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Food Research International 40 (2007) 1212–1219

Bioavailability and storage stability of vitamin A fortificant(retinyl acetate) in fortified cookies

Masood Sadiq Butt a, Muhammad Umair Arshad b,*, Muhammad Shahzad Alam a,Muhammad Tahir Nadeem a

a Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistanb Department of Food Science and Technology, University of Sargodha, Sargodha, Pakistan

Received 4 April 2007; accepted 21 July 2007

Abstract

Vitamin A deficiency is one of the major nutritional deficiencies affecting population in developing regions. In Pakistan, 60% of thechild deaths are due to diarrhea and respiratory infection, which are associated with vitamin A deficiency. This study was to assess thebioavailability and stability of vitamin A fortificant in cookies fortified with retinyl acetate. Cookies fortified with Vitamin A (retinylacetate) were manufactured and physically, chemically and sensorial analyzed. Efficacy studies were carried and Serum retinol concen-trations were measured to assess the bioavailability of Vitamin A (retinyl acetate), different organ weights were also measured to checkthe effects of Vitamin A (retinyl acetate) on organ weights and body growth. The treatment T4 containing (257.85 lg) 45% RDA forti-fication level of retinyl acetate was judged best considering, physical, chemical and sensory characteristics. Baking losses were lowest inT4. Efficacy study revealed that Serum retinol concentrations were 125.19 lg/dl in control group and it was raised to 148.64 lg/dl in T4.Liver, lungs, left kidney, right kidney, heart, spleen, brain, left testicles and right testicles increased in their weights when fed with vitaminA fortified diet as compared to control. Exceptions were only brain and spleen, in spleen it remained stable and brain weight reduced ascompared to control. Fortification of cookies with 257.85 lg (45% RDA) of retinol acetate improved nearly all quality attributes andthrough efficacy studies it was concluded that vitamin A significantly affects growth and body functioning of rats. Concludingly cookiesfortified with 257.85 lg of retinol acetate can be used effectively to overcome vitamin A deficiency in the children.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Bioavailability; Cookies; Fortification; HPLC; Retinyl acetate; VAD

1. Introduction

The nutrients in food, required in balanced amounts toproduce and maintain optimum health, belong to thebroad groups of carbohydrates, proteins, fats, vitaminsand minerals (Potter & Hotchkiss, 1997). Vitamins areorganic chemicals that must be supplied in smaller amountsto maintain health. These are conveniently divided into twogroups; water-soluble vitamins including thiamine, ribofla-vin, folic acid, etc. and fat-soluble vitamins comprising

0963-9969/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2007.07.002

* Corresponding author.E-mail address: umairfood1@gmail.com (M.U. Arshad).

vitamins A, D, E and K (Awan, 2006). All people needvitamin A to maintain strong bodies, mental sharpness,fight off disease and bear healthy children (Gibson, 2006).

Vitamin A also acts as antimalarial agent (Hamzah,Skinner-Adams, & Davis, 2003), plays key role in embryo-genesis (Mizuno et al., 2003). Liver development also needsvitamin A because of its ability to turn off a particular liverpromoting gene. The high dietary intake of vitamin Areduces the risk of cancer, cardiovascular disease and otherdiseases (Ghoshal, Pasham, Odom, Furr, & McGrane,2003). According to WHO/UNICEF/IVACG (1997), rec-ommended dietary allowance (RDA) for vitamin A inPakistan are for pregnant women 750 RE, lactatingwomen, 1200 RE, for adults 700 RE and for children

M.S. Butt et al. / Food Research International 40 (2007) 1212–1219 1213

300–600 RE or 60.00 IU/kg of body weight. 1.0 lg of reti-nol is equivalent to retinol equivalent (RE).

In Pakistan, deficiency of vitamin A was found to be18.95% among rural adult females of age group 20–23years and age group 24–27 years was also found to be defi-cient. Among the urban adult females age group 28–31years was the most deficient with deficiency of 24.16%.Age groups of 20–23 years and 24–27 years were also founddeficient. Deficiency of vitamin A can be correlated withhigher child death in Pakistan due to diarrhea and respira-tory infection as they are highly associated with vitamin Adeficiency (Hussain & Shah, 1999). Nearly 48% of Paki-stani children are suffering from vitamin A deficiency(VAD) and enhancing its supplement can help to decreasechild mortality by 25%. About 1–2 million pregnant moth-ers suffer from VAD in South Asia and nearly 60,000women die of childbirth-related disorders, mostly causedby complications, which can be reduced through betternutrition, including provision of vitamin A. Every year,200,000 children die, in Pakistan, of diarrhea, 250,000 ofpneumonia and vitamin A is the best remedy to counterthese severe consequences. Based on the above mentionedfacts, it is highly probable that Pakistani females and chil-dren both in rural and urban areas are at significant risk ofvitamin A deficiency. Enhancing vitamin A supplement canhelp to reduce child mortality by 25% (Sher, 2004).

There are three major strategies to combat vitamin Adeficiency, which are as food fortification, supplementationwith vitamin A and food diversification (Bloem, De Pee, &Darton-Hill, 1998; Chakarvarty, 2000; Filteau & Tomi-kins, 1999). Food fortification can be an economical, flex-ible and socially acceptable way to improve the nutrientintake of groups at risk in order to ensure nutritionallyadequacy of the diet (Hoffpauer & Wright, 1994). Foodfortification is an option where people have access to milledor processed food (Mason, Lotfi, Dalmiya, Sethuraman, &Deitchel, 2001). Fortification can also reinstate the naturalvitamin A content of a foodstuff if it has been lost duringprocessing. Vitamin A fortification requires special atten-tion in regard that vitamin A is fat-soluble and provenvehicles for vitamin A fortification include sugar, oils andfats, and cereals flours (Arroyave, Aguilar, Flores, & Guz-man, 1979; Dary, Arroyave, Flores, Campose, & Lins,1996; Lotfi, Manner, Merx, & Heuvel, 1996). Bauernfeindand De Ritter (1991) described cereals as best for fortifica-tion because in developing countries 95% of the populationconsumes cereals as dietary staple and as staples, milledcereals are relatively inexpensive (Ranum, 2000); they aregrown and consumed worldwide by all economic classes,versatile in preparation and use, and generally processedin large, centralized plants. In poor households, the over-whelming part of the diet might consist of staple grainsand vegetables (Graebner, Siqueira, Arruda, & de-Souza,2004). Cookies, crackers and other bakery products arewidely used in sub-continents and especially in Pakistan.High amount of fat in cookies increases vitamin A absorp-tion in the human body, as vitamin A absorption is in

direct proportionality with fat contents, cookies are alsocomparatively cheap and liked by all age groups and clas-ses of the society; cookies, therefore, are considered as thebest product for the fortification purpose with retinyl ace-tate or retinyl palmitate (Ranum, 2000; Stringer, 2000).For example, in a programme in South Africa, fortified bis-cuits containing about half the RDA for vitamin A weregiven to children at school. They proved to be successfulin ensuring a sufficient daily vitamin A intake (IVACG,2003).

Regarding the stability of vitamin A in cookies the pre-vious studies of Rice, Black, Carlin, and Robinson (1941)revealed that in baked products such as bread, biscuits,and cake, which are baked under moderate conditions, itappears that 80–100% of the vitamin A survives the bakingprocess.

Present research was also planned to assess the ability ofretinyl acetate to act as possible vitamin A fortificant. For-tification of cookies with retinyl acetate could be bettervehicle for increasing vitamin A status of Pakistani chil-dren (Lotfi et al., 1996). In the developing countries, forti-fication is increasingly recognized as a measure to improvenutritional status of large populations. Active governmentparticipation must be ensure for continue for food fortifica-tion programs to succeed in developing countries. Foodfortification can be implemented and sustained over longperiods.

2. Materials and methods

The present study was undertaken in the BiotechnologyLab, Institute of Food Science and Technology, Universityof Agriculture, Faisalabad.

2.1. Procurement of raw materials

Wheat was purchased from the Ayub AgriculturalResearch Institute (AARI), Faisalabad. Straight gradeflour was prepared by UDY cyclone mill in the Instituteof Food Science and Technology, University of Agricul-ture, Faisalabad. After milling of wheat, flour samples werepacked in polypropylene bags and stored at room temper-ature for further study. Ingredients required for the prepa-ration of cookies were purchased from the local market.The fortificant, i.e. retinyl acetate was purchased fromFluka Chemicals, Switzerland.

2.2. Chemical composition of raw materials

The moisture, crude protein, crude fat, total ash andcrude fiber contents of flours and cookies were determinedby AACC (2000). Nitrogen free extract (NFE) was calcu-lated by difference. The factors, n = 5.70 (for wheat flour),and n = 6.25 (for cookies) were used for conversion ofnitrogen to crude protein. Vitamin A content of the flourand commercially available shortening was determinedaccording to the method given in AOAC (1995).

Table 2Composition of experimental diets fed to rats

Dietsa Ingredients of diets (g/100 g)

Cookies Cornoil

Mineralmix

Vitaminmix

Cornstarch

T1 (0%) 69.1 10 2.5 1 17.4T4 (45% RDA) 69.1 10 2.5 1 17.4

a Prepared from cookies containing.

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2.3. Vitamin A fortification and preparation of cookies

Vitamin A is used in varying concentration according toRDA basal recommendations (1 retinol equiva-lent = 1.146 lg retinyl acetate). The treatment plan accord-ing to the RDA basal recommendations for the study isgiven in Table 1.

Cookies were prepared according to the proceduredescribed by McWatters (1985) with slight modifications.The basic ingredients used were 380 g of flour blend,100 g vegetable shortening, 225 g of granulated cane sugar,21 g of beaten whole egg, 3.75 g of salt, and 1.8 g of bakingpowder. The dry ingredients were weighed and mixed thor-oughly in a bowl by hand for 3–5 min. Shortening wasadded and rubbed-in until uniform. The egg was addedand dough thoroughly kneaded in a mixer for 5 min. Thedough was rolled thinly on a sheeting board to a uniformthickness (8.0 mm) and cut out using a round scorn cutterto a diameter of 35.0 mm. The cut out dough pieces werebaked on greased pans at 160 �C for 15 min in baking oven.The prepared cookies were cooled at room temperature(30 ± 2 �C). They were then packed in high density poly-ethylene bags, sealed and stored in a freezer (0–5 �C) untilrequired.

2.4. Storage studies

The cookies containing different levels of vitamin A for-tificant were packed in the airtight bags and stored at roomtemperature. The storage studies include physical (width,thickness and spread factor), chemical (moisture, crudeproteins, crude fat, crude fiber and ash contents) accordingto the methods described in AACC (2000). The cookieswere analyzed by a panel of judges for color, tastes, flavor,texture and overall acceptability by following the proce-dures of Larmond (1977).

2.5. Baking and storage stability of fortificant

Vitamin A contents of cookies were determined accord-ing to the method of AOAC (1995) at 0, 15, and 30 days toevaluate the stability of fortificant during storage. VitaminA contents were determined through spectrophotometerbefore and after baking and above said storage period toevaluate the storage stability of the fortificant. Vitamin Alosses were computed in percentages to estimate bakinglosses.

Table 1Treatment plan

Treatments % RDA Retinyl acetate (lg)

T1 0 (control) 0T2 15 85.95T3 30 171.90T4 45 257.85

2.6. Efficacy studies

The young female Sprague Dawley rats with an initialweight of 148–159 g (7–9 week of age) were purchased fromNational Institute of Health (Veterinary Division), Islama-bad and brought to the laboratory at Institute of FoodScience and Technology, University of Agriculture Faisal-abad. The efficacy study was conducted according to themodel adopted by Levrat-Verny et al. (1999). The rats weredivided in to eight groups (eight per group) for each testdiet and housed in separate cages (wire bottomed) andplaced it under control temperature maintained at25–30 �C with a dark period of 12 h a day. The rats werefed-basal diet for three days. The deionized water wasoffered ad libitum. Test diets were prepared (Table 2) tomeet essential requirements and the nutritional standardsset for rats (Reeves, Nielelsen, & Fahey, 1993). The animalswere divided in to eight groups (eight rats per group) foreach test diet and housed in separate cages (wire bottomed)and placed it under control temperature maintained at25–30 �C with a dark period of 12 h a day. The best-selected treatment along with control was given to selectedgroups of rats and organs weights and serum retinol levelwas estimated after 40 days.

2.6.1. Organ weights

At the end of feeding trials of 40 days, the rats weredecapitated, and weight of various organs (liver, lungs, leftkidney, right kidney, heart, spleen, brain, left testicles andright testicles) were measured on electronic balance.

2.6.2. Estimation of retinol in serum by HPLC

At the end of feeding trials the rats were decapitated,blood was taken in the centrifuge tubes and stored inrefrigerator for 2–3 h. After coagulation of blood, it wascentrifuged at 3000 rpm for 5 min to get serum. Thenserum was collected in the eppendorf tubes with the helpof a micropipette and stored at �20 �C. The vitamin A con-tents were determined according to procedure adopted byDriskell and Neese (1982).

3. Results and discussion

3.1. Chemical composition of raw materials

Straight grade flour with protein contents under limit of9.5% was used in the present study. It was chemically

Table 3Chemical composition of raw wheat flour used in the present study

Components Mean (g/100 g)

Moisture 11.46 ± 0.152Ash 0.50 ± 0.023Protein 9.98 ± 0.075Fat 1.11 ± 0.029Fiber 0.36 ± 0.012NFE 76.60 ± 0.122

M.S. Butt et al. / Food Research International 40 (2007) 1212–1219 1215

analyzed to check different quality characteristics like mois-ture, protein, fat, crude fiber and ash contents. Resultsregarding the composition of raw materials are given inTable 3.

Proximate composition is function of wheat varietiesand milling fraction. Wheat quality is dependent uponmoisture content and protein contents (Halverson & Zele-ny, 1998). Ash and fiber tends to decreases with increase inthe extraction rate, while higher quantities of proteins andlower quantities of lipids results in increased water bindingcapacity of flour, which is undesirable characteristic forcookies manufacturing (Pomeranz, 1998). Similar chemicalcomposition of flour has been reported earlier in theliterature.

3.2. Analysis of cookies

The cookies prepared after fortification with vitamin Awere subjected to physical, chemical, and sensory analysis.

3.2.1. Physical analysis

Physical tests includes width, thickness and spread fac-tor of the cookies and it was revealed that non-significantvariation exists among treatments and storage periods,but T4 (45% RDA, 257.85 lg) gave maximum width(2.45 cm), minimum thickness (49.20 mm) and highestspread factor (50.48 mm). Spread potential and size ofcookies depends on flour particle size and moisture con-tents (Gaines & Donelson, 1985). Generally increasing

Table 4Effect of retinyl acetate fortification and storage on physical attributes (scores

Parameters Storage intervals Treatments

T1 T2

Width (cm) 0 day 2.43.000 ± 2.890 2.43.000 ±15 days 2.41.000 ± 1.150 2.44.000 ±30 days 2.42.000 ± 1.150 2.42.000 ±Mean 2.42.000 ± 1.000 2.43.000 ±

Thickness (mm) 0 day 4.9890 ± 0.243 4.9760 ±15 days 4.9980 ± 0.317 4.9910 ±30 days 4.9950 ± 0.224 4.9850 ±Mean 4.9940 ± 0.133 4.9840 ±

Spread factor 0 day 48.450 ± 0.187 48.830 ±15 days 49.110 ± 0.009 49.280 ±30 days 48.360 ± 0.320 48.540 ±Mean 48.640 ± 0.160 48.883 ±

the concentration of vitamin A (retinyl acetate) resultedin decrease of thickness of cookies while an increasingtrend was observed for width and spread factor of cookies(Table 4).

3.2.2. Chemical analysis

Regarding chemical composition, minimum moisturecontents were found at 0 days (2.34%) and highest after30 days (3.92%). Similarly ash, proteins, fat, and fiber con-tents were higher at 0 days were observed as 0.53, 6.71,22.80 and 0.26, respectively (Table 5). During storage,these values were reduced to 0.49, 6.41, 21.62 and 0.24,respectively. NFE contents were found maximum after 15days of storage while minimum NFE contents wererecorded at the start of storage. The moisture content usu-ally varies from 8.14 to 12.17% in different wheat cultivarsand is significantly affected by milling fractions and culti-vars (Butt, 1997) and decreasing trend in protein contentswas observed as a function of storage. Fats content signif-icantly decreased as a function of storage and was higher at0 day. The decrease could be due to lipase activity whichresults in fat deterioration during storage. The conditionsconducive to deterioration of fat were storage of cookiesat higher moisture contents that could result in reductionof crude fat contents. The results in present study are alsoin accordance with the outcomes of Anjum, Ahmad,Pasha, and Butt (2003), who reported 21.64% fat contentsin biscuits. The mean values for crude fiber and ash of dif-ferent wheat varieties were non-significantly reduced dur-ing storage, while Treatments did not impartedsubstantial differences on ash and fiber contents. Anjumet al. (2003) also observed similar non-significant resultsregarding crude fiber contents. Ash is dependent on extrac-tion rate and amount of bran present in the flour (Pomer-anz, 1998) (Table 6).

Vitamins A contents were determined prior to bakingand after baking. Results showed that minor losses occurduring baking. Retinyl acetate losses ranged 8.69–11.11%.The results obtained showed that as the dose of retinyl

) of cookies

T3 T4 Mean

2.310 2.44.000 ± 1.730 2.45.000 ± 2.310 2.43.50 ± 1.0601.150 2.45.000 ± 1.150 2.46.000 ± 0.580 2.44.50 ± 0.5601.150 2.44.000 ± 2.890 2.44.000 ± 1.150 2.42.70 ± 0.8400.870 2.44.333 ± 1.040 2.45.000 ± 0.82.0

0.014 4.9320 ± 0.301 4.8980 ± 0.589 4.905 ± 0.1050.035 4.9200 ± 0.503 4.8860 ± 0.019 4.905 ± 0.2010.010 4.9140 ± 0.004 4.9760 ± 1.001 4.967 ± 0.2360.023 4.9220 ± 0.191 4.9200 ± 0.363

0.006 49.020 ± 0.008 49.850 ± 0.177 49.04 ± 0.1640.168 49.460 ± 0.314 51.970 ± 0.056 49.95 ± 0.7450.017 49.650 ± 0.583 49.630 ± 0.413 49.04 ± 0.2460.188 49.377 ± 0.213 50.483 ± 0.967

Table 5Effect of retinyl acetate fortification and storage on proximate composition (%) of cookies

Parameters Storage intervals Treatments

T1 T2 T3 T4 Mean

Moisture 0 day 2.320 ± 0.055 2.400 ± 0.029 2.340 ± 0.047 2.280 ± 0.093 2.34 ± 0.02915 days 3.470 ± 0.076 3.380 ± 0.067 3.390 ± 0.071 3.410 ± 0.067 3.41 ± 0.03230 days 3.950 ± 0.173 3.890 ± 0.092 3.940 ± 0.138 3.890 ± 0.092 3.92 ± 0.055Mean 3.247 ± 0.248 3.223 ± 0.221 3.223 ± 0.240 3.193 ± 0.242

Ash 0 day 0.530 ± 0.015 0.520 ± 0.021 0.530 ± 0.021 0.520 ± 0.021 0.53 ± 0.00815 days 0.500 ± 0.017 0.500 ± 0.018 0.510 ± 0.009 0.500 ± 0.009 0.50 ± 0.00630 days 0.490 ± 0.012 0.490 ± 0.201 0.480 ± 0.015 0.490 ± 0.120 0.49 ± 0.006Mean 0.507 ± 0.010 0.503 ± 0.011 0.507 ± 0.010 0.503 ± 0.009

Crude protein 0 day 6.670 ± 0.174 6.700 ± 0.127 6.720 ± 0.133 6.760 ± 0.197 6.71 ± 0.06915 days 6.560 ± 0.173 6.570 ± 0.115 6.570 ± 0.131 6.600 ± 0.203 6.58 ± 0.06930 days 6.420 ± 0.116 6.370 ± 0.067 6.390 ± 0.104 6.450 ± 0.191 6.41 ± 0.055Mean 6.550 ± 0.086 6.547 ± 0.071 6.560 ± 0.078 6.603 ± 0.108

Crude fat 0 day 22.710 ± 0.193 22.700 ± 0.157 22.920 ± 0.311 22.850 ± 0.289 22.80 ± 0.10915 days 22.120 ± 0.339 22.180 ± 0.189 22.050 ± 0.268 22.200 ± 0.130 22.144 ± 0.10630 days 21.790 ± 0.283 21.530 ± 0.176 21.600 ± 0.205 21.560 ± 0.094 21.62 ± 0.091Mean 22.207 ± 0.194 22.137 ± 0.190 22.190 ± 0.234 22.203 ± 0.209

Fiber contents 0 day 0.250 ± 0.023 0.270 ± 0.012 0.260 ± 0.015 0.260 ± 0.015 0.26 ± 0.00815 days 0.240 ± 0.012 0.250 ± 0.012 0.250 ± 0.009 0.250 ± 0.009 0.25 ± 0.00530 days 0.220 ± 0.012 0.240 ± 0.009 0.230 ± 0.012 0.240 ± 0.018 0.24 ± 0.006Mean 0.237 ± 0.009 0.253 ± 0.007 0.247 ± 0.007 0.250 ± 0.008

NFE 0 day 67.520 ± 0.173 67.420 ± 0.143 67.240 ± 0.246 67.330 ± 0.241 38.5867.38315 days 67.110 ± 0.111 67.110 ± 0.308 67.240 ± 0.319 67.040 ± 0.089 67.13 ± 0.10230 days 67.130 ± 0.577 67.470 ± 0.168 67.350 ± 0.408 67.360 ± 0.191 67.33 ± 0.165Mean 67.253 ± 0.189 67.333 ± 0.123 67.277 ± 0.167 67.243 ± 0.106

Table 6Effect of treatments and storage on storage stability of retinyl acetate (lg/100 dl)

Storage intervals Treatments

T1 T2 T3 T4 Mean

0 day 0.160 ± 0.003 78.150 ± 0.005 155.970 ± 1.173 235.620 ± 1.091 117.48 ± 26.44115 days 0.150 ± 0.003 73.200 ± 0.949 146.640 ± 0.971 224.090 ± 1.141 111.02 ± 25.12830 days 0.140 ± 0.003 67.740 ± 0.874 136.510 ± 0.751 214.530 ± 0.767 104.73 ± 24.016Mean 0.150 ± 0.003 73.030 ± 0.566 146.373 ± 2.878 224.747 ± 3.098

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acetate increased the percent baking losses decreased signif-icantly and vitamin A losses were lowest (8.69%) for T4

0.00

50.00

100.00

150.00

200.00

250.00

300.00

T1 T2 T3 T4

Vit

amin

A C

onte

nt

Before baking

After Baking

% Loss

Fig. 1. Vitamin A contents before and after baking.

(45% RDA, 257.85 lg) among the fortified cookies(Fig. 1) and highest (9.36%) for T1 (control 0%). Storageof cookies at ambient temperature significantly reducedvitamin A contents. After 15 days, the vitamin losses were5.89% while among the treatments losses were statisticallynon-significant. However, after 30 days, the losses weredoubled as compared to these at 15 days (Fig. 2). VitaminA is sensitive to heat and light and its losses occur whenexposed to heat and light. The higher storage temperaturecaused such losses, as it may increase oxidation of thevitamin and results in loss of ability of binding with lipidsthat also results in its lower absorption capacities. Maxi-mum losses of 10.85% occurred after 30 days of storage(Table 7).

In a similar work Emodi and Scialei (1980) studied theretention of vitamin A during baking of bread andobserved minor loss of 7–10% during baking. In anotherstudy (Bauernfeind, 2006), 90–100% retention of vitaminA was observed during baking. He also observed a minimal

0day 15days 30daysStorage intervals

Per

cen

tag

e (%

)

T1 T2 T3 T4

0

2

4

6

8

10

12

Fig. 2. Storage losses of vitamin A contents of cookies at different storageintervals.

M.S. Butt et al. / Food Research International 40 (2007) 1212–1219 1217

loss of vitamin A after 6-month storage. The difference inresults could be due to storage temperature. Increasingstorage temperature decreases retention of vitamin A.Wheat flour stored at 45 �C has been found to show 72%loss after 3 months of storage.

3.2.3. Sensory evaluation

Sensory evaluation of fortified cookies was carried outfortnightly. Among the treatments generally maximumscores in all parameters were gained by T1 (control 0%)and T4 (45% RDA, 257.85 lg), while lower scores wererecorded for T2 (15% RDA, 85.95 lg) and T3 (30%RDA, 171.90 lg). T4 (45% RDA, 257.85 lg) scored maxi-mum mean score of 6.73, 6.80, 6.93 and 6.73 for taste, tex-ture appearance and acceptability, respectively (Table 8).Difference in these parameters is not so much to be rejectedfor further use, but comparatively these two treatmentswere scored better by the panelists due to which these wereselected for further studies. However, Vitamin A showedno effect on color and flavor of cookies. Only significantvariation in color and flavor of cookies were observed withrespect to storage intervals. Color and flavor significantlyaffected due to Millard reaction (Bender, 1996). The colorchanges during storage might be due to acceleration of Mil-lard reaction, absorption of moisture contents during stor-age stimulates Millard reaction in cookies. In anotherstudy (Elahi, 2006), gradual decrease in color and flavorof biscuits as a function of storage was observed which sup-ports the findings of present study. During storage of cook-ies absorption of moisture causes flavor deteriorationduring storage of cookies for longer periods (Wada, 1998).

In a similar fashion, taste, texture, appearance and over-all acceptability also decreased as a function of storage

Table 7Effect of treatments and storage on baking losses (% ages) of retinyl acetate

Storage intervals Treatments

T1 T2

0 day 0.000 ± 0.000 0.000 ± 0.00015 days 6.390 ± 0.140 6.330 ± 0.81930 days 12.770 ± 0.277 13.280 ± 2.004Mean 6.387 ± 1.846 6.537 ± 2.017

(Table 8). Oxidation of fats and Millard reaction are mightbe the main causes for such deterioration. Similar resultsare presented by other researchers (Bender, 1996; Elahi,2006; Wada, 1998) who reported the same decreasingtrends in taste, texture, appearance and overall acceptabil-ity with increase in storage time.

3.2.4. Efficacy studies

After the physical and chemical analysis as well as sen-sory evaluation T4 (45% RDA, 257.85 lg) was selectedfor bioavailability trials in rats. Efficacy studies were car-ried out to check the uplift of serum retinol level in rat.Organs of the rats were weighed after the study period tocheck the ultimate effect of fortificant on the growth ofrats. The reason behind this analysis was also to know ifthere is any negative effect of the fortified diets on rats.Most interestingly the organs weight of rats improved withthe addition of vitamin A in diet. Results for the organsweight of rats are presented in Table 8, which indicatedthat organ weight increased was more pronounced in vita-min A fortified cookies fed diets as compared to control,which clearly demonstrated that vitamin A affect growthand body functioning. Liver, lungs, left kidney, right kid-ney, heart, spleen, brain, left testicles and right testiclesweighed 1.47, 0.35, 0.17, 0.17, 0.17, 0.09, 1.26, 0.42,0.44 g respectively, in control as compared to 1.83, 0.40,0.20, 0.20, 0.21, 0.09, 1.24, 0.44, 0.47, respectively, in vita-min A fortified diet (T4 (45% RDA, 257.85 lg)). Organweight significantly increased with vitamin A fortification,exceptions were brain and spleen, in spleen it remained sta-ble at 0.09 g and in brain it reduced 0.02 g when comparedwith control. Liver and heart recorded 24.49 and 23.53%more weight gain in groups fed with vitamin A fortifieddiets as compared to control groups.

Serum retinol concentration was measured throughHPLC. The results for serum retinol concentration of ratsare presented in Table 9. In control groups (T1) (control0%) it was observed to be 125.19 lg/dl and it was raisedto 148.64 lg/dl in T4 (45% RDA, 257.85 lg) (Retinyl ace-tate group) which showed that an increase of 18.93% inserum retinol level was observed in groups fed with forti-fied diets. Meija, Bunge, and Deluce (1977) reported anincrease in serum and liver vitamin A levels in two differentexperiments and observed that rats fed on supplementeddiets had serum concentration from 131.61 to 139.50 lg/dl. Present results are in close agreement with the previous

T3 T4 Mean

0.000 ± 0.000 0.000 ± 0.000 0.00c ± 0005.950 ± 1.453 4.890 ± 0.587 5.98b ± 5.980

12.450 ± 1.365 8.940 ± 0.731 11.86a ± 11.8606.133 ± 1.887 4.610 ± 1.321

Table 8Effect of treatments and storage on sensory evaluation of vitamin fortified cookies

Parameters Storage intervals Treatments

T1 T2 T3 T4 Mean

Color 0 day 6.800 ± 0.120 7.000 ± 0.160 7.000 ± 0.160 6.800 ± 0.200 6.900 ± 0.08015 days 6.800 ± 0.200 6.600 ± 0.240 6.800 ± 0.200 6.600 ± 0.240 6.700 ± 0.10030 days 6.600 ± 0.240 6.400 ± 0.240 6.400 ± 0.240 6.600 ± 0.240 6.500 ± 0.110

Mean 6.730 ± 0.110 6.670 ± 0.140 6.730 ± 0.130 6.670 ± 0.130Flavor 0 day 7.000 ± 0.320 7.000 ± 0.320 6.800 ± 0.200 7.200 ± 0.370 7.000 ± 0.150

15 days 6.600 ± 0.240 6.800 ± 0.200 6.600 ± 0.240 7.000 ± 0.320 6.750 ± 0.12030 days 6.400 ± 0.240 6.000 ± 0.320 6.400 ± 0.240 6.400 ± 0.240 6.300 ± 0.130Mean 6.670 ± 0.160 6.600 ± 0.190 6.600 ± 0.130 6.870 ± 0.190

Taste 0 day 6.800 ± 0.200 7.000 ± 0.320 6.800 ± 0.370 6.800 ± 0.370 6.850 ± 0.15015 days 6.800 ± 0.200 6.600 ± 0.240 6.600 ± 0.240 6.800 ± 0.200 6.700 ± 0.11030 days 6.200 ± 0.200 6.200 ± 0.200 6.400 ± 0.240 6.600 ± 0.240 6.350 ± 0.110Mean 6.600 ± 0.130 6.600 ± 0.160 6.600 ± 0.160 6.730 ± 0.150

Texture 0 day 6.800 ± 0.200 7.200 ± 0.370 7.000 ± 0.320 7.200 ± 0.200 7.050 ± 0.14015 days 6.600 ± 0.240 7.000 ± 0.320 6.800 ± 0.200 6.800 ± 0.370 6.800 ± 0.14030 days 6.400 ± 0.240 6.200 ± 0.200 6.200 ± 0.370 6.400 ± 0.240 6.300 ± 0.130Mean 6.600 ± 0.130 6.800 ± 0.200 6.670 ± 0.190 6.800 ± 0.170

Appearance 0 day 6.800 ± 0.200 7.000 ± 0.320 6.800 ± 0.200 7.200 ± 0.200 6.950 ± 0.11015 days 6.800 ± 0.200 6.600 ± 0.240 6.800 ± 0.370 7.000 ± 0.320 6.800 ± 0.14030 days 6.400 ± 0.240 6.200 ± 0.200 6.200 ± 0.200 6.600 ± 0.240 6.350 ± 0.110Mean 6.670 ± 0.130 6.600 ± 0.160 6.600 ± 0.160 6.930 ± 0.150

Over all acceptability 0 day 7.000 ± 0.320 6.800 ± 0.200 7.000 ± 0.320 6.800 ± 0.200 6.900 ± 0.12015 days 6.800 ± 0.200 6.600 ± 0.240 6.800 ± 0.200 7.000 ± 0.320 6.800 ± 0.12030 days 6.200 ± 0.200 6.000 ± 0.320 6.000 ± 0.370 6.400 ± 0.240 6.200 ± 0.140Mean 6.670 ± 0.160 6.470 ± 0.170 6.670 ± 0.190 6.730 ± 0.150

Table 9Effect of treatments on serum retinol concentration and organs weight of rats

Treatments Retinol (lg/dl) Organs weight (g)

Liver Lungs Kidney (L) Kidney (R) Heart Spleen Brain Testicles (L) Testicles (R)

T1 (0%) 125.19 ± 15.81 1.47 0.35 0.17 0.17 0.17 0.09 1.26 0.42 0.44T4 (45% RDA) 148.64 ± 19.01 1.83 0.40 0.20 0.20 0.21 0.09 1.24 0.44 0.47

1218 M.S. Butt et al. / Food Research International 40 (2007) 1212–1219

findings of Meija et al. (1977), who observed highly signif-icant difference in serum retinol levels of rats fed on vita-min A supplemented and vitamin A deficient diets. Hereported that rats fed on vitamin A supplemented dietshowed a 3–4 folds increase in serum retinol concentrationto those fed on deficient diet. Serum retinol concentrationwas directly proportionality with weight gain of liver, kid-ney and heart, while it was negatively correlated with thebrain weight. The serum retinol plays an important rolein maintaining LDL/HDL ratio that plays an importantand crucial role in the prevention of cardiovasculardiseases.

4. Conclusion

Vitamin A deficiency is prevalent in many developingcountries and children and pregnant women are at partic-ular risk. Different food-based strategies are consideredto be effective in the combat against vitamin A deficiency.The present work shows that different food items like cook-

ies fortified with vitamin A are very helpful in ambientbody growth. Keeping in view the physiological require-ments and responses of rats and humane body it is statedthat the controlled study on rats exhibit almost same effectson humane body. So, this strategy can be used to increasevitamin A level of serum required for healthier body. A for-tification level of 45% of RDA for 4–6 years of children isrecommended, which also resulted in baking and storagelosses up to permissible levels and same level of retinyl ace-tate as fortificant produced better results regarding bio-availability of fortified vitamin A and thus can be used asfortificant.

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