Fortifying Bread with Each of Three Antioxidants

White bread was fortified individually with fat-coated l -ascorbic acid (AsA), cold-water-dispersible (CWD) β-carotene, and CWD all-rac-α-tocopheryl acetate (ToAc) at levels of 64, 5, and 100 mg, respectively, of active ingredient per 100 g of flour (14% mb). The freshly baked puploaves retained 76, 67, and 96% of added antioxidant, respectively. To extract ToAc quantitatively from bread or dough, dimethyl sulfoxide (DMSO) gave better results than hexane, which indicated that ToAc was in a partially bound state. “Protein-encased” (PE) β-carotene did not impart a yellow color to bread crumb and had one-fourth higher retention in fresh bread when compared to CWD β-carotene. In loaves stored at 25°C for one to seven days, AsA disappeared rapidly and PE β-carotene disappeared slowly; CWD β-carotene and ToAc were stable. In spite of storage loss, bread fortified with PE β-carotene retained significantly higher levels of β-carotene when compared to CWD β-carotene. One serving size (one slice, 28 g) of three-day-old bread fortified with one of the three antioxidants was calculated to provide 7, 120–150, and 13–16%, respectively, of the adult recommended daily allowances (RDA) for vitamins C, E, and A. When bread was fortified with both fat-coated AsA and CWD β-carotene and stored for five days, no protecting effect on the retention of the antioxidants was found.     

Effects of preparation and cooking of folic acid-fortified foods on the availability of folic acid in a folate depletion/repletion rat model

The practice of food fortification with folic acid offers the potential to increase the folate intake of the general population. To fully exploit the potential of fortification for raising folate nutriture, appropriate food vehicles need to be selected. Selection should involve determination of the availability of folic acid as affected by characteristics of the carrier food, food matrix, food preparation, and cooking. The present study investigated the effects of preparation and cooking of a range of folic acid-fortified foods on the folate status of folate-deficient rats. Fifty-six weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1% succinyl sulfathiazole for 28 days. Following depletion, six rats were randomly assigned to each of eight repletion diets containing cooked or uncooked meringue mix, quick bread mix, brownie mix, or pizza base mix. The test foods were fortified with 1400 microg of folic acid/kg of food and incorporated as 19% of the repletion diets. Each of the first four groups was pair-fed a diet containing a cooked fortified food with another group fed the corresponding uncooked fortified food. After a further 28 days, plasma, liver, and kidney folate concentrations were determined by microbiological assay. Mean plasma and liver folate concentrations of rats fed diets containing cooked fortified foods were similar to those of rats fed uncooked fortified foods. Preparation and cooking did not affect the availability of folic acid from the selected cereal-based convenience foods in this rat model system, suggesting that these foods are appropriate vehicles for fortification with folic acid.     

Phytate, calcium, iron, and zinc contents and their molar ratios in foods commonly consumed in China

A total of 60 food samples commonly consumed in China were analyzed for phytate using the anion-exchange method and for calcium, iron, and zinc using atomic absorption spectrophotometry. The foods analyzed included those based on cereal grains and soybean. Phytate contents expressed on a wet weight basis ranged from 0 for foods made from starches to 1878 mg/100 g for dried stick-shaped soybean milk film. The calcium contents were between 2.08 mg/100 g for ground corn and 760.67 mg/100 g for diced fried soybean curd. The lowest values of iron and zinc were 0.04 mg/100 g for Panjin pearl rice cooked with discarding extra water and 0.08 mg/100 g for potato and bean starches, while the highest values of iron and zinc were observed in dried stick-shaped soybean milk film. Although many foods were relatively rich in calcium, zinc, and iron, many also contained a higher level of phytate. Of the 60 food samples, 34 foods had a phytate/calcium molar ratio >0.24, 53 foods had a phytate/iron molar ratio >1, 31 foods had a phytate/zinc molar ratio >15, and only 7 foods had a phytate x calcium/zinc >200. Phytate in foods impair the bioavailability of calcium, iron, and zinc, which to some extent depends upon food processing and cooking methods.     

Stability of Native Folate and Added Folic Acid in Micronutrient‐Fortified Corn Masa and Tortillas

Degradation of added folic acid and native folates in micronutrient‐fortified corn masa and tortillas was evaluated using masa prepared from either nixtamalized corn flour or fresh nixtamal. Variations in masa pH, masa holding time at an elevated temperature, and iron source failed to show significant differences in folate loss in corn flour masa prepared in the laboratory. Masa was subsequently prepared from fresh nixtamal in a commercial mill in Mexico, and fortified with one of two different micronutrient premixes containing iron, zinc, B‐vitamins, and either unencapsulated or lipid‐encapsulated folic acid. Folate loss in commercial masa increased significantly with prebake masa holding time for both premixes. Unencapsulated folic acid showed a 73% loss after 4 hr of holding, compared to 60% loss for encapsulated. The difference was statistically significant, indicating a protective effect from the lipid coating. No significant differences in folate levels were found between prebake masa and baked tortillas. Holding baked tortillas for up to 12 hr also had no effect on folate levels. Native folate showed no significant losses throughout the process. Results from the commercial tortilla mill indicate that most of the loss in added folic acid occurs during prebake holding of masa, possibly from microbial degradation.     

HPLC Determination of Stability and Distribution of Added Folic Acid and Some Endogenous Folates During Breadmaking

Bread flour was spiked with folic acid (1.40 mg/lb or 3.08 μg/g of flour) and processed into bread by the sponge and dough method. Changes that occurred to added folic acid and endogenous folate contents through different processing stages, including sponge formation, proofing, and baking, were assessed by reversed‐phase ion‐pair HPLC combined with UV and fluorometric detection. Sample extraction required α‐amylase and rat plasma deconjugase digestion, and sample preparation required purification by solid‐phase extraction. Added folic acid was measured by monitoring UV absorption at 280 nm. Four selected forms of endogenous folates including tetrahydrofolate (THF), 5‐formyl‐THF, 10‐formylfolate, and 5‐methyl‐THF were identified and quantified throughout the bread processing using a fluorescence excitation wavelength of 290 nm and emission wavelength of 350 or 450 nm. Data indicate a relatively good stability of added folic acid and native folates to the baking process, and increased endogenous folate contents in dough and bread as compared with the flour from which they were made.     

Commercial Evaluation of a Continuous Micronutrient Fortification Process for Nixtamal Tortillas

The corn tortilla plays an integral role in the Mexican diet and is an ideal vehicle for micronutrient fortification. Approximately 60% of corn tortillas in Mexico are produced from nixtamal, with the remainder prepared from masa flour. A process for continuous fortification of nixtamal tortillas was evaluated in two commercial mills in Mexico. A commercial powder dosifier was used to add micronutrient premix containing iron, zinc, folic acid, niacin, riboflavin, and thiamin to nixtamal (1 g/kg) as it was milled. After training and preliminary sampling, mills produced fortified tortillas unassisted for four weeks. Masa flow rates over a four‐day period were 10–12 kg/min in both plants. Premix flow from the dosifier showed good stability, with an average coefficient of variation of 1.6%. Initial results indicated consistency in the fortification process, with significantly increased variation during the four‐week production period. Fortified tortillas had significantly higher levels of all nutrients tested. Micronutrient losses were <11% in all cases except folic acid, which showed an 80% loss. Despite processing losses, fortification resulted in a nearly fivefold increase in folic acid compared with control tortillas. The new fortification process is technically viable and was well received by millers.     

Effect of Digestive Enzyme Treatment on Antioxidant Properties of Germinated Green Gram Fortified with Minerals

The antioxidant activity (AA) of green gram germinated in mineral‐fortified water (with iron at 100 or 200 mg/100 mL or zinc at 50 or 100 mg/100 mL), cooked under pressure (PC), or microwaved and subjected to digestive enzyme treatment was determined by using three assay methods. Total AA showed that dehulled grains (DG) had higher AA than whole grains (WG). Cooking reduced the AA in both methods compared with raw grains with a lower percent retention of AA, although significant variations were not found between methods. The percent retention of AA in postdigestion samples was 22.68–51.39 (DG) and 15.67–44.0 (WG). Free radical scavenging activity indicated a different pattern, in which WG exhibited higher AA than DG and, among cooking methods, PC grains had higher activity. A reducing‐power assay showed similar results: DG had lower and PC samples had higher AA. Postdigestion AA of WG was higher for cooked than raw samples, although DG showed the opposite trend. In all cases, samples fortified with iron showed less AA because of prooxidant action of iron. In all assays, postdigestion samples had less activity than predigested extracts. In conclusion, germinated grains showed high AA, influenced by the presence of minerals, dehulling process, and cooking.     

Folic Acid Content of Ready‐to‐Eat Cereals Determined by Liquid Chromatography‐Mass Spectrometry: Comparison to Product Label and to Values Determined by Microbiological Assay

Twelve popular ready‐to‐eat breakfast cereals fortified with folic acid were sampled in the United States in 2006, and the data have been incorporated into the USDA National Nutrient Database for Standard Reference. Cereals were collected from three statistically selected retail outlets in each of four primary census regions, and four composites of each product were prepared using random groupings of three locations each. Folic acid was determined using a validated LC‐MS method, with ¹³C‐folic acid as an internal standard, after trienzyme treatment and solid phase extraction. A cereal reference material (AACC VMA399) was analyzed as a control. Selected samples were also assayed using the standard microbiological method, with and without trienzyme extraction, to generate an estimate of endogenous folate. On average, as shown on the label, folate content was underestimated. In seven cereals, folate was within 5% of the declared value; in four cereals, it was 5–20% higher; and in two cereals, it was >20% greater, representing –75 to +69 μg/serving (mean 17) of the label value, equivalent to –19% to +17% of the 400 μg/daily value. The microbiologically determined folic acid was higher than LC‐MS by 10–67% (mean 40%). Therefore, use of label values might underestimate folate intake from some breakfast cereals.     

Effect of Micronutrient Fortification on Nutritional and Other Properties of Nixtamal Tortillas

Nixtamalization is the process of steeping dried corn in hot water with calcium hydroxide (lime) with subsequent removal of all or most of the pericarp through washing. The resulting product is called nixtamal. Approximately 60% of corn tortillas in Mexico are produced from nixtamal, with the remainder prepared from nixtamalized corn flour. Nixtamal was fortified with micronutrient premix containing iron, zinc, folic acid, niacin, riboflavin, and thiamin. Premix composition followed a proposed Mexican regulation for corn flour fortification, adjusted for moisture. Effects of premix on masa adhesiveness, hardness, and pH, as well as tortilla sensory properties, stretchability, rollability, and color were measured. Micronutrient levels were tested in the dry corn, nixtamal, masa, and tortillas. There were no significant differences in masa texture or pH, tortilla rollability, or consumer acceptance of tortillas when comparing unfortified control and fortified treatments. Added thiamin was almost entirely degraded during processing. Folic acid and riboflavin decreased 26 and 45%, respectively, through the masa‐tortilla manufacturing process. Niacin showed no significant loss. Despite processing losses, fortification resulted in significant nutrient increases compared with control tortillas. Folic acid increased 974%, riboflavin increased 300%, niacin increased 141%, iron increased 156%, and zinc increased 153% in fortified tortillas.     

Pressurized liquid extraction and HPLC quantification of folic Acid in fortified wheat flours

A pressurized liquid extraction (PLE) method using phosphate buffer as solvent was applied for folic acid (FA) extraction from fortified wheat flours and was compared to a standard solid-liquid extraction (SLE) method. Extracted FA was quantified by reverse phase high-performance liquid chromatography (RP-HPLC) hyphenated with a phenyl column and an absorption photometric detector (λ = 280 nm). Detection and quantification limits were 0.12 and 0.4 ng, respectively, corresponding to 0.06 and 0.2 μg g(-1) of analyzed wheat flour. Equivalent FA contents were found by both extraction methods, but a single PLE allowed a total recovery of FA content, whereas at least three successive SLEs were needed to achieve a total recovery of FA. The obtained results indicated that PLE is a rapid and efficient technique for FA extraction from fortified wheat flour.     

Folate content in commercial white and whole wheat sandwich breads

After the U.S. mandate of folic acid fortification of enriched grain products, a report indicated higher than expected fortification. Limited information is available on folic acid in enriched products. We measured the folate content in 92 sandwich breads (46 white breads and 46 whole wheat breads) in Birmingham, Alabama, during 2001-2003. The mean folate content in white bread declined significantly from 2001 to 2002 or 2003, whereas the decline in folate content in whole wheat bread containing enriched flour was not significant. White bread contained significantly more folate than whole wheat bread containing enriched flour in 2001 and 2003. In 2002 and 2003, >40% of breads made of enriched flour contained <115 microg of folate/100 g and >70% contained <160 microg/100 g. These percentages were markedly higher than those in 2001. Our data suggest that folic acid in breads containing enriched flour declined after 2001 and monitoring of fortification may be necessary.     

Quantitation of N2-[1-(1-carboxy)ethyl]folic acid, a nonenzymatic glycation product of folic acid, in fortified foods and model cookies by a Stable isotope dilution assay

A stable isotope dilution assay (SIDA) for the quantitation of N(2)-[1-(carboxy)ethyl]folic acid (CEF) has been developed by using [(2)H(4)]CEF as the internal standard. After sample cleanup by anion exchange chromatography, the three-dimensional specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the nonenzymatic glycation product of folic acid (FA). When CEF was added to cornstarch, the detection limit for CEF was found to be 0.4 microg/100 g, and a recovery of 98.5% was determined. In analyses of cookies, the intra-assay coefficient of variation was 8.0% (n = 5). Application of the SIDA to commercial cookies produced from wheat flour fortified with FA revealed CEF contents of up to 7.1 microg/100 g, which accounted for approximately 10-20% of the cookies' FA content. In baby foods, multivitamin juices, and multivitamin sweets, however, CEF was not detectable. Further studies on CEF formation during baking of cookies made from fortified flour and different carbohydrates revealed that fructose was most effective in generating CEF followed by glucose, lactose, and sucrose with 12.5, 3.9, 2.5, and 2.5 microg/100 g of dry mass, respectively. During baking, approximately 50% of FA was retained for both monosaccharides fructose and glucose, and 77% as well as 85% of its initial content was retained for the disaccharides lactose and sucrose, respectively. Of the degraded amount of FA, CEF comprised 28% for fructose as well as 18, 12, and 8% for sucrose, lactose, and glucose, respectively. Therefore, CEF can be considered an important degradation product of FA in baked foods made from fructose. To retain a maximum amount of FA, products should rather be baked with sucrose than with reducing carbohydrates.     

Impact of Iron Source and Concentration on Rice Flavor Using a Simulated Rice Kernel Micronutrient Delivery System

An extruded grain designed to look like a rice kernel fortified with one of two sources of iron (elemental iron and ferrous sulfate), with and without multiple fortificant (zinc, thiamin, and folic acid), was mixed with milled Calrose rice at low (1:200), medium (1:100), and high (1:50) concentrations. The intensities of water‐like, sour taste, hay‐like musty, and alfalfa/grassy/green bean flavors were enhanced by the addition of ferrous sulfate (FeSO₄) or FeSO₄ plus multiple fortificants. Astringent mouthfeel was likewise affected by addition of FeSO₄ or FeSO₄ plus multiple fortificants. Overall, the elemental iron with multiple fortificants least affected the oxidation of the extruded kernals. Lipid oxidation products in stored fortificant increased the first two to three months and concentrations were higher in samples with FeSO₄ as the iron source.     

Fortifying Bread with a Mixture of Wheat Fiber and Psyllium Husk Fiber Plus Three Antioxidants

A 7:3 (w/w) mixture of wheat fiber (WF) and psyllium husk fiber (PHF) was substituted for 10wt% of flour on a 14% mb, and the protein in the blend was restored to 10.3% by incorporating vital wheat gluten. After adding 0.5% sodium stearoyl 2-lactylate, the blend (100 g) was fortified with a combination of fat-coated ascorbic acid (AsA), proteinencased (PE) β-carotene, and cold-water-dispersible (CWD) all-rac-α-tocopheryl acetate (ToAc) at levels of 72, 5.6, and 115 mg, respectively, of active material. Adding the fiber ingredients to the pup loaf formula increased water absorption 25% and mixing time 50% and imparted stickiness to the dough. The fiber and antioxidant bread showed a 10% reduction in loaf volume and a somewhat inferior crumb grain with an off-color caused by small, black specks on a dark gray background. The crumb of the fiber and antioxidant bread remained much softer than control bread during one to seven days of storage at room temperature. Caramel coloring masked the off-color. AsA was lost significantly faster in the fiber and antioxidant bread than in antioxidant bread; the losses of AsA were 97 and 86%, respectively, after three days at 25°C. Approximately 25% of β-carotene was lost from the fiber and antioxidant bread after three days, and 33% after seven days, but the loss of ToAc was <10%. One serving size (one slice, 28 g) of fiber and antioxidant bread was calculated to provide 2.1 g of dietary fiber, or ~8% of daily value, of which ~30% was soluble. The three-day-old slice also contained vitamin E and vitamin A (as β-carotene) at 120–150% and 12–15%, respectively, of the adult recommended daily allowances, but with 16% fewer calories than white pan bread.     

A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods

A stable-isotope liquid chromatography-mass spectrometry (LC-MS) assay was developed for the quantitative determination of folic acid in fortified foods. Folic acid was extracted from food samples into a phosphate buffer, purified on a C-18 Sep-Pak cartridge, and analyzed by LC-MS in the negative ion mode using electrospray ionization. The analyte was quantified using (13)C(5)-folic acid as an internal standard. The coefficient of variation for the precision of the method was 5.6% based on the analysis of four sample replicates. The accuracy of the method was assessed using a standard method of addition of folic acid to a shredded whole-wheat cereal. The quantitative determination of folic acid in this matrix was linear over 1 order of magnitude having a concentration range of 2.4 to 24 microg/g of food (or 0.05 to 0.5 microg of analyte injected into the LC-MS). The overall quantitative efficiency of the method was evaluated using a standard reference material (infant formula SRM 1846). The method was applied to the determination of folic acid in several test samples (fortified breakfast cereals), and the values were in accord with the manufacturer's claim. This method advances a LC-MS technique for the determination of folic acid in fortified foods based on stable-isotope dilution methodology. The specificity of the technique and quantitative accuracy of the method in various food substrates suggests that the method may be adapted for routine analysis in other fortified foods.     

Oxido-Reductases and Lipases as Dough-Bleaching Agents

To replace benzoyl peroxide as a bread dough-bleaching agent, pure and commercial oxido-reductases (peroxidases, catalases, glucose oxidases, lipoxygenase, and laccase) were screened based on degradation of β-carotene in a liquid system (5 μg of β-carotene/mL of 0.1M citrate phosphate buffer at pH 5.5 or 6.5) or dough. Peroxidases had the best bleaching activity; some catalases also showed bleaching potential in a liquid system but not in bread dough, suggesting that screening enzymes in liquid media has limited application for dough. In 100 g of flour, combinations of peroxidase (3,000 U), lipase (815–1,630 U), and linoleic acid (0–300 mg) completely bleached bread dough.     

Synchronous fluorescence as a rapid method for the simultaneous determination of folic acid and riboflavin in nutritional beverages

A rapid synchronous spectrofluorimetric method was first developed for the simultaneous determination of folic acid and riboflavin in nutrimental beverages. Folic acid could be detected by using H(2)O(2) plus Cu(II) as oxidation system to produce pterine-6-carboxylic acid, which had strong fluorescence in aqueous solution, and riboflavin itself was obviously fluorescent. Various operational parameters were thoroughly discussed in terms of their effects on the fluorescence signals, including instrumental parameters, concentration of the oxidation system, and pH. Under optimum conditions, the calibration curves were linear in the ranges of 100-250 μg/L for folic acid and 1-250 μg/L for riboflavin, and the detection limits were 2.0 and 0.014 μg/L, respectively. In addition, this method was applied to the determination of folic acid and riboflavin in nutrimental beverages with satisfactory results.     

Stability of tea catechins in the breadmaking process

A green tea extract (GTE) was incorporated into bread as a source of tea catechins. The stability of tea catechins in the breadmaking process including unfrozen and frozen dough was studied. A method was developed for the separation and quantification of tea catechins in GTE, dough, and bread samples using a RP-HPLC system. The separation system consisted of a C18 reversed-phase column, a gradient elution system of water/methanol and formic acid, and a photodiode array UV detector. Tea catechins were detected at 275 nm. GTEs at 50, 100, and 150 mg per 100 g of flour were formulated. The results obtained showed that green tea catechins were relatively stable in dough during freezing and frozen storage at -20 degrees C for up to 9 weeks. There were no further detectable losses of tea catechins in bread during a storage of 4 days at room temperature. It was also revealed that (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) were more susceptible to degradation than (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC). (-)-EGCG and (-)-ECG were normally selected as the quality indices of green tea catechins, and their retention levels in freshly baked bread were ca. 83 and 91%, respectively. One piece of bread (53 g) containing 150 mg of GTE/100 g of flour will provide 28 mg of tea catechins, which is approximately 35% of those infused from one green tea bag (2 g).     

Evaluation of Bacterial Effects on Folic Acid Loss in Fortified,Nixtamalized Corn Masa Flour

Many Mexican women are deficient in folic acid. Fortification of the corn tortilla could be an effective way to help increase the folic acid levels among the Mexican population. Previous studies have shown significant folic acid losses in the masa dough as it is held before baking. This loss in folic acid could be owing to utilization by lactic acid bacteria naturally present in the masa. The objective of this study was to determine the effect of bacteria native to corn masa on the folic acid content in masa. Bacteria in dough samples from six mills in Guadalajara, Mexico, were isolated and identified. Bacterial isolates were inoculated into sterile fortified corn masa flour, which was converted to masa and held at 56°C for 0, 3, and 6 h, replicating the conditions of freshly milled masa as held before baking. All samples, including the control, showed losses of folic acid between 66 and 79% w/w in the first 3 h of incubation. Because folic acid degradation in the sterile control sample was not different than the inoculated sample results, the decline in folic acid was not owing to bacteria (mainly Streptococcus spp.) present in the masa flour but appeared to be a chemical degradation related to time and temperature.     

Effects of Boiling,Refrigerating, and Microwave Heating on Cooked Quality and Stability of Lipids in Macaroni Containing Ground Flaxseed

Research was conducted to determine the effects of boiling, refrigerating, and microwave heating on the cooked quality and stability of lipids in macaroni containing 15% (w/w) ground flaxseed. Boiling increased brightness but decreased redness and yellowness of macaroni containing ground flaxseed. Boiled macaroni and boiled‐refrigerated‐microwave heated (BRMH) macaroni had similar appearance. After boiling, cooking loss was lower and cooked firmness was greater for macaroni dried at ultra‐high temperature (90°C) than at low temperature (40°C). Macaroni fortified with ground flaxseed had lower cooking loss than did nonfortified macaroni. Firmness was greatest with boiled, intermediate with boiled‐refrigerated (BR), and least with BRMH macaroni. Cooking in boiling water reduced extractable lipid content of macaroni with flaxseed. Boiled‐refrigerated and BRMH macaroni with flaxseed had similar extractable lipid contents and were lower than that for boiled macaroni with flaxseed. Free fatty acid content was greatest with dried, intermediate with boiled, and least with BR and BRMH macaroni with flaxseed. Boiling, refrigerating, and microwave heating did not affect conjugated diene content in lipid extracted from macaroni with flaxseed, regardless of drying temperature.