Folates in Asian Noodles: III. Fortification, Impact...

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Folates in Asian Noodles: III. Fortification, ImpactofProcessing,andEnhancementofFolate IntakesLAN T. T. BUI AND DARRYL M. SMALL

ABSTRACT: Asian noodles, a widely consumed staple food, were evaluated as potential vehicles for fortificationwith folic acid. Samples of white salted, yellow alkaline, and instant noodles, prepared under controlled laboratoryconditions, were fortified and folates were measured at each stage of processing using a microbiological assay.Although the 3 styles showed differing patterns of retention, overall losses were slightly more than 40% and weresimilar for all styles. White salted and yellow alkaline noodles showed no significant decrease in total folate contentduring production. In contrast, significant losses occurred for instant noodles during steaming and deep-fryingof the noodle strands. In all cases, substantial losses occurred during subsequent cooking of the dried noodles.Fortification at a rate of 50% of the reference value per serving resulted in retention of folate at levels correspondingto 30% following cooking, whereas unfortified noodles contributed less than 4% per serving. It is concluded thatfortifying Asian noodles provides an effective means for enhancing folate intake.

Keywords: Asian noodles, folate, fortification, nutrients, processing

Introduction

Folates are found in plants and some animal tissues, largely inthe form of conjugates with more than 1 molecule of glutamic

acid. The simplest naturally occurring folate is pteroyl glutamate; itcontains only 1 glutamate unit. This molecule is commonly referredto as folic acid and is the form of folate that is commercially avail-able and permitted for use in fortifying food products (Bailey 1991;FSANZ 2006).

Folates are generally thought of as belonging to the B group ofvitamins. However, folic acid has low water solubility under condi-tions of neutral pH. It is reported to be more soluble in acetic acid,phenol, pyridine, and solutions of alkaline hydroxides and carbon-ates as well as a limited number of organic solvents (Budavari 2001).Folates are essential for the synthesis of deoxyribonucleic acid andribonucleic acid, the genetic materials of cells, as well as for proteinmetabolism. As a part of the role in protein metabolism, folates areinvolved in the conversion of homocysteine to methionine. Highlevels of homocysteine have been linked to an increased risk of car-diovascular disease (Lucock 2000). In addition to being importantduring pregnancy, folates are essential for healthy red blood cells,bones, and hair, as well as nervous, digestive, and immune systems(Reavley 1998) and may reduce the incidence of cataract formationin the eyes (Kuzniarz and others 2001). There is increasing evidencethat folates play a role in protection against carcinogenesis (Kim1999).

Folate deficiency is associated with negative health effects; itresults from inadequate intake, defective absorption, abnormalmetabolism, or drug therapy conditions. When the broad issue offolate fortification of foods was reviewed (NHMRC 1995), it wasconcluded that further studies of retention are required. Pasta wasspecifically identified as a product requiring study, particularly the

MS2007055 Submitted 1/25/2007, Accepted 3/30/2007. Authors were withSchool of Life Sciences and Technology, Victoria Univ., Werribee Campus,PO Box 14428 MCMC, Victoria 8001, Australia. Direct inquiries to authorBui, now with the Australian Defence Organisation, Scottsdale, Tasmania7260 Australia (E-mail: [email protected]).

effect of cooking on pasta and noodles that have been fortified withfolate.

The stability of vitamins during food processing is influenced byvarious factors, including light, temperature, pH, moisture, and thepresence of certain food additives and ingredients (Gubler 1991;Bender 1997). Although Asian noodles might be expected to be agood source of thiamin, it was recently reported that it is relativelyunstable during the processing of Asian noodles. The amounts lostwere found to vary for different styles, and in the case of yellowalkaline noodles, the overall losses were as high as 97% (Bui andSmall 2007a).

In an investigation of commercial Asian noodles, no clear rela-tionship was established between the level of folate retained andproduct pH (Bui and Small 2007c). These findings are now extendedand the objective of this study was to provide an understanding ofthe stability of folate during the processing of the 3 predominantstyles of Asian noodles. The study monitored fortification and pro-duction of noodles at the various stages from flour to final product,thus encompassing the impact of cooking.

Materials and Methods

MaterialsThe chemicals, including enzyme preparations and vitamins,

used in product formulations and analytical procedures were of ana-lytical grade or of the highest purity available, unless otherwise spec-ified. A commercial flour (P-farina, Allied Defiance, NSW, Australia)was selected as it is unfortified and suited to noodle preparation.Folic acid (4-7866; Sigma Chemical Co., St. Louis, Mo., U.S.A.) wasused as chemical standard for analytical purposes as well as fortifica-tion. The culture of Lactobacillus casei (L. casei) subsp. Rhamnosus(ATCC 7469) was obtained from the Dept. of Microbiology, the Univ.of New South Wales, Sydney. α-Amylase (A-3176) was from Sigma.Lactobacilli broth AOAC (Nr 0901-15), folic acid casei medium (Nr0822-15), and chicken pancreas (Nr 0459-12) came from Difco Lab-oratories, Detroit, Mich., U.S.A.; and protease (subtilisin A, 10401)came from Megazyme Intl., Bray Co., Wicklow, Ireland.

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Folates in Asian noodles: III . . .

Preparation of noodle samplesNoodles were prepared using procedures based on those de-

scribed by Moss and others (1987). All steps were carried out un-der subdued lighting conditions in order to minimize the potentialimpact of light on vitamin retention. In general, the noodle makingmethods for the 3 styles have 4 common steps: mixing, sheeting,cutting, and drying. However, for instant noodles, after the cuttingstep, steaming and frying steps were also required. The preparationof each of the 3 styles of noodles is briefly described below.

Preparation of white salted noodlesThe ingredient formulation used for making white salted noodles

was 300.0 g flour, 96.0 g distilled water, and 9.0 g common table salt(sodium chloride). The salt was first dissolved in the water and thissolution was added to the flour during mixing. After mixing, theresultant dough had a crumbly consistency. Then the dough wasfirst formed into a dough sheet by a process of folding and passingthe crumbly dough through the rollers of the noodle machine (MOD150-1048534; Imperia, Ambrogio di Torino, Italy) several times. Thethickness of the sheet was reduced stepwise by passing between therollers of the noodle machine before cutting into strands. The freshnoodles were arranged upon trays lined with aluminum foil. Thenoodles were placed loosely in order to facilitate effective drying andthe trays were stored in a dehydrator at 40 ◦C for 24 h. The productwas then allowed to cool for 30 min in the ambient conditions of thelaboratory prior to being placed in airtight plastic bags or containersfor storage.

Preparation of yellow alkaline noodlesThe ingredient formulation was 300.0 g flour, 96.0 g distilled wa-

ter, and 3.0 g alkaline salts (often referred to as lye water or kan swi[kansui] and typically mixtures of sodium and potassium carbon-ates). The procedure for making yellow alkaline noodles was thesame as that for white salted noodles.

Preparation of instant noodlesThe ingredients used to make instant noodles were 200.0 g flour,

60.0 g distilled water, 0.2 g alkaline salts (Moss and others 1987). Palmoil was used to deep-fry the noodles. The procedure for making in-stant noodles was the same as that for yellow alkaline noodles at themixing and rolling steps. However, additional steps were steaming(2 min) over vigorously boiling water, frying (45 s/150 ◦C), draining(30 s), and cooling in an air flow for 20 min prior to packing andstorage.

Fortification of noodle samples with folic acidFolic acid was prepared as an aqueous solution of ethanol (20%),

which also contained NaOH (0.01 M). Dried folic acid (20 mg) wasdissolved in 500 mL of the solvent and incorporated into the formu-lation at a rate of 7.15 mL per 100 g fresh weight flour, correspondingto a level of 286 µg/100 g flour.

A further experiment was carried out for noodles fortified withfolate in which varying levels of fortification were compared. Forthis purpose, flour was fortified using a standard folic acid solutionat a concentration of 40 µg/mL. This solution was prepared by dis-solving 20 mg of dried folic acid into 500 mL of the solvent describedabove. Fortified noodles were made in the laboratory by adding dif-ferent volumes of the folic acid solution (3.57, 7.15, and 10.0 mL per100 g fresh weight flour) into the typical formulation of white saltednoodles. These corresponded to levels of fortification of 143, 286,and 400 µg/100 g, respectively.

Preparation of samples for folate analysesFor analysis purposes, flours did not require a pretreatment, while

noodle products were prepared as follows:

Grinding. All dry samples were ground to pass a 35-mesh screen(sieve aperture: 500 µm) prior to extraction of folate.

Cooking. Samples were cooked by placing noodles into asaucepan of gently boiling water (approximately 30 g of noodle/600 mL distilled water). After every minute, a strand of noodle wasremoved and immediately placed into water at ambient tempera-ture. The strand was then squeezed between 2 microscope slides.Noodles were considered to be fully cooked at the point where theuncooked core had just disappeared, that is, when a uniform colorand appearance could be observed upon squeezing.

Analysis proceduresThe moisture contents of all samples were measured by the air

oven method of the American Assn. of Cereal Chemists (AACC2005a). The pH values of samples were determined by the AACCstandard procedure (AACC 2005b). Extraction of samples was basedon the method described by Shrestha and others (2000), and the mi-crobiological assay was based on that by Arcot and others (2002a);both were used as modified by Bui and Small (2007b). Each samplewas analyzed for total folate (trienzyme treatment) and free folate(without enzyme treatment). Samples from replicate trials were an-alyzed at least in triplicate. All results were calculated to a dry weightbasis in order to facilitate the direct comparison of the results, par-ticularly for different sample types.

Results and Discussion

Fortification of flour and noodle samplesPrior to fortifying noodles, analysis of the flour confirmed that the

native folate content was relatively low in both total folate (18.2 ±0.8 µg/100 g) and free folate (13.8 ± 1.2 µg/100 g). In order to assessthe suitability of noodles as a potential vehicle for fortification, con-sideration was given to selection of appropriate levels of fortifica-tion. Within current Australian regulatory standards (FSANZ 2006),it is permissible to fortify a range of food products with folic acid.In addition, there is no designated limit on the amount that can beadded to flour or flour-based foods. In the labeling of fortified flour,the maximum level that can be claimed for the addition of folic acidis 100 µg per 35 g flour (equivalent to 286 µg folic acid/100 g flour).This level is based upon current recommended dietary intake (RDI)values and represents 50% of the RDI for folates. Consideration ofU.S. requirements (Rader and others 2000) indicates that levels ofaddition are utilized similar to those described for Australia. Thus, indesigning experiments to study folate fortification of Asian noodlesthe level referred to in the standard was used. Controls of unfortifiedflour were compared with 3 levels of addition corresponding with25%, 50%, and 75% RDI.

Another issue in the fortification of flours and noodles is thechemical form of folate that might be added. Folic acid is the formgenerally used for fortification (NHMRC 1995; Ranhotra and Keagy1995), including in Australia (FSANZ 2006), and it is known to bemore stable than either of the 2 derivatives tetrahydrofolic acid or5-methyltetrahydrofolic acid with respect to pH, temperature, andmoisture content (Hawkes and Villota 1989; Gregory 1989; Ball 1994;Eitenmiller and Landen 1999).

Impact of varying levels of fortificationFlour to which folic acid had been added was used to prepare

white salted noodles. The prepared noodles were dried for 24 h at40 ◦C and then cooked to the optimum point. The folate contentswere determined and the results are shown in Table 1.

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The proportion of total folate lost during cooking of noodles wasfound to lie in the range of 23% to 52%. The loss for the controlnoodles was lower than for the fortified noodles. For those noodlesto which folic acid was added the proportional losses did not appearto vary greatly and were around 45%. Effectively, for this style ofnoodles, fortification with folate appears to be both practical andeffective because the addition of folate results in increased levels inthe product and therefore would contribute to dietary folate intake.

There are a number of possible ways that folate might be lostand these include leaching of folate into the cooking water. Alterna-tively, the folate molecules may be subject to chemical degradationdue to the high temperatures, due to the pH of the cooking medium,or related to the time of cooking. In relation to the results obtainedhere the same cooking time was used in all cases. It is not clear inwhich way losses did occur. However, most white salted noodlesare cooked in boiling water and the water is then discarded. There-fore, if losses are occurring by leaching, then the vitamin will not beavailable to the consumer of the noodles.

There have been a variety of studies reporting losses in folate infoods and earlier studies were reviewed (Gregory 1989). The resultstypically indicate good retention of folate during storage of foods.The use of heat during processing did not appear to cause large de-creases in folate contents. The greatest losses were observed duringboiling and the evidence is that the losses occur due to leachingrather than by any other mechanism.

Foods based on cereal grain have also been investigated and forbreakfast cereals it appears that the method of manufacture andpoint of addition of folate critically affect the retention (Hawkes andVillota 1989). Losses have been found in the range of 39% to 79%for popped cereals and 18% to 22% for extruded cereals. A study offolate added at levels of 1 to 5 µg/g wheat flour showed only smalllosses after 1 y of storage at 120 ◦F (49 ◦C), while losses of nativefolate were 40% after 12 wk. For bread, the losses of folate averaged11%, whereas endogenous folate decreased by 31% during baking(Keagy and others 1975; Osseyi and others 2001; Arcot and others2002c).

Folate like many water-soluble vitamins is subject to leachingfrom foods into surrounding cooking water, in addition to chemical

Table 1 --- Analysis of folate contents of cooked white salted noodles following fortification with different levels offolic acid

Total folate Free folateFolate levelFortification following After Proportion After Proportionlevel fortification cooking lost cooking lost

0 18.2 13.9 ± 1.8 23 10.2 ± 1.0 44143 161.2 77.4 ± 9.7 52 40.3 ± 5.9 75286 304.2 170.3 ± 18.8 44 76.1 ± 4.7 75400 418.2 239.1 ± 17.0 43 186.9 ± 19.7 55

Folate results are presented as mean ± SD. Units for folate contents are µg/100 g expressed on a dry weight basis. The levels of fortification correspond to 0%, 25%,50%, and 70% RDI per serving, respectively, based upon current Australian regulations (FSANZ 2006). The proportional values are calculated as the ratio of folatecontent after cooking to that prior to cooking and expressed as percentages.

Table 2 --- A comparison of the total folate supplied in a serving of various noodles

Before cooking After cooking

Folate in Proportion of RDI Folate in Proportion of RDISample Folate in 1 serving in 1 serving (%) serving in 1 serving (%)

Unfortified noodles 7.5 3.8 4.9 2.4White salted 112.2 56.1 68.0 34.0Yellow alkaline 111.6 55.8 67.7 33.9Instant noodles 76.5 38.3 63.7 31.8

The unfortified sample used in this comparison was of white salted noodles prepared in the laboratory without incorporation of additional folate. In the calculation ofthese data only total folate values were used. The folate contents of 1 serving are calculated on the basis of a serving of noodles corresponding to 35 g of product aspurchased and expressed in units of µg.

modes of degradation (Gregory 1989; Eitenmiller and Landen 1999).Losses of folate have been reported in boiled foods (Hurdle andothers 1968). The boiling of vegetables causes quite variable lossesof folate (Leichter and others 1978) and from 22% to 84% of the initialfolate is typically recovered in the cooking water. The blanchingof spinach resulted in a decrease of 33% of total folate (Chen andothers 1983), 22% in asparagus, and 84% loss in cauliflower (Leichter1980). The reasons for the differences between these vegetables arenot apparent. In addition, the sum of folate retained in the cookedvegetable plus that of the cooking water was nearly equivalent tothe total folate of the raw vegetable in most cases. This indicatedthat aqueous extraction is frequently responsible for the losses offolate during cooking, rather than oxidation or thermal degradation(Leichter and others 1978).

In considering the results of stability and retention studies re-ported in the literature, it is noted that most of the data were ob-tained using analytical methodology, which may underestimate thefolate values. When samples were subjected to different prepara-tion and extraction treatments, quite different results were obtained(Table 1 and 2). The folate contents measured with and without en-zyme treatments indicate that for different noodle samples and forvarying treatments the proportion of folate recovered in untreatedextracts shows considerable variation. This confirms that the valueof earlier published data for folate contents of foods is now limited.

The loss of folate in fortified noodles was twice that of the control.This might be due to the folate content in the control being low,although another issue is that the native folate in the flour maybe present in molecular forms, which are less readily leached orreleased from the food matrix following cooking. Although almosthalf of the amount of fortified folate appeared to have been lostafter the sample was cooked to an optimum time, the results clearlyindicated the potential of fortification to enhance the nutrient intakeof this vitamin.

The stability of folic acid duringthe processing of 3 types of noodles

Samples of the 3 types of noodles were prepared in the labora-tory, and for this it was decided to use only 1 kind of flour in order to

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facilitate direct comparisons between the types of noodles and theprocessing conditions. In addition, all of the samples were fortifiedto the same extent. From the data in Table 2 it can be seen that thelosses found for white salted noodles were similar regardless of thelevel of fortification; and so the level of fortification selected wasthat corresponding to 50% RDI (FSANZ 2006). Samples were takenat the various stages of processing and folate contents analyzed(Figure 1 and 2).

The patterns of results observed for white salted and yellow alka-line noodles were very similar. No significant decrease in total folatecontent occurred during the processing from fortified flour to thedried product. The step of drying at 40 ◦C had no impact on totalfolate contents. Since the 2 types of noodles were made using thesame conditions, as well as the same flour and folate fortificationlevel, it can be concluded that up until the cooking step, pH is notinfluencing the retention of folate in these products.

Relatively large losses occurred when dried white and yellownoodles were cooked in boiling water with 38% losses in whitesalted noodles and 40% in yellow alkaline noodles. The results ob-tained here are similar to that found earlier (Table 2) for white saltednoodles when varying fortification levels were considered. It is notedthat the cooking times for the 2 noodle types were chosen such thatuncooked core was no longer observed. Thus the times were 3.50min for white and 6.10 min for yellow noodles. This indicates thatpH may have been a factor during boiling. Folate appeared to bemore stable in the yellow alkaline noodles (pH 10) than at the lowervalues of the white salted product (pH 6.12) because similar lossesoccurred despite the differing cooking times.

For instant noodles (Figure 2), the overall loss in total folate was44%, which appears to be similar to the other 2 types (Figure 1). How-ever, the pattern was different with losses occurring during steaming

Figure 1 --- Folate content in white salted (A) and yellowalkaline (B) noodles during different stages of processing.Folate contents are presented as mean ± SD expressedin units of µg/100 g on a dry weight basis.

and deep-frying of the noodle strands, as well as upon cooking. Theformulation of instant noodles prepared in the laboratory includedsufficient kansui to raise the pH of the deep-fried product to 7.4.These results confirm that the incorporation of 0.2% alkaline saltdid not increase the loss of folate (Figure 1B) over that observed inthe other 2 types.

Observations on the significance of free and totalfolate contents of noodles during processing

During the analysis of the laboratory noodles reported here, bothfree and total folate contents were measured. This approach re-flects that used in many recent studies (Rader and others 1998, 2000;Shrestha and others 2000; DeVries and others 2001; Arcot and others2002a, 2002b, 2002c; Johnston and others 2002a, 2002b). The pur-pose of monitoring free folates has been to evaluate the impact ofprocessing on the amount of this form measured.

In summarizing the data, the proportion of total folate found tobe free in unfortified flour was 76%. The changes for each of the 3types of noodles prepared in the laboratory (Figure 1 and 2) showthat the free folate was not significantly lower than the total folatelevels in the flours or during dough mixing. However, with furtherprocessing involving drying or heating, the proportions of free folatedeclined. The most likely explanation of these observations is thatduring processing of the noodles some of the folates are becomingtrapped within the food matrix and cannot be effectively extractedunless the sample is treated with amylase and protease. This expla-nation is also consistent with the further observation of decreasedproportions of free folate for the various commercial noodles, whichwere analyzed before and after cooking (Bui and Small 2007c).

The measurement of total folate in Asian noodles is more useful.In terms of nutritional availability, the release of folates from a foodmatrix, particularly one in which starch has gelatinized, is expectedto be similar to that occurring during analysis of total folate. It istherefore recommended that enzymatic digestion by amylase andprotease should be routinely used in the analysis of folates of cereal-based foods.

The potential contributions of Asiannoodles to dietary folate intakes

In order to evaluate Asian noodles as a potential source of folate,the results for samples prepared in the laboratory were recalculatedto facilitate comparisons in terms of the RDI (Table 3). White saltednoodles were used as the example of unfortified noodles because

Figure 2 --- Folate content in instant noodles during differ-ent stages of processing. Folate contents are presentedas mean ± SD expressed in units of µg/100 g on a dryweight basis.




Table 3 --- Relative losses of total folate during processing of laboratory noodles

Noodle Folate Cumulativestyle Processing stage loss folate loss

White salted Flour → dough 1.3 1.3Dough → dried noodles 0 1.3Dried noodles →cooked noodles 40 41.3

Yellow alkaline Flour → dough 0.93 0.93Dough → dried noodles 0.63 1.55Dried noodle → cooked noodles 39.3 40.9

Instant Flour → dough 0.94 0.94Dough → steamed noodles 19.4 20.3Steamed → fried noodles 11.6 31.9Fried noodles →cooked noodles 11.6 43.4

All values are relative losses of folate expressed as percentages and calculated in comparison with the levels originally present in the flour after fortification. Basedon the data presented in Figure 1 and 2.

similar relative losses were observed for commercial white saltedand yellow alkaline noodles (Bui and Small 2007c).

For the unfortified noodles, the amount of folate in the driednoodles represented a relatively small proportion of the amount offolate required on a daily basis. The losses during cooking furtherdecreased the amount provided, so that a serving contains less than3% of the RDI value. Therefore, unfortified Asian noodles are nota good source of folate. However, the data also show that noodlescan provide an effective means of enhancing folate intake. The addi-tion of folate at a rate equivalent to 50% RDI per serving resulted incooked noodles retaining folate corresponding to one-third of theRDI. As all 3 types of noodles were fortified to the same level, theresults clearly demonstrate that all are equally effective as sourcesof folate and can be used to supply folate in the diet (Table 3).

In summary, the use of enzymatic treatment was demonstratedto be essential in the analysis of folate in the 3 styles of Asian noo-dles. The folate contents at each processing stage of fortified noodles(Table 3) demonstrated good retention in white salted and yellowalkaline noodles. Drying at a temperature of 40 ◦C did not affectthe folate content. In these styles, the only significant losses oc-curred subsequently when the dried products were cooked and thefolate contents decreased regardless of the alkaline or acid condi-tions in the noodles. In contrast, for instant noodles, losses of folateappeared to occur at each step during processing, including the ul-timate cooking of the product. The losses were primarily associatedwith any step involving higher temperatures.

When the 3 styles of noodles were cooked to their individual op-timum cooking times, the same overall losses were found (approx-imately 40%). When white salted noodles were fortified to differentextents, the relative losses were higher than for the unfortified con-trol. However, when folate was added at levels of up to 70% RDIper serving, the relative losses were around 40% and the vitaminretained in the cooked noodles was approximately 42% of the RDIper serving.

Conclusion

Asian noodles fortified with folate can be considered as a goodsource of this important micronutrient. Each of the 3 predom-

inant styles of Asian noodles can be used effectively for fortificationpurposes in order to enhance human dietary intakes of folate.

AcknowledgmentWe gratefully acknowledge the financial support provided by an Aus-tralian Postgraduate Award for Lan T. T. Bui. We are particularlygrateful to Dr. Jeanne Rader (U.S. Food and Drug Administration,Washington, D.C.) for her advice and kind encouragement duringthe critical stage of validating the folate determination.

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