- Email: [email protected]
Effect of folic acid fortification on the characteristics of lemon yogurt
ARTICLE IN PRESS
LWT 41 (2008) 1335–1343 www.elsevier.com/locate/lwt
Effect of folic acid fortification on the characteristics of lemon yogurt C.A. Boeneke, K.J. Aryana School of Animal Sciences, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, 111 Dairy Science Bldg., Baton Rouge, LA 70803, USA Received 16 August 2006; received in revised form 24 August 2007; accepted 27 August 2007
Abstract Development of dairy products with new flavors and health benefits helps the dairy industry increase sales of products as well as provide consumers with products they enjoy. Folic acid is used in the prevention of neural tube defects, heart defects, facial clefts, urinary tract abnormalities, and limb deficiencies. The objective of this study was to determine the effect of different concentrations and stage of addition of folic acid on the physico-chemical and sensory characteristics of lemon yogurt over a storage period. Lemon yogurts were manufactured with 0%, 25%, 50%, 75%, and 100% of the RDA of 400 mg folic acid per 224 ml cup. Folic acid was added before and after pasteurization of yogurt mix. Moisture, ash, fat, and protein concentrations were measured at week 1 only. Folic acid concentration was measured at weeks 1 and 5. Viscosity, pH, TA, syneresis, color, and sensory analysis were measured at weeks 1, 3, and 5. Mean folic acid content values were higher when folic acid was added post-pasteurization. Average mean viscosity values were lower when folic acid added post-pasteurization. Greater syneresis was seen in samples where folic acid was added post-pasteurization. Less viscous yogurts had more free whey resulting in higher syneresis values. Level of folic acid impacted flavor scores. As level of folic acid increased, flavor scores decreased. Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. Keywords: Folic acid; Lemon yogurt; Fortification
1. Introduction Development of dairy products with new flavors and products with health benefits has the potential to increase sales. Dairy products in the market place are targeted to different consumer groups. For example, fat free dairy products are targeted to consumers with cardiovascular problems. Lactose free products target lactose intolerant people. Consumers want dairy products that taste good and have increased health benefits. Yogurt is a dairy product which is consumed as a snack and dessert. There has been a steady increase in total sales of yogurt from 779 Gg in 1999 to 1083 Gg in 2003 (Milk Facts, 2004). Studies have shown that folic acid when taken during initial stages of pregnancy can prevent neural tube defects Corresponding author. Tel.: +1 225 578 4411; fax: +1 225 578 4008.
E-mail addresses: [email protected] (C.A. Boeneke), [email protected] (K.J. Aryana).
such as spina bifida and ancencephaly, heart defects, facial clefts, urinary tract abnormalities, and limb deficiencies (Hall & Solehdin, 1998). Sources of folic acid include liver, avocado, black beans, spring greens, asparagus, and soybean nuts. Dairy products are not a good source of folic acid. Cow’s milk contains 5–7 mg of folic acid per 100 g of milk (Renner, 1983; Scott, 1989). The major form of folate in milk is 5-methyl-tetrahydrofolate (Forseen, Jagerstad, Wigertz, & Witthoft, 2000; Gregory, 1996). Folic acid itself is not reduced and is the most stable form. Folic acid has also been shown to reduce the risk of colorectal and breast cancers (Langenohl, Fohr, & Pietrzik, 2001). Folic acid can also act as a cofactor for the conversion of homocysteine to methionine (Maxwell, 2000). Epidemiological studies have shown increased concentrations of homocysteine to be associated with vascular disease (Maxwell, 2000). Folic acid fortification of breads and cereals is commonly practiced. Direct addition of vitamins A and D
0023-6438/$34.00 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. doi:10.1016/j.lwt.2007.08.010
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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during fluid milk processing is a common practice. Previous work conducted at the Louisiana State University Agricultural Center has shown folic acid can be added successfully in plain yogurt up to the recommended daily allowance of 400 mg (Boeneke & Aryana, 2007). The objective of this study was to determine the effect of
different concentrations and stage of addition of folic acid on the physico-chemical and sensory characteristics of lemon flavored yogurt over a 5-week storage period.
2. Materials and methods 2.1. Experimental design
Table 1 Formulae for-fat free lemon yogurt folic acid amounts up to 400 mg per 224 ml yogurt Composition (mg)
0
100
200
300
400
Skim milk (l) NFDM (g) Starch (g) Aspartame (g) Folic acid (mg) Starter culture (g) Lemon flavor (ml)
3.78 114.00 23.00 1.14 0.00 1.30 9.0
3.78 114.00 23.00 1.14 1.67 1.30 9.0
3.78 114.00 23.00 1.14 3.35 1.30 9.0
3.78 114.00 23.00 1.14 5.03 1.30 9.0
3.78 114.00 23.00 1.14 6.70 1.30 9.0
Fat free lemon flavored yogurts were manufactured with 0, 100, 200, 300, and 400 mg folic acid per 224 ml cup. Folic acid was added before or after pasteurization of the yogurt mix. Moisture, ash, fat, and protein concentrations were determined at week 1. Folic acid concentration was measured at weeks 1 and 5. Viscosity, pH, syneresis, color, and sensory properties were measured at weeks 1, 3, and 5. The data was analyzed by a randomized complete block with repeated measures in time analysis. The replications were the blocks. The experiment was replicated three times with three determinations per replication.
5
4.5
4
3.5
Folic acid
3
2.5
2
1.5
1
0.5
0 100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
400ug After Pasteruization
Fig. 1. Mean (+S.E.) peak areas (mg/ml) of folic acid in fat-free lemon flavored yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: week 1; &, week 5.
,
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2.2. Yogurt manufacture
2.3. Analytical procedures
Fat free lemon yogurt was manufactured at the Louisiana State University Department of Dairy Science. The dry ingredients (Table 1) were incorporated in an 18.9 l stainless steel container. Each mix was pre-heated to 60 1C and homogenized at 12.4 and 3.4 MPa on the first and second stages using a Gaulin 300 DJF 4 2PS homogenizer (APV Gaulin, Wilmington, MA), and batch pasteurized at 85 1C for 30 min. Each mix was cooled to 40 1C before addition of a culture containing Lactobacilus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Chr. Hansens, Milwaukee, WI) at a rate of 1.3 g per 3.78 l of the mix. Inoculated mixes were poured in 224 ml plastic cups and incubated at 40 1C until pH reached 4.5. Samples were cooled immediately and stored at 4 1C. Folic acid powder (Lakeshore Tech., Norton Shores, MI) was added with dry ingredients for the pre-pasteurization runs and prior to culture addition for post-pasteurization runs. Lemon flavorings were obtained from Target Flavors, Brookfield, CT, and added at a manufacture’s suggested usage rate of 9.0 ml/gal. Flavoring was added prior to incubation.
Protein concentration was determined 1 week after yogurt was manufactured. Samples were prepared by drying 15 g of sample for 48 h at 100 1C in a convection oven (Fisher Scientific, Houston, TX). Dry sample was ground using a mortar and pestle, 0.2 g was loaded into tin cups, folded and loaded into a Leco FP428 (Leco Corp., St. Joseph, MI) nitrogen analyzer. Sample was incinerated and results expressed as percent nitrogen. Results were multiplied by a protein correction factor of 6.38. Fat, moisture, and ash contents were also determined 1 week after manufacture according to Richardson (1985). Folic acid concentration was determined by using high performance liquid chromatography (HPLC) as modified from Albala´-Hurtado, Vecina-Nogue´s, Izquierdo-Pulido, and Marine´-Fone (1997). The HPLC system comprised of a Waters (Waters Corp., Milford, MA) 501 pump, Waters 717 Plus auto-sampler, and Waters 486 tunable UV detector set at 282 nm. Peak areas were calculated using the Waters Millenniums software. The separation was conducted isocratically using a Waters Spherisorb 5 mm ODS2 4.6 250 mm column with guard cartridge. Run
20000 19000 18000 17000 16000 15000 14000 13000
Viscosity [cp]
12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
Fig. 2. Mean viscosity (cp) of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: 3; and , week 5.
400ug After Pasteruization
, week 1;
, week
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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time for samples was 20 min using a flow rate of 1 ml per minute and an injection volume of 10 ml per sample. A standard curve was prepared by dissolving known amounts (1.67, 3.35, 5.03, and 6.70 mg) of folic acid in 3.78 l of HPLC grade double distilled water, i.e. the same amount of folic acid that was used in 3.78 l of yogurt mix and analyzed using HPLC-UV. Peak area results from HPLC analysis were correlated to the standard curve to determine folic acid concentrations. Viscosity, pH, syneresis, and color were determined and sensory analysis conducted according to Boeneke and Aryana (2007).
2.4. Statistical analysis Data were analyzed in the following manner: concentration of folic acid, viscosity, pH, syneresis, color, and sensory analysis were analyzed by the Statistical Analysis System using the General Linear Model procedure with a repeated measure in time. Data from moisture, ash, and protein concentration were analyzed using the General Linear Model with Tukey’s Studentized Range Test. Significant differences were determined at Po0.05.
3. Results and discussion Protein contents on dry matter basis ranged from 34.09 to 38.38 g/100 g. Fat content for all samples were o0.5 g/ 100 g. Moisture content ranged from 88.46 to 89.77 g/100 g. Ash content ranged from 0.5667 to 0.9367 g/100 g. No significant (Po0.05) differences were found for protein, fat, moisture, and ash contents. Folic acid as expected did not alter the composition of the yogurts. Folic acid peak area concentrations for lemon flavored yogurts are reported in Fig. 1. Mean folate values in fortified plain yogurt were1.39 and 1.35 mg/ml for weeks 1 and 5, respectively. Mean folate values for lemon yogurts were 2.24 and 1.73 mg/ml for weeks 1 and 5, respectively, although no significant (Po0.05) interaction for time was found. The higher values for lemon yogurts is due to the folic acid content in the lemon extract added for flavoring. Lemon extract contained approximately 0.86 mg/ml of folic acid. Although no significant effect of (Po0.05) pasteurization was observed in the lemon flavored yogurts, mean values were higher where folic was added after pasteurization. Ristow, Gregory, and Damron (1982) found folic acid to be stable at processing temperatures of 120 1C for 20 min. However, Wigertz, Hansen, HoierMadisen, Holm, and Jagerstad, (1996) examined folate and
200 190 180 170 160 150 140 130
Syneresis [ml]
120 110 100 90 80 70 60 50 40 30 20 10 0 Control
100ug Before 200ug Before 300ug Before 400ug Before 100ug After 200ug After 300ug After 400ug After Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 3. Mean syneresis of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
5-methyl-tetrahydrofolate found naturally in milk and yogurt and reported significantly reduced levels of both 5-methyl-tetrahydrofolate and total folate in pasteurized milk, UHT treated milk, and yogurt made from a mix pasteurized at 90 1C for 10 min. Boeneke and Aryana (2007) found mean folic acid peak area values to be higher in plain yogurts when folic acid was added prior to pasteurization of the yogurt mix. This discrepancy can possibly be explained by interactions of added folic acid and the folic acid content of the lemon flavor. Folic acid has a fully oxidized ring system and has greater stability than other forms. Perhaps this stability protects the folic acid from heat treatments and helps it to disperse in the product. Lemon extract could be interacting with the increased folic acid content and causing an opposite effect of lowering the mean values. Culture addition probably did not affect folic acid concentrations. Rao and Shahani, (1987) found skim milk inoculated with Lactobacillus delbrueckii subsp. bulgaricus lowered folate levels after 36 h incubation. Milk fermented by S. thermophilus and Lactobacullus acidophilus increased folate levels significantly. The S. thermophilus and L. delbrueckii subsp. bulgaricus culture used in this experiment had little effect on total folate content in the finished product. Viscosity values of lemon yogurts are reported in Fig. 2. Although overall level of folic acid addition was found to have no significant effect on viscosity (P40.05) in lemon
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yogurts, average mean viscosity values were lower for folic acid added after pasteurization. Mean peak area values were higher where folic acid was added after pasteurization meaning greater folic acid content within these samples. This may be due to altered localized casein micelle aggregation, as yogurt is a casein micelle network (Kalab, Allan-Wojtas, & Phillips-Todd, 1983) and viscosity is an indication of the strength of this network. Perhaps increased folic acid concentrations in the lemon yogurt helps decrease casein micelle aggregation resulting in decreased viscosity in samples with increased concentration of folic acid. The mean pH values for the lemon yogurt ranged from 4.06 to 4.22. Level of pasteurization and folic acid addition showed no significant interaction for lemon yogurts. Syneresis values are reported in Fig. 3. The mean values for syneresis ranged from 92.67 to 137.33 ml. There were significant (Po0.05) differences in syneresis with folic acid added before verses after pasteurization. Mean values for syneresis were higher when folic acid was added after pasteurization. Lemon yogurts were also less viscous when folic acid was added after pasteurization. Less viscous yogurts had more free whey resulting in higher syneresis values. This again would be due to the casein micelle network. There were no significant (Po0.05) differences in syneresis with different concentrations of folic acid. There were significant (Po0.05) differences in syneresis over
50
45
40
35
L*
30
25
20
15
10
5
0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before 400ug Before Pasteurization Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After 400ug After Pasteruization Pasteruization
Fig. 4. Mean L* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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weeks 1, 3, and 5. Values decreased 25 g/100 g at week 3 and increased 2 g/100 g at week 5. No explanation for this decrease then increase could be found. L* values are reported in Fig. 4. Folic acid addition before or after pasteurization did not significantly (Po0.05) impact the L* (lightness) values of yogurt. There were no overall significant (Po0.05) differences in lightness of yogurts attributable to concentration of folic acid. A* values are reported in Fig. 5. Pasteurization did not have any significant (Po0.05) effect on a* (redness) values. Significant differences were found in the level of folic acid added and overall time for lemon yogurts. Values decreased by 10 g/100 g at week 3 and increased 83 g/ 100 g at week 5 indicating that the extent of redness was much less at week 3 compared to week 5. B* values are reported in Fig. 6. Level of folic acid addition and time had a significant (Po0.05) effect on b*
(yellowness) values. Mean values decreased 28.6 g/100 g at week 3 and increased 42 g/100 g at week 5. Pasteurization did not have a significant (Po0.05) effect. Generally, as folic acid content increased the more yellow the yogurts became. Syneresis values decreased at week 3 then increased at week 5. The riboflavin in the whey probably influenced a* and b* values. Flavor scores are reported in Fig. 7. No significant (Po0.05) differences in flavor scores were found with folic acid addition before vs. after pasteurization; however, the level of folic acid addition and time did have a significant (Po0.05) effect on flavor scores. As levels of folic acid increased, mean flavor scores decreased. Mean flavor score was 6.72 for lemon flavored yogurts compared with 6.11 for plain yogurts. Flavor scores for lemon yogurts decreased with time. Mean flavor scores were higher for lemon yogurts than for plain yogurt.
5
4
a*
3
2
1
0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After Pasteurization Pasteurization Pasteurization
200ugAfter Pasteruization
300ug After Pasteruization
Fig. 5. Mean a* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
400ug After Pasteruization
, week 1;
, week 3;
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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30
25
b*
20
15
10
5
0 Control
100ug Before 200ug Before 300ug Before 400ug Before Pasteurization Pasteurization Pasteurization Pasteurization
100ug After 200ug After 300ug After 400ug After Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 6. Mean b* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
10
9
8
7
Flavor Score
6
5
4
3
2
1
0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
400ug After Pasteruization
Fig. 7. Mean flavor scores of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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9
8
Body & Texture Score
7
6
5
4
3
2
1
0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After 200ug After 300ug After Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization
400ug After Pasteruization
Fig. 8. Mean body and texture scores of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: , week 3; and , week 5.
Body and texture scores are reported in Fig. 8. Level of folic acid addition did not significantly (Po0.05) impact body and texture scores. Pasteurization and overall time were found significant (Po0.05) for lemon yogurts. Mean values were higher where folic acid was added prior to pasteurization. Mean viscosity values for lemon yogurts were also higher when folic acid was added prior to pasteurization. Consumers preferred the more viscous samples. Mean values appeared to decrease over the storage period. Body and texture scores are from 1 to 5 with 5 being no criticism. A mean value of 5 indicates no defect in body and texture of yogurts. Most values were approximately 4 indicating little defect in body and texture of yogurts. Appearance and color scores are reported in Fig. 9. Level of folic acid addition and overall time was found non-significant (P40.05) for appearance and color. Appearance and color values were significantly (Po0.05) higher when folic acid was added before pasteurization. Yogurts where folic acid was added to the mix then
, week 1;
pasteurized had less free whey, probably impacting appearance and color scores. Mean value of lemon yogurt appearance and color was 3.92 compared with 3.29 for plain yogurt.
4. Conclusions Folic acid addition had no effect on protein, fat, moisture, or ash content of lemon yogurts. Mean values for folic acid were higher for lemon yogurts when folic acid was added prior to pasteurization. Level of folic acid impacted flavor scores. As level of folic acid increased, flavor scores decreased. Mean viscosity values for lemon yogurts were higher when folic acid was added prior to pasteurization. Consumers preferred the more viscous samples. Body and texture values of lemon yogurts decreased over the 5-week storage period. Yogurts in which folic acid was added to the mix then pasteurized had less free whey. Lemon flavored, folic acid fortified yogurts
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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10
9
8
Appearance & Color Score
7
6
5
4
3
2
1
0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After 200ug After 300ug After 400ug After Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 9. Mean appearance and color scores of folic acid fortified fat-free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: week 1; , week 3; and , week 5.
had high flavor and body and texture scores which is of use to the dairy industry. References Albala´-Hurtado, S., Vecina-Nogue´s, M. T., Izquierdo-Pulido, M., & Marine´-Fone, A. (1997). Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography. Journal of Chromatography A, 778, 247–253. Boeneke, C. A., & Aryana, K. J. (2007). Effect of folic acid fortification on the characteristics of yogurt. Milchwissenschaft. Forseen, K. M., Jagerstad, M. I., Wigertz, K., & Witthoft, C. M. (2000). Folates and dairy products: A critical update. Journal of the American College of Nutrition, 19(90002), 100S–110S. Gregory, J. F. (1996). In F. Owen (Ed.), Food chemistry (3rd ed., pp. 590–599). New York: Marcel Dekker Inc. Hall, J., & Solehdin, F. (1998). Folic acid for the prevention of congenital anomalies. European Journal of Pediatrics, 157, 445–450. Kalab, M., Allan-Wojtas, P., & Phillips-Todd, B. (1983). Development of microstructure in set-style nonfat yoghurt — A review. Food Microstructure, 2, 51–66. Langenohl, R., Fohr, I., & Pietrzik, K. (2001). Beneficial role for folate in the prevention of colorectal and breast cancer. European Journal of Nutrition, 40, 98–105.
,
Maxwell, S. R. (2000). Coronary artery disease-free radical damage, antioxidant protection and the role of homocysteine. Journal of Basic Research Cardiology, 95(Suppl. 1), I/65–I/71. Milk Facts. (2004). International Dairy Foods Association. Washington, DC, pp. 33–35. Rao, D. R., & Shahani, K. M. (1987). Vitamin content of cultured milk products. Cultured Dairy Products Journal, 6–10. Renner, E. (1983). Milk and dairy products in human nutrition. VV Gmbh Volkswirtschaftlicher Verlag, Federal Republic of Germany. Richardson, G. H. (1985). Standard methods for the examination of dairy products (15th ed). Washington, DC: American Public Health Association. Ristow, K. A., Gregory, J. F., & Damron, B. L. (1982). Thermal processing effects on folacin bioavailability in liquid model systems, liver and cabbage. Journal of Agriculture and Food Chemistry, 30, 801. Scott, K. J. (1989). Micronutrients in milk and milk based products. London: Elsevier Applied Science pp. 71–123. Wigertz, K., Hansen, I., Hoier-Madisen, M., Holm, J., & Jagerstad, M. (1996). Effect of milk processing on the concentration of folate binding protein (FBP), the folate-binding capacity and the retention of 5methyltetrahydrofolate. International Journal of Food Sciences and Nutrition, 47, 315–322.
LWT 41 (2008) 1335–1343 www.elsevier.com/locate/lwt
Effect of folic acid fortification on the characteristics of lemon yogurt C.A. Boeneke, K.J. Aryana School of Animal Sciences, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, 111 Dairy Science Bldg., Baton Rouge, LA 70803, USA Received 16 August 2006; received in revised form 24 August 2007; accepted 27 August 2007
Abstract Development of dairy products with new flavors and health benefits helps the dairy industry increase sales of products as well as provide consumers with products they enjoy. Folic acid is used in the prevention of neural tube defects, heart defects, facial clefts, urinary tract abnormalities, and limb deficiencies. The objective of this study was to determine the effect of different concentrations and stage of addition of folic acid on the physico-chemical and sensory characteristics of lemon yogurt over a storage period. Lemon yogurts were manufactured with 0%, 25%, 50%, 75%, and 100% of the RDA of 400 mg folic acid per 224 ml cup. Folic acid was added before and after pasteurization of yogurt mix. Moisture, ash, fat, and protein concentrations were measured at week 1 only. Folic acid concentration was measured at weeks 1 and 5. Viscosity, pH, TA, syneresis, color, and sensory analysis were measured at weeks 1, 3, and 5. Mean folic acid content values were higher when folic acid was added post-pasteurization. Average mean viscosity values were lower when folic acid added post-pasteurization. Greater syneresis was seen in samples where folic acid was added post-pasteurization. Less viscous yogurts had more free whey resulting in higher syneresis values. Level of folic acid impacted flavor scores. As level of folic acid increased, flavor scores decreased. Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. Keywords: Folic acid; Lemon yogurt; Fortification
1. Introduction Development of dairy products with new flavors and products with health benefits has the potential to increase sales. Dairy products in the market place are targeted to different consumer groups. For example, fat free dairy products are targeted to consumers with cardiovascular problems. Lactose free products target lactose intolerant people. Consumers want dairy products that taste good and have increased health benefits. Yogurt is a dairy product which is consumed as a snack and dessert. There has been a steady increase in total sales of yogurt from 779 Gg in 1999 to 1083 Gg in 2003 (Milk Facts, 2004). Studies have shown that folic acid when taken during initial stages of pregnancy can prevent neural tube defects Corresponding author. Tel.: +1 225 578 4411; fax: +1 225 578 4008.
E-mail addresses: [email protected] (C.A. Boeneke), [email protected] (K.J. Aryana).
such as spina bifida and ancencephaly, heart defects, facial clefts, urinary tract abnormalities, and limb deficiencies (Hall & Solehdin, 1998). Sources of folic acid include liver, avocado, black beans, spring greens, asparagus, and soybean nuts. Dairy products are not a good source of folic acid. Cow’s milk contains 5–7 mg of folic acid per 100 g of milk (Renner, 1983; Scott, 1989). The major form of folate in milk is 5-methyl-tetrahydrofolate (Forseen, Jagerstad, Wigertz, & Witthoft, 2000; Gregory, 1996). Folic acid itself is not reduced and is the most stable form. Folic acid has also been shown to reduce the risk of colorectal and breast cancers (Langenohl, Fohr, & Pietrzik, 2001). Folic acid can also act as a cofactor for the conversion of homocysteine to methionine (Maxwell, 2000). Epidemiological studies have shown increased concentrations of homocysteine to be associated with vascular disease (Maxwell, 2000). Folic acid fortification of breads and cereals is commonly practiced. Direct addition of vitamins A and D
0023-6438/$34.00 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. doi:10.1016/j.lwt.2007.08.010
ARTICLE IN PRESS C.A. Boeneke, K.J. Aryana / LWT - Food Science and Technology 41 (2008) 1335–1343
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during fluid milk processing is a common practice. Previous work conducted at the Louisiana State University Agricultural Center has shown folic acid can be added successfully in plain yogurt up to the recommended daily allowance of 400 mg (Boeneke & Aryana, 2007). The objective of this study was to determine the effect of
different concentrations and stage of addition of folic acid on the physico-chemical and sensory characteristics of lemon flavored yogurt over a 5-week storage period.
2. Materials and methods 2.1. Experimental design
Table 1 Formulae for-fat free lemon yogurt folic acid amounts up to 400 mg per 224 ml yogurt Composition (mg)
0
100
200
300
400
Skim milk (l) NFDM (g) Starch (g) Aspartame (g) Folic acid (mg) Starter culture (g) Lemon flavor (ml)
3.78 114.00 23.00 1.14 0.00 1.30 9.0
3.78 114.00 23.00 1.14 1.67 1.30 9.0
3.78 114.00 23.00 1.14 3.35 1.30 9.0
3.78 114.00 23.00 1.14 5.03 1.30 9.0
3.78 114.00 23.00 1.14 6.70 1.30 9.0
Fat free lemon flavored yogurts were manufactured with 0, 100, 200, 300, and 400 mg folic acid per 224 ml cup. Folic acid was added before or after pasteurization of the yogurt mix. Moisture, ash, fat, and protein concentrations were determined at week 1. Folic acid concentration was measured at weeks 1 and 5. Viscosity, pH, syneresis, color, and sensory properties were measured at weeks 1, 3, and 5. The data was analyzed by a randomized complete block with repeated measures in time analysis. The replications were the blocks. The experiment was replicated three times with three determinations per replication.
5
4.5
4
3.5
Folic acid
3
2.5
2
1.5
1
0.5
0 100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
400ug After Pasteruization
Fig. 1. Mean (+S.E.) peak areas (mg/ml) of folic acid in fat-free lemon flavored yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: week 1; &, week 5.
,
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2.2. Yogurt manufacture
2.3. Analytical procedures
Fat free lemon yogurt was manufactured at the Louisiana State University Department of Dairy Science. The dry ingredients (Table 1) were incorporated in an 18.9 l stainless steel container. Each mix was pre-heated to 60 1C and homogenized at 12.4 and 3.4 MPa on the first and second stages using a Gaulin 300 DJF 4 2PS homogenizer (APV Gaulin, Wilmington, MA), and batch pasteurized at 85 1C for 30 min. Each mix was cooled to 40 1C before addition of a culture containing Lactobacilus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Chr. Hansens, Milwaukee, WI) at a rate of 1.3 g per 3.78 l of the mix. Inoculated mixes were poured in 224 ml plastic cups and incubated at 40 1C until pH reached 4.5. Samples were cooled immediately and stored at 4 1C. Folic acid powder (Lakeshore Tech., Norton Shores, MI) was added with dry ingredients for the pre-pasteurization runs and prior to culture addition for post-pasteurization runs. Lemon flavorings were obtained from Target Flavors, Brookfield, CT, and added at a manufacture’s suggested usage rate of 9.0 ml/gal. Flavoring was added prior to incubation.
Protein concentration was determined 1 week after yogurt was manufactured. Samples were prepared by drying 15 g of sample for 48 h at 100 1C in a convection oven (Fisher Scientific, Houston, TX). Dry sample was ground using a mortar and pestle, 0.2 g was loaded into tin cups, folded and loaded into a Leco FP428 (Leco Corp., St. Joseph, MI) nitrogen analyzer. Sample was incinerated and results expressed as percent nitrogen. Results were multiplied by a protein correction factor of 6.38. Fat, moisture, and ash contents were also determined 1 week after manufacture according to Richardson (1985). Folic acid concentration was determined by using high performance liquid chromatography (HPLC) as modified from Albala´-Hurtado, Vecina-Nogue´s, Izquierdo-Pulido, and Marine´-Fone (1997). The HPLC system comprised of a Waters (Waters Corp., Milford, MA) 501 pump, Waters 717 Plus auto-sampler, and Waters 486 tunable UV detector set at 282 nm. Peak areas were calculated using the Waters Millenniums software. The separation was conducted isocratically using a Waters Spherisorb 5 mm ODS2 4.6 250 mm column with guard cartridge. Run
20000 19000 18000 17000 16000 15000 14000 13000
Viscosity [cp]
12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
Fig. 2. Mean viscosity (cp) of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: 3; and , week 5.
400ug After Pasteruization
, week 1;
, week
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time for samples was 20 min using a flow rate of 1 ml per minute and an injection volume of 10 ml per sample. A standard curve was prepared by dissolving known amounts (1.67, 3.35, 5.03, and 6.70 mg) of folic acid in 3.78 l of HPLC grade double distilled water, i.e. the same amount of folic acid that was used in 3.78 l of yogurt mix and analyzed using HPLC-UV. Peak area results from HPLC analysis were correlated to the standard curve to determine folic acid concentrations. Viscosity, pH, syneresis, and color were determined and sensory analysis conducted according to Boeneke and Aryana (2007).
2.4. Statistical analysis Data were analyzed in the following manner: concentration of folic acid, viscosity, pH, syneresis, color, and sensory analysis were analyzed by the Statistical Analysis System using the General Linear Model procedure with a repeated measure in time. Data from moisture, ash, and protein concentration were analyzed using the General Linear Model with Tukey’s Studentized Range Test. Significant differences were determined at Po0.05.
3. Results and discussion Protein contents on dry matter basis ranged from 34.09 to 38.38 g/100 g. Fat content for all samples were o0.5 g/ 100 g. Moisture content ranged from 88.46 to 89.77 g/100 g. Ash content ranged from 0.5667 to 0.9367 g/100 g. No significant (Po0.05) differences were found for protein, fat, moisture, and ash contents. Folic acid as expected did not alter the composition of the yogurts. Folic acid peak area concentrations for lemon flavored yogurts are reported in Fig. 1. Mean folate values in fortified plain yogurt were1.39 and 1.35 mg/ml for weeks 1 and 5, respectively. Mean folate values for lemon yogurts were 2.24 and 1.73 mg/ml for weeks 1 and 5, respectively, although no significant (Po0.05) interaction for time was found. The higher values for lemon yogurts is due to the folic acid content in the lemon extract added for flavoring. Lemon extract contained approximately 0.86 mg/ml of folic acid. Although no significant effect of (Po0.05) pasteurization was observed in the lemon flavored yogurts, mean values were higher where folic was added after pasteurization. Ristow, Gregory, and Damron (1982) found folic acid to be stable at processing temperatures of 120 1C for 20 min. However, Wigertz, Hansen, HoierMadisen, Holm, and Jagerstad, (1996) examined folate and
200 190 180 170 160 150 140 130
Syneresis [ml]
120 110 100 90 80 70 60 50 40 30 20 10 0 Control
100ug Before 200ug Before 300ug Before 400ug Before 100ug After 200ug After 300ug After 400ug After Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 3. Mean syneresis of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
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5-methyl-tetrahydrofolate found naturally in milk and yogurt and reported significantly reduced levels of both 5-methyl-tetrahydrofolate and total folate in pasteurized milk, UHT treated milk, and yogurt made from a mix pasteurized at 90 1C for 10 min. Boeneke and Aryana (2007) found mean folic acid peak area values to be higher in plain yogurts when folic acid was added prior to pasteurization of the yogurt mix. This discrepancy can possibly be explained by interactions of added folic acid and the folic acid content of the lemon flavor. Folic acid has a fully oxidized ring system and has greater stability than other forms. Perhaps this stability protects the folic acid from heat treatments and helps it to disperse in the product. Lemon extract could be interacting with the increased folic acid content and causing an opposite effect of lowering the mean values. Culture addition probably did not affect folic acid concentrations. Rao and Shahani, (1987) found skim milk inoculated with Lactobacillus delbrueckii subsp. bulgaricus lowered folate levels after 36 h incubation. Milk fermented by S. thermophilus and Lactobacullus acidophilus increased folate levels significantly. The S. thermophilus and L. delbrueckii subsp. bulgaricus culture used in this experiment had little effect on total folate content in the finished product. Viscosity values of lemon yogurts are reported in Fig. 2. Although overall level of folic acid addition was found to have no significant effect on viscosity (P40.05) in lemon
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yogurts, average mean viscosity values were lower for folic acid added after pasteurization. Mean peak area values were higher where folic acid was added after pasteurization meaning greater folic acid content within these samples. This may be due to altered localized casein micelle aggregation, as yogurt is a casein micelle network (Kalab, Allan-Wojtas, & Phillips-Todd, 1983) and viscosity is an indication of the strength of this network. Perhaps increased folic acid concentrations in the lemon yogurt helps decrease casein micelle aggregation resulting in decreased viscosity in samples with increased concentration of folic acid. The mean pH values for the lemon yogurt ranged from 4.06 to 4.22. Level of pasteurization and folic acid addition showed no significant interaction for lemon yogurts. Syneresis values are reported in Fig. 3. The mean values for syneresis ranged from 92.67 to 137.33 ml. There were significant (Po0.05) differences in syneresis with folic acid added before verses after pasteurization. Mean values for syneresis were higher when folic acid was added after pasteurization. Lemon yogurts were also less viscous when folic acid was added after pasteurization. Less viscous yogurts had more free whey resulting in higher syneresis values. This again would be due to the casein micelle network. There were no significant (Po0.05) differences in syneresis with different concentrations of folic acid. There were significant (Po0.05) differences in syneresis over
50
45
40
35
L*
30
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20
15
10
5
0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before 400ug Before Pasteurization Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After 400ug After Pasteruization Pasteruization
Fig. 4. Mean L* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
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weeks 1, 3, and 5. Values decreased 25 g/100 g at week 3 and increased 2 g/100 g at week 5. No explanation for this decrease then increase could be found. L* values are reported in Fig. 4. Folic acid addition before or after pasteurization did not significantly (Po0.05) impact the L* (lightness) values of yogurt. There were no overall significant (Po0.05) differences in lightness of yogurts attributable to concentration of folic acid. A* values are reported in Fig. 5. Pasteurization did not have any significant (Po0.05) effect on a* (redness) values. Significant differences were found in the level of folic acid added and overall time for lemon yogurts. Values decreased by 10 g/100 g at week 3 and increased 83 g/ 100 g at week 5 indicating that the extent of redness was much less at week 3 compared to week 5. B* values are reported in Fig. 6. Level of folic acid addition and time had a significant (Po0.05) effect on b*
(yellowness) values. Mean values decreased 28.6 g/100 g at week 3 and increased 42 g/100 g at week 5. Pasteurization did not have a significant (Po0.05) effect. Generally, as folic acid content increased the more yellow the yogurts became. Syneresis values decreased at week 3 then increased at week 5. The riboflavin in the whey probably influenced a* and b* values. Flavor scores are reported in Fig. 7. No significant (Po0.05) differences in flavor scores were found with folic acid addition before vs. after pasteurization; however, the level of folic acid addition and time did have a significant (Po0.05) effect on flavor scores. As levels of folic acid increased, mean flavor scores decreased. Mean flavor score was 6.72 for lemon flavored yogurts compared with 6.11 for plain yogurts. Flavor scores for lemon yogurts decreased with time. Mean flavor scores were higher for lemon yogurts than for plain yogurt.
5
4
a*
3
2
1
0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After Pasteurization Pasteurization Pasteurization
200ugAfter Pasteruization
300ug After Pasteruization
Fig. 5. Mean a* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
400ug After Pasteruization
, week 1;
, week 3;
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30
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b*
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0 Control
100ug Before 200ug Before 300ug Before 400ug Before Pasteurization Pasteurization Pasteurization Pasteurization
100ug After 200ug After 300ug After 400ug After Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 6. Mean b* values of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
10
9
8
7
Flavor Score
6
5
4
3
2
1
0 Control
100ug Before Pasteurization
200ug Before Pasteurization
300ug Before Pasteurization
400ug Before Pasteurization
100ug After Pasteurization
200ug After Pasteruization
300ug After Pasteruization
400ug After Pasteruization
Fig. 7. Mean flavor scores of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: and , week 5.
, week 1;
, week 3;
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9
8
Body & Texture Score
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6
5
4
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0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After 200ug After 300ug After Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization
400ug After Pasteruization
Fig. 8. Mean body and texture scores of folic acid fortified fat free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: , week 3; and , week 5.
Body and texture scores are reported in Fig. 8. Level of folic acid addition did not significantly (Po0.05) impact body and texture scores. Pasteurization and overall time were found significant (Po0.05) for lemon yogurts. Mean values were higher where folic acid was added prior to pasteurization. Mean viscosity values for lemon yogurts were also higher when folic acid was added prior to pasteurization. Consumers preferred the more viscous samples. Mean values appeared to decrease over the storage period. Body and texture scores are from 1 to 5 with 5 being no criticism. A mean value of 5 indicates no defect in body and texture of yogurts. Most values were approximately 4 indicating little defect in body and texture of yogurts. Appearance and color scores are reported in Fig. 9. Level of folic acid addition and overall time was found non-significant (P40.05) for appearance and color. Appearance and color values were significantly (Po0.05) higher when folic acid was added before pasteurization. Yogurts where folic acid was added to the mix then
, week 1;
pasteurized had less free whey, probably impacting appearance and color scores. Mean value of lemon yogurt appearance and color was 3.92 compared with 3.29 for plain yogurt.
4. Conclusions Folic acid addition had no effect on protein, fat, moisture, or ash content of lemon yogurts. Mean values for folic acid were higher for lemon yogurts when folic acid was added prior to pasteurization. Level of folic acid impacted flavor scores. As level of folic acid increased, flavor scores decreased. Mean viscosity values for lemon yogurts were higher when folic acid was added prior to pasteurization. Consumers preferred the more viscous samples. Body and texture values of lemon yogurts decreased over the 5-week storage period. Yogurts in which folic acid was added to the mix then pasteurized had less free whey. Lemon flavored, folic acid fortified yogurts
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9
8
Appearance & Color Score
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6
5
4
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2
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0 Control
100ug Before 200ug Before Pasteurization Pasteurization
300ug Before 400ug Before 100ug After 200ug After 300ug After 400ug After Pasteurization Pasteurization Pasteurization Pasteruization Pasteruization Pasteruization
Fig. 9. Mean appearance and color scores of folic acid fortified fat-free lemon yogurt at 100, 200, 300, and 400 mg before and after pasteurization at: week 1; , week 3; and , week 5.
had high flavor and body and texture scores which is of use to the dairy industry. References Albala´-Hurtado, S., Vecina-Nogue´s, M. T., Izquierdo-Pulido, M., & Marine´-Fone, A. (1997). Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography. Journal of Chromatography A, 778, 247–253. Boeneke, C. A., & Aryana, K. J. (2007). Effect of folic acid fortification on the characteristics of yogurt. Milchwissenschaft. Forseen, K. M., Jagerstad, M. I., Wigertz, K., & Witthoft, C. M. (2000). Folates and dairy products: A critical update. Journal of the American College of Nutrition, 19(90002), 100S–110S. Gregory, J. F. (1996). In F. Owen (Ed.), Food chemistry (3rd ed., pp. 590–599). New York: Marcel Dekker Inc. Hall, J., & Solehdin, F. (1998). Folic acid for the prevention of congenital anomalies. European Journal of Pediatrics, 157, 445–450. Kalab, M., Allan-Wojtas, P., & Phillips-Todd, B. (1983). Development of microstructure in set-style nonfat yoghurt — A review. Food Microstructure, 2, 51–66. Langenohl, R., Fohr, I., & Pietrzik, K. (2001). Beneficial role for folate in the prevention of colorectal and breast cancer. European Journal of Nutrition, 40, 98–105.
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Maxwell, S. R. (2000). Coronary artery disease-free radical damage, antioxidant protection and the role of homocysteine. Journal of Basic Research Cardiology, 95(Suppl. 1), I/65–I/71. Milk Facts. (2004). International Dairy Foods Association. Washington, DC, pp. 33–35. Rao, D. R., & Shahani, K. M. (1987). Vitamin content of cultured milk products. Cultured Dairy Products Journal, 6–10. Renner, E. (1983). Milk and dairy products in human nutrition. VV Gmbh Volkswirtschaftlicher Verlag, Federal Republic of Germany. Richardson, G. H. (1985). Standard methods for the examination of dairy products (15th ed). Washington, DC: American Public Health Association. Ristow, K. A., Gregory, J. F., & Damron, B. L. (1982). Thermal processing effects on folacin bioavailability in liquid model systems, liver and cabbage. Journal of Agriculture and Food Chemistry, 30, 801. Scott, K. J. (1989). Micronutrients in milk and milk based products. London: Elsevier Applied Science pp. 71–123. Wigertz, K., Hansen, I., Hoier-Madisen, M., Holm, J., & Jagerstad, M. (1996). Effect of milk processing on the concentration of folate binding protein (FBP), the folate-binding capacity and the retention of 5methyltetrahydrofolate. International Journal of Food Sciences and Nutrition, 47, 315–322.