The Bioaccessibility of Folate in Breads and the Stability of Folate Vitamers during in vitro Digestion

Abstract

Both the liberation and stability of endogenous folate are relevant to the bioaccessibility of folate. Since folates are unstable, in addition to studying the natural folate content in foods, bioaccessibility should be considered. To understand folate changes during digestion, a mixture of standard folate compounds was subjected to a static in vitro gastrointestinal digestion assay. Next, different types of bread were analysed to study how food matrices influence folate bioaccessibility. Folates were identified and quantitated by a UHPLC-PDA/FL method. Folic acid and 10¬formylfolic acid were stable throughout the digestion, and the conversions among formyl folates and 5,10-methenyltetrahydrofolate were triggered at the gastric phase. Tetrahydrofolate began to degrade during the oral phase and was lost completely during the gastric phase. During the intestinal phase, 5-methyltetrahydrofolate began to degrade and suffered a 60% loss. With bread matrices, folate conversions and the decrease of reduced folates were also common, but the extent of changes varied. Generally, rye breads had the highest (80–120%) bioaccessibility of folate, while oat breads had the lowest (31–102%). The high proportion of 5-methyltetrahydrofolate could result in low bioaccessibility because of its relatively low stability during digestion in bread matrices. An increase in 10-formylfolic acid content was observed for all the breads, but 10-formyldihydrofolate seemed to be more stable in rye breads than in oat and wheat breads. The results showed that folates undergo significant changes during digestion and that food matrices could be modified to affect these changes towards better folate bioaccessibility.

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Food &
Function
PAPER
Cite this: DOI: 10.1039/d1fo03352b
Received 6th October 2021,
Accepted 18th February 2022
DOI: 10.1039/d1fo03352b
rsc.li/food-function
The bioaccessibility of folate in breads and the
stability of folate vitamers during in vitro
digestion†
Fengyuan Liu, * Minnamari Edelmann, Vieno Piironen and Susanna Kariluoto
Both the liberation and stability of endogenous folate are relevant to the bioaccessibility of folate. Since
folates are unstable, in addition to studying the natural folate content in foods, bioaccessibility should be
considered. To understand folate changes during digestion, a mixture of standard folate compounds was
subjected to a static in vitro gastrointestinal digestion assay. Next, different types of bread were analysed
to study how food matrices influence folate bioaccessibility. Folates were identified and quantitated by a
UHPLC-PDA/FL method. Folic acid and 10-formylfolic acid were stable throughout the digestion, and the
conversions among formyl folates and 5,10-methenyltetrahydrofolate were triggered at the gastric phase.
Tetrahydrofolate began to degrade during the oral phase and was lost completely during the gastric
phase. During the intestinal phase, 5-methyltetrahydrofolate began to degrade and suffered a 60% loss.
With bread matrices, folate conversions and the decrease of reduced folates were also common, but the
extent of changes varied. Generally, rye breads had the highest (80–120%) bioaccessibility of folate, while
oat breads had the lowest (31–102%). The high proportion of 5-methyltetrahydrofolate could result in low
bioaccessibility because of its relatively low stability during digestion in bread matrices. An increase in
10-formylfolic acid content was observed for all the breads, but 10-formyldihydrofolate seemed to be
more stable in rye breads than in oat and wheat breads. The results showed that folates undergo signifi-
cant changes during digestion and that food matrices could be modified to affect these changes towards
better folate bioaccessibility.
1. Introduction
Folate is essential for amino acid and nucleotide metabolism.
Folate deficiency results in health disorders, such as megalo-
blastic anaemia and neural tube defects.
1
The mandatory forti-
fication of flours with folic acid (a form of synthesised folate)
is carried out in many countries, such as the USA, Canada and
Australia.
2
However, most European countries do not practice
mandatory folate fortification. Additionally, since adverse
effects may occur with a high intake of folic acid, such as the
masking of vitamin B12 deficiency,
3
it is important to study
folate from natural sources.
Bread is one of the most common staple foods in many
regions. The ingredients as well as the processing methods
used for bread-making, play an important role in the charac-
teristics of bread products. In addition, breads differ widely in
size, shape, and texture. Regardless of the differences, bread is
the main source of energy for many people and a rich source
of B vitamins.
Since bread is consumed in high quantities, even moderate
enhancement of folate in bread is relevant to the folate intake
of the population. Popular approaches to improving natural
folate levels in bread are germination and fermentation, which
have been proven to be effective for compensating for folate
loss during bread-making.
4
Furthermore, some researchers
have enhanced folate content in bread by including spinach or
Swiss chard as one of the ingredients.
5
However, due to the
instability of natural folate, whether folate could be delivered
to the small intestine without degradation remains a question
to be answered. This question relates to the bioaccessibility or
bioavailability of folate, meaning the proportion of folate that
can be absorbed or utilised by human beings.
There is a wide range of human studies supporting the idea
that folic acid or 5-methyltetrahydrofolate in fortified cereal
products is well bioavailable,
6–9
and bread has been used as a
good vehicle to deliver folic acid. However, information about
the bioaccessibility or bioavailability of endogenous folate in
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1fo03352b
Department of Food and Nutrition, University of Helsinki, Agnes Sjöbergin katu 2,
FI-00014, Finland. E-mail: fengyuan.liu@helsinki.fi,
minnamari.edelmann@helsinki.fi, vieno.piironen@helsinki.fi,
susanna.kariluoto@helsinki.fi
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bread is rare, and the results vary due to the different study
approaches. Vahteristo et al.
10
reported good bioavailability of
folate from rye meals consisting of rye products (breads and
muesli) and orange juice, similar to that of folic acid from for-
tified white bread. Ohrvik et al.
11
reported over 75% folate
bioaccessibility in different types of whole-meal bread using
the TIM (TNO Gastro-Intestinal Model) and over 90% in a
breakfast meal (with wheat bread, fruits and other foods).
Furthermore, a paper reported 100% bioaccessibility of
endogenous folic acid and 5-methyltetrahydrofolate in bread
samples using a Caco-2 cell absorption model.
12
Both the liberation and stability of endogenous folate are
relevant to the bioaccessibility of folate in food matrices. Our
previous study showed that both ingredient and thermal treat-
ments can affect the bioaccessibility of folate in simple faba
bean and cereal matrices.
13
It is hypothesised that the bioac-
cessibility of folate in breads could be linked to their ingredi-
ents. Antioxidants from foods may protect folate during diges-
tion.
14
Additionally, folate composition can be critical because
of the varied stability of endogenous folate vitamers.
15–17
A
recent comprehensive study reported around 20–50% folate
bioaccessibility in wheat germ, spinach and cheese.
14
With the
information on folate vitamers, they showed that the bioacces-
sibility of food folate depends on folate stability during in vitro
digestion. Another hypothesis is that the processing method
could affect the bioaccessibility of folate by modifying the
structure of the food. Bationo et al.
18
studied the bioaccessibil-
ity of folate in seven types of African cereal-based products and
found that those with dense structures had the highest folate
bioaccessibility (57–81%). Moreover, a recent report by Hiolle
et al.
19
stated that the release of added folic acid during the
gastric phase was faster for biscuit and sponge cake than for
custard and pudding, although they were made from the same
materials. Their further research showed that food structure
can significantly affect folate bioavailability in human adults
and that custard had the lowest folate bioavailability, followed
by biscuits.
20
In the case of custard, they suspected that inter-
actions of folate with the matrix components took place in the
gastrointestinal tract, causing a delayed plasma folate peak.
For biscuits, they suggested that their relatively low folate bio-
availability could be due to the overall inefficient digestion of
nutrients. Indeed, folate can bind to macronutrients, such as
starch and protein. Thus, efficient digestion of food com-
ponents could release folate from food matrices, improving
folate bioavailability or bioaccessibility.
Since bioavailability studies are expensive and time-con-
suming, bioaccessibility studies are useful for generating
hypotheses and for sample screening. Hence, this study aimed
to compare the bioaccessibility of endogenous folate in twelve
types of commercial breads using a standardised static in vitro
digestion model.
21
The breads were made from different cereal
ingredients and varied in shape and structure. Additionally,
the bioaccessibility of folate was investigated in oat breads,
which were baked in the laboratory using different oat culti-
vars, but the same baking process. Finally, since we have pre-
viously reported that in vitro digestion induces the interconver-
sion and degradation of folate in faba bean and cereal
matrices,
13
in vitro digestion of the standard mixture was
carried out to understand the changing patterns of folate vita-
mers throughout digestion and analysis. Thus, this study will
provide insights on the bioaccessibility of folate from the
aspects of folate stability and folate liberation during the
in vitro digestion.
2. Materials and methods
2.1 Enzymes and calibrants
The following enzymes and bile extract were purchased from
Sigma-Aldrich (St Louis, MO, USA): αamylase from Aspergillus
oryzae (A9857), pepsin (P7125), bile from bovine and ovine
(B8381), trypsin (T0303), chymotrypsin (C4129) and protease
(P8811). For the calibrants, (6S)-tetrahydrofolate (H
4
folate,
sodium salt), (6S)-5-methyltetrahydrofolate (5-CH
3
-H
4
folate,
calcium salt), (6R,S)-5,10-methenyltetrahydrofolate hydro-
chloride (5,10-CH
+
-H
4
folate) and (6S)-5-formyltetrahydrofolate
(5-HCO-H
4
folate, sodium salt) were obtained from Eprova AG
(Schaffhausen, Switzerland). 10-Formylfolic acid (10-HCO-PGA)
and folic acid (PGA) were obtained from Schirck’s Laboratories
(Jona, Switzerland). 10–formyldihydrofolate (10-HCO-H
2
folate)
was synthesised from 5,10-CH
+
-H
4
folate according to Kariluoto
et al.
22
Calibrants were prepared into 50 mM sodium borate
solution with 0.4% 2-mercaptoethanol (approximately 1 mg
mL
−1
), and ultrasonic agitation was applied when necessary.
The concentrations of the standards were determined and con-
firmed spectrophotometrically (Table S1†), according to
Kariluoto et al.
22
Stock solutions for each folate standard were
prepared by diluting (1 :10) the calibrant solution into 50 mM
sodium borate solution with 1% ascorbic acid and stored at
−20 °C.
2.2 Samples
2.2.1 Standard mixture for digestion tests. The folate stan-
dard mixture was prepared freshly before the in vitro digestion
assay. A mixture of individual standard compounds with spec-
trophotometrically determined concentrations was prepared
from stock solutions to Milli-Q water. The concentration of
total folate was 1.5 µg mL
−1
, containing PGA (0.2 µg mL
−1
),
10-HCO-PGA (0.2 µg mL
−1
), 10-HCO-H
2
folate (0.4 µg mL
−1
),
H
4
folate (0.2 µg mL
−1
), 5-CH
3
-H
4
folate (0.2 µg mL
−1
),
5-HCO-H
4
folate (0.2 µg mL
−1
) and 5,10-CH
+
-H
4
folate (0.1 µg
mL
−1
).
2.2.2 Commercial breads. Twelve types (one package of
each type) of commercial bread were purchased from the local
markets, and their information is provided in Table 1 and
Fig. 1. They were toast breads, flat portion breads, round
breads and crispbread. Their abbreviations are as follows:
toast breads included white wheat toast (WT), whole-grain
wheat toast (WGT), whole-grain rye toast (RT) and whole-grain
oat toast (OT). Flat portion breads included oat flat portion
bread (OF), rye flat portion bread (RF) and rye flat portion
bread with germinated rye (RGF). Round breads comprised rye
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breads with a hole in the centre, including traditional Finnish
round rye bread (RR), round rye bread with a softer texture
because wheat flour was added (RRW), round rye bread
without added yeast (RRNY) and round rye bread baked in the
afterheat of the oven (RRAO). For crispbread, only rye crisp-
bread (RC) was studied. Bread samples were taken from
different parts of the bread and torn into small pieces by hand
and ground for 5 seconds using a four-bladed coffee grinder
(EGK 200, Rommelsbacher, Dinkelsbühl, Germany). The
ground samples were stored at room temperature in the dark
until analysis within 2 days upon arrival.
2.2.3 Whole-grain oat breads. Whole-grain oat breads were
baked in the laboratory. Grains of three different oat cultivars
were de-hulled, kilned, flaked and milled by Vääksyn Mylly Ltd
(Asikkala, Finland) to produce whole-grain oat flours, coded
LOA, LOB and LOC, which were then used to bake breads on a
laboratory scale (Fig. 1). The contents of major components in
the oat flours were as follows (% dry matter basis): 61.5–62.8%
starch, 13.1–17.3% protein, 6–8.1% lipid and 3.7–4.6% beta-
glucan (data adapted from Jokinen et al.,
23
and LOA, LOB
and LOC are referred to F16, F25 and F19). The same recipe
was used for all cultivars, except that the amount of water
was optimised for each cultivar by test baking. The mass
ratios to flour mass were as follows: 4% light syrup, 3%
baker’s yeast, 2% salt and 2% psyllium. The psyllium was
mixed with part of the water and allowed to stand for 10 min,
after which all ingredients were mixed. After a floor time of
15 min at room temperature, the dough was divided into
three pieces of 400 g each that were moulded by hand,
panned and proofed for 30 min at 35 °C and 100% relative
humidity. The breads were baked at 215 °C for 30 min (three
bread replicates from each cultivar). After cooling down, the
breads were cut into slices, and three slices from different
parts of the breads were selected and pooled. The samples
were ground and stored in the same way as the commercial
breads.
Table 1 Information on commercial breads from package labels
Bread Fat
Carbo-
hydrates
Dietary
fibre Protein Grain ingredients Note
Whole-grain rye toast (RT) 5.1 38 8.8 11 Whole-grain rye (flour, groats, malt), endosperm rye
flour (lestyruisjauho), sunflower seed, linseed, barley
malt extract, 100% rye, 52% whole-grain
Added yeast
Added wheat
gluten
Whole-grain oat toast (OT) 3.5 37 6 13 Whole-grain oats (flour, groats, flake), wheat flour, oat
bran, oat fibre, wheat germ, 51% whole-grain oats
Added yeast
Added wheat
gluten
White wheat toast (WT) 2.2 49 4 8.4 Wheat flour Added yeast
Added wheat
gluten
Dried wheat
sourdough
Rye flat portion bread (RF) 1.7 43 12 10 Whole-grain rye (groats, flour, malt), wheat flour,
potato flake, 87% whole-grain rye
Added yeast
Added wheat
gluten
Sourdough
process
Oat flat portion bread (OF) 5.7 36 7.2 8.6 Whole-grain oats (flake, flour, grain), potato flake, oat
bran, 100% oats
Added yeast
Whole-grain wheat toast
(WGT)
3.6 43 7.3 9.3 35% wholemeal wheat flour, wheat flour, wheat bran,
56% whole-grain
Added yeast
Added wheat
gluten
Dried wheat
sourdough
Rye flat portion bread with
germinated rye (RGF)
1.7 42 11 9.4 Whole-grain rye (germinated grain, flour, groats,
malt), wheat flour, potato flake, 82% whole-grain rye,
36% germinated rye
Added yeast
Added wheat
gluten
Sourdough
process
Traditional Finnish round rye
bread (RR)
1.3 40.4 9.7 6.6 100% whole-grain, rye sourdough, rye flour Added yeast
Sourdough
process
Round rye bread with softer
texture where wheat flour was
added (RRW)
1.7 46.6 12 6.8 Whole-grain 80% of grain raw materials, 57% whole-
grain rye flour, wheat flour, psyllium fibre
Rye and wheat
Added yeast
Sourdough
process
Rye crispbread (RC) 2.5 58 20 10 88% whole-grain rye flour, water, 4% rye bran Added yeast
Round rye bread without
added yeast (RRNY)
1.5 42.8 13.3 7.4 73% wholemeal rye flour No added yeast
Round rye bread baked in the
afterheat of the oven (RRAO)
2.1 51 15 8.2 85% organic whole-grain rye flour, gluten, 100%
whole grains
Added yeast
After oven
Sourdough
process
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2.3 In vitro digestion
A static in vitro digestion model, as described by Minekus
et al.,
21
was applied in this study with small modifications.
Individual enzymes were used for intestinal digestion, and
α-amylase from Aspergillus oryzae was used for starch hydrolysis
instead of human salivary α-amylase and porcine pancreatic
α-amylase. Lipase was not included in this assay, as in a prelimi-
nary experiment, the exclusion of lipase in the intestinal phase
did not have a significant effect on the bioaccessibility of folate
in the selected samples with a relatively high lipid level
(Fig. S1†). The simulated salivary fluid (SSF, pH 7), simulated
gastric fluid (SGF, pH 3) and simulated intestinal fluid (SIF, pH
7) were prepared according to the protocol described by
Minekus et al.
21
Additionally, the concentration of bile acid and
the activities of individual enzymes were determined before the
assay (Table S2†) according to the INFOGEST protocol.
21
The static in vitro digestion included three steps: the oral
phase, gastric phase and intestinal phase. First, 5 g of the
sample (or 5 mL of the standard mixture) was mixed with
4 mL of SSF with α-amylase. The total volume was brought to
10 mL by adding CaCl
2
and Milli-Q water (W/V = 5/5). The
mixture was incubated at 37 °C for 2 min under shaking.
Second, 8 mL of SGF with pepsin was added. After the pH was
adjusted to 3, CaCl
2
and Milli-Q water were added to bring the
total volume to 20 mL. The solution was then incubated at
37 °C for 2 h with constant shaking. Thirdly, 6 mL of SIF with
α-amylase and 10 mL of SIF with bile extract were added to the
tube, and the pH was adjusted to 7 subsequently. The chymo-
trypsin and trypsin were added, and the final volume was
determined to be 40 mL by adding CaCl
2
and Milli-Q water.
The digesta was obtained after centrifugation and kept at
−20 °C until analysis. A blank control was carried out to deter-
mine the amount of folate in the enzymes in each batch of the
digestion. The amounts of enzymes added were based on the
analysed enzyme activities and the reference values (units per
mL) described by Minekus et al.
21
2.4 Extraction and purification of folate
A tri-enzyme treatment described previously was applied to the
extraction of folate.
24
In brief, 2 g of the ground bread sample
was mixed with 15 mL of CHES/HEPES buffer (pH 7.85) with
sodium ascorbate (2%) and 2-mercaptoethanol (10 mM).
Then, the mixture was brought to a boiling water bath for
10 min. After cooling down on the ice, the pH was adjusted to
4.9, and 20 mg of α-amylase, as well as 1 mL of hog kidney
conjugase, were added. The extract was then incubated by
shaking at 37 °C for 3 h. After incubation, the pH was adjusted
to 7, and 4 mg of protease was added. The solution was again
incubated by shaking at 37 °C for 1 h. The enzymes were then
inactivated by 5 min boiling in a water bath. The final extract
was collected by centrifugation (12 000 rpm, 10 min) and fil-
tered through a 0.45 µm syringe filter.
The extraction of digesta was carried out similarly except
for not using α-amylase and protease. In short, 10 mL of
digesta was mixed with 10 mL of extraction buffer and placed
in a boiling water bath for 10 min. The pH was then adjusted
to 4.9, and 1 mL of hog kidney conjugase was added. The
mixture was incubated at 37 °C for 3 h, after which the
enzymes were inactivated for 5 min in a boiling water bath,
and the final extract was obtained in the same ways as pre-
viously described.
The purification of the extracts was achieved by affinity
chromatography, as described previously.
24
Affinity columns
with folate-binding protein (Scripps Laboratories, San Diego,
CA) bound with Affinity agarose gel (Affi-Gel 10, Bio-Rad
Laboratories, Richmond, CA) were prepared. Folates were
eluted with 0.02 M trifluoracetic acid/0.01 M dithiothreitol
into a 5 mL volumetric flask with piperazine (60 µmol mL
−1
),
ascorbic acid (2 mg mL
−1
) and 2-mercaptoethanol (0.1%). The
concentrated folate extracts were filtered through 0.2 µm
Fig. 1 Images of the bread. The radius of the white plate is about six
centimetres. The round rye bread was torn into smaller pieces. a)–l)
were were commercial breads. m)–o) were whole-grain oat breads pre-
pared in the laboratory using different oat cultivars, labeled as LOA, LOB
and LOC.
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syringe filters, flushed with nitrogen and kept at −20 °C for no
more than 7 days.
The extraction was carried out in triplicate for non-digested
samples. For the digesta, since triplicate digestion was carried
out, the extraction process was performed in duplicate.
Furthermore, duplicate blank controls during extraction were
also carried out in each batch to determine the endogenous
folate in the enzymes.
2.5 Stability test with the folate standard mixture
Folate standards were subjected to in vitro assay and extraction
processes to understand their stability during the analysis. The
standard mixture was exposed to different treatments before,
during and after the digestion (Fig. 2): no treatments or
control (N); a heat treatment in a boiling water bath for 5 min
(B); a heat treatment in a boiling water bath for 5 min and
purification by affinity columns (A); in vitro oral digestion fol-
lowed by 5 min of boiling and purification by gastric affinity
columns (O); in vitro oral and gastric digestion followed by
5 min of boiling and purification by affinity columns (G);
in vitro oral, gastric and intestinal digestion followed by 5 min
boiling and purification by affinity columns (I); in vitro oral,
gastric and intestinal digestion, followed by the folate extrac-
tion process, 5 min boiling and purification by affinity
columns (IE). The folate extraction process included 10 min of
boiling in a water bath, pH adjustment to 4.9, 3-hour incu-
bation with shaking at 37 °C (flushed with nitrogen) and
5 min of boiling in a water bath. All the standard mixture
samples were filtered through 0.2 µm syringe filters, flushed
with nitrogen, and kept at −20 °C for no more than 7 days.
2.6 Quantification of folate
A reversed-phase ultra-high-performance liquid chromato-
graphy (UHPLC) method, which was developed and validated
by our laboratory,
24
was applied to determine the folate
vitamer contents. The column was HSS T3 manufactured by
Waters (1.8 µm, 2.1 × 150 mm; Waters, Milford, MA). During
the UHPLC analysis, the samples were kept in a dark autosam-
pler at 4 °C. The mobile phases were 30 mM potassium phos-
phate buffer (A, pH 2.2) and acetonitrile (B). The following gra-
dient elution was applied: 95% A from 0 to 2.16 min,
95–93.1% A from 2.16 to 4.71 min, 93.1–84.6% A from 4.71 to
7.47 min, 84.6% A from 7.47 to 7.87 min and, finally, to initial
conditions, 84.6%–95% A from 7.87 to 8.3 min, with recondi-
tioning of the column to 95% A from 8.3 to 11 min. The flow
rate was 0.4 mL min
−1
, and the column temperature was
30 °C.
Folate vitamers were quantified using a combination of a
fluorescence (FL) detector and a photodiode array (PDA) detec-
tor. In specific, H
4
folate (tetrahydrofolate) and 5-CH
3
-H
4
folate
(5-methyltetrahydrofolate) were detected by FL (excitation
wavelength: 290 nm; emission wavelength: 356 nm);
10-HCO-PGA (10-formylfolic acid) was determined by FL (exci-
tation wavelength: 360 nm; emission wavelength: 465 nm);
10-HCO-H
2
folate (10-formyldihydrofolate), PGA (folic acid) and
5-HCO-H
4
folate (5-formyltetrahydrofolate) were determined by
PDA (290 nm); and 5,10-CH
+
-H
4
folate (5,10-methenyltetrahy-
drofolate) was determined by PDA (360 nm). The construction
of standard curves was illustrated by Edelmann et al.
24
The
identification of the monoglutamate folates was carried out by
comparing the retention times of the sample peaks to those of
the standard peaks (Fig. S2†). Additionally, the ultraviolet (UV)
spectra of the standard peaks and the sample peaks were
checked to confirm the vitamers. Quantification was achieved
using external calibration curves, with peak areas on the x-axis
and the folate amount (ng) on the y-axis. For rye breads,
5-HCO-H
4
folate and PGA peaks were often masked by
unknown impurities, hindering the accurate quantification of
these vitamers.
2.7 Calculation and statistical analysis
The folate content was expressed as the mean ± standard devi-
ation (µg/100 g, n= 3) on a fresh matter (FM) basis. The total
folate content was calculated as the sum of folate vitamers.
Folate bioaccessibility (%) was obtained by dividing the total
folate content after digestion (µg/100 g FM) by the total folate
content before digestion (µg/100 g FM). Data visualisation and
analysis were carried out using the R Studio platform. Two
sample t-tests were selected to study the differences between
folate contents before and after in vitro digestion. For multi-
group comparisons, one-way analysis (ANOVA) of variance and
Tukey’s honestly significant difference (HSD) post hoc test were
used. Principle component analysis (PCA) was carried out to
Fig. 2 Flow chart of the digestion of standard mixture solution. Folate
extraction process included 10 min in a boiling water bath, pH adjust-
ment, 3-hour incubation with shaking under 37 °C (flushed with nitro-
gen), and 5 min boiling. Seven groups of the standard mixture subjected
to different treatments are represented by the capital letters: N, no treat-
ments; B, 5 min in a boiling water bath; A, 5 min in a boiling water bath
and purification by affinity columns; O, treated by in vitro oral digestion
followed by 5 min in a boiling water bath and purification by affinity
columns; G, treated by in vitro oral and gastric digestion followed by
5 min in a boiling water bath and purification by affinity column; I, treated
by in vitro oral, gastric and intestinal digestion followed by 5 min in a
boiling water bath and purification by affinity columns; IE, treated by
in vitro oral, gastric and intestinal digestion followed by the folate extrac-
tion process, 5 min in a boiling water bath and purification by affinity
columns. UHPLC, ultra-high performance liquid chromatography.
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provide insight into the factors that could influence folate
bioaccessibility. The contents of fat, protein, carbohydrates
and dietary fibre were obtained from the package labels.
5-CH
3
-H
4
folate, 10-HCO-H
2
folate and 10-HCO-PGA were
selected because they were the predominant folate vitamers of
the commercial breads in this study. The total folate and folate
bioaccessibility are important indexes for evaluating the nutri-
tional value of folate in the samples. Before the analysis, the
data were centred by subtracting the column means of xfrom
their corresponding columns and scaled by dividing the
centred columns of xby their standard deviations.
3. Results and discussion
3.1 The stability of folate vitamers without food matrices
To understand the baseline of the changes in folates through-
out the analysis, the in vitro digestion of the standard mixture
solution was carried out, and the results are shown in Table 2.
Samples N, B, A and IE were used to study the recovery of
folates during the extraction, while samples O, G and I were
used to study folate interconversion or degradation during
in vitro digestion.
The recovery of folates during extraction was high. The
levels of total folate in sample N (7.50 ± 0.21 µg) and B (7.31 ±
0.11 µg) were similar, indicating that 5-minute boiling was safe
for most of the folate vitamers. The folate distributions were
similar, too, except for H
4
folate, which showed a significant
decrease by 24% (p< 0.05) in sample B. H
4
folate is inherently
unstable. A previous study reported that H
4
folate could be
totally degraded within one hour under 37 °C, even with the
presence of antioxidants.
23
Therefore, rapid degradation of
H
4
folate in a boiling water bath in this study was expected.
Nevertheless, the 5-minute boiling, which was used to inacti-
vate the enzymes (α-amylase, hog kidney conjugase and pro-
tease) in this study, should not cause any significant loss of
most of the vitamers. Similarly, the extraction process, includ-
ing 10-minute boiling, adjustment of pH and 3-hour incu-
bation under 37 °C, had little impact on the total folate
amount as well as the folate distribution, which can be seen by
comparing samples I and IE. Six folate vitamers were detected
in these samples, and only 5-CH
3
-H
4
folate showed a signifi-
cant difference (p< 0.05), with a higher amount in sample IE
(0.51 ± 0.01 µg) than in sample I (0.32 ± 0.01 µg). Since ascor-
bate and mercaptoethanol were included in the extraction
process, these antioxidants could have protected 5-CH
3
-
H
4
folate from oxidation. Indeed, the stabilizing effect of anti-
oxidants, such as ascorbate and ascorbic acid, on 5-CH
3
-
H
4
folate has been demonstrated in previous research.
25,26
Therefore, the extraction method used in this study can
efficiently protect folate from being degraded and simul-
taneously prevent interconversions among folate vitamers.
This is consistent with the results published earlier by our
group, which showed folate recoveries of 85–99% using a
similar extraction process.
24
Table 2 Folate amounts (and distributions, %) of the standard mixture subjected to different treatments
Folates N B A O G I IE
PGA 0.69 ± 0.02 (9%) a 0.70 ± 0.01 (10%) a 0.58 ± 0.02 (9%) a 0.69 ± 0.02 (11%) a 0.55 ± 0.02 (11%) a 0.59 ± 0.01 (12%) a 0.53 ± 0.01 (10%) a
10-HCO-H
2
folate 2.27 ± 0.13 (30%) a 2.52 ± 0.10 (34%) a 2.13 ± 0.17 (34%) a 2.28 ± 0.17 (35%) a 1.21 ± 0.01 (24%) b 3.02 ± 0.01 (58%) c 2.91 ± 0.01 (56%) c
10-HCO-PGA 0.91 ± 0.01 (12%) a 0.88 ± 0.01 (12%) a 0.75 ± 0.02 (12%) a 0.77 ± 0.02 (12%) a 0.77 ± 0.02 (16%) a 0.88 ± 0.01 (17%) a 0.86 ± 0.01 (17%) a
H
4
folate 0.88 ± 0.03 (12%) a 0.67 ± 0.01 (9%) b 0.36 ± 0.04 (6%) c 0.25 ± 0.04 (4%) d ———
5-CH
3
-H
4
folate 1.00 ± 0.01 (13%) a 0.98 ± 0.01 (13%) a 0.79 ± 0.01 (13%) b 0.79 ± 0.01 (12%) b 0.70 ± 0.02 (14%) b 0.32 ± 0.01 (6%) c 0.51 ± 0.01 (10%) d
5-HCO-H
4
folate 1.10 ± 0.02 (15%) a 1.06 ± 0.01(15%) a 0.86 ± 0.01 (14%) b 0.87 ± 0.01 (14%) b 0.51 ± 0.02 (10%) c 0.27 ± 0.01 (5%) d 0.25 ± 0.01 (5%) d
5,10-CH
+
-H
4
folate 0.65 ± 0.07 (9%) a 0.50 ± 0.01 (7%) a 0.71 ± 0.08 (12%) b 0.79 ± 0.08 (12%) b 1.24 ± 0.02 (25%) c 0.11 ± 0.01 (2%) d 0.13 ± 0.01 (2%) d
Total 7.50 ± 0.21 a 7.31 ± 0.11 a 6.19 ± 0.05 a 6.43 ± 0.05 a 4.97 ± 0.09 b 5.19 ± 0.02 b 5.18 ± 0.01 b
Folate contents are expressed as mean ± standard deviation (µg), n= 3; values within the same row with different letters differed significantly (p< 0.05). Seven groups of the standard mixture
subjected to different treatments are represented by the capital letters: N, no treatments; B, kept in a boiling water bath for 5 min; A, kept in a boiling water bath for 5 min and purified by
affinity columns; O, treated by in vitro oral digestion followed by 5 min in a boiling water bath and purification by affinity columns; G, treated by in vitro oral and gastric digestion followed by
5 min in a boiling water bath and purification by affinity columns; I, treated by in vitro oral, gastric and intestinal digestion followed by 5 min in a boiling water bath and purification by
affinity columns; IE, treated by in vitro oral, gastric and intestinal digestion followed by the folate extraction process, 5 min in a boiling water bath and purification by affinity columns.
Paper Food & Function
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In vitro digestion induced folate interconversion and degra-
dation. Folates were stable during the oral phase, except for
H
4
folate, as shown in Table 2 (samples A and O). H
4
folate
underwent further degradation and was completely degraded
during gastric digestion due to its inherent lability (samples O
and G). Additionally, the interconversion of formyl folates
began at the gastric stage. A considerably higher amount (p<
0.05) of 5,10-CH
+
-H
4
folate was observed in sample G (1.24 ±
0.02 μg), compared to sample O (0.79 ± 0.08 µg), while the
amount of 5-HCO-H
4
folate and 10-HCO-H
2
folate decreased sig-
nificantly (p< 0.05).
Consequently, the distribution of folate was altered. After
oral digestion, 10-HCO-H
2
folate (34%) was the dominant
vitamer (sample O), but 5,10-CH
+
-H
4
folate (35%) became the
dominant folate during gastric digestion (sample G) due to the
change in pH from the oral phase (pH = 7) to the gastric phase
(pH = 3). 5-HCO-H
4
folate is stable in a neutral environment,
but it will convert to 5,10-CH
+
-H
4
folate easily in an acidic solu-
tion.
17
Since there was only a trace amount of reducing agent
(coming from the stock solution) in the standard mixture, con-
version from 10-HCO-H
2
folate to 5,10-CH
+
-H
4
folate was un-
likely. Rather, a part of 10-HCO-H
2
folate could have been oxi-
dised or cleaved into biologically inactive products.
The intestinal digestion triggered further changes to the
folate vitamers, although the total folate amount remained
similar. Considerably decreases (p< 0.05) were observed for
5-HCO-H
4
folate and 5,10-CH
+
-H
4
folate. Especially for 5,10-
CH
+
-H
4
folate, a plummet of 91% was detected. Meanwhile, an
increase in the amount of 10-HCO-H
2
folate from 1.21 ±
0.01 µg (sample G) to 3.02 ± 0.01 µg (sample I) was observed,
making it the dominant vitamer (58%) in sample I. Seemingly,
most 5,10-CH
+
-H
4
folate was converted to 10-HCO-H
2
folate
during the intestinal phase (pH 7), as 5,10-CH
+
-H
4
folate is
unstable at neutral pH.
16
The amount of 5-CH
3
-H
4
folate
started to decrease significantly (p< 0.05) in the intestinal
phase, from 0.70 ± 0.02 µg (sample G) to 0.32 ± 0.01 µg
(sample I), indicating that 5-CH
3
-H
4
folate was less sensitive
to an acidic pH in the gastric phase than formyl folates, as pre-
viously reported by Lucock et al.
27
Further, De Brouwer et al.
28
reported that 5-CH
3
-H
4
folate was stable at 37 °C for
2 hours under a wide range of pH with the protection of
antioxidants.
Additionally, unlike formyl folates, 5-CH
3
-H
4
folate has no
interconversions but can be degraded to para-aminobenzoyl-
glutamate (pABG)
29
or be oxidised to an oxidation product: a
pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-
tetrahydrofolate called MeFox.
30,31
However, neither pABG nor
MeFox bound to the folate binding protein in the affinity
column, and therefore we could not identify these degradation
products with our current method. At the stage of intestinal
digestion, the contribution of reduced folates (H
4
folate, 5-CH
3
-
H
4
folate, 5-HCO-H
4
folate and 5,10-CH
+
-H
4
folate) was merely
13% (sample I), whereas it had been 45% (sample A) before
the in vitro digestion. On the other hand, the fully oxidised
folates (PGA, 10-HCO-PGA) were stable throughout the diges-
tion and folate analyses.
The interconversion and degradation of single vitamers
have been studied by Ringling and Rychlik (2017),
14
but with a
different in vitro digestion model. Overall, our results were in
line with their results when digestion was carried out without
the added ascorbic acid: the recoveries of PGA and
10-HCO-PGA were high (around 100%), and those of 5-CH
3
-
H
4
folate and H
4
folate were low (0.2–4%). Moreover, they con-
firmed that the increases of 5,10-CH
+
-H
4
folate and
10-HCO-H
2
folate during the gastric phase and intestinal phase
originated mainly from the conversion of 5-HCO-H
4
folate and
5,10-CH
+
-H
4
folate, respectively, which is also consistent with
our results. One discrepancy was that we observed a decrease
of 5-HCO-H
4
folate during intestinal digestion, whereas this
vitamer was reported to be stable in their study. The reason for
this could be the different pH adjustment methods used.
Ringling and Rychlik
14
used an automatic titrator in their
model, and the pH was adjusted manually in our study. Since
5-HCO-H
4
folate is extremely sensitive to pH, more
5-HCO-H
4
folate could have been lost due to a less controllable
pH adjustment in this study.
3.2 Total folate and folate bioaccessibility in bread
Table 3 summarises the total folate content and bioaccessibil-
ity of folate in the bread samples. Eight were rye breads, with
total folate levels ranging from 14.0 ± 0.6 to 41.6 ± 1.8 µg/100 g
FM. Four were oat (2) or wheat (2) breads, and their total folate
content was between 11.9 ± 0.7 and 26.7 ± 3.2 µg/100 g FM.
Three oat breads were baked in the laboratory, with the
highest total folate content in LOB (18.8 ± 3.9 μg/100 g FM),
followed by LOA (17.8 ± 3.6 μg/100 g FM) and LOC (11.5 ±
0.9 μg/100 g FM). Rye crispbread (RC), which contained 88%
whole-grain rye flour and 4% rye bran (Table 1), had the
highest total folate content, while white wheat toast (WT) had
the lowest. Previous research has shown that folate in cereal
grains is concentrated in the germ and bran.
32
Therefore,
whole-grain breads contain higher total folate levels than
those with white flour. Comparable results can be found in a
study by Patring et al.,
33
who analysed the folate contents of 32
commercial bread samples from Norway and Sweden. They
reported that the folate content ranged from 14 to 40 μg folic
acid equivalents/100 g FW in soft breads and from 40 to 80 μg
folic acid equivalents/100 g FW in crispbreads.
The bioaccessibility of folate in the commercial breads
varied from 60% ± 12% to 120% ± 4%, with 77% ± 4% to 120 ±
4% in rye breads, 60% ± 12% to 78% ± 2% in oat breads and
79% ± 10% to 94% ± 4% in wheat breads (Table 3). As for
wheat bread, whole-grain wheat toast (WGT) had a higher
bioaccessibility of folate than white wheat toast (WT).
Similarly, rye breads with a higher proportion of whole-grain
flour had higher folate bioaccessibility. For example, round rye
bread baked in the afterheat of the oven (RRAO) contained
85% whole-grain rye flour (Table 1) and had the highest folate
bioaccessibility of 120 ± 4%, while round rye bread without
added yeast (RRNY) contained 73% wholemeal rye flour
(Table 1) and had the lowest folate bioaccessibility among rye
breads (77% ± 4%). Whole-grain bread was reported to have a
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higher content and bioaccessibility of phenolic compounds
than white bread, and phenolic compounds are the main anti-
oxidant in many cereal grains.
35
Phenolic compounds could
act as antioxidants and protect folates from oxidation during
digestion, consequently resulting in a higher bioaccessibility
of folate in bread.
Oat flat portion bread (OF) had the lowest folate bioacces-
sibility among commercial breads: 60% ± 12%. Additionally,
the bioaccessibility of folate in whole-grain oat toast (OT) was
not high (78% ± 2%). As for the oat bread prepared in the lab-
oratory, significantly (p< 0.05) lower total folate contents
were measured in LOA and LOB digesta, which resulted in the
lowest folate bioaccessibility values: 31% ± 1% and 46% ±
3%, respectively. However, the bioaccessibility of folate in
LOC was high with 102% ± 11%. Data about the baking
quality of LOA, LOB and LOC has been reported by
Sammalisto et al. (referred to F16, F25, F19 respectively),
36
and LOC had the lowest crumb hardness value. Low bioacces-
sibility of folate in oat bread compared to the other breads
could be linked to the existence of beta-glucan, which can
slow down the in vitro digestion of starch by hindering the
efficiency of amylase.
37
This could influence the extractability
of folate from bread matrices during digestion. Furthermore,
as shown in Fig. 1(m–o), LOC had a more porous crumb
structure compared to LOA and LOB. However, the differences
between LOA and LOB were less obvious. Porous bread has
been reported to be more accessible to digestive enzymes,
which is related to thinner cell walls in porous bread than in
bread with dense crumbs.
38,39
The in vitro digestion of LOC
could have been more complete than that of LOA and LOB.
Therefore, more folate could have been released from the
LOC than from the LOA and LOB, resulting in high folate
bioaccessibility in the LOC.
Previous studies on folate bioaccessibility or bioavailability
in bread have mainly focused on folate-fortified wheat bread,
and the results showed that added PGA or 5-CH
3
-H
4
folate was
well bioaccessible or bioavailable.
9,12,40,41
For endogenous
folate, folate from rye products and orange juice was shown to
have a good bioavailability that was similar to folic acid from
fortified white bread.
10
Similarly, Ohrvik et al.
11
reported 76%
and 77% bioaccessibility of endogenous folates in wholemeal
bread and rye bran bread, respectively, but 94% in a breakfast
meal that contained bread, orange juice and kiwifruit. These
results, together with the results of this study, imply that
endogenous antioxidants in foods could stabilise folate during
digestion and thus improve bioaccessibility or bioavailability.
3.3 Folate stability in breads during in vitro digestion
Due to the variation in the stability of folate vitamers, the dis-
tribution of folate in the commercial breads underwent signifi-
cant changes during in vitro digestion, as shown in the pie
charts of Fig. 3. The proportion of reduced folates (H
4
folate,
5-CH
3
-H
4
folate, 5-HCO-H
4
folate or 5,10-CH
+
-H
4
folate) in the
commercial breads varied from 24% to 61%. 5-CH
3
-H
4
folate
was the main contributor in whole-grain rye toast (RT, 36%),
whole-grain oat flat portion bread (OF, 39%) and rye crisp-
bread (RC, 32%), while in whole-grain oat flat portion bread
(OT), it was 5-HCO-H
4
folate (28%). For the rest of the eight
types of bread, the main vitamer was 10-HCO-H
2
folate or
10-HCO-PGA. In the bread digesta, the contribution of oxi-
dised folates (10-HCO-H
2
folate, 10-HCO-PGA or PGA) was
72–97%, and 10-HCO-PGA became the dominant vitamer in all
Table 3 Total folate and folate bioaccessibility of bread
Bread
Total folate content
(µg/100 g FM)
Folate
bioaccessibility (%)
Contribution (%) of bioaccessible
folate in 100 g bread to RDA
Before
digestion
After
digestion
Whole-grain rye toast (RT) 14.0 ± 0.6 11.5 ± 0.7** 82 ± 5 2.9
Whole-grain oat toast (OT) 18.0 ± 2.5 14.1 ± 0.3 78 ± 2 3.5
White wheat toast (WT) 11.9 ± 0.7 9.4 ± 1.2* 79 ± 10 2.4
Rye flat portion bread (RF) 15.9 ± 2.3 16.4 ± 1.1 103 ± 7 4.1
Oat flat portion bread (OF) 26.7 ± 3.2 16.1 ± 3.1* 60 ± 12 4.0
Whole-grain wheat toast (WGT) 13.5 ± 1.1 12.7 ± 0.6 94 ± 4 3.2
Rye flat portion bread with germinated rye (RGF) 16.2 ± 2.8 15.3 ± 1.4 94 ± 9 3.8
Traditional Finnish round rye bread (RR) 18.2 ± 1.6 14.7 ± 1.2* 81 ± 6 3.7
Round rye bread with softer texture where wheat flour was
added (RRW)
15.9 ± 1.4 15.4 ± 2.2 97 ± 14 3.9
Rye crispbread (RC) 41.6 ± 1.8 33.1 ± 2.5* 80 ± 6 8.3
Round rye bread without added yeast (RRNY) 18.5 ± 7.8 14.2 ± 0.8 77 ± 4 3.6
Round rye bread baked in the afterheat of the oven (RRAO) 15.7 ± 0.7 18.8 ± 0.6** 120 ± 4 4.7
LOA 17.8 ± 3.6 5.5 ± 0.2* 31 ± 1 1.4
LOB 18.8 ± 3.9 8.7 ± 0.6* 46 ± 3 2.2
LOC 11.5 ± 0.9 11.7 ± 1.3 102 ± 11 2.9
Values are expressed as mean ± standard deviation. For total folate contents before digestion, the standard deviations represent variation among
three analytical replicates; for total folate contents after digestion and folate bioaccessibility, the standard deviations represent variation among
triplicate digestions. Additionally, statistical differences between the folate contents before and after digestion are marked with * (p< 0.05) or **
(p< 0.01). LOA, LOB and LOC are whole-grain oat breads prepared in the laboratory using different oat cultivars. RDA means recommended
dietary allowance, and RDA of folate is 400 µg for healthy adults.
34
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the bread samples (40–67%), except for rye crispbread (RC),
where 10-HCO-H
2
folate was the main vitamer (53%).
The decrease of reduced folates (H
4
folate, 5-CH
3
-H
4
folate,
5-HCO-H
4
folate or 5,10-CH
+
-H
4
folate) was common during the
digestion for the commercial breads (Fig. 3). H
4
folate was
detected only in rye crispbread (RC). 5-CH
3
-H
4
folate was
detected in all commercial breads, but its content decreased
significantly (p< 0.05) during digestion in most of the bread
samples. Similarly, 5-HCO-H
4
folate was present in all the
studied samples, but a decreasing trend was observed, except
for whole-grain rye toast (RT) and round rye bread baked in
the afterheat of the oven (RRAO), where 5-HCO-H
4
folate was
shown to be stable. 5,10-CH
+
-H
4
folate was detected in all the
bread samples, and for most samples, it was unstable during
the digestion. The patterns of the reduced vitamers were in
line with those from the experiment with the standard
mixture. This indicated that potential endogenous antioxi-
dants from the bread could not completely protect the reduced
folates from degradation or conversion during in vitro diges-
tion. 5-CH
3
-H
4
folate in bread matrices was stable in other
studies,
11,12
possibly because of the inclusion of sodium ascor-
bate or ascorbic acid in the digestion models. Although
ascorbic acid is secreted into the human stomach,
42
its capa-
bility as an antioxidant to protect folate during digestion is
uncertain. Ringling and Rychlik
14
found that the protective
effect of the physiological concentration of ascorbic acid
depended on the food matrices, and its protective effect
towards 5-CH
3
-H
4
folate in wheat germ was limited.
Additionally, our previous study showed that the physiological
concentration of ascorbic acid did not significantly protect
folates in cereal and legume flours during in vitro digestion.
13
Therefore, the role of ascorbic acid in protecting endogenous
folates in bread should be elucidated in future studies.
The changing patterns of oxidised folates
(10-HCO-H
2
folate, 10-HCO-PGA or PGA) during in vitro diges-
tion varied among the commercial breads (Fig. 3).
10-HCO-PGA in commercial bread was stable, which agrees
with the conclusion from the digestion of the standard
mixture. However, for several samples, significantly larger
amounts of 10-HCO-PGA were detected in the bread digesta
than in bread. This may be caused by the catalysation effect of
metal ions, such as iron, in bread matrices. Iron compounds
can catalyse the oxidation of formyl folates, for example,
10-HCO-H
4
folate to 10-HCO-H
2
folate.
43
10-HCO-H
2
folate could
be further oxidised to 10-HCO-PGA, resulting in an increase of
10-HCO-PGA in the digesta samples. On the other hand, the
content of 10-HCO-H
2
folate in wheat and oat breads decreased
during digestion, while in rye bread it was stable, and its
content increased significantly (p< 0.05) in some samples (RC
and RRAO). One possible explanation is the rich formyl folate
pool in the rye. 10-HCO-H
2
folate could originate from the con-
version of 5,10-CH
+
-H
4
folate or the oxidation of
Fig. 3 (a) Folate vitamer contents and (b) folate vitamer distributions in the commercial breads before and after digestion. The error bars of breads
and bread digesta represent the standard deviation among triplicate analysis and triplicate digestions, respectively. In addition, * or ** indicates a sig-
nificant difference in vitamer contents before and after digestion at a level of p< 0.05 or p< 0.01, respectively. A, RT, whole-grain rye toast; B, OT,
whole-grain oat toast; C, WT, white wheat toast; D, RF, rye flat portion bread; E, OF, oat flat portion bread; F, WGT, whole-grain wheat toast; G, RGF,
rye flat portion bread with germinated rye; H, RR, traditional Finnish round rye bread; I, RRW, round rye bread with a softer texture where wheat
flour was added; J, RC, rye crispbread; K, RRNY, round rye bread without added yeast; L, RRAO, round rye bread baked in the afterheat of the oven.
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10-HCO-H
4
folate. Furthermore, since the determination of
5-HCO-H
4
folate from rye samples was influenced by impurities
in this study, its content could have been underestimated, as
previous studies have reported that the level of 5-HCO-H
4
folate
in rye bread was higher than that in wheat or oat bread.
22,33
As
for PGA, no significant difference was found between oat
bread and digesta. In wheat bread, it seems to be degraded
during digestion. However, the amount of PGA in wheat bread
was low. In rye breads, PGA was not determined due to the
masking by the impurities. Nevertheless, these material-
dependent changing patterns of oxidised folates during
in vitro digestion should be studied more in the future.
The changes in folate vitamers in whole-grain oat bread
baked in the laboratory were consistent with those of the com-
mercial breads (Fig. 4). The reduced folates decreased con-
siderably (p< 0.05) during the digestion. 5,10-CH
+
-H
4
folate
was relatively stable, but its level was low in the oat bread.
10-HCO-H
2
folate decreased in different degrees, with a
plummet (p< 0.05) in LOA, but only a slight decrease in LOB
and LOC. PGA was not detected in LOB, but its content was sig-
nificantly (p< 0.05) higher in the digesta of LOA and LOC than
in the respective bread samples. 10-HCO-PGA was stable as
expected, and a significantly higher (p< 0.05) content of this
vitamer was observed in the LOC digesta than in the bread,
possibly due to a better release of folate from the LOC matrix
than LOA and LOB. Oxidised folates accounted for 39–42% of
the total folates in these breads, and their contribution was
raised to 71–75% in the bread digesta.
3.4 Implications of macronutrient content and folate
distribution for folate bioaccessibility
The commercial breads were separated into five main groups
according to the PCA (Fig. 5). Rice crispbread (RC) was separ-
ated solely from other breads, mainly because of its high total
folate level. In contrast, wheat breads (WT and WGT) were
grouped mainly because of their relatively low total folate
levels. Oat breads (OT and OF) formed a cluster mainly
because of their higher fat content compared to the other
breads in this study. Interestingly, whole-grain rye toast bread
(RT) was also included in this cluster, which could be due to
the inclusion of sunflower seeds as one of the ingredients
(Table 1), yielding extra fat in the bread. Round rye breads (RR,
RRW, RRNY and RRAO) were also clustered as a group, mainly
because of their relatively high 10-HCO-PGA content and folate
bioaccessibility. Rye flat portion breads (RF and RGF) were
grouped in the middle of the biplot, with no significant corre-
lation to any variables.
The directions of the arrows indicate the correlations
among the variables (Fig. 5). Folate bioaccessibility and fat or
protein content were negatively associated, while carbohydrate
Fig. 4 (A–C) (a) folate vitamer contents and (b) folate vitamer distributions in the whole-grain oat bread baked in the laboratory before and after
digestion. The error bars of breads and bread digesta represent the standard deviation among triplicate analysis and triplicate digestions, respectively.
The results are expressed as means ± standard deviation. In addition, * or ** indicates a significant difference in folate contents before and after
digestion at a level of p< 0.05 or p< 0.01, respectively.
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(starch) and dietary fibre content seemed to have a positive
influence on folate bioaccessibility. Starch is the major com-
ponent of bread. The presence of proteins and lipids in food
matrices can decrease the digestibility of starch compared to
those matrices with pure starch.
44
Therefore, bread with high
lipid or protein contents would be less digestible, and folate
cannot be fully liberated during digestion, resulting in lower
folate bioaccessibility. On the other hand, bioaccessibility was
independent of total folate content. This echoes the findings
of Seyoum and Selhub
45
that foods with high folate contents
did not necessarily have high folate bioavailability and vice
versa.
5-CH
3
-H
4
folate correlated to the total folate content in the
commercial bread to some extent but could impair folate
bioaccessibility (Fig. 5). 5-CH
3
-H
4
folate was the main vitamer
in whole-grain oat flat portion bread (OF), and this vitamer
was lost completely during digestion, causing lower folate
bioaccessibility (60% ± 12%) than that of whole-grain oat toast
(OT, 78% ± 2%). Similarly, the folate bioaccessibility of whole-
grain rye toast (RT, 82% ± 5%) and rye crispbread (RC, 80% ±
6%) was relatively low, and both had 5-CH
3
-H
4
folate as their
dominant vitamer. Furthermore, rye flat portion bread (RF)
and rye flat portion bread with germinated rye (RGF) were
similar products in terms of their shape and textural character-
istics. The latter bread contained more 5-CH
3
-H
4
folate than
the former one. Previous research reported that germination
improved the level of 5-CH
3
-H
4
folate in rye seeds.
46
However,
the bioaccessibility of folate in RGF (94% ± 9%) was lower
than in RF (103% ± 7%), as most of the 5-CH
3
-H
4
folate was
degraded during the digestion. From the results with the stan-
dard mixture, it is known that the loss of 5-CH
3
-H
4
folate
occurred in the intestinal phase, where the absorption of
folate takes place.
47
5-CH
3
-H
4
folate is the most abundant
folate vitamer in many plant materials.
48
Based on the lability
of 5-CH
3
-H
4
folate observed in this study, if this vitamer could
not be protected before absorption, the bioavailability of folate
would be undermined. An early study pointed out that 5-CH
3
-
H
4
folate might be of less nutritional value due to its instabil-
ity.
49
To the best of our knowledge, studies on the changes in
endogenous folate distribution during in vitro digestion are
rare. Ringling et al.
14
reported that the stability of 5-CH
3
-
H
4
folate affected the bioaccessibility of folate in wheat germ.
Therefore, combined with the results of this study, foods that
are rich in 5-CH
3
-H
4
folate may have low bioaccessibility or
bioavailability.
10-HCO-PGA and folate bioaccessibility were positively cor-
related (Fig. 5). Furthermore, breads with high folate bioacces-
sibility had a small proportion of reduced folates (H
4
folate,
5-CH
3
-H
4
folate, 5-HCO-H
4
folate or 5,10-CH
+
-H
4
folate), but on
the other hand, a small proportion of reduced folates in the
folate composition did not guarantee high folate bioaccessibil-
ity in the bread matrices. All the round rye breads in this study
had small reduced-folate content, with percentages ranging
from 28% to 38%. Round rye bread is a traditional Finnish
bread with a dense and hard structure. It has a larger heating
surface area than other breads in the oven; therefore, the
reduced folates could have been oxidised or degraded during
the processing. Nevertheless, this could improve folate bioac-
cessibility, as the folates would be stable enough to survive
digestion. However, the round rye bread with a softer texture
where wheat flour was added (RRW) had a relatively low folate
bioaccessibility of 77% ± 4% in this study, and the traditional
Finnish round rye bread (RR) had a bioaccessibility of 81% ±
6%. In these two types of bread, the content of 10-HCO-PGA
Fig. 5 The biplot (dim 1–2) from the PCA of commercial bread. Dimensions 1 and 2 represent 39.1% and 32.7% of the total variances of the vari-
ables, respectively; cos2 comprises how well the individual was represented in this dim 1–2 biplot; contrb comprises the contribution of the variable
to the dim 1–2 biplot. RT, whole-grain rye toast; OT, whole-grain oat toast; WT, white wheat toast; RF, rye flat portion bread; OF, oat flat portion
bread; WGT, whole-grain wheat toast; RGF, rye flat portion bread with germinated rye; RR, traditional Finnish round rye bread; RRW, round rye bread
with softer texture where wheat flour was added; RC, rye crispbread; RRNY, round rye bread without added yeast; RRAO, round rye bread baked in
the afterheat of the oven.
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did not increase during digestion as it did in the round rye
bread without added yeast (RRNY) and round rye bread baked
in the afterheat of the oven (RRAO), as shown in Fig. 3 (H, I, K
and L). 10-HCO-PGA was stable during the in vitro digestion
(Table 2); therefore, the increase in 10-HCO-PGA could be due
to the different formyl folate pools in the round rye bread. The
folates that were not studied in this paper, such as
10-HCO-H
4
folate,
49
could contribute to the total folate,
although it was determined to be 10-HCO-H
2
folate and
10-HCO-PGA in this study. In this case, for the bread with
10-HCO-PGA increasing during digestion, folate bioaccessibil-
ity could be overestimated.
3.5 Hypothesised pathway of folate interconversion and
degradation during digestion
Based on the results from this study, together with those from
previous reports,
14–17,50
the possible interconversion and oxi-
dation pathways of food folates are summarised in Fig. 6.
H
4
folate in foods could undergo losses during mastication and
then complete degradation in the gastric phase. Since the pH
will drop significantly in the stomach, interconversions of
formyl folates will be triggered. 5-HCO-H
4
folate would be con-
verted to 5,10-CH
+
-H
4
folate. 10-HCO-H
4
folate (not studied in
this paper) would also be converted to 5,10-CH
+
-H
4
folate.
10-HCO-H
2
folate could be oxidised to 10-HCO-PGA or degraded
during this stage. However, with the presence of antioxidants
from food matrices, 10-HCO-H
2
folate could be reduced to
10-HCO-H
4
folate, which would be further converted to 5,10-
CH
+
-H
4
folate. Likewise, the original 5-CH
3
-H
2
folate present in
food matrices could be reduced to 5-CH
3
-H
4
folate by endogen-
ous antioxidants. In the intestinal phase, where the pH is
neutral, 5,10-CH
+
-H
4
folate would be converted back to
5-HCO-H
4
folate or 10-HCO-H
4
folate, and 10-HCO-H
4
folate
couldbedegradedoroxidisedto10-HCO-H
2
folate and, sub-
sequently, to 10-HCO-PGA. In some food matrices with high
levels of metal ions, more 10-HCO-H
2
folate would be oxidised
to 10-HCO-PGA because of the catalytic effect. Additionally,
10-HCO-H
4
folate could also undergo isomerisation to
5-HCO-H
4
folate under long-term incubation at 37 °C. Losses of
5-CH
3
-H
4
folate would begin in the intestine, and 5-CH
3
-H
4
folate
would be degraded or oxidised to 5-CH
3
-H
2
folate and MeFox.
Many food matrices have been reported to be rich in MeFox,
which is the main oxidation product of 5-CH
3
-H
4
folate.
51,52
However, MeFox does not have bioactivity. PGA, which has been
reported to be the most stable folate, could also suffer from
losses in some food matrices during the intestinal phase.
4. Conclusion
This study investigated the bioaccessibility of folate in bread
and changes in vitamer patterns during in vitro digestion.
Folate bioaccessibility varied among bread types, ranging from
31 ± 1 to 120 ± 4%. The data of the individual folate vitamers
revealed three major trends in terms of folate stability during
in vitro digestion: (1) the decrease of reduced folates (5-CH
3
-
H
4
folate, 5-HCO-H
4
folate and 5,10-CH
+
-H
4
folate); (2) the inter-
conversion among formyl folates (5-HCO-H
4
folate,
10-HCO-H
2
folate and 5,10-CH
+
-H
4
folate; and (3) the high stabi-
lity of the oxidised folates (PGA and 10-HCO-PGA). These
trends could be universal for all samples, but the degree of
change would vary among the different materials, as demon-
strated by the data from the bread matrices in this study.
Therefore, to improve the bioaccessibility of folate, we could
(1) stabilise the reduced folates during digestion and (2)
increase the contribution of oxidised folates to the total folate
in foods. Additionally, the results from the whole-grain oat
breads prepared in the laboratory indicated that the digesti-
bility of foods can play an important role in the bioaccessibility
of endogenous folates.
Although the prediction of folate bioaccessibility is
complex, we discovered some common changing patterns of
folate vitamers during in vitro digestion. Factors that could
influence the stability of folates during digestion should be
studied, such as the antioxidant capacity and buffer capacity of
foods. Furthermore, the food structure can affect the digesti-
bility of foods, which would further influence the extractability
of endogenous folates during digestion. In the future, more
studies should be carried out to systematically investigate the
effects of the above-mentioned factors on folate bioaccessibil-
ity or bioavailability.
Author contributions
Fengyuan Liu: conceptualization, investigation, data curation,
visualization, writing –original draft. Susanna Kariluoto: con-
ceptualization, writing –review & editing, supervision.
Minnamari Edelmann: writing –review & editing, supervision.
Vieno Piironen: writing –review & editing, supervision.
Conflicts of interest
There are no conflicts to declare.
Fig. 6 Hypothesized pathway of the folate interconversion and degra-
dation during digestion.
Paper Food & Function
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Acknowledgements
The authors would like to thank Miikka Olin for his kind help
in UHPLC analysis, Saara Sammalisto for baking oat bread in
the laboratory, the China Scholarship Council (CSC) for finan-
cial support for the PhD project of Fengyuan Liu, and the
Finnish Food Research Foundation for a research grant.
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