Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

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

Item Metadata

Download

Media
831-UBC_1985_A6_7 P36.pdf [ 3.04MB ]
Metadata
JSON: 831-1.0096224.json
JSON-LD: 831-1.0096224-ld.json
RDF/XML (Pretty): 831-1.0096224-rdf.xml
RDF/JSON: 831-1.0096224-rdf.json
Turtle: 831-1.0096224-turtle.txt
N-Triples: 831-1.0096224-rdf-ntriples.txt
Original Record: 831-1.0096224-source.json
Full Text
831-1.0096224-fulltext.txt
Citation
831-1.0096224.ris

Full Text

EVALUATION OF METHODS FOR FORTIFYING SKIM MILK POWDER WITH VITAMIN A By GAETAN MARC ANDRE PAQUETTE B.Sc. (Agr.), University 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 this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1985 (c) Gae'tan Marc Andre Paquette, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of FOOD SCIENCE  The University of B r i t i s h Columbia 1956 Main Mall Van couve r, Canada V6T 1Y3 Date SEPTEMBER. 1985 ABSTRACT Evaluation of Methods for Fortifying 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 in e f f e c t i v e with available methods. The purpose of this study was to assess new methods and materials for thei r effectiveness in providing s t a b i l i t y to vitamin A in 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 in P i l o t Plants which evaluated 14 different treatments for vitamin A s t a b i l i t y during storage periods of twelve months at 22°C and s i x months at 37°C. The second and th i r d phases of the experiment consisted of primary and instant powder t r i a l s in commercial plants using the most stable 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 project, eight treatments were tested for primary powder and ten for instant type of powder. Results show that levels of antioxidants were important to control the oxidative degradation of vitamin A in the milk powder. The vitamin A concentrate containing BHA (5 mg), BHT (55 mg) and ct-tocopherol (12.5 mg) antioxidants produced the best results for primary powder. Ascorbyl palmitate-ct-tocopherol combination of antioxidants was found to be more effective than the BHA-BHT-ct-tocopherol blend for 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 for s t a b i l i t y , 0.2% being s l i g h t l y better than 0.1% in primary powder. A 12% emulsion injected at such a rate as to add 0.027% o i l in milk solids was the best treatment of the instant powder t r i a l s . Hay-like flavour in reconstituted skim milk powder was correlated with vitamin A destruction. ACKNOWLEDGEMENTS The author wishes to express appreciation to the following: Dr. S. Nakai, Supervisor of my thesis, for his advice and guidance during my graduate program and to Dr. D.B. Emmons for his patience, constructive c r i t i c i s m s and for many helpful suggestions with regard to interpretation of the results and the review of this manuscript. Company Representatives for 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 Allan Chia, Gay Lea Foods for the i r technical assistance and for the cooperation of their companies. Laboratory and Research s t a f f : Michelle Murray, Linda Kwan and panelists 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 , for the i r assistance in laboratory analysis and plant work. Mrs. Gail Butler, Engineering and S t a t i s t i c a l Research I n s t i t u t e , for her advice and assistance in the s t a t i s t i c a l analysis of the data. The Secretarial s t a f f of Food Research Institute for their efforts in the typing and corrections of the manuscript. The Canadian Dairy Commission for funding of the project and to my employer, Agriculture Canada for 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 v i i LIST OF FIGURES v i i i LIST OF APPENDICES ix INTRODUCTION 1 REVIEW OF LITERATURE 3 Vitamin A deficiency 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 Off-flavour from vitamin A destruction 13 NEED FOR THE STUDY 14 PROJECT OUTLINE s 16 PHASE I - PILOT PLANT TRIALS 18 Introduction 18 Materials and Methods 18 Method of drying and addition of the vitamins 18 Storage and analysis 20 Treatment description 20 -V-Page Results and Discussion Conclusions PHASE II - COMMERCIAL PLANT TRIALS - PRIMARY POWDER Introduction 29 Materials and Methods 30 Addition of the vitamins 30 Treatment description 30 Storage and sampling 32 Vitamin A analysis 32 Sensory evaluation 33 Moisture analysis 33 Method for graphing 34 S t a t i s t i c a l analysis 37 Results and Discussion 39 Vitamin A st a b i 1 i t y 39 Haylike flavour 42 Comparison of production pi ants 42 Comparison of analytical methods of vitamin A 45 Conclusions 46 24 28 - v i -Page Phase III - COMMERCIAL PLANT TRIALS - INSTANT POWDER Introduction 47 Materials and Methods 48 Treatment description 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 in 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 for Phase I - P i l o t Plant T r i a l . 3. I n i t i a l levels of vitamin A and percent vitamin A losses during storage in primary skimmilk powder f o r t i f i e d by different treatments in P i l o t Plants A and D after 3 and 12 months of storage at 22°C and after 3 and 6 months at 37°C. 4. Levels of antioxidants contained in 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 for Phase II - Primary Powder. 6. Estimated regression coefficients for Multiple Comparison Analysis for Primary Powder stored at 22 and 37°C. 7. Losses of vitamin A in Primary Skimmilk Powder f o r t i f i e d by different treatments in 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 for Phase III Instant Powder. 9. Estimated regression coefficients for Multiple Comparison Analysis for Instant Powder stored at 22 and 37°C. 10. Losses of vitamin A in Instant Skimmilk Powder f o r t i f i e d by different treatments in Plants A and B after 12 months of storage at 22°C and 6 months at 370C. - v i i i -LIST OF FIGURES Pac£ 1. Formulas of most common forms of vitamin A and provitamin 5 A. 2. Vitamin A depletion in Primary Skimmilk Powder during 12 months of storage at 22°C. 35 3. Vitamin A depletion in Primary Skimmilk Powder during 6 months of storage at 370C. 35 4. Vitamin A depletion in Instant Skimmilk Powder during 12 54 months of storage at 22°C. 5. Vitamin A depletion in Instant Skimmilk Powder during 6 months 55 of storage at 37°C. LIST OF APPENDICES Regression equations for the Primary Powder treatments when stored at 22 and 37°C. Regression equations for the Instant Powder treatments during storage at 22 and 37°C. - 1 -INTRODUCTION Despite outstanding advances in vitamin f o r t i f i c a t i o n of food, vitamin A deficiency s t i l l occurs in endemic proportions in many developing countries and is occasionally seen in technologically developed societies (WHO, 1982; McLaren, 1980). Interest in the f o r t i f i c a t i o n of nonfat dry milk (NDM) with vitamin-A has inten s i f i e d in recent years as a result of growing concerns for n u t r i t i o n a l l y deficient populations resulting in the export of increasing quantities of this nutritious product to Third World Countries. In 1984, the Food Aid Program of the Canadian International Development Agency (CIDA) shipped approximately 22,000 tonnes of f o r t i f i e d NDM to food-deficient areas of the world. Since 1979, following recommendations from the Protein Advisory Group of the U.N. Agency (Anonymous, 1976) and Marquardt (1979), CIDA has adopted the recommendation to f o r t i f y a l l NDM for Food Aid Programs with vitamin A to 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 instantized nonfat dry milk for consumers with vitamin A. Section B.08.014 of the Act (1979) stipulates that "the added vitamin A shall be in such amount that a reasonable daily intake of the milk contains not less than 1200 I.U. and not more than 2500 I.U. of vitamin A". The reasonable daily intake of mi-Ik is determined further in Schedule K of the Act as 852 ml. - 2 -Therefore the addition of vitamin A to instantized nonfat dry milk is usually between 1400 and 2900 I.U. lOOg allowing for overage. In spite of extensive research in developing methods to produce a stable vitamin A f o r t i f i e d NDM, this process has remained less than satisfactory. In an attempt to improve the s t a b i l i t y of vitamin A in 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 primary and instant NDM 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 storage. The development of haylike flavour was also examined. - 3 -REVIEW OF LITERATURE Vitamin A deficiency Vitamin A deficiency has been studied extensively in experimental animals and in humans. Early signs of vitaminosis A include loss of appe-t i t e , growth f a i l u r e and impaired immune response with lowered resistance to in 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 deficiency, is the most common cause of blindness in young children throughout the world (WHO, 1976; Sommer et a l . , 1981). The Protein-Calorie 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 is considered that a vitamin A deficiency problem of public health significance occurs (Anonymous, 1976). That report recognized xerophthalmia to be endemic in many of the countries in southern and eastern Asia and parts of Latin Ame-r i c a . In recent years, attention has also been drawn to i t s frequent occurrence in many countries in A f r i c a and the Middle East. In well-nourished societies, requirements are more than adequately met by an ample intake of both vitamin A and carotenoids from milk, vege-tables and f r u i t s , but in developing countries, requirements are frequent-ly not met. The shipping of high protein foods alone to these n u t r i t i o n a l -ly deficient populations is not s u f f i c i e n t . It is known that with diets having protein 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 precipitates xerophthalmia (Srikantia, 1975; Pereira and Begum, 1976). - 4 -Skim milk powder being consumed in large quantities may be used as a vehicle to provide deficient populations with vitamin A and especially children who are most frequently in need of the antixerophthalmic and an 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 is in vision. Other less defined nu 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 effective in the treatment of many skin disorders and various kinds of cancer (Sporn and Newton, 1979; Bollag, 1979; McLaren, 1984). The term "vitamin A" includes a l l B-ionone derivatives, other than the provitamin A carotenoids. Figure 1 shows the formulas for the more common forms of vitamin A which include: all-trans r e t i n o l (Figure IA); esters of all - 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 al 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 IE), the acidic 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, all-trans 3-carotene is shown in Figure IF. Vitamin A is f a i r l y stable when heated to moderate temperatures in an inert atmosphere in the absence of l i g h t , but i t is unstable in 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 . Trace metals may also accelerate the oxidation of vitamin A. Moisture and pH are somewhat more c r i t i c a l for certain forms of the vitamin (acetate ester)(Bauernfeind, 1978). Therefore, some precautions in handling - 5 -Figure 1. Formulas of most common forms of vitamin A and Provitamin A. -6 -CH2OH all-trans retinol CH 20C0C 1 5H 3 1 retinyl palmitate CH20C0CH3 retinyl acetate retinaldehyde or retinal COOH retinoic acid all-trans g-carotene - 7 -vitamin A include:, (a) exclusion of oxygen; (b) protection against l i g h t ; (c) avoidance of pro-oxidant trace metals and strongly acidic environment. The principal methods of s t a b i l i z i n g vitamin A involve (a) sealing under vacuum or inert gas, (b.) storage at low temperatures, (c) the addition of antioxidants, (d) coating and sealing vitamin containing oil-droplets with a protective matrix such as gelatin or vegetable gums. The f i r s t two methods are common methods of storing 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 is common practice. However, users are confined to those antioxidants that are acceptable for food addition. In the preparation of dry forms of vitamin A, powders, granulates, microspheres, beadlets and agglomerates have been prepared by a variety of processing methods involving absorption, granulation, spray congealing and encapsulating procedures as detailed 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 is not new;trials were reported in the l i t e r a t u r e over 30 years ago (Olson et a l . , 1949; Bauernfeind et a l . , 1953) soon after the successful synthesis of vitamin A in 1947. 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. The fact that the added vitamin A in NDM generally oxidizes e a s i l y can be attributed to factors, such as the rigors 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 possibly 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 practical vitamin f o r t i f i c a t i o n of NDM by examining the following factors: a) c a r r i e r formulation for the vitamin A (Conochie and Wilkinson, 1956), b) influence of homogenization (Shroff et a l . , 1954), c) effect of heat (Wilkinson and Conochie, 1958), d) effect of light (Dalle et a l . , 1969; Lerner et a l . , 1970; Sattar et a l . , 1976; Sattar et a l . , 1977; Thompson and Erdody, 1974; Smith and MacLeod, 1957; Stu11, 1951), e) influence of reconstitution (Anantakrishan 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 and Allen (1963) to improve methods of vitamin A f o r t i f i c a t i o n to NDM. In 1963, these researchers outlined two new methods for vitamin A f o r t i f i c a t i o n of NDM. - 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 in 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 prior 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 la t e r , Bauernfeind and Parman (1964) demonstrated that both "wet" 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. The key to their success now appears to be in the use of antioxidant-stabilized vitamin A. I t has been suggested that added synthetic vitamin A is less stable than naturally-occurring vitamin A (Thompson and Erdody, 1974). This is possibly 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 into a non-lipid solution resulting in the synthetic vitamin A being in fewer dispersed particles than naturally-occurring vitamins. Another p o s s i b i l i t y is 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 certain substances i n h i b i t oxidation or antioxidant action. Various substances have been established as safe and effective as antioxidant; they can be divided into two categories comprised of primary antioxidants and of synergists. Primary antioxidants are those substances which function by i n h i b i t i n g or interrupting the free radical 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 oxidized, the antioxidants either prevent direct oxidation or provide indirect protection by breaking the oxidation chain reaction. - 10 -A l i s t of commonly used primary antioxidants in vegetable o i l include the tocopherols, gallates, 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 in vitamin A concentrates is limited to BHA,BHT and dl-ct-tocopherol. In Canada, the Food and Drugs Act, Division B.16.100 under Class IV Preservatives (1979) stipulates that the addition of BHA and BHT to vitamin A preparations is limited to 5 mg of each BHA and BHT per m i l l i o n I.U. of vitamin A. There is no r e s t r i c t i o n on dl-a-tocopherol. Synergists are substances which, in combination with other subs-tances, result in a mixture whose a c t i v i t y is greater than the sum of the a c t i v i t y of the individual components. Synergists may possess no a c t i v i t y of their 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-tion 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 for 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 in o i l and are also effective. Lecithin is another substance that appear to exhibit an antioxidant effect. However, i t i s suggested that this effect may be a synergistic effect on the true antioxidant action of primary antioxidants (Sherwin, 1976). - 11 -Antioxidants and synergists thus protect not only fats from oxidizing, but also other substances of an "unsaturated" nature that are contained in foods such as vitamin A. Evidence of the antioxidative effect of tocopherol in vegetable o i l s was observed over 40 years ago (Greenbank et a l . , 1944) and research for i t s possible use in dairy products has since been extensive (Abbot and Waite, 1965; Abbot and Waite, 1962; Battna et a l . , 1982; Timmen, 1978; Merzametov and Gadzhieva, 1982; Erickson et a l . , 1963; King, 1968). Dl-o>tocopherol is one of the most widely used antioxidants for i t s action as a primary antioxidant and vitamin E a c t i v i t y , but at least 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 gallate in spray-dried whole milk powder. No tocopherol preparation was eff e c t i v e , but dodecyl gallate was. Previously, Lea and Ward (1959) and Lea (1960) found that out of seven tocopherols, ^-tocopherol gave a consistently good performance but a-tocopherol was not effective. Abbot and Waite (1965) reported that the tocopherols and dodecyl gallate did not impart any off - f l a v o r to the powder. They were used at the 0.01% level in the powder. Several other primary antioxidants and synergists have been used in the dairy industry. Abbot and Waite (1962) tested flavones, gallates, BHA and NDGA in spray-dried whole milk powder stored for up to 400 days at 20 and 37°C. Dodecyl gallate was shown to be a very effective antioxidant and, propyl gallate and NDGA, whilst gave good protection were less so; - 12 -BHA did l i t t l e to improve keeping quality. A l l antioxidants were used at the 0.01% level in the powder. When powder is f o r t i f i e d at 5000 I.U./lOOg this level of antioxidant (0.01%) is equivalent to 20 mg/million units of vitamin A. Pont (1964) showed that dodecyl gallate 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 in prolonging the induction period of oxidation in butter. Hammond (1970) found BHT, propyl gallate and NDGA effective antioxidants for b u t t e r o i l . S t u l l (1951) showed the effectiveness of NDGA at retarding 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 synergist. Timmen (1978) tested in pure butterfat the antioxidative effect of gallates, BHA, BHT, a-tocopherol and ascorbyl palmitate singly or in combination. Simultaneous addition of a-tocopherol and ascorbyl palmitate d r a s t i c a l l y retarded oxidation; this effect being further i n t e n s i f i e d by l e c i t h i n and propyl gallate. As demonstrated in 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 to ensure a proper s t a b i l i z a t i o n should be the smallest possible; for most food 100-500 ppm are s u f f i c i e n t . The addition of larger quantities may in certain cases produce the opposite r e s u l t , i . e . , accelerate oxidation. For example, i t is important to pay attention to the o r i g i n a l tocopherol content of food - 13 -because in general, the total tocopherol should not exceed 1000 ppm or 1 mg per gram of o i l (Schuler, 1980). Others (Klaui, 1979; Timmen, 1978) have also reported that a great excess of tocopherol no longer has an antioxidative effect on ret 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 to foods for the i r dual purposes by acting as antioxidants and for their biological a c t i v i t y as vitamins when not oxidized. Ascorbyl palmitate has the f u l l biological a c t i v i t y of vitamin C; i t breaks down in the digestive tract releasing ascorbic acid. 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 to foods. Off-flavour from vitamin A destruction In addition to loss in vitamin A a c t i v i t y when destroyed by oxidation, the by-products of this reaction have been reported to cause an of f - f l a v o r called "haylike flavor". As described in the term, this o f f - f l a v o r i s comparable to the smell coming from a drying hay f i e l d on a sunny summer day. Haylike flavor may be described as a l f a l f a or ca r r o t - l i k e and is d i s t i n c t l y different than the oxidized flavor from fat oxidation or the activated flavor caused by u l t r a v i o l e t lig h t on milk protein (Nakai et a l . , 1983). Many researchers have published the association of hay-like flavor development with destruction of added vitamin A in f o r t i f i e d dairy products (Weckel and Chicoye, 1954; Bauernfeind and Alle 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 recently, the li t e r a t u r e has cited several incidences in which researchers have observed rapid loss of vitamin A in 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 flavor (Nakai et a l . , 1983; Thomas et a l . , 1965; Weckel and Chicoye, 1954; Suyama et a l . , 1983). In a previous Canadian Dairy Commission (CDC) contract conducted at the University of B r i t i s h Colombia, Nakai et a l . (1983) found from a market survey that 27% of instantized NDM samples obtained from various manufacturers in 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. This indicated that there was s t i l l 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 ins t a n t i z i n g process was the best method and the "wet" method adding the vitamin A in a hydrogenated coconut o i l emulsion was the next best method. The result of the above survey spelled out the need for further research. The need for an investigation 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 Agriculture Canada researchers. Research was also requested by the milk powder industry as a result of the findings from the above research project. The Canadian International Development Agency (CIDA) - 15 -which buys large quantities of f o r t i f i e d NDM annually was another body requesting recommendations regarding this subject. The major portion of t h e i r milk powder purchase is 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 , this method has been shown to be effective in producing powder with reasonable vitamin A s t a b i l i t y during storage, but is p r o h i b i t i v e l y expensive, i.e. $145/tonne. Since the major portion of this cost is due to the labour required for the debagging, batch-blending and re-bagging of the powder, the discovery of an equally effective method of f o r t i f i c a t i o n during or prior to production has s i g n i f i c a n t saving potent 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 is 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 for CIDA's Food Aid Program alone would be $2.75 millions for one year. - 16 -PROJECT OUTLINE In an attempt to solve this seemingly unsolved problem of vitamin A i n s t a b i l i t y , a research project was undertaken with the following objectives: 1. Prepare in P i l o t Plants and test the storage s t a b i l i t y of vitamin A added to skim milk by different methods before drying. 2. Prepare in Commercial Plants and test 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 for instant and primary powder production. A major U.S. powder manufacturer was v i s i t e d in 1982 to obtain the latest U.S. technology on vitamin A f o r t i f i c a t i o n to NDM. For the f o r t i f i c a t i o n of instant powder, vitamin A in a beadlet form was metered with a dry vitamin feeder into the powder ahead of the agglomerator. They had developed this process following 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 involving 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 v a r i a b i l i t y in levels. The regular powder was f o r t i f i e d using a vitaminized coconut-oil concentrate following 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 to contain vitamin A in a range of 2200-4400 I.U./lOOg for thei r Food Aid Program. This method appeared to easily 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 in this project are based on the "wet" method of f o r t i f i c a t i o n . This basic procedure is of particular interest 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 this project was divided into three phases; Phase I (Objective #1) involved the testing of several treatments at the P i l o t Plant level in order to determine the best possible treatments for the subsequent phases of the project. Phases II and III were carried out in Commercial Plants, by applying the treatments chosen from the results 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 for this phase of the project were conducted in two different P i l o t Plants. Those P i l o t Plants w i l l be referred to as P i l o t Plant A and P i l o t Plant D in this report. Each treatment was replicated twice during different work weeks and at alternating P i l o t Plants. Materials and Methods Methods of drying milk and addition of vitamins Enough condensed skimmmik of 36-44% to t a l solids was obtained to spray dry at least 20 kg of powder for 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 for a batch, was heated to 50-52°C. As the generally accepted minimum Vitamin A level in f o r t i f i e d NDM is 22 I.U./g, to ensure th i s amount is found in the dried product, i t is necessary to include a certain percentage of overage to 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 in an o i l c a r r i e r , was then added with constant agi-tation to the heated portion of the condensed milk which was immediately homogenized with a double stage homogenizer at pressures of 2000 and 500 ps i . 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 for both P i l o t Plants are l i s t e d in Table 1. Following the spray-drying of each treatment, Table 1. Processing conditions in 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 Concentrate Preparation Total solids of Condensed Milk Holding temperature of Condensed Milk (when held overnight) Temperature of O i l -Vitamin mix Temperature of Oil -Vitamin concentrate at homogenization Temperature of vitaminized batch prior to drying 40-44% 2°C 49°C 49°C n ° c 36-38% 2°C 49°C 49°C 11°C Drying Conditions Name of dr i e r Type of drier Anhydro Compact Tower co-current with cyclone c o l l e c t o r Rogers Inverted tear-drop co-current with cyclone c o l l e c t o r . Capacity (Kg powder/hr) Inlet a i r temperature Outlet a i r temperature Type of nozzle Method of removing powder from d r i e r 20 200°C 98°C Centrifugal Cyclone c o l l e c t o r emptied every 15 min. 25 155°C 80°C #72-220 Scrape bottom box each 15 min plus cyclone c o l l e c t o r . - 20 -the dryer box was swept clean to minimize carry-over from one treatment to another. Each lot of vitaminized powder was then blended for one hour in 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 lot of powder was subsequently divided into 250-g samples in 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 for 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. Moisture content was also determined on each lot at the beginning and at the end of the storage period. Treatment description a) The l i s t of the treatments and processing conditions tested in this t r i a l are shown in Tables 1 and 2. The following are brief descriptions of the materials used in 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 (cholecalciferol) per gram intended for non-instantized NDM. In addition, this product contains polysorbate 80 with BHA and BHT as antioxidant in a base of hydrogenated coconut o i l (HCO). Due to i t s HCO content, this concentrate i s not l i q u i d at room temperature. a)Trade names of commercial products were not used throughout this report to protect the identity of the implicated companies. - 21 -Table 2. Vitamin A f o r t i f i c a t i o n treatments for Phase I - P i l o t Plant T r i a l . A. Commercial HCO-based product B. Commercial milkfat-based product C. Mi l k f a t c a r r i e r -0.1% (no antioxidant) D. Milkfat c a r r i e r -0.1% (500 ppm commercial antioxidant mixture) E. Commercial Vitamin A concentrate (vegetable oil-based) 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) - 22 -Treatment B uses a commercial preparation containing the same vitamin A and D concentrations as treatment A, but is in a base of reconstituted non-fat dry milk and butter o i l . This product is meant for instant skim-milk powder f o r t i f i c a t i o n and has no declared antioxidants in 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 0.1% of the dry weight. Vitamins A ( a l l trans-retinyl palmitate) and D 3 were added to the butter o i l from a highly concentrated source (1 m i l l i o n I.U. per gram in vegetable o i l ) . Treatment E uses a U.S. commercial preparation with vitamins A and D con-centrations of 50,000 I.U. and 10,000 I.U. per gram, respectively. The vitamin c a r r i e r in this product is a blend of hydrogenated coconut o i l and vegetable o i l containing a food-grade emulsifier; i t is orange in 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 dl-a-tocopherol (7.5%) with c i t r i c acid as synergist in a mono- and d i -glycerides matrix. This product can be described as off-white to tan in 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 o i l (Peroxide Value = 0.20 and antioxidant-free) as the vitamins c a r r i e r to the respective percent (0.05, 0.1 or 0.2%) of the dry weight of conden-sed milk. The antioxidant and vitamin source used for these treatments is the same materials as described under Treatments C and D section, except - 23 -for Treatments K and L for which the vitamin A source contained i t s own antioxidants. The antioxidant content for these two treatments (K and L) is 5 mg of BHA, 55 mg BHT and 12.5 mg of dl -a-tocopherol per m i l l i o n I.U. of vitamin A. A l l expressions of the level of antioxidants in t h i s thesis, thereafter, are based on milligrams per m i l l i o n I.U. of vitamin A. - 24 -Results and Discussion Table 3 presents i n i t i a l levels of vitamin A in 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 in levels of more than 20 I.U. at the beginning of storage. One treatment (D) using vitamin A in milk fat with no antioxidant resulted in an i n i t i a l level of 11.6 I.U./g. A similar laboratory-prepared mix adding 0.05% HCO to the powder (treatment F) and containing no antioxidant resulted in a level of 14.5 I.U./g. These treatments resulted in 90% or more destruction of the remaining vitamin A after three months of storage at both temperatures. After 3 months at 22°C, treatments E,K,L,N and 0 had less than 25% destruction. These were respectively, a commercial preparation, antioxidant s t a b i l i z e d vitamins at 0.1 and 0.2% HCO in powder and vitamin A with no antioxidant and 500 ppm of a commercial antioxidant mixture at 0.2% HCO. It is apparent that higher levels of hydrogenated coconut o i l and antioxidants are beneficial during i n i t i a l storage at 22°C. Further evidence of the effect of antioxidants l i e s in comparing treatments H,I,J and K, a l l at 0.1% HCO. Vitamin A losses after 3 months at 22°C were 80, 77, 52 and 15% respectively. Types and levels of antioxidants for non-commercial treatments are given on Table 4. Antioxidant levels were: zero (H); 1.3 and 5.2 mg a-tocopherol and 4 and 16 mg of ascorbyl palmitate (treatments I and J respectively); - 25 -Table 3. I n i t i a l levels of vitamin A and percent of vitamin A losses during storage in primary skimmilk powder f o r t i f i e d by different treatments in P i l o t Plants A and D after 3 and 12 months of storage at 22°C and after 3 and 6 months at 37°C. Treatment I n i t i a l - / Percent vitamin A loss—^ Level of Room Temp (22°C) High Temp (37°C) Vitamin A (I.U.) 3 mo 12 mo 3 mo 12 mo A 46.8 67.6 95.8 92.3 97.3 B 11.6 89.7 91.7 90.8 100 C 27.7 93.0 96.0 93.8 100 D 23.2 92.6 94.1 90.3 100 E 36.8 22.6 57.8 49.1 88.3 F 14.5 91.3 94.4 92.8 100 G 20.2 95.5 96.2 95.3 100 H 22.1 80.7 92.0 79.4 92.7 I 24.9 77.4 93.0 78.7 93.2 J 28.2 52.5 93.6 81.1 94.8 K 32.1 15.4 27.5 22.6 31.5 L 29.9 21.2 30.2 24.0 29.0 N 24.6 24.5 85.0 55.9 80.4 0 27.6 23.7 80.4 34.2 74.6 — - Mean of two t r i a l s in each of two P i l o t Plants. - 26 -Table 4. Levels of antioxidants contained in P i l o t Plant treatments Treatment Antioxidant Concentration (mg/million I.U.Vit.A) A (Commercial) Undeclared B (Commercial) Undeclared C None 0 D Ascorbyl Palmitate 4.0mg a-tocopherol 1.3mg E (Commercial) Undeclared F None 0 G Ascorbyl Palmitate 2.0mg a-tocopherol 0.65mg H None 0 I Ascorbyl Palmitate 4.0mg a-tocopherol 1.3mg J Ascorbyl Palmitate 16.0mg a-tocopherol 1.3mg K BHA 5.0mg BHT 55.-pmg a-tocopherol 12.5mg L BHA 5.0mg BHT 5'5.0mg a-tocopherol 12.5mg N None • 0 0 Ascorbyl Palmitate 8.0mg a-tocopherol 2.6mg - 27 -and-5 mg BHA, 55 mg BHT and 12.5 mg a-tocopherol (K). HCO levels of 0.05, 0.1 and 0.2% in the powder with no antioxidant were in treatments F,H and N; destruction after 3 months at 22°C for these treatments were 91, 81 and 24% respectively. Levels of HCO with 500 ppm of commercial antioxidant were 0.05, 0.1 and 0.2% in the powder for treatments G,I and 0; destruction after 3 months at 22°C were 96, 77 and 24% respectively. Clearly higher levels of HCO resulted in greater s t a b i l i t y of vitamin A. This is in 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% level 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 in vegetable o i l (A,E) showed poor and medium s t a b i l i t y respectively. The commercial emulsion (B) was one of the least stable treatments. It is l i k e l y that the large difference in s t a b i l i t y exhibited by the commercial preparations is due to th e i r contents of antioxidants. The crucial test for s u i t a b i l i t y of the treatments l i e s in the storage survival of the vitamin after 12 months at 22°C or 6 months at 37°C. Only treatments K and L resulted in about 30% destruction. A l l others resulted in 75% or more destruction for the same storage condition except treatment E which resulted in 58% destruction at 22°C, but-88% at 37°C. - 28 -C o n c l u s i o n s 1. The presence of antioxidant materials d i s t i n c t l y aided s t a b i l i t y during storage. 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. The antioxidant content of the commercial products were unknown. Different levels and combinations of antioxidants are used in the subsequent t r i a l s in Phases II and I I I . 2. Higher levels of the o i l carrier (HCO) increased s t a b i l i t y . The levels 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 levels. 3. HCO resulted in s l i g h t l y better s t a b i l i t y than milkfat after three months of storage, but showed no difference in 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 in the subsequent t r i a l s . - 29 -PHASE II-COMMERCIAL PLANTS TRIALS-PRIMARY POWDER  Introduction This phase of the project involves the testing of methods of vitamin A f o r t i f i c a t i o n to regular or primary type of skimmilk powder (SMP) when produced at the commercial scale. A brief review of the process is given in the following paragraph. For primary powder production, milk is treated in an evaporator to remove much of the water before the drying process. There are a number of different types of evaporators in use, but regardless of which, the discharged f l u i d usually contains approximately 40-42% soli d s . It has probably been in a pa r t i a l vacuum at a temperature of around 65°C. The condensed milk is then pumped with a high-pressure pump through a small o r i f i c e into the drier box. The a i r temperature in the spray drier is 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 to 50°C for several hours. - 30 -Materials and Methods Addition of the vitamins The vitamin concentrate was injected into the condensed skimmilk at a point just prior to the high-pressure pump leading to the spray-nozzle of the spray d r i e r . A positive-pressure pump (Bran & Luebbe Inc., Model No. N-P31, Des Plaines, I l l i n o i s ) was used to inject the vitamin mixture. It was necessary to heat the vitamin concentrate above 50°C (52-53) prior to injection in order to ensure good f l u i d i t y since most treatments contained hydrogenated coconut o i l (HCO) which is not f l u i d at room temperature (22-25°C). The vitamin-concentrate injection was performed continuously for at least one hour, during which time, the f i r s t one-half hour production was not collected to ensure s u f f i c i e n t time for the powder to travel to the exit and proper equilibration of the system. Spread over the next one-half hour, at least 20 kg of powder was collected by drawing at f i v e minutes intervals from the production line before bagging. Each treatment was done twice at both plants on separate days. In both cases, the dryers were not shut-down between treatments, because they operate continuously for three or four days. Treatment description The treatments tested for these t r i a l s are l i s t e d with a brief description in Table 5. Those formulations were either 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 for the lab-made preparations were described in Phase I. Treatments E2 and H were additional treatments conducted as follow-up - 3:1 -Table 5. Description of vitamin A f o r t i f i c a t i o n treatments for Phase II - Primary Powder. A - Commercial Vitamin A concentrate (45,400 I.U. Vit.A/ml in 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 in the powder with 2000 ppm of a commercial antioxidant mixture in the o i l . i . e . 1 6 mg Ascorbyl palmitate and 5.2 mg a-tocopherpl. D - 0.2% HCO in the powder with 2000 ppm of a commercial antioxidant mixture in the o i l . i . e . 3 2 mg Ascorbyl palmitate and 10.4 mg a-tocopherol. E, - 0.1% HCO in the powder with a commercial s t a b i l i z e d vitamin concentrate containing 5 mg of BHA, 55 mg of BHT and 12.5 mg of a-tocopherol. E 2 - Repeat of Treatment E-j. F - 0.2% HCO in the powder with the same vitamin concen-trate as in Ep E^. H - Same as Treatment F, plus 2000 ppm of the commercial antioxidant mixture used in Treatments C and D. i.e. 5mg BHA, 55mg BHT, 23mg a-tocopherol and 32mg ascorbyl palmitate. Note: Treatments Ep and H were additional treatments conducted after eight months of storage of the other treatments. - 32 -t r i a l s from the most promising results obtained after 6 months of storage. The purpose of these additional treatments (E2 and H) were to confirm treatment Ei results and to determine the effect of higher levels of antioxidants on vitamin A s t a b i l i t y . Samples from freshly 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 for Treatment B in this t r i a l . The results 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 different (P<0.05) than the results for Treatment B; for that reason only Treatment B results w i l l be shown in this paper. Storage and sampling The powder samples produced for both Primary and Instant T r i a l s were handled the same way as described for Phase I, i . e . , they were transported to the Food Research Institute in Ottawa immediately after production for blending, bagging and storage at 22°C for 12 months and at 37°C for s i x months. Sub-samples were sent at regular intervals for vitamin A analysis, sensory evaluation and moisture analysis. Vitamin A analysis The determination of vitamin A content was conducted by three different laboratories at 0,1,2,3,6,9 and 12 months of storage at 22°C and by one of those laboratories at 0,1,2,3,4 and 6 months of storage at 37°C. Each of the three laboratories used a different method of analysis. In the past, the Carr-Price method (AOAC, 1975; Carr and Price, 1926) has t r a d i t i o n a l l y been used to estimate the Vitamin A content in dried milk products. More recently 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 laboratories (Lab A - HPLC method and Lab C - Spectrof1uorometric method). Data from the thir d laboratory (Lab B - Carr-Price method) was obtained for powder samples from only one plant; this data was used for the comparison of results obtained by the different methods of analysis. The powder samples stored at 37°C were analyzed by only Lab C using the spectrof1uorometric method of vitamin A analysis. Sensory evaluation Haylike flavour 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 reconstituted milk (10 g powder; 90 ml water) by f i v e or six selected panelists who indicated the intensity of the haylike flavour using the following scale: 0 = no haylike flavour 1 = doubtful 2 = s l i g h t 3 = moderate 4 = strong 5 = extreme Moisture analysis The moisture content of powder samples for 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 for the experiment was within commercially acceptable levels. - 34 -Method of graphing The vitamin A depletion in powder can best be visualized graphically over the storage period. The method of graphing is as follows. Figures 2 and 3 show the estimated quadratic curves for "the best f i t " of vitamin A levels in powders plotted against storage time when stored at 22°C and 37°C respectively. The plotted line for each treatment accounts for the actual vitamin A content of the two replicate productions at two different plants as determined by one, two or three different methods. To obtain those curves, the data was f i t t e d in a regression equation.of the form: y = a + bix + b?x 2 where: y = I.U. of vitamin A x = storage time (month) a = regression intercept bi = slope b2 = quadratic term (curvature) The regression equations so produced for the curves shown on Figures 2 and 3 are given in Appendix 1. In order to f a c i l i t a t e the visual comparison of individual treatment, i t is desirable for a l l treatments to have the same i n i t i a l value. For that reason, the regression intercepts (a) in the regression equations were assigned the value of 30 as being the i n i t i a l vitamin A content for a l l treatments in Figures 2 and 3, even though the actual regression intercepts were different for each treatment as shown in Appendix 1. Figure 2. Vitamin A depletion in Primary Skim milk Powder during 12 months of storage at 22°C. STORAGE TIME (mo) Figure 3 . Vitamin A depletion in 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 to the c u r v i l i n e a r i t y of the relationship 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 establish the reaction order of the vitamin A oxidation. A computer program was used for 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 to search for the best f i t values of A and B to find the highest r^ values in linear regression analysis after data transformation using t= (y + A ) D for l i n e a r i z a t i o n . This approach was abandoned when i t was found that B value or the reaction order was very different among treatments. Unless a common transformation (B value) could have been found, i t is not possible to use this elaborate technique to compare treatment results. Considering that the various treatments contained different types and levels of antioxidants and that antioxidants do affect the oxidation reaction, i t is not surprising to obtain different reaction orders for individual treatments. Due to the d i f f i c u l t y of comparing such a large number of treatments, a Multiple Comparison Analysis using the protected Least Significant Difference (LSD) test was employed (Snedecor and Cochran, 1967) on the estimated slopes from the linear regression equations. The quadratic terms of the second order equation were also treated in the same manner. This procedure is equivalent to using orthogonal polynomials and results in an independent test for l i n e a r i t y and the curvature. (Rowel 1 and Walters, 1976). To study the parellelism of the li n e s , the results of - 38 -both tests were combined. A probable grouping was then subjected to the analysis of variance. 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 in the analysis of variance are discussed in the results. Note that the protected LSD test i s considered to generally err on the side of too many sig n i f i c a n t differences. This was considered desirable in a screening experiment such as t h i s . Upon examination of the raw data, i t was noted that results of vitamin A analysis from laboratory C were erroneous for powder samples of the additional t r i a l s (E2 and H) at time 0 ( i n i t i a l ) . For that reason, the s t a t i s t i c a l analysis was performed on the data with missing values at time 0 for treatments E2 and H. To confirm that the results of the Multiple Comparison Analysis (LSD test) were not affected by the omission of the missing values for E2 and H, the same test was performed on the data without time 0 values for a l l treatments. Ranking and grouping of non-significant treatments remained unchanged for either set of data. Therefore, results of the s t a t i s t i c a l analysis with missing values for treatments E2 and H are reported. - 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 in the powder during storage for 12 months at 22°C and for 6 months at 37°C. These plots are from the regression equations "of best f i t " for the two t r i a l s in each of two plants. From the s t a t i s t i c a l analysis, Table 6 l i s t s P a r t i a l Regression Coefficients (linear and quadratic) in order of decreasing magnitude for each treatment for the two temperature storage conditions. It i l l u s t r a t e s the grouping of non-significant differences among treatments based on the Multiple Comparison Analysis. The P a r t i a l Regression Coefficient (PRC) for the linear analysis is 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 indicate that the treatments f a l l into four general levels of increasing s t a b i l i t y : C; A; B,D; E j , E2,F,H. This grouping resulted in a highly s i g n i f i c a n t difference between group sum of squares and i n s i g n i f i c a n t differences within groups. A brief description of the treatments is 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 for more d e t a i l s ) . . The results show that the levels of BHA-BHT-a-tocopherol used in 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 in treatments C and D. However treatment C contained 15mg AP and 5 mg a-t and 1 2 Table 6. Estimated regression coefficients ' for Multiple Comparison Analysis (LSD test) for Primary Powder stored at 22 and 37°C 22°C Storage L S D Linear 3 T -1.95 -1.04 -.91 -.52 -.47 -.43 -.40 -.39 .50 Treatment C A D B H E x E 2 F Grouping p<.001 Quadratic p 2 0.153 0.088 0.068 0.042 0.041 0.011 0.000 -.007 .050 Treatment C A B E x D F E 2 H Grouping ' P ^ 0 0 1 37°C Storage Linear Ql -4.51 -4.14 -1.64 -1.59 -1.33 -1.27 -1.21 -1.21 .84 Treatment C A D B H E 2 E 2 F Grouping : P ^ 0 0 1 Quadratic B 2 0.476 0.437 0.236 0.215 0.087 0.001 ,-.042 -.073 .23 Treatment C A B F D £ 1 E 2 H Grouping p<.001 Equations for the 22°C storage are of the form y = 8 0 + & i x i + 8 2 x 2 where x ^ 0,1,2,3,6,9,12 and x 2=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 straight l i n e and 8 2 the coefficient of the quadratic term in the f i t t e d second order equation. Equations for the 37°C storage are of the form y = B 0 + 8 i X 1 + 8 2 x 2 where x x = 0,1,2,3,4,6 and x 2 = X! 2-6x!= 0,-5,-8,-9,-8,0. - 41 -treatment D contained double those amounts with 30mg AP and lOmg a-t. The lower level of AP-a-t antioxidants present in treatment D compared to the BHA-BHT-at antioxidants level present in treatment F (at the same HCO level) 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 additional improvement in s t a b i l i t y of vitamin A (H versus F). The 0.2% of HCO (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 the 0.1% level (C) with a-tocopherol and ascorbyl palmitate, whereas with the BHA-BHT-a-tocopherol antioxidant mixture ( E i , E 2 and F) there was no difference. The Food and Drugs Act of Canada stipulates a maximum level of 0.1% HCO in NDM. Due to this l i m i t a t i o n , when excluding treatments containing HCO levels above 0.1% the BHA-BHT-a-tocopherol antioxidants at the levels in treatments E i , E 2, F and H gave the higher s t a b i l i t y at 0.1% HCO in the powder. In summary, i t appears that a vitamin A concentrate in HCO containing BHA, BHT and a-tocopherol at the levels in treatments Ex,E2,F and H 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 is s i g n i f i c a n t l y different (P< 0.05) than E 2 and H, but the linear analysis did not find s i g n i f i c a n t difference between these treatments. Treatments E i , E 2 , F and H were also - 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 in 0.1% HCO. However, these same antioxidants used at the higher levels in 0.2% HCO (D) produced a non-significant difference (P< 0.05) in s t a b i l i t y over Ei,E 2,F and H. Therefore, i t would appear that when u t i l i z i n g 0.2% HCO with the antioxidants tested in these t r i a l s or 0.1% HCO with the BHA-BHT-at antioxidants combination and level used in these t r i a l s , with a-level of 30 I.U. of vitamin A per gram present in the powder after production, one could expect to retain approximately 26 I.U./gm when stored under conditions equivalent to 22°C for 12 months. Approximately 23 I.U. can be expected to be retained from these same treatments when stored under conditions equivalent to 37°C for six months. Haylike flavour Sensory evaluation was used to assess the development of haylike flavour in powder samples during the storage period. On a scale from 0 to 5, 0 being no HLF detected and 5 being extreme HLF, the panelists generally detected increasing levels of HLF during storage. S t a t i s t i c a l l y , the correlation between units of vitamin A losses and HLF was found to be si g n i f i c a n t at the 1% level for this type of powder (r=0.592 d.f.=82). Those results are consistent with the previous findings of Nakai et al.(1983). Comparison of Production Plants Table 7 shows the percent vitamin A loss for each treatment from the two different production plants during 12 months of storage at 22°C and 6 months at 37°C. Results indicate that the powder produced at Plant A had - 43 -Table 7. Losses of vitamin A in Primary Skimmilk Powder f o r t i f i e d by different treatments in Plants A and B after 12 months of storage at 22°C and 6 months at 37°C. Treatment Storage Temperature 22°C 37°C Plant A Plant B Plant A Plant B % loss % 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 E l 26.8 8.2 32.0 20.0 E2 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 - 44 -roughly twice the percentage vitamin A loss of Plant B for the same treatments when stored at 22°C for twelve months. The losses were also generally higher for Plant A powders when stored at 37°C for 6 months. However, the difference between the two plants was less marked at the higher temperature storage. The reasons for the lower losses in Plant B are not immediately apparent. One p o s s i b i l i t y is the s l i g h t l y higher temperature in the dryer of Plant B (400°F; 200°F outlet) than in Plant A (290-310°F i n l e t ; 190-195°F outlet; and the difference in the pre-heat treatments, both contributing to greater whey protein denaturation in Plant B powder. The hi-heat powder thereby produced in Plant B would contain more free sulphydryl groups which provides a natural antioxidant effect later in the stored product. This phenomena has previously been recognized to occur in 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 in skim milk powder (Pyenson and Tracy,1948). The relevance of this hypothesis to our situation 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 in Plant A samples; the lower whey protein nitrogen level indicating the greater extent of denaturation. Further t r i a l s would be required to confirm this hypothesis. -45 -Comparison of analytical 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 different laboratories. A r a t i o of results to be expected' among methods of analysis was then calculated to be 0.82: 1.0: 1.16 for the Carr-Price, spectrofluorometric and HPLC methods respectively. A similar r a t i o was obtained throughout the storage periods and for various vitamin A levels. - 46 -C o n c l u s i o n s 1. It was demonstrated in these t r i a l s that the use of antioxidants is important to control the oxidation of vitamin A in primary skim milk powder. The antioxidants BHA-BHT-a-tocopherol at levels of 5,55 and 12.5mg/106I.U. respectively in 0.1 or 0.2% HCO permitted the retention of approximately 80% of the vitamin A when stored at 22°C for 12 months and approximately 70% when stored at 37°C for 6 months. 2. Haylike flavour development was correlated with vitamin A destruction. 3. A large difference in vitamin A s t a b i l i t y was observed between production plants. This difference was attributed to the type of dryer used and pre-heat treatment given to the milk prior to spray drying. 4. The three different methods of vitamin A analysis u t i l i z e d in these t r i a l s consistently produced results proportionally different from each other. Their respective degree of accuracy was not determined. The HPLC method consistently produced higher values and the Carr-Price method produced lower values than the spectrofluorometric method. 5. The level of HCO added to the powder was important for 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 level (0.2%) did not affect s t a b i l i t y . - 47 -Phase Ill-Commercial Plant Trials-Instant Powder  Introduction Experiments for this phase of the project involved the testing of methods of vitamin A f o r t i f i c a t i o n to instant type of skim milk powder (SMP). T r i a l s for this type of powder f o r t i f i c a t i o n were carried out simultaneously with the Commercial Primary Powder t r i a l s (Phase II) since both types of powder were produced at the same plants. A brief review of the instant powder manufacturing process is given in the following paragraph. For instant powder production, the instantizers used are a tunnel-like construction where the dry powder t r a v e l l i n g in a stream of ai r i s sprayed at i t s entrance with either a fine jet of water or steam to cause the milk particles to agglomerate with a moisture content of 10-15%. These par t i c l e s passing through the tunnel or tube structure are subjected to temperatures of 150 to 175°C to reduce the moisture content to 4-5%. They are then sized, with the fines being recycled back to the start of the inst a n t i z i n g process and the rest i s bagged. T r i a l s for this phase of the project were conducted by two different commercial skim milk powder manufacturers. Each treatment was replicated twice at alternating work weeks and plants. - 48 -Materials and Methods Treatment description The treatments tested for these t r i a l s are l i s t e d with a brief des-c r i p t i o n in Table 8. Treatments A, H and K are commercial vitamin concen-trates, the rest are lab-made formulations containing different combinations of kinds and levels of antioxidants blended in 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 . Its 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 in Phase I t r i a l s . Commercially, this product is added to the primary NDM on a dry basis by metering i t to the powder on the way to the instan t i z e r . In th i s experiment, this product was dissolved in water to make a 20% solution for treatment H and a 10% solution for treatment K. These dilutions caused a f i v e - or ten-fold reduction in vitamin concentration and made i t possible to i n j ect this material on a "wet" basis using the same procedure as the other treatments. Preparation of vitamin mixes The lab-made preparations (treatments B to J) were prepared by melt-ing HCO at 50°C to which the vitamins and the antioxidants were, a d d e d . >• This vitaminized HCO was then added to 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. Description of Vitamin A f o r t i f i c a t i o n treatments for Phase I I I . - Instant Powder. A. - Commercial vitamin A and D concentrate (45,400 I.U. vitamin A/ml in 6% milkfat) 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 in B. D. _ 25% HCO - Skimmilk emulsion with the same commercial s t a b i l i z e d vitamin concentrate as in 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 of a commercial antioxidant mixture added separately. i.e.l.2mg AP and 0.4mg a-tocopherol. G. _ 12% HCO - Skimmilk dispersion (same ingredients as i n C, except homogenization was not used during preparation). H. - Commercial Beadlets dissolved in water to make a 20% solution. J - ^ - 12% HCO - Skimmilk emulsion with the same ingredients as in C, but with lower vitamin concentration to inject four times more HCO into the powder. K— - Commercial Beadlets dissolved in water to make a 10% solution. T7 ; -Treatments J and K were additional treatments conducted af t e r eight months of storage results from other treatments. - 50 -and 500 psi of pressure in a laboratory homogenizer. The vitamins added to these preparations were such to create a product of similar vitamin concentration (50,00 I.U. Vitamin A/ml) as the commercial products (treatments A,H and K). The percentage of o i l stated for each treatment indicates the fat content in the vitamin concentrate which unlike primary powder represents a very small addition to the dried powder. In f a c t , for example, the 12% oil-skim 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 circulated through the homogenizer at very low pressure (circa 100-200 p s i ) . Because this preparation was not homogenized, i t was necessary to agitate constantly during injection to prevent the vitamin-containing o i l from r i s i n g to the surface. The purpose of this treatment was to determine the effect of homogenization on the vitamin s t a b i l i t y . As in Phase I I , additional treatments were conducted as follow-up t r i a l s based on results obtained after 6 months of storage. They were treatments J and K; J was to evaluate the effect 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 in water) in a more di l u t e solution. - 51 -Addition of the vitamins For this type of powder, the vitamin concentrate was injected d i r e c t l y into the instantizing chamber through an opening at the middle top section of the cylinder. A small pulsating pump (Waltham Chemical Pump Model No. 10611-361) was used to transfer the material through a nozzle into the instan t i z e r . This pump has the capacity to deliver 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 for an even d i s t r i b u t i o n of vitamins to the powder. This method of injection was used in both plants for t h e i r normal operation. The use of a different pump was the only modification to the existing system. The powder sample co l l e c t i o n was conducted the same way as described for Primary Powder; vitamin 'concentrate was injected for one hour and 20 Kg of powder was collected d i r e c t l y from the bagging l i n e , during the last half-hour. Storage and sampling- see Phase II Vitamin A analysis - see Phase II Sensory evaluation - see Phase II S t a t i s t i c a l analysis The same method of analysis was used as for the primary powder t r i a l s - Phase II i e . Multiple Comparison Analysis(LSD) (see Phase II for description). As observed in Phase II t r i a l s , results of vitamin A analysis from laboratory C were also erroneous for 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 Multiple Comparison - 52 -Analysis was performed on the data with missing values at time 0 for treatments J and K and on the data omitting time 0 values for a l l treatments. However, in this case the ranking and grouping of non-significant treatments remained unchanged for the two sets of data of the 22°C storage but was different for the 37°C storage. The omission of time 0 values of laboratory C has a more important effect 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 results of treatments J and K for the 37°C storage. The adjustment i s reflected in the results discussed l a t e r . 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 in the powder during storage for 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 in each of two plants. Appendix 2 l i s t s the regression equations for each treatment at the two temperature storages. From the s t a t i s t i c a l analysis, Table 9 l i s t s both P a r t i a l Regression Coefficients (linear & quadratic) in order of decreasing magnitude for 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 Multiple Comparison Analysis. The P a r t i a l Regression Coefficient ( B 2 ) analysis indicate the degree of curvature. For this type of powder, Figures 4 and 5 show that only s l i g h t differences were found among treatments, especially at 22°C storage. The s t a t i s t i c a l analysis determining the grouping of non-significant difference among treatments shown on Table 9 supports this observation. Only treatment A (Commercial milkfat emulsion) was shown to be s i g n i f i c a n t l y (P<0.05) different than a l l other treatments and this 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 si g n i f i c a n t differences (P<0.05) among treatments for the 22°C storage which indicate that one can rely solely on the results of the linear analysis for the evaluation of treatments at that temperature. Figure 4. Vitamin A depletion in Instant Skim milk Powder during 12 months of storage at 22°C. T 1 1 1 1 r 0 1 2 3 6 9 STORAGE TIME (mo) Hgure 5. Vitamin A depletion in Instant Ski, U K Powder during 6 »nths of storage at 37°C. 10 Table 9. Estimated regression coefficients ' for Multiple Comparison Analysis (LSD) for Instant Powder stored at 22°C and 37°C. 22°C Storage LSD Linear Bi -1.58 -1.10 -.97 -.85 -.79 -.78 -.75 -.70 -.67 -.55 .32 Treatment A K B H G C D F E J Grouping p<.05 Quadratic .064 .055 .036 .033 .028 .026 .004 -.010 -.020 -.021 Treatment F E B K G C J H A D Grouping 37°C Storage Linear -3.13 -3.10 -3.00 -2.76 -2.72 -2.44 -1.89 -1.70 -1.58 -1.56 .60 Treatment A K H B C G D F E J Grouping p<.05 3 2 .844 .802 .648 .634 .511 .487 .340 .263 .207 -.050 .36 Treatment J K H A B G F C E D Grouping p<.05 Equations for the 22°C storage are of the form y =3o+3i-X1 + 8 2X 2 where Xi = 0,1,2,3,6,9,12 and X2= Xi -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 straight l i n e and e 2 the coefficient of the quadratic term i n the f i t t e d second order equation. Equations for the 37°C storage are of the form y = g 0 + 3 i X x + e 2 x 2 where Xx = 0,1,2,3,4,6 and X2= X x -6X1 = 0,-5,-8,-9,-8,0. - 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 for description of treatments). To assess the effect of HCO on vitamin s t a b i l i t y one must examine the results 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 in Table 9 ranked those treatments as higher in s t a b i l i t y with increasing HCO levels for both storage temperatures. This d i s t i n c t i o n is also noticeable in 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 effect of different types of antioxidants, treatment B(6%HC0,BHA-BHT-at) is compared with E(6%HC0,at,AP) and C(12%HC0,BHA-BHT-at) against F (12%HC0, at,AP). The results of the linear 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 different (P<0.05) for 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 results, we may conclude that the commercial antioxidant mixture containing a-tocopherol and ascorbyl palmitate was more effective than the BHA-BHT-a-tocopherol - 58 -mixture for s t a b i l i z i n g the powder especially when used in the lower HCO emulsion (6%). Only a sl i g h t difference was found between the two mixes of antioxidants in the 12% HCO emulsion for the two temperature storages. Considering that the antioxidant levels present in treatments E and F (0.6mg AP, 0.2mga-t and 1.2mg AP, 0.4mga-t), were much lower than the level used in treatments B and C (5mg BHA, 55mg BHT and 1.2mga-t) i t may be concluded that ascorbyl palmitate is required in smaller quantities than BHA-BHT to s t a b i l i z e instantized powder. Treatments H and K were Commercial Beadlets dissolved in water. Results of those treatments were among the poorest for 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) to produce a stable powder when i t is incorporated by dry blending. The dissolving of this product is water which remove the protective gelatin 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 in these t r i a l s relates to the effect of homogenization of the emulsion (HCO-Skimmilk) on vitamin s t a b i l i t y . To evaluate the effect of this 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 analysis, both linear and quadratic for the two temperature storages did not find 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 injection 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) to 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 after production, one could expect to retain approximately 23 I.U./gm when stored under conditions equivalent to 22°C for 12 months. About 21 I.U./gm can be expected to be retained when stored under conditions equivalent to 37°C for s i x months. Haylike flavour The results of the sensory evaluation for this type of powder indicated that the correlation between haylike flavour 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 to 5, haylike flavour being 0=no haylike flavour; l=doubtful; 2=slight; 3=moderate; 4=strong; 5=extreme, panelists judged that approximately 9.5 I.U. of vitamin A had to be destroyed before a powder sample was labelled doubtful(1) in haylike flavour. Only an additional 2.5 I.U. had to be lost before the powder was judged s l i g h t (2) in haylike flavour. 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 haylike flavour and none were strong or extreme in that flavour. Comparison of production plants Table 10 shows the percent vitamin A losses for each treatment from two different production plants during 12 months of storage at 22°C and 6 months at 37°C. Results of vitamin A losses for the same treatments were very si m i l a r with variations generally lower than 20% between plants - 60 " Table 10. Losses of vitamin A in Instant Skimmilk Powder f o r t i f i e d by different treatments in Plants A and B after 12 months of storage at 22°C and 6 months at 37°C. Treatment Percent vitamin A loss-^ 22°C 37°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 of two replicate t r i a l s analyzed by two laboratories for 22°C and by one laboratory for 37°C storage. - 61 -under both storage conditions. In fact an analysis of variance of the data indicated that no si g n i f i c a n t difference existed between the two plants. Those results are different than for the primary powder t r i a l s (Phase II) in which powder produced at Plant B resulted in 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 for this change in s t a b i l i t y between plants for instant type of powder is not obvious. Perhaps an explanation for this discrepancy l i e s in the difference in micro-environments of the o i l droplets containing the vitamins and antioxidants for 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 particles 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 oxidation. 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 particles and the vitamin-containing o i l droplets w i l l probably adhere to the exterior of the agglomerates. In this case, the vitamins have greater exposure to oxygen and are not surrounded by proteins from the milk as for primary powder; therefore, free sulphydryl groups would have l i t t l e effects on vitamin s t a b i l i t y . - 62 -Conclusions 1. These t r i a l s demonstrated the positive effect of higher levels of hydrogenated coconut o i l on vitamin A s t a b i l i t y . The highest HCO level tested (treatment J) limited 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 milkfat as the vitamin c a r r i e r in an emulsion form did not produce a stable f o r t i f i e d powder. The poor results of this treatment are consistent with previous work of Nakai et al.(1983). 3. Antioxidants were again shown to be important additives to vitamin concentrates in order to delay vitamin A degradation during storage. Ascorbyl palmitate in conjunction with a-tocopherol was shown to be required in smaller quantities than BHA-BHT to s t a b i l i z e instantized powder. 4. It was found that when the dry beadlet vitamin concentrate was dissolved in water for the wet method of f o r t i f i c a t i o n , this product did not produce a stable powder. 5. Homogenization of the vitamin concentrate did not affect the s t a b i l i t y of the added vitamin to powder. However, when only dispersion is used, the need for constant s t i r r i n g during injection of th i s material makes i t impractical in commercial operations. - 63 -6. Hay-like flavour development during storage was again correlated with vitamin A destruction. 7. The large difference 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 II) was not found for 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 effective for 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 is f o r t i f i e d at a higher vitamin A level than powder intended for Canadian market and is generally submitted to storage conditions conducive to faster vitamin A destruction. Therefore, unless higher levels of antioxidants are used for this powder, greater losses of vitamin A can be expected. - 65 -LITERATURE CITED Abbot, J. and Waite, R. 1962. The effect of antioxidants on the keeping qu a l i t y of whole milk powder. J. Dairy Res. 29: 55. Abbot, J. and Waite, R. 1965. The effect of antioxidants on the keeping qu a l i t y of whole milk powder. I I . Tocopherols. J. Dairy Res. 32, 143. Agriculture Canada. 1977. Manual on Skim Milk Powder Analysis. Moisture, p. 8. Anantakrishnan, CP. and Conochie, J. 1958. Some observations on vitamin A in reconstituted f o r t i f i e d non-fat milk s o l i d s . Australian 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. Association of O f f i c i a l Analytical 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 Analytical 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 in the presence of Ronoxan A and other antioxidants. Internat. J. Vit. Nutr. Res. 52:241. Bauernfeind, J.C and Al l e n , L.E. 1963. Vitamin A and D enrichment of non fat dry milk. J. Dairy Sci. 46:245. 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. Synthetic vitamin A aids food f o r t i f i c a t i o n . Food Eng. 25:(6) 81-82, 85, 87. Bauernfeind, J.C. 1978. The technology of vitamin A. Proc. of the XI Int. Congress of Nutr., Rio de Janeiro. Bollag, W. 1979. Retinoids and cancer. Cancer Chemother. Pharmacol. 3:207. Carr, F.H. and Price, E.A. 1926. Colour reactions attributed to vitamin A. Biochem. J. 20: 497. Conochie, J. and Wilkinson, R.A. 1956. The f o r t i f i c a t i o n of nonfat milk solids with vitamin A. XlVth Intern. Dairy Congr. Proc. I (II):357. Dalle, 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 carotinoides. B u l l . Soc. Chim. Fr. (1):232. - 66 -de Boer, M., deMan, L. and deMan, J.M. 1984. Effect of time and storage conditions on vitamin A in instantized 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. Erickson, D.R., Dunkley, W.L. and Ronning, M. 1963. Effect of intravenously injected tocopherol on oxidized flavor in milk. J. Dairy S c i . 46, 911. Food and Drugs Act. 1979. Department of National Health and Welfare, Canada, Ottawa, Ont. F u j i i , S. and Nakai, S. 1980. Optimization of data transformations for 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 quality of commercial dried whole milk packaged in air and nitrogen. J. Dairy Sc i . 27:686. Hammond, E.J. 1970. S t a b i l i z i n g milk fat with antioxidants. American Dairy Review, June 1970. Hollender, H.A. and Tracy, P.H. 1942. The r e l a t i o n of the use of certain antioxidants and methods of processing to the keeping quality 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 effect of separating and c l a r i f y i n g and pasteurization of a milk upon the keeping quality 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. Direct addition of tocopherol to milk for control of oxidized flavor. J. Dairy S c i . 51, 1705. Klaui, H., Hausheer, W., Huschke, G. 1970. Technological aspects of the use of fat-soluble vitamins and carotenoids and the development of s t a b i l i z e d marketable forms. Int. Encyclopedia Food and Nutrition. (Pergamon Press) 9:113. Klaui, H. 1979. Inactivation 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. Sci. Fd. Agric. 11: 212. - 67 -Lea, C.H. and Ward, R.J. 1959. Relative antioxidant a c t i v i t i e s of the seven tocopherols. J. Sci. Fd. Agric. 10: 537. LeMaguer, I. and Jackson, H. 1983. S t a b i l i t y of vitamin A in pasteurized and ultra-high-temperature processed milks. J. Dairy Sci. 66:2452. Lerner, D.A., Mani, J.C. and Mousseron-Canet, M. 1970. Luminescence et photoreactivite dans la serie de l a vitamine A. Etude quantitative. B u l l . Soc. Chim. Fr. (5):1968. Marquardt, H.G. 1979. Distribution of dried skim milk as food r e l i e f measure and new findings on vitaminization of dried skim milk. Deutsche Milchwirtschaft 30(4) 118. Mattick, A.T.R., Hiscox, E.R., Crossley, E.L., Lea, C.H., Thompson, S.Y., Kon, S.K. and Egdell, J.W. 1945. The effect of temperature of preheating, of c l a r i f i c a t i o n and bacteriological quality 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. Nutritional Ophthalmology. Academic Press, London. McLaren, D.S. 1984. Vitamin A deficiency and t o x i c i t y . In "Present Knowledge in Nutrition". The Nutrition Foundation, F i f t h Edition, Washington, D.C. p. 203. Merzametov, M.M. and Gadzhieva, L.I. 1982. Antioxidants for milk f a t . Pishchevaya Tekhnologiya. No. 5, 35. Merzametov, M.M. and Gadzhieva, L.I. 1982. Cystine and tocopherol as milk fat antioxidants. 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 in nonfat dry milk and pasteurized low fat.milk. Can. Inst. Food S c i . Technol. J. 16:116. Olson, F.C., Gruber, G.W., Kozlik, 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 relationship between the swift test time and the keeping quality of butterfat. Aust. J. Dairy Tech. 19:108. Pereira, S.M. and Begum, A. 1976. Vitamin A deficiency in Indian children. 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 quality of spray-dried ice cream mix. J. Dairy Sci. 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 unit. J. Agric. S c i . , Camb. 87:423. Sattar, 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 fats to fluorescent l i g h t i r r a d i a t i o n . J. Am. Oil Chem. Soc. 52:473. Sattar, A., deMan, J.M. and Alexander, J.C. 1977. Wavelength effect on light-induced decomposition of vitamin A and 3-carotene in solutions of milk f a t . Can. Inst. Food Sci. Technol. J. 10:56. Schuler, P. 1980. Roche antioxidants. Roche Information Service, Food Industries Department. Publication #270-82535-1737. Schroff, N.B., Narayawan, K.M., Anantakrishnan, CP. and Sen, K.C. 1954. Studies on vitamin A in milk. Part VII. Effect of processing on the s t a b i l i t y of vitamin A in f o r t i f i e d milk. Indian J. Dairy S c i . 7:40. Sherwin, E.R. 1976. Antioxidants for vegetable o i l s . J. Assoc. Off. Anal. Chem. 53:430. Smith, A.C and MacLeod, P. 1957. Effect of pasteurization temperatures and exposure to light 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. Sixth Edition. Iowa State University Press, Ames, Iowa. Sommer, A., Hussaini, I., Tarwatjo, D., Susanto, D. and Soegiharto, T. 1981. Incidence, prevalence and scale of blinding malnutrition. Lancet 1:1407. Sporn, M.B. and Newton, D.L. 1979. Chemoprevention of cancer with retinoids. Fed. Proc. 38:2528. Sri k a n t i a , S.G. 1975. Human vitamin A deficiency. World Rev. Nutr. Dietet. 20:184. S t u l l , J.W. 1951. The effect of li 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 for haylike flavour production in 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 light of vitamin A added to milk. Can. Inst. Food Sci. 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 Bu 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 butterfat by antioxidants. Proc. 20th Int. Dairy Congress, Paris, June p. 865. Waite, J.D.C., Smith, J.A.B. and Lea, C.H. 1947., The effects of a highpreheating temperature with and without ethyl gal late on the storage l i f e of whole milk powder spray-dried on a Gray-Jensen Drier. J. Dairy Res. 15:127. Weckel, K.G. and Chicoye, E. 1954. Factors responsible for the development of a hay-like flavour in 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 deficiency 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 in reconstituted f o r t i f i e d non-fat milk s o l i d s . Part I. The effect of heat. Australian J. Dairy Technol. 13:29. Wodsak, W. 1953. Keeping qu a l i t i e s of vitamin A in 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 in 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 lard, 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 Sci. Technol. 16: 99. - 70 -Appendix 1 - Quadratic equations for the Primary Powder treatments when stored at 22°C and 37°C. Regression Equation Treatment 22°C 37°C A y = 27.26 - 2 .04x + 0.088x2 y = 29.22 - 6.76x + 0.437x2 B y = 20.31 - 1 .29x + 0.068x2 y = 20.21 - 3.01x + 0.068x2 C y = 28.94 - 3 .70x + 0.153x2 y = 31.40 - 7.36x + 0.476x2 D y 27.5.5 - 1 .27x + 0.041x2 y 28.55 - 2.16x + 0.087x2 E l y = 25.75 - 0 .87x + 0.042x2 y = 26.02 - 1.22x + O.OOlx2 E2 y = 28.55 - 0 .47x + O.Ollx2 y - 25.69 - 0.98x + 0.215x2 F y = 25.78 - 0 .32x - O.OOlx2 y = 30.11 - 2.41x - 0.042x2 H y = 28.69 - 0 .52x - 0.007x2 y = 27.07 - 0.81x - 0.073x2 - 71 -Appendix 2 - Regression equations for 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 - 0.020x2 y = 28.68 - 6 .94x + 0.634X2 B y = 25.46 - 1.34x + 0.036x2 y = 24.80 - 5 .81x + 0.509x2 C y = 26.46 - 1.03x + 0.026x2 y = 26.78 - 4 .30x + 0.263x2 D y = 27.68 - 0.48x - 0.021x2 y = 25.30 - 1 .59x - 0.050x2 E y = 16.96 - 1.29x + 0.055x2 y = 16.38 - 2 .82x + 0.207x2 F y = 16.59 - 1.41x + 0.064x2 y = 16.43 - 3 .74x + 0.340x2 G y = 24.97 - 1.07x + 0.028x2 y = 24.21 - 5 . 36x + 0.487x2 H y = 33.29 - 0.66x - O.OlOx2 y = 33.93 - 6 .89x + 0.648X 2 J y = 24.63 - 0.53x + 0.004x2 y = 25.06 - 4 .36x + 0.554X2 K y = 23.75 - 1.34'x-+ 0.033x2 y = 25.09 - 5 .64x + 0.512x2 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096224/manifest

Comment

Related Items