1-1 by liwenting


									 1                   Microencapsulated Iron
 2              for Drink Yogurt Fortification


 5                 S. J. Kim, J. Ahn and H. S. Kwak
 6                   Department of Food Science and Technology

 7                Sejong University, 98 Kunja-dong, Kwangjin-ku,

 8                              Seoul, 143-747, Korea



11                 Running head: Iron fortification in drink yogurt


13   Corresponding author : H.S. Kwak, Dept. of Food Science and Technology,

14   Sejong University, 98 Kunja-dong, Kwangjin-ku, Seoul, 143-747, Korea.

15   E-mail: kwakhs@sejong.ac.kr

16   Tel : (822) 3408-3226

17   Fax : (822) 497-8931

18   ABSTRACT : This study was designed to examine the effect of micro
19   encapsulated iron fortified drink yogurt and vit C as a bioavailable helper of

20   iron on chemical and sensory aspects during 20 d storage. Coating material

21   was PGMS, and ferric ammonium sulfate and vit C were selected as core m
 1   aterials. The highest efficiency of microencapsulation of iron and vit C were

 2   73% and 76%, respectively, with 5:1:50 ratio (w/w/v) as coating to core mat

 3   erial to distilled water. Iron fortification did not affect to the fermentation ti

 4   me required for the drink yogurt to reach pH 4.2. The addition of uncapsulat

 5   ed iron decreased the pH during storage. TBA absorbance was significantly

 6   lower in capsulated treatments than those in uncapsulated treatments during

 7   storage. In sensory aspect, the yogurt sample added with uncapsulated iron a

 8   nd vit C, regardless of capsulation, showed a significantly high score of astri

 9   ngency, compared with those of control and other groups. A significantly str

10   ong sourness was observed in treatment containing capsulated iron and unca

11   psulated vit C at every time intervals. The present study provides evidence t

12   hat microencapsulation of iron with PGMS is effective for iron fortification

13   in drink yogurt.

14   Key words: Microencapsulation, Iron, PGMS, Yogurt, Vitamin C

15   Abbreviation key: PGMS = polyglycerol monostearate.



18                             INTRODUCTION

20      Yogurt has gained widespread consumer acceptance in the U.S. (Otto, 19

21   88) and other developed countries, primarily by women, children and teenag

 1   ers, who consume yogurt as a luncheon or snack food. These populations ha

 2   ve high calcium requirements and are also frequently deficient in iron (Dall

 3   man et al., 1984). Even though yogurt is an excellent source of calcium and

 4   protein (United States Department of Agriculture, 1982), it contains very litt

 5   le iron (Blanc, 1981).

 6      Fortification of iron in yogurt would help meet this nutritional need. Usin

 7   g dairy foods as a vehicle for supplementing iron seems to be an advantage

 8   because people who consume diets with low iron density usually consume

 9   more dairy products (Hekmat and McMahon, 1997). Futhermore, iron-fortifi

10   ed dairy foods have a relatively high iron bioavailiability (Woestyne et al., 1

11   991). However, before any such fortification is undertaken in yogurt, the eff

12   ects of iron fortification on microbial physiology during manufacture and sh

13   elf-life of yogurt, oxidation of milk fat, and the effect of iron on sensory cha

14   racteristics must be ascertained.

15      Iron in food is absorbed by the intestinal mucosa and especially, nonhem

16   e iron, the major dietary pool, is greatly influenced by meal composition. It i

17   s well known that vit C is a powerful enhancer of nonheme iron absorption (

18   Lynch and Cook, 1980). Its influence may be pronounced in meals of iron a

19   vailability. Vit C facilitates iron absorption by forming a chelate with ferric i

20   ron at acid pH that remains soluble at the alkaline pH of the duodenum. Ho

21   wever, the addition of vit C influences on the quality of yogurt due to its hig

 1   h acid. Therefore, iron and vit C need microencapsulation.

 2      Microencapsulation, which shows potential as carriers of enzymes in the

 3   food industry, could be a good vehicle for the addition of iron to milk (Jacks

 4   on and Lee, 1991; Bersen’eva et al., 1990). Currently there is a considerable

 5   interest in developing encapsulated flavors and enzymes. Among several fac

 6   tors to be considered, choice of coating material is the most important and d

 7   epends on the chemical and physical properties of the core material, the proc

 8   ess used to form microcapsules, and the ultimate properties desired in micro

 9   capsules.

10      For microencapsulation – although several researchers have used coating

11   materials such as milk fat, agar, and gelatin, etc. responsible for enzyme, fla

12   vor and iron microencapsulation in foods (Braun and Olson, 1986; Magee a

13   nd Olson, 1981a,b), no study has measured the efficiency of iron microenca

14   psulation using fatty acid esters, and the stability of microcapsule itself and i

15   nside the body. Therefore, the objective of this study was to examine the eff

16   ect of microencapsulated iron and/or vit C added yogurt on chemical and se

17   nsory aspects during storage.





















17                 MATERIALS AND METHODS

19   Materials

20     For the microencapsulation of iron complex, polyglycerol monostearate (

21   PGMS) was used as a coating material. It was purchased from Il-Shin Emuls

1    ifier Co., LTD. (Seoul, Korea). As core materials, water-soluble iron comple

 2   x, ferric ammonium sulfate (FeNH4(SO4) 2 4H2O) and L-ascorbic acid were

3    purchased from Sigma Chemical Co. (St. Louis, MO, USA) and Shinyo Pur

4    e Chemical Co. LTD (Osaka, Japan) and were in food grade.


6    Preparation of microcapsule

7       Microcapsules of iron were made by polyglycerol monostearate (PGMS),

8    which was selected as a major coating material from our previous study (K

 9   wak et al., 2001). Also, ferric ammonium sulfate and L-ascorbic acid were s

10   elected (Kwak et al., 2002). Other experimental factors were as follows: the

11   ratio of coating material to core material was 5:1, and 50 mL distilled water

12   was additionally added because PGMS solution was highly viscous. The spr

13   ay solution was heated at 55C for 20 min, and stirred with 1,200 rpm for 1

14   min during spraying. An airless paint sprayer (W-300, Wagner Spray Tech.

15   Co., Markdorf, Germany) nebulized a coating material-iron emulsion at 45

16   C into a cyclinder containing a 0.05% polyethylene sorbitan monostearate (

17   Tween 60) solution at 5C. The diameter of the nozzle orifice was 0.33 mm.

18   The chilled fluid was centrifuged at 2,490 x g for 10 min to separate unwash

19   ed microcapsule suspension. Microcapsules were formed as lipid solidified i

20   n the chilled fluid. The microencapsulation of iron and ascorbic acid were d

21   one in triplicate.


 2   Yogurt preparation

 3       A commercial homogenized and pasteurized milk containing 3.4% fat a

 4   nd 13.4% total solids was fortified with 2.7% (w/v) skim milk powder to inc

 5   rease viscosity of yogurt and then homogenized (HC-5000 Homogenizer, M

 6   icrofluidics Corp., Newton, MA, USA) at 150 kg/cm2 (60C) and cooled to

 7   42C. A 0.2% commercial YC-380 starter culture (Chr. Hansen Pty. Ltd. Ba

 8   yswater, Autralia) in freeze-dried direct-to vat set form containing Lactobac

 9   illus delbrueckii ssp. bulgaricus and Streptococcus thermophilus was inocul

10   ated and fermented at 42C to be reached at pH 4.3. After fermentation, som

11   e yogurt samples were initially removed and incubated at 4C for 24 h. The

12   remaining samples were stored at 4C for 20 d to study the changes in chem

13   ical, microbial and sensory aspects during prolonged fermentation of yogurt

14   at 5 d intervals.


16   Treatments

17      Five different groups in this experiment were as followed: 1) no addition

18   as control (Trt 1), 2) 20 ppm uncapsulated iron added (Trt 2), 3) 20 ppm cap

19   sulated iron added (Trt 3), 4) 20 ppm capsulated iron and 100 ppm uncapsul

20   ated vit C added, and 5) 20 ppm capsulated iron and 100 ppm capsulated ad

21   ded vit C.


 2   Efficiency of microencapsulation

 3      For iron measurement, the dispersion fluid was assayed for untrapped iro

 4   n during microencapsulation. One milliliter of the dispersion fluid was taken

 5   and diluted ten times and total iron content was measured at 259.94 nm wav

 6   e- length by inductively coupled plasma spectrometer (ICP). Lactam 8440

 7   Model spectrometer (Plasmalab, Victoria, Austrailia) was used. A sample m

 8   easurement was run in triplicate.

 9     Total vit C was analyzed spectrophotometrically using DNP (2,4-dinitrop

10   henyl hydrazine) test described (Korea Food Code, 2002). Samples were pre

11   pared immediately before analyses and kept cold and protected against dayli

12   ght during analysis. A vit C stock solution was prepared daily by dissolving

13   10 mg of vit C in 100 mL of deionized water (100g/mL). It was diluted wit

14   h deionized water to obtain the final concentration of 10, 20, 30, 40 and 50

15   g/mL. Total vit C was determined using the calibration graph based on conc

16   entration (g/mL) vs absorbance, prepared daily running fresh standard solu

17   tions:


19   Chemical analyses

20   pH and titratable acidity (TA)

21      pH and titratable acidity (determined by titration to pH 8.3) of the yogurt

 1   samples were measured at a room temperature using pH meter (Sartorius, G

 2   ermany). The TA was determined after mixing the 9 mL yogurt sample with

 3   18 mL distilled water and titrating with 0.1N NaOH using a 0.5% phenolpht

 4   halein indicator to an end point of faint pink color.


 6   Thiobarbituric acid (TBA) test

 7      Oxidation products were analyzed spectrophotometrically using the thiob

 8   arbituric acid (TBA) test (Hegenauer et al., 1979). The TBA reagent was pre

 9   pared immediately before use by mixing equal volumes of freshly prepared

10   0.025M TBA (brought into by neutralized with NaOH) and 2M H3PO4/2M c

11   itric acid. Reactions were terminated by pipetting 5.0 mL of yogurt sample c

12   ontaining iron microcapsules into a glass centrifuge tube and mixed through

13   ly with 2.5 mL TBA reagent. The mixture was heated immediately in a boili

14   ng water bath for exactly 10 min, and then cooled on ice. Then 10 mL cyclo

15   hexanone and 1 mL of 4M ammonium sulfate were added and centrifuged at

16   2,490 x g for 5 min at room temperature. The orange-red cyclohexanone sup

17   ernatant was decanted and its absorbance at 532 nm was measured spectrop

18   hotometically in an 1-cm light path. All measurements were run in triplicate.


20   Viscosity

21     Viscosity measurement was made with Bostwick consistometer (CSC Sci

 1   entific Company, Seoul, Korea). Sample (100g) was placed and flow distanc

 2   e (cm) for 1 min was expressed as viscosity.


 4   Microbiological analyses

 5     Lactic acid bacteria were determined from the colony counts on specific l

 6   actic agar: MRS agar (pH 5.4) for Lactobacillus delbrueckii subsp. bulgaric

 7   us and M17 agar for Streptococcus thermophilus. A 1-g yogurt samples stor

 8   ed for 0, 5, 10, 15, and 20 d were diluted with 9 mL of sterile peptone and w

 9   ater diluent. Subsequent serial dilutions of each sample were plated in triplic

10   ate and plated were incubated at 41C for 48 h.


12   Sensory evaluation

13      For the storage test, 10 mL drink yogurt containing capsulated or uncaps

14   ulated iron and vit C was stored at 4C for 0, 5, 10, 15 and 20 d. An eleven-

15   person panel, semi-experienced in judging dairy products were recruited fro

16   m faculty and graduate students in the Department of Food Science and Tec

17   hnology at Sejong University and evaluated the yogurt samples throughout t

18   he study.

19      The intensity of taste aspects (bitterness, astringency, and sourness) were

20   scored on a nine-point scale (1 = none, 3 = slight, 5 = moderate, 7 = strong a

21   nd 9 = very strong) and overall preference were scored on a nine-point scale

 1   (1 = dislike extremely, 3 = dislike moderate, 5 = neither like or dislike, 7 = l

 2   ike moderate, and 9 = like extremely). A randomized, balanced, complete bl

3    ock design was used (Cochran and Cox, 1957) that resulted in two replicatio

4    ns for all samples.


 6   Statistical analysis

 7      Data from each experiment were analyzed by analysis of variance (ANO

8    VA) using a SAS program (1985) and differences among treatments were de

 9   termined by Student-Newman-Keuls comparison test at p < 0.05, unless oth

10   erwise stated.





15                    RESULTS AND DISCUSSION

17   Microencapsulation

18      In the present study, the yield of iron and vit C microencapsulation were

19   73% and 76%, respectively. In our laboratory, PGMS was appeared to be ha

20   rd to spray, therefore, we found the optimum ratio of PGMS to deionized wa

21   ter to reduce the viscosity of PGMS solution. In our previous study, the ratio

 1   of PGMS to iron to distilled water was 5:1:50 (w/w/v), efficiency of the mic

2    roencapsulation was 75% as the highest value (Kwak et al., in press).

 3      The size of microencapsulated iron or vit C with PGMS was irregular fro

 4   m nano to micrometer, and the average size was in the range of 2 to 5 m (p

 5   ictures not shown). Microscopic examination of microcapsules revealed sph

 6   erical particles. Microcapsules containing iron or vit C had smooth surfaces

 7   and evenly distributed pockets. The shape of the microcapsules was likely af

 8   fected by encapsulated conditions.

 9      Magee and Olson (1981a), and Braun and Olson (1986) found that lipid a

10   nd cooling fluid temperatures affected the shape of microcapsule by controll

11   ing the cooling rate of lipid coatings. They observed that microcapsules wer

12   e cylindrical when the lipid coating was rapidly cooled and spherical when t

13   he lipid was slowly cooled.

14   The change of pH and titratable acidity (TA)

15      Iron fortifications did not affect fermentation time required for the yogurt

16   mixes to reach pH 4.10  4.20 (Fig. 1). After 5 h fermentation, further trend

17   of pH changes during storage were also similar: the pH values of control an

18   d fortified samples reached 4.00  4.07 after 1 d and 3.95  4.07 after 20 d.

19      The treatment was divided into 5 different groups as followed: 1) no addi

20   tion as control (Trt 1), 2) 20 ppm uncapsulated iron (Trt 2), 3) 20ppm capsul

21   ated iron (Trt 3), 4) 20 ppm capsulated iron and 100ppm uncapsulated vit C

 1   (Trt 4), and 5) 20 ppm capsulated iron and 100 ppm capsulated vit C (Trt 5).

 2      The change of pH was shown as in Fig 1. pH was the highest in control g

 3   roup among 5 different groups. pH was 4.20 at 0 d and decreased to 4.07 at

 4   5 d and plateaued thereafter upto 20 d storage in control.

 5      When compared with uncapsulated iron added group (Trt 2) and capsulat

 6   ed iron groups (Trt 3), pH was significantly lower in Trt 2 at every time inte

 7   rvals. Uncapsulated iron resulted in high level of acidity during fermentation

 8   (0 d storage) and further storage, which indicating that iron capsulation sho

 9   wed a profound effect on pH at every time intervals.

10      When compared with capsulated iron with uncapsulated vit C (Trt 4) and

11   capaulated iron with capsulated vit C (Trt 5), there was no difference at ever

12   y time intervals from the beginning to the end, especially, until 10 d storage.

13   In results, uncapsulated vit C showed a certain protective effect on pH decre

14   ase. Above result indicated that uncapsulated iron addition decreased the pH

15   of yogurt during storage. Addition of iron to skim milk led to a decrease in p

16   H. This decrease is related to the acidities of iron solution and to exchange b

17   etween iron ions and micellar bound H+ (Gaucheron, 2000).

18      Titratable acidity (TA) increased with the storage time (Fig. 2). The TA o

19   f control was the lowest and that of Trt 2 containing uncapsulated iron was t

20   he highest.


 1   TBA test during storage

 2      The effect of iron fortification in yogurt on chemical oxidation (as measu

 3   red by the TBA test) during 20 d storage is shown in Fig 3. When compared

 4   with uncapsulated (Trt 2) and capsulated (Trt 3) iron added group, TBA val

 5   ue was slightly higher in uncapsulated iron added groups at 0, 5, and 20 d st

 6   orage. The difference of TBA value between 2 groups increased dramaticall

 7   y at 10 and 15 d storage.

 8      When compared with capsulated iron with uncapsulated vit C (Trt 4) and

 9   capsulated iron with capsulated vit C (Trt 5), the TBA value of Trt 5 was no

10   t significantly lower than that of Trt 4 in the early stage of storage (0, 5 and

11   10 d). However, the TBA value of Trt 5 containing capsulated vit C was sig

12   nificantly lower at 15 d storage.

13      In this experiment, TBA absorbance was significantly lower in capsulate

14   d groups than those in uncapsulated group, regardless of iron and vit C, duri

15   ng storage. These data indicated that oxidation process may be faster in yog

16   urt samples containing uncapsulated iron than in those containing capsulated

17   iron.

18      Another study (Kwak et al., in press) showed the effect of iron fortificati

19   on in milk on chemical oxidation during 15 d storage. They reported that TB

20   A absorbance was significantly lower in capsulated group than that in uncap

21   sulated group at 15 d.

 1      Jackson and Lee (1991) indicated that samples containing uncapsulated ir

 2   on (ferrous sulfate and ferric chloride) showed 2-3 times high in fatty acid p

 3   roduction, compared with those containing capsulated iron complex when m

 4   ilk fat was used as a coating material. The reason why iron fortification caus

 5   ed several modifications in milk and yogurt could be explained that added ir

 6   on may interact with casein, resulting in iron-casein complexes and the pres

 7   ence of O2 acts as a prooxidant, therefore, lipid oxidation in yogurt can be a

 8   ccelerated.



11   Change of Microbial counts during storage

12      The change of Lactobacillus delbrueckii ssp. bulgaricus in microencapsu

13   lated iron fortified drink yogurt at 4C for 20 d storage is shown in Table 1.

14   At 0 d, the mean counts of L. delbrueckii ssp. bulgaricus for control and oth

15   er groups were not significantly different. Also, the mean counts in all group

16   s showed a decrease trend during 20 d storage. L. delbrueckii ssp. bulgaricu

17   s counts were about 107 cfu/ml.

18      The change of viable cells of Streptococcus salivaries ssp. thermophilus i

19   n microencapsulated iron fortified drink yogurt at 4C for 20 d storage is sh

20   own in Table 2. At 0 d, the mean counts of S. salivaries ssp. thermophilus fo

21   r control and other groups were not significantly different. Also, the mean c

 1   ounts in all groups did not show any change during 20 d storage. Meanwhile

 2   , S. salivaries ssp. thermophilus counts were about 108 cfu/ml.

 3      Hekmat and McMahon (1997) reported that counts of L. delbrueckii ssp.

 4   bulgaricus and S. salivaries ssp. thermophilus after 1 d of storage in iron-for

 5   tified skim yogurt were 7.0 x 108 cfu/ml, which were not significantly differ

 6   ent from counts in unfortified yogurts. Counts decreased to 2.5 x 108 cfu/ml

 7   and 1.9 x 108 cfu/ml for L. delbrueckii ssp. bulgaricus and S. salivaries ssp.

 8   thermophilus, respectively, after 30 d storage. Fortifying yogurt with iron di

 9   d not affect the growth of Pseudomonas flurescens or Escherichia coli.


11   Viscosity

12      When measured viscosity by consistometer, flowing longer distance indic

13   ated lower viscosity, as shown in Fig 4. Four treatments except Trt 2 showe

14   d a slightly decreasing trend of viscosity during 20 d storage. However, Trt

15   2 containing uncapsulated iron only showed no difference to other treatment

16   s until 10 d storage, however, a dramatic decreasing was found from 15 d up

17   to 20 d. The dramatic decrease of viscosity in uncapsulated iron fortified tre

18   atment (Trt 2) may be explained that an interaction of casein and whey prote

19   in with iron complex was developed in yogurt during storage (Sadler et al.,

20   1973).


 1   Sensory analysis

 2         The sensory characteristics of drink yogurts in five treatments were show

3       n in Table 3. For bitterness, it was not significantly different among treatme

4       nts during 20 d storage. However, in Trt 2, uncapsulated iron fortified yogur

5       t revealed a slightly higher score than others. Also, no difference was found

6       in all treatments during storage.

 7         For astringency, uncapsulated or capsulated iron containing drink yogurt

 8      (Trts 2 and 3) showed a higher score, compared with those of control and ot

 9      her treatments at 0 d. At 5 d storage, groups containing uncapsulated iron (T

10      rt 2) and capsulated iron with vit C regardless of capsulation (Trts 4 and 5) s

11      howed a significantly higher astringency, compared with other groups. No d

12      ifference was found among groups thereafter.

13         For sourness, a significantly strong sourness was observed in Trt 4, whic

14      h was added with capsulated iron and uncapsulated vit C at every periods ex

15      cept 10 d storage. Sourness increased with storage time in all groups. If we c

16      ompared Trt 2 with Trt 3, we may find the effect of iron microencapsulation

17      on sourness score, and no significant difference was found.

18         The only difference we found was between Trt 4 and Trt 5, and it was ca

19      used by vit C encapaulation or not. Therefore, a dramatically high sourness

20      score in Trt 4 was resulted from not capsulated iron, but uncapsulated vit C i

21      n the drink yogurt.

 1      For overall preference, control (Trt 1) and capsulated iron (Trt 3) and/or

 2   vit C (Trt 5) containing treatments showed a high consumer preference in all

 3   storage periods. The scores of Trts 2 and 4 (uncapsulated iron added, and ca

 4   psulated iron and uncapsulated vit C added groups) were dramatically lower

 5   compared with those of other three treatments. This result indicated that mic

 6   roencapsulation process was very effective to mask off-taste and flavor of ir

 7   on and vit C in this experiment.

 8      The sensory quality of iron-fortified dairy foods has been shown to be eff

 9   ective by the capsulation of both iron and vit C. Two major off-flavors have

10   been associated with dairy products: oxidized flavor resulted from catalysis

11   of lipid oxidation by iron, and sourness contributed by vit C.

12      Iron is known to catalyze lipid oxidation resulting in rancidity with devel

13   opment of an unpleasant odor and flavor. The TBA test has been extensively

14   applied to food in which the absorbance of TBA reaction products correlates

15   positively with sensory evaluation. Fortification with iron complex causes o

16   xidized off-flavor and high TBA number. To avoid oxidized and metallic fla

17   vors and color changes, microencapsulation techniques were needed. (Gauc

18   heron, 2000).











 8                               CONCLUSION

10      The present study demonstrated that the ratio of 5:1:50 (w/w/v) as coatin

11   g (PGMS) to core material (iron complex) to distilled water showed a high e

12   fficiency of microencapsulation of iron and vit C such as 73% and 76%, res

13   pectively. Our results indicated that lipid oxidation process measured by TB

14   A test was significantly slower in capsulated iron than in uncapsulated iron f

15   ortified yogurt. In sensory, we need to point out that no significantly adverse

16   effects was found in microcapsulated iron and vit C fortified drink yogurt du

17   ring 20 d in this experiment. Therefore, we may suggest that this study provi

18   de an important evidence that microcapsules of iron and vit C were an effect

19   ive means of fortification, and can be applied to drink yogurt without any ch

20   anges in sensory aspects.









 8                     ACKNOWLEDGEMENT

10     This research was supported by the Small & Medium Business Administr

11   ation (SMBA) in Seoul, Korea.


















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 8   Figure Legends

 9   Fig 1. Changes of pH in microencapsulated iron fortified drink yogurt stored at 4C

10         for 20 d. Trt 1, control (no addition); Trt 2, 20 ppm uncapsulated

11         iron; Trt 3, 20 ppm capsulated iron; Trt 4, 20 ppm capsulated iron and 10

12         ppm uncapsulated vit C; Trt 5, 20 ppm capsulated iron and 100 ppm

13         capsulated vit C.

14   Fig 2. Changes of titratable acidity (TA) in microencapsulated iron fortified drink

15         yogurt stored at 4C for 20 d. Trt 1, control (no addition); Trt 2, 20 ppm

16         uncapsulated iron; Trt 3, 20 ppm capsulated iron; Trt 4, 20 ppm capsulated

17         iron and 10 ppm uncapsulated vit C; Trt 5, 20 ppm capsulated iron and 100

18         ppm capsulated vit C.

19   Fig 3. Changes of TBA absorbance in microencapsulated iron fortified drink yogurt

20         stored at 4C for 20 d. Trt 1, control (no addition); Trt 2, 20 ppm

21         uncapsulated iron; Trt 3, 20 ppm capsulated iron; Trt 4, 20 ppm capsulated

1         iron and 10 ppm uncapsulated vit C; Trt 5, 20 ppm capsulated iron and 100

2         ppm capsulated vit C.

3   Fig 4. Changes of viscosity in microencapsulated iron fortified drink yogurt

4         stored at 4C for 20 d. Trt 1, control (no addition); Trt 2, 20 ppm

5         uncapsulated iron; Trt 3, 20 ppm capsulated iron; Trt 4, 20 ppm capsulated

6         iron and 10 ppm uncapsulated vit C; Trt 5, 20 ppm capsulated iron and 100

7         ppm capsulated vit C.


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