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Evaluation of methods for fortifying skim milk powder with vitamin A Paquette, Gaëtan Marc Andre 1985

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EVALUATION OF METHODS FOR FORTIFYING SKIM MILK POWDER WITH VITAMIN A By GAETAN MARC ANDRE PAQUETTE B.Sc.  (Agr.), U n i v e r s i t y of Guelph, 1977  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES Department of Food Science  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1985 (c) Gae'tan Marc Andre Paquette, 1985  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 of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  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 copying o f 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 o f  department o r by h i s o r her r e p r e s e n t a t i v e s .  my  It i s  understood t h a t 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  permission.  Department o f  FOOD SCIENCE  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Van couve r , Canada V6T  1Y3  Date  SEPTEMBER. 1985  written  ABSTRACT  Evaluation of Methods f o r F o r t i f y i n g  Skim Milk Powder with Vitamin A  The f o r t i f i c a t i o n of skim milk powder with vitamin A has been found to be i n e f f e c t i v e with a v a i l a b l e methods.  The purpose of t h i s study was  to assess new methods and materials f o r t h e i r effectiveness i n providing s t a b i l i t y to vitamin A i n f o r t i f i e d skim.milk powder. The f i r s t phase of the project involved t r i a l s i n P i l o t Plants which evaluated 14 d i f f e r e n t treatments f o r vitamin A s t a b i l i t y during periods of twelve months at 22°C and s i x months at 37°C.  storage  The second and  t h i r d phases of the experiment consisted of primary and instant powder t r i a l s i n commercial plants using the most s t a b l e methods from the P i l o t Plant t r i a l s .  In the l a t t e r phases of the p r o j e c t , eight treatments were  tested f o r primary powder and ten f o r instant type of powder. Results show that levels of antioxidants were important the o x i d a t i v e degradation concentrate  of vitamin A i n the milk powder.  to control The vitamin A  containing BHA (5 mg), BHT (55 mg) and ct-tocopherol  antioxidants produced the best r e s u l t s f o r primary powder.  (12.5 mg)  Ascorbyl  palmitate-ct-tocopherol combination of antioxidants was found to be more e f f e c t i v e than the BHA-BHT-ct-tocopherol blend f o r instant powder.  The  level of hydrogenated coconut o i l (HCO) used as the vitamin c a r r i e r was also found to be important 0.1% i n primary powder.  f o r s t a b i l i t y , 0.2% being s l i g h t l y better than  A 12% emulsion i n j e c t e d at such a rate as to add  0.027% o i l i n milk s o l i d s was the best treatment of the instant powder t r i als. Hay-like f l a v o u r i n r e c o n s t i t u t e d skim milk powder was c o r r e l a t e d with vitamin A d e s t r u c t i o n .  ACKNOWLEDGEMENTS  The author wishes to express appreciation t o the f o l l o w i n g : Dr. S. Nakai, Supervisor of my t h e s i s , f o r his advice and guidance during my graduate program and t o Dr. D.B. Emmons f o r his patience, constructive c r i t i c i s m s and f o r many helpful suggestions with regard t o i n t e r p r e t a t i o n of the r e s u l t s and the review of t h i s  manuscript.  Company Representatives f o r providing f o r t i f i c a t i o n materials and product information:  Dr. R. Wasik, Hoffman LaRoche; Ralph Murray,  Kingsway Chocolate; B i l l Woodford, Land O'Lakes. Dr. R. Giroux, Agropur and A l l a n Chia, Gay Lea Foods f o r t h e i r technical assistance and f o r the cooperation of t h e i r companies. Laboratory and Research s t a f f :  M i c h e l l e Murray, Linda Kwan and  p a n e l i s t s at U.B.C, Connie Martin and Janice Cox at Gay Lea Foods, Real Archambeault at Agropur, and Don Beckett, Ralph Cooligan, Duncan Stewart at the Food Research  I n s t i t u t e , f o r t h e i r assistance i n laboratory  analysis and plant work. Mrs. Gail B u t l e r , Engineering and S t a t i s t i c a l Research  Institute,  for her advice and assistance i n the s t a t i s t i c a l analysis of the data. The S e c r e t a r i a l s t a f f of Food Research  Institute for their efforts  in the typing and corrections of the manuscript. The Canadian Dairy Commission f o r funding of the project and to my employer, A g r i c u l t u r e Canada f o r f i n a n c i a l support during my graduate work.  - i v-  TABLE OF CONTENTS  Page  ABSTRACT  i i  ACKNOWLEDGEMENTS  i i i  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF APPENDICES  ix  INTRODUCTION  1  REVIEW OF LITERATURE  3  Vitamin A d e f i c i e n c y  3  Function and technology of vitamin A  4  Vitamin A f o r t i f i c a t i o n of NDM  8  Antioxidant technology  9  O f f - f l a v o u r from vitamin A destruction  13  NEED FOR THE STUDY PROJECT OUTLINE PHASE I - PILOT PLANT TRIALS  14 s  16 18  Introduction  18  Materials and Methods  18  Method of drying and addition of the vitamins  18  Storage and analysis  20  Treatment d e s c r i p t i o n  20  -V-  Page  Results and Discussion  24  Conclusions  28  PHASE I I - COMMERCIAL PLANT TRIALS - PRIMARY POWDER Introduction  29  M a t e r i a l s and Methods  30  Addition of the vitamins  30  Treatment d e s c r i p t i o n  30  Storage and sampling  32  Vitamin A analysis  32  Sensory evaluation  33  Moisture  33  analysis  Method f o r graphing  34  Statistical  37  analysis  Results and Discussion  39  Vitamin A s t a b i 1 i t y  39  Haylike flavour  42  Comparison of production pi ants  42  Comparison of a n a l y t i c a l methods of vitamin A  45  Conclusions  46  -vi-  Page  Phase I I I - COMMERCIAL PLANT TRIALS - INSTANT POWDER Introduction  47  M a t e r i a l s and Methods  48  Treatment d e s c r i p t i o n  48  Preparation of vitamin mixes  48  Addition of the vitamins  51  Storage and sampling  51  Vitamin A analysis  51  Sensory evaluation  51  S t a t i s t i c a l analysis  51  Results and Discussion  53  Vitamin A s t a b i l i t y  53  Haylike flavour  59  Comparison of production  plants  59  Conclusions  62  OVERALL CONCLUSIONS  64  LITERATURE CITED  65  APPENDIX -Additional table  70  - V I  1 -  LIST OF TABLES  1.  Processing conditions i n P i l o t Plants A and D.  2.  Vitamin A f o r t i f i c a t i o n treatments f o r Phase I - P i l o t Plant Trial.  3.  Initial  levels of vitamin A and percent vitamin A losses  during storage i n primary skimmilk powder f o r t i f i e d by d i f f e r e n t treatments i n P i l o t Plants A and D after 3 and 12 months of storage at 22°C and a f t e r 3 and 6 months at 37°C. 4.  Levels of antioxidants contained i n P i l o t Plant treatments  5.  Description of vitamin A f o r t i f i c a t i o n treatments f o r Phase II - Primary Powder.  6.  Estimated regression c o e f f i c i e n t s f o r M u l t i p l e Comparison Analysis f o r Primary Powder stored at 22 and 37°C.  7.  Losses of vitamin A i n Primary Skimmilk Powder f o r t i f i e d by d i f f e r e n t treatments i n Plants A and B after 12 months of storage at 22°c and 6 months at 370C.  8.  Description of vitamin A f o r t i f i c a t i o n treatments f o r Phase III Instant Powder.  9.  Estimated regression c o e f f i c i e n t s f o r M u l t i p l e Comparison Analysis f o r Instant Powder stored at 22 and 37°C.  10.  Losses of vitamin A i n Instant Skimmilk Powder f o r t i f i e d by d i f f e r e n t treatments i n Plants A and B a f t e r 12 months of storage at 22°C and 6 months at 370C.  -vi i i -  LIST OF FIGURES  Pac£  1.  Formulas of most common forms of vitamin A and provitamin  5  A.  2.  Vitamin A depletion i n Primary Skimmilk Powder during 12 months of storage at 22°C.  3.  4.  35  Vitamin A depletion i n Primary Skimmilk Powder during 6 months of storage at 3 7 0 C .  35  Vitamin A depletion i n Instant Skimmilk Powder during 12  54  months of storage at 22°C.  5.  Vitamin A depletion i n Instant Skimmilk Powder during 6 months of storage at 37°C.  55  LIST OF APPENDICES  Regression equations f o r the Primary Powder treatments when stored at 22 and 37°C.  Regression equations f o r the Instant Powder treatments during storage at 22 and 37°C.  - 1 -  INTRODUCTION  Despite outstanding advances i n vitamin f o r t i f i c a t i o n of food, vitamin A d e f i c i e n c y s t i l l occurs i n endemic proportions i n many developing countries and i s o c c a s i o n a l l y seen i n t e c h n o l o g i c a l l y developed s o c i e t i e s (WHO, 1982; McLaren, 1980). nonfat dry milk (NDM)  Interest i n the f o r t i f i c a t i o n of  with vitamin-A has i n t e n s i f i e d i n recent years as a  r e s u l t of growing concerns f o r n u t r i t i o n a l l y d e f i c i e n t populations r e s u l t i n g i n the export of increasing q u a n t i t i e s of t h i s n u t r i t i o u s product to Third World Countries. In 1984,  the Food Aid Program of the Canadian International  Development Agency (CIDA) shipped approximately NDM to f o o d - d e f i c i e n t areas of the world.  22,000 tonnes of f o r t i f i e d  Since 1979, f o l l o w i n g  recommendations from the Protein Advisory Group of the U.N. Agency (Anonymous, 1976)  and Marquardt (1979), CIDA has adopted the  recommendation t o f o r t i f y a l l NDM f o r Food Aid Programs with vitamin A t o a level of 5000 I.U./100 g. In Canada, the Food and Drugs Act requires f o r t i f i c a t i o n of i n s t a n t i z e d nonfat dry milk f o r consumers with vitamin A.  Section  B.08.014 of the Act (1979) s t i p u l a t e s that "the added vitamin A s h a l l be in such amount that a reasonable d a i l y intake of the milk contains not less than 1200 I.U. and not more than 2500 I.U. of vitamin A". The reasonable d a i l y intake of mi-Ik i s determined f u r t h e r i n Schedule K of the Act as 852 ml.  - 2 -  Therefore the addition of vitamin A to i n s t a n t i z e d nonfat dry milk i s usually between 1400 and 2900 I.U. lOOg allowing f o r overage. In s p i t e of extensive research i n developing methods to produce a stable vitamin A f o r t i f i e d NDM, satisfactory.  t h i s process has remained less than  In an attempt to improve the s t a b i l i t y of vitamin A i n skim  milk powder, a research project funded by the Canadian Dairy Commission was undertaken to evaluate the effectiveness of vitamin A f o r t i f i c a t i o n methods. The main objective was to test proposed new methods of instant NDM storage.  primary  and  f o r t i f i c a t i o n by determining the vitamin A s t a b i l i t y during  The development of haylike f l a v o u r was  also examined.  - 3 -  REVIEW OF LITERATURE Vitamin A deficiency Vitamin A d e f i c i e n c y  has been  studied extensively  i n experimental  animals and i n humans. Early signs of vitaminosis A include loss of appet i t e , growth f a i l u r e and impaired immune response with lowered resistance to i n f e c t i o n .  In humans the signs of c l i n i c a l importance are the ocular  manifestations  (McLaren, 1984). Xerophthalmia, the term generally used to  cover a l l the ocular manifestations of vitamin A d e f i c i e n c y , i s the most common cause of blindness in young c h i l d r e n throughout the world 1976;  Sommer et a l . , 1981).  (WHO,  The P r o t e i n - C a l o r i e Group of the United  Nations has l i s t e d 73 countries and t e r r i t o r i e s where i t i s considered that a vitamin A d e f i c i e n c y problem of public health s i g n i f i c a n c e occurs (Anonymous, 1976).  That report recognized xerophthalmia  to be endemic i n  many of the countries i n southern and eastern A s i a and parts of L a t i n Amer i c a . In recent years, attention has also been drawn to i t s frequent occurrence i n many countries i n A f r i c a and the Middle  East.  In well-nourished s o c i e t i e s , requirements are more than adequately met by an ample intake of both vitamin A and carotenoids from milk, vegetables and f r u i t s , but i n developing countries, requirements are frequently not met. The shipping of high protein foods alone to these n u t r i t i o n a l ly d e f i c i e n t populations  i s not s u f f i c i e n t . I t i s known that with diets  having p r o t e i n supplements without adequate vitamin A f o r t i f i c a t i o n , vitamin A depletion occurs in the l i v e r and p r e c i p i t a t e s xerophthalmia ( S r i k a n t i a , 1975;  P e r e i r a and Begum, 1976).  - 4 -  Skim milk powder being consumed i n large q u a n t i t i e s may be used as a vehicle to provide d e f i c i e n t populations with vitamin A and e s p e c i a l l y c h i l d r e n who are most frequently in need of the antixerophthalmic and a n t i - i n f e c t i v e vitamin. Function and technology of vitamin A The best-defined role of vitamin A i s i n v i s i o n .  Other less defined  n u t r i t i o n a l roles are with growth and d i f f e r e n t i a t i o n of c e l l s , i . e . , gene expression.  C l i n i c a l l y , large doses of vitamin A have been shown to be  e f f e c t i v e i n the treatment of many skin disorders and various kinds of cancer (Sporn and Newton, 1979; B o l l a g , 1979; McLaren, 1984).  The term  "vitamin A" includes a l l B-ionone d e r i v a t i v e s , other than the provitamin A carotenoids. Figure 1 shows the formulas  f o r the more common forms of  vitamin A which include: a l l - t r a n s r e t i n o l (Figure IA); esters of a l l - t r a n s r e t i n o l such as r e t i n y l palmitate (Figure IB) and r e t i n y l acetate (Figure IC); the aldehyde form of a l l - t r a n s r e t i n o l commonly designated  as retinaldehyde or r e t i n a l  (Figure ID); and r e t i n o i c acid  (Figure I E ) , the a c i d i c form of vitamin A. Among the more than 400 characterized carotenoids, only 30 possess provitamin A a c t i v i t y . The most active carotenoid, a l l - t r a n s 3-carotene i s shown i n Figure IF. Vitamin A i s f a i r l y stable when heated to moderate temperatures in an i n e r t atmosphere i n the absence of l i g h t , but i t i s unstable i n the presence of oxygen or a i r , or when exposed to u l t r a v i o l e t l i g h t . metals may also accelerate the oxidation of vitamin A.  Trace  Moisture and pH  are somewhat more c r i t i c a l f o r c e r t a i n forms of the vitamin (acetate ester)(Bauernfeind, 1978).  Therefore, some precautions i n handling  - 5 -  Figure 1.  Formulas of most common forms of vitamin A and Provitamin A.  -6-  CH OH 2  all-trans retinol  CH 0C0C H 2  15  retinyl palmitate  CH 0C0CH 2  3  retinyl acetate  retinaldehyde or retinal  COOH  retinoic acid  all-trans g-carotene  31  - 7vitamin A include:, (a) exclusion of oxygen; (b) protection against l i g h t ; (c) avoidance of pro-oxidant trace metals and strongly a c i d i c environment. The p r i n c i p a l methods of s t a b i l i z i n g vitamin A involve (a) s e a l i n g under vacuum or i n e r t gas, (b.) storage at low temperatures, (c) the addition of antioxidants, (d) coating and s e a l i n g vitamin containing o i l - d r o p l e t s with a p r o t e c t i v e matrix such as g e l a t i n or vegetable gums. The f i r s t two methods are common methods of s t o r i n g any unstable compound, but they are not always convenient  and p r a c t i c a l .  The use of antioxidants to protect vitamin A i s common p r a c t i c e . However, users are confined t o those antioxidants that are acceptable f o r food a d d i t i o n . In the preparation of dry forms of vitamin A, powders, granulates, microspheres,  beadlets and agglomerates have been prepared by  a v a r i e t y of processing methods i n v o l v i n g absorption, granulation, spray congealing and encapsulating procedures as d e t a i l e d by Klaui. et al.(1970).  1  - 8 -  Vitamin A f o r t i f i c a t i o n of  NDM  The addition of vitamin A to nonfat dry milk i s not new;trials were reported i n the l i t e r a t u r e over 30 years ago (Olson et a l . , 1949; Bauernfeind i n 1947.  et a l . , 1953)  soon a f t e r the successful synthesis of vitamin A  Since then, attempts to f o r t i f y dry milk products with vitamin A  have been discouraging due to o f f - f l a v o r and s t a b i l i t y problems. that the added vitamin A i n NDM  generally oxidizes e a s i l y can  The f a c t  be  a t t r i b u t e d to f a c t o r s , such as the r i g o r s of the process to dry the product; the degree of a i r exposure and the frequently long length of storage at high temperature which a l l p o s s i b l y contributes to the d i f f i c u l t y of producing  a product with stable vitamin A.  Several studies contributed greatly to the p r a c t i c a l f o r t i f i c a t i o n of NDM  vitamin  by examining the f o l l o w i n g f a c t o r s : a) c a r r i e r  formulation f o r the vitamin A (Conochie and Wilkinson, 1956), b) influence of homogenization (Shroff et a l . , 1954), c) e f f e c t of heat (Wilkinson Conochie, 1958), d) e f f e c t of l i g h t (Dalle et a l . , 1969; 1970;  Sattar et a l . , 1976;  Smith and MacLeod, 1957; (Anantakrishan  and  Lerner et a l . ,  Sattar et a l . , 1977; Thompson and Erdody,  1974;  Stu11, 1951), e) influence of r e c o n s t i t u t i o n  and Conochie, 1958)  and f ) importance of packaging (Wodsak,  1953). Some of these e a r l i e r works were basis of studies Bauernfeind A l l e n (1963) to improve methods of vitamin A f o r t i f i c a t i o n to NDM. 1963,  these researchers o u t l i n e d two new methods f o r vitamin A  f o r t i f i c a t i o n of  NDM.  and In  - 9 -  The f i r s t , the wet method, involved the addition of  concentrated,  s t a b i l i z e d vitamin A palmitate i n a c a r r i e r of l i q u i f i e d hydrogenated coconut o i l (HC0-) to condensed skim milk p r i o r to spray drying.  The  dry  method involved the blending of dry, s t a b i l i z e d vitamin A palmitate beadlets to NDM.  One year l a t e r , Bauernfeind  demonstrated that both "wet"  and Parman (1964)  and "dry" methods offered a high degree of  vitamin A s t a b i l i t y and low o f f - f l a v o r to the NDM. success now  The key to t h e i r  appears to be i n the use of a n t i o x i d a n t - s t a b i l i z e d vitamin A.  I t has been suggested that added s y n t h e t i c vitamin A i s less stable than n a t u r a l l y - o c c u r r i n g vitamin A (Thompson and Erdody, 1974).  This i s  p o s s i b l y due to the d i f f i c u l t y of blending small amounts of a l i p i d soluble substance i n t o a n o n - l i p i d s o l u t i o n r e s u l t i n g i n the synthetic vitamin A being i n fewer dispersed p a r t i c l e s than n a t u r a l l y - o c c u r r i n g vitamins.  Another p o s s i b i l i t y i s that skim milk might have lost i t s  natural antioxidants that protect native vitamin A.  Antioxidant technology I t has long been observed that c e r t a i n substances i n h i b i t oxidation or antioxidant a c t i o n .  Various substances have been established as safe  and e f f e c t i v e as antioxidant; they can be divided into two comprised of primary antioxidants and of s y n e r g i s t s .  categories  Primary  antioxidants  are those substances which function by i n h i b i t i n g or i n t e r r u p t i n g the free r a d i c a l stage or the i n i t i a t i o n step of autoxidation (Sherwin, 1976). Thus by being p r e f e r e n t i a l l y o x i d i z e d , the antioxidants e i t h e r prevent d i r e c t oxidation or provide i n d i r e c t protection by breaking the oxidation chain reaction.  - 10 -  A l i s t of commonly used primary antioxidants i n vegetable o i l include the tocopherols, g a l l a t e s , nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole  (BHA), butylated hydroxytoluene (BHT) and t e r t i a r y butylhy-  droquinone (TBHQ). The l i s t of permitted primary antioxidants i n vitamin A concentrates  i s l i m i t e d to BHA,BHT and dl-ct-tocopherol. In Canada, the  Food and Drugs Act, D i v i s i o n B.16.100 under Class IV Preservatives (1979) s t i p u l a t e s that the addition of BHA and BHT to vitamin A preparations i s l i m i t e d to 5 mg of each BHA and BHT per m i l l i o n I.U. of vitamin A. There i s no r e s t r i c t i o n on dl-a-tocopherol. Synergists are substances which, i n combination with other substances, r e s u l t i n a mixture whose a c t i v i t y i s greater than the sum of the a c t i v i t y of the i n d i v i d u a l components. Synergists may possess no a c t i v i t y of t h e i r own (e.g. c i t r i c acid) or may themselves be antioxidants (e.g. ascorbyl palmitate). Their actual mode of action has not yet been f u l l y explained but probably  involves chelation of prooxidant metals, regenera-  t i o n or sparing of primary antioxidants and/or i n h i b i t i o n of peroxide decomposition which interrupt the autoxidation process (Sherwin,  1976).  Synergists f o r primary antioxidants are acids such as c i t r i c , phosphoric,  ascorbic and t a r t a r i c . Derivatives of the acids such as  ascorbyl palmitate have better s o l u b i l i t y i n o i l and are also e f f e c t i v e . L e c i t h i n i s another substance that appear t o e x h i b i t an antioxidant effect.  However, i t i s suggested that t h i s e f f e c t may be a s y n e r g i s t i c  e f f e c t on the true antioxidant action of primary antioxidants 1976).  (Sherwin,  - 11 -  Antioxidants and synergists thus protect not only f a t s from o x i d i z i n g , but also other substances of an "unsaturated"  nature that are  contained i n foods such as vitamin A. Evidence of the a n t i o x i d a t i v e e f f e c t of tocopherol i n vegetable was  observed over 40 years ago (Greenbank et a l . , 1944)  oils  and research f o r  i t s p o s s i b l e use in dairy products has since been extensive (Abbot and Waite, 1965; Abbot and Waite, 1962; Merzametov and Gadzhieva, 1982;  Battna et a l . , 1982; Timmen, 1978;  Erickson et a l . , 1963; King, 1968).  Dl-o>tocopherol i s one of the most widely used antioxidants f o r i t s action as a primary antioxidant and vitamin E a c t i v i t y , but at l e a s t seven other types of tocopherol have been used as antioxidant with varying degrees of effectiveness. Abbot and Waite (1965) tested dl-y-tocopherol and mixtures of a,y,6 -tocopherols  along with dodecyl  g a l l a t e i n spray-dried whole milk powder.  No tocopherol preparation was e f f e c t i v e , but dodecyl  g a l l a t e was.  P r e v i o u s l y , Lea and Ward (1959) and Lea (1960) found that out of seven tocopherols, ^-tocopherol gave a c o n s i s t e n t l y good performance but a-tocopherol tocopherols  was not e f f e c t i v e . Abbot and Waite (1965) reported that the and dodecyl  g a l l a t e did not impart any o f f - f l a v o r t o the  powder. They were used at the 0.01%  level i n the powder.  Several other primary antioxidants and synergists have been used i n the d a i r y industry. Abbot and Waite (1962) tested flavones, g a l l a t e s , BHA and NDGA i n spray-dried whole milk powder stored f o r up to 400 days at 20 and 37°C.  Dodecyl g a l l a t e was shown to be a very e f f e c t i v e antioxidant  and, propyl g a l l a t e and NDGA, w h i l s t gave good protection were less so;  - 12 -  BHA  did l i t t l e t o improve keeping q u a l i t y . A l l antioxidants were used at  the 0.01%  l e v e l i n the powder. When powder i s f o r t i f i e d at 5000 I.U./lOOg  t h i s level of antioxidant (0.01%) i s equivalent to 20 mg/million  units of  vitamin A. Pont (1964) showed  that dodecyl g a l l a t e and NDGA are two times  better than BHA, BHT and ascorbyl palmitate and three times better than a-tocopherol  and l e c i t h i n i n prolonging the induction period of oxidation  in butter. Hammond (1970) found BHT, propyl g a l l a t e and NDGA e f f e c t i v e antioxidants f o r b u t t e r o i l . S t u l l  (1951) showed the effectiveness of NDGA  at r e t a r d i n g the oxidation of whole milk powder during 12 months of storage at 22°C.  Those researchers  also showed the enhanced antioxidant  power of NDGA when used with c i t r i c acid as s y n e r g i s t . Timmen (1978) tested i n pure b u t t e r f a t the a n t i o x i d a t i v e e f f e c t of g a l l a t e s , BHA, BHT, a-tocopherol  and ascorbyl palmitate s i n g l y or i n  combination. Simultaneous addition of a-tocopherol  and ascorbyl  palmitate  d r a s t i c a l l y retarded o x i d a t i o n ; t h i s e f f e c t being f u r t h e r i n t e n s i f i e d by l e c i t h i n and propyl g a l l a t e . As demonstrated i n the above, the action of synergists in combinations such as ascorbyl palmitate, dl-a-tocopherol and l e c i t h i n have been widely studied. (Battna et a l . , 1982;  Schuler, 1980;  S t u l l , 1951). The  amount of antioxidant used t o ensure a proper s t a b i l i z a t i o n  should be the smallest p o s s i b l e ; f o r most food 100-500 ppm are sufficient.  The addition of larger q u a n t i t i e s may i n c e r t a i n cases  produce the opposite r e s u l t , i . e . , accelerate o x i d a t i o n . is important to pay attention t o the o r i g i n a l tocopherol  For example, i t content of food  - 13 -  because i n general, the t o t a l tocopherol should not exceed 1000 ppm or 1 mg per gram of o i l (Schuler, 1980).  Others ( K l a u i , 1979; Timmen, 1978)  have also reported that a great excess of tocopherol no longer has an a n t i o x i d a t i v e e f f e c t on r e t i n o l or r e t i n y l acetate; 50 ppm being considered optimum. Ascorbyl palmitate, dl-a-tocopherol (vitamin E) and ascorbic acid (vitamin C) are often added t o foods f o r t h e i r dual purposes by acting as antioxidants and f o r t h e i r b i o l o g i c a l a c t i v i t y as vitamins when not oxidized.  Ascorbyl palmitate has the f u l l b i o l o g i c a l a c t i v i t y of vitamin  C; i t breaks down i n the d i g e s t i v e t r a c t r e l e a s i n g ascorbic a c i d . For that reason, Health and Welfare Canada has no r e s t r i c t i o n on the levels of a-tocopherol, ascorbyl palmitate and ascorbic acid added t o foods. Off-flavour from vitamin A destruction  In addition t o loss i n vitamin A a c t i v i t y when destroyed by o x i d a t i o n , the by-products of t h i s reaction have been reported to cause an o f f - f l a v o r c a l l e d "haylike f l a v o r " . As described i n the term, t h i s o f f - f l a v o r i s comparable t o the smell coming from a drying hay f i e l d on a sunny summer day. Haylike f l a v o r may be described as a l f a l f a or c a r r o t - l i k e and i s d i s t i n c t l y d i f f e r e n t than the o x i d i z e d f l a v o r from f a t oxidation or the activated f l a v o r caused by u l t r a v i o l e t l i g h t on milk protein (Nakai et a l . , 1983).  Many researchers have published the  a s s o c i a t i o n of hay-like f l a v o r development with destruction of added vitamin A i n f o r t i f i e d dairy products Bauernfeind  (Weckel and Chicoye, 1954;  and A l l e n , 1963; Thomas et a l . , 1965, Suyama et a l . , 1983;  Nakai et a l . , 1983).  -  14 -  NEED FOR THE STUDY  Despite e a r l i e r t r i a l s demonstrating the effectiveness of Bauernfeind's suggested methods of f o r t i f i c a t i o n , s t i l l r e c e n t l y , the l i t e r a t u r e has c i t e d several incidences i n which researchers have observed rapid loss of vitamin A i n NDM during storage (Woollard and Edmiston, 1983; Nakai et a l . , 1983; Suyama et a l . , 1983; deBoer et al.,1984) and simultaneous development of haylike f l a v o r (Nakai et a l . , 1983; Thomas et al.,  1965; Weckel and Chicoye, 1954; Suyama et a l . , 1983). In a previous Canadian Dairy Commission (CDC) contract conducted at  the U n i v e r s i t y of B r i t i s h Colombia, Nakai et a l . (1983) found from a market survey that 27% of i n s t a n t i z e d NDM samples obtained from various manufacturers i n Canada and the U.S. had vitamin A levels less than the accepted minimim (14 I.U./g). Of the r e t a i l samples of both U.S. and Canadian o r i g i n , 73% had vitamin A levels less than 20 I.U./g. indicated that there was s t i l l  This  a problem with NDM f o r t i f i c a t i o n at the  r e t a i l l e v e l . This same study found that dry-blending vitamin A beadlets with the powder at the agglomeration chamber during the i n s t a n t i z i n g process was the best method and the "wet" method adding the vitamin A i n a hydrogenated coconut o i l emulsion was the next best method. The r e s u l t of the above survey spelled out the need f o r f u r t h e r research.  The need f o r an i n v e s t i g a t i o n into the effectiveness of  proposed new methods of vitamin f o r t i f i c a t i o n was also suggested by A g r i c u l t u r e Canada researchers. Research was also requested by the milk powder industry as a r e s u l t of the findings from the above research project. The Canadian International Development Agency (CIDA)  - 15 -  which buys large q u a n t i t i e s of f o r t i f i e d NDM  annually was  requesting recommendations regarding t h i s subject.  another body  The major portion of  t h e i r milk powder purchase i s f o r t i f i e d using the dry  batch-blending  method of incorporating a beadlet vitamin concentrate to the powder. As mentioned e a r l i e r , t h i s method has been shown to be e f f e c t i v e i n producing powder with reasonable is p r o h i b i t i v e l y expensive,  vitamin A s t a b i l i t y during storage, but  i . e . $145/tonne. Since the major portion of  t h i s cost i s due to the labour required f o r the debagging, and re-bagging  batch-blending  of the powder, the discovery of an equally e f f e c t i v e method  of f o r t i f i c a t i o n during or p r i o r to production has s i g n i f i c a n t saving p o t e n t i a l . The cost of such a procedure ( i n - l i n e addition) including the f o r t i f i c a t i o n materials i s estimated at approximately  $15-20/tonne of  powder. For the f o r t i f i c a t i o n of 22,000 tonnes the saving f o r CIDA's Food Aid Program alone would be $2.75 m i l l i o n s f o r one year.  - 16 -  PROJECT OUTLINE  In an attempt t o solve t h i s seemingly unsolved problem of vitamin A i n s t a b i l i t y , a research project was undertaken with the f o l l o w i n g objectives: 1.  Prepare i n P i l o t Plants and t e s t the storage s t a b i l i t y of vitamin A  added t o skim milk by d i f f e r e n t methods before drying. 2.  Prepare i n Commercial Plants and t e s t the storage s t a b i l i t y of  vitamin A added by the best p i l o t plant methods f o r instant and primary powder production. A major U.S. powder manufacturer was v i s i t e d i n 1982 to obtain the l a t e s t U.S. technology fortification  on vitamin A f o r t i f i c a t i o n t o NDM.  For the  of instant powder, vitamin A i n a beadlet form was metered  with a dry vitamin feeder into the powder ahead of the agglomerator.  They  had developed t h i s process f o l l o w i n g a problem of poor r e c o n s t i t u t a b i l i t y which arose from a previous method i n v o l v i n g the spraying of a coconut-oil-based  product into the agglomerator.  The vitamin A s t a b i l i t y  of the beadlet method was claimed to be good, but produced some variability  i n levels.  The regular powder was f o r t i f i e d using a vitaminized coconut-oil concentrate f o l l o w i n g Bauernfeind's method except that approximately 0.06% was added to the powder instead of 0.2%.  The USDA requires the f o r t i f i e d  NDM t o contain vitamin A i n a range of 2200-4400 I.U./lOOg f o r t h e i r Food Aid Program.  This method appeared to e a s i l y meet those requirements at  the time of manufacture; they f e l t that i t was the best and the simplest  - 17 -  method of adding vitamins to regular powder. The methods of f o r t i f i c a t i o n tested i n t h i s project are based on the "wet"  method of f o r t i f i c a t i o n . This basic procedure i s of p a r t i c u l a r  i n t e r e s t to both instant and primary powder manufacturers since i t has the advantage of being added on a continuous on-line operation. The experimental  work of t h i s project was divided i n t o three phases;  Phase I (Objective #1) involved the t e s t i n g of several treatments at the P i l o t Plant level i n order to determine the best possible treatments f o r the subsequent phases of the project. Phases I I and I I I were c a r r i e d out in Commercial P l a n t s , by applying the treatments chosen from the r e s u l t s of Phase I , as most l i k e l y to produce powder with stable vitamin A.  - 18 -  PHASE I - PILOT PLANTS TRIALS  Experiments f o r t h i s phase of the project were conducted i n two different P i l o t Plants.  Those P i l o t Plants w i l l be referred t o as P i l o t  Plant A and P i l o t Plant D i n t h i s report.  Each treatment was r e p l i c a t e d  twice during d i f f e r e n t work weeks and at a l t e r n a t i n g P i l o t P l a n t s . Materials and Methods  Methods of drying milk and addition of vitamins Enough condensed skimmmik of 36-44% t o t a l s o l i d s was obtained to spray dry at least 20 kg of powder f o r each treatment.  To f a c i l i t a t e the  incorporation of the vitamins, about 10% (6 kg) of the amount of condensed skimmilk  required f o r a batch, was heated t o 50-52°C.  As the generally  accepted minimum Vitamin A level i n f o r t i f i e d NDM i s 22 I.U./g, t o ensure t h i s amount i s found i n the dried product, i t i s necessary t o include a c e r t a i n percentage of overage t o the level of added vitamin A.  Therefore  30 I.U. of vitamin A and 6 I.U. of vitamin D2 per gram of dry powder were added. The vitamins concentrate containing vitamins A and D, and the a n t i oxidants formulation i n an o i l c a r r i e r , was then added with constant  agi-  t a t i o n t o the heated portion of the condensed milk which was immediately homogenized with a double stage homogenizer at pressures of 2000 and 500 psi.  This vitaminized portion (6 kg) was then thoroughly blended with the  rest of the batch by manual s t i r r i n g and immediately  spray-dried. The  various processing conditions and types of dryer f o r both P i l o t Plants are l i s t e d i n Table 1. Following the spray-drying of each  treatment,  Table 1.  Processing conditions i n P i l o t Plant A and D.  Processing Parameter  P i l o t Plant A  P i l o t Plant D  40-44%  36-38%  2°C  2°C  Temperature o f O i l Vitamin mix  49°C  49°C  Temperature of Oil Vitamin concentrate at homogenization  49°C  49°C  Temperature o f vitaminized batch p r i o r to drying  n°c  11°C  Concentrate  Preparation  Total s o l i d s o f Condensed Milk Holding temperature of Condensed Milk (when held overnight)  Drying Conditions Name o f d r i e r  Anhydro Compact  Rogers  Type of d r i e r  Tower co-current with cyclone collector  Inverted teardrop co-current with cyclone collector.  Capacity (Kg powder/hr)  20  25  I n l e t a i r temperature  200°C  155°C  Outlet a i r temperature  98°C  80°C  Type o f nozzle  Centrifugal  #72-220  Method o f removing powder from d r i e r  Cyclone c o l l e c t o r emptied every 15 min.  Scrape bottom box each 15 min plus cyclone collector.  - 20 -  the dryer box was swept clean to minimize carry-over from one treatment to another.  Each l o t of vitaminized powder was then blended f o r one hour i n  a butter-churn to ensure an homogeneous d i s t r i b u t i o n of the vitamins. Storage and analysis Each l o t of powder was subsequently divided into 250-g samples i n foi1-1 aminated bags.  A number of those powder bags from each treatment  were then stored at 22°C (room temperature) and 37°C (high temperature) to be analysed f o r vitamin A content  at 0,1,2,3,4,6,9 and 12 months (22°C)  and 0,1,2,3,4 and 6 months (37°C) of storage.  The spectrofluorometric  method as described by Thompson et a l . (1978) was used by laboratory C to determine the vitamin A content. each l o t at the beginning  Moisture content was also determined on  and at the end of the storage period.  Treatment d e s c r i p t i o n ) a  The l i s t of the treatments and processing conditions tested i n t h i s trial  are shown i n Tables 1 and 2. The f o l l o w i n g are b r i e f descriptions of  the materials used i n the treatment preparations.  Treatment A uses a  commercial preparation with a vitamin concentration of 45,400 I.II. of vitamin A palmitate and 9090 I.U. of vitamin D3 ( c h o l e c a l c i f e r o l ) per gram intended f o r non-instantized NDM.  In a d d i t i o n , t h i s product contains  polysorbate 80 with BHA and BHT as antioxidant i n a base of hydrogenated coconut o i l (HCO).  Due to i t s HCO content, t h i s concentrate  i s not l i q u i d  at room temperature. )Trade names of commercial products were not used throughout t h i s report t o protect the i d e n t i t y of the implicated companies. a  - 21 -  Table 2.  Vitamin A f o r t i f i c a t i o n treatments f o r Phase I - P i l o t Plant T r i a l .  A.  Commercial HCO-based product  B.  Commercial milkfat-based product  C.  M i l k f a t c a r r i e r -0.1% (no antioxidant)  D.  M i l k f a t c a r r i e r -0.1% (500 ppm commercial antioxidant mixture)  E.  Commercial Vitamin A concentrate  F.  HCO  c a r r i e r -0.05% (no antioxidant)  G.  HCO  c a r r i e r -0.05% (500 ppm commercial antioxidant mixture)  H.  HCO  c a r r i e r -0.10% (no antioxidant)  I.  HCO  c a r r i e r -0.10% (500 ppm commercial antioxidant mixture)  J.  HCO  c a r r i e r -0.10% (2000 ppm commercial antioxidant mixture)  K.  HCO  c a r r i e r -0.10% (Commercial s t a b i l i z e d vitamin  concentrate)  L.  HCO  c a r r i e r -0.20% (Commercial s t a b i l i z e d vitamin  concentrate)  N.  HCO  c a r r i e r -0.20% (no antioxidant)  0.  HCO  c a r r i e r -0.20% (500 ppm commercial antioxidant mixture)  (vegetable  oil-based)  - 22 -  Treatment B uses a commercial preparation containing the same vitamin A and D concentrations  as treatment A, but i s i n a base of reconstituted  non-fat dry milk and butter o i l .  This product i s meant f o r instant skim-  milk powder f o r t i f i c a t i o n and has no declared antioxidants i n i t s content. Treatments C and D are lab-made preparations using butter o i l from melted butter as the vitamin c a r r i e r added to condensed milk at a level of of the dry weight.  Vitamins A ( a l l t r a n s - r e t i n y l palmitate) and D  added to the butter o i l from a highly concentrated per gram i n vegetable Treatment E uses a U.S.  3  0.1% were  source (1 m i l l i o n  I.U.  oil). commercial preparation with vitamins A and D con-  centrations of 50,000 I.U. and 10,000 I.U. per gram, r e s p e c t i v e l y .  The  vitamin c a r r i e r i n t h i s product i s a blend of hydrogenated coconut o i l and vegetable  o i l containing a food-grade e m u l s i f i e r ; i t i s orange i n colour  and remains almost l i q u i d (very soft paste) at room temperature. The  antioxidants used for Treatments D,G,I,J and 0 are from a commer-  c i a l mixture containing a blend of ascorbyl palmitate (23%) and d l - a tocopherol  (7.5%) with c i t r i c acid as synergist i n a mono- and d i -  glycerides matrix.  This product can be described as off-white to tan i n  colour and odourless with a waxy and l a r d - l i k e consistency.  Treatments F  to 0 are a l l lab-made preparations using f u l l y hydrogenated pure coconut oil  (Peroxide Value = 0.20  to the respective percent sed milk.  and a n t i o x i d a n t - f r e e ) as the vitamins (0.05, 0.1  or 0.2%)  carrier  of the dry weight of conden-  The antioxidant and vitamin source used f o r these treatments i s  the same materials as described under Treatments C and D s e c t i o n , except  - 23 -  f o r Treatments K and L f o r which the vitamin A source contained i t s own antioxidants. i s 5 mg of BHA, of vitamin A.  The antioxidant content f o r these two treatments 55 mg BHT  (K and  and 12.5 mg of dl-a-tocopherol per m i l l i o n  L) I.U.  A l l expressions of the level of antioxidants i n t h i s  t h e s i s , t h e r e a f t e r , are based on milligrams per m i l l i o n I.U. of vitamin A.  - 24 -  Results and Discussion  Table 3 presents  initial  levels of vitamin A i n powders from the 14  treatments and the percentage loss of the i n i t i a l  level during storage at  22 and 37°C. Considering that the target f o r t i f i c a t i o n level was 30 IU/g, 12 treatments resulted i n l e v e l s of more than 20 I.U. at the beginning of storage.  One treatment (D) using vitamin A i n milk f a t with no  antioxidant resulted i n an i n i t i a l laboratory-prepared  level of 11.6 I.U./g.  A similar  mix adding 0.05% HCO to the powder (treatment F) and  containing no antioxidant resulted i n a l e v e l of 14.5 I.U./g.  These  treatments resulted i n 90% or more destruction of the remaining vitamin A a f t e r three months of storage at both temperatures. A f t e r 3 months at 22°C, treatments E,K,L,N and 0 had less than 25% destruction.  These were r e s p e c t i v e l y , a commercial preparation,  antioxidant s t a b i l i z e d vitamins at 0.1 and 0.2% HCO i n powder and vitamin A with no antioxidant and 500 ppm of a commercial antioxidant mixture at 0.2% HCO.  I t i s apparent that higher l e v e l s of hydrogenated coconut o i l  and antioxidants are b e n e f i c i a l during i n i t i a l storage at 22°C. Further evidence of the e f f e c t of antioxidants l i e s i n comparing treatments H,I,J and K, a l l at 0.1% HCO.  Vitamin A losses a f t e r 3 months  at 22°C were 80, 77, 52 and 15% r e s p e c t i v e l y .  Types and levels of  antioxidants f o r non-commercial treatments are given on Table 4. Antioxidant l e v e l s were:  zero (H); 1.3 and 5.2 mg a-tocopherol  16 mg of ascorbyl palmitate  (treatments  I and J r e s p e c t i v e l y ) ;  and 4 and  - 25 -  Table 3.  Treatment  A B C D E F G H I J K L N 0  I n i t i a l l e v e l s o f vitamin A and percent o f vitamin A losses during storage i n primary skimmilk powder f o r t i f i e d by d i f f e r e n t treatments i n P i l o t Plants A and D a f t e r 3 and 12 months o f storage a t 22°C and a f t e r 3 and 6 months a t 37°C. Initial-/ Level o f Vitamin A (I.U.) 46.8 11.6 27.7 23.2 36.8 14.5 20.2 22.1 24.9 28.2 32.1 29.9 24.6 27.6  Percent vitamin A l o s s — ^ Room Temp (22°C) High Temp (37°C) 3 mo  12 mo  3 mo  67.6 89.7 93.0 92.6 22.6 91.3 95.5 80.7 77.4 52.5 15.4 21.2 24.5 23.7  95.8 91.7 96.0 94.1 57.8 94.4 96.2 92.0 93.0 93.6 27.5 30.2 85.0 80.4  92.3 90.8 93.8 90.3 49.1 92.8 95.3 79.4 78.7 81.1 22.6 24.0 55.9 34.2  — - Mean o f two t r i a l s i n each o f two P i l o t Plants.  12 mo 97.3 100 100 100 88.3 100 100 92.7 93.2 94.8 31.5 29.0 80.4 74.6  - 26 Table 4.  Treatment  Levels o f antioxidants contained i n P i l o t Plant treatments  Antioxidant  Concentration (mg/million I.U.Vit.A)  A  (Commercial)  Undeclared  B  (Commercial)  Undeclared  C  None  0  D  Ascorbyl Palmitate a-tocopherol  E  (Commercial)  Undeclared  F  None  0  G  H  4.0mg 1.3mg  Ascorbyl Palmitate a-tocopherol  2.0mg 0.65mg  None  0  I  Ascorbyl Palmitate a-tocopherol  4.0mg 1.3mg  J  Ascorbyl Palmitate a-tocopherol  16.0mg 1.3mg  K  BHA BHT a-tocopherol  5.0mg 55.-pmg 12.5mg  L  BHA BHT a-tocopherol  5.0mg 5'5.0mg 12.5mg  N  None  0  Ascorbyl Palmitate a-tocopherol  •  0 8.0mg 2.6mg  - 27 -  and-5 mg BHA, 55 mg BHT and 12.5 mg a-tocopherol (K). HCO l e v e l s of 0.05, 0.1 and 0.2% i n the powder with no antioxidant were i n treatments F,H and N; destruction a f t e r 3 months at 22°C f o r these treatments were 91, 81 and 24% r e s p e c t i v e l y .  Levels of HCO with 500 ppm  of commercial antioxidant were 0.05, 0.1 and 0.2% i n the powder f o r treatments G,I and 0; destruction a f t e r 3 months at 22°C were 96, 77 and 24% r e s p e c t i v e l y .  C l e a r l y higher l e v e l s of HCO resulted i n greater  s t a b i l i t y of vitamin A.  This i s i n agreement with the findings of  Bauernfeind and Parman (1964). There was l i t t l e difference between the milkfat (C,D) and HCO (H, I ) at 0.1% l e v e l as the vitamin c a r r i e r except at three months of storage at which time the l a t t e r showed less destruction. The two commercial products with the vitamin i n vegetable o i l (A,E) showed poor and medium s t a b i l i t y r e s p e c t i v e l y . (B) was one of the least stable treatments.  The commercial emulsion  I t i s l i k e l y that the large  difference i n s t a b i l i t y exhibited by the commercial preparations  i s due t o  t h e i r contents of antioxidants. The c r u c i a l t e s t f o r s u i t a b i l i t y of the treatments l i e s i n the storage s u r v i v a l of the vitamin a f t e r 12 months at 22°C or 6 months at 37°C.  Only treatments K and L resulted i n about 30% d e s t r u c t i o n . A l l  others resulted i n 75% or more destruction f o r the same storage condition except treatment E which resulted i n 58% destruction at 22°C, but-88% at 37°C.  - 28 Conclusions  1.  The presence of antioxidant materials d i s t i n c t l y  during storage.  aided  stability  The most stable product used 5 mg of BHA, 55 mg of BHT 6  and 12.5 mg of a-tocopherol/10  I.U. of vitamin A.  content of the commercial products were unknown.  The antioxidant D i f f e r e n t l e v e l s and  combinations of antioxidants are used in the subsequent t r i a l s i n Phases II and I I I . 2.  Higher l e v e l s of the o i l c a r r i e r (HCO) increased s t a b i l i t y .  The  l e v e l s of 0.05% and 0.1% were unsuitable; the HCO level of 0.2% resulted in much better s t a b i l i t y than the lower l e v e l s . 3.  HCO resulted i n s l i g h t l y better s t a b i l i t y than m i l k f a t after three  months of storage, but showed no d i f f e r e n c e i n s t a b i l i t y after six(37°C) and 12 (22°C) months of storage when l i t t l e or no antioxidants are used. HCO i s used i n the subsequent t r i a l s .  - 29 -  PHASE II-COMMERCIAL PLANTS TRIALS-PRIMARY POWDER  Introduction  This phase of the project involves the t e s t i n g of methods of vitamin A f o r t i f i c a t i o n t o regular or primary type of skimmilk powder (SMP) when produced at the commercial scale. A b r i e f review of the process i s given i n the f o l l o w i n g paragraph. For primary powder production, milk i s treated i n an evaporator t o remove much of the water before the drying process. There are a number of d i f f e r e n t types of evaporators i n use, but regardless of which, the discharged f l u i d usually contains approximately 40-42% s o l i d s . I t has probably been i n a p a r t i a l  vacuum at a temperature of around 65°C. The  condensed milk i s then pumped with a high-pressure pump through a small o r i f i c e into the d r i e r box. The a i r temperature i n the spray d r i e r i s lowered from 200 to 85°C between the i n l e t and the outlet of the powder. "From the d r i e r , the powder i s usually sacked or binned u n t i l needed and the temperature could be 40 t o 50°C f o r several hours.  - 30 -  M a t e r i a l s and Methods Addition of the vitamins The vitamin concentrate was injected into the condensed skimmilk at a point j u s t p r i o r t o the high-pressure pump leading t o the spray-nozzle of the spray d r i e r .  A p o s i t i v e - p r e s s u r e pump (Bran & Luebbe Inc., Model No.  N-P31, Des P l a i n e s , I l l i n o i s ) was used t o i n j e c t the vitamin mixture. I t was necessary t o heat the vitamin concentrate above 50°C (52-53) p r i o r t o i n j e c t i o n i n order to ensure good f l u i d i t y since most treatments contained hydrogenated  coconut o i l (HCO) which i s not f l u i d at room temperature  (22-25°C).  The vitamin-concentrate i n j e c t i o n was performed continuously  for at least one hour, during which time, the f i r s t one-half hour production was not c o l l e c t e d to ensure s u f f i c i e n t time f o r the powder to t r a v e l t o the e x i t and proper e q u i l i b r a t i o n of the system.  Spread over  the next one-half hour, at least 20 kg of powder was c o l l e c t e d by drawing at  f i v e minutes i n t e r v a l s from the production l i n e before bagging.  treatment was done twice at both plants on separate days.  Each  In both cases,  the dryers were not shut-down between treatments, because they operate continuously f o r three or four days. Treatment d e s c r i p t i o n The treatments tested f o r these t r i a l s are l i s t e d with a b r i e f d e s c r i p t i o n i n Table 5. Those formulations were e i t h e r  commercial  preparations or lab-made preparations chosen from the most successful P i l o t Plant (Phase I ) treatments; the commercial  preparations and the  ingredients f o r the lab-made preparations were described i n Phase I . Treatments E2 and H were a d d i t i o n a l treatments conducted as follow-up  - 3:1 -  Table 5.  D e s c r i p t i o n of vitamin A f o r t i f i c a t i o n f o r Phase I I - Primary Powder.  treatments  A  -  Commercial Vitamin A concentrate (45,400 I.U. Vit.A/ml i n hydrogenated coconut o i l )  B  -  Commercial Vitamin A concentrate (50,000 I.U. Vit.A/ml i n vegetable o i l )  C  - 0.1% HCO i n the powder with 2000 ppm o f a commercial antioxidant mixture i n the o i l . i . e . 1 6 mg Ascorbyl palmitate and 5.2 mg a-tocopherpl.  D  -  0.2% HCO i n the powder with 2000 ppm of a commercial antioxidant mixture i n the o i l . i . e . 3 2 mg Ascorbyl palmitate and 10.4 mg a-tocopherol.  E, - 0.1% HCO i n the powder with a commercial s t a b i l i z e d vitamin concentrate containing 5 mg o f BHA, 55 mg o f BHT and 12.5 mg o f a-tocopherol. E  2  -  Repeat of Treatment E-j.  F  - 0.2% HCO i n the powder with the same vitamin concent r a t e as i n E p E^.  H  -  Note:  Same as Treatment F, plus 2000 ppm o f the commercial antioxidant mixture used i n Treatments C and D. i . e . 5mg BHA, 55mg BHT, 23mg a-tocopherol and 32mg ascorbyl palmitate.  Treatments Ep and H were additional treatments conducted a f t e r eight months o f storage o f the other treatments.  - 32 -  t r i a l s from the most promising r e s u l t s obtained after 6 months of storage.  The purpose of these additional treatments  (E2 and H) were t o  confirm treatment E i r e s u l t s and to determine the e f f e c t of higher levels of antioxidants on vitamin A s t a b i l i t y . Samples from f r e s h l y produced powder was obtained from a U.S. manufacturer. This powder was f o r t i f i e d using the same method and vitamin concentrate used f o r Treatment B i n t h i s t r i a l . The r e s u l t s of the vitamin A s t a b i l i t y from the U.S.-manufactured powder were not s i g n i f i c a n t l y d i f f e r e n t (P<0.05) than the r e s u l t s f o r Treatment B; f o r that reason only Treatment B r e s u l t s w i l l be shown i n t h i s paper. Storage and sampling The powder samples produced f o r both Primary and Instant T r i a l s were handled the same way as described f o r Phase I , i . e . , they were transported to the Food Research  I n s t i t u t e i n Ottawa immediately a f t e r production f o r  blending, bagging and storage at 22°C f o r 12 months and at 37°C f o r s i x months. Sub-samples were sent at regular i n t e r v a l s f o r vitamin A a n a l y s i s , sensory evaluation and moisture a n a l y s i s . Vitamin A a n a l y s i s The determination of vitamin A content was conducted  by three  d i f f e r e n t l a b o r a t o r i e s at 0,1,2,3,6,9 and 12 months of storage at 22°C and by one of those l a b o r a t o r i e s at 0,1,2,3,4 and 6 months of storage at 37°C.  Each of the three l a b o r a t o r i e s used a d i f f e r e n t method of  analysis.  In the past, the C a r r - P r i c e method (AOAC, 1975; Carr and P r i c e ,  1926) has t r a d i t i o n a l l y been used to estimate the Vitamin A content i n d r i e d milk products.  More r e c e n t l y a spectrofluorometric method  - 33 (Thompson et a l . , 1978, Indyk, 1982) and an HPLC method (Thompson et a l , 1980; Wool lard and Woolard, 1981; de Vries et a l . 1979) have been developed  and shown to produce more accuracy than the Carr-Price method.  The powder samples stored at 22°C were analyzed by two l a b o r a t o r i e s (Lab A - HPLC method and Lab C - Spectrof1uorometric method).  Data from the  t h i r d laboratory (Lab B - C a r r - P r i c e method) was obtained f o r powder samples from only one plant; t h i s data was used f o r the comparison of r e s u l t s obtained by the d i f f e r e n t methods of analysis. The powder samples stored at 37°C were analyzed by only Lab C using the spectrof1uorometric method of vitamin A a n a l y s i s . Sensory evaluation Haylike f l a v o u r usually i n t e n s i f i e s with increasing vitamin A destruction which i s p a r t i c u l a r l y noticeable and objectionable in a bland product such as skim milk. Sensory evaluation was performed on the r e c o n s t i t u t e d milk (10 g powder; 90 ml water) by f i v e or s i x selected panelists who indicated the i n t e n s i t y of the haylike flavour using the f o l l o w i n g scale: 0 = no haylike f l a v o u r  1 = doubtful  2 = slight  3 = moderate  4 = strong  5 = extreme  Moisture analysis The moisture content of powder samples f o r the various treatments was determined  by the forced-air-oven method (Agriculture Canada, 1977).  This determination was performed immediately after production and at the end of each storage period  (6 months at 37°C and 12 months at 22°C). This  analysis was performed to ensure that the moisture content of the powder produced f o r the experiment  was w i t h i n commercially acceptable l e v e l s .  - 34 -  Method of graphing The vitamin A depletion i n powder can best be v i s u a l i z e d g r a p h i c a l l y over the storage period.  The method of graphing i s as f o l l o w s .  Figures 2 and 3 show the estimated quadratic curves f o r "the best f i t " of vitamin A levels i n powders plotted against storage time when stored at 22°C and 37°C r e s p e c t i v e l y . The p l o t t e d l i n e f o r each treatment accounts f o r the actual vitamin A content of the two r e p l i c a t e productions at two d i f f e r e n t plants as determined by one, two or three d i f f e r e n t methods. To obtain those curves, the data was f i t t e d i n a regression equation.of the form: y = a + bix + b?x where:  2  y = I.U. of vitamin A x = storage time (month) a = regression intercept bi = slope b2 = quadratic term (curvature)  The regression equations so produced f o r the curves shown on Figures 2 and 3 are given i n Appendix 1.  In order to f a c i l i t a t e the visual  comparison  of i n d i v i d u a l treatment, i t i s d e s i r a b l e f o r a l l treatments to have the same i n i t i a l value.  For that reason, the regression intercepts (a) i n the  regression equations were assigned the value of 30 as being the i n i t i a l vitamin A content f o r a l l treatments i n Figures 2 and 3, even though the actual regression intercepts were d i f f e r e n t f o r each treatment as shown i n Appendix 1.  Figure 2.  Vitamin A depletion i n Primary Skim milk Powder during 12 months of storage at 22°C.  STORAGE TIME (mo)  Figure 3 .  Vitamin A depletion i n Primary Skim milk Powder during 6 months of storage at 37°C.  STORAGE TIME (mo)  - 37 -  S t a t i s t i c a l analysis Due t o the c u r v i l i n e a r i t y of the r e l a t i o n s h i p between vitamin A content and time of storage (months)  i t was f e l t that l i n e a r i z a t i o n  through data transformation would be useful to compare treatments and e s t a b l i s h the reaction order of the vitamin A o x i d a t i o n . A computer program was used f o r l i n e a r i z a t i o n of data. The super simplex optimization program of Routh et a l . (1977) which was modified by F u j i i and Nakai (1980) was used t o search f o r the best f i t values of A and B t o f i n d the highest r ^ values i n l i n e a r regression analysis a f t e r data transformation using t= (y + A ) f o r l i n e a r i z a t i o n . This approach was abandoned when i t D  was found that B value or the reaction order was very d i f f e r e n t among treatments. Unless a common transformation (B value) could have been found, i t i s not possible to use t h i s elaborate technique to compare treatment r e s u l t s . Considering that the various treatments  contained  d i f f e r e n t types and levels of antioxidants and that antioxidants do affect the o x i d a t i o n r e a c t i o n , i t i s not s u r p r i s i n g to obtain d i f f e r e n t r e a c t i o n orders f o r i n d i v i d u a l treatments. Due t o the d i f f i c u l t y of comparing such a large number of treatments, a M u l t i p l e Comparison Analysis using the protected Least S i g n i f i c a n t Difference (LSD) t e s t was employed (Snedecor and Cochran, 1967) on the estimated slopes from the l i n e a r regression equations.  The quadratic  terms of the second order equation were also treated i n the same manner. This procedure  i s equivalent to using orthogonal polynomials and r e s u l t s  in an independent t e s t f o r l i n e a r i t y and the curvature. (Rowel 1 and Walters, 1976).  To study the p a r e l l e l i s m of the l i n e s , the r e s u l t s of  - 38 -  both t e s t s were combined. analysis of variance.  A probable grouping was then subjected to the  The groupings which explained a l l but an  i n s i g n i f i c a n t part of the treatment sum of squares i n the analysis of variance are discussed i n the r e s u l t s .  Note that the protected LSD  test  i s considered to generally err on the side of too many s i g n i f i c a n t differences.  This was considered d e s i r a b l e i n a screening experiment  such  as t h i s . Upon examination of the raw data, i t was noted that r e s u l t s of vitamin A analysis from laboratory C were erroneous f o r powder samples of the a d d i t i o n a l t r i a l s (E2 and H) at time 0 ( i n i t i a l ) . the s t a t i s t i c a l  For that reason,  analysis was performed on the data with missing values at  time 0 f o r treatments E2 and H.  To confirm that the r e s u l t s of the  M u l t i p l e Comparison Analysis (LSD t e s t ) were not affected by the omission of the missing values f o r E2 and H, the same t e s t was performed on the data without time 0 values f o r a l l treatments. Ranking and grouping of n o n - s i g n i f i c a n t treatments remained unchanged f o r e i t h e r set of data. Therefore, r e s u l t s of the s t a t i s t i c a l treatments E2 and H are reported.  analysis with missing values f o r  - 39 -  Results and  Discussion  Vitamin A s t a b i 1 i t y Figures 2 and 3 show the estimated levels of vitamin A remaining i n the powder during storage f o r 12 months at 22°C and f o r 6 months at 37°C. These plots are from the regression equations "of best f i t " f o r the two t r i a l s i n each of two plants. From the s t a t i s t i c a l a n a l y s i s , Table 6 l i s t s P a r t i a l Regression C o e f f i c i e n t s ( l i n e a r and quadratic) i n order of decreasing magnitude f o r each treatment f o r the two temperature storage conditions. I t i l l u s t r a t e s the grouping of non-significant differences among treatments based on the M u l t i p l e Comparison A n a l y s i s . The P a r t i a l Regression C o e f f i c i e n t (PRC) f o r the l i n e a r analysis i s the slope and the PRC f o r the quadratic analysis indicates the degree of curvature. An overview of the Figures 2 and 3 and Table 6 i n d i c a t e that the treatments f a l l B,D;  i n t o four general levels of increasing s t a b i l i t y : C; A;  E j , E ,F,H. This grouping r e s u l t e d i n a highly s i g n i f i c a n t difference 2  between group sum of squares and i n s i g n i f i c a n t differences w i t h i n  groups.  A b r i e f d e s c r i p t i o n of the treatments i s as follows: C(0.1%HC0,a-t,AP); A(Commercial); B(Commercial) and D(0.2%HC0,a-t,AP); E i and E  2  (0.1%HC0,  BHA-BHT-at), F(0.2%HC0, BHA-BHT-at) and H(0.2%HC0, BHA-BHT-ctt + at,AP)(see Table 5 f o r more d e t a i l s ) . . The r e s u l t s show that the levels of BHA-BHT-a-tocopherol used i n treatments E's and F (5mg BHA,55mg BHT,12.5mga-t) gave higher s t a b i l i t y than the levels of a-tocopherol and ascorbyl palmitate (AP) contained i n treatments C and D. However treatment C contained 15mg AP and 5 mg a-t and  1 2 Estimated regression c o e f f i c i e n t s ' f o r M u l t i p l e Comparison Analysis (LSD t e s t ) f o r Primary Powder stored at 22 and 37°C  Table 6.  22°C Storage Linear  L S D  3  T  Treatment  -1.95  -1.04  -.91  -.52  C  A  D  B  -.47  -.43  H  -.40  -.39  E  F  E  x  2  .50  Grouping Quadratic  p  2  Treatment  p<.001 0.153  0.088  0.068  0.042  0.041  C  A  B  E  D  x  0.011  0.000  -.007  E  H  F  2  Grouping  .050 '  P^  0 0 1  37°C Storage Linear  Q  -4.51  -4.14  -1.64  -1.59  Treatment  C  A  D  B  l  Grouping Quadratic  B  2  Treatment  -1.33 -1.27 H  E  -1.21  -1.21  E  F  2  2  :  .84 P^  0.476  0.437  0.236  0.215  0.087  C  A  B  F  D  0.001 ,-.042 -.073 £  E  1  Grouping  2  0 0 1  .23  H p<.001  Equations f o r the 22°C storage are of the form y = 8 & i i + 8 x where x ^ 0,1,2,3,6,9,12 and x =x -11.83X1 = 0,-10.83,-7.83,-2.83,24.17,69.17,132.17. Thus 3a corresponds to the slope of the f i t t e d s t r a i g h t l i n e and 8 the c o e f f i c i e n t of the quadratic term i n the f i t t e d second order equation. +  x  0  2  2  2  2  Equations f o r the 37°C storage are of the form y = B x = X! -6x!= 0,-5,-8,-9,-8,0. 2  2  0  + 8iX + 8 x 1  2  2  where x = 0,1,2,3,4,6 and x  x  - 41  -  treatment D contained double those amounts with 30mg AP and lOmg a - t .  The  lower level of AP-a-t antioxidants present i n treatment D compared to the BHA-BHT-at antioxidants level present in treatment F (at the same HCO l e v e l ) do not permit the comparison of the effectiveness of those antioxidant mixtures.  Perhaps equivalent levels of AP-a-t and BHA-BHT-at  would produce comparable vitamin s t a b i l i t y . The addition of a-tocopherol and ascorbyl palmitate to the BHA-BHT-a-t mixture gave no s i g n i f i c a n t  a d d i t i o n a l improvement i n  s t a b i l i t y of vitamin A (H versus F ) . The 0.2% of HCO the 0.1% level  (D) gave s i g n i f i c a n t l y (P*0.05) higher s t a b i l i t y than  (C) with a-tocopherol and ascorbyl palmitate, whereas with  the BHA-BHT-a-tocopherol antioxidant mixture ( E i , E d i f f e r e n c e . The Food and Drugs Act of 0.1% HCO  i n NDM.  containing HCO  and F) there was  2  of Canada s t i p u l a t e s  no  a maximum level  Due to t h i s l i m i t a t i o n , when excluding treatments  levels  above 0.1% the BHA-BHT-a-tocopherol antioxidants at  the levels i n treatments E i , E , F and H gave the higher s t a b i l i t y at 2  0.1% HCO  i n the powder.  In summary, i t appears that a vitamin A concentrate i n HCO containing BHA,  BHT  and a-tocopherol at the levels  i n treatments  Ex,E ,F and H 2  produced the highest s t a b i l i t y , being s i g n i f i c a n t l y better -(P<0.05) than one commercial product (A) and only s l i g h t l y better than the other commercial product (B). As shown on Table 6, the quadratic analysis at both temperature storages of treatment B i s s i g n i f i c a n t l y d i f f e r e n t 0.05) than E  2  and H, but the l i n e a r analysis did not f i n d  (P<  significant  d i f f e r e n c e between these treatments. Treatments E i , E , F and H were also 2  - 42 -  s i g n i f i c a n t l y better (P<0.05) than C which contained  ct-tocopherol and  ascorbyl palmitate i n 0.1% HCO. However, these same antioxidants used at the higher l e v e l s i n 0.2% HCO (D) produced a n o n - s i g n i f i c a n t difference (P< 0.05) i n s t a b i l i t y over Ei,E ,F 2  Therefore,  and H.  i t would appear that when u t i l i z i n g 0.2% HCO with the  antioxidants tested i n these t r i a l s or 0.1% HCO with the BHA-BHT-at antioxidants combination and level used i n these t r i a l s , with a-level of 30 I.U. of vitamin A per gram present i n the powder a f t e r production, one could expect to r e t a i n approximately 26 I.U./gm when stored under conditions equivalent t o 22°C f o r 12 months. Approximately 23 I.U. can be expected t o be retained from these same treatments when stored under conditions equivalent t o 37°C f o r s i x months. Haylike f l a v o u r Sensory evaluation was used t o assess the development of h a y l i k e f l a v o u r i n powder samples during the storage period. On a scale from 0 t o 5, 0 being no HLF detected and 5 being extreme HLF, the p a n e l i s t s generally detected  increasing l e v e l s of HLF during  storage.  S t a t i s t i c a l l y , the c o r r e l a t i o n between units of vitamin A losses and HLF was found to be s i g n i f i c a n t at the 1% level f o r t h i s type of powder (r=0.592 d.f.=82).  Those r e s u l t s are consistent with the previous  f i n d i n g s of Nakai et al.(1983). Comparison of Production  Plants  Table 7 shows the percent vitamin A loss f o r each treatment from the two d i f f e r e n t production plants during 12 months of storage at 22°C and 6 months at 37°C.  Results i n d i c a t e that the powder produced at Plant A had  - 43 -  Table 7. Losses o f vitamin A i n Primary Skimmilk Powder f o r t i f i e d by d i f f e r e n t treatments i n Plants A and B a f t e r 12 months of storage at 22°C and 6 months a t 37°C. Treatment  Storage Temperature 22°C  37°C  Plant A Plant B % loss  Plant A  Plant B % loss  A  61.0  36.3  89.0  80.8  B  43.8  22.6  54.9  44.0  C  84.2  71.4  88.2  82.7  D  49.2  13.2  41.1  27.1  26.8  8.2  32.0  20.0  19.4  11.4  26.8  30.8  F  23.8  7.8  25.3  18.7  H  27.7  14.7  27.8  28.3  E  l  E  2  - 44 -  roughly twice the percentage vitamin A loss of Plant B f o r the same treatments when stored at 22°C f o r twelve months.  The losses were also  generally higher f o r Plant A powders when stored at 37°C f o r 6 months. However, the difference between the two plants was less marked at the higher temperature storage. The reasons f o r the lower losses i n Plant B are not immediately apparent.  One p o s s i b i l i t y i s the s l i g h t l y higher temperature i n the dryer  of Plant B (400°F; 200°F o u t l e t ) than i n Plant A (290-310°F i n l e t ; 190-195°F o u t l e t ; and the difference i n the pre-heat treatments,  both  c o n t r i b u t i n g to greater whey protein denaturation i n Plant B powder. The hi-heat powder thereby produced i n Plant B would contain more free sulphydryl groups which provides a natural antioxidant e f f e c t l a t e r i n the stored product. This phenomena has previously been recognized t o occur i n products such as highly-heated milk (UHT) (LeMaguer and Jackson,1983), whole milk powder (Hollender and Tracy,1942; Holm et al.,1926; Mattick et al.,1945; Waite et al.,1947) and i n skim milk powder (Pyenson and Tracy,1948). The relevance of t h i s hypothesis t o our s i t u a t i o n was supported when several powder samples from Plant B were found to contain less than half the undenatured whey protein nitrogen (WPN of 3 versus 7 mg N/g) found i n Plant A samples; the lower whey protein nitrogen level i n d i c a t i n g the greater extent of denaturation. Further t r i a l s would be required to confirm t h i s  hypothesis.  -45 -  Comparison of a n a l y t i c a l methods of vitamin A The s t a t i s t i c a l  analysis of the data indicated that the difference  among methods of vitamin A analysis was s i g n i f i c a n t (P*0.05). Values were tabulated with the data produced from each of the three methods of analysis done on the same powder samples at d i f f e r e n t l a b o r a t o r i e s . A r a t i o of r e s u l t s to be expected' among methods of analysis was then calculated to be 0.82: 1.0: 1.16 f o r the C a r r - P r i c e , spectrofluorometric  and HPLC methods r e s p e c t i v e l y .  A s i m i l a r r a t i o was  obtained throughout the storage periods and f o r various vitamin A l e v e l s .  - 46 -  Conclusions  1. I t was demonstrated i n these t r i a l s that the use of antioxidants i s important  to control the oxidation of vitamin A i n primary skim milk  powder. The antioxidants BHA-BHT-a-tocopherol at levels of 5,55 and 12.5mg/10 I.U. r e s p e c t i v e l y i n 0.1 or 0.2% HCO permitted the retention of 6  approximately  80% of the vitamin A when stored at 22°C f o r 12 months and  approximately  70% when stored at 37°C f o r 6 months.  2. Haylike f l a v o u r development was c o r r e l a t e d with vitamin A destruction.  3. A large d i f f e r e n c e i n vitamin A s t a b i l i t y was observed between production plants. This difference was a t t r i b u t e d t o the type of dryer used and pre-heat treatment given to the milk p r i o r t o spray drying.  4. The three d i f f e r e n t methods of vitamin A analysis u t i l i z e d i n these t r i a l s c o n s i s t e n t l y produced r e s u l t s p r o p o r t i o n a l l y d i f f e r e n t from each other. Their respective degree of accuracy was not determined.  The HPLC  method c o n s i s t e n t l y produced higher values and the C a r r - P r i c e method produced lower values than the spectrofluorometric method.  5.  The level of HCO added t o the powder was important f o r vitamin  s t a b i l i t y when low levels of antioxidants were used.  In treatments with  properly s t a b i l i z e d vitamins with antioxidants, the higher HCO l e v e l (0.2%) d i d not a f f e c t s t a b i l i t y .  - 47 -  Phase Ill-Commercial Plant T r i a l s - I n s t a n t Powder  Introduction  Experiments f o r t h i s phase of the project involved the t e s t i n g of methods of vitamin A f o r t i f i c a t i o n t o instant type of skim milk powder (SMP). T r i a l s f o r t h i s type of powder f o r t i f i c a t i o n were c a r r i e d out simultaneously with the Commercial Primary Powder t r i a l s (Phase I I ) since both types of powder were produced at the same plants. A b r i e f review of the i n s t a n t powder manufacturing process i s given i n the f o l l o w i n g paragraph. For instant powder production, the i n s t a n t i z e r s used are a t u n n e l - l i k e construction where the dry powder t r a v e l l i n g i n a stream of a i r i s sprayed at i t s entrance with e i t h e r a f i n e j e t of water or steam t o cause the milk p a r t i c l e s to agglomerate with a moisture content of 10-15%. These p a r t i c l e s passing through the tunnel or tube structure are subjected t o temperatures of 150 to 175°C t o reduce the moisture content to 4-5%. They are then s i z e d , with the fines being recycled back t o the s t a r t of the i n s t a n t i z i n g process and the rest i s bagged. T r i a l s f o r t h i s phase of the project were conducted by two d i f f e r e n t commercial skim milk powder manufacturers. Each treatment was r e p l i c a t e d twice at a l t e r n a t i n g work weeks and plants.  - 48 -  M a t e r i a l s and Methods Treatment d e s c r i p t i o n The treatments  tested f o r these t r i a l s are l i s t e d with a b r i e f des-  c r i p t i o n i n Table 8. Treatments A, H and K are commercial vitamin concent r a t e s , the rest are lab-made formulations containing d i f f e r e n t combinations of kinds and levels of antioxidants blended i n an emulsion of HCO, l i q u i d skimmilk  and the vitamins A and D.  Treatment A i s a commercial preparation containing vitamins A and D in a base of reconstituted non-fat dry milk and butter o i l . I t s content of antioxidants was not revealed. This product was part of Phase I t r i a l s . Treatments H and K are a commercial dry beadlet vitamin  concentrate  containing 250,000 I.U. of vitamin A and 50,000 of vitamin D per gram on a dry basis. This product was not previously tested i n Phase I t r i a l s . Commercially, t h i s product i s added to the primary NDM on a dry basis by metering  i t t o the powder on the way t o the i n s t a n t i z e r . In t h i s  experiment, t h i s product was dissolved i n water t o make a 20% s o l u t i o n f o r treatment H and a 10% s o l u t i o n f o r treatment K. These d i l u t i o n s caused a f i v e - or t e n - f o l d reduction i n vitamin concentration and made i t possible to i n j e c t t h i s material on a "wet" basis using the same procedure as the other  treatments.  Preparation of vitamin mixes The  lab-made preparations  (treatments B t o J) were prepared by melt-  ing HCO at 50°C t o which the vitamins and the antioxidants were,  a d d e d . >•  This vitaminized HCO was then added t o l i q u i d skim milk to make 6,12 or 25% mixtures which was subsequently  homogenized with 52-55°C with 2500  -49 Table 8.  T7  Description o f Vitamin A f o r t i f i c a t i o n f o r Phase I I I . - Instant Powder.  treatments  A.  -  Commercial vitamin A and D concentrate (45,400 I.U. vitamin A/ml i n 6% m i l k f a t )  B.  _  6% HCO - Skimmilk emulsion with a commercial s t a b i l i z e d vitamin A and D concentrate containing 5 mg BHA, 55 mg BHT and 12.5 mg a-tocopherol per gram.  C.  _  12% HCO - Skimmilk emulsion with the same commercial s t a b i l i z e d vitamin concentrate as i n B.  D.  _  25% HCO - Skimmilk emulsion with the same commercial s t a b i l i z e d vitamin concentrate as i n B and C.  E.  _  6% HCO - Skimmilk emulsion with vitamins A and D and 2000 ppm of a commercial antioxidant mixture added separately. i.e.0.6mg AP and 0.2mg a-tocopherol.  F.  _  12% HCO - Skimmilk emulsion with vitamins A and D and 2000 ppm o f a commercial antioxidant mixture added separately. i.e.l.2mg AP and 0.4mg a-tocopherol.  G.  _  12% HCO - Skimmilk d i s p e r s i o n (same ingredients as i n C, except homogenization was not used during preparation).  H.  -  Commercial Beadlets d i s s o l v e d i n water to make a 20% solution.  J-^  -  12% HCO - Skimmilk emulsion with the same ingredients as i n C, but with lower vitamin concentration t o i n j e c t four times more HCO i n t o the powder.  K—  -  Commercial Beadlets d i s s o l v e d i n water t o make a 10% solution.  ;  -Treatments J and K were a d d i t i o n a l treatments conducted a f t e r eight months o f storage r e s u l t s from other treatments.  - 50 -  and 500 psi of pressure i n a laboratory homogenizer. The vitamins added to these preparations were such to create a product of s i m i l a r vitamin concentration (50,00 I.U. Vitamin A/ml) (treatments A,H  as the commercial products  and K).  The percentage  of o i l stated f o r each treatment indicates the f a t  content i n the vitamin concentrate which u n l i k e primary powder represents a very small addition to the dried powder.  In f a c t , f o r example, the 12%  o i l - s k i m emulsion w i l l add only 0.0067% or 67 ppm o i l to the f o r t i f i e d powder. Treatment G was prepared d i f f e r e n t l y than other lab-made treatments, in that the mixture was not homogenized; the l i q u i d was simply c i r c u l a t e d through the homogenizer at very low pressure ( c i r c a 100-200 p s i ) . Because t h i s preparation was not homogenized, i t was necessary to a g i t a t e constantly during i n j e c t i o n to prevent the vitamin-containing o i l from r i s i n g to the surface. The purpose of t h i s treatment was to determine e f f e c t of homogenization  the  on the vitamin s t a b i l i t y .  As i n Phase I I , additional treatments were conducted  as follow-up  t r i a l s based on r e s u l t s obtained after 6 months of storage. They were treatments J and K; J was to evaluate the e f f e c t of using more HCO  on the  vitamin s t a b i l i t y and K was a repeat of treatment H (beadlets i n water) i n a more d i l u t e s o l u t i o n .  - 51 -  Addition of the vitamins For t h i s type of powder, the vitamin concentrate was i n j e c t e d d i r e c t l y into the i n s t a n t i z i n g chamber through an opening at the middle top section of the c y l i n d e r .  A small p u l s a t i n g pump (Waltham Chemical  Pump Model No. 10611-361) was used to t r a n s f e r the material through a nozzle into the i n s t a n t i z e r .  This pump has the capacity to d e l i v e r small  amounts of l i q u i d (range 12 to 80 ml/min) with a maximum number of strokes per minute which produces a more constant flow f o r an even d i s t r i b u t i o n of vitamins to the powder.  This method of i n j e c t i o n was used i n both plants  f o r t h e i r normal operation.  The use of a d i f f e r e n t pump was the only  modification t o the e x i s t i n g system. The powder sample c o l l e c t i o n was conducted the same way as described f o r Primary Powder; vitamin 'concentrate was i n j e c t e d f o r one hour and 20 Kg of powder was c o l l e c t e d d i r e c t l y from the bagging l i n e , during the l a s t half-hour. Storage and sampling- see Phase II Vitamin A analysis - see Phase I I Sensory evaluation - see Phase I I Statistical  analysis  The same method of analysis was used as f o r the primary powder t r i a l s - Phase II i e . M u l t i p l e Comparison Analysis(LSD) (see Phase II f o r description). As observed i n Phase II t r i a l s , r e s u l t s of vitamin A analysis from laboratory C were also erroneous f o r the additional t r i a l s (treatments J and K) at time 0 ( i n i t i a l l e v e l ) . Again, the M u l t i p l e  Comparison  - 52 -  Analysis was performed on the data with missing values at time 0 f o r treatments J and K and on the data omitting time 0 values f o r a l l treatments.  However, i n t h i s case the ranking and grouping of  n o n - s i g n i f i c a n t treatments remained unchanged f o r the two sets of data of the 22°C storage but was d i f f e r e n t f o r the 37°C storage.  The omission of  time 0 values of laboratory C has a more important e f f e c t on the 37°C storage temperature.  Therefore an adjustment  based on slopes and  quadratic terms differences between the two sets of data was executed on the s t a t i s t i c a l adjustment  r e s u l t s of treatments J and K f o r the 37°C storage.  i s r e f l e c t e d i n the r e s u l t s discussed l a t e r .  The  Results and Discussion Vitamin A s t a b i l i t y Figures 4 and 5 show the estimated levels of vitamin A remaining i n the powder during storage f o r 12 months at 22°C and 6 months at 37°C. As for the primary powder t r i a l s (Phase I I ) , these plots are from the regression equations obtained from the data of two duplicate t r i a l s i n each of two plants. Appendix 2 l i s t s the regression equations f o r each treatment at the two temperature storages. From the s t a t i s t i c a l  a n a l y s i s , Table 9 l i s t s both P a r t i a l Regression  C o e f f i c i e n t s ( l i n e a r & quadratic) i n order of decreasing magnitude f o r each treatment at the two temperature storage conditions. This table also i l l u s t r a t e s the grouping of non-significant differences among treatments based on the M u l t i p l e Comparison A n a l y s i s . The P a r t i a l Regression C o e f f i c i e n t ( B ) analysis i n d i c a t e the degree of curvature. 2  For t h i s type of powder, Figures 4 and 5 show that only s l i g h t differences were found among treatments, e s p e c i a l l y at 22°C storage. The statistical  analysis determining the grouping of n o n - s i g n i f i c a n t  difference among treatments shown on Table 9 supports t h i s observation. Only treatment A (Commercial  m i l k f a t emulsion) was shown to be  s i g n i f i c a n t l y (P<0.05) d i f f e r e n t than a l l other treatments and t h i s only at the 22°C storage. The s t a t i s t i c a l  analysis of the quadratic components  revealed no s i g n i f i c a n t differences (P<0.05) among treatments f o r the 22°C storage which i n d i c a t e that one can r e l y s o l e l y on the r e s u l t s of the l i n e a r analysis f o r the evaluation of treatments at that temperature.  Figure 4.  Vitamin A depletion i n Instant Skim milk Powder during 12 months of storage at 22°C.  T  0  1  1  1  2  1  3  1  r  6  S T O R A G E TIME  9 (mo)  Hgure 5.  Vitamin A depletion i n Instant S k i , U K of storage at 37°C.  10  Powder during 6 » n t h s  Table 9.  Estimated regression c o e f f i c i e n t s ' f o r M u l t i p l e Comparison Analysis (LSD) f o r Instant Powder stored at 22°C and 37°C.  22°C Storage Linear  LSD Bi Treatment  -1.58  -1.10  -.97  -.85  -.79  -.78  -.75  -.70  -.67  A  K  B  H  G  C  D  F  E  -.55  .32  J  Grouping p<.05 Quadratic Treatment  .064  .055  .036  .033  .028  .026  .004  F  E  B  K  G  C  J  H  -.010  -.020  -.021  A  D  Grouping 37°C Storage Linear Treatment  -3.13  -3.10  -3.00  -2.76  -2.72  -2.44  -1.89  -1.70  -1.58  -1.56  A  K  H  B  C  G  D  F  E  J  .60  Grouping 3  2  Treatment  p<.05 .844  .802  .648  .634  .511  .487  .340  .263  .207  -.050  J  K  H  A  B  G  F  C  E  D  .36  Grouping  p<.05  Equations f o r the 22°C storage are of the form y =3o 3i-X + 8 X where X i = 0,1,2,3,6,9,12 and X = X i -11.83Xi= 0,-10.83,-7.83,-2.83,24.17,69.17,132.17. Thus Bi corresponds to the slope of the f i t t e d s t r a i g h t l i n e and e the c o e f f i c i e n t of the quadratic term i n the f i t t e d second order equation. +  1  2  2  2  2  Equations f o r the 37°C storage are of the form y = g + 3 i X -6X1 = 0,-5,-8,-9,-8,0. 0  + e 2 where X x  x  2  x  = 0,1,2,3,4,6 and X = X 2  x  - 57 -  In overview, the treatments containing higher HCO and/or the antioxidant mixture of a-tocopherol and ascorbyl palmitate produced the highest s t a b i l i t y during both temperature storages. Those treatments were J(4xHC0,BHA-BHT-at), D(25%HC0,BHA-BHT-at), E(6%HC0, at,AP) and F(12%HC0,at,AP) (see Table 8 f o r d e s c r i p t i o n of treatments). To assess the e f f e c t of HCO on vitamin s t a b i l i t y one must examine the r e s u l t s of treatments B, C, D and J which contained increasing levels of HCO with the same types and level of antioxidants(5mg BHA,55mg BHT,12.5mg a-t). The s t a t i s t i c a l analysis shown i n Table 9 ranked those treatments as higher i n s t a b i l i t y with increasing HCO levels f o r both storage temperatures. This d i s t i n c t i o n i s also noticeable i n Figures 4 and 5. There was generally no s i g n i f i c a n t difference among HCO levels at 22°C but the treatment with 25% HCO level (treatment J) showed s i g n i f i c a n t l y higher s t a b i l i t y than the 6 and 12% HCO emulsions (B and C) when stored at 37°C. To evaluate the e f f e c t of d i f f e r e n t types of antioxidants, treatment B(6%HC0,BHA-BHT-at) i s compared with E(6%HC0,at,AP) and C(12%HC0,BHA-BHT-at) against F (12%HC0, at,AP). The r e s u l t s of the l i n e a r s t a t i s t i c a l analysis show that at 37"C, treatments E and F produced s i g n i f i c a n t l y (P<0.05) higher s t a b i l i t y than treatments B and C. Although those treatments were not found to be s i g n i f i c a n t l y d i f f e r e n t (P<0.05) f o r the 22°C storage, however treatments E and F were ranked with s l i g h t l y higher s t a b i l i t y than treatments B and C.  Based on these r e s u l t s , we may  conclude that the commercial antioxidant mixture containing a-tocopherol and ascorbyl palmitate was more e f f e c t i v e than the BHA-BHT-a-tocopherol  - 58 -  mixture f o r s t a b i l i z i n g the powder e s p e c i a l l y when used i n the lower HCO emulsion ( 6 % ) . Only a s l i g h t difference was found between the two mixes of antioxidants i n the 12% HCO emulsion f o r the two temperature storages. Considering that the antioxidant l e v e l s present i n treatments E and F (0.6mg AP, 0.2mga-t  and 1.2mg AP, 0.4mga-t), were much lower than the  level used i n treatments B and C (5mg BHA, 55mg BHT and 1.2mga-t) i t may be concluded that ascorbyl palmitate i s required i n smaller quantities than BHA-BHT to s t a b i l i z e i n s t a n t i z e d powder. Treatments H and K were Commercial Beadlets dissolved i n water. Results of those treatments were among the poorest f o r both temperature storages as i l l u s t r a t e d on Figures 4 and 5.  Dry beadlets have been found  by Nakai et a l . (1983) t o produce a stable powder when i t i s incorporated by dry blending. The d i s s o l v i n g of t h i s product i s water which remove the p r o t e c t i v e g e l a t i n coating over the o i l droplets i s l i k e l y the reason f o r i t s lower s t a b i l i t y . Another parameter which was studied i n these t r i a l s relates t o the e f f e c t of homogenization of the emulsion (HCO-Skimmilk) on vitamin stability.  To evaluate the e f f e c t of t h i s process, treatment C i s  compared against G; both contain the same ingredients but G was prepared prepared without homogenization. The s t a t i s t i c a l a n a l y s i s , both l i n e a r and quadratic f o r the two temperature storages did not f i n d s i g n i f i c a n t (P<0.05) differences between these two treatments. However, a vitamin concentrate prepared without proper homogenization produces a very weak emulsion which requires constant s t i r r i n g during i n j e c t i o n to prevent separation.  - 59 -  Therefore,  based on these t r i a l s , i t would appear that when using  treatment J (4xHC0,BHA-BHT-at) t o f o r t i f y skimmilk  powder with vitamin A,  i f the powder contains 30 I.U. of vitamin A per gram a f t e r production, one could expect t o r e t a i n approximately  23 I.U./gm when stored under  conditions equivalent to 22°C f o r 12 months. About 21 I.U./gm can be expected to be retained when stored under conditions equivalent to 37°C f o r s i x months. Haylike f l a v o u r The r e s u l t s of the sensory evaluation f o r t h i s type of powder indicated that the c o r r e l a t i o n between haylike f l a v o u r and units of vitamin A losses was s i g n i f i c a n t at the 5% l e v e l . (r=0.194,d.f.=111) On a scale of 0 t o 5, h a y l i k e f l a v o u r being 0=no h a y l i k e f l a v o u r ; l=doubtful; 2=slight; 3=moderate; 4=strong; 5=extreme, p a n e l i s t s judged that approximately  9.5 I.U. of vitamin A had t o be destroyed  before a powder  sample was l a b e l l e d doubtful(1) i n haylike f l a v o u r . Only an a d d i t i o n a l 2.5 I.U. had to be l o s t before the powder was judged s l i g h t (2) i n h a y l i k e f l a v o u r . However, despite the r e l a t i v e l y large number of samples loosing higher amounts of vitamin A, only a few were judged as high as "moderate" in h a y l i k e f l a v o u r and none were strong or extreme i n that f l a v o u r . Comparison of production  plants  Table 10 shows the percent  vitamin A losses f o r each treatment from  two d i f f e r e n t production plants during 12 months of storage at 22°C and 6 months at 37°C.  Results of vitamin A losses f o r the same treatments were  very s i m i l a r with v a r i a t i o n s generally lower than 20% between plants  - 60 "  Table 10. Losses of vitamin A i n Instant Skimmilk Powder f o r t i f i e d by d i f f e r e n t treatments i n Plants A and B a f t e r 12 months of storage at 22°C and 6 months a t 37°C.  Treatment  Percent vitamin A l o s s - ^ 37°C  22°C Plant A  Plant B  Plant A  Plant  A  43.4  55.5  67.4  72.1  B  44.3  42.9  61.6  74.3  C  40.2  29.6  62.7  64.1  D  39.9  22.3  49.5  58.6  E  48.8  50.4  58.5  65.1  F  57.4  55.7  61.6  74.8  G  36.2  35.2  63.3  68.4  H  27.7  26.1  57.9  54.8  J  22.4  16.7  30.5  26.3  K  42.6  50.2  57.9  54.4  — Values are means o f two r e p l i c a t e t r i a l s analyzed by two l a b o r a t o r i e s f o r 22°C and by one laboratory f o r 37°C storage.  - 61 under both storage conditions.  In fact an analysis of variance of the  data indicated that no s i g n i f i c a n t d i f f e r e n c e existed between the two plants. Those r e s u l t s are d i f f e r e n t than f o r the primary powder t r i a l s (Phase II) i n which powder produced at Plant B resulted i n s i g n i f i c a n t l y higher vitamin A s t a b i l i t y during storage.  The reason f o r t h i s change i n  s t a b i l i t y between plants f o r instant type of powder i s not obvious. Perhaps an explanation f o r t h i s discrepancy l i e s i n the difference i n micro-environments of the o i l droplets containing the vitamins and antioxidants f o r the two types of powder.  Presumably, during spray drying  of f o r t i f i e d condensed milk, the vitamin containing o i l would be incorporated within powder p a r t i c l e s which would provide some protection to the vitamins against oxidation by r e s t r i c t i n g oxygen access and being in close contact with the protein component of the milk.  As previously  suggested, highly-heated milk would have higher protein denaturation, hence more free sulphydryl groups acting as antioxidants which would provide close-by vitamins greater protection against o x i d a t i o n . For instant powder f o r t i f i c a t i o n , the vitamin containing emulsion i s sprayed on the agglomerated powder p a r t i c l e s and the vitamin-containing o i l droplets w i l l probably adhere to the e x t e r i o r of the agglomerates. t h i s case, the vitamins have greater exposure to oxygen and are not surrounded by proteins from the milk as f o r primary powder; therefore, free sulphydryl groups would have l i t t l e e f f e c t s on vitamin  stability.  In  - 62 Conclusions 1.  These t r i a l s demonstrated the p o s i t i v e e f f e c t of higher levels of  hydrogenated coconut o i l on vitamin A s t a b i l i t y .  The highest HCO l e v e l  tested (treatment J) l i m i t e d the vitamin A loss to approximately 20% during 12 months of storage at 22°C and approximately  30% during 6 months  at 37°C. 2.  The commerical vitamin concentrate containing m i l k f a t as the vitamin  c a r r i e r i n an emulsion form did not produce a s t a b l e f o r t i f i e d powder. The poor r e s u l t s of t h i s treatment  are consistent with previous work of  Nakai et al.(1983). 3.  Antioxidants were again shown to be important  concentrates  additives to vitamin  i n order to delay vitamin A degradation during storage.  Ascorbyl palmitate i n conjunction with a-tocopherol was shown to be required in smaller q u a n t i t i e s than BHA-BHT to s t a b i l i z e i n s t a n t i z e d powder. 4.  I t was found that when the dry beadlet vitamin  concentrate  was dissolved i n water f o r the wet method of f o r t i f i c a t i o n , t h i s  product  did not produce a stable powder. 5.  Homogenization of the vitamin concentrate d i d not a f f e c t the  s t a b i l i t y of the added vitamin to powder.  However, when only dispersion  i s used, the need f o r constant s t i r r i n g during i n j e c t i o n of t h i s material makes i t impractical in commercial operations.  - 63 6.  Hay-like flavour development during storage was again correlated with  vitamin A d e s t r u c t i o n . 7.  The large d i f f e r e n c e in vitamin A s t a b i l i t y between production  plants  in the primary powder t r i a l s (Phase I I ) was not found f o r the instant type of powder.  - 64 OVERALL CONCLUSION  The previously reported poor s t a b i l i t y of presently used commercial vitamin concentrate was reproduced in these t r i a l s .  The "wet" method of  f o r t i f y i n g NDM with vitamin A can be e f f e c t i v e f o r both regular and instant types of powder providing s u f f i c i e n t antioxidants and hydrogenated coconut o i l are added with the vitamins. Powder purchased for Food Aid Programs i s f o r t i f i e d at a higher vitamin A level than powder intended f o r Canadian market and i s generally submitted  to storage conditions conducive to f a s t e r vitamin A  destruction.  Therefore, unless higher levels of antioxidants are used for  t h i s powder, greater losses of vitamin A can be expected.  - 65 LITERATURE CITED Abbot, J. and Waite, R. 1962. q u a l i t y of whole milk powder.  The e f f e c t of antioxidants on the J. Dairy Res. 29: 55.  Abbot, J. and Waite, R. 1965. q u a l i t y of whole milk powder.  The e f f e c t of antioxidants on the keeping I I . Tocopherols. J. Dairy Res. 32, 143.  A g r i c u l t u r e Canada. Moisture, p. 8.  1977.  keeping  Manual on Skim Milk Powder A n a l y s i s .  Anantakrishnan, C P . and Conochie, J. 1958. Some observations on vitamin A in r e c o n s t i t u t e d f o r t i f i e d non-fat milk s o l i d s . A u s t r a l i a n J. Dairy Technol. 13:151. Anonymous. 1976. F o r t i f i c a t i o n of skimmilk powder with vitamins A and D. Protein Advisory Group B u l l . 6(4):2. A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists. 1975. In O f f i c i a l Methods of Analysis of the Association of O f f i c i a l A n a l y t i c a l Chemists. (Horwitz Ed.) Ch. 43, 816-821. Battna, J . , Parizkova, H. and Kucerova, Z. 1982. Fat and vitamin A s t a b i l i t y i n the presence of Ronoxan A and other antioxidants. Internat. J. V i t . Nutr. Res. 52:241. Bauernfeind, J.C and A l l e n , L.E. 1963. fat dry milk. J. Dairy S c i . 46:245.  Vitamin A and D enrichment of non  Bauernfeind, J.C and Parman, G.K. 1964. Restoration of nonfat dry milk with vitamins A and D. Food Technol. 18:52. Bauernfeind, J . C , Rokosny, D. and Siemers, G.F. 1953. A aids food f o r t i f i c a t i o n . Food Eng. 25:(6) 81-82, 85, Bauernfeind, J.C. 1978. The technology of vitamin A. Int. Congress of Nutr., Rio de Janeiro. B o l l a g , W. 1979. 3:207.  Retinoids and cancer.  Carr, F.H. and P r i c e , E.A. A. Biochem. J. 20: 497.  1926.  Synthetic vitamin 87. Proc. of the XI  Cancer Chemother.  Pharmacol.  Colour reactions a t t r i b u t e d to vitamin  Conochie, J. and Wilkinson, R.A. 1956. The f o r t i f i c a t i o n of nonfat milk s o l i d s with vitamin A. XlVth Intern. Dairy Congr. Proc. I (II):357. D a l l e , J.P., Mousseron-Canet, M. and Mani, J.C. 1969. Photo-oxydation s e n s i b i l i t e de composes apparentes aux c a r o t i n o i d e s . B u l l . Soc. Chim. Fr. (1):232.  - 66 de Boer, M., deMan, L. and deMan, J.M. 1984. E f f e c t of time and storage conditions on vitamin A in i n s t a n t i z e d nonfat dry milk. J. Dairy S c i . 67:2188. deVries, J.W., Egherg, D.C. and Heroff, J.C. 1979. Liquid Chromatographic Analysis of Food and Beverages. Charalambous, G. Ed. Vol. 2. pp. 477-497. Academic Press, New York. E r i c k s o n , D.R., Dunkley, W.L. and Ronning, M. 1963. E f f e c t of intravenously injected tocopherol on oxidized f l a v o r in milk. J. Dairy S c i . 46, 911. Food and Drugs Act. 1979. Canada, Ottawa, Ont.  Department of National Health and  Welfare,  F u j i i , S. and Nakai, S. 1980. Optimization of data transformations f o r l i n e a r i z a t i o n . Can. Inst. Food S c i . Technol. J. 13:188. Greenbank, G.R., Wright, P.A. and Deysher, E.J. 1944. The keeping q u a l i t y of commercial dried whole milk packaged in a i r and nitrogen. J. Dairy S c i . 27:686. Hammond, E.J. 1970. S t a b i l i z i n g milk f a t with antioxidants. Dairy Review, June 1970.  American  Hollender, H.A. and Tracy, P.H. 1942. The r e l a t i o n of the use of c e r t a i n antioxidants and methods of processing to the keeping q u a l i t y of powdered whole milk. J. Dairy S c i . , 25:249. Holm, G.E., Greenbank, G.R. and Deysher, E.J. 1926. Results of preliminary experiments upon the e f f e c t of separating and c l a r i f y i n g and p a s t e u r i z a t i o n of a milk upon the keeping q u a l i t y of i t s powder. J. Dairy S c i . 9:512. Indyk, H. 1982. The routine determination of vitamin A in f o r t i f i e d milk powder products. N.Z. J. Dairy S c i . Technol. 17:257. King, R.L. 1968. oxidized f l a v o r .  Direct addition of tocopherol to milk f o r control of J. Dairy S c i . 51, 1705.  K l a u i , H., Hausheer, W., Huschke, G. 1970. Technological aspects of the use of f a t - s o l u b l e vitamins and carotenoids and the development of s t a b i l i z e d marketable forms. Int. Encyclopedia Food and N u t r i t i o n . (Pergamon Press) 9:113. K l a u i , H. 1979.  I n a c t i v a t i o n of vitamins.  Proc. Nutr. Soc. 38:  135.  Lea, CH. 1960. Influence of substrate temperature and level of oxidation on the antioxidant a c t i v i t i e s of the tocopherols. J. S c i . Fd. Agric. 11: 212.  - 67 Lea, C.H. and Ward, R.J. 1959. R e l a t i v e antioxidant a c t i v i t i e s of the seven tocopherols. J. S c i . Fd. Agric. 10: 537. LeMaguer, I. and Jackson, H. 1983. S t a b i l i t y of vitamin A i n pasteurized and ultra-high-temperature processed milks. J. Dairy S c i . 66:2452. Lerner, D.A., Mani, J.C. and Mousseron-Canet, M. 1970. Luminescence et p h o t o r e a c t i v i t e dans l a s e r i e de l a vitamine A. Etude q u a n t i t a t i v e . B u l l . Soc. Chim. Fr. (5):1968. Marquardt, H.G. 1979. D i s t r i b u t i o n of dried skim milk as food r e l i e f measure and new findings on v i t a m i n i z a t i o n of dried skim milk. Deutsche M i l c h w i r t s c h a f t 30(4) 118. M a t t i c k , A.T.R., Hiscox, E.R., Crossley, E.L., Lea, C.H., Thompson, S.Y., Kon, S.K. and E g d e l l , J.W. 1945. The e f f e c t of temperature of preheating, of c l a r i f i c a t i o n and b a c t e r i o l o g i c a l q u a l i t y of the raw milk on the keeping properties of whole milk powder dried by the Kestner Spray Process. J. Dairy Res. 14:116. McLaren, D.S. 1980. N u t r i t i o n a l Ophthalmology.  Academic Press, London.  McLaren, D.S. 1984. Vitamin A d e f i c i e n c y and t o x i c i t y . In "Present Knowledge i n N u t r i t i o n " . The N u t r i t i o n Foundation, F i f t h E d i t i o n , Washington, D.C. p. 203. Merzametov, M.M. and Gadzhieva, L.I. 1982. Antioxidants f o r milk f a t . Pishchevaya Tekhnologiya. No. 5, 35. Merzametov, M.M. and Gadzhieva, L.I. 1982. Cystine and tocopherol as milk f a t a n t i o x i d a n t s . Pishchevaya Tekhnologiya. No. 6, 20. Nakai, S., Amantea, G. Eugster, K., Jung, L., Ma, C.Y., Nielson, K., Suyama, K. and Emmons, D.B. 1983. Vitamin A and haylike flavour i n nonfat dry milk and pasteurized low f a t . m i l k . Can. Inst. Food S c i . Technol. J . 16:116. Olson, F.C., Gruber, G.W., K o z l i k , R., and Brown, K. 1949. The s t a b i l i t y to drying of added vitamin A to spray-dried milk. J. Dairy S c i . 32:695. Pont, E.G. 1964. The r e l a t i o n s h i p between the s w i f t test time and the keeping q u a l i t y of b u t t e r f a t . Aust. J. Dairy Tech. 19:108. P e r e i r a , S.M. and Begum, A. 1976. Vitamin A d e f i c i e n c y i n Indian c h i l d r e n . World Rev. Nutr. Dietet. 24:192. Pyenson, H. and Tracy, P.H. 1946. Relation of the heat treatment given the skim milk to the keeping q u a l i t y of spray-dried ice cream mix. J. Dairy S c i . 29:371.  - 68 Routh, M.W., Swarty, P.A. and Denton, M.B. 1977. Performance of the super modified simplex. Anal. Chem. 49:1422. Rowel 1, J.G. and Walters, D.E. 1976. Analyzing data with repeated observations on each experimental u n i t . J . Agric. S c i . , Camb. 87:423. S a t t a r , A., deMan, J.M. and Alexander, J.C. 1976. S t a b i l i t y of edible o i l s and f a t s to fluorescent l i g h t i r r a d i a t i o n . J. Am. O i l Chem. Soc. 52:473. Sattar, A., deMan, J.M. and Alexander, J.C. 1977. Wavelength e f f e c t on light-induced decomposition of vitamin A and 3-carotene i n solutions of milk f a t . Can. Inst. Food S c i . Technol. J. 10:56. Schuler, P. 1980. Roche antioxidants. Roche Information Service, Food Industries Department. P u b l i c a t i o n #270-82535-1737. S c h r o f f , N.B., Narayawan, K.M., Anantakrishnan, C P . and Sen, K.C. 1954. Studies on vitamin A i n milk. Part VII. E f f e c t of processing on the s t a b i l i t y of vitamin A i n f o r t i f i e d milk. Indian J. Dairy S c i . 7:40. Sherwin, E.R. 1976. Antioxidants f o r vegetable o i l s . J . Assoc. Off. Anal. Chem. 53:430. Smith, A.C and MacLeod, P. 1957. E f f e c t of p a s t e u r i z a t i o n temperatures and exposure to l i g h t on homogenized milk. J. Dairy S c i . 40:862. Snedecor, G.W. and Cochran, W.G. 1967. S t a t i s t i c a l Methods. E d i t i o n . Iowa State U n i v e r s i t y Press, Ames, Iowa.  Sixth  Sommer, A., Hussaini, I., Tarwatjo, D., Susanto, D. and Soegiharto, T. 1981. Incidence, prevalence and scale of b l i n d i n g m a l n u t r i t i o n . Lancet 1:1407. Sporn, M.B. and Newton, D.L. 1979. Chemoprevention of cancer with r e t i n o i d s . Fed. Proc. 38:2528. S r i k a n t i a , S.G. 1975. Human vitamin A d e f i c i e n c y . World Rev. Nutr. Dietet. 20:184. S t u l l , J.W. 1951. The e f f e c t of l i g h t on activated flavour development and on the constituents of milk and i t s products. A review. J. Dairy S c i . 36:1153. Suyama, K., Yeow, T. and Nakai, S. 1983. Vitamin A oxidation products responsible f o r haylike flavour production i n nonfat dry milk. J. Agric. Food Chem. 31:22-26. Thomas, E.L., Coulter, S.T. and Kudale, J.M. 1965. Influence of vitamin A and D f o r t i f i c a t i o n on the flavour of instant nonfat dry milk. J. Dairy S c i . 48:1561.  - 69 Thompson, J.N. and Erdody, P. 1974. Destruction by l i g h t of vitamin A added to milk. Can. Inst. Food S c i . Technol. J. 7:157. Thompson, J.N., Erdody, P., Maxwell, W.B. and Murray, T.K. 1978. The fluorometric determination of vitamin A in dairy products. Research Laboratories, Health Protection Br., Health and Welfare Canada, Ottawa, Ont. Amended B u l l e t i n VT-5. June. Thompson, J.N., Hatina, G. and Maxwell, W.B. 1980. High performance l i q u i d chromatographic determination of vitamin A in margarine, milk, p a r t i a l l y skimmed milk and skimmed milk. J. Assoc. Off. Anal. Chem. 63:894-898. Timmen, H. 1978. Improvement of oxidation s t a b i l i t y of pure b u t t e r f a t by antioxidants. Proc. 20th Int. Dairy Congress, P a r i s , June p. 865. Waite, J.D.C., Smith, J.A.B. and Lea, C.H. 1947., The e f f e c t s of a highpreheating temperature with and without ethyl gal l a t e on the storage l i f e of whole milk powder spray-dried on a Gray-Jensen D r i e r . J. Dairy Res. 15:127. Weckel, K.G. and Chicoye, E. 1954. Factors responsible for the development of a hay-like flavour i n vitamin A f o r t i f i e d low-fat milk. J. Dairy S c i . 37:1346. WHO. 1976. Vitamin A d e f i c i e n c y and xerophthalmia. Report of a j o i n t WHO/AID meeting. Technical Report Series No. 590, Geneva. WHO. 1982. Vitamin A Deficiency and Xerophthalmia. World Health Organization, Technical Report Series No. 672, Geneva. Wilkinson, R.A. and Conochie, J. 1958. The s t a b i l i t y of vitamin A i n r e c o n s t i t u t e d f o r t i f i e d non-fat milk s o l i d s . Part I. The e f f e c t of heat. A u s t r a l i a n J. Dairy Technol. 13:29. Wodsak, W. 1953. Keeping q u a l i t i e s of vitamin A i n foods f o r t i f i e d with vitamin A. Fette Seifen Anstrichm. 55:32. Wool l a r d , D.C. and Edmiston, A.D. 1983. S t a b i l i t y of vitamins i n f o r t i f i e d milk powders during a two-year storage period. N.Z. J. Dairy S c i . Technol. 18: 21. Woollard, D.C. and Wool l a r d , G.A. 1981. Determination of vitamin A in f o r t i f i e d milk powders using high performance l i q u i d chromatography. N.Z. J. Dairy S c i . Technol. 16: 99.  - 70 -  Appendix 1 - Quadratic equations f o r the Primary Powder treatments when stored a t 22°C and 37°C.  Regression Equation Treatment  22°C  37°C  A  y = 27.26 - 2 .04x + 0.088x  B  y = 20.31 - 1 .29x + 0.068x  C  y = 28.94 - 3 .70x + 0.153x  2  D  y  27.5.5 - 1 .27x + 0.041x  2  2  2  2  E  l  y = 25.75 - 0 .87x + 0.042x  E  2  y = 28.55 - 0 .47x + O . O l l x  2  F  y = 25.78 - 0 .32x - O.OOlx  H  y = 28.69 - 0 .52x - 0.007x  2  2  y = 29.22 - 6.76x + 0.437x  2  y = 20.21 - 3.01x + 0.068x  2  y = 31.40 - 7.36x + 0.476x 28.55 - 2.16x + 0.087x  y  2  y = 26.02 - 1.22x + O.OOlx y  -  25.69 - 0.98x + 0.215x  2  2  2  y = 30.11 - 2.41x  -  0.042x  2  y = 27.07 - 0.81x  -  0.073x  2  - 71 -  Appendix 2 - Regression equations f o r the Instant Powder treatments during storage at 22°C and 37°C.  Treatment  Regression Equation 22°C  37°C  A  y = 30.93 - 1.26X - 0.020x  B  y = 25.46 - 1.34x + 0.036x  C  y = 26.46 - 1.03x + 0.026x  D  y = 27.68 - 0.48x - 0.021x  E  y = 16.96 - 1.29x + 0.055x  F  y = 16.59 - 1.41x + 0.064x  G  y = 24.97 - 1.07x + 0.028x  H  y = 33.29 - 0.66x - O.OlOx  J  y = 24.63 - 0.53x + 0.004x  K  y = 23.75 - 1.34'x-+ 0.033x  2  2  2  2  2  2  2  2  2  2  y = 28.68 - 6 .94x + 0.634X  2  y = 24.80 - 5 .81x + 0.509x y = 26.78 - 4 .30x + 0.263x  2  2  y = 25.30 - 1 .59x - 0.050x y = 16.38 - 2.82x + 0.207x y = 16.43 - 3 .74x + 0.340x y = 24.21 - 5 . 36x + 0.487x y = 33.93 - 6 .89x + 0 . 6 4 8 X y = 25.06 - 4 .36x + 0.554X y = 25.09 - 5 .64x + 0.512x  2  2  2  2  2  2  2  

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