Journal of Food Composition and Analysis 38 (2015) 27–31

Original Research Article

Thiamin fortification of bread-making flour: Retention in bread andlevels in Australian commercial fortified bread varieties

Sue Ann Tiong, Maria V. Chandra-Hioe *, Jayashree Arcot *

Food Science and Technology, School of Chemical Engineering, The University of New South Wales, Chemical Science Building (F10),

High Street (Enter via Gate 2), Sydney 2052, Australia

A R T I C L E I N F O

Article history:

Received 26 March 2014

Received in revised form 31 October 2014

Accepted 1 November 2014

Available online 18 November 2014

Keywords:

Thiamin

Wheat flour

Bread making

Fortified bread

HPLC

Food analysis

Food composition

A B S T R A C T

In Australia, thiamin is mandatorily added to bread-making flour with the main purpose of fortification

and reducing the prevalence of Wernicke–Korsakoff syndrome. This study aims to measure the retention

of added thiamin through laboratory-scale bread processing and provide an update on thiamin contents

in commercially fortified bread and flatbread varieties since the introduction of the program in

1991. Even though baking caused degradation loss of thiamin (approximately 16%), the laboratory

fortified white bread had a 25% higher thiamin content than its corresponding flour, and laboratory

fortified wholemeal bread showed a 16% increase (p < 0.05). Thiamin levels in commercially fortified

bread and flat bread varieties ranged between 0.24 and 1.9 mg/100 g (dry weight basis). It can be

suggested that most of the bread varieties were made from flour fortified at the minimum mandated

level (0.64 mg/100 g flour). Samples of flat bread varieties (white without yeast, wholemeal with yeast

and wholemeal without yeast) showed low thiamin levels (0.24–0.49 mg/100 g, dry weight basis). The

results suggest that the flat bread varieties were likely made from either commercially under-fortified

flour or unfortified general-purpose flour, as only bread-making flour is fortified with thiamin.

� 2014 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

jo u rn al ho m epag e: ww w.els evier . c om / lo cat e/ j fc a

1. Introduction

Thiamin, a common term for 3-[(4-amino-2-methyl-5-pyrimi-dinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium, is a watersoluble B1 vitamin (Combs, 2012). In plant based foods, thiaminpredominantly occurs in free form, while in animal based foods itexists in phosphorylated forms as thiamin monophosphate,thiamin diphosphate and thiamin triphosphate. Thiamin diphos-phate, also known as thiamin pyrophosphate, is the biologicallyactive form. It plays an important role as a co-factor for several keyenzymes involved in the carbohydrate metabolism and defencemechanisms (Martin et al., 2003). Thiamin triphosphate isessential in neural function (Combs, 2012). Thiamin found in foodis sensitive to pH and high temperatures (Butterworth, 2003). It isstable between pH 2.0 and 4.0, but unstable in alkaline solutions(Mihhalevski et al., 2013). Several studies reported that heat duringbaking caused loss of endogenous thiamin that ranged from 20% to56% (Batifoulier et al., 2005; Martinez-Villaluenga et al., 2009;Mihhalevski et al., 2013).

* Corresponding authors. Tel.: +61 2 93854337/2 93855360.

E-mail addresses: m.v.chandra-hioe@unsw.edu.au (M.V. Chandra-Hioe),

J.Arcot@unsw.edu.au (J. Arcot).

http://dx.doi.org/10.1016/j.jfca.2014.11.003

0889-1575/� 2014 Elsevier Inc. All rights reserved.

Whole grain cereals are typically rich in thiamin. However, thescutellum and germ are removed during milling, which results inthe production of grains and flour with lower thiamin content.Beri-beri, a condition of severe thiamin deficiency, has beendocumented to be prevalent in countries where polished rice was astaple (Lonsdale, 2006). Severe thiamin deficiency leads tocardiovascular and neurological disease (Harper, 2006; Rapala-Kozik, 2011) including Wernicke–Korsakoff Syndrome (WKS),which is commonly associated with alcohol abuse (Harper et al.,1989). Australia had a higher incidence of WKS (2.1%) than othercomparable countries (Truswell, 2000). Based on this evidence, in1991 the mandatory thiamin fortification program was introducedwith the purpose of reducing the prevalence of WKS. This is unlikeother countries, where fortification programs are being imple-mented to replace the thiamin loss due to milling (Truswell, 2000).The Australian fortification program mandates the addition of0.64 mg (minimum) thiamin hydrochloride per 100 g bread-making flour.

The objective of this study was to determine the actual thiaminlevels in selected commercially fortified bread and flat breadvarieties being sold in the Sydney metropolitan area (Australia).Specifically, the study measured thiamin in bread samples madefrom laboratory-scale fortified flour. The results presented herewould provide an update on thiamin levels after 22 years of the

S.A. Tiong et al. / Journal of Food Composition and Analysis 38 (2015) 27–3128

fortification program, as thiamin values are not being declared onthe nutrition labels of most bread and flat-bread varieties.

2. Materials and methods

2.1. Reagents and plant materials

Thiamin hydrochloride was obtained from DSM NutritionalProducts (Kaiseraugst, Switzerland). Potassium ferricyanide usedto oxidise thiamin hydrochloride was purchased from AjaxFinechem (Sydney, NSW, Australia). HPLC grade acetonitrile andmethanol were obtained from Honeywell Burdick and Jackson(Sydney, NSW, Australia). Enzyme taka-diastase (100 U/mg,product no 86247) from Aspergillus oryzae was acquired fromSigma–Aldrich (Sydney, Australia). Purified water (conductance�0.1 mS/cm) was sourced from a Milli-Q-system (Millipore, Bedford,MA, USA).

Commercial thiamin-fortified flour (white and wholemeal),unfortified white flour, wheat bran and wheat germ were providedby a flour miller in Australia and stored in 20 L air-tight containers.Bread ingredients including instant dry yeast, canola oil, table saltand bread improver were purchased from a local supermarket inSydney.

2.2. Laboratory-made fortified flour

Wholemeal flour was prepared by substituting 20% of unforti-fied white flour with wheat bran (10%) and wheat germ (10%). Tounfortified flour (white and wholemeal) thiamin hydrochloridewas added at the minimum fortification level of 0.64 mg/100 gflour. Both types of flour were then homogenised using a rotaryshaker for at least 18 h. Flour samples (white flour, n = 4 and wholemeal flour, n = 4) were then obtained for thiamin analysis andmoisture determination according to AOAC 925.09-1925 method(AOAC, 2000). The laboratory-fortified flour was used for breadmaking.

2.3. Laboratory-made fortified bread

Bread loaves (wholemeal, n = 2 and white, n = 2) were madefrom the laboratory fortified flour previously described. Baking ofthese bread types was repeated on different day to obtain a total of8 bread loaves. Initially, bread ingredients consisting of 1 kg flour,15 g table salt, 15 g bread improver, 20 g canola oil, 13 g instant dryyeast and 600 g water (Chandra-Hioe et al., 2013b) were mixedusing Vorwerk Thermomix (Wuppertal, Germany). The dough washand-kneaded, proofed for 60 min at 37 8C and then baked at200 8C in a pre-heated oven for 30 min. Samples were collectedafter mixing (dough), 60 min of proofing and stored in the freezer.On the day of baking, the samples including bread were cut andground (Vorwek Thermomix, Wuppertal, Germany) for moisturedetermination and thiamin analysis. Ingredients used for breadmaking in this study (excluding salt, oil and water) were alsoanalysed for thiamin contents.

2.4. Bread sampling

A variety of bread was selected according to the A.C. Nielsen TopBrands Report 2009 that achieved sales exceeding 66 millionAustralian dollars since 2003 (Nielsen, 2010). Bread varieties fromleading brands including private labels (n = 84 loaves) werepurchased in August 2013 from the supermarkets and localbakeries from 2 different suburbs of Sydney. They were thengrouped into 10 types: white bread (n = 14 loaves), white breadwith added soy fibre (n = 4 loaves), white bread containing soyfibre and resistant starch (n = 3 loaves), wholemeal bread

(n = 9 loaves), multigrain bread (n = 16 loaves), white flatbreadwith yeast (n = 6 packs) and without yeast (n = 6 packs), whiteflatbread (wheat fibre added) with yeast (n = 3 packs), wholemealflatbread with yeast (n = 6 packs) and without yeast (n = 6 packs).In the flatbread category sample composites were prepared fromLebanese bread, pita and wraps. On the day of purchase, 6 sliceswere taken from each pack (different brands within the samecategory), then diced, ground and homogenised using VorwerkThermomix (Wuppertal, Germany) to obtain bread composites.

2.5. Preparation of standard solutions

The standard stock solution (100 mg/mL) was prepared bydissolving thiamin hydrochloride in Milli-Q water. Fresh workingstandard solution (5 mg/mL) was made on the day of use bydilution of the stock solution in Milli-Q water. For the purpose ofdetection, thiochrome was prepared by oxidising the workingstandard solution (4 mL) with alkaline potassium ferricyanide(3 mL). The concentrations of the calibration curve (10 points)ranged between 0.025 and 1.75 mg/mL.

2.6. Thiamin extraction

Extraction of thiamin was performed according to a publishedmethod (Ndaw et al., 2000). Samples (at least in duplicate) of instantdry yeast (0.5 g), bread improver, wheat bran, wheat germ, flour,dough and bread (2.5 g) were weighed in the centrifuge tubes. Afteradding 0.1 M hydrochloric acid (25 mL) samples were placed in ashaking water bath (100 8C) for 30 min, and then cooled down. Hotacid digestion was carried out to denature protein complexes andrelease protein-bound thiamin. The pH of the samples was thenadjusted to 4.5 with 2.5 M sodium acetate, followed by taka-diastase (250 mg) treatment in a 37 8C shaking water bath for 18 h.Taka-diastase was used to dephosphorylate thiamin to its free formfor quantitative analysis (Ndaw et al., 2000; Tang et al., 2006). MilliQ water was consequently added into the samples to make up to50 mL volume. Samples were centrifuged at 10,000 � g for 10 minat 4 8C and the supernatants were filtered through a 0.45 mm PTFEsyringe filter (Grace Davison, Chicago, IL, USA). Similar to theworking standard solutions, to 4 mL of the filtered samples 3 mL ofalkaline potassium ferricyanide (1:25 of 1% (w/v) potassiumferricyanide in 15% (w/v) sodium hydroxide) was added, vortexedand equilibrated for exactly 1 min (Arella et al., 1996).

Solid phase extraction was performed according to Arella et al.(1996) using a vacuum manifold (Supelco, St. Louis, MO, USA).Initially, Sep-Pak C18 cartridges (Waters, Milford, MA, USA) wereactivated with 5 mL of Milli-Q water and conditioned with 2 mL ofmethanol. The oxidised samples (7 mL) were then passed throughthe cartridges. After washing with 10 mL of 0.05 M sodium acetate(pH 6.0), the analyte was eluted with 8 mL of 70% (v/v) methanol inMilli-Q water. The eluents were then made up to 10 mL and filteredprior to chromatographic analysis.

2.7. Liquid chromatography

The liquid chromatography system (Shimadzu Co., Kyoto,Japan) was coupled with a fluorescence detector (RF-10A XL) andemployed a Synergi Hydro-RP column (150 mm � 4.6 mm, 4 mm,Phenomenex, Torrance, CA, USA). The method was adopted fromArella et al. (1996) with some modifications. The mobile phaseconsisted of 70% 0.05 M sodium acetate (pH 6.0) and 30%methanol (isocratic), the flow rate and the injection volume were1 mL min�1 and 20 mL, respectively. The column temperaturewas set at 30 8C. The excitation and emission wavelengths usedfor detecting thiochrome were set at 365 nm and 435 nm,respectively.

Table 1Thiamin contents in the bread ingredients.

Sample (in duplicate) Thiamin content dwba

(wwbb), mg/100 g

Wheat bran 0.35 � 0.007 (0.31)

Wheat germ 0.33 � 0.03 (0.30)

Unfortified white flour 0.33 � 0.001 (0.29)

Instant dry yeast 14.8 � 0.004 (14.0)

Bread improver Not detected

a dwb = dry weight basis.b wwb = wet weight basis.

Each sample was injected twice to obtain mean (� standard deviation).

S.A. Tiong et al. / Journal of Food Composition and Analysis 38 (2015) 27–31 29

2.8. Statistical analysis

SAS Enterprise Guide 5.1 (SAS institute Inc., Cary, NC, USA) wasused to analyse the experimental data. Mean values werecompared using one-way variance (ANOVA) analysis, where p

value of 0.05 was chosen to report the level of significance.

3. Results and discussion

As shown in Table 1, there was no significant differencebetween mean thiamin concentrations, expressed on a dry weightbasis (dwb), in the unfortified white flour (0.33 mg/100 g), wheatbran (0.35 mg/100 g) and wheat germ (0.33 mg/100 g) using one-way ANOVA (p > 0.05). The mean thiamin in the unfortified whiteflour presented here was higher in comparison to another studythat reported a range between 0.13 and 0.22 mg/100 g, dwb(Batifoulier et al., 2006).

Mean thiamin in the wheat bran (0.31 mg/100 g) was similar tothose in the wheat germ with 0.30 mg/100 g; both values areexpressed on a wet weight basis (wwb). Other studies reportedthat the range of thiamin (wwb) in the bran and germ were 1.1–1.3 mg/100 g (Jakobsen, 2008) and 1.5–2.3 mg/100 g (Brandoliniand Hidalgo, 2012), respectively and these values were higher thanour findings. Factors that may lead to varying thiamin contents aredifference in wheat cultivars, year of growth, growing location andthe type of soil (Batifoulier et al., 2006).

Ingredients used for bread making (excluding salt, oil andwater) were also determined (Table 1). This study found thatinstant dry yeast contained 14 mg thiamin/100 g, while otherstudies reported different values; 7.8–8.1 mg/100 g (Jakobsen,2008), 16.2–17.2 mg/100 g (Yamanaka et al., 1994); 44.9–52.5 mg/100 g (Ndaw et al., 2000) and 1.3 mg/100 g (Arella et al., 1996). Thedifference in thiamin contents may be due to various yeast strainsanalysed in other studies.

Table 2 shows that the laboratory fortified wholemeal flour(n = 4) had 0.91 � 0.034 mg thiamin per 100 g flour (� standarddeviation) and was higher than the laboratory fortified white flour(n = 4) with 0.74 � 0.061 mg thiamin per 100 g (expressed as dwb);thiamin levels in bread samples baked on 2 different days wereconsistent. The finding was consistent with a previous study that

Table 2Mean thiamin at different stages of bread making.

Sample

(in duplicate)

Thiamina (mg/100 g) dwbb Moisture (%)

White Wholemeal White Wholemeal

Flour 0.74 � 0.06 0.91 � 0.03 12.9 12.1

Dough 1.1 � 0.006 1.3 � 0.06 41.9 42.7

Proofed dough 1.2 � 0.01 1.3 � 0.07 43.2 43.1

Bread 1.0 � 0.02 1.1 � 0.02 36.4 36.7

a Mean � standard deviation, obtained from analytical quadruplicate.b dwb refers to dry weight basis.

showed that addition of non-endosperm fractions increased thiaminin flour (Batifoulier et al., 2006). This implied that the wheat bran andwheat germ added to unfortified white flour contributed to higherthiamin in wholemeal flour. Mean thiamin in samples of commer-cially fortified white flour was 1.1 � 0.04 mg/100 g and in samples ofcommercially fortified wholemeal was 1.2 � 0.06 mg per 100 g. Themean differences between laboratory and commercially fortifiedflour samples were significant at 95% confidence interval.

White and wholemeal dough samples showed 30% higherthiamin than their corresponding flour samples, and the differencewas significant (p < 0.05). This was because yeast synthesisedthiamin de novo (Xu et al., 2012), which contributed to higherendogenous thiamin measured here. Also, the instant dry yeastused for the bread making contained 14.8 � 0.04 mg thiamin/100 g(dwb). Our result was in contrast to a previous study that found a 40%loss of thiamin in kneaded dough (Batifoulier et al., 2005). However, itis unclear whether the thiamin value reported here is expressed asdry weight basis.

After proofing mean thiamin in white dough was significantlyhigher than before proofing (p < 0.05), conversely the meandifference in wholemeal was not significant (p > 0.05). Increasedthiamin in wholemeal dough samples was 2% lower than in whitedough samples (9%). Proofing is a process of anaerobic fermenta-tion of carbohydrates by yeast to ethanol and carbon dioxide. Yeastrequires thiamin diphosphate for fermentation (Xu et al., 2012),but also synthesise thiamin (Bettendorff, 2012). By replacing 20%of white flour with wheat bran and wheat germ to make wholemeal bread, there was less glucose available for yeast growth andthiamin synthesis, as shown in thiamin level measured here. Yeastwas deactivated during baking when the temperature of the doughreached 45 8C (Rosell, 2011). In comparison to its flour, thiamin inproofed (white) dough was 43% higher, in wholemeal proofeddough 31% higher, and the differences were significant (p < 0.05).

Bread samples (white and wholemeal) showed significantlylower thiamin than their respective proofed dough samples(p < 0.05), because the heat during baking elicited thermaldegradation of thiamin. White bread had 18% lower thiaminthan its proofed dough and wholemeal bread 15%. Even thoughbaking caused thiamin loss, the results suggested that endoge-nous thiamin in yeast contributed to higher thiamin level in breadthan its corresponding flour (p < 0.05). Mean thiamin in whitebread was 1.0 � 0.028 mg/100 g, dwb that was 25% highercompared to its corresponding flour. In wholemeal bread meanthiamin was 1.1 � 0.025 mg/100 g, dwb and was 16% more than itsrespective flour. The findings presented here seemed to beinconsistent with previous studies that reported loss of endogenousthiamin (30–58%) in bread than its corresponding flour (Batifoulieret al., 2005, 2006). This study measured only endogenous thiamin, incontrast our study determined the levels of added and endogenousthiamin in bread. Also, there are differences in the bread-makingprocess, bread formulation, yeast used for fermentation, endoge-nous thiamin in flour, extraction rate of flour, thus direct comparisonis not possible.

Fig. 1 shows that commercially fortified multigrain breadsamples had the lowest thiamin (0.69 mg/100 g dwb) in compari-son to white and wholemeal bread varieties. This was likely due toa smaller fraction of fortified flour present in the mixed flour(Chandra-Hioe et al., 2011), since multigrain flour could contain14–24% of other grains such as rye, buckwheat, barley and oats.

Commercially fortified wholemeal bread had 0.90 mg thiamin/100 g (dwb) and white bread contained 0.89 mg/100 g (dwb,Figure 1). The data presented here are comparable with thiaminlevels measured in bread made from laboratory-fortified flour.Adding back the wheat bran and germ (as per manufacturingpractice) would have a dilution effect reducing the fortificantconcentration in flour (Chandra-Hioe et al., 2013a), however high

0.0 0.5 1. 0 1.5 2. 0 2.5

White bread

White bread+soy fibre

White+soy fibre plus resista nt starch

Wholemeal bread

Mul�grain bread

White flatbread+ yeast

White flatbread+yeast_added fibre

White flatbread no yeast

Whomeal flatbread+yeast

Wholemeal flatbread no yeast

Thiamin (mg /100g, dry weight basis )

Fig. 1. Mean thiamin in 10 bread categories (dwb). Each category represents the following bread composites: white bread (n = 14 loaves), white bread with added soy fibre

(n = 4 loaves), white bread containing soy fibre and resistant starch (n = 3 loaves), wholemeal bread (n = 9 loaves), multigrain bread (n = 16 loaves), white flatbread with yeast

(n = 6 packs) and without yeast (n = 6 packs), white flatbread (wheat fibre added) with yeast (n = 3 packs), wholemeal flatbread with yeast (n = 6 packs) and without yeast

(n = 6 packs). Error bars represent standard deviation.

S.A. Tiong et al. / Journal of Food Composition and Analysis 38 (2015) 27–3130

levels of endogenous thiamin in bran and germ compensated forthe dilution effect.

The highest thiamin is found in white bread containing soy fibrewith 1.9 mg thiamin/100 g (dwb). This bread also contained soyflour that has been reported to have 0.71 mg thiamin/100 g(Lebiedzinska and Szefer, 2006), and might contribute to a higherthiamin level presented here.

Mean thiamin in white (0.68 � 0.1 mg/100 g, dwb) and whole-meal flatbread with yeast (0.49 � 0.1 mg/100 g, dwb) differedsignificantly as opposed to white (0.43 � 0.05 mg/100 g, dwb) andwholemeal (0.24 � 0.05 mg/100 g, dwb) without yeast. The resultsshow that yeast contributes to higher thiamin levels presented here.

Among the flat bread varieties, mean thiamin in white withyeast (0.68 � 0.1 mg/100 g, dwb) as well as in white with yeast andadded wheat fibre (0.75 � 0.1 mg/100 g, dwb) were the highest,suggesting that they were made from flour fortified with theminimum mandated level of thiamin. Wholemeal flatbread withoutyeast showed the lowest thiamin (0.24 � 0.05 mg/100 g, dwb), whichdiffered significantly with white flatbread without yeast (p < 0.05).The wholemeal flat bread was likely made from unfortified flour,because mean thiamin in our experimental bread made fromunfortified flour was 0.57 � 0.02 mg/100 g (dwb). Mean thiamin inwhite flat bread without yeast was 0.43 mg, which indicates thatunder-fortified flour was used for baking.

This study revealed that thiamin levels in the majority of breadand flatbread varieties were greater than 0.64 mg/100 g, which isthe minimum level added to bread-making flour, suggesting thatbread were made from fortified flour.

4. Conclusion

The experimental data showed that laboratory fortified breadhad significantly higher thiamin than its corresponding flour.Increased thiamin in white bread (25%) was higher than inwholemeal bread (16%). White dough had 43% higher thiamin afterproofing than its respective flour, while wholemeal dough had 31%(p < 0.05). This is because yeast synthesises thiamin de novo duringproofing (Bettendorff, 2012). The thiamin in bread varieties rangedbetween 0.69 and 1.9 mg/100 g (dwb) and in flatbread varietiesthiamin ranged between 0.24 and 0.75 mg/100 g (dwb). Thefindings of this study suggest that most of commercially fortified

bread varieties were made from flour fortified at the minimummandated level (0.64 mg/100 g flour), under-fortified flour orunfortified general purpose flour also seemed to be used in someflat bread varieties. This study also raises the possibility of usingthe most appropriate strain of yeast that can increase thiamin inbread instead of fortifying bread flour with thiamin.

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