Tween type e m u l s i f i e r s because of t h e i r i n s o l u b i l i t y i n pet. ether. D i s p e r s i b i l i t y Test The method of Sinnamon et a l (38) was s l i g h t l y modified as follows: 4 gm. of milk powder was added to 40 ml. water at 4°C i n a 250 ml. beaker. The mixture was s t i r r e d by hand with a spatula f o r a predetermined time i n t e r v a l , 10, 20, 30, 40, and 50 seconds, dispersing as much material as possible, and poured through a c l o t h layer over a 200 mesh funnel with the a i d of about 15" vacuum. Five m i l l i t e r s of the f i l t r a t e was d r i e d i n an oven 93°C f o r 5 hours. Percent d i s p e r s i b i l i t y was c a l c u l a t e d . E x t r a c t a b i l i t y of L e c i t h i n from a Mixture of Casein and L e c i t h i n The mixture of a c i d casein and l e c i t h i n of d i f f e r e n t con-centrations was prepared and l e f t overnight at room temperature, * D i s t r i b u t e d by N u t r i t i o n a l Biochemicals Corporation, p u r i t y : Veg. L e c i t h i n 95% > Bovine L e c i t h i n 90%, Soybean L e c i t h i n ( o i l not removed ). ** KLIM Trade Mark, 28% b u t t e r f a t , 26.5% p r o t e i n , 37.2% l a c t o s e , 5.8% minerals and 2.5% moisture. -13-then freeze-dried. The mixture was prepared as follows: A. Acid casein, 2% was dissolv e d i n an imidazole buffer, pH 7.0. B. L e c i t h i n 0.2% was dispersed i n the same buffer by 1 min. sonication with Biosonik (Bronwill S c i e n t i f i c ) . C. Imidazole buffer, pH 7.0. Appropriate amounts of A,B and C were mixed to make casein-l e c i t h i n r a t i o s of 5, 10, 20, 30, 40, 50, 70, and 90 (mg/mg). The e x t r a c t a b i l i t y of l e c i t h i n from the freeze-dried mixture was measured by using three 4 ml portions of hexane-acetone mixture (4:1). The phosphorus content of the extracts was measured according to A l l e n ( 1 ) , with a s l i g h t modification as follows: The hexane-acetone solvent was transf e r r e d into a 10 ml Kjeldahl f l a s k , evaporated with gentle heat, and digested with 2.0 ml of 60% p e r c h l o r i c a c i d . The digest was then d i l u t e d with 2 ml of the amidol reagent and 1 ml of the ammonium molybdate s o l u t i o n and tra n s f e r r e d q u a n t i t a t i v e l y to a 25 ml v o l . f l a s k with d i s t i l l e d water. The blue c o l o r produced was measured a f t e r 12 minutes i n a Beckman DB Spectrophotometer at 690 mu.. The amount of phosphorus present was read from a c a l i b r a t i o n curve prepared by the same procedure f o r a l i q u o t s of a standard phosphate s o l u t i o n containing from 0.01 to 0.2 mg P. Reagents Pe r c h l o r i c a c i d , 60% s o l u t i o n sp. g r a v i t y 1.54-. Ammonium molybdate s o l u t i o n , 8.3% s o l u t i o n . A small quantity of concentrated ammonium hydroxide was added to f a c i l i t a t e s o l u t i o n . Amidol reagent, 2 gm. of amidol and 4 0 gm. of sodium b i -sulphite dissolved i n g l a s s - d i s t i l l e d water and di l u t e d to 200 ml. The reagent was stored i n a well-stoppered b o t t l e painted black outside. This solu t i o n was used within 10 days. Standard phosphate solution, a stock solution containing 1 mg. P per ml prepared by di s s o l v i n g I.O967 gm KH2P0^ (dried:, in an a i r oven) i n d i s t i l l e d water and d i l u t i n g to 250 ml. and stored at 4°C 0 Ultracentrlfugatlon Schlieren patterns of acid casein with and without l e c i t h i n were obtained i n a Beckman L2-65B preparative ultracentrlfuge at 20°C with Schlieren optics. One percent acid caseins with and without 0.35$ l e c i t h i n were prepared i n 0.08 M Imidazole buffer, pH 7 . 0 . Centrifuge speed was 60,000 rpm. Polyacrylamide Gel Electrophoresis Polyacrylamide g e l electrophoresis was performed by a modi-f i c a t i o n of Aschaffenburg's method ( 2 ) c The gel was prepared as follows; 10 .5 gm acrylamide and 0.52 gm N, N-methylenebis-acrylamide were dissolved i n 0 o l M T r i s - g l y c i n e buffer (pH 9»1) and made up to a f i n a l volume of 150 ml. This solution was filterm-ed through No. 1 f i l t e r paper into a vacuum f l a s k . A f t e r the. deaer ation 1.0 ml. TMED (30$ N„ N, N ' , N'-tetramethylene ethylene diamine In 95$ ethanol) and 1.25 ml of 10$ ammonium persulfate were added to the solution, mixed gently, poured immediately into a mold, covered with a perspex plate and weighed down to expel a l l the a i r . The gel was allowed to stand f o r 30 to 6 0 minutes -15-to permit completion of polymerization. A horizontal electro-phoresis apparatus, with double containers at either end of the gel was used. A sodium chloride solution, 0.1M and 0o175M Tris-glycine buffer were placed i n the outer and the inner con-tainers, respectively. The containers were connected with eight folds of cheesecloth. The gel plate was placed on the electro-phoresis apparatus and equilibrated by running the current through the gel for 20 hours at 4 °C. The amperage was maintained at 20 mA on a power supply. Preparation of Sample A mixture of 2% acid casein and 0o5% l e c i t h i n was prepared i n 0.175M Tris-glycine buffer i n addition to the control (2% acid casein in buffer only) 0 Strips of Wahtman 3Mrl f i l t e r paper 1.5 x 10mm, which were soaked i n the casein-lecithin solution after 24 hours, or the control sample and the excess blotted, were inserted in the slot on the equilibrated gel plate. The gel plate was covered with Saran f i l m to prevent drying. Elec^ trophoresis was carried out under the same conditions as the equilibration of the gel, for 24 hours i n a cold room,, The gel removed from the mold was stained i n 0.2% amide black 10 B, solution containing 45% methanol and 9°8% acetic acid for 5 minutes and then destained by washing i n 10% acetic acid u n t i l a clear gel background was obtained. Sinkability test The s i n k a b i l i t y was determined according to Bullock and Winder ( 9 ) with a s l i g h t modification as follows? Materials: A. A 15 by 20 cm. funnel having an outlet of 1 cm. In diameter to which a short rubber tubing with a pinch clamp was attached. B. A glass bottle 2.5 cm. i n diameter and 5 cm. deep. A piece of screen soldered on the open end of the bottle to sprinkle dried milk on water surface. C. A stop watch. Method: Five hundred milligrams-of dried milk were weighed Into the small glass b o t t l e . F i f t y m i l l i l i t e r s of d i s t i l l e d water at 25°C were placed in the funnel with a pinch clamp i n place. A 500 ml. Erlenmeyer f l a s k was positioned beheath the o u t l e t . The dried milk was sprinkled quickly and evenly onto the quies-cent water surface. A f t e r a predetermined time i n t e r v a l , usually 15-30 seconds, the pinch clamp was opened and the portion of the dried milk which had sunk was drawn o f f quickly with the water onto the Erlenmeyer flask, leaving behind that f r a c t i o n of the sample which had not sunk. After complete solution of the sample i n the f l a s k , a 5 nil. aliquot was removed and t o t a l solids were determined by drying at 93°C for 5 hours, to ascertain the f r a c t i o n of the sample which sank. The results were expressed as percent s i n k a b i l i t y and were calculated as follows: weight of s o l i d s i n 5 ml. a l i q u o t x50xl00 percent s i n k a b i l i t y = 5x weight of o r i g i n a l sample Free-flowability of the Powder The static measurement of free-flowability for the milk powders has been done according to Sjollema (40): A funnel with a narrow stem was mounted exactly 2 cm. above a piece of uniform paper lying on a horizontal f l a t table. Powder having uniform particle size, 40-60 mesh, was moved down with an ele c t r i c a l vibrator feeder in a very fine stream into the funnel from which i t f e l l on the paper, so that a conic heap was formed. When the top of the powder met the end of the funnel stem, the powder stream was stopped, and the base of the powder heap was outlined with a pencil. After removing the powder, the outlined paper c i r c l e was cut out and weighed. As weight per unit area of paper i s known, the area of the circular base, and thus the radius were calculated. Then the angle of repose was calculated according to the following formulaJ tan. &L = h/( r-|a) where: @t = angle of repose h = height of stem above base = 2 cm. r = radius of the base of the powder heap a = diameter of the funnel stem = 0.3 cm. Iodine Value of Lecithin Iodine value was measured by the o f f i c i a l method of A.O.AoC. ( 5 ) . Interfacial Tension of Butter Oil-(I.T.) Interfacial tension of butter o i l at 40°C was measured accord-ing to Baker et a l (7) using DuNoy tensiometer. DuNoy tensiometer was used also for measuring the surface tension of water. RESULTS Effec t of L e c i t h i n on the D i s p e r s i b i l i t y of Dried Whole Milk  Powder L e c i t h i n of d i f f e r e n t o r i g i n s (bovine, vegetable, soybean —>• refined and unrefined) was added to commercial spray dried whole milk powder, and the d i s p e r s i b i l i t y t e s t was performed,, Figure 1 shows the d i s p e r s i b i l i t y curve f o r the powder with and without l e c i t h i n . A l l the samples containing d i f f e r e n t l e c i -thins reached almost the maximum s o l u b i l i t y within 1/5 of the time required f o r the control„ The e f f e c t of treatment with l e c i t h i n s on d i s p e r s i b i l i t y of whole milk powder did not s i g n i f i -cently d i f f e r , except In the case of powder treated with refined soy l e c i t h i n which showed a s l i g h t l y increased d i s p e r s i b i l i t y compared to powder treated with other l e c i t h i n s (Appendix Table 1 and Figure 1)„ E f f e c t of Commercial Emulslfler on the D i s p e r s i b i l i t y of Dried Whole Milk In an attempt to f i n d the re l a t i o n s h i p between the effect, of adding l e c i t h i n and commercial emulsiflers on the reco n s t i t u -t a b i l i t y of dried whole milk. Span and Tween type emulsiflers were used on spray dried whole milk i n the same manner as i n the case of l e c i t h i n . Figures 2 and 3 indicate the d i s p e r s i b i l i t y curves of dried whole milk treated with Span and Tween type emulsiflers, respectively. I t has been found that Span 8 5 , 80 and 20 improved the d i s p e r s i b i l i t y greatly without any s i g n i f i c a n t difference between emulsiflers. However, Span 40 did not show any improvement instant skim milk powder. whole milk powder containing 2% refined s o y ~ l e c i t h i n . whole milk powder containing 2% bovine l e c i t h i n , whole milk powder containing 2% soy l e c i t h i n , whole milk powder containing 2% vegetable lecithin,, whole milk powder without adding l e c i t h i n . Pig. 1 . D i s p e r s i b i l i t y rates f o r whole milk powder, Instant skim milk powder, and whole milk powder containing 2% l e c i t h i n s of d i f f e r e n t o r i g i n s . 40 SECS. -20-and i t s treated powder showed no s i g n i f i c a n t d i f f e r e n c e with the control (Appendix Table II and Figure 2). On the other hand the d i s p e r s i b i l i t y was improved by Tween 20, Tween 80, and Tween 40, almost to the same extent'with no s i g n i f i c a n t d i f f e r e n c e , where-as Tween 60 showed no improvement when compared with the c o n t r o l (Appendix Table I I I and Figure 3)» E f f e c t of D i f f e r e n t L e c i t h i n s and E m u l s i f i e r s on the I n t e r f a c i a l  Tension of Butter O i l and the Surface Tension of Water In order to study ./the mechanism involved i n the improvement of the r e c o n s t i t u t a b i l i t y of d r i e d whole milk by l e c i t h i n , butter o i l containing d i f f e r e n t concentrations of l e c i t h i n s and emulsifiers was prepared and the i n t e r f a c i l a tension (I.T.) be-tween o i l and water f o r butter o i l at 40°C was measured (Tables I and I I ) o I t i s obvious from these tables that d i f f e r e n t l e c i t h i n s show d i f f e r e n t e f f e c t s on the I.T. of butter o i l . The one from bovine decreased remarkably the LT, The decrease by l e c i t h i n s from other sources eras l e s s than bovine l e c i t h i n , the surface tension (S.T.) of water containing l e c i t h i n and emulsifiers was also measured at 25°C and i s presented i n Tables X I and IIo Bovine l e c i t h i n decreased the surface tension of water more than other l e c i t h i n s d i d , whereas, d i f f e r e n t Span and Tween emulsifiers had almost the same e f f e c t on the I.T. of butter o i l , and the S.T. of water *(Table I I ) . 21-( o O ) Span 80 ( A — A ) span 85 ( O — O ) Span 20 (O O) Span 40 ( e O) Control F i g . 2. D i s p e r s i b i l i t y rates f o r whole milk powder containing 2% d i f f e r e n t Span type emulsifiers. 100 10 20 30 40 STIRRING T I M E (SECS.) o—-® o — 0 G— a 0 # Tween 20 Tween 80 Tween 40 Tween 60 Control F i g . 3. D i s p e r s i b i l i t y rates f o r whole milk powder containing 2% d i f f e r e n t Tween type emulsifiers. TABLE I The iodine value of d i f f e r e n t l e c i t h i n s and t h e i r e f f e c t on the i n t e r f a c i a l tension ( o i l -water) of butter o i l , the surface tension of water and the s i n k a b i l i t y of dried whole milk Surface Tension of Water Containing I n t e r f a c i a l Tension % S i n k a b i l i t y 0.050% (oil-water) dyne/cm at tO°C of Powder Iodine L e c i t h i n Concentration of L e c i t h i n i n Butter Containing Type of Value of (dyne/cm.) O i l (%) 2% L e c i t h i n No. L e c i t h i n L e c i t h i n at 25°C .0000 .0312 .0625 .1250 .2500 (30 sec, t e s t time) 1 Bovine 40.9 42.8 24.5 18.8 5.3 4,1 3.8 98 Le c i t h i n I 2 Vegetable 62.5 63.3 24.5 20.4 19.3 17.2 14.6 34 '/«. Le c i t h i n Soybean 85.9 65,0 24.5 21.6 20.4 17.5 16.0 26 Le c i t h i n Refined 67.0 68.0 24.5 21.9 20.1 16.4 15.4 16 soy L e c i t h i n Control 72.0 a.5 I TABLE II E f f e c t of Span and Tween type e m u l s i f i e r s on the I.T. of butter o i l and the S.T. of water and the d i s p e r s i b i l i t y of powder Surface tension of water % disper-containing I n t e r f a c i a l tension ( o i l - s i b i l i t y Melting 0.10% water) dyne/cm. at 40°C of powder containing 2% emulsi-Type of emsulsi- HLB 2 value point^ of emulsi-e m u l s i f i e r dyne/cm. 25°C Cone. of e m u l s i f i e r (%) i n butter o i l f i e r No. f i e r s C 0.000 0.031 0.062 0 .125 0.250 f i e r l 20 8.6 l i q u i d 3 33.2 24.8 21.3 20.0 17.5 13.4 6 7.8 21.5 |; 91.-6 Spans 40 80 6.7 4.3 46 l i q u i d 43.5 33.4 24.8 24.8 21.4 21.5 18.6 21.4 17.2 18.5 12.5 15.7 85 1.8 l i q u i d 34.5 24.8 21.0 21.0 20.5 19.3 70 .4 Cone, of e m u l s i f i e r i n butter o i l (%) 0.000 0.015 0.02 0.030 0.060 20 16 .7 l i q u i d 39.5 24. 8 14.9 11.5 11.9 4.6 87.3 40 15.6 l i q u i d 39.1 24.8 15.0 11.2 8.1 4.0 61. 3 60 14.9 24 42.4 24.8 14.7 10.0 7.5 4.2 52.8 80 15.0 l i q u i d 39 .7 24.8 13.2 8.0 8.6 3.8 75.5 % d i s p e r s i b i l i t y as shown on F i g . 3 and 4 a f t e r 10 sec. s t i r r i n g time Reference No. (35) -25-Iodine Value of L e c i t h i n and i t s E f f e c t on the F r e e - f l o w a b i l i t y  of Whole Milk Powder The angle of repose f o r whole milk powder treated with d i f f e r e n t l e c i t h i n s having uniform p a r t i c l e s i z e was determined as a measure of f r e e - f l o w a b i l i t y of the powder. Table I I I shows the angle of repose of the powders treated with d i f f e r e n t l e c i t h i n s . I t has been found that the sa.mple treated with bovine l e c i t h i n y e i l d e d an angle of repose very close to that of instant skim milk powder while l e c i t h i n s from other o r i g i n s did not improve the f r e e - f l o w a b i l i t y of the powder extensively except vegetable l e c i t h i n . There was an inverse r e l a t i o n s h i p between the iodine value which r e f l e c t s the melting point of the l e c i t h i n and the f r e e - f l o w a b i l i t y of the powder (Figure 4). Hydrophylic-lypophilic Property of Milk Constituents The hydrophilic property of the protein i s very important f o r the d i s p e r s i b i l i t y of milk powder ( 1 6 ) . Attempts were made to check whether there i s any i n t e r a c t i o n between the casein and l e c i t h i n which might improve the h y d r o p h i l i c property of the protein. The i n t e r a c t i o n was investigated by polyacrylamide gel electrophoresis, u l t r a c e n t r i f u g a t i o n and the e x t r a c t a b i l i t y of l e c i t h i n from the casein and from the d r i e d skim milk treated with l e c i t h i n . However, no i n d i c a t i o n of i n t e r a c t i o n between the casein and the l e c i t h i n was detected. The mobility on electrophoretogram and the sedimentation coefficient'.of acid casein did not change a f t e r adding l e c i t h i n . And no decrease i n the e x t r a c t a b i l i t y of the l e c i t h i n was observed f o r the c a s e i n - l e c i t h i n mixture and f o r the d r i e d m i l k - l e c i t h i n mixture (Figure 5)<> -26-( O ) soy l e c i t h i n ( O ) refined, soy l e c i t h i n ( D ) vegetable l e c i t h i n ( A ) bovine l e c i t h i n F i g . 4. Iodine value of l e c i t h i n and i t s e f f e c t on the free-f l o w a b i l i t y (angle of repose) of whole milk powder containing 2% l e c i t h i n 41 45 4 9 53 5 7 A N G L E OF REPOSE IN DEGREES -2?-TABLB I I I Angle of repose of ivhole milk powder treated with 2% d i f f e r e n t l e c i t h i n s L e c i t h i n Treatment A n g l e of Repose (degrees) Control Bovine l e c i t h i n Vegetable l e c i t h i n Soybean l e c i t h i n Ref. soybean l e c i t h i n Inst, skim milk a 41* b 46^ a' 5H a 5H a 40^ b 'a 8 and 8b' are two groups s i g n i f i c a n t l y d i f f e r e n t (Appendix Table V). -28-E f f e c t of P a r t i c l e Size on the S i n k a b i l i t y of the Treated Powder Powders treated with l e c i t h i n s or commercial e m u l s i f i e r s were fract i o n a t e d into d i f f e r e n t size p a r t i c l e s (130-500 ) by sieve permeametry as i t i s possible that, these ad d i t i v e s change the p a r t i c l e s i z e of the powder (Figure 9 ) . The sink-a b i l i t y of the fr a c t i o n a t e d powders treated with these additives was measured. Figure 6 shows the r e s u l t s of t h i s t e s t . I t i s noticed that the p a r t i c l e s i z e did not a f f e c t the s i n k a b i l i t y of the control sample. The additive that d i d not improve the d i s p e r s i b i l i t y of the powder, Span 40, revealed no e f f e c t as we l l . However, the p a r t i c l e s i z e d i s c l o s e d a marked e f f e c t on the s i n k a b i l i t y of powder treated with the additives capable to improve the d i s p e r s i b i l i t y of the powder (Span 20, Tween 20, bovine l e c i t h i n ) . The p a r t i c l e s i z e of powder treated with additives which improved the d i s p e r s i b i l i t y to the l e s s e r extent revealed less e f f e c t on the s i n k a b i l i t y of the powder (Tween 60). Concentration of Vegetable L e c i t h i n and the S i n k a b i l i t y and  D i s p e r s i b i l i t y of the Powder The e f f e c t of d i f f e r e n t concentrations of vegetable l e c i t h i n on the s i n k a b i l i t y and d i s p e r s i b i l i t y of the powder i s seen i n Figure 7. The s i n k a b i l i t y was e f f e c t i v e l y improved at concentra-tions higher than 1%. On the other hand, l e c i t h i n has a most pronounced e f f e c t upon d i s p e r s i b i l i t y at lower concentrations, e.g., 0.25%, followed by a plateau (Figure 7). The data f o r the s i n k a b i l i t y t e s t f i t the equation y = a + b log x with a -29-F i g . 5» E x t r a c t a b i l i t y of l e c i t h i n from c a s e i n - l e c i t h i n mixtures of d i f f e r e n t weight ratio(mg/mg)• 30 50 70 C A S E I N / L E C I T H I N nig/,™ -30-Pig. 6 . E f f e c t of p a r t i c l e s i z e on the s i n k a b i l i t y (15 sees, test time) of whole milk powder containing 2% Span 2 0 ( &= — © ) , Tween 2 0 (^—^ ), bovine l e c i t h i n (O—S 3 ) s Tween 6 0 ( 0 — O ) . refined soy l e c i t h i n ( &—ffl ), Span 4-0 (^—A.), and control ( ). -31-c o r r e l a t i o n c o e f f i e i e n t of 0.97 (Appendix Table IV). The l e c i t h i n content i n p a r t i c l e s separated from powder treated with 2% vegetable l e c i t h i n increased c u r v i l i n e a r l y with p a r t i c l e s i z e from 100 to 300/*(then declined s l i g h t l y to 500/ f^o L e c i t h i n content ranged between 1.27% and 2.4% (Figure 8). - 32 -F i g . 7 , E f f e c t of concentration of l e c i t h i n (vegetable l e c i t h i n ) on the s i n k a b i l i t y (30 secs 0 test time) and the d i s p e r s i b i l i t y of whole milk powder. F i g . 8. D i s t r i b u t i o n of l e c i t h i n as related to p a r t i c l e s i z e of dried whole milk powder treated with 2% vegetable l e c i t h i n . M L E C I T H I N t o 00 b KJ t o § t o to O o I o o - 3 4 -Powder treated with 2% vegetable l e c i t h i n Control Figure 9» D i s t r i b u t i o n of p a r t i c l e s i z e of the dried whole milk before and a f t e r t r e a t i n g v/ith 2% vegetable l e c i t h i n . -35-DISCUSSION Asworth and Bendixen (3 ) reported that the physical state of casein is the l i m i t i n g factor during the dispersion of dried whole milk. J u l i e n and Baker (16) were able to improve the w e t t a b i l i t y of dried whole milk by l i m i t e d proteolysis of the whole milk before drying (with Rhozyme P 11). These research-ers suggested that t h i s enhancement i n w e t t a b i l i t y was due to a modification of the protein on the m l c e l l a r surface which resulted i n increased hydrophilic character. The r e c o n s t i t u t a b i l i t y of dried whole milk powder was improved considerably: a f t e r the addition of l e c i t h i n . L e c i t h i n , being an amphiphile, is capable of interacting with milk proteins through hydrogen bonding, e l e c t r o s t a t i c i n t e r a c t i o n or hydrophobic i n t e r -action. Such interaction should lead to a more hydrophilic ca-sein micelle. The properties of mixture of various l e c i t h i n and dried, whole milk powder were investigated to determine the l e v e l and type of interaction, i f any, and also to evaluate the e f f e c t of the various l e c i t h i n s on the physical properties of the dried whole milk powder. For measuring the interaction between l e c i t h i n and acid casein, d i f f e r e n t techniques were used, such as the sedimentation analysis, gel electrophoresis and e x t r a c t a b i l i t y of l e c i t h i n from a mixture of acid casein and l e c i t h i n . However, there was no difference i n the sedimentation c o e f f i c i e n t of acid casein and a mixture of acid casein and l e c i t h i n . Their m o b i l i t i e s on - 3 6 -the polyacrylamide gel electrophoretegram were i d e n t i c a l . There was no v a r i a t i o n i n e x t r a c t a b i l i t y of l e c i t h i n from d i f f e r e n t c a s e i n - l e c i t h i n mixtures (Figure 5). From these r e s u l t s i t was concluded there was no chemical i n t e r a c t i o n between l e c i t h i n and acid casein. Therefore, the physical c h a r a c t e r i s t i c s of the powder were investigated. The d i s p e r s i b i l i t y t e s t f a i l e d to show any s i g n i f i c a n t differences among the milk powders treated with d i f f e r e n t l e c i t h i n s except i n the case of powder treated with r e f i n e d soy l e c i t h i n which showed a s l i g h t l y increased d i s p e r s i b i l i t y com-pared to powders treated with other l e c i t h i n s . Thus the sink-a b i l i t y t est was used (Figure 7) which does not require the use of mechanical force i n the t e s t . S i n k a b i l i t y i s the ease with which the powder p a r t i c l e s make contact with water. I t i s generally accepted that an adequate s i n k a b i l i t y i s the pre-r e q u i s i t e f o r good d i s p e r s i b i l i t y and sometimes the r e c o n s t i -t u t a b i l i t y i s measured i n terms of s i n k a b i l i t y (17). When l e c i t h i n s were added to milk powder, a change i n the secondary structure* was observed, i n which the size d i s t r i -bution of the powder p a r t i c l e s was changed and p a r t i c l e s having a diameter between 100-500/4were produced (Figure 9). However, most of the published data gave an average p a r t i c l e s i z e i n the *According to King(17), dried milk e x h i b i t s a well-pronounced dual physical structure, the structure of the i n d i v i d u a l p a r t i c l e containing the milk s o l i d s and small amounts of moisture and a i r d i s t r i b u t e d i n the state (primary s t r u c t u r e ) , and the structure of the bulk of the dried p a r t i c l e s , which represents a t y p i c a l powder, a system of cl o s e l y packed s o l i d p a r t i c l e s i n a gas (secondary s t r u c t u r e ) . - 3 7 -range of 50-110,4 f o r spray d r i e d whole milk powder (15,17). When the s i n k a b i l i t y t e s t f o r d i f f e r e n t s i z e p a r t i c l e s of l e c i t h i n treated powder was performed, the p a r t i c l e s i z e showed a great e f f e c t on the s i n k a b i l i t y of the powder i n that the greater the p a r t i c l e s i z e , the higher was the s i n k a b i l i t y (Figure 6). However, the same range of p a r t i c l e size (100-500/V) of the whole milk powder without the addition of l e c i t h i n (pre-pared by moistening the powder to 10% water and re-drying i n a vacuum oven at 40°C) d i d not show any e f f e c t on the s i n k a b i l i t y of the powder (Figure 6). 'This was i n agreement with the r e s u l t s obtained by Swanson (4-2) who found no c o r r e l a t i o n between the average p a r t i c l e - s i z e of dried whole milk powder and i t s r e -const i t u t a b i l i t y . On the other hand, when the amount of l e c i t h i n i n the d i f f e r e n t s i z e p a r t i c l e s was measured, i t was found that the amount of l e c i t h i n i n these f r a c t i o n s was i n the range of 1.27 and 2.35% i n d i c a t i n g that the greater the p a r t i c l e s i z e , the higher was i t s l e c i t h i n content (Figure 8). Moreover, high-er concentrations of l e c i t h i n i n the powder Increased the sink-a b i l i t y and d i s p e r s i b i l i t y (Figure 7)» Thus, i t i s obvious that there are reasons other than the change i n the p a r t i c l e s i z e involved i n the improvement of the r e c o n s t i t u t a b i l i t y of the powder by adding l e c i t h i n . The f a t content of dried milk may a f f e c t the r e c o n s t i t u t a -b i l i t y of the powder. Ashworth (4) found that milk powders with low, medium and high f a t content (12%, 25% and 34%),;showed d i f -ferent a b i l i t y i n maintaining t h e i r w e t t a b i l i t y during storage. A considerable d e t e r i o r a t i o n i n w e t t a b i l i t y was noticed i n medium and high f a t powders. However, the low f a t powder did not show any change In i t s w e t t a b i l i t y . S e l f - d i s p e r s i o n i s also affected by the fat content. Stone et al_ (4l) shows that s e l f - d i s p e r s i o n of milk powder containing 1.4$ f a t was diminished from 4.559 gm to 0.527 gm when the f a t content increased to 3^*9$. Reink et a l (33) also found, that reducing the f a t content of the powder increas-ed the se l f - d i s p e r s i o n . With instantized dried whole milk powder, the d i s p e r s i b i l i t y was lowest at 26$, when compared with f a t content of 10$, 5$, 2$ and 1$ (23). As to the e f f e c t of the com-position of milk f a t on the d i s p e r s i b i l i t y of the powder, i t appears that the w e t t a b i l i t y and the d i s p e r s i b i l i t y increased when the melting point was lower. This phenomena was observed by Stone et a l (4l), Baker et a l ( 6),. and Nelson et a l (25). Bullock and Winder (9 ) found that the s i n k a b i l i t y of dried milk powder was greatly affected by the temperature treatment immediately a f t e r drying. They devised a temperature condi-tioning treatment which secured the maximum s i n k a b i l i t y . The assumption was that a r e d i s t r i b u t i o n of solids and l i q u i d fractions of the milk f a t takes place on the surface of the p a r t i c l e s . By heating milk powder, surface f a t i s melted, and tri g l y c e r i d e s rearrange themselves i n a random manner. Rapid c h i l l i n g then s o l i d i f i e s f a t and the s o l i d and l i q u i d t r i g l y c e r -ides are fixed i n a random d i s t r i b u t i o n . Therefore, the nature of the surface of p a r t i c l e s and the kind of compounds present on the surface i s considered to be a l i m i t i n g f a c t o r on the re-c o n s t i t u t a b i l i t y of dried whole milk powder. When d i f f e r e n t commercial emulsifiers(Span and Tween) having an HL3 value i n the range of 1.8-l6.7were added to milk powder, -39-i t was observed that a l l emulsifiers having a low melting point which existed i n a l i q u i d form at 20°C showed greater improvement in the d i s p e r s i b i l i t y of the powder (Table I I ) . However, d i s -p e r s i b i l i t y of the powder treated with Span 40 , an emulsifier having a melting point of 46°C did not indicate s i g n i f i c a n t d i f -ference with the control. Tween 60, which has the highest melting point, 2 4°c , i n the group also did not show a s i g n i f i c a n t d i f -ference with the control. These results suggest that the degree of unsaturation i n the fa t t y acid constituent of the l e c i t h i n i s probably important for i t s a b i l i t y to improve the d i s p e r s i b i l i t y of the powder. However, i t was found that l e c i t h i n s having lower iodine values (less unsaturated f a t t y acid) revealed greater a b i l i t y to improve the s i n k a b i l i t y of the powder (Table I ) . Since refined soy l e c i t h i n showed s i g n i f i c a n t l y a greater ef f e c t i n improving the d i s p e r s i b i l i t y of milk powder than other l e c i t h i n s (Figure 1), the low s i n k a b i l i t y of i t s treated powder may be due to a cohesion of powders re s u l t i n g in a poor flow c h a r a c t e r i s t i c . The angle of repose of the powder was 5 1 ^(Table I I I ) . The same was true for powder treated with soy l e c i t h i n . As soon as cohesive powder is moistened, portions of the powder become coated with the l i q u i d outside, leaving a pocket of trapped a i r inside which delays i t s s i n k a b i l i t y . Bovine l e c i t h i n which has the lowest iodine value, improved s i g n i f i c a n t l y the free-flowingness of the powder of which angle of repose was very close to that of instant skim milk powder (Table I I I ) . This powder exhibited, the best s i n k a b i l i t y . It is necessary for surface active agents to locate on the surface -40-of the powder p a r t i c l e s i n order to improve the d i s p e r s i b i l i t y (14). A method f o r adding l e c i t h i n or commercial emulsifier to milk powder i n this study w i l l comply with this requirement. Gibson and Halthby (L2 ) ascribed effects of surfactants either to lowering of the surface tension of water, thus f a c i l i -tating i t s penetration into the p a r t i c l e spaces of milk powder, or to an orientation of these substances into a layer on the powder p a r t i c l e surface a t t r a c t i n g the water into the powder. Furthermore, the findings of Baker and Samuel ( ? ) indicate that the lower melting f a t f r a c t i o n has lower i n t e r f a c i a l tension towards water than the higher ones suggesting a possible r e l a t i o n -ship between the i n t e r f a c i a l tension of f a t and the w e t t a b i l i t y of milk powder. In a more detailed investigation, they came to the conclusion that rather the combination of two factors, the physical state and the i n t e r f a c i a l tension of the fat,are of importance. Being aware of these assumptions the experiments were conducted to investigate the e f f e c t of l e c i t h i n s and commer-c i a l emulsifiers on the surface tension of water and the i n t e r -f a c i a l tension of butter o i l , and the rela t i o n s h i p with the s i n k a b i l i t y or d i s p e r s i b i l i t y of milk powder,, I t was found that the l e c i t h i n having a greater a b i l i t y to improve the s i n k a b i l i t y of milk powder alters'considerably the i n t e r f a c i a l tension of butter f a t and the surface tension of water (Table I ) . When the same test was done with commercial emulsifiers (Tween and Span) the a b i l i t y of these emulsifiers to a l t e r the i n t e r f a c i a l tension of the butter o i l and the surface tension of water was not s i g -n i f i c a n t l y correlated with t h e i r improving a b i l i t y i n the d i s --41 p e r s i b i l i t y of milk powder. I t was also noticed that the a l t e r a -tion i n the i n t e r f a c i a l tension was dependent on the concentra-tion. Generally the Span type was l e s s e f f e c t i v e than the Tween type emulsifiers. At a concentration of 0.06$ Tween 20, 40, 60, and 80 decreased the i n t e r f a c i a l tension of butter o i l from 24.8 to 4.6, 4.0, 4.2 and 3.8 dyne/cm respectively (Table I I ) . Mean-while a concentration of 0.25$ was required f o r Span 20, 40, 80 and 85 to decrease the i n t e r f a c i a l tension of butter o i l from 24.8 to 13.4, 12.5, 15»7 and 19«3 dyne/cm, respectively. However, there was almost no c o r r e l a t i o n observed between the e f f e c t of commercial emulsifiers on the surface tension of water and t h e i r a b i l i t y to improve the d i s p e r s i b i l i t y of milk powder. A l l the Tween and the Span type emulsifiers decreased the surface tension of water to almost the same extent at a concentration of 0.1$ (Table II). On the other hand, the l e c i t h i n s that were more eff e c t i v e in decreasing the surface tension of water showed greater improvement i n the s i n k a b i l i t y of milk powder (Table I ) . As l e c i t h i n s dissolved i n an organic solvent was added to milk powder i t i s possible that the l e c i t h i n was d i s t r i b u t e d on the surface of powder p a r t i c l e s enhancing the surface cohesiveness of powder upon evaporation of the solvent. The p a r t i c l e s cohered forming porous aggregates which contain more l e c i t h i n than the unaggregated p a r t i c l e s . During the reconstitution of t h i s powder the l e c i t h i n orienting on the p a r t i c l e surface attracts the water into the powder, and a l t e r s the S.T. of water and I.T. of butter o i l . Other factors might be involved i n this improve-ment such as the degree of unsaturation of the f a t t y acid content -42-of the surface active agents which seems to be the predominant factor i n improving the d i s p e r s i b i l i t y of the powder treated with Tween and Span type emulsifiers. -43-SUMMARY Studies were conducted to observe the changes i n the r e c o n s t i t u t a b i l i t y of dried whole milk when d i f f e r e n t l e c i t h i n s or commercial Span and Tween type emulsifiers were added to the powder at ?:% concentration. D i s p e r s i b i l i t y and s i n k a b i l i t y tests were used as a measure of r e c o n s t i t u t a b i l i t y . In the d i s p e r s i -b i l i t y test the powder containing d i f f e r e n t l e c i t h i n s reached almost the maximum s o l u b i l i t y within 1/5 the time required for the control. D i f f e r e n t Span or Tween type emulsifiers re-vealed d i f f e r e n t effects on the d i s p e r s i b i l i t y of the powder. However, a d i f f e r e n t degree of improvement i n the s i n k a b i l i t y was observed when the powder was treated with 2% l e c i t h i n from d i f f e r e n t sources, or with 2% Span and Tween type emulsifiers. No evidence f o r casein l e c i t h i n interaction was observed. However, i t was found that the powder p a r t i c l e s were agglomerated p a r t i a l l y and a change i n the p a r t i c l e size d i s t r i b u t i o n was observed. Moreover, the p a r t i c l e size i t s e l f showed no e f f e c t on the s i n k a b i l i t y of the control powder (without adding l e c i -t h i n ) . It was found that those l e c i t h i n s which improve the s i n k a b i l i t y of the powder remarkably decrease the i n t e r f a c i a l tension of butter o i l and surface tension of water more than do those which show very l i t t l e improvement i n the s i n k a b i l i t y . Several Span and Tween type emulsifiers revealed almost the same ef f e c t on the i n t e r f a c i a l tension of butter o i l , and the -44-surface tension of water. I t i s concluded that the state of emulsifiers, whether i t i s l i q u i d or s o l i d , i s the c o n t r o l l i n g factor i n improving the d i s p e r s i b i l i t y of the powder. But the a l t e r a t i o n i n the t e r f a c i a l tension of butter o i l and the surface tension of water by l e c i t h i n s would play an important role i n the sinka-b i l i t y of the powder. -45-APPSNDIX -46-TABLE I Analysis of Variance i n D i s p e r s i b i l i t y of Powder Treated with Lecithins Source of variation Degree of freedom Mean square F-ratio Lecithins 4 194-5.7 627.19** S t i r r i n g time 4 570.45 183.79** V x T 16 209.23 67.41** Error 25 3.1038 Total 49 ** Significant at probability 0.01 Duncan New Multiple Range Test's— D i s p e r s i b i l i t y of powder treated with bovine l e c i t h i n , vegetable l e c i t h i n and soy l e c i t h i n were homogeneous (did not d i f f e r by more than the shortest significant range). Lecithin treatment Control r e f. soy. veg. bov. soy. lec, lec. lec. Mean value 63.19 97.81 97.67 91.58 93.70 S t i r r i n g time(secs) 10 20 50 40 50 Mean, value 75.67 85.99 89.72 92.68 94.89 -47-TABLE II Analysis of Variance i n D i s p e r s i b i l i t y of Powder Treated with Span Type Emulsifiers Source of v a r i a t i o n Degree of freedom Mean square P-ratio Span emulsifiers 4 1455.500 15.85** S t i r r i n g time 4 960.970 9.80** Error 16 98.022 TOTAL 24" ** S i g n i f i c a n t at p r o b a b i l i t y 0.01 Duncan:.New Multiple Range Test:-Two homogeneous sets were shown: 1. D i s p e r s i b i l i t y of powder treated with Span 20, 80, and 85. 2. D i s p e r s i b i l i t y of powder treated with Span 40 and contro l . Span treatment Control 20 4-0 80 8 5 Mean value 53.48 82.78 54.12 94.00 88.68 S t i r r i n g time (sees) 10 20 30 40 5 0 Mean value 54.78 72.04 80.80 85.86 89.58 - 4 8 -T A B L E I I I Analysis of Variance i n D i s p e r s i b i l i t y of Powder Treated with Tween Type Emulsifiers Source of v a r i a t i o n . Degree of freedom Mean square F-r a t l o Tween emulsiflers 4 397.92 8 . 2 4 * * S t i r r i n g time k • . 7 1 5 . 7 8 1 4 . 8 2 * * Error 1 6 4 8 . 3 0 TOTAL 2 4 " ' " ~~ ** S i g n i f i c a n t at p r o b a b i l i t y 0.01 Duncan New Multiple Range Test:-Two homogeneous sets were shown: 1. D i s p e r s i b i l i t y of powder treated with Tween 20, 4 0 , and 8 0 . 2. D i s p e r s i b i l i t y of powder treated with Tween 60 and con t r o l . Tween treatment Control 20 40 60 80 Mean value 70.04 92.46 84.14 79.16 89.70 S t i r r i n g time (sees) 10 20 30 40 50 Mean value 63.26 81.24 86.92 91.30 ;:)2.78 -k9-TABLB IV Curve F i t t i n g by Computing UBC TRIP Program (Triangular Regression Package) Ef f e c t of L e c i t h i n Concentration on the S i n k a b i l i t y of Dried Whole Milk Type of Regression Regression Equation Correlation C o e f f i c i e n t Polynomlnal Linear Equation y = 0.109+0.0383x 0.93 Exponential Linear Equation y = -2.563+2.834 log x* 0.97 Recriprocal Equation y = 2.778-25.6(l/x) 0.89 Polynomlnal Quadra-t i c Equation y = -0.276+0.0686x-0.000365x2 O.85 Polynomlnal Cubic 0 Equation y = -1.122+0.17Q5x-0.00332x^ +0.0000237X-3 0.78 *The equation is only v a l i d over the range of the data, where x = % s i n k a b i l i t y and y = % vegetable l e c i t h i n . - 5 0 -TABLE V Analysis of variance i n angle of repose of powder treated with l e c i t h i n Source of variance Degree of freedom Mean square F-ratio Lecithin treatment 5 50.538 33.249** Rep. 1 1.02 3 0.673 T x R 5 1.52 Total 11 ** S i g n i f i c a n t at p r o b a b i l i t y 0.01 Duncan New Multiple Range Test: Angle of repose for the control fpowder treated with refined soy l e c i t h i n and soybean l e c i t h i n were homogeneous. Angle of repose f o r powder treated with Bovine l e c i t h i n , vegetable l e c i t h i n and instant skimmilk powder were homogeneous. Angle of SSR LSR Soy Refined Control repose l e c . soy, l e c . Instant skimmilk 40 1/2 6.25 8.85 11.0 10.75 10.75 Bovine l e c i t h i n 41 3/4 6.18 8.74 9.75 9.5 9.5 Vegetable l e c i t h i n 46 1/2 6.11 8.64 NS NS NS Control (spray dried whole milk 51 1/4 5.96 8.42 Refined soy l e c i t h i n 51 1/4 5.70 8.06 Soy l e c i t h i n 51 1/2 -51-BIBLIOGRAPHY 1. A l l e n , R.J.L., (1940) "The Estimation of Phosphorus." Biochem. J. 858. 2. Aschaffenburg, R. (1964) "Protein Phenotyping by Direct Polyacrylamide-Gel Electrophoresis of Whole Milk." Biochem. Biophys. Acta 82, 188. 3. Ashworth, U.S. and Bendixen, H. A. (194?) "Factors Af f e c t i n g The Ease of Reconstitution of Milk Powders." J. Dairy Science 2P_, 528. 4. Ashworth, U.S. (1955) "Dry Milk Symp. QMFd. Cont. Inst., U n i v . Chicago, Sept. , 1954. 5. A.O.A.C. 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