Folic acid retention evaluation in preparations with wheat flour and corn submitted to different cooking methods by HPLC/DAD.

Folic acid content was evaluated in food preparations containing wheat and corn flour submitted to baking, deep-frying, and steaming. Commercially fortified flours showed the absence of folic acid. Flours with laboratory folic acid fortification showed 487 and 474 μg of folic acid in 100 g of wheat and corn flours, respectively. In the corn flour preparations, the cake had the highest retention (99%) when compared to couscous (97%). Besides, the cake showed higher retention when compared to the wheat flour preparations due to the interactions of the folic acid with the hydrophobic amino acids of the Zein, a protein found in corn. In wheat flour preparations, vitamin retention was 87%, 80% and 57% in bread, cake, and White sauce respectively. These findings relate to the change of the physicochemical properties of food components that occurs during mixing and cooking of the ingredients.

1. Introduction

Folic acid is a yellow crystalline compound synthetically produced by the food industry which is used in nutritional supplements and fortification of cereals and flours [1, 2]. Also found in food, vitamin B9 plays an important role in the synthesis of proteins and haemoglobin, cell growth, hypertension control, and prenatal development. Its deficiency has been pointed out as a factor that induces the appearance of serious diseases that affect human beings such as congenital malformations, cardiovascular diseases, cerebrovascular diseases, among others [3, 4].

The high amount of problems related to folic acid deficiency led several countries to initiate the fortification of cereals and flours voluntarily or through legislation [1, 5]. In Brazil, the requirements for the fortification of wheat and corn flour were updated in April 2017 through the publication of RDC (Collegiate Board Resolution) No. 150 of April 13, 2017 [6], which revoked Ordinance SVS/MS (Department of Health Surveillance–Ministry of Health) n. 344/2002 [7] and established, among other updates, a maximum limit (220 μg.100g-1) for the addition of the vitamin in order to avoid exposure of the population to unnecessary risk [6].

The fortification of commonly consumed food items, such as wheat flour and corn flour, has been proposed as the best method to ensure the increase in folic acid intake in order to reduce the risks associated with micronutrient deficiency [8, 9]. However, after food enrichment, nutrient behavior studies are necessary to evaluate the present vitamin content after processing, storage and cooking of the fortified food since such conditions may alter the concentration of the nutrients [10]. As to cooking, the thermolabile nature of the vitamins contributes to the fact that the heat treatment is an important factor for the degradation of these nutrients, and its effect may vary according to the type of food, the chosen method and cooking time [11].

Folic acid is an unstable molecule, it is light sensitive and undergoes oxidative degradation, which can be reinforced by pH, oxygen, heat and acidic conditions [3], but little is known about its stability when facing different cooking methods.

Therefore, the aim of the present study was to investigate the folic acid content and the folic acid retention in food preparations made with corn flour and wheat flour, submitted to cooking methods usually applied in a household.

2. Material and methods

2.1. Samples and reagents

Samples of flours and ingredients used to prepare the food preparations were acquired from supermarkets (Assaí wholesaler, Hiper Bompreço and Extra supermarkets) located in the city of Recife–PE, Brazil. All ingredients used in the preparation, including flour, are produced and marketed in Northeastern Brazil and comply with current legislation. The brands available for analysis were, wheat flour: Dona Bente, Rosa Branca, Finna, Boa Sorte and Sarandi and Cornflour: Coringa, Novomilho, Santa Clara and Vitamilho. The sample quota related to commercially fortified flours was composed of six different brands of wheat flour (WF) and three different brands of corn flour (CF) that were collected from two different batches and evaluated in different months of the year (July and September), totalizing the value of 18 samples. The laboratory folic acid fortified wheat flour and corn flour had the addition of the vitamin following the fortification protocol described in item 2. and they were also used in the elaboration of the different preparations for the study of folic acid retention.

Methanol was purchased from Merck®, Brazil; monobasic potassium phosphate by Sigma-Aldrich®, Germany; orthophosphoric acid by Fluka®, Switzerland and ammonium acetate and ascorbic acid by Vetec®, Brazil, all of them in analytical grade (greater than 99%). The standard for folic acid identification and quantification was obtained from Sigma-Aldrich®, U.S.A. The water used was obtained from a Milli-Q ultra-purification water system (Millipore, Bedford, MA, U.S.A).

2.2. Protocol for laboratory folic acid fortification of wheat and corn flour

Approximately 5,000 μg (0.0050 g) of the folic acid standard were weighed and added to 500 g of wheat flour or 500 g of corn meal in order to achieve a concentration of 1000 μg of folic acid for every 100 g of wheat or corn flour. For a good homogenization of the vitamin in the farinaceous matrix, two procedures of homogenization were used: (1) Folic acid (0.0050 g) was added to the total content (500 g) of the farinaceous matrices, shaken in a cutter (Metvisa) for 8 minutes and sieved using a domestic sieve; (2) The 500 g of the wheat and corn flour to be fortified were first shaken in a cutter (Metvisa) for 1 minute and then sieved, discarding the unsifted residue. This procedure was performed three times. After that, the total content of the flours (500 g) was fractionated into five 100 g portions. The total amount of folic acid (0.0050 g) to be added to the farinaceous matrices was also weighed and divided into fractions (5 fractions of 0.0010 g). In another essay,Each folic acid fraction (0.0010 g) was added to each portion (100 g) of the wheat and corn flours and the mixtures (flour and vitamin) were first homogenized manually for 1 min using a spatula, and then homogenized in a cutter (Metvisa) also for 1 minute. This procedure was repeated for each of the five portions (100 g each) from the fractionation of the total content (500 g) of the wheat and corn flours that would be fortified. After each portion of the flour was added folic acid and homogenized, manually and mechanically, the total content (500 g) was mixed and homogenized in cutter for 2 minutes. At the end of the fortification procedure, the total time used for the homogenization of the vitamin into the farinaceous matrix, including manual (1 min for each 100 g) and mechanical homogenization (1 min for every 100 g + 2 minutes for 500 g), was of 12 minutes.

The fortified flours were then packed under the same conditions as those applied in industry and were used afterward to determine folic acid content as well as to prepare the different food preparations used to evaluate folic acid retention.

2.3. Food preparations

The preparations were prepared with laboratory folic acid fortified wheat flour and corn flour, from pre-tested recipes, following the preparation techniques described below:

2.4. Wheat flour cake

The egg whites of two large eggs (50 g each) were separated from the yolks and beat until firm peaks were obtained. The egg yolks from the two eggs, 40 g of margarine and 160 g of sugar were mixed with a domestic food mixer until a homogeneous mixture was obtained. 240 mL of whole milk and 240 g of wheat flour were gradually added to the egg yolk, margarine and sugar mixture as it was still being mixed. Finally, 30 g of baking powder and the whipped egg white were added and mixed. The cake mixture was poured into a greased and floured Bundt pan. The conventional oven was used for baking. It was preheated and the cake was baked at 180°C for 40 minutes.

2.5. White cream sauce

One medium garlic (5 g) and ½ of one small onion (25 g) were grated and fried in 10 g of margarine. The 240 mL of whole milk, 20 g of wheat flour and salt to taste were added gradually and stirred until a thick cream was obtained. The heat was lowered from about 100°C to 60°C and the white sauce was left to cook for five minutes, stirring when necessary. The pan was removed from the heat, followed by the addition of 50 g of table cream to the sauce. The white cream sauce was cooked in the pan, in a conventional stove at 60°C for 5 minutes.

2.6. Bread Loaf

In a small bowl, 10 g of yeast was mixed with 1/4 cup of warm water until they were dissolved. The mixture was left to rest for about 5 minutes until bubbles started to rise. In a large bowl, 240 g of the laboratory fortified wheat flour and 8g of salt was mixed leaving a well in the center of the mixture. The dissolved yeast was gradually added to the mixture of flour and salt, from the center outwards. The dough was brought together using the hands and 300 mL of milk was also added as the ingredients were being mixed. As soon as the flour absorbed the liquids, the procedure was followed by the incorporation of butter to the dough and more mixing. The dough was pressed, kneaded, stretched and kneaded again for 10 minutes, until it obtained a soft and moist texture. It was then shaped in the shape of a ball and returned to the bowl, covering it with cling film, and left to rest for 1 hour until it doubled in size. After that, the dough was transferred to the workbench and divided in half, flattening each half into a rectangle. To make the bread loaves, one of the two larger sides of the rectangles was folded to the center of the dough and tightened gently; it was then covered by its opposite side and tightened well to seal. The two remaining sides of the rectangle (the smaller ones) were then folded 4 cm from the end of the dough and the seams were pressed tightly.

The dough was transferred, with the dough seam side down, to a butter greased baking pan. It was covered with cling film and set to rest and prove for another 40 minutes. Exactly 20 minutes before the proofing time was finished, the oven was preheated to 180°C. The dough was baked in a conventional oven for about 40 minutes.

2.7. Corn cake

In a domestic blender, 75 mL of whole milk, 85 mL of corn oil and 2 large eggs were put together and blended well. After that, the blender was turned off, 80 g of white sugar was added and the mixture was blended a bit more. Following the sugar, the blender was turned off once more for the addition of 240 g of fortified corn flour and the mixture was blended once again. Salt to taste and 5 g of baking powder were added to the mixture and it was blended again. The mixture was poured into a greased and floured Bundt pan. The cake was baked in a preheated conventional oven at a temperature of 180°C for 40 minutes.

2.8. Couscous

120 g of folic acid laboratory fortified corn flour and 2 g of salt were placed in a glass bowl. 200 mL of water was gradually added and mixed well with the aid of a spatula. After mixing everything, the couscous mixture was placed in a couscous steamer and taken to the heat. The couscous was cooked in a conventional stove at an initial temperature of 100°C, being reduced to 60°C when the couscous steamer water began to boil. At this temperature, it was allowed to steam for 9 minutes before the heat source was turned off.

3. Folic acid analysis by HPLC

3.1. Sample extraction

The folic acid was extracted from the samples (fortified wheat flour and corn flour, raw preparations, and cooked preparations) following the methods of Paiva et al. [12]. Approximately 2 grams of the sample was weighed and put into a falcon test tube and 20 mL of the ammonium acetate extracting solution (50 mmol L-1) was added, which were then vortexed for one minute. The extracts were submitted to a thermostated water bath treatment at 40°C for 10 minutes. Then the samples were centrifuged in at 5000 g rotation at a temperature of 4°C for 15 minutes with subsequent filtration on cellulose acetate membranes of 0.22 μm porosity. The amount equivalent to 1 mL of each extract was filtered once more into polyvinylidene fluoride membranes of 0.22 μm porosity into vials which were then brought to the chromatograph. All determinations were performed in quintuplet.

3.2. Chromatographic conditions

All High-Performance Liquid Chromatography (HPLC) analyses were performed on a Shimadzu liquid chromatography (Shimadzu®, Japan), consisting of the following modules: controller system, model CBM-20A; diode array detector, model SPD-20AV; quaternary pump, model LC-20TA; column oven, model CTO-20AC; autosampler, model SIL-20AC. All commands were performed using LC Solution software (version 1.25, Shimadzu®, Japan).

The mobile phase, used in isocratic mode, was composed of a mixture of monobasic potassium phosphate buffer (pH 2.0) and 85% methanol (85:15 v/v). The flow rate was 0.5 mL/min, with an injection volume of 20 μL and a total run time of 20 min. Folic acid was eluted with a retention time of approximately 18.2 minutes. The column was washed with water and conditioned with acetonitrile (Merck-grade HPLC) at the end of the analyses (PAIVA et al., 2012).

3.3. Identification and quantification

Peak identification was performed by spectral similarity (an equipment feature that allows the comparison between the standards’ spectra and samples’ spectra) and by comparing the retention time in both standards and the samples. Quantification was performed using external standardization by constructing an analytical curve with 10 different concentrations of folic acid ranging from 0.05 μg.mL-1 to 100 μg.mL-1. The solutions used to draw the calibration curve were individually prepared by diluting the stock solution (1000 μg.mL-1 folic acid) in ammonium acetate (8 mmol.L-1) from the individual standard.

The quantification limit (QL) was defined as the lowest concentration of the analyte that reached a signal 10 times above the baseline noise [13], using the following equation: QL = (SD x 10) / SC. The detection limit (DL) was defined as the lowest concentration of the analyte that reached a signal 3 times above the baseline noise [13], using the following equation: DL = (SD x 3) / SC. SD is the standard deviation of the intercept with the y-axis of at least three calibration curves and SC is the slope of the calibration curve.

3.4. True retention

The retention of folic acid was calculated by the percentage of vitamin retention in the food preparations made with the laboratory fortified wheat flour and corn flour, using different cooking methods. The percentage was obtained considering the weight changes after the raw mixture was cooked. For this, the true or real retention formula (% RR) [14] was used as described below:

3.5. Statistical test

Initially, normality and homoscedasticity tests were carried out (Bartlet's test). In the absence of such parameters for a few samples, the information was transformed into a log(x+1) to meet the requirements of analysis of variance (ANOVA) to observe the differences between treatments. For the independent sample treatments classified as dry-heat and moist-heat, the Kruskal-Wallis test was used. Statistica 7.0 software was used for all statistical analyses.

4. Results and discussion

As can be seen in Fig 1, in samples analysed in July and September, there was no signal in time and absorptivity matching the folic acid standard, making us conclude that the samples obtained from the local markets did not have the vitamin. It is important to emphasize that other (unpublished) studies were done in previous years and folic acid was not found in samples from the Northeast of Brazil. The chromatograms shown have signals, other than folic acid, that couldn’t be identified in this study. Yet, they have a high similarity with the folates database available in our software.

Fig 1

HPLC chromatogram of commercially fortified wheat flour and corn flour evaluated in July (A) and September (B). Chromatograms for one of the samples of commercially fortified wheat flour and corn flour evaluated in July (A) and one of the wheat flour and corn flour samples evaluated in September (B).

Determination of folic acid in commercially fortified and laboratory fortified wheat and corn flours

In Fig 2 the chromatograms of the laboratory fortified wheat and corn flours are illustrated in the chromatograms and in Table 1 the content of laboratory fortified folic acid in the corn and wheat flours are shown.

Fig 2

Chromatograms of the laboratory fortified wheat and corn flours.

Chromatograms referring to the samples of wheat and corn flour laboratory fortified with folic acid.

Table 1

Content of folic acid in laboratory fortified.

Samples	μg.100g-1 of folic acid ± SD	Similarity with the folic acid standard	Peak Purity	Analytical parameters	Calibration curve
WF	487 ± 0,5	1,00	1,00	LOD—230,56	y = 4389.1x - 869.42R2 = 0.9996
CF	474 ± 0,2	0,99	1,00	LOQ—698,66

WF—wheat flour; CF—corn flour; SD—standard deviation. LOD and LOQ

n = 5

The laboratory fortified folic acid flour samples had a medium concentration of 487 μg.100g-1 for the wheat flour and 474 μg.100g-1 for the corn flour as seen in Table 1. The folic acid content found has shown to be inferior to the one added to the farinaceous matrices through the fortification protocol (item 2). Although we performed two test procedures for homogenization, the first being more efficient due to a shorter exposure time of the material to environmental codes, the recovery rate was still between 51 and 53%.

There is a high variation in the folic acid content in commercialized corn and wheat flours and this may occur, probably, due to problems during the homogenization process, which can be related to the tendency the vitamin has to agglomerate, making its dispersion more difficult [15, 16, 17]. The significant variation in the folic acid content of the laboratory fortified corn and wheat flours may be related to the homogenization process performed on the workbench. In the present study, the time (12 minutes) used to mix the vitamin with the flour, and the equipment used for the homogenization (cutter) may have contributed to the low recovery of the added vitamin as the contents found after the fortification process, when compared to the added value, were lower for both types of flours. Studies evaluating folic acid content in Brazilian corn and wheat flours show that the amount of the vitamin varies considerably from one region to another.

Soeiro et al. [18], when investigating the quality of fortified flours sold in the state of São Paulo, found that the folic acid concentration in wheat flour was close to the value determined by the Brazilian legislation (150 μg.100g-1), however, the corn flours had a much higher content (twice as much) than the one established. In a study carried out by Alaburda et al. [19] the results for the analysis of 33 samples of fortified wheat flour showed that 17 of them (51%) had concentration below the minimum level stated by the Brazilian legislation (1.50 μg.g-1).

The quality of commercially fortified flours has been evaluated in other countries around the world and, therefore, a couple of papers on folic acid content have been published. Chandra-hioe, Bucknalll, Arcot [20] found that folic acid levels in commercially fortified flours were below the average concentrations (200μg.100g-1 of flour) required by the Australian legislation. For flours sold in supermarkets in Poland, the average value reported for folic acid concentration was 237 μg.100g-1 [21].

When analyzing commonly commercialized flours in the United States, whose fortification regulation stipulates a minimum of 140 μg.100g-1, Rader et al. [22] found folic acid levels ranging from 33 μg.100g-1 to 229 μg.100g-1. Arcot, Shrestha and Gusanov et al. [23] reported values ranging from 82 μg.100g-1 to 95 μg.100g-1 in the same food matrix also commercialized in the United States.

In the preparations made with wheat flour, the folic acid content found in the raw mixture varied from 218 μg.100g-1 to 431 μg.100g-1. The lowest content was found in the white cream sauce and the highest content was found in the bread loaf. In the corn flour preparations, the folic acid content ranged from 272 μg.100g-1 in the corn cake to 310 μg.100g-1 in couscous, as it can be seen in Table 2.

Table 2

Content of folic acid in the raw preparations.

Farinaceous matrix	Preparations	The Folic acid content in a raw preparation (μg.100g-1) ± SD
Wheat flour	Cake	335 ± 0,3
	White Cream Sauce	218 ± 0,2
	Bread Loaf	431 ± 0,4
Corn flour	Corn cake	272 ± 0,4
	Couscous	310 ± 0,3

SD–Standard deviation.

n = 5

The results for the true retention of folic acid in the preparations of wheat flour cake, white cream sauce, bread loaf, corn cake, and couscous after cooking are shown in Table 3. In the food preparations formulated with fortified wheat flour, the highest retention in folic acid content was observed for the bread loaf (87%), followed by the wheat flour cake (80%) and the white cream sauce (57%). In the preparations made with laboratory fortified corn flour, vitamin retention was high, being the highest value observed for the corn cake (99%) followed by the couscous (97%).

Table 3

True retention of folic acid in food preparations made with wheat and corn flour fortified in the laboratory.

Samples	Cooking conditions		The Folic acid content in cooked preparation (mcg/g) ± SD	The Folic acid content in cooked preparation (g/100g) ± SD	Weight of cooked preparation (g)	The Folic acid content in a raw preparation (mcg/g) ± SD	Weight of the raw preparation (g)	RR (%) ± SD
	Time (min)	Temp. (°C)
Wheat flour cake	40	180	3,0 ± 0,001	0,30 ± 0,057	516	3,4 ± 0,0003	576	80 ± 17
Bread loaf	40	180	4,3 ± 0,0004	0,43 ± 0,032	335	4,3 ± 0,0004	383	87 ± 8
White cream sauce	5	100–60	2,3 ± 0,001	0,23 ± 0,008	117	2,2 ± 0,0001	218,42	57 ± 3
Corn cake	40	180	2,9 ± 0,001	0,29 ± 0,038	511	2,7 ± 0,0004	546	99 ± 9
Couscous	5	100–40	3,1 ± 0,0002	0,31 ± 0,011	250	3,1 ± 0,0003	260	97 ± 11

RR–real retention; SD–standard deviation. n = 5.

Fig 3 shows statistical analysis of variance (ANOVA), a posteriori test mean (+- 95% confidence interval), Dunnet with the value of p <0.0001, applied to fortified wheat flour and its preparations.

Fig 3

Comparison between treatments of wheat flour fortified with folic acid.

Different letters indicate differences between means (p <0.0001). 1—folic acid fortified wheat flour (1,000μg.100g-1); 2—wheat flour cake before cooking; 3—wheat flour cake after cooking; 4 –bread dough before cooking; 5 –bread loaf after cooking.

Samples 4 and 5 do not differ from each other not even when compared to the control (1—fortified wheat flour). Samples 2 and 3 also do not differ from each other, but they differ from the control (1) and samples (4) and (5).

Fig 4 shows the statistical analysis of variance (ANOVA), mean (± 95% confidence interval), Dunnet a posteriori test with the value of p <0.000, applied for evaluation of the folic acid content between the fortified corn flour and its preparations. Comparing to the control, sample 12, all other samples (8, 9, 10 and 11) showed a significant difference in folic acid content. Samples 8 and 9, 10 and 11 showed no significant difference in folic acid content: when compared to the raw mixture and after cooking of the same preparation and also when comparing the samples with each other.

Fig 4

Comparison between treatments of corn flour fortified with folic acid.

Different letters indicate differences between means (p <0.0001). 8—couscous before cooking; 9 couscous after cooking; 10—corn cake before cooking; 11—corn cake after cooking; 12—corn flour fortified with folic acid.

Fig 5 shows the statistical treatment, T-Test (Kruskal-Wallis) for independent samples (t = 6.42; gl = 33; p <0.00001), which was applied for the evaluation of the folic acid content among the preparations considering the heat treatment used; dry-heat (1) and moist-heat (2).

Fig 5

Comparison between dry-heat and moist-heat cooked preparations 1 –cooking using dry-heat; 2—cooking using moist heat.

The comparison between the two cooking methods let the authors conclude that there was a significant statistical difference in the content of folic acid in the preparations due to the cooking method. The preparations cooked using the dry-heat method (1) showed a higher concentration of folic acid as compared to the preparation cooked using the moist-heat method (2) which had the lowest content of the retained vitamin.

The differences in folic acid concentration in the different raw preparations (Table 2) compared to the amount of folic acid added to the farinaceous matrices used in the preparations suggest that the food preparation itself contributes to losses in the vitamin content. These observations were rectified when the statistical treatment applied (Figs 3 and 4) showed significant differences in the folic acid content between the control (fortified flour) and its preparations.

The results for the true retention of folic acid in the preparations of wheat flour cake, white cream sauce, bread loaf, corn cake and couscous after cooking are shown in Table 3.

The higher retention of folic acid in the preparations (Table 3) submitted to the dry-heat cooking method suggests that this technique is more appropriate when you want to effectively retain folic acid in the prepared food. Samples cooked using the dry-heat method (wheat flour cake, bread loaf, couscous and corn cake) were statistically higher in the folic acid content as compared to the preparation cooked using moist-heat (white cream sauce) (Fig 5). The highest losses were observed in the white cream sauce (43%), and even before heat treatment, it showed the lowest concentration of folic acid suggesting that the direct contact of the food with the cooking water promotes considerable losses in the content of this vitamin that continues to be lost during cooking.

The decrease in the folic acid content after cooking by immersion in water can be explained by the water-soluble nature of the vitamin that contributes to the losses occurring mainly due to the leaching to the cooking water in food preparations. However, in this case, it is more likely that the loss of folic acid in the white cream sauce is related to the hydrolysis of the vitamin in the cooked food matrix than to leaching, since the loss of the vitamin to the cooking water is unlikely as the water remained incorporated into the food preparation while it was being made and even after the cooking process was finished.

The variation in folic acid concentration in the food preparations made with wheat flour (Fig 4) also showed a relationship with the cooking method used; however, it is further believed that the preparation stage of the mixture can also contribute to a higher or lower concentration of the vitamin. The observed folic acid retention for bread loaf (87%) was higher when compared to the wheat flour cake (80%) and white cream sauce (57%). When comparing the wheat flour cake and bread loaf preparations, it is observed that both were submitted to the same heat treatment conditions (180° C / 40 minutes), however, the preparation method used for the bread dough, which uses the mechanical energy passed on to the dough during the kneading to induce the breakage and formation of bonds, results in the formation of a gluten network [24]. This network plays an important role, in affecting the water absorption capacity (which, in this case, is higher), cohesion, viscosity, and elasticity of the dough [25].

It is believed that the formation of the gluten network in the bread loaf, which also happens on the wheat flour cake (in a smaller degree), had an influence in the retention of folic acid in the food matrix, being the retention higher on the bread loaf due to the greater mechanical work applied when preparing the dough that was, therefore able to absorb more water than the cake and thus have a better connection of the vitamin with the water that was retained in the gluten network formed. Folic acid has a water-soluble nature and, if during the cooking process by immersion in water it is lost by being diffused in the cooking water, in the case of bread and wheat cake, it can be retained by the water present in the gluten network.

Regarding the preparations made with fortified corn flour, both preparations, corn cake and couscous, showed the best folic acid retentions in the present study. The statistical test (Fig 5) showed that the differences between the two preparations were not significant and, therefore, there were no significant losses of folic acid.

The higher folic acid retention in the corn flour cake can be explained by the formation of a protein network, similar to the one found on the bread loaf and the wheat flour cake, which allows for there to be an interaction between the network components and the folic acid, entrapping the vitamin in the food matrix. The protein network observed in the corn flour preparations is formed by the Zein protein which, when exposed to temperatures higher than 35°C, loses its native state, turning into a new structural arrangement with structural changes similar to the ones observed in the viscoelastic polymers of gluten [26].

According to Penalva et al. [9] there is no information that suggests the presence of protein-ligand binding sites for the folic acid with the Zein, however, according to the results of the study involving the creation of Zein nanoparticles for the oral delivery of folic acid, the authors suggest that a fraction of the folic acid was stabilized inside the Zein protein matrix by the non-covalent bonds. Therefore, the higher folic acid retention obtained in the corn flour cake can be explained by the interaction between the folic acid with the hydrophobic aminoacids of the Zein which prevents the oxidation of the vitamin.

The evaluation of folic acid stability in preparations developed with enriched flours was also studied by Phillips [27]. When evaluating tortillas and chip tortillas made from fortified cornmeal, the author found a 13% loss of folic acid. in cooked tortillas, but no loss during frying leading him to conclude that folic acid is relatively stable to the dry heat cooking process.

In Ireland, a study conducted to quantify folic acid reduction during baking of four different types of commercial breads [16] found that the thermal degradation of folic acid was between 21.9% and 32, 1%, is the percentage of vitamin degradation similar for white bread, baguettes, and brown soda and significantly higher for whole-grain bread when compared to other types of bread tested.

In another study by Silveira et al. [28] the content and stability of folic acid were verified in fortified rice after four different cooking methods (frying, boiling, microwave and boiling in food service). After analysis the authors concluded that the lowest folic acid content (0.17mg / kg) was found in food service rice and the highest retention (96.11%) in fried rice.

In spite of the limitation observed in paper availability concerning the subject, it is acknowledged that the methods that best preserve the content of folic acid are those that use dry-heat, since the moist-heat methods, especially those that immerse the food in the cooking water, contribute to higher losses. In this way, the use of cooking techniques that better preserve the folic acid content in foods, or even the ingestion of the cooking water from the food prepared by immersion, contribute to its greater preservation/retention in cooked food and, consequently, an adequate ingestion of the required amount of the vitamin, thus avoiding the manifestation of pathologies due to its deficiency or low intake.

Food fortification has to be considered as an important tool for the population to reach its minimum nutrient requirements as recommended in food policy interventions [19], however, the monitoring and controlling of the amount of the micronutrient added to foods that go through this technological process are of extreme importance to guarantee the commercialization of these products containing the actual amount of micronutrients required by legislation and stated on the food labels.

It should also be emphasized that enriched flours are used as ingredients for the preparation of different foods and that, during food preparation for consumption, vitamin losses may occur due to degradation or leaching of the vitamin, which will have a direct impact on the consumption of the added micronutrient [18].

Therefore, good control of how micronutrients are added in the final food products is necessary to guarantee the quality of processed foods and for the control and regulation of public policies [19]. Although the determination of the folic acid content in fortified wheat and corn flours has a significant effect on the practice of fortification performed by industries worldwide, it has received little attention in Brazil.

The results reported in the present study provide important information about the practice of folic acid fortification of wheat and corn flours carried out in Brazilian industries, especially those flours commercialized in the Northeast of the country, contributing to reinforcing the necessity of the Brazilian authorities to better monitor the fortification process of farinaceous matrices.

5. Conclusions

Commercially fortified wheat and corn flours showed the absence of industrially added folic acid. That is believed to happen due to the homogenization process carried out in the industries. The results reported show a need to monitor the folic acid fortification programs of farinaceous matrices, especially when considering no detection of the vitamin in all the food products analysed.

Different cooking techniques cause significant losses of folic acid, but these losses are related to the complexity of the food matrix as well as the cooking method applied. Additional studies investigating the stability of folic acid in other preparations commonly consumed by the population and using various cooking methods are necessary not only to improve the techniques in order to increase the folic acid retention in the cooked food to be consumed, but also to update the food composition databases and thus provide further useful information for calculations of dietary intake and nutritional adequacy.

The information on the stability of folic acid in fortified foods that have been submitted to different cooking techniques is limited and the majority of the studies are focused on investigating the influence of different cooking methods under the folate content in vegetables. Therefore, there is a need for more research on evaluations of the stability of folic acid in foods prepared from fortified matrices, especially wheat and corn flours that are commonly used as ingredients for the preparation of different foods regularly consumed by the population.

15 Oct 2019

PONE-D-19-26781

Determination of folic acid by HPLC in fortified wheat and corn flour and evaluation of its retention in preparations submitted to different cooking methods

PLOS ONE

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3.  Please include in your Methods section vendor information about the ingredients used and the flours tested, in order to ensure reproducibility of the results by other researchers. 2

4.  Please consider whether the graphs in figures 3 and 4 should have the data points connected by lines or not.

5.  Thank you for stating the following financial disclosure:  "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please provide an amended Funding Statement that declares *all* the funding or sources of support received during this specific study (whether external or internal to your organization) as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.

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Additional Editor Comments (if provided):

I have completed my evaluation of your manuscript. Although I am recommending revision, you have a major task to radically improve the content of your paper and its organization and interpretation. Each of the reviewer comments need to be addressed individually and listed. The paper is really a borderline for rejection at this stage.

other comments:

The statistical analysis is missed in different illustrations (Table 2 and Table3).

The manuscript includes several grammar and syntax errors and should be revised by a specialist.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Partly

Reviewer #6: No

Reviewer #7: Yes

Reviewer #8: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: Yes

Reviewer #5: I Don't Know

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

Reviewer #6: No

Reviewer #7: Yes

Reviewer #8: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: • The Title: clearly describe the article

• Abstract: it reflect the content of the article

it describe what the author hoped to achieve accurately, and clearly state the problem being investigated and summarize relevant research to provide context, and explain what other authors’ findings, It describe the experiment, the hypothesis(es) and the general experimental design or method.

The author explain how the data was collected and analyzed there is sufficient information present for the readers, the article identify the procedures the method are advanced and new.

• The author/s explain in words what he discovered in the research. It should be clearly laid out and in a logical sequence. the statistics are correct

• Conclusion/Discussion: the claims in this section supported by the results, they seem reasonable, the authors indicated how the results relate to expectations and to earlier research.

It is well integrated with current research

• Language: Article is good written

• The Tables and figures describe the data accurately

Accepted

Reviewer #2: it is my pleasure to review the manuscript entitles "Determination of folic acid by HPLC in fortified wheat and corn flour and evaluation of its retention in preparations submitted to different cooking methods", this paper should become acceptable for publication. So my decision is accept the publication.

Reviewer #3: 1-Line 54 till 55:(Your low ingestion) not giving a clear meaning that lack of folic acid or its deficiency can cause series diseases, I recommend re-writing these lines.

2-page 9 in Identification and quantification:The authors didn't mention the detector type, is it Diode array or UV detector to perform such spectral similarities.

3-Page 13 (line 326) the word maize has to be corrected to corn.

4- Page 20 (line 437): biting site has to be corrected as binding site.

5-In Fig. 3 & 4 in the supporting information: you have to name the Y-axis.

Reviewer #4: Reviewer Comment

EMMANUELA AZEVEDO and his coauthors wrote a manuscript about their investigation on Determination of folic acid by HPLC in fortified wheat and corn flour and evaluation of its retention in preparations submitted to different cooking methods. The above mentioned goal has been achieved in a clear way. However, the manuscript requires revision though it may eventually be publishable in this journal.

The manuscript needs English editing; some sentences are not clear due to mistakes (e.g. punctuation, grammatical or typos errors).

General comments:

• The authors must follow the formatting of the Journal as indicated in the authors guidelines.

This implies for reference formatting and numbering of sections of the manuscript.

• The authors should further compare their results with other works on fortified wheat and corn flour

• Another method of preparation using moist heat should have been added to strengthen your results not just only one.

• Tables and figures have to be self-explanatory. All abbreviations have to be explained in each table/figure.

Specific comments:

In the whole manuscript: μg.mL-1 must be changed to μg mL-1 (without a dot inbetween and -1 should be superscript) or change to μg/mL

Why do you change from μg to g and not to mg as in line 106 and other places ‘Approximately 5,000 μg (0.0050 g) of folic acid’??? why not be “Approximately 5,000 μg (5 mg) of folic acid”…etc.

Line 38 add ‘to’ after ‘submitted’

Line 44 remove the before zein and make it capital ‘Zein’

Line 45 remove ‘of’ before 87% and change ‘the on bread’ to ‘on the bread’

Line 47 change ‘the mixture’ to ‘mixing

Line 54: remove ‘Your’

Line 75 and 76: remove ‘the’ before ‘the method’ and ‘the cooking time’.

Line 159: add a space between the 8 and g in ‘8 g’

Line 205: what is the meaning of ‘Then the samples were centrifuged in 5000 g rotation’ is it ‘Then the samples were centrifuged at 5000 rpm rotation’

Line 256: ‘The methodology applied allowed for there to be retention of folic acid’ change ‘for there to be’ to ‘for the’.

Line 257: why was 290 nm used for measuring the absorptivity and not 280 nm, was it in the literature.

Line 268: Fig. 1 Chromatogram o commercially fortified wheat flour and corn flour. I think this ‘o’ is ‘of’

In Fig. 3 and 4 what does mcg/g stand for, if it is microgram then it has to be written as ‘μg/g’.

There were newer versions of ICH guidelines than 1994 please consider.

• It is better to switch the paragraph starting at line 444-450 to the end of the discussion.

The manuscript covers an interesting topic with interesting results. I would recommend this manuscript for publication after major revision.

Reviewer #5: please find it also as the attached file for author

Dear authors

Please find the following suggestions and I hope to enhance your paper for good publication ‎

Introduction ‎

‎1-‎ Please highlight the important of folic acid for the human health and the negative effect ‎of its deficiency to spotlight the important of your work ‎

‎2-‎ Please highlight‎ some of the previous reports discussed thermal effect on the retention of ‎folic acid and other micronutrients through the different cooking methods

Material and methods

‎1.‎ Samples and reagents

‎1-‎ Lines (88-90): Commercially fortified wheat and corn flours and laboratory fortified ‎wheat and corn flours were evaluated in the laboratory. (what the added value of this ‎sentence please delete it)‎

‎2-‎ ‎Lines (91-92): ‎ different brands of wheat flour (WF); different brands of corn flour (CF) ‎‎(please add the name of used brands).‎

‎3-‎ Lines (95): ‎item 2.2. (there is no 2.2. please indicate it as 2)‎

‎2.‎ Protocol for laboratory folic acid fortification of wheat and corn flour

‎1-‎ Lines (108): ‎a concentration of 1000 μg of folic acid for every 100 g of wheat or corn ‎flour. (Why you exceeded the Brazilian recommended dose; a maximum limit (220 ‎μg.100g-1) for addition of the vitamin in order to avoid exposure of the population to an ‎unnecessary risk (BRAZIL, 2017). ‎

‎2-‎ ‎Lines (109-110): ‎two procedures of homogenization were used (I recommended the first ‎procedure only because it is the simplest and applicable one either for individual or ‎industrial use, so please delete the second procedure).‎

‎3-‎ ‎ Lines (124-127): ‎At the end of the fortification procedure, the total time used for the ‎homogenization of the vitamin into the farinaceous matrix, including manual (1 min for ‎each 100 g) and mechanical homogenization (1 min for every 100 g + 2 minutes for 500 ‎g), was of 12 minutes. (Why you mentioned this sentence to compare the spent time and ‎what about the multiple steps and tools is it ok for industrial application??)‎

‎4-‎ Lines (128): ‎packages to prevent degradation (please describe the full conditions (temp. ‎container material and color, humidity, etc...) you used to prevent degradation ‎ for ‎storage until used).‎

‎3.‎ Food preparations

‎1-‎ Lines (133): ‎laboratory folic acid fortified wheat flour and corn flour, (which one you ‎used? Homogenized with procedure 1 or 2).‎

‎4.1. Sample extraction‎

‎1- Lines (200-201): ‎The methodological procedure was a solid-liquid extraction developed ‎and used for plant matrices by Paiva et al. (PAIVA et al., 2012). (Please replace it to; The ‎folic acid was extracted from the samples (fortified wheat flour and corn flour, raw ‎preparations, and cooked preparations) following the methods of PAIVA et al. (2012).‎

‎2-‎ Lines (205): ‎ in 5000 g rotation (please replace in with at)‎

‎4.2. Chromatographic conditions‎

‎1-‎ Please indicate the full description of HPLC equipment used in this analysis with the ‎country of made, the type of the detector used and wavelength at which the folic acid ‎was detected. Also, indicate the type and specification of the used column.‎

‎2-‎ Line (218); The column was washed with water and conditioned with acetonitrile ‎‎(Merck-grade HPLC) at the end of the analyses (PAIVA et al., 2012). (Please delete ‎this sentence and move the reference in the right place) ‎

‎4.3.Identification and quantification‎

‎1-‎ ‎(1000 μg.mL-1 folic acid) (please replace it with (1000 μg.mL-1 folic acid)‎

‎5.True retention ‎

‎(MURPHY; CRINER, GRAY, 1975) (Please correct it to Murphy‎‏ ‏et al‏., 1975)‏

Results and discussion

‎1-‎ ‎Line (256-257);‎The methodology applied allowed for there to be retention of folic acid in ‎‎18.2 minutes at maximum absorptivity in 290nm. (Please delete it because you mentioned ‎it in the methodology section).‎

‎2-‎ Lines (265-266);‎‏ ‏‎1. Determination of folic acid in commercially fortified and laboratory ‎fortified wheat and corn flours‎‏ ‏‎(please delete it) ‎

‎3-‎ ‎‏ ‏Line (268); ‎Fig. 1 Chromatogram o commercially fortified wheat flour and corn flour. ‎‎(Please make it: HPLC chromatogram of commercially fortified wheat flour and corn ‎flour evaluated in July (A) and September (B)).‎

‎4-‎ ‎‏ ‏Lines (269-274);‎ ‎ Chromatograms for one of the samples of commercially fortified wheat ‎flour and corn flour evaluated in July (A) and one of the wheat flour and corn flour ‎samples evaluated in September (B) Chromatographic conditions: C18 (150 x 4.6 mm ‎‎5μm) chromatographic column (Allcrom Phenomenex) with the oven at 40 °C, isocratic ‎elution using potassium phosphate buffer (pH 2.0) and methanol (85:15) as mobile phase ‎at a flow of 0.5 mL/min-1 and UV detection with wavelength of 290 nm. (Please delete it ‎and mentioned it in the methodology section‎‏ ‏as general condition for all HPLC analysis‏), ‏and apply it for the other figures‏.‏

‎5-‎ Lines (276);‎ (1.000μg.100g-1) ( Please correct it to ‎1000μg.100g-1‎‏) ‏and check it through ‎the paper‏ ‏

‎6-‎ ‎ the presentation of the results is not acceptable because of:‎

‎ 1- Using different units for folic acid in different tables and figures

‎2- Content of folic acid in the raw preparations in table 1 is different than in table 3 for ‎example for Bread Loaf is 431 ± 0,4(μg.100g-1) ± SD in table 1 and is 0,43 ± 0,030 (g/100g) ‎‎± DP even 431μg equal 0.000431‬g and what is the meaning of DP. ‎

‎3- Repeated data on the tables and figures, figures 3, 4 derived from table 3, so table 3 is ‎enough

‎4- Table 1 contains extra statistical data have no effect on the study

‎5- Cooking conditions columns in Table 3 did not give any information as it as the same ‎mentioned in materials and methods and are not variable studied conditions ‎

‎7-‎ The folic acid peak in figure 2 is not strong peak for trusted quantification of folic acid as ‎the other peaks in the chromatogram are at so near or at the same height. (I highly ‎recommended the preparation of standard curve for folic acid inside each raw ‎preparation. After that, you can use this standard curve for quantification of folic acid ‎inside the raw and cooked preparations. ‎

‎8-‎ Using of standard curve for folic acid inside each raw preparation will verify the retention ‎time for folic acid on the chromatogram and will give you the recovery percent of the ‎solvent used for folic acid extraction from the preparation and it will answer you about ‎the big variation on the folic acid quantities on your different preparations as the ‎extraction method has a strong effect on the recovery content of folic acid. ‎

‎9-‎ The interpretation of discussion needs more supporting results for discussing the ‎variable ‎results ‎against the different conditions of cooked preparation of temperature ‎and ‎foodstuff added per each ‎preparation. ‎ ‎

Reviewer #6: The MS needs depth revision and English editing. The aims of the study are clear, but the Introduction does not review in depth the problematic and the hypothesis of the MS. I think that the MS is not sufficiently novel and interesting to warrant publication. The work is not original.

Reviewer #7: The manuscript concerned with determination of Folic acid content in fortified wheat and corn flours and their folic acid

retention in food preparations by using cooking methods: baking, deep

frying and steaming.

In the corn flour preparations, Folic acid retention is high compared to Wheat flour preparations Why?

The author should do another experiment in which a mixture of folic acid fortified corn and wheat flours are mixed together to testify or investigate the effect of mixing corn with wheat in Foods on the stability or retention of folic acid.

Reviewer #8: The idea is good.

There is no matching research

The language of the manuscript is good with some simple mistakes.

The methods do not have technical errors that spoil the search.

But:

Failure to standardize the expression of concentrations. Makes it difficult to review the results

Data transformation has reduced the variance between treatments, which requires statistical re-analysis

This has led to a change in results but will not affect the overall direction of the findings or final conclusions.

There are some problems with references (in formulation and completeness only).

**********

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Reviewer #1: No

Reviewer #2: Yes: Doha H Abou Baker

Reviewer #3: No

Reviewer #4: Yes: Marwa Y. Issa

Reviewer #5: No

Reviewer #6: No

Reviewer #7: No

Reviewer #8: Yes: Sabry A. AbdAllah

Lecturer of pesticides chemistry and toxicology

plant protection department, Faculty of Agriculture

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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Submitted filename: Report for Editor ,PONE-D-19-26781.docx

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Submitted filename: Manuscript.docx

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3 Jan 2020

Dear editorial below I send as replies of the comments and suggestions of the reviewers. For each comment below follows an answer. We appreciate all the considerations you have made and we seek the ultmost from all that has been requested.

All line locations were made by the document "Revised manuscript with track changes"

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Answer: The manuscript was reviewed by an expert in the English language.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

Answer: We fulfill this request.

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

Answer: We fulfill this request.

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

Answer: We fulfill this request.

3. Please include in your Methods section vendor information about the ingredients used and the flours tested, in order to ensure reproducibility of the results by other researchers.

Answer: We fulfill this request. Line 96 to 98

4. Please consider whether the graphs in figures 3 and 4 should have the data points connected by lines or not.

Answer: In fact the points should not be linked as the treatments are independent. Figures 3 and 4 have been changed.

5. Thank you for stating the following financial disclosure: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

6. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical.

Answer: Was made

7. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

Answer: Was made

Additional Editor Comments (if provided):

I have completed my evaluation of your manuscript. Although I am recommending revision, you have a major task to radically improve the content of your paper and its organization and interpretation. Each of the reviewer comments need to be addressed individually and listed. The paper is really a borderline for rejection at this stage.

Answer: We appreciate that you have considered this manuscript for further revision.

other comments:

The statistical analysis is missed in different illustrations (Table 2 and Table3).

Answer: For the construction of tables 2 and 3, only the mean and standard deviation evaluation was used.

The manuscript includes several grammar and syntax errors and should be revised by a specialist.

Answer: The manuscript was reviewed by an expert in the English language.

Reviewer #1:

The Title: clearly describe the article

• Abstract: it reflect the content of the article

it describe what the author hoped to achieve accurately, and clearly state the problem being investigated and summarize relevant research to provide context, and explain what other authors’ findings, It describe the experiment, the hypothesis(es) and the general experimental design or method.

The author explain how the data was collected and analyzed there is sufficient information present for the readers, the article identify the procedures the method are advanced and new.

• The author/s explain in words what he discovered in the research. It should be clearly laid out and in a logical sequence. the statistics are correct

• Conclusion/Discussion: the claims in this section supported by the results, they seem reasonable, the authors indicated how the results relate to expectations and to earlier research.

It is well integrated with current research

• Language: Article is good written

• The Tables and figures describe the data accurately

Accepted

Answer: Thank you for your considerations.

Reviewer #2:

it is my pleasure to review the manuscript entitles "Determination of folic acid by HPLC in fortified wheat and corn flour and evaluation of its retention in preparations submitted to different cooking methods", this paper should become acceptable for publication. So my decision is accept the publication.

Answer: Thank you for your considerations.

Reviewer #3:

1-Line 54 till 55:(Your low ingestion) not giving a clear meaning that lack of folic acid or its deficiency can cause series diseases, I recommend re-writing these lines.

Answer: The paragraph has been rewritten, check lines 60 through 62.

2-page 9 in Identification and quantification:The authors didn't mention the detector type, is it Diode array or UV detector to perform such spectral similarities.

Answer: All equipment information including the detector description has been entered in item 4.2. Chromatographic conditions, lines 224 to 229.

3-Page 13 (line 326) the word maize has to be corrected to corn.

Answer: Change made, check line 344

4- Page 20 (line 437): biting site has to be corrected as binding site.

Answer: Change made, check line 455 and 456

5-In Fig. 3 & 4 in the supporting information: you have to name the Y-axis.

Answer: Y axis has been named, check the changes in figures 3 and 4

Reviewer #4:

Reviewer Comment

EMMANUELA AZEVEDO and his coauthors wrote a manuscript about their investigation on Determination of folic acid by HPLC in fortified wheat and corn flour and evaluation of its retention in preparations submitted to different cooking methods. The above mentioned goal has been achieved in a clear way. However, the manuscript requires revision though it may eventually be publishable in this journal.

The manuscript needs English editing; some sentences are not clear due to mistakes (e.g. punctuation, grammatical or typos errors).

Answer: The article was reviewed by an English language expert. We hope to have better manuscript quality

General comments:

• The authors must follow the formatting of the Journal as indicated in the authors guidelines.

This implies for reference formatting and numbering of sections of the manuscript.

Answer: We reviewed the article regarding the format of the magazine and met the two items mentioned.

The authors should further compare their results with other works on fortified wheat and corn flour

Answer: We seek to discuss our results using references that have also worked with these matrices, as can be seen from Lines 323 to 329.

Another method of preparation using moist heat should have been added to strengthen your results not just only one.

Answer: Answer: In fact, during a statistical assessment it is observed that the data are stronger if there are more matrices for heat humid. However, our findings have a significant value for the scarce information on cooked and prepared foods, thus maintaining the results of the co-study method in the study.

Tables and figures have to be self-explanatory. All abbreviations have to be explained in each table/figure.

Answer: The tables presented contain the values obtained in the analyzed material. Table 1 refers to the method parameters. We seek to address the discussion of results by focusing on the most relevant data and discussing them, so we hope that the reader can make better use of the material.

Specific comments:

In the whole manuscript: μg.mL-1 must be changed to μg mL-1 (without a dot inbetween and -1 should be superscript) or change to μg/mL

Answer: A requested correction has been made.

Why do you change from μg to g and not to mg as in line 106 and other places ‘Approximately 5,000 μg (0.0050 g) of folic acid’??? why not be “Approximately 5,000 μg (5 mg) of folic acid”…etc.

Answer: We seek to present the results also in grams because we are dealing with food prepared for consumption. In this sense, the reader could extrapolate a faster interpretation of nutrient availability in food per gram of food.

Line 38 add ‘to’ after ‘submitted’

Answer: A requested correction has been made.

Line 44 remove the before zein and make it capital ‘Zein’

Answer: A requested correction has been made.

Line 45 remove ‘of’ before 87% and change ‘the on bread’ to ‘on the bread’

Answer: A requested correction has been made.

Line 47 change ‘the mixture’ to ‘mixing

Answer: A requested correction has been made.

Line 54: remove ‘Your’

Answer: A requested correction has been made.

Line 75 and 76: remove ‘the’ before ‘the method’ and ‘the cooking time’.

Answer: A requested correction has been made.

Line 159: add a space between the 8 and g in ‘8 g’

Answer: A requested correction has been made.

Line 205: what is the meaning of ‘Then the samples were centrifuged in 5000 g rotation’ is it ‘Then the samples were centrifuged at 5000 rpm rotation’

Answer: Samples were centrifuged at 5000 g as described in line 218.

Line 256: ‘The methodology applied allowed for there to be retention of folic acid’ change ‘for there to be’ to ‘for the’.

Answer: A requested correction has been made.

Line 257: why was 290 nm used for measuring the absorptivity and not 280 nm, was it in the literature.

Answer: corrected to 280nm, it was a typo.

Line 268: Fig. 1 Chromatogram o commercially fortified wheat flour and corn flour. I think this ‘o’ is ‘of’

Answer: A requested correction has been made.

In Fig. 3 and 4 what does mcg/g stand for, if it is microgram then it has to be written as ‘μg/g’.

Answer: A requested correction has been made.

There were newer versions of ICH guidelines than 1994 please consider.

Answer: Answer: In fact there is a new version, but the one we had access to was 1994.

It is better to switch the paragraph starting at line 444-450 to the end of the discussion.

Answer: A requested correction has been made.

The manuscript covers an interesting topic with interesting results. I would recommend this manuscript for publication after major revision.

Answer: thank you for your considerations.

Reviewer #5:

Dear authors

Please find the following suggestions and I hope to enhance your paper for good publication ‎

Introduction ‎

‎1-‎ Please highlight the important of folic acid for the human health and the negative effect ‎of its deficiency to spotlight the important of your work ‎

Answer: We add one more highlight about the value of vitamin in human food.

‎2-‎ Please highlight‎ some of the previous reports discussed thermal effect on the retention of ‎folic acid and other micronutrients through the different cooking methods

Answer: We did not find articles where flour preparations were produced for the evaluation of retained folic acid content.

Material and methods

‎1.‎ Samples and reagents

‎1-‎ Lines (88-90): Commercially fortified wheat and corn flours and laboratory fortified ‎wheat and corn flours were evaluated in the laboratory. (what the added value of this ‎sentence please delete it)‎

Answer: A requested correction has been made.

‎2-‎ ‎Lines (91-92): ‎ different brands of wheat flour (WF); different brands of corn flour (CF) ‎‎(please add the name of used brands).‎

Answer: A requested correction has been made.

‎3-‎ Lines (95): ‎item 2.2. (there is no 2.2. please indicate it as 2)‎

Answer: A requested correction has been made.

‎2.‎ Protocol for laboratory folic acid fortification of wheat and corn flour

‎1-‎ Lines (108): ‎a concentration of 1000 μg of folic acid for every 100 g of wheat or corn ‎flour. (Why you exceeded the Brazilian recommended dose; a maximum limit (220 ‎μg.100g-1) for addition of the vitamin in order to avoid exposure of the population to an ‎unnecessary risk (BRAZIL, 2017). ‎

Answer: We had a lot of difficulty with a homogenization step (this was described in the material and methods item) One of the strategies we found was to increase the amount of vitamin added to flour to reduce the effects of this step.

‎2-‎ ‎Lines (109-110): ‎two procedures of homogenization were used (I recommended the first ‎procedure only because it is the simplest and applicable one either for individual or ‎industrial use, so please delete the second procedure).‎

Answer: In this study the two homogenization protocols were applied in order to verify the process difficulties and better condition. We do not find it useful to remove one of these descriptions since these tests are part of the effort made in the study.

‎3-‎ ‎ Lines (124-127): ‎At the end of the fortification procedure, the total time used for the ‎homogenization of the vitamin into the farinaceous matrix, including manual (1 min for ‎each 100 g) and mechanical homogenization (1 min for every 100 g + 2 minutes for 500 ‎g), was of 12 minutes. (Why you mentioned this sentence to compare the spent time and ‎what about the multiple steps and tools is it ok for industrial application??)‎

Answer: We describe in detail the homogenization step due to the absence of folic acid in commercial samples. Our homogenization protocol is experimental and its viability in the industry needs to be evaluated.

‎4-‎ Lines (128): ‎packages to prevent degradation (please describe the full conditions (temp. ‎container material and color, humidity, etc...) you used to prevent degradation ‎ for ‎storage until used).‎

Answer: we replaced the text with: The fortified flours were packaged under the same conditions as those applied in industry.

‎3.‎ Food preparations

‎1-‎ Lines (133): ‎laboratory folic acid fortified wheat flour and corn flour, (which one you ‎used? Homogenized with procedure 1 or 2).‎

Answer: We opted for the first homogenization assay. We clarify this on lines 128 and 309 to 312.

‎4.1. Sample extraction‎

‎1- Lines (200-201): ‎The methodological procedure was a solid-liquid extraction developed ‎and used for plant matrices by Paiva et al. (PAIVA et al., 2012). (Please replace it to; The ‎folic acid was extracted from the samples (fortified wheat flour and corn flour, raw ‎preparations, and cooked preparations) following the methods of PAIVA et al. (2012).‎

Answer: A requested correction has been made.

‎2-‎ Lines (205): ‎ in 5000 g rotation (please replace in with at)‎

Answer: A requested correction has been made.

‎4.2. Chromatographic conditions‎

‎1-‎ Please indicate the full description of HPLC equipment used in this analysis with the ‎country of made, the type of the detector used and wavelength at which the folic acid ‎was detected. Also, indicate the type and specification of the used column.‎

Answer: This description was made on lines 228 through 233.

‎2-‎ Line (218); The column was washed with water and conditioned with acetonitrile ‎‎(Merck-grade HPLC) at the end of the analyses (PAIVA et al., 2012). (Please delete ‎this sentence and move the reference in the right place) ‎

Answer: A requested correction has been made.

‎4.3.Identification and quantification‎

‎1-‎ ‎(1000 μg.mL-1 folic acid) (please replace it with (1000 μg.mL-1 folic acid)‎

Answer: A requested correction has been made.

‎5.True retention ‎

‎(MURPHY; CRINER, GRAY, 1975) (Please correct it to Murphy‎‏ ‏et al‏., 1975)‏

Answer: A requested correction has been made.

Results and discussion

‎1-‎ ‎Line (256-257);‎The methodology applied allowed for there to be retention of folic acid in ‎‎18.2 minutes at maximum absorptivity in 290nm. (Please delete it because you mentioned ‎it in the methodology section).‎

Answer: A requested correction has been made.

‎2-‎ Lines (265-266);‎‏ ‏‎1. Determination of folic acid in commercially fortified and laboratory ‎fortified wheat and corn flours‎‏ ‏‎(please delete it) ‎

Answer: A requested correction has been made.

‎3-‎ ‎‏ ‏Line (268); ‎Fig. 1 Chromatogram o commercially fortified wheat flour and corn flour. ‎‎(Please make it: HPLC chromatogram of commercially fortified wheat flour and corn ‎flour evaluated in July (A) and September (B)).‎

Answer: A requested correction has been made.

‎4-‎ ‎‏ ‏Lines (269-274);‎ ‎ Chromatograms for one of the samples of commercially fortified wheat ‎flour and corn flour evaluated in July (A) and one of the wheat flour and corn flour ‎samples evaluated in September (B) Chromatographic conditions: C18 (150 x 4.6 mm ‎‎5μm) chromatographic column (Allcrom Phenomenex) with the oven at 40 °C, isocratic ‎elution using potassium phosphate buffer (pH 2.0) and methanol (85:15) as mobile phase ‎at a flow of 0.5 mL/min-1 and UV detection with wavelength of 290 nm. (Please delete it ‎and mentioned it in the methodology section‎‏ ‏as general condition for all HPLC analysis‏), ‏and apply it for the other figures‏.‏

Answer: A requested correction has been made.

‎5-‎ Lines (276);‎ (1.000μg.100g-1) ( Please correct it to ‎1000μg.100g-1‎‏) ‏and check it through ‎the paper‏ ‏

Answer: A requested correction has been made.

‎6-‎ ‎ the presentation of the results is not acceptable because of:‎

‎1- Using different units for folic acid in different tables and figures

Answer: All material has been reviewed and as standard units of measurement.

‎2- Content of folic acid in the raw preparations in table 1 is different than in table 3 for ‎example for Bread Loaf is 431 ± 0,4(μg.100g-1) ± SD in table 1 and is 0,43 ± 0,030 (g/100g) ‎‎± DP even 431μg equal 0.000431‬g and what is the meaning of DP.

‎ Answer: All material has been reviewed and as standard units of measurement. Standard deviation was added.

‎3- Repeated data on the tables and figures, figures 3, 4 derived from table 3, so table 3 is ‎enough

Answer: Chromatogram figures do not allow quantitative measurement. This information is contained only in the tables.

‎4- Table 1 contains extra statistical data have no effect on the study

Answer: We could not identify which statistical information did not belong to the study and which was presented in table 1.

‎5- Cooking conditions columns in Table 3 did not give any information as it as the same ‎mentioned in materials and methods and are not variable studied conditions ‎

Answer: In table 3, we highlight the temperatures for each cooking process already known, the longer or the higher the temperature that decreases the nutrients. Therefore, we consider this information useful for a better interpretation of the data obtained.

‎7-‎ The folic acid peak in figure 2 is not strong peak for trusted quantification of folic acid as ‎the other peaks in the chromatogram are at so near or at the same height. (I highly ‎recommended the preparation of standard curve for folic acid inside each raw ‎preparation. After that, you can use this standard curve for quantification of folic acid ‎inside the raw and cooked preparations.

Answer: It is very common for a color chromatogram to have higher or lower intensity signals for what was not part of the study. The important thing is that the analyte of interest is totally separate from any other substance. In our chromatograms we have the isolated folic acid peak and the other peaks obtained.

‎

‎8-‎ Using of standard curve for folic acid inside each raw preparation will verify the retention ‎time for folic acid on the chromatogram and will give you the recovery percent of the ‎solvent used for folic acid extraction from the preparation and it will answer you about ‎the big variation on the folic acid quantities on your different preparations as the ‎extraction method has a strong effect on the recovery content of folic acid. ‎

Answer: In fact the extraction method has significant effect on the success of the analysis. In this sense, we made the best effort to investigate all situations that could make the analysis difficult and we bring this discussion throughout the work.

‎9-‎ The interpretation of discussion needs more supporting results for discussing the ‎variable ‎results ‎against the different conditions of cooked preparation of temperature ‎and ‎foodstuff added per each ‎preparation. ‎ ‎

Answer: we add one more paragraph and more references on the subjects cited.

Reviewer #6:

The MS needs depth revision and English editing. The aims of the study are clear, but the Introduction does not review in depth the problematic and the hypothesis of the MS. I think that the MS is not sufficiently novel and interesting to warrant publication. The work is not original.

Answer: Thank you for your considerations.

Reviewer #7:

The manuscript concerned with determination of Folic acid content in fortified wheat and corn flours and their folic acid

retention in food preparations by using cooking methods: baking, deep

frying and steaming.

In the corn flour preparations, Folic acid retention is high compared to Wheat flour preparations Why?

Answer: We hypothesized for this condition on lines 448 through 455.

The author should do another experiment in which a mixture of folic acid fortified corn and wheat flours are mixed together to testify or investigate the effect of mixing corn with wheat in Foods on the stability or retention of folic acid.

Answer: The description and necessity of this new experiment has not been clarified for us.

Reviewer #8:

The idea is good.

There is no matching research

The language of the manuscript is good with some simple mistakes.

The methods do not have technical errors that spoil the search.

Answer: Thank you for your considerations.

But: Failure to standardize the expression of concentrations. Makes it difficult to review the results

Data transformation has reduced the variance between treatments, which requires statistical re-analysis

Answer: The necessary corrections have been made regarding the expression of the folic acid concentration obtained

This has led to a change in results but will not affect the overall direction of the findings or final conclusions.

There are some problems with references (in formulation and completeness only).

Answer: A requested correction has been made.

Submitted filename: Response to Reviewers.docx

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28 Jan 2020

PONE-D-19-26781R1

Folic acid retention evaluation in preparations with wheat flour and corn submitted to different cooking methods by HPLC/DAD.

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

Reviewer #7: Partly

Reviewer #8: Yes

**********

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Reviewer #3: I Don't Know

Reviewer #7: I Don't Know

Reviewer #8: Yes

**********

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Reviewer #3: Yes

Reviewer #7: No

Reviewer #8: Yes

**********

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Reviewer #7: Yes

Reviewer #8: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: the authors have addressed my comments and explained how they collected their data but the type of column and its specifications used in HPLC analysis weren't mentioned in the chromatographic conditions (3.2) and were deleted in page12 so it has to be added.

Reviewer #7: (No Response)

Reviewer #8: The search is good, its language is intact. Statistical analysis is good. The idea is good.

But there are a few linguistic mistakes. And some references do not match the text or vice versa. Good job in the end.

**********

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Reviewer #3: No

Reviewer #7: No

Reviewer #8: Yes: Sabry AbdElMonem AbdAllah

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Submitted filename: Manuscript-with track changes.docx

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29 Feb 2020

Dear editorial below I send as replies of the comments and suggestions of the reviewers. For each comment below follows an answer. We appreciate all the considerations you have made and we seek the ultmost from all that has been requested.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

Answer: Thank you for your considerations.

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

Reviewer #7: Partly

Reviewer #8: Yes

Answer: Thank you for your considerations.

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

Reviewer #7: I Don't Know

Reviewer #8: Yes

Answer: Thank you for your considerations.

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #7: No

Reviewer #8: Yes

Answer: Thank you for your considerations.

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Answer: Thank you for your considerations.

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: the authors have addressed my comments and explained how they collected their data but the type of column and its specifications used in HPLC analysis weren't mentioned in the chromatographic conditions (3.2) and were deleted in page12 so it has to be added.

Reviewer #7: (No Response)

Reviewer #8: The search is good, its language is intact. Statistical analysis is good. The idea is good.

But there are a few linguistic mistakes. And some references do not match the text or vice versa. Good job in the end.

Answer: References have been revised to meet the journal's standards. Please check the manuscript with markings.

________________________________________

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Reviewer #3: No

Reviewer #7: No

Reviewer #8: Yes: Sabry AbdElMonem AbdAllah

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Mar 2020

Folic acid retention evaluation in preparations with wheat flour and corn submitted to different cooking methods by HPLC/DAD.

PONE-D-19-26781R2

Dear Dr. AZEVEDO,

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Additional Editor Comments (optional):

Reviewers' comments:

27 Mar 2020

PONE-D-19-26781R2

Folic acid retention evaluation in preparations with wheat flour and corn submitted to different cooking methods by HPLC/DAD.

Dear Dr. de Paiva Azevedo:

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