UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Membrane processing of cheese whey and preparation of ferric whey protein by heating Amantea, Gerald F. 1973

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1973_A6_7 A44_7.pdf [ 4.39MB ]
Metadata
JSON: 831-1.0101301.json
JSON-LD: 831-1.0101301-ld.json
RDF/XML (Pretty): 831-1.0101301-rdf.xml
RDF/JSON: 831-1.0101301-rdf.json
Turtle: 831-1.0101301-turtle.txt
N-Triples: 831-1.0101301-rdf-ntriples.txt
Original Record: 831-1.0101301-source.json
Full Text
831-1.0101301-fulltext.txt
Citation
831-1.0101301.ris

Full Text

MEMBRANE PROCESSING OF CHEESE WHEY, AND PREPARATION OF FERRIC WHEY PROTEIN BY HEATING by GERALD F. AMANTEA B.Sc, University of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of FOOD SCIENCE Faculty of A g r i c u l t u r a l Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 19 7 3 .In p r e s e n t i n g 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 o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e 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 r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e Head of my D e p a r t -ment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . G e r a l d F. Amantea Department o f Food S c i e n c e , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , Vancouver 8, Canada. ABSTRACT A concentrate containing up to 7 3% protein N was recovered from cheese whey by using c e l l u l o s e acetate u l t r a -f i l t r a t i o n membrances designed to r e j e c t solutes larger than 30,000 molecular weight by a continuous washing procedure. Conditions necessary f o r increasing the u l t r a -f i l t r a t i o n process f o r cheese whey are reported. Variables include pressure, membrane porosity, feed rate, c l a r i f i c a t i o n , temperature and pH. The objective was to prepare whey products with a minimum concentration of monovalent s a l t s and maximum concentration of protein while s t i l l maintaining a high f l u x r a t e . As expected pH adjustment to 7.0 and c l a r i -f i c a t i o n at 2000 X g f o r 5 min were c r i t i c a l i n increasing f l u x rate. However, membrane blockage occurred and gel electrophoresis indicated that (3-casein and a g-casein were the major components responsible yet s a l t s and lactose may also be implicated to a le s s e r degree. Flux rate increased with temperature but was not affected by pressure. Results indicate that concentrating 3-4X would be p r a c t i c a l but higher l e v e l s would be uneconomical due to the accumulation of viscous materials on the membrane. Gel f i l t r a t i o n showed that whey proteins are retained almost q u a n t i t a t i v e l y i n the concentrate while low molecular weight nitrogen containing material pass the membrane i n t o the permeate. i i . TABLE OF CONTENTS PAGE ABSTRACT ' i TABLE OF CONTENTS i i LIST OF TABLES i i i LIST OF FIGURES i v LIST OF PLATES v ACKNOWLEDGEMENTS vi. INTRODUCTION 1 LITERATURE REVIEW 4 PART I. OPTIMIZATION OF CONDITIONS FOR THE ULTRAFILTRATION OF CHEESE WHEY Method and Materials 13 Results 2 5 • Discussion 40 PART I I . PREPARATION OF FERRIC WHEY PROTEIN BY HEATING Introduction 51 Methods and Materials 5 3 Results and Discussion 55 LITERATURE CITED 6 6 APPENDIX 7? i i i . LIST OF TABLES TABLE PAGE II I I I Rates of the u l t r a f i l t r a t i o n process using d i f f e r e n t membranes Chemical analysis of f r a c t i o n s and l e v e l s of retention of the membranes i n u l t r a -f i l t r a t i o n Analysis of whey protein concentrate and crude soluble lactose (freeze dried) 26 27 28 PART I I IV VI E f f e c t of pH on the s o l u b i l i t y and the recovery of protein i n f i n a l product Comparison of heating methods f o r pre-paration of soluble whey protein powder Analysis of whey protein powder and crude soluble lactose (freeze dried) 57 59 61 VII Mean terminal values and standard devia-tions f o r hemoglobin, hemtocrit and l i v e r i r o n of chicks fed d i f f e r e n t sources of i r o n 65 APPENDIX I Analysis of variance i n hemoglobin a f t e r r e p l e t i o n II Analysis of variance i n hematocrit a f t e r r e p l e t i o n I I I Analysis of variance of ir o n i n l i v e r samples a f t e r r e p l e t i o n IV , LIST OF FIGURES FIGURE PAGE 1 Decline i n permeate f l u x during u l t r a -f i l t r a t i o n of whey at pH 4.6-. . .30 2 Relationship between f l u x and pH 31 3 E f f e c t of centri f u g a t i o n on protein p r e c i p i t a t i o n .  ' 3 2 4 Polyacrylamide gel electrophoresis of whey protein 34 5 Agarose gel electrophoresis of whey proteins 3 5 6 Sephadex G-50 gel f i l t r a t i o n of whey and u l t r a f i l t e r e d - whey 36 7 E f f e c t of temperature on f l u x rate 36a 8 Graph showing the best washing conditions necessary to increase protein concentration to greater than 8 0% 3 8 9 The e f f e c t of pH adjustment of the wash water on permeate f l u x 3 9 10 Flow diagram f o r the preparation of whey, protein, crude soluble lactose and the reuse of curd r i n s i n g water 47 11 Flow diagram of preparation of f e r r i c whey protein and crude soluble lactose 56 12 Gel electrophoretogram of whey proteins 62 LIST OF PLATES PLATE I U l t r a f i l t r a t i o n apparatus — laboratory scale. II U l t r a f i l t r a t i o n assembly I I I AC1 agarose f i l m cassette system IV 1. Convection oven; 2. Heated agarose plate support ACKNOWLEDGEMENTS The author wishes to express his deepest g r a t i -tude to Dr. S. Nakai, Associate Professor, Department of Food Science, University of B r i t i s h Columbia, under whose supervision t h i s project was undertaken and for his constant advice and encouragement i n the preparation of t h i s t h e s i s . Appreciation i s also extended to Dr. W.D. Powrie, Head of the Department of Food Science, Drs. J.F. Richards and P.M. Townsley, Department of Food Science, f o r t h e i r discussion and c r i t i c i s m during the period of research and i n the preparation of t h i s t h e s i s . Suggestions from senior graduate students Thomas Beveridge and Sadiq Toma were greatly appreciated. A p a r t i c u l a r thanks i s also extended to the Fraser Valley Milk Producers Association f o r f i n a n c i a l assistance during the period of research. INTRODUCTION T r a d i t i o n a l l y , whey,the by-product of cheese manu-facture has been considered a waste product to be disposed of as cheaply as possible. However, as a n t i - p o l l u t i o n laws have become more r i g i d , processors have been forced to think of whey i n a u t i l i z a t i o n context. This i n turn has led to the recognition of the food value of whey protein i n human n u t r i t i o n . As pointed out by Mann (31) the most s i g n i f i c a n t of a l l f or the future manufacture and u t i l i z a t i o n of whey proteins and whey protein products involves the modern developments i n membrane separation technology. The two re l a t e d membrane processes -- reverse osmosis (RO) and u l t r a f i l t r a t i o n (UF) -- have received considerable attention as new tools f o r economically t r e a t i n g whey. Both reverse osmosis and u l t r a f i l t r a t i o n are based on the a b i l i t y of polymeric membranes to discriminate between molecules on the basis of s i z e . Although RO and UF are often used synonymously there are c e r t a i n differences. In RO the membranes have a much more c l o s e l y knit structure which blocks the passage of most solute molecules whilst solvent i s free to pass through. _2 Working pressures are usually high, i n the region of 4MNjn (600 l b / i n ^ ) . U l t r a f i l t r a t i o n i s distinguished by the use of membranes having r e l a t i v e l y open structures which allow the passage of molecules of a l l sizes up to the pore size of t h e membrane. P r e s s u r e s employed a r e u s u a l l y low i n t h e r e g i o n o f 0.2-0.5 MNrrT2 (30 - 70 l b / i n 2 ) . McDonough (.35) and R o u a l e y n (-54) showed t h a t by c o n t r o l l i n g pore s i z e d u r i n g f a b r i c a t i o n o f t h e membranes and-by u s i n g d i f f e r e n t c o m b i n a t i o n s o f membranes and d i f f e r e n t s equences o f p r o c e s s i n g , a v a r i e t y o f u s e f u l p r o d u c t s concen-t r a t e d f r o m whey c o u l d be o b t a i n e d . Numerous r e p o r t s c o n c e r n i n g c o n c e n t r a t i o n o f cheese whey by RO and UF have appeared i n t h e l i t e r a t u r e ( 1 3 , 24, 32, 34, 35, 36, 37, 3 8 ) . More r e c e n t l y t h e s e p r o c e s s e s have been a p p l i e d t o whole m i l k and s k i m m i l k f o r t h e p r o d u c t i o n o f v a r i o u s d a i r y p r o d u c t s ( 7 , 13, 15, 33, 4 7 ) . A l s o a t r e a t m e n t p r o c e s s based on a t w o - s t e p a p p l i c a t i o n o f UF and RO has shown t o r e d u c e t h e BOD on t h e o r d e r o f 99 p e r c e n t (19, 20, 2 1 ) . Y e t , two m ajor problems s t i l l r e m a i n t h a t l i m i t t h e p e r m e a t i o n r a t e a c h i e v e d d u r i n g p r o c e s s i n g o f whey by b o t h RO and UF. These a r e c o n c e n t r a t i o n p o l a r i z a t i o n and a g r a d u a l f o u l i n g o f t h e membrane d u r i n g o p e r a t i o n . C o n c e n t r a t i o n and d r y i n g a r e e x p e n s i v e because of t h e low c o n c e n t r a t i o n (6 - 7%) o f s o l i d s i n t h e f l u i d whey. A l s o t h e h i g h p r o p o r t i o n s o f l a c t i c a c i d and s a l t s i n t h e s o l i d s l i m i t t h e u t i l i z a t i o n o f d r i e d whey i n f o o d p r o d u c t s . T h u s , an e c o n o m i c a l p r o c e s s t h a t would c o n c e n t r a t e whey and a t t h e same t i m e remove a t l e a s t p a r t o f t h e l a c t i c a c i d and s a l t s would g r e a t l y i n c r e a s e t h e f e a s i b i l i t y o f p r o c e s s i n g whey f o r food uses. I t was the purpose of t h i s project to study the e f f e c t of varying operating parameters through the manipulation of temperature, pH, pressure, membrane charac-t e r i s t i c s and a l t e r a t i o n of the feed (whey) i t s e l f i n the * hope of developing an e f f i c i e n t method f o r t r e a t i n g whey, making i t f e a s i b l e f o r i n d u s t r i a l a p p l i c a t i o n . LITERATURE REVIEW Whey Concentration In recent years'the concentration of whey by RO and UF has been studied extensively (13, 24, 32, 34, 35, 36, 37, 38 45 ,. 46). Rei'd et a l . (36) showed c e l l u l o s e acetate to be an e f f e c t i v e material f o r desalination of brackish and sea water but these membranes and low permeabilities. Loeb (36) improved upon them by a sophisticated f i l m casting technique, thus making i t possible to produce durable p l a s t i c films with high hydraulic permeability, coupled with the a b i l i t y to block the passage of quite small solute molecules. Michaels (41) has l i s t e d the presently a v a i l a b l e membranes, t h e i r manufactures, c h a r a c t e r i s t i c s and l i m i t a t i o n s . Included i n the l a t t e r are membrane hydrolysis outside the range of pH 3.5 to 8.0 and a change i n membrane properties above 35 - 40°C. These l i m i t a t i o n s have been overcome by poly-e l e c t r o l y t e membranes, manufactured by Amicon Corporation, which are e f f e c t i v e over a wide range of pH and temperature. Along with the marked increase i n avail a b l e membranes there has been an i n t e r e s t i n RO and UF f o r research and i n d u s t r i a l use. In the dairy and food i n d u s t r i e s , however, i t i s of greater i n t e r e s t as a low cost a l t e r n a t i v e f o r evaporation and has an a d d i t i o n a l advantage of not requiring any heat (38). Marshall et a l . (32) applied RO to the f r a c t i o n a t i o n and concentration of whey. The whey containing 26.0% s o l i d s was concentrated to 53.3% then cooled to 3°C then agitated 24 hr to c r y s t a l l i z e the lactose. A f t e r . c e n t r i f u g i n g at 3000 rpm for 24 hr three f r a c t i o n s were obtained -- a p r e c i p i t a t e , infranate and supernatant comprising respectively 56.6, 19.7 and 2 3.7% of the t o t a l weight. The p r e c i p i t a t e was l a r g e l y lactose while 7 5% of the infranate s o l i d s contained protein. However, they indicated two problems that l i m i t e d the per-meation rate achieved during processing of whey by t h i s method -- concentration p o l a r i z a t i o n and gradual f o u l i n g of the membrane during operation. McDonough et a l . (36) showed that a f o u r f o l d concentration was possible but higher l e v e l s would be uneco-nomical due to clogging problems from an accumulation of viscous materials and insoluble s o l i d s . Glover (15) without seeking the optimum conditions f o r the process showed that protein forms a gel which adheres to the membrane and i n e f f e c t adds another f i l t e r i n g layer. Lim e t a l . (28) showed that the major cause of blockage was due to casein but a-lactalbumin and 3-lactoglobulin were also present. Flux decline has been att r i b u t e d to such factors as accumulation of fo u l i n g material on the membrane, concen-t r a t i o n p o l a r i z a t i o n , decrease i n d r i v i n g force with increase i n concentration and compaction of the membrane (32, 36, 48, 50). P e r i e_t al_. (50) working with whey and a simulated milk showed that the permeability and s e l e c t i v i t y of the membranes were attributed to the differences i n the r e l a t i v e importance of osmosis and pore flow. They showed that by using a c l a r i f i e d whey with increase i n pressure that there was a subsequent increase i n retention. Blockage again was found to be due to protein and other high molecular weight substances. However, the e f f e c t was r e a d i l y r e v e r s i b l e by washing with water which restored the o r i g i n a l permeability and s e l e c t i v i t y of the membrane.. Only by seeking optimum conditions for RO and UF would t h e i r i n d u s t r i a l a p p l i c a t i o n be f e a s i b l e . Forbes (14) showed that by the s e l e c t i o n of the correct operating tempera-ture, pH and feed v e l o c i t y that the protein layer responsible f o r the clogging of the membrane i s t o t a l l y r e d i s p e r s i b l e so that equilibrium and steady output are obtained. P e r i and Dunkley (48) reported the influence of composition of the feed on the performance of c e l l u l o s e acetate reverse osmosis membranes using varied operating pressures but constant flow-conditions. They found that f o u l i n g decreased permeation rate but i t s influence on retention was variable and depended p r i n c i p a l l y on the feed, the solute and the avail a b l e d r i v i n g force. In a subsequent paper by the same workers (49) they found that by modifying the flow condition to increase turbulence,improved performance of RO membranes. Roualeyn et al_. (54) described methods to produce a var i e t y of useful product concentrates from raw whey using d i f f e r e n t combinations of UF and Ro membranes and d i f f e r e n t sequences of processing operations. They showed that the protein: lactose r a t i o i n the concentrate was a function of the. permeability and s e l e c t i v i t y c h a r a c t e r i s t i c s of the membrane, as well as the system design and operating conditions Ratios ranged from 1:8 raw whey through 3:5 to 2:1 or higher. McDonough et a l . (34) using Calgin-Havens type 215 membranes and optimum conditions have shown r e j e c t i o n values of 97.4% f o r p rotein, 4.8% f o r ash, 3.9% f o r l a c t i c acid and 13.4% f o r lactose, the l a t t e r being s l i g h t l y higher than desired. Recently Horton et_ a l . (19) have set up the f i r s t commercial scale UF/RO plant f o r f r a c t i o n a t i n g cottage cheese whey into protein and lactose concentrates capable of handling 300,000 lb of cottage cheese whey oer day using sanitary clean-in-place equipment. Concentration and Fractionation of Milk and Skimmilk  by RO and UF There are two main reasons f o r concentrating milk: f i r s t to f a c i l i t a t e the transport of l i q u i d milk and second to reduce the volumes of l i q u i d involved i n cheese making. Glover (15) was able to concentrate milk 2X without any f l a v o r changes while r e t a i n i n g a l l the main constituents of the milk. Although f o u l i n g was i n e v i t a b l e , t h i s could be overcome by a two-step process. U l t r a f i l t r a t i o n could be used f i r s t to concentrate the f a t and protein, followed by RO to concentrate the remainder of the components, a f t e r which the products of both processes would be recombined to produce a concentrated milk. U l t r a f i l t r a t i o n of skim milk has been made i n a concentration r a t i o of 6:1 by Maubois and Mocquot (33) and the concentration was used f o r the production of Camembert cheese whose organoleptic q u a l i t i e s were found to be equivalent to the cheese prepared according to the conventional procedure. In addition to the increase i n y i e l d , reduction i n p o l l u t i o n due to the whey and c e r t a i n technological advantages are claimed; such as improvements i n factory design, less hand work and less weight f l u c t u a t i o n from one cheese to another. Fenton-May et aJL. (13) were able to concentrate skim milk to 22% t o t a l s o l i d s by RO or to fractionate i t by UF to produce l i q u i d concentrates with 50 to 80% protein (dry b a s i s ) . Proteinaceous deposits on the membranes resulted i n a measurable resistance to transport and consequently the permeate f l u x . They suggested therefore that systems be designed to operate with high feed v e l o c i t i e s to minimize t h i s boundary layer thickness. Bundgaard et a l . (7) succeeded i n producing by u l t r a f i l t r a t i o n a skim milk concentrate of such a composition that i t could be used f o r the production of fresh cheese, white mould cheese and a semi-hard cheese. Moreover, by a RO treatment of the UF permeate the concentrates would be made suitable f o r c a l f feed as well as enabling u t i l i z a t i o n of the lactose f o r yogurt production. More recently P e r i et a l . (47), by combining u l t r a -f i l t r a t i o n and washing, were able to obtain concentrations of skim milk proteins up to values of 80% on a dry weight basis. A p p l i c a t i o n of RO and UF to other areas Applications of RO to foods can be c l a s s i f i e d into three areas: concentation, p u r i f i c a t i o n or f r a c t i o n a t i o n and waste disposal (26, 40). As proposed by Morgan et a l . (42) these methods can be used f o r the concentration of l i q u i d food products economically without phase change or •the a p p l i c a t i o n of heat. Lowe et al_. (30) using a Wurstack RO arrangement, developed at the Western U t i l i z a t i o n Research D i v i s i o n successfully concentrated egg white 2.5 times. In a d d i t i o n other products processed with varying degrees of success include: lemon, orange, tomato, whole milk, grape, coffee and apple. A 30% s o l i d s egg white concentrate with ex c e l l e n t functional properties equal to that of fresh egg white was also produced (29). Apple and orange j u i c e were concentrated f o u r f o l d by RO (34). In spite of the loss of some water soluble aroma compounds the f l a v o r of the concentrate was judged to be exc e l l e n t . In commercial evaporation of orange j u i c e , a l l of the water-soluble aroma compounds are completely 10. stripped o f f . To compensate f o r this,commercial practice i s to add peel o i l and to over-concentrate. Therefore, u l t r a -f i l t r a t i o n overcomes these problems. Although the cost i s s l i g h t l y more expensive than thermal evaporation equipment, 7 5% of the water was removed from maple sap by RO (59), with about 1 part i n two thousand sap s o l i d s l o s t i n the by-product water. Experiments i n applying UF to enzyme recovery have been encouraging. It has been possible to concentrate up to sevenfold or more at f l u x rates of 10 - 20 gal/day f t with enzyme retentions of 90 - 95% while s t i l l r e t a i n i n g 95 - 98% of the enzyme a c t i v i t y (9). UF can also be applied to the concentration of viruses and t h i s has a wide p o t e n t i a l implication f o r the manufacture of sera and vaccines. Concentration r a t i o s of 18 - 19 were achieved i n various media with l i t t l e measured loss of a c t i v i t y (9). Membrane' Separation Process f o r the Abatement of P o l l u t i o n The establishment of new standards and requirements for e f f l u e n t q u a l i t y water has accentuated the need f o r new and improved routes f o r waste treatment (56). Moreover, a n t i - p o l l u t i o n concern i s f o r c i n g processors to look towards u t i l i z a t i o n rather than disposal (35). UF and RO treatments eliminates the need f o r high capacity s e t t l i n g equipment and use of coagulants and 11. f l o c c u l a n t s as s e t t l i n g aids and c l a r i f i e r s i n waste treatment operations (41). RO equipment and- operating procedures are r e l a t i v e l y expensive f o r processing spent liqu o r s from the pulp and paper industry i n amounts of several m i l l i o n gallons per day compared to conventional methods (57). Yet, the treatment (through high-flux , low pressure membranes) of primary and secondary sewage e f f l u e n t s y i e l d s c r y s t a l c l e a r , microorganism-free f i l t r a t e s from which v i r t u a l l y a l l b i o l o g i c a l l y active t a s t e , color and odor-producing contaminants have been removed (41). Moreover, e f f l u e n t s are of a low BOD suitable f o r discharge without p o l l u t i o n and the concentrate can be r e a d i l y desiccated and incinerated or processed economically f o r by-product recovery. Recoveries of excellent q u a l i t y water, well i n excess of 80%, have been obtained. Thus c a p a b i l i t i e s of s u b s t a n t i a l l y extending the r e c y c l i n g of water within pulp m i l l systems are possi b l e (58). A whey treatment process which uses a two-step a p p l i c a t i o n of UF f o r the f i r s t step and RO f o r the second has shown to be able to reduce the BOD i n the order of 99% (19, 20, 21). Also the BOD of raw whey has been reduced from 50,000 - 60,000 ppm to 1000 ppm a f t e r UF treatment (9). At present the Crowleys Milk Company Inc. i s able to process 300,000 lbs of cottage cheese whey per day (19). Besik 'et a l . (4) pointed out that a 39.3% return on c a p i t a l i s possible by processing whey through a combination of RO with spray drying and at the same time solving the unpleasant p o l l u t i o n problem. METHODS AND MATERIALS U l t r a f i l t r a t i o n U l t r a f i l t r a t i o n was done on a small laboratory-scale apparatus (Model 52, Amicon Corporation, Lexington, Massachusetts, U.S.A.) i l l u s t r a t e d i n plates I and I I , having a maximum i n t e r n a l volume of 65 ml. It consisted of a c y l i n d r i c a l container 130 mm high, 43 mm i n t e r n a l diameter, with a h o r i z o n t a l porous support disc near the bottom 2 . supporting a c e l l u l o s e acetate membrane 12 50 mm i n area. Immediately above the membrane was a magnetic s t i r r e r . The membranes used were i n the form of f l a t sheets, and designated as types UM05, PM10, PM30, XM100 having minimal cutoffs at molecular weights 500, 10,000, 30,000 and 100,000 res p e c t i v e l y . These values may have been only approximate since i t was found that another membrane having the same molecular cutoff point had d i f f e r e n t permeation rates. Therefore, i n order to standardize the membrane, the permeation rate of d i s t i l l e d water was measured over a h a l f hour period. Pressure was - 2 2 applied from an a i r c y l i n d e r at 0.H MNm (60 l b / i n ) and the experiments were c a r r i e d out at temperatures between 25 and 45°C. Turbulence was maintained at a constant speed i n the UF c e l l by means of the magnetic s t i r r e r . The f i l t r a t e was taken out through a tube just below the porous support disc and c o l l e c t e d i n a graduated cyli n d e r . In order to supply l i q u i d to the u l t r a f i l t r a t i o n c e l l when the volume of l i q u i d to be processed exceeded the 14. Pressure S M F Cj> Fill rate Plate I. U l t r a f i l t r a t i o n apparatus -- laboratory scale, C, concentrate; F, f i l t r a t e , M, membrane mounted on porous p l a s t i c supporting disc: S, magnetic s t i r r e r : r e l i e f valve. V, f i l l i n g tube and 15. Plate I I . U l t r a f i l t r a t i o n Assembly 1. A i r cylin d e r 2. Fiberglass r e s e r v o i r 3. U l t r a f i l t r a t i o n chamber with membrane 4. Magnetic s t i r r e r ; • 5. Graduated c o l l e c t i n g c y l i n d e r c e l l capacity, the u l t r a f i l t r a t i o n c e l l was connected to a fi b e r g l a s s l i q u i d r e s e r v o i r (Model RG-3 Amicon Corporation, Lexington, Massachusetts, -U.S.A.), i l l u s t r a t e d i n plate II having a maximum i n t e r n a l volume of 27 50 ml. Membranes were conditioned before using by r i n s i n g i n a beaker of d i s t i l l e d water f o r at least one hour with one change to remove the glycerine added. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed according to Aschaffenburg's method (2) with modifications. The gel was prepared as follows: 10.5 g: acrylamide, 0.52 gm N,N-methylenebis acrylamide and 40 g. urea were dissolved i n 0.17 5M 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 s o l u t i o n was f i l t e r e d through No.1 f i l t e r paper into a vacuum f l a s k . A f t e r deaeration 0.45 ml mercaptoethanol, 1.2 5 ml TMED (3 0% N, N, N, N, - t e t r a -methylene ethylenediamine i n 95% ethanol) and 1.25 ml of 10% ammonium persulfate were added to the solution i n that order, mixed gently, poured immediately into a mold, covered with a pl e x i g l a s s plate and weighed down to exclude the a i r . The gel was allowed to stand 30 - 60 minutes to permit complete polymerization. A ho r i z o n t a l electrophoresis apparatus with two troughs at ei t h e r end of the gel was used with a sodium chloride s o l u t i o n 0.1 M i n the outer and 0.175 M T r i s - g l y c i n e buffer i n the inner. The two buffers were connected with cheesecloth bridges. The gel was placed 17. on the electrophoresis apparatus and covered with saran wrap extending over the cheesecloth bridges to avoid drying. The gel was eq u i l i b r a t e d for 20 hr at 4°C by running a current through i t . The amperage was maintained at 2 0 mA by a power supply. Preparation of Sample A 2% soluti o n of each of the following samples was prepared i n 0.175 M T r i s - g l y c i n e buffer: 1. 8-lactoglobulin 2. K-casein 3. whey pasteurized at 72 C f o r 10 min a f t e r adjusting to pH 7.0 4. acid casein 5. the supernatant a f t e r centrifuging No. 3 at 1,50 0 X g f o r 10 min at 0°C. 6. the sediment of No. 5 7. f e r r i c whey protein containing 224 ug/ml whey. To each sample except No. 7 was added one drop of mercapto-ethanol. Strips of Whatman No.3 MM f i l t e r paper 1.5 cm X 0.2 cm wide were saturated with each sample and blotted on a clean t i s s u e . With the aid of a razor blade they were then inserted into the s l o t on the eq u i l i b r a t e d gel plate. E l e c t r o -phoresis was ca r r i e d out under the same conditions as the e q u i l i b r a t i o n of the gel . A f t e r migration was completed the gel was removed from the mold and stained f o r 10 min i n a 1% amido black dye solution i n 10% a c e t i c acid. Destaining was accomplished by continuously washing the gel i n 5% a c e t i c acid u n t i l the protein bands appeared blue against a cle a r background. 18. Agarose Gel Electrophoresis Agarose gel electrophoresis was done using the ACI Cassette Electrophoresis C e l l and Power Supply and the ACI Agarose Universal Electrophoresis Film ( A n a l y t i c a l Chemists Inc., Palo A l t o , C a l i f o r n i a , U.S.A.) i l l u s t r a t e d i n Plate I I I . A l l procedures were c a r r i e d out at 4°C. The electrophoresis f i l m was removed from i t s p l a s t i c support and placed i n the s t i r dish (plate III) and conditioned for one hour i n 200 ml of HM urea made up i n T r i s - g l y c i n e buffer pH 9.1. A f t e r conditioning,the f i l m was removed and dryed so that no buffer remained i n the sample s l o t s . ' Preparation of Sample A 3% s o l u t i o n of each of the following was prepared ' i n T r i s - g l y c i n e b u f f e r pH 9.1 plus one drop of mercaptoethanol. 1. 3 - l a c t o g l o b u l i n 2. K-casein 3. a c i d casein 4. the sediment of No.6 5. the supernatant a f t e r centrifuging No.6 at 1,50 0 X g f o r 10 min at 0°C. 6. ' whey pasteurized at 72°C fo r 10 min a f t e r adjusting to pH 7.0. Samples were applied to the f i l m s l o t s by using the micro-l i t e r dispensor with disposable sample t i p s . Electrophoresis was then c a r r i e d out by placing the f i l m i n the cassette cover, which was f i t t e d into p o s i t i o n on the cassette holder containing about 200 ml of sodium b a r b i t a l buffer, pH 8.6, 0.5 M with 0.035% EDTA. The cassette assembly was then attached to the power supply and run f o r one hour. Following Plate I I I . ACI Agarose Film Cassette System 1. Power supply 2 . Cassette 3. Cassette cover H. Agarose gel plate 5. Quantitative m i c r o l i t e r sample dispensor 6. Disposable sample t i p s 7 . ' S t i r - s t a i n dish 2 electrophoresis the proteins were simultaneously fi x e d and stained by immersing the f i l m i n approximately 200 ml of 0.2 g % Amido Black i n 5% acetic acid for 15 min. The excess Amido Black was removed by placing the f i l m i n a st a i n dish of 5% a c e t i c acid f o r 30 seconds with a g i t a t i o n . The f i l m was then dried at a temperature between 7 5 - 8 5°C i n a convection oven (plate IV). Destaining was accomplished by placing the f i l m i n 5% ac e t i c acid u n t i l the majority of the s t a i n was cleared. This required approximately one minute. The remaining background s t a i n was removed by placin; the f i l m i n a dish containing cl e a r 5% ac e t i c acid f o r one minute. The f i l m was then dried i n the oven as before. The en t i r e procedure including conditioning of the f i l m takes approximately three hours. Acid Whey Cottage cheese whey was obtained from a l o c a l dairy (Fraser Valley Milk Producers Association, Vancouver, Canada) The whey was pasteurized at 7 2°C for 10 min, adjusted to pH 7.0 and centrifuged at 1,500 X g for 5 min at 0°C using a S o r v a l l RC2-B centrifuge. The • supernatant was then used f o r the u l t r a f i l t r a t i o n experiments. Whey samples were held at U°C for no longer than 2 days. Preparation of F e r r i c Whey Protein As described i n Part I I . S o l u b i l i t y A suspension of 0.2 g freeze dryed whey product 21. Plate IV. 1. 2. Convection oven Heated agarose plate support i n 20 ml of water at 25 C was blended i n a Lourdes Model MM - 1A multimixer (Lourdes Instrument Corporation, New York) at 18,000 rpm. Protein i n the supernatant a f t e r centrifuging at 2,000 X g f o r 19 min was determined. . The s o l u b i l i t y was expressed as a percentage of t o t a l protein i n the o r i g i n a l sample. Nitrogen Determi nation s Nitrogen was determined according to the dye binding procedure (12) f o r routine an a l y s i s . A micro-kjeldahl method (3) was used as a standard method. Tot a l protein was deter-mined by the factor 6.3 8 X t o t a l nitrogen. La c t o s e Lactose was analyzed by an anthrone method (57). ' T o t a l Solids T o t a l s o l i d s were determined according to the O f f i c i a l AOAC method (3). • Ash Ash analysis was done according to the O f f i c i a l AOAC method (3). Iron A co l o r i m e t r i c method using ferrozine was applied a f t e r deproteinization (8) or Jarrel-Ash Atomic Absorption Spectrophotometer 82 - 800 was used a f t e r dry ashing. Agreeable r e s u l t s were obtained between both methods. Calcium and Magnesium A back t i t r a t i o n using calcium chloride and a calcium i n d i c a t o r (44) was used d i r e c t l y or Jarrel-Ash Atomic Absorption Spectrophotometer 82 - 800 was used a f t e r dry ashing. Phosphorous Phosphorous was determined spectrophotometrically a f t e r wet ashing using sulphuric acid (43). B i o l o g i c a l Oxidation Demand BOD was analyzed using the O f f i c i a l Standard Method (56) with a 5 day incubation period of 20°C. A l l whey samples were fresh and stored at 4°C f o r no longer than 2 days. Samples including acid whey, u l t r a f i l t e r e d concentrates and permeates were d i l u t e d 10 0.times and 5 ml of t h i s d i l u t i o n covering a BOD range of 12,000 - 36,000 ppm was used f o r analysis. Gel F i l t r a t i o n Gel f i l t r a t i o n was c a r r i e d out i n Sephadex G-50 i n a column f i t t e d with flow adaptors and operated i n an upward flow manner. The e l u t i n g buffer was 0.05 M ammonium acetate pH 4.5 and the sample was applied by means of a pipette (52). M emb'rane Re gen e r a t i o n Membrane regeneration was accomplished by back-washing f i r s t with approximately 60 ml of NaOH pH 11.5 followed by the same amount of 0.5 N HC1. Regeneration was repeated i f the permeation rate of d i s t i l l e d water at 25°C over a h a l f hour period did not approach the same rate as that of a new membrane under the same conditions. Membranes were replaced i f the permeation rates of d i s t i l l e d water a f t e r regeneration were low. Temperature The e f f e c t of temperature on permeate flux was studied by placing the f i l t r a t i o n apparatus i n a cont r o l l e d c i r c u l a t i n g water bath. Temperature ranged from 2 5 - 4 5 ± 0.5°C. The process was not started u n t i l the desired tempera-ture of the whey was reached. • RESULTS Rates Table I gives the rates of the u l t r a f i l t r a t i o n pro-cess at 2 5°C. Average permeate fluxes f o r whey ranged from 2 2 10 ml per m per hr f o r UM 0 5 membrances to 34 ml per m hr f o r XM 100 membranes. Generally the permeate fl u x decreased with time as the process continued. However, there was an increase i n permeate fl u x with temperature increase (Figure 7) . F e r r i c whey protein was prepared according to the method o u t l i n e d i n Part I I , omitting the preconcentration step. Although u l t r a f i l t r a t i o n of the supernatant was 83% lower i n p r o t e i n i t did not s u b s t a n t i a l l y increase permeate f l u x (Table I ) . Membrahe Se1ect ion The f i r s t part of the work undertaken was concerned with s e l e c t i o n of a membrane. Table II gives the chemical a n a l y s i s of the f r a c t i o n s and the l e v e l s of retention of the various membranes. These r e s u l t s were obtained from unwashed concentrates and permeates. I t can be seen that with the PM 3 membrane 94% of the protein and 53% of the lactose remained i n the concentrate. Various protein : lactose r a t i o s were obtained with d i f f e r e n t membranes. Washing of the protein concentrate removed r e s i d u a l lactose and made i t possible to increase the p r o t e in concentration to greater than 7 0% (Table I I I ) . TABLE I Rates of the u l t r a f i l t r a t i o n process using d i f f e r e n t membranes at 25°C Memb. Sample Orig. F i n a l Time Cone. Temp. Permeate type v o l . Vol. hr. factor °C fl u x hr. by v o l . ml ml ml ml/hr UM0;5 whey 60 50 1.0 1. 20 25 10 PM10 whey 60 28 1.0 2.14 25 32 PM30 whey 60 29 T. 0 2.06 25 31 XM100 whey 60 26 1.0 2.30 25 34 XM1001 Fe whey 60 27 1.0 2 . 22 25 33 1 F e m e whey protein containing 2 24 mg Fe/ml Whey + Fe 90°C f o r 10" + centrifuge at 3 00 0 rpm fo r 10" adjusts pH of supernatant to 7.0 centrifuge same speed ->- UF supernatant. . TABLE I I E f f e c t pf membrane type on retention of selected f r a c t i o n s i n the u l t r a f i l t r a t i o n of whey _ R E T E N T I O N % Memb. Sample Protein Lactose Total s o l i d s Ash type-UM05 whey 92.71 89.35 90 . 22 61 PM10 whey 93. 81 51.60 55.42 70 PM30 whey 94.44 52 .61 58 . 86 70 XM10 0 whey 89.80 47.87 50.48 52 - XM10 0 Fe whey 82.. 39 46.81 53.26 56 (Membrane re t e n t i o n stated as amount retained i n the concen-t r a t e expressed as percentage of the component i n the o r i g i n a l sample). 28 TABLE III Analysis of whey protein concentrate and crude soluble lactose (freeze dried) Product Moisture T o t a l Protein Lactose Ash Ca Mg Phosphorous s o l i d s 9- % % o, 9. o. -6 -8 -6 % Cottage cheese whey . protein 3.45 96 72.8 18.0 69 .059 .023 1.3 1st lactose 4.55 95 6.5 86 91 .061 .026 56 2nd lactose 4.04 96 6.5 83 13 .073 .030 .70 Whey concentrated 3X then washed continuously with 166 ml P^O. Protein % N X 6.38. 29, . A l l subsequent experiments were done using a PM 3 0 membrane with molecular cutoff at .30 ,000. Decline i n Permeate Flux Figure 1 shows the decline i n permeate f l u x during the u l t r a f i l t r a t i o n of pasteurized cheese whey pH 4.6 at 25°C using a PM 30 membrane at an operating pressure of 60 p s i . From the graph i t can be seen that a f t e r 2 5 hours the membrane i s so clogged that the permeate flux i s e s s e n t i a l l y zero. Relationship Between Flux and pH The e f f e c t of pH on membrane flux i s rather dramatic as i l l u s t r a t e d i n Figure 2. There i s no substantial difference i n permeate rate between centrifuged and uncentrifuged samples i n the pH range 3 - 5 . However, the difference i s large i n . the pH range 5 - 8 . Maximum f l u x was obtained with the c e n t r i f u g a l samples at pH 7.0. E f f e c t of Cen'trifuging From the r e s u l t s obtained i n Figure 2 i t can be seen that c e n t r i f u g i n g has a great e f f e c t on increasing f l u x rate. P r o t e i n determination done on the supernatants a f t e r c e n t r i -fuging (Figure 3) show that maximum fl u x i s associated with the minimum concentration of protein. Centrifuging at pH 6 -8 produced a s l i g h t p r e c i p i t a t e which was assumed to be p a r t l y responsible f o r the decrease i n f l u x . Po1yacry1amide Gel Electrophoresis Polyacrylamide and Agarose gel electrophoresis were done to determine what proteins were responsible for ure 1. Decline i n permeate fl u x during u l t r a f i l t r a t i o n of whey at pH 4.6. 3 4 5 6 7 8 pH Figure 2. Relationship between permeate fl u x and pH of the whey © © No cen t r i f u g a t i o n a f t e r pH adjustment B S Centrifuged at 1500 X g f o r 5 min af t e r pH adjustment. Figure 3. E f f e c t of c e n t r i f u g a t i o n on protein p r e c i p i t a t i o n the decrease i n f l u x (Figure 4). From the r e s u l t s i t can be seen that 3-casein and a -casein are responsible to a c e r t a i n s r degree i n the clogging of-the membrane. Also, the a s-casein band disappears a f t e r centrifuging (Figure 4 (5)) while i t i s s t i l l present i n the uncentrifuged sample. Agarose gel electrophoresis did not give good separation (Figure 5). I t was used because of i t s speed and s i m p l i c i t y and answered question which would otherwise have to be obtained through polyacrylamide gel electrophoresis. Gel Permeation Determination of the r e l a t i o n between t o t a l nitrogen and true protein retention was accomplished by performing a series of gel permeation analyses, t y p i c a l data from which are i l l u s t r a t e d i n Figure 6. Two curves are shown: one f o r whey and one f o r permeate. I t can be seen that the lower molecular weight nitrogen-containing material such as free amino acids, peptides and urea pass through the membrane into the permeate while the. proteins are retained almost q u a n t i t a t i v e l y i n the concentrate. E f f e c t of Washing Washing the concentrate removes r e s i d u a l lactose and thereby increases the concentration of proteins up to values of 70% on a dry weight basis (Table I I I ) . The c a l c u l a t i o n adopted was that of Stahl (55). By increasing the number of washings i t was possible to decrease the amount of water needed 34. | <xs-Ca oc- La j B-Lg & B-Ca > K -Ca Figure 4. Polyacrylamide gel electrophoresis of whey proteins. 1. 3-lactoglobulin; 2. K-casein; 3. whey pasteurized at 72 C f o r 10 min a f t e r adjusting the pH 7.0; 4. acid casein; 5. the supernatant a f t e r centrifuging No.3 at 1,50 0 X g f o r 10 min at 0 C; the sediment of No. 5; 7. f e r r i c whey protein. 35. • m -oi-La G- Lg & B - C a K-Ca Figure 5. Agarose gel electrophoresis of whey proteins 1. 3-lactoglobulin; 2. K-casein; 3. acid casein; 4. the sediment of No.6; 5. the supernatant a f t e r centrifuging No.6 at 1,50 0 X g f o r 10 min at 0 C; 6. whey pasteurized at 72 C f o r 10 min a f t e r adjusting to pH 7.0. 10 20 30 40 50 60 FRACTION N O . Figure 6. Sephadex G<-5 0 gel f i l t r a t i o n of whey (. .) and u l t r a f i l t e r e d whey . Conditions: gel bed size 20 X 33 cm, flow rate 85 ml/hr, el u t i n g buffer 0.0 5 M NHu0Ac, pH 4.5, column operated i n an upward flow manner. a. casein f r a c t i o n ; b. g-lactoglobulin; c. a-lactalbumin; d. low molecular weight nitrogen containing material. 150 J L_. __j L . L I L. 25 30 35 40 45 50 TEMPERATURE ,°C Figure 7. E f f e c t of temperature on f l u x rate (Figure 8). Continuous washing was the most e f f i c i e n t method to increase the protein concentration. pH Adjustment of Wash Water The pH adjustment of the wash water i s very c r i t i c a l i n optimizing permeate f l u x and extending membrane l i f e . Pre-liminary studies (Figure 9) indicate that the pH of the water adjusted to the basic side affords best r e s u l t s . 38. Figure 8. Graph showing washing conditions necessary to increase protein concentration to greater than 80%. 36 30 £ i X i u_ 1 8 1 2 conc .3x by vol. pH8.5 pH 6.0 pH 5.5 • pK 7.0 8 10 12 TIME IN HOURS Figure 9. The e f f e c t of pH adjustment of the wash water on permeate f l u x . Whey pasteurized at 7 2°C for 10 min and l e f t • a a t p H 4 _ 6 # Whey pasteurized at 72°C f o r 10 min, adjusted to pH 7.0 and centrifuged at 1,500 X g for 5 ^ min at 0°C the supernatant of which was used f o r • u l t r a f i l t r a t i o n . pH adjusted wash water as indicated. DISCUSSION Concentration and f r a c t i o n a t i o n of whey by u l t r a -f i l t r a t i o n has been studied extensively (13, 32, 34, 35, 36, 37, 38), yet few workers have sought optimum conditions f o r the process. The retentive properties of various membranes under d i f f e r e n t conditions were investigated to determine maximum flu x rates with minimum f o u l i n g . Data summarized i n Tables I and II show that per-meation rates varied with each membrane. Since optimum protein retention was desired with maximum flux a PM 30 membrane with molecular cutoff at 30,000 was chosen. Although lactose retention i s high, 100% removal of lactose from whey could be achieved only by removing 100% of the water. Data i n Table II show a lactose retention of 52.6% for PM 30 at 48% volume reduction. Whereas, with a 80% volume reduction, 87% of the lactose was removed. Therefore, the protein-to-lactose r a t i o i s d i r e c t l y r e l a t e d to the permeate removed. Figure 1 shows that the permeation rate of whey decreased with time. This i s a d i r e c t r e s u l t of the phenomenon, concentration p o l a r i z a t i o n which i s the accumula-t i o n of rejected dissolved solutes at the membrane surface. The accumulation of these at the boundary r a p i d l y r e s u l t s i n the-' formation of a viscous concentrated or gelatinous layer which o f f e r s resistance to flow. Concurrent with t h i s i s the increase i n osmotic pressure opposing the process as the concentration increases. The components mainly responsible are p r o t e i n , lactose and the s a l t s . Fouling of a t h i r d type termed pore plugging also -occurs and unlike the formation of the g e l - l i k e layer occurs more ra p i d l y (47). This i s so because of the early predominance of pore flow and the l i m i t e d opportunity f o r convection flow to remove macrosolute or c o l l o i d a l p a r t i c l e s that become lodged i n the pores. This r e s u l t s i n a decrease i n permeation rate. We found that t h i s problem could be p a r t i a l l y solved by centrifuging ( c l a r i f y i n g ) whey samples p r i o r to u l t r a f i l t r a t i o n such that c o l l o i d a l p a r t i c l e s would be p r e c i p i t a t e d (Figure 3). Carrying out the process at a high rate of shear r e s u l t e d i n greater permeation rates. Yet, as pointed out 'by Forbes (14) high shearing rates carry an energy penalty and any system optimization requires compromise between f l u x improvement and energy expenditure. I t was found that opera-t i n g at a pressure of 60 p s i s a t i s f i e d both conditions. Data reported i n Figure 1 are s i m i l a r to that obtained by other workers where optimum conditions f o r the . • process were not investigated. Figure 2 shows that pH adjustment of the whey accompanied by c l a r i f i c a t i o n greatly increases the permeate f l u x . The minimum i s associated with pH 6.0 due to the p r e c i p i t a t i o n of denatured proteins and/ or calcium and magnesium s a l t s not removed by centrifugation p r i o r to u l t r a f i l t r a t i o n . Maximum permeate f l u x i s at pH 7.0. This arises since on the a l k a l i side the protein denatures and a f t e r c e n t r i f u g i n g i s p r e c i p i t a t e d . Minimum fl u x i s between pH 3 - 5 since t h i s i s the i s o e l e c t r i c point of the p r o t e i n , thus aggregation occurs tending to give lower d i f f u s i o n rates. Centrifuging at pH 7.0 r e s u l t s i n a decrease of protein i n the supernatant (Figure 3) and p a r t i a l l y explains the increase i n f l u x rate i f protein i s assumed to be responsible for membrane blockage (28). Experimentation has shown that i f whey i s u l t r a f i l t e r e d at pH 4.6 at a protein concentration consistent with the centrifuged sample at pH 7.0 the f l u x rate i s considerably lower. Therefore, pH adjustment followed by centrifugation i s very c r i t i c a l i n 'optimizing the process. Centrifuging at neutral pH removes macrosolutes or c o l l o i d a l p a r t i c l e s which would otherwise become lodged i n the membrane pores and decrease the f l u x rate through f o u l i n g . Lim et a l . (28) on the basis of estimation of^the molecular weight together with electrophoretic patterns showed that the major components responsible f o r clogging were.: 1) Casein; 2) B-lactoglobulin; 3) a-lactalbumin, and 4) a nonprotein f r a c t i o n . Polyacrylamide gel e l e c t r o -phoresis (Figure 4) shows that only B-casein and a s-casein are responsible f o r blockage. The protein forms a gel which adheres to the membrane and i n e f f e c t adds another f i l t e r i n g layer. Further analysis of the p r e c i p i t a t e suggested that Ca and Mg s a l t s as well as lactose may also be responsible, but to a much smaller degree. Gel f i l t r a t i o n (Figure 6) indicated that by using a PM30 membrane a l l the proteins are retained while low molecular weight nitrogen-containing materials pass through. These r e s u l t s are consistent with those i n Table II where protein retention was 9 4.4%. Moreover, peaks b and c (Figure 6) represented by B-lactoglobulin and oclactalbumin r e s p e c t i v e l y (21) appeared i n the gel electrophoresis studies (Figure 4) as did peak a which i s assumed to be r e s i d u a l casein. This l a t t e r f r a c t i o n was probably not affected by rennet and or b a c t e r i a l action during the cheese making process or possibly may be casein fines from the cottage cheese i t s e l f . Although Figure 6 indicates that a l l the low molecular weight nitrogen containing material passes through, some remains behind i n the concentrate. These f r a c t i o n s tend to penetrate and become lodged i n the membrane and therefore decrease flow rate through pore plugging. Generally, increasing pressure does not appear to increase f l u x s i g n i f i c a n t l y (19, 20). A l l that happens i s a thickening of the boundary layer u n t i l the rate of material a r r i v a l equals the rate of back-diffusion (14,16). Operating at 60 p s i was used as a means of creating shear i n the passage of whey through the membrane and thereby increasing flow rate. Although i d e n t i c a l f l u x rate was obtained at lower pressures i f turbulence by means of the magnetic s t i r r e r was increased,.it was found that by operating at a constant pressure of 60 p s i and maintaining turbulence at a constant speedy r e s u l t e d i n s a t i s f a c t o r y flow r a t e s . Operating at high temperatures increases permeate f l u x (Figure 7) due to v i s c o s i t y decrease. As pointed out by Roualeyn et a l . (54) operating at the highest possible temperature compatible with the membrane gives best r e s u l t s . They found that every 20° r i s e i n temperature i s accompanied by an increase i n permeate f l u x of approximately 40%, consistent with our r e s u l t s . Moreover, operating at elevated temperatures above 50°C was found to decrease m i c r o b i o l o g i c a l a c t i v i t y (54). K i s s i n g e r et_ a l . (25) showed that by i n o c u l a t i n g the membranes with a mixed b a c t e r i a l culture followed by incubation had no d e s t r u c t i v e e f f e c t yet severe sliming of the membrane " occurred. In our work mi c r o b i a l growth was not a problem since a l l samples were pasteurized before u l t r a f i l t r a t i o n . Although standard plate counts were not done i t was assumed that b a c t e r i a l growth was minimum since a f t e r 20 hours of u l t r a f i l t e r i n g the pH of the whey remained the same. Generally the optimum temperature appears to be around 50°C f o r maximum f l u x (20). This temperature has a d d i t i o n a l advantages from the standpoint of plant operation since the whey comes o f f the cheese vats about t h i s temperature. Only r e c e n t l y has washing of the concentrate been implemented to increase i t s protein concentration (47). Protein values of about 80% on a dry weight basis were obtained by P e r i et a l . (51) but t h e i r washing technique involved four p u r i f i c a t i o n steps. As pointed out by these same workers t h i s s o - c a l l e d d i a f i l t r a t i o n step may be started from the beginning of the operation or a f t e r a preliminary step of simple u l t r a f i l t r a t i o n . The correct combination of u l t r a f i l -t r a t i o n and d i a f i l t r a t i o n i s the key fa c t o r i n defining the optimum process. Our t h e o r e t i c a l c a l c u l a t i o n s (Figure 8) indicated that continuous washing would y i e l d high protein concentrates while minimizing water consumption. According to the data reported i n Table III the optimum process f o r obtaining p r o t e i n concentrates with greater than 7 0% protein on a dry weight basis as well as being low i n ash would be accomplished by continuous washing. The retention of calcium i s high because t h i s f r a c t i o n i s associated with the proteins or i s present as c o l l o i d a l calcium phospahte. It i s evident therefore that . the f r a c t i o n of calcium that can be permeate decreases during the process as protein concentration increases. Moreover, pH adjustment of the wash water (Figure 9) toward the basic side increases f l u x rate since the protein remains denatured and aggregation cannot occur. I t therefore becomes economically p o s s i b l e to obtain a product with a high protein-to-lactose r a t i o . I f membranes can be improved to permit 0% retention of lactose rather than 18% reported here, concentrates with s i g n i f i c a n t l y higher protein could be produced. Per i et a l . (51) showed that d i a f i l t r a t i o n of skim milk increased permeation rate whereas those for whey decreased. Data reported i n Figure 9 also showed a decrease i n permeation rates during the d i a f i l t r a t i o n process. The explanation of t h i s i s that microbial and enzymatic degradation of proteins r e s u l t s during the cheese making process. This tends to increase the proportion of non-protein nitrogen and of proteoses and peptones i n the whey. These f r a c t i o n s have dimensions s i m i l a r to those of the membrane pores and therefore tend to penetrate into the membrane structure and plug i t . Results showed that the BOD of whey can be reduced i n the order of 20%. Although t h i s i s not a substantial decrease from a p o l l u t i o n standpoint, i t i s possible to couple u l t r a f i l t r a t i o n with reverse osmosis and thereby reduce the BOD by 99% (20, 21). Moreover, the use of sanitary RO equipment makes i t possible to control the microbiological' q u a l i t y of water. Thus, reuse of the curd r i n s i n g water i n cottage cheese manufacture would be possible according to the scheme s i m i l a r to that i l l u s t r a t e d i n Figure 10. Combination of the two processes of u l t r a f i l t r a t i o n and reverse osmosis i s now under i n v e s t i g a t i o n i n t h i s lab. High protein concentrates are a t t r a c t i v e from the 47 . Curd * Skim.—-9» Cheese *• 1st Wash * 2nd Wash > 3rd Wash ? Curd Whey -Adjust pH to 7.0 -Pasteurize 72°C f o r 10 min. Separator 1500Xg fo r 5 min. Separator 1500Xg for 5 min. 1 $ Dilute Whey -Adjust pH to 8 . 5 Sediment Water -t I r> Reverse Osmosis Lactose Sediment Whey —> U l t r a f i l t r a t i o n — Protein Concentrate L-* UF Permeate - lactose - water - minerals - s a l t s J, Spray Dry <-or Spray Dry or use as a concentrated Enzyme treatment solut i o n . I 73% protein on a dry 86% lactose on a basis dry basis Figure 10. Flow diagram for the preparation of whey, protein, crude soluble lactose and the reuse of curd r i n s i n g water. 48. standpoint of increased u t i l i z a t i o n of whey (18), but a p o l l u t i o n problem involving disposal of the permeate remains. The lactose recovered i n the permeate i s greater than 8 5%, e a s i l y soluble i n water and therefore has possible a p p l i c a -tions i n the food area (5, 19). As s p e c i f i e d e a r l i e r i n t h i s paper the data 9 reported r e f e r to cheese whey and a 12 50 ram membrane area. The time would be proportionally reduced or the capacity proportionally increased by an increase of membrane area. Optimum conditions suggested i n t h i s work are r e l a t i v e to our experimental conditions and apparatus and are fundamentally dependent on membrane type, temperature, pH and flow conditions. 49 . PART II PREPARATION OF FERRIC WHEY PROTEIN BY HEATING 50. ABSTRACT Soluble f e r r i c whey protein containing more than 80% protein was prepared by an improved heating method at acid pH. Whey was concentrated 3 to 4 times and heated to 92°C f o r 15 min a f t e r adding FeCl and adjusting to pH 2.5. A f t e r centrifugation,the sediment was washed with d i l u t e HC1 at pH 2.5, dissolved and dried. Lactose was c o l l e c t e d from the supernatant. Preconcentration of whey before heating increased the y i e l d of f e r r i c protein to 6 0% of t o t a l N. The optimum pH f o r obtaining soluble products with the highest y i e l d was 2.5 to 3.5 depending on the amount of FeClg added. Washing the p r e c i p i t a t e d protein at pH 2.5 to remove excess s a l t s and high speed blending to dissolve washed p r e c i p i t a t e s at neutral pH were necessary f o r obtaining high s o l u b i l i t y of the dried protein. Iron i n f e r r i c whey protein was available to growing chicks. Results showed no s i g n i f i c a n t difference i n a v a i l a b i l i t y between f e r r i c whey protein and the FeSO^ standard. INTRODUCTION Since the success i n manufacturing f e r r i l a c t i n from cheese whey by Block et a l . (6) i n 19 53, several modifications (22, 23) have been submitted f o r separation of whey proteins with i r o n s a l t s . In the f e r r i l a c t i n manufacturing, FeClg i s added to whey and the pH adjusted a f t e r which i t i s frozen to crys-t a l l i z e the protein and then f i l t e r e d . However, t h i s procedur e s p e c i a l l y f r e e z i n g and f i l t r a t i o n , i s lengthy and tedious. Imado et a l . (22) u t i l i z e d whole whey without separating f e r r i l a c t i n a f t e r addition of FeClg. They added polyphosphate to prevent brown colour due to f e r r i c i r o n i n ion-exchanged f e r r i c whey. The product was successfully used to f o r t i f y ' f l u i d milk, dry milk and dry baby formula without causing vitamin destruction or f l a v o r d e t e r i o r a t i o n during storage. Jones et a l . (23) developed a method to separate whey pr o t e i n u t i l i z i n g a ferripolyphosphate. Although t h e i r cold process i s a d i s t i n c t advantage, the protein content i n the dry products was r e l a t i v e l y low with a considerably higher phosphate content that may disturb normal kidney function e s p e c i a l l y i n babies. We have been t r y i n g to e s t a b l i s h methods to separate f e r r i c whey proteins for f o r t i f i c a t i o n of ir o n to food products. Emphasis was placed on higher y i e l d and s o l u b i l i t y of the dry products. The acid washing method of Harwalker and Emmons (17)was e f f e c t i v e l y employed to maintain high s o l u b i l i t y f o r whey proteins separated by heating. A study on cold processes using u l t r a f i l t r a t i o n f o r unde --natured f e r r i c whey protein i s described i n Part I. This paper reports the r e s u l t s of the preparation of f e r r i c whey protein with a protein content greater than 80%. The conventional heating method at an acid pH was modified to obtain soluble products with high y i e l d s . MATERIALS AND METHODS Preparation of F e r r i c Whey ' Whey was obtained from a l o c a l dairy (Fraser Valley Milk Producers Association, Vancouver, Canada) and c l a r i f i e d by c e n t r i f uging at 1,500 X g f o r 10 min at 0°C i n a S o r v a l l RC2-B c e n t r i f u g e . The supernatant was concentrated three times by volume on a Flash Evaporator (Buckler Instruments, Fort Lee, New Jersey) held at 50°C. Reverse Osmosis Reverse Osmosis experiments were conducted on a lar g e r assembly made by Fre-Del -Engineering Corporation, Santa Ana, C a l i f o r n i a , U.S.A. The membrane unit i s of a sandwich c o n s t r u c t i o n which d i r e c t s the flow of l i q u i d between two c e l l u l o s e acetate membranes 2 5 inch i n area, supported by s t a i n l e s s s t e e l sintered metal back up plates. As whey flowed through the membranes under a pressure of 10 0 0 p s i , water passed out of the hose bibs at the base of the membrane unit and the concentrate was re*-circulated. Pressure, was maintained- constant by the aid of a surge suppressor and an adjustable back, pressure c o n t r o l valve. Volumes of 1500 ml were used. The membrane was s p e c i f i e d as KP9 6 type with s a l t r e j e c t i o n a t 9 5%. A l l experiments were conducted at tempera-tures not exceeding 2 5°C. Iron' Depletion The-method of Pla and F r i t s (53) was followed using one-day-old cockerels. The chicks were housed i n galvanized cages. The basal d i e t and de-ionized water were supplied ad l i b i t u m . Water troughs were painted with a non leaded paint so that rusting would be i n h i b i t e d . A f t e r two weeks on the basal d i e t hemoglobin and hematocrit were determined by the cyanmethemoglobin method of Crosby et a l . (11) and by a m i c r o c a p i l l a r y centrifuge method (10)Respectively. Hemoglobin was determined using Cyanmethemoglobin Standards and Cyanmethemoglobin Reagent (Hycel, Inc., Houston, Texas). Blood samples were drawn from the wing vein and also by heart puncture. Both techniques gave s i m i l a r r e s u l t s . A f t e r chicks were s a t i s f a c t o r i l y depleted (hemoglobin < 5 gm/100 ml and/or hematocrit < 24%) they were divided into three groups of ten. Groups I, II and III received diets containing 20 mg Fe/kg i n the forms of standard FeSO^, f e r r i c whey protein prepared by the heating method described i n t h i s paper, and undenatured whey protein, i . e . dried cottage cheese whey containing 2 24 ug Fe/ml whey re s p e c t i v e l y . A f t e r 2 weeks on the t e s t diets and each subsequent week thereafter f o r 4 weeks hemoglobin and hematocrit analyses were done. The responses were compared to the FeSO^ standard by analysis of variance and Duncan's Multiple Range Test. RESULTS AND DISCUSSION The outline of the procedure f i n a l l y adopted i s shown i n Figure 11. E f f e c t of Preconcentration To increase the y i e l d , whey was concentrated 3 times and heated to 80, 9 0 or 10 0°C for 10 min at pH 4.5. Protein was analyzed i n the supernatant a f t e r centrifuging at 2,000 X g f o r 10 min. The control was boiled f o r 15 min without preconcentration. The protein sedimented was 44, 56 and 61% by heating preconcentrated whey to 80, 90 and 100°C compared to 52% f o r the co n t r o l . The higher y i e l d can be expected by preconcentration under the same heating conditions. However, preconcentrating greater than 4 times decreased the y i e l d due to excessive v i s c o s i t y which prevents sedimentation of coagulated proteins. E f f e c t of pH The pH was varied from 4.5 to 2.5 f o r concentrated wheys, which were subsequently heated to 8 5°C f o r 10 min (Table IV). On average the highest y i e l d was obtained at pH 2.5. However, at t h i s pH the s o l u b i l i t y of the whey with low i r o n content was markedly depressed. The pH optimum f o r obtaining soluble products with high y i e l d s , were 2.5 and 3.5 with and without i r o n r e s p e c t i v e l y . E f f e c t of Washing To decrease ash content and improve s o l u b i l i t y , the Cheese Whey, 2 kg Evaporate 3 to 1 times Concentrate (500 to 650 g) Add .21 ml of. 30% FeClg 1 , adjust to pH 2.5, heat to 92°C f o r 15 min and centrifuge Sediment. Wash with d i l u t e HC1 at pH 2.5. The 1st wash-1 Washed p r e c i p i t a t e Dissolve i n d i l u t e NaOH at pH 7, blend or homogenize and spray dry Protein powder 1 4 g 60% of t o t a l N i n the s t a r t i n g whey 1 -> Supernatant «-Evaporate, cool and centrifuge Sediment Dissolve at pH 7 and centrifuge Sediment (Discard) Supernatant Supernatant Spray dry r Crude soluble o lactose 65 g The retention of iro n i n the f i n a l product was approximately 7 0%. T o t a l protein i n 2 kg of whey, appr. 18 g; 60% y i e l d , Appr. 11 g. .21 ml of 30% FeClg contains .3 X 55.85/ 162.2 X .21 = .0216 g; the retention 70%, appr. 15 mg Fe. Fe i n protein i n f i n a l product, appr. .145. Figure 11. Flow diagram of preparation of f e r r i c whey protein and crude soluble lactose. 57. Table IV. E f f e c t of pH on the s o l u b i l i t y and the recovery of protein i n f i n a l product. pH Fe S o l u b i l i t y Protein Recovery ug/ml whey % % 4.5 224 99 . 5 42.4 56 100 36 . 7 10.8 98 33.9 0 '' 100 28.2 3.5 224 100 55.7 56 100 40.4 10.8 98 39.3 0 100 35.7 2 . 5 224 99 60 . 3 56 100 55.3 10. 8 64 57 .9 0 70.3 57 . 5 Concentrated wheys were heated at 8 5°C f o r 10 rain and the protein p r e c i p i t a t e s were washed twice. 58. washing method of Harwalker and Emmons (17)was employed except that the pH was adjusted to 2.5 instead of 3.5. The r e f r a c t o -metric observation of s o l i d s i n the wash water indicated that washing 2 to 3 times was usually adequate to eliminate most of lactose and s a l t s . Protein i n the t o t a l s o l i d was increased from 40% to 75, 80, and 90 and even 95% by washing one, two and three times r e s p e c t i v e l y . It was also found that blending washed protein p r e c i p i t a t e s at high speeds or homogenizing to disintegrate coagulated proteins when d i s s o l v i n g at neutral pH was important f o r obtaining soluble dry products. Comparison with a conventional heating method The combination of selected conditions (preconcen-t r a t i o n , pH, temperature and washing) was applied and compared to a control prepared by the conventional heating method under an" acid condition. The pH during washing was maintained at 2.5 except f o r the c o n t r o l , - f o r which p l a i n water was used. The r e s u l t s are shown i n Table V. S a t i s f a c t o r y protein recovery and s o l u b i l i t y were obtained by heating cottage cheese whey without i r o n to 9 2°C f o r 10 min at pH 3.5. For whey with i r o n at the l e v e l of 2.9% i n protein (224 yg/ml whey), heating to 92°C at pH 2.5 did not decrease the s o l u b i l i t y or the protein content i n the dry product. Temperatures higher than 92°C increased gelatinous sediments which decreased the s o l u b i l i t y a f t e r drying. The q u a l i t y of the control product was poor with low protein recovery and s o l u b i l i t y . The protein recovery f o r the conventional method was considerably lower than the preconcentration control (52%) probably r e s u l t i n g Table V. Comparison of heating methods f o r preparation of soluble whey protein powder. Whey pH Temper-ature Fe Protein i n dry weight Protein recovery S o l u b i l i t y C ug/ml whey % % % Unconcen-trated 4.5 98 0 76 .4 15.6 25.9 Concen- 3.5 85 0 84.4 39. 2 96.7 trated 4 times 3.5 92 0 90.2 61.0 92 .5 2 . 5 85 224 60 .0 60.3 99.0 2.5 92 224 84.2 60 .1 91. 2 Heated f o r 10 min. 60 . from a loss of protein during washing with p l a i n water. As seen i n Tables V and VI, the dry products contained more than 80% protein with s o l u b i l i t i e s greater than 90% i n water at 2 5°C. The procedure was equally well applied to both cottage cheese whey and cheddar cheese whey. A good y i e l d of crude lactose that was e a s i l y soluble i n water was obtained from the supernatant a f t e r sedimentation of the proteins (Figure 10 and Table VI). Gel' electrophcresis Block' et 'al. (6) showed by paper electrophoresis of f e r r i l a c t i n that both B-lactoglobulin and a-lactalbumin migrated while one spot remained at the o r i g i n . Jones et a l . (2 3) using both urea and nonurea gels found that the addition of ferripolyphosphate to whey caused p r e c i p i t a t i o n of 3-lactoglobulin and a-lactalbumin. F e r r i c whey protein pre-pared by our method exhibited bands s i m i l a r to those of whole whey, implying the p r e c i p i t a t i o n of both B-lactoglobulin and a-lactalbumin by heating with F e C l 3 (Figure 12). Undenatured f e r r i c whey pr o t e i n , separated by u l t r a f i l t r a t i o n , showed the same r e s u l t . Reverse Osmosis Treatment of reconstituted f e r r i c whey supernatant increased f l u x rate greater than 2X as compared to conventional cheese whey. This. increase i s a t t r i b u t e d to the complete removal of whey proteins as shown by gel f i l t r a t i o n studies. Table VI. Analysis of whey protein powder and crude soluble lactose (freeze d r i e d ) . Product Moisture Protein Lactose Ash Fe" 9- -6 % Cottage cheese whey protein 4.1 87 . 6 1.7 7.0 .3 Cheddar cheese whey protein 3.9 81. 0 4.0 4.2 . l i 1st lactose 1.7 4.4 84.3 4.5 2nd lactose 4.9 4.4 87.0 7.5 Adjustable depending on purpose 62. I I } o t s - C a <x- La } G-Lg & B - C a K - C a J Figure j2. Polyacrylamide gel electrophoresis of whey proteins. 1. 3-lactoglobulin; 2. K-casein; 3. whey pasteurized at 72 C f o r 10 min a f t e r adjusting the pH 7.0; 4-. acid casein; 5. the supernatant a f t e r centrifuging No.3 at 1,500 X g f o r 10 min at 0 C; 6. the sediment of No. 5; 7. f e r r i c whey protein. Unlike UF, pH adjustment to 7.0 followed by c e n t r i -fugation decreased rather than increased permeate f l u x . This i s assumed to be due to the formation of insoluble c o l l o i d a l p a r t i c l e s which become lodged i n the membrane pores or accumu-l a t e on the surface. In UF these c o l l o d i a l p a r t i c l e s are small enough to pass through and protein becomes the major cause of blockage. Calcium was removed from the f e r r i c whey supernatant by potassium oxalate and RO treatment s u b s t a n t i a l l y increased f l u x rate as compared to the untreated f e r r i c whey. This c l e a r l y implicates the involvement of s a l t s i n the blockage of RO membranes and further studies are presently underway to elucidate these complex s i t u a t i o n s . Ava11ab1e Iron Block' et a l . (.6) found that the i r o n of f e r r i l a c t i n was r e a d i l y a v a i l a b l e to aenemic rats f o r regeneration of hemoglobin. Imado' et_ a l . (22) reported that there was no d i f f e r e n c e i n the hemoglobin value and the serum iron among r a t s fed with FeSO^, f e r r i c c i t r a t e and t h e i r f e r r i c whey products. However, the iron deposits e s p e c i a l l y the hemo-s i d e r i n i r o n were higher f o r the groups with whey, s u l f a t e , and whey i n t h i s order. Because of frequent disagreement among researchers of feeding te s t r e s u l t s , a n a l y t i c a l pro-cedures f o r avai l a b l e i r o n have been under intensive re-examination. . Recently, a more r e l i a b l e method was submitted (1, 53) and the a v a i l a b i l i t y of iron i n our f e r r i c whey protein was estimated by t h i s new procedure (Table VII). Iron present i n f e r r i c proteins both denatured and undenatured was r e a d i l y a v a i l a b l e to chicks and there was no s i g n i f i c a n t difference from ava i l a b l e i r o n i n the FeSO,. standard. Table VII. Mean terminal values and standard deviations f o r hemoglobin, hematocrit and l i v e r iron of chicks fed d i f f e r e n t sources of i r o n . Iron Source Iron dose Hemoglobin Hematocrit Liver iron -mg/kg d i e t - -g/100 ml- -yg/g wet wt-Basal diet 0 7 .69+1.49a 28 . 24+5. 27 a FeSO^ 20 9 .72+1.18b 41 .7614. 2 2 b 84.5+12. 5 a Denatured f e r r i c whey-proteins 20 9 .76± .59 b 41 .2511. 49 b 63.0117. 5 a Undenatured f e r r i c whey Proteins 20 10 .89+1.23° 40 .63 + 2 . 9 5 b 111,9139. 2a 'a', 'b' and 'c' are s i g n i f i c a n t l y d i f f e r e n t by Duncan's multiple range t e s t (P < .05) 66. LITERATURE CITED 1.. Amine, E.K., R. Neff and D.M. Megsted, 1972 . B i o l o g i c a l estimation of ava i l a b l e i r o n using chicks or r a t s . J. Agr. Food Chem., 20: 24 6. 2. Aschaffenburg, R., 19 64. Protein phenotyping by d i r e c t polyacrylamide-gel electrophoresis of whole milk. Biochim. Biophys. Acta., 82: 188. 3. Association of O f f i c i a l A g r i c u l t u r a l Chemists O f f i c i a l Methods of Analysis, 1970. 11th ed. Washington, D.C. *+. Besik, F. , G. Zarnett, V. Pancuska and A. Mlynarczyk ,1971. Food Engineering, 43(7): 72. 5 . Birch, G.G., 1971. Lactose: One of Nature's Paradoxes. J . Milk Food Tech. 35(1) : 32. 6 . Block, R.J., D. B o i l i n g , K.W. Weiss and G. Zweig, 1953. Studies on bovine whey proteins. I. Preparation of the f e r r i c derivates of whey proteins. Arch. Biochem. Biophys., 47: 88. 7. Bundgaard, A.G., O.J. Olsen and R.F. Madsen, 1972. U l t r a f i l t r a t i o n and h y p e r f i l t r a t i o n of skim milk f o r production of various dairy products. Dai. Ind. , October 1972. 8. Carter, P., 1971. Spectrophotometric determination of serum ir o n at the submillogram l e v e l with a new reagent (Ferrozine) Anal..Biochem., 40: 450. 9 . Chian, E.S.K. and J.T. S e l l d o r f f , 1969. U l t r a f i l t r a t i o n of b i o l o g i c a l materials. Process Biochem.., Sept., 1969. 1 0 . Cohen, R.R..,1967. Anticoagulation centrifugation time and sample r e p l i c a t e number i n the microhematocrit method for avian blood. Poultry S c i . , 46: 214. 1 1 . Crosby, W.H., J.I. Munn and F.W. Furth, 1954. Standardizing a method fo r c l i n i c a l hemaglobin-ometry. U*S. Armed Forces Med. J, 5: 69 3. 67 12. Dolby, R.M., 1961. Dye-binding methods f o r estimation of protein i n milk. J . Dai. Res., 28: 4 3. 13. Fen-ton-May, R.I., C.G'. H i l l , J r . , CM. Amundson, M.H. Lopez and P.D. Auclau, 1972. Concentration and f r a c t i o n a t i o n of skim milk by reverse osmosis and u l t r a f i l t r a t i o n . J. Dai. S c i . , 55(11): 1561. 14. Forbes, F., 1972. Considerations i n the optimization of u l t r a -f i l t r a t i o n . The Chemcial Engineer, January, 197 2. 15. Glover, F.A., 1971. Concentration of milk by u l t r a f i l t r a t i o n and reverse osmosis. J . Dai. Res., 38: 373. 16. Goldsmith, R.L., 1971. Macromolecular u l t r a f i l t r a t i o n with microporous membranes. Ind. Eng. Chem. Fundam., 10(1): 113. 17. Harwalker, V.R. and D.B. Emmons, 1969. Low-ash lactalbumin as a by-product of lactose production. Can. Inst. Food Tech. J . , 2: 9. 18. Holsinger, V.H., L.P. Posate, E.D. DeVilbiss and M.J. Pallansch, 1973. F o r t i f y i n g s o f t drinks with cheese whey protein. Food Tech., 27: 59. 19. Horton, B.S., R.L. Goldsmith and R.R. Z a l l , 1972. Membrane processing of cheese whey reaches commer-c i a l s c ale. Food Tech., 26(2): 30. 20. Horton, B.S., 1970. Prevents whey p o l l u t i o n recovers p r o f i t a b l e by-products. Food Engineering, July 1970. 21. Horton, B.S., R.L. Goldsmith, S. Hossain and R.R. Z a l l , 1970. Membrane separation processes f o r the abatement of p o l l u t i o n from cottage cheese whey. The Milk Ind.. 22. Imado, M., A. Yajima and S. Nakai, 1962. Improvement i n method of f o r t i f i c a t i o n of i r o n for infa n t feeding. Jap. J. Dai . S c i . , 11: A17 7. 68. 23. Jones, S.B., E.B.'Kalan, T.C. Jones and J.F. Hazel, 1972. "Ferripolyphosphate" as a whey protein p r e c i p i t a n t . J . Agr. Food Chem., 20: 229. 24. K i r k p a t r i c k , K.J., 1970. Membrane separation processes. International Dairy Congress (18th, Sydney)lE: 443 (1970) (En) (Dairy Res. Inst., Palmerston North, New Zealand. 25. K i s s i n g e r , J.C. and CO. W i l l i t s , 1970. Preservation of reverse osmosis membranes from microbial attack. Food Tech., 24: 481. 26. L e i g h t e l l , B., 1972. Reverse osmosis i n the concentration of food. Process Biochem.., March 1972 . 27. L i Jerome, C.R., 1964. S t a t i s t i c a l Inference I. Edwards Brothers Inc., Ann Arbor, Michigan. 28. Lim, Toh Hoy, W.L. Dunkley and R.L. Merson, 1970. Role of protein i n reverse osmosis of cottage cheese whey. J . Dai. S c i . , 54: 306. 29. Lowe, E. , E.L. Durkee, R.L. Merson, K. I j i c h i and S.L. Cimino, 1969. Egg white concentrated by reverse osmosis. Food Tech., 23: 45. 30. Lowe, E. , E.L. Durkee and A.I. Morgan, Jr., 1968. A reverse osmosis unit f o r food use. Food Tech., 22: 915. 31. Mann, J . , 1971. Lactalbumin and i t s uses. Dai. Ind., Sept., 1971. 32. Marshall, P.G., W.L. Dunkley and E. Lowe, 1968. Fractionation and concentration of whey by reverse osmosis. Food Tech., 22: 969. 33. Maubois, J.L. and G. Mocq\iot, 19 71. UF of skim milk f o r cheese making. Le L a i t 51: 495. 34. McDonough, F.E., W.A. Mattingly, J.H. Vestal, 1971. Prote i n concentrate from cheese whey by u l t r a -f i l t r a t i o n . J . Dai. S c i . , 54(10): 1406. 69. 35. McDonough, F.E., 1971. Membrane processing a new t o o l f o r whey disposal. Dai. Ind., 36(9): 507. 36. McDonough, F.E. and W.A. Mattingly, 1970. P i l o t - p l a n t concentration of cheese whey by reverse osmosis. Food Tech., 24: 88. 37. McDonough, F.E., 196 8. Whey concentration by reverse osmosis. Food Engineering 40: 124. 38. McKenna, M. , 1970 . Reverse osmosis a new concept i n concentration f o r the dairy industry. Dairy Ind., Nov. 1970. 39. Merson, R.L. and A.I. Morgan, J r . , 1968. Juice concentration by reverse osmosis. Food Tech.. , 22( 5) : 97 . 40. Merson, R.L. and L.F. Ginnette, 1971 Improved processing of foods by reverse osmosis. Food Tech., 25(3) 41. 41. Michaels, A.S., 1968. New separation techniques f o r the CP1. Chemical Engineering Progress, 64: 31 (December). 42. Morgan, A.I. J r . , E. Lowe, R.L. Merson and E.L. Durkee, 1965. Reverse osmosis. Food Tech., 19(2): 52. 43. Morrison, W.R., 1964. A f a s t , simple and r e l i a b l e method for the micro-determination of phosphorous i n b i o l o g i c a l materials. Anal. Biochem., 7: 218. 44. N t a i l i a n a s , H.A. and R. McL. Whitney, 1964. Calcein as an in d i c a t o r f o r the determination of t o t a l calcium and magnesium and calcium alone i n the same aliquot of milk. J . Dai. S c i . , 47: 19. 45. O'Sullivan, A.C., 1972. Whey processing by RO, UF and gel f i l t r a t i o n . Dai. Ind., Dec. 1972. 46. 0;Sullivan, A.C., 1971. Whey processing by reverse osmosis, u l t r a f i l t r a t i o n and gel f i l t r a t i o n . Dai. Ind. , Nov. , 1971. 70 . P e r i , C., Pompei, C. and F. Rossi, 1973. Process optimization in skim milk protein recovery and p u r i f i c a t i o n by u l t r a f i l t r a t i o n . J . of Fd. S c i . , 38: 135-140. P e r i , C. and W. L. Dunkley, 1971. Reverse osmosis of cottage cheese whey. I. Influence of composition of the feed. J . of Fd. S c i . , 36: 25. P e r i , C. and W.L. Dunkley, 1971. Reverse osmosis of cottage cheese whey. I I . Influence of flow conditions. J. of Fd. S c i . , 36: 395. P e r i , C. and W.L. Dunkley, 196 8. Semi-permeable membrane f o r reverse osmosis: tests on permeability and s e l e c t i v i t y with a model system and with whey. Annali d e l l a Facolta d i Agraria d e l l a U n i v e r s i t a d e g l i Studi d i Perugia 23: 19-45 (1968). . P e r i , C. and C. Pompei, Concentration and p u r i f i c a t i o n of milk and whey proteins by u l t r a f i l t r a t i o n . I n s t i t u t o d i Tecnologie Alimentari, U n i v e r s i t a d i Milano, I t a l y . Pharmacia Fine Chemicals Handbook, 1966. Sephadex-gel f i l t r a t i o n i n theory and p r a c t i c e . Pl a , G.W. and J.C. F r i t z , 1970. A v a i l a b i l i t y of iro n . J. Assoc. O f f i c . Agr. Chem. 53: 791. Roualeyn, I., Fenton-May and C.G. H i l l , J r . , 1971. Use of u l t r a f i l t r a t i o n / r e v e r s e osmosis systems for the concentration and f r a c t i o n a t i o n of whey. J. of Fd. S c i . , 36: 14. Stahl, Egon, 1965. Thin-layer chromatography a laboratory handbook. Academic Press Inc., Publishers, New York, London. Standard Methods f o r the Examination of Water and Waste Water, 1965. Twelfth E d i t i o n , APHA . AWWA . WPCF. Trevelyan, W.E. and J.S. Harrison, 1952. Fractionation and microdetermination of c e l l carbohydrates. Biochem. J . 50: 298. 71. 58. Wiley, A.J., A.C.F. Ammerlaan and G.A. Dubey, 1967. A p p l i c a t i o n of reverse osmosis to processing of spent l i q u i r s from the pulp arid paper industry. Tappi, 50(9): 455. 59. W i l l i t s , CO., J'.C. Underwood and U. Mertin, 1967 . Concentration by reverse osmosis of maple sap. Food Tech., 21: 24. 72. APPENDIX 73. TABLE I Analysis of variance i n hemoglobin a f t e r r e p l e t i o n : Source diets error t o t a l d.f. 3 36 39 S.S. 53.213 42.672 95.885 M.S . 17 .738 1.185 Duncan's Multiple Range Test (P < .05) A B D TABLE II Analysis o f variance i n hematocrit a f t e r r e p l e t i o n : Source di e t s error t o t a l d.f. 3 36 39 S.S. 1264.320 563.039 1827.359 M.S. 421.440 15.639 F. 14.964 F. 26.946 Duncan's Multiple Range Test (P < .05) B D 74 TABLE III Analysis of variance of i r o n i n l i v e r samples a f t e r r e p l e t i o n Source d.f. S.S. M.S. F. diets 2 .00239 .00119 .8959 error 3 .00400 .00134 t o t a l 4 .00540 Duncan's Multiple Range Test (P < .0 5) No s i g n i f i c a n t difference 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101301/manifest

Comment

Related Items