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Profiling of naturally occurring folates in a diverse soybean germplasm by
HPLC-MS/MS
Article in Food Chemistry · February 2022
DOI: 10.1016/j.foodchem.2022.132520
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Profiling of naturally occurring folates in a diverse soybean germplasm by
HPLC-MS/MS
Kwadwo Gyapong Agyenim-Boateng, Shengrui Zhang, Md Shariful Islam,
Yongzhe Gu, Bin Li, Muhammad Azam, Ahmed M. Abdelghany, Jie Qi,
Suprio Ghosh, Abdulwahab S. Shaibu, Berhane Sibhatu Gebregziabher, Yue
Feng, Jing Li, Yinghui Li, Chunyi Zhang, Lijuan Qiu, Zhangxiong Liu, Qiuju
Liang, Junming Sun
PII:
DOI:
Reference:
S0308-8146(22)00482-4
https://doi.org/10.1016/j.foodchem.2022.132520
FOCH 132520
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
7 December 2021
27 January 2022
17 February 2022
Please cite this article as: Gyapong Agyenim-Boateng, K., Zhang, S., Shariful Islam, M., Gu, Y., Li, B., Azam,
M., Abdelghany, A.M., Qi, J., Ghosh, S., Shaibu, A.S., Sibhatu Gebregziabher, B., Feng, Y., Li, J., Li, Y., Zhang,
C., Qiu, L., Liu, Z., Liang, Q., Sun, J., Profiling of naturally occurring folates in a diverse soybean germplasm by
HPLC-MS/MS, Food Chemistry (2022), doi: https://doi.org/10.1016/j.foodchem.2022.132520
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Profiling of naturally occurring folates in a diverse soybean germplasm by HPLCMS/MS
Kwadwo Gyapong Agyenim-Boateng1#, Shengrui Zhang1#, Md Shariful Islam2#, Yongzhe
Gu3#, Bin Li1#, Muhammad Azam1, Ahmed M. Abdelghany1,4, Jie Qi1, Suprio Ghosh1,5,
Abdulwahab S. Shaibu1,6, Berhane Sibhatu Gebregziabher1,7, Yue Feng1, Jing Li1, Yinghui
Li3, Chunyi Zhang2, Lijuan Qiu3, Zhangxiong Liu3*, Qiuju Liang2*, Junming Sun1*
1
The National Engineering Laboratory for Crop Molecular Breeding, MARA Key
Laboratory of Soybean Biology (Beijing), Institute of Crop Sciences, Chinese Academy of
Agricultural Sciences, Beijing 100081, China
2 Biotechnology
Research Institute, Chinese Academy of Agricultural Sciences, Beijing
100081, China
3 The
National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI)/
Key Laboratory of Germplasm and Biotechnology (MARA), Institute of Crop Sciences,
Chinese Academy of Agricultural Sciences, Beijing 100081, China
4 Crop
Science Department, Faculty of Agriculture, Damanhour University, Damanhour
22516, Egypt
5 Bangladesh
Agricultural Research Institute, Gazipur 1701, Bangladesh
6 Department
of Agronomy, Bayero University, Kano 700001, Nigeria
7 Crop
Sciences Research Department, Mehoni Agricultural Research Center, Maichew 7020,
Ethiopia
#These
authors contributed equally to this work.
*Corresponding
authors
Email: sunjunming@caas.cn, liangquiju@caas.cn, liuzhangxiong@caas.cn
Tel. Fax: 0086-10-82105805
Abstract
Soybean is a rich source of folates. We optimised the extraction and detection of folates from
soybean seeds by HPLC-MS/MS and analysed the folate content and composition of 1074
accessions. Total folate content ranged from 64.51–691.24 μg/100 g fresh weight, with 10fold variation, and 60 elite accessions with over 400 μg/100 g of total folate were identified.
The most abundant component was 5-CHO-H4folate, which accounted for an average of 60%
of total folate. Seed-coat colour, seed weight, ecoregion, and cultivar type significantly
affected soybean folate content. Furthermore, 5-CH3-H4folate correlated positively with seed
protein (r = 0.24***) and negatively with oil (r = -0.26***). The geographical distribution of
folate according to accession origin revealed that accessions from Northeast China contain
higher amounts of total folate and 5-CHO-H4folate. This study provides comprehensive and
novel insights into the folate profile of soybean, which will benefit soybean breeding for
folate enhancement.
Keywords: Soybean (Glycine max L. Merrill); Folate; HPLC-MS/MS; Germplasm; Natural
variation; Elite accessions
Chemical compounds
10-Formyl-folic acid
(PubChem
CID:
135405023);
5,10-Methenyl-tetrahydrofolate
(PubChem CID: 135398657); 5-Formyl-tetrahydrofolate (PubChem CID: 135403648); 5Methyl-tetrahydrofolate (PubChem CID: 135483998); Dihydrofolate (PubChem CID:
135398604); Folic acid (PubChem CID: 135398658); Tetrahydrofolate (PubChem CID:
135444742); Methotrexate (PubChem CID: 126941); Sodium phosphate monobasic
(PubChem CID: 23672064); Sodium phosphate dibasic (PubChem CID: 24203); Sodium
ascorbate (PubChem CID: 23667548); β-Mercaptoethanol (PubChem CID: 1567); α-Amylase
(PubChem CID: 62698); Acetonitrile (PubChem CID: 6342); Formic acid (PubChem CID:
284)
1. Introduction
Folates (vitamin B9) are essential water-soluble vitamins that function as co-enzymes in
numerous metabolic processes by mediating one-carbon transfer reactions (Strobbe &
Dominique, 2017). Humans cannot synthesise folates de novo and depend entirely on dietary
sources. Folate deficiency causes severe health disorders, including neural tube defects,
anaemia, cardiovascular disease, and certain cancers (Blancquaert et al., 2010). Due to the
severe risks associated with folate deficiency, folic acid fortification has been mandated in
certain countries. Notwithstanding the financial challenges and difficulties in public
education associated with synthetic folic acid fortification, chronic intake of synthetic folic
acid has been linked to adverse health effects. These effects include increased cancer risks,
hepatoxicity and masked vitamin B12 deficiencies (Patel & Sobczyńska-Malefora, 2017).
Thus, folate biofortification to enhance the natural folate content in crops by metabolic
engineering and conventional breeding stands as cost-effective, efficient and promising
alternatives.
Generally, legumes are considered as rich sources of folates with diversity in variation,
making them good candidates for folate improvement. Analysis of the folate content of 29
wild and 4 cultivated lentil accessions revealed that wild and cultivated lentils contained
197.00–497.00 μg/100 g and 174.00–364.00 μg/100 g dry weight (DW) of total folate,
respectively (Zhang et al., 2019). A study of an 85-pea germplasm panel showed that folate
contents ranged from 14.00–55.00 μg/100 g DW (Jha et al., 2020). The total folate contents
of 96 common bean accessions were 113.0–222.00 μg/100 g (Martin, Torkamaneh, & Pauls,
2021). According to studies, soybean total folate content ranges from 202.90–450.00 μg/100
g (Shohag, Wei, & Yang, 2012; Rychlik, Englert, &Kirchhoff, 2007; Ginting & Arcot, 2004;
Mo et al., 2013). However, few soybean cultivars were used in these studies and the natural
variation of folates in a large soybean population has not been investigated. Soybean is a
major economic crop, containing averagely 40% protein, 20% oil, and 15% carbohydrates
(Azam et al., 2020). Therefore, studying the folate composition of a large soybean germplasm
population and the further selection of elite accessions will be beneficial towards soybean
breeding and combatting folate deficiencies. Furthermore, through conventional breeding, the
natural variation of folates in a crop population can be exploited to identify QTL or genes for
folate biofortification.
The objectives of this study were to (i) optimise the extraction protocol for folate
monoglutamates in soybean, focusing on the extraction buffer pH, enzyme treatment and
boiling time; (ii) investigate the natural variation of folate in a diverse soybean germplasm;
(iii) assess the effect of seed coat colour and seed weight on folate content; and (iv) evaluate
the association of folate with protein, oil content and geographical factors. The findings of
this study would provide essential information for folate improvement in soybean. The elite
cultivars identified can be used in food industries and can also be used as donor parents in
developing soybean cultivars for folate improvement.
2. Materials and methods
2.1. Materials
For folate extraction optimisation, we used Zhonghuang 203 (ZH203), a soybean cultivar
developed by the Soybean High-Yield and Quality Breeding Research Group of the Institute
of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS), which was harvested
in Autumn 2019. Three technical replicates were used for method development. The soybean
germplasm composed of 590 landraces and 382 cultivars and was collected from the three
major soybean ecoregions in China, including Northern Region (NR, 187 accessions),
Huanghuaihai Region (HR, 252 accessions) and Southern Region (SR, 343 accessions), as
well as cultivars from outside China, including East Asia (19 accessions), Russia (15
accessions), North America (15 accessions), South America (9 accessions) and Europe (8
accessions). The accessions were planted in 2018 in Sanya, Hainan province (18°24′ N and
109°5′ E), China. The experimental details, climatic conditions and agronomic practices have
been previously reported (Abdelghany et al., 2020). The harvested seeds were air-dried to a
low moisture content (4–8%), pulverised (IKA, A10 basic, Rheinische, Germany), and stored
at -20°C until folate analysis. Hundred-seed weight was taken after soybean seeds were dried.
Seed-coat colours were visually determined as black (102 accessions), brindled (19
accessions), brown (73 accessions), green (86 accessions), purple-red (8 accessions) and
yellow (757 accessions).
2.2. Reagents
Folate monoglutamates including folic acid (PteGlu; FA), 5-methyltetrahydrofolate (5-CH3H4folate; 5MTHF), tetrahydrofolate (H4folate; THF), 5,10-methenyltetrahydrofolate (5,10CH=H4folate; 5,10MTHF), 5-formyltetrahydrofolate (5-CHO-H4folate; 5FTHF), 10formylfolic acid (10-CHO-PteGlu; 10FFA), dihydrofolate (H2folate; DHF), and Methotrexate
(MTX) were purchased from Schirks Laboratories (Jona, Switzerland). A pyrazino-s-triazine,
MeFox, the oxidation product of 5-CH3-H4folate, was obtained from Toronto Research
Chemicals (Toronto, Canada). Methotrexate (MTX) was used as the internal standard to
correct errors during sample preparation and quantification. The purity of all folate standards
was over 95%.
Sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), sodium
ascorbate, β-mercaptoethanol, α-amylase (from Aspergillus oryzae, ∼ 30 units/mg), and
protease (Type XIV, from Streptomyces griseus, ≥ 3.5 units/mg) were sourced from SigmaAldrich (St. Louis, MO, USA). Ultra-pure water was purified on a Heal Force ultra-pure
water system (Shanghai, China). Acetonitrile and formic acid (HPLC-MS grade) were
purchased from Fisher Scientific (Geel, Belgium). Rat serum, chicken pancreas, and βmercaptoethanol were sourced from Beijing Solarbio Science and Technology Company
(Beijing, China). A certified reference material (BCR485) was purchased from the Institute
for Reference Materials and Measurements (Geel, Belgium) and was stored at -80°C.
2.3. Stock solutions and folate standards
All standard stock solutions (100 g/mL) were prepared under subdued light to prevent
photo-oxidation using methanol and 20 mM ammonium acetate (pH6.2) (Riaz et al., 2019),
containing 0.5% L-ascorbic acid and 0.5% -mercaptoethanol. 5,10-CH=H4folate was
prepared using the same buffer as above at pH4.5. Standard stock solutions were stored in 1
mL aliquots at -80°C.
2.4. Folate extraction
Parameters including pH of extraction buffer (pH 4.5, 5.5, 6.5, 7, 7.5, 8.5, and 9), enzyme
treatment and dosage (α-amylase, protease, chicken pancreas and rat serum), and boiling time
(5, 10, 15 and 20 min) were optimised for the best folate recovery from soybean seeds.
Sample extraction was carried out under subdued light to prevent photo-oxidation. However,
the initial extraction protocol adopted for our experiment is as follows:
Fifty milligrams of fine powder was transferred into a 1.5 mL screw-cap tube (ST-150;
Axygen, Union City, CA, USA) and was mixed with 1 mL of 50 mM phosphate buffer
(containing 0.5% L-ascorbic acid and 0.2% -mercaptoethanol as antioxidants, and 30 ng/mL
MTX as internal standard) on a Miulab multi-tube vortex mixer (Miulab, Hangzhou, China).
The mixture was immediately boiled for 10 min in a boiling water bath and cooled on ice for
10 min. Twenty microlitres of -amylase was added and incubated at 37°C with shaking for
30 min. After incubation, the sample was boiled for 5 min to deactivate the enzymes. The
sample was then cooled on ice for 10 min. Thereafter, 15 μL of protease was added, and the
tube was incubated for 1 h at 37°C. After incubation, the sample was boiled for 5 min to
deactivate enzymes. The deconjugation of polyglutamylated tails was carried out by adding
30 μL of rat serum to the sample and incubating for 4 h at 37°C. Subsequently, the sample
was boiled for 10 min, cooled on ice for 10 min, and centrifuged twice at 13,000 rpm for 10
min at 4°C. For sample clean-up, 400 µL of the supernatant was taken into a 3KDa MWCO
ultra-filtration tube (Millipore, Burlington, MA, USA) and centrifuged at 13,000 rpm for 20
min at 4°C. Finally, 100 μL of the filtrate was taken for analysis, and the remaining was
stored at -80°C. Extracts were kept in low-actinic HPLC vials to avoid exposure to light.
2.5. HPLC-MS/MS
For folate separation and quantification, all parameters were the same as described by Liang
et al. (2020). Folate separation was performed using an Agilent 1260 HPLC system with a
Kromasil 100-5 C18 column (Akzo Nobel, Sweden), Agilent SB-C18 pre-column (Agilent
Technologies, USA), and an injection volume of 15 μL, which was controlled using the Mass
Hunter software. The injector and column oven temperatures were maintained at 4°C and
25°C, respectively. The mobile phases included 0.1% (v/v) formic acid in water (phase A)
and 0.1% (v/v) formic acid in acetonitrile (phase B). The gradient program was run for 16.5
min (Supplementary Table S1). Electrospray ionisation in a positive mode was performed
using a triple-quadrupole tandem mass spectrometry (Agilent 6420, Palo Alto, CA, USA).
Multiple reaction monitoring (MRM) parameters, including the precursor ion, product ion
and collision energy were optimised for fragmentation and one major product ion for each
folate vitamer was selected for subsequent analysis.
2.6. Method validation
For method validation, the sensitivity, linearity, precision, trueness, absolute recovery, and
matrix effect (ME) were evaluated. Sensitivity and linearity parameters were determined
based on an eight-point calibration curve prepared in a blank soybean matrix (n = 3). For
trueness, the folate content of BCR485 was quantified in triplicate. Precision was calculated
based on the relative standard deviation (RSD) of the content and peak areas of BCR485 and
standards on the same and different days. Intra-day repeatability was assessed in one batch,
while inter-day repeatability was evaluated by running samples on three different days (n = 6).
The RSD of the retention time of the folate standards relative to the analytes was also
calculated to evaluate the stability of the method. Absolute recovery and matrix effect were
calculated as described by Matuszewski, Constanzer, & Chavez-Eng (2003).
To prepare a blank soybean matrix, 2 g of seed powder of ZH203 was weighed and mixed
with 40 mL of 50 mM phosphate buffer (without antioxidants). The mixture was boiled for 1
h at 100°C while being exposed to direct sunlight to degrade endogenous folates and was
centrifuged at 13,000 rpm for 30 min at 4°C. Ten per cent activated charcoal was added to the
supernatant and was incubated with shaking for 1 h and centrifuged at 13,000 rpm for 30 min.
The supernatant was filtered through 3KDa MWCO ultra-filtration tubes, and the filtrate was
confirmed for folates below detection limits and was stored at -20°C until use.
2.7. Folate composition of soybean in a wide soybean germplasm
The determination of the folate composition in 1074 soybean accessions was carried out
using the final optimised protocol and BCR485 was analysed in triplicate with every batch as
quality control, with a rejection threshold of 10%. Briefly, 50 mg of fine powder in a 1.5 mL
screw-cap tube was mixed with a 1 mL 50 mM phosphate buffer (pH5.5, 0.5% ascorbic acid,
0.2% -mercaptoethanol and 30 ng/mL MTX) using a vortex mixer and was placed in a
boiling water bath for 15 min. After cooling on ice for 10 min, 200 μL each of rat serum and
chicken pancreas was added to the sample and was incubated for 4 h at 37°C. After
incubation, the sample was boiled for 10 min, cooled on ice for 10 min, and centrifuged twice
at 13,000 rpm for 10 min at 4°C. For sample clean-up, 400 µL of the supernatant was taken
into a 3KDa MWCO ultra-filtration tube and centrifuged at 13,000 rpm for 20 min at 4°C.
One hundred microlitres of the filtrate was taken for analysis with HPLC-MS/MS, and the
remaining was stored at -80°C. Extracts were kept in low-actinic HPLC vials to avoid
exposure to light. Samples were not stored for more than a week.
2.8. Statistical analysis
Data were subjected to analysis of variance (ANOVA) with agricolae package (https://cran.rproject.org/web/packages/agricolae)
and
boxplots
with
the
ggplot2
package
(https://www.rdocumentation.org/packages/ggplot2/versions/3.3.5) in R 3.4.5 (R Foundation
for Statistical Computing, Vienna, Austria). Post-hoc mean separation was done using
Tukey’s HSD at (P < 0.05). Figures for method development were produced using GraphPad
Prism version 9.00 for Windows. Accessions were grouped into accession types (cultivar and
landrace), ecoregions (NR, HR, and SR), and seed morphological traits (seed coat colour and
seed weight) to evaluate their effect on folate content via ANOVA. Pearson’s correlation
analysis between folates from this study and quality traits (protein and oil) from the same
soybean accessions in our previous study (Azam et al., 2021) was conducted and visualised
using the corrplot package (https://www.rdocumentation.org/packages/corrplot/versions/0.92)
in R. The geographical distribution maps of soybean seed folates were drawn using ArcGIS
10.0 (ESRI, Redlands, CA, USA, http://destktop.arcgis.com/en/arcmap/) using ordinary
kriging interpolation. Folate concentrations were calculated as μg/100 g based on fresh
weight (FW). The sum of individual folates, excluding MeFox, was calculated as total folate.
3. Results and Discussion
3.1. Optimisation of folate extraction from soybean seeds
To enhance the efficiency of our method, folate monoglutamate extraction from soybean
seeds was optimised by considering three major factors, the pH value of extraction buffer,
enzyme treatment and dosage, and boiling time.
The stability of certain folate vitamers is greatly affected by pH and it is sample-dependent
(De Brouwer et al., 2007). In this study, the effects of seven pH levels of the extraction buffer
(4.5, 5.5, 6.5, 7, 7.5, 8.5, and 9) on folate monoglutamate recovery were evaluated. Folate
monoglutamate extraction includes enzymatic treatments that require complex and timeconsuming pH adjustments, which may lead to oxidation. Therefore, we investigated the
stability of folate monoglutamate vitamers from homogenisation. Total folate differed
significantly (P < 0.001) among the pH treatments (Fig. 1A). Due to the variability in the
stability of folate vitamers, having an optimal pH for all vitamers for extraction is very
difficult. The highest total folate content (144.32 μg/100 g FW) was observed at pH5.5,
followed by pH4.5 (119.88 μg/100 g FW) and pH6.5 (116.13 μg/100 g FW). Remarkably, the
stability of folate vitamers, H4folate, 5-CH3-H4folate and 5-CHO-H4folate was highest at
pH5.5 (Fig.1A, Supplementary Table S2). These results were consistent with a previous
study in mungbean, where the highest folates were extracted at pH4.5-5.5 (Monch & Rychlik,
2012). Therefore, an extraction buffer of pH5.5 was used for folate extraction in this study.
Enzymes, including α-amylase, protease, and conjugases are commonly used for folate
extraction from different food matrices. In this experiment, different combinations and
dosages of enzymes were evaluated (Fig. 1B, Supplementary Table S3). It was observed that
folate yield increased with increased amounts of rat serum. Using 30 μL rat serum yielded
113.58 μg/100 g total folate, whereas 100 μL of rat serum yielded 198 μg/100 g total folate.
The single-use of 100 μL chicken pancreas yielded 129.58 μg/100 g of total folate. The three
or four-enzyme treatments resulted in folate yield from 155.80–200.95 μg/100 g but were
time-consuming and may not be ideal, especially for a larger sample size. Moreover,
increasing protease volumes increases background noise and affects quantification, consistent
with other studies (Zhang et al., 2005). On the other hand, the combination of higher amounts
of rat serum and chicken pancreas (200 μL of each) resulted in an optimal folate yield
(220.54 μg/100 g). Consistently, higher doses of conjugases have been reported to improve
deconjugation and folate extraction efficiency from black bean (Ramos-Parra, Urrea-López,
& de la Garza, 2013). The current study revealed that a two-step extraction process involving
heating and deconjugation was sufficient for the optimal release of folates. This was
consistent with a previous study in chickpea, where higher amounts of chicken pancreas and
rat serum resulted in similar total folate amounts (407 μg/100 g) as compared to the
combination of four enzymes (422 μg/100 g) (Zhang et al., 2018). Therefore, for further
experiments in our study, the two-step extraction protocol including boiling and the
combined treatment with 200 μL of rat serum and 200 μL chicken pancreas was used.
Heating samples at 100°C aids in cell lysis and inactivates endogenous enzymes, inducing
greater folate release from the matrix and preventing further folate conversion (Zhang et al.,
2005). The effect of boiling at 100°C for 5, 10, 15, and 20 min was investigated in this study,
and the total folate contents observed were 201.06, 224.19, 290 and 275.49 μg/100 g,
respectively (Fig. 1C, Supplementary Table S4). As shown in Fig.1C, total folate content was
the highest at 15 min. However, folate content decreased at 20 min, which may have resulted
from folate degradation induced by long-time heating. This observation follows earlier
reports that boiling at 100°C for 15 min and 12 min was optimal for folate recovery from
food matrices and spinach, respectively (Czarnowska-Kujawska, Gujska, & Michalak, 2017;
Shohag et al., 2017). Hence, boiling at 100°C for 15 min was adopted for subsequent
experiments in this study.
3.2. Method validation
The LOD, LOQ, and linearity were determined for each folate standard and MTX from a
multi-point calibration curve prepared in a blank soybean matrix in triplicate. The correlation
coefficients (R2) for all folates were > 0.99, indicating good linearity of the massspectrometric response within the concentration ranges (Table 1). The MRM transition of
seven folate vitamers, MeFox and internal standard MTX is shown in Supplementary Fig. S1.
For matrix effects, folate standards and MTX (10 ng/mL) were added to the extraction
buffer and blank soybean matrix. Matrix effect ranged from 81.86–104.87% (Supplementary
Table S5). The intra-day and inter-day precision of all folates were within the acceptable
ranges of 3.81–7.01% and 6.26–11.09% RSD, respectively (Supplementary Table S6). RSDs
of retention times ranged from 0.06–0.33%, indicating run-to-run precision and robustness of
the method. Similarly, we observed no different peak shapes between the analytes and the
standards, indicating no co-eluting compounds with the target analyte.
The absolute recoveries of each folate and MTX are listed in Supplementary Table S5. The
major folate vitamers showed acceptable absolute recoveries (70.26–126.06%). The low
recoveries of H2folate (11.43–19.90%) and 5,10-CH=H4folate (44.22–48.51%) were caused
by their instability to heat and pH conditions. H2folate is labile under heat, and its recovery is
always low during folate extraction. Despite this, our method provided a relatively higher
recovery for this minor component than the absolute recovery of H2folate in lentils (4.00–
13.00%) (Zhang et al., 2019). The recovery of 5,10-CH=H4folate found in this study may
indicate conversions from 5,10-CH=H4folate to 5-CHO-H4folate. Moreover, the lower
recoveries of these two vitamers do not significantly affect major components and total folate
content because they are minor components accounting for lesser than 3% of total folate in
soybean. Nevertheless, pH plays much significance in folate quantification and must be
critically evaluated.
Absolute recovery of H4folate ranged between 35.27–38.48%. The low absolute recovery
of this folate is due to its high lability to pH and heating conditions (Strandler et al., 2015).
However, the changes of H4folate in a sample matrix may be different from a standard
solution, as observed in our pH and boiling time experiments (Supplementary Table S2 and
S4). This indicates that the combined use of thiols and ascorbic acid may reduce H4folate
degradation in a soybean matrix. Moreso, a similar pH value of 6 has been reported to
improve H4folate stability in plant and food matrix (Zhang et al., 2005; Loznjak et al., 2019).
Precision and all other validation parameters for H4folate were within the acceptable ranges.
For trueness, the certified reference material, BCR485, was analysed and compared to its
certified value and results from other studies (Supplementary Table S7). In our study, the
total folate content of BCR485 (350.08 μg/100 g) was slightly higher than the certified total
folate content (315±28 μg/100 g) using the microbiological assay (MA) (Finglas et al., 1998).
Recent studies using HPLC-MS/MS have also reported higher total folate contents (336–375
μg/100 g) than the certified value (Vishnumohan, Arcot, & Pickford, 2011; Ringling &
Rychlik, 2017), which is similar to our results. In this study, 5-CH3-H4folate was higher than
the indicative value (non-certified value) and close to reported values using HPLC-MS/MS.
However, the proportion of 5-CH3-H4folate (~ 90%) in total folate was consistent with
previous studies. The discrepancies between MA and HPLC methods and the differences in
sample pre-treatment methods may be responsible for the varying results in the reference
material. Additionally, the content of other folate vitamers, H4folate, 5-CHO-H4folate, 10CHO-PteGlu, H2folate, PteGlu, 5,10-CH=H4folate, and MeFox were also determined in this
study.
3.3. Folate profiling and vitamer distribution among 1074 soybean accessions
The final optimised folate method was applied to determine the folate composition of 1074
diverse soybean accessions. Total folate levels ranged from 64.51–691.24 g/100 g, with an
average of 262.01 g/100 g (Supplementary Table S8). The wide natural variation in folates
in this soybean panel can be utilised to map QTL and identify candidate genes for folate
accumulation. The average total folate in this study falls within the folate contents previously
reported in soybean between 188–450 μg/100 g (Shohag et al., 2012; Mo et al., 2013).
Consequently, higher diversity was discovered and 60 folate-rich soybean accessions with >
400 g/100 g were identified, of which four accessions (ZDD14672, ZDD12910, ZDD12830
and ZDD14683) contained > 600 g/100 g of total folate (Supplementary Table S9). The
soybean accessions rich in folates could be used as genetic material for folate breeding
programs or serve as good dietary sources. Compared with other crops, the highest folate
levels obtained in this study show that total folate contents of soybean are many folds higher
than that of potato, wheat and pea (Riaz et al., 2019; Jha et al., 2015; Jha et al., 2020). Thus, a
100 g serving of soybeans could provide a significant amount of the recommended daily
allowance of folates.
Seven folate monoglutamates were identified in soybean seeds: 5-CHO-H4folate, PteGlu,
5-CH3-H4folate, H4folate, 10-CHO-PteGlu, 5,10-CH=H4folate, and H2folate, with the first
five contributing 95–98% of the total folate content (Supplementary Table S8). The most
abundant vitamer, 5-CHO-H4folate, contributed 60% of total folate. PteGlu, 5-CH3-H4folate
and H4folate accounted for an average of 12%, 10% and 7.6% of total folate, respectively.
Meanwhile, 10-CHO-PteGlu accounted for about 6% of total folate, while 5,10-CH=H4folate
and H2folate were the least abundant folate vitamers, collectively contributing about 3% of
total folate. MeFox, the oxidative product of 5-CH3-H4folate, was also identified in this study,
ranging from 110.00–1601.71 g/100g in soybean seeds. Studies on folate vitamer
distribution in soybean are scanty. Whereas studies reported 5-CHO-H4folate as the most
dominant folate vitamer (Ginting & Arcot, 2004; Shin et al., 1975), other studies have
contrastingly reported H4folate as the most dominant (Rychlick et al., 2007; Shohag et al.,
2012). The discrepancies in folate vitamer distributions may be caused by storage, cultivar
type, environment, and analytical method. H4folate, being one of the most labile vitamers,
one would assume will be oxidised to PteGlu at a low pH condition. However, this was not
observed in the current study. Moreover, the analytical method used in this study had high
specificity and this enabled us to quantify seven folate monoglutamates and MeFox in
soybean. Furthermore, 5-CHO-H4folate was determined to be the most dominant vitamer.
Therefore, subsequent studies on the effects of location and storage on soybean folate vitamer
distribution will be helpful to understand soybean vitamer distribution.
3.4. Soybean folate content varies among accession types and ecoregions
To evaluate the variation of folates among accession types, the accessions were grouped into
landraces (590 accessions) and improved cultivars (382 accessions). Total folate contents
varied significantly (P < 0.05) between the accession types, with landraces having a wider
range (64.51–691.24 g/100 g) and a higher mean of 268.99 g/100 g than the improved
cultivars (77.54–515.93 g/100 g with a mean of 253.60 g/100 g) (Fig. 2). This suggests
that past breeding efforts have not focused on folate improvement in soybean. Consistent
with our results, cultivated lentils had 174–361 g/100 g of total folate compared to
undomesticated lentils (195–497 g/100 g) (Zhang et al., 2019). Past breeding efforts geared
towards enhancing crop yield and appearance may have contributed to a decline in nutritional
values and genetic diversity of modern cultivars. A recent study of the Glycine spp.
pangenome revealed a significant reduction of the average number of protein-coding genes
per individual during domestication and selection (Bayer et al., 2021). Soybean landraces,
representing a tremendous genetic diversity, are adapted to various environments and
climatic conditions, exhibit tolerance to biotic stresses and harbour genes and gene
complexes for quality traits. Previous studies from our research group have also shown that
soybean seed quality traits, including isoflavone, fatty acids, and tocopherols are also
influenced by accession type (Azam et al., 2020; Abdelghany et al., 2020; Ghosh et al., 2021).
Landraces are valuable germplasm to broaden the genetic base of modern soybean cultivars.
Thus, the introgression of genes from soybean landraces in breeding programs will help
enhance the nutritional value of soybean.
Total folate (P < 0.05) and individual vitamers (P < 0.001) differed significantly among
the ecoregions (NR, HR and SR), except 10-CHO-PteGlu (P > 0.05) (Fig. 3). Total folate
was highest in NR (277.00 g/100 g), followed by SR (267.85 g/100 g) and HR (255.54
g/100 g). The concentration of 5-CHO-H4folate was highest in the NR (181.18 g/100 g).
As shown earlier, 5-CHO-H4folate accounted for over 60 % of total folate in this study,
which explains why total folate was the highest in NR. Among cultivars, the first eight
accessions with the highest folates were from NR whereas, among the landraces with the
highest folates, 70% were from the SR ecoregion. This showed a tendency for ecoregion to
affect folate levels, indicating a genetic component of variation.
3.5. Association of folates with protein and oil
The analysis of the correlation between quality traits is essential in breeding and selection
programs. Therefore, we analysed the correlation between folates, protein and oil contents
(Fig. 4).
Generally, significant positive correlations existed within folates. The highest correlation
was observed between 5-CHO-H4folate and total folate (r = 0.90***), whereas the lowest
positive correlation was between H4folate and 10-CHO-PteGlu (r = 0.07*). The high
correlation of 5-CHO-H4folate with total folate may be due to the high abundance of this
vitamer in soybean. However, 5-CHO-H4folate and 5,10-CH=H4folate had a low negative
correlation (r = -0.11***), which may have been caused by the interconversion relationship
between these two vitamers.
The folate vitamer, 5-CH3-H4folate had a positive correlation with protein (r = 0.24***)
but was negatively correlated with oil (r = -0.26***). Soybean seed proteins have a
significant negative correlation with oil. In this study, 5-CH3-H4folate negatively correlated
with oil, but the contrary trend was observed with protein. Folates are protected from photooxidation by binding to proteins (folate binding proteins; FBPs) (Liang et al., 2013) and the
photo-oxidation of folates induces protein damage (Wusigale et al., 2021). In Arabidopsis,
high folate accumulation was associated with the up-regulation of FBPs (Puthusseri et al.,
2018), and the transgenic expression of bovine FBP increased folate content in rice
(Blancquaert et al., 2015). As already known, folates are involved in many key metabolic
functions, including amino acid synthesis, and thus can explain to a higher degree the
positive correlations identified in this study. However, further studies will be necessary to
confirm this relationship in soybean.
3.6. Effects of seed morphology and agronomic traits on folate content
The relationship between seed morphological traits and nutritional content will help in seed
selection and soybean genetic improvement. However, the effect of these traits on soybean
folates is unknown. A previous study in pulses reported that seed morphological traits did not
significantly correlate with folate content (Jha et al., 2015). In this study, we evaluated the
effect of seed weight and seed-coat colour on soybean folate monoglutamates. We observed
highly significant differences for all folates due to variations in seed-coat colour. The blackseeded soybean contained the highest amounts of total folate, 5-CHO-H4folate and other
folate vitamers (Supplementary Fig. S2). Studies have shown that the black-seeded soybean
contains higher levels of anthocyanins and valuable nutrients with great antioxidant and
carcinogenic properties (Slavin et al., 2009). Soybean seed coat colour is controlled by
multiple loci, with most of these loci involved in flavonoid-based pigmentation pathways. It
has been reported that the chalcone synthase gene, which catalyses the first step of the
flavone and flavonol branch of phenylpropanoid biosynthesis was up-regulated more than
two-fold in transgenic high-folate tomato (Waller et al., 2010). This suggests a possible
crosstalk between folates and the flavonoid pathway. The current study is the first to study
the association of folates with seed coat colour, and thus, further studies are needed to
confirm this in soybean.
To study the effect of seed weight on folates, the accessions in this study were grouped
into five categories based on 100-seed weight, namely: A (<10.0 g), B (≥ 10 g and < 15 g), C
(≥ 15 g and < 20 g), D (≥ 20 g and < 25 g), and E (≥ 25 g). Total folate and most individual
vitamer levels were significantly (P < 0.001) affected by seed weight (Supplementary Fig.
S3). Accessions with seed weight in category A had the highest amounts of total folate
(average, 304.03 g/100 g) and most folate vitamers, indicating that the smaller the seed size,
the higher the folate content. In pea, the folate content was higher in the embryo than in the
cotyledon (Jabrin et al., 2003). Smaller soybean seeds contain higher proportions of the
embryo and may contribute to the higher folate concentrations. However, little is known
about the detailed distribution of folates in the soybean seed and thus needs further studies.
3.7. Geographical effects and distribution of folates in soybean in China
The geographical distribution of soybean folates, based on their ecoregion is shown in Fig. 5.
The correlation between the geographical factors and soybean folates revealed a significant
association (Supplementary Table S10). Significant positive correlations were observed
between longitude and MeFox, 10-CHO-PteGlu, 5-CHO-H4folate and total folate. On the
other hand, longitude had significant negative correlations with other vitamers. There was a
negative correlation between latitude with H4folate, 5-CH3-H4folate, H2folate, and PteGlu,
while MeFox, 10-CHO-PteGlu, 5-CHO-H4folate, and 5,10-CH=H4folate positively correlated
with latitude. Altitude had a positive correlation with H4folate and a negative correlation with
5,10-CH=H4folate, MeFox and 10-CHO-H4folate.
The geographical map revealed a distinction between the distribution of total folate, 5CHO-H4folate, and other folate vitamers in China. The highest contents of total folate and 5CHO-H4folate were concentrated in the NR part of China. Conversely, the highest amounts
of 5-CH3-H4folate could be seen widely distributed in the three ecoregions with more
intensity at the HR followed by the SR, with the lowest contents observed in the NR.
4. Conclusion
In conclusion, we conducted a novel study investigating the folate monoglutamate
composition of 1074 diverse soybean germplasm using HPLC-MS/MS. First, we optimised
the extraction method and found optimal recoveries for the most important folate vitamers at
pH5.5, boiling for 15 min and a combined use of 200 L of both rat serum and chicken
pancreas. The extraction method was time-saving and cost-effective compared to the
traditional tri-enzyme treatment methods and reduced the complications associated with
longer extraction times. By profiling the 1074 soybean germplasm, we identified a
significantly wide variation among the folate contents, with an over 10-fold variation for total
folate content and found elite accessions with total folate contents > 400 g/100 g.
Furthermore, we observed that soybean folate monoglutamates are affected by a plethora of
factors, including ecoregion, accession type, seed-coat colour, seed size, and geographical
factors. Finally, correlation analysis revealed folate had positive and negative relationships
with protein and oil, respectively. Overall, this study provides the basis for the understanding
of folates in soybean and shows that soybean is a strong candidate for folate biofortification.
Supplementary Data
Supplementary Fig. S1. MRM transition of seven folate vitamers, MeFox and internal
standard (MTX).
Supplementary Fig. S2. Folate distribution (μg/100 g FW) among different seed coat
colours in soybean.
Supplementary Fig. S3. Folate distribution (μg/100 g FW) among different seed weights in
soybean.
Supplementary Table S1. Gradient program for folate analysis on HPLC MS/MS in
soybean seeds.
Supplementary Table S2. Folates extracted (μg/100 g FW) after using different pH
conditions in soybean seeds.
Supplementary Table S3. Folates extracted (μg/100 g FW) after using different enzyme
treatments and amounts in soybean seeds.
Supplementary Table S4. Folates extracted (μg/100 g FW) using different boiling times in
soybean seeds.
Supplementary Table S5. Matrix effect and absolute recovery of folate vitamers.
Supplementary Table S6. Precision measurements of folates standards and BCR485.
Supplementary Table S7. Comparison of the folate values (μg/100 g) of BCR485 detected
in our study with previous studies and certified value.
Supplementary Table S8. Descriptive statistics of 7 folate monoglutamates, total folate and
MeFox among soybean accessions from Hainan 2018 extracted using our optimised
extraction protocol.
Supplementary Table S9. List of soybean accessions containing > 400 g/100 g FW of total
folate.
Supplementary Table S10. Correlations between geographical factors and soybean folates.
Author contributions
K.G.A-B, S.Z, S.I., R.G. and B.L. - Formal analysis, Investigation; Methodology, Software,
Writing - original draft, review & editing, Data curation. M.A., A.M.A., A.S., J.Q., S.G,
B.S.G., Y.F., J.L., Y.L - Investigation, Methodology. C.Z., L.Q.- Formal analysis, Resources,
Writing - review & editing. J.S., Q.L. and Z.L. - Conceptualization, Funding acquisition,
Project administration, Supervision, Resources, Writing - review & editing.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Ministry of Science and Technology (2021YFD1201605),
National Natural Science Foundation of China (32161143033 and 32001574), and CAAS
(Chinese Academy of Agricultural Sciences) Agricultural Science and Technology
Innovation Project (2060302-2, CAAS-ZDRW202004 and SWJSZD2020-001).
We thank the public laboratory of the Biotechnology Research Institute, Chinese Academy
of Agricultural Sciences, for providing us with access to the HPLC and triple-quadrupole
MS/MS instruments and for providing technical assistance. Sincere gratitude goes to Dr
Benjamin Karikari for his assistance in proofreading this manuscript and to all who
contributed to making this manuscript fit for publication.
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Figure Legends
Fig. 1. Extraction optimisation of folate monoglutamates from ZH203. A. Stability of
soybean folate monoglutamates under seven different pH treatments. pH test was conducted
using tri-enzyme treatment consisting of 20 μL -amylase, 15 μL protease and 30 μL rat
serum. B. Folates extracted from ZH203 under different enzyme treatments and amounts; A30 L rat serum, B- 50 L rat serum, C- 100 L rat serum, D- 150 L rat serum, E- 100 L
chicken pancreas, F- 200 L rat serum + 200 L chicken pancreas, G- 20 L -amylase +15
L protease + 30 L rat serum, H- 20 L -amylase +150 L protease + 100 L rat serum, I20 L -amylase +150 L protease + 100 L rat serum +150 L chicken pancreas; C. Folate
recovery of ZH203 at different boiling times (n = 3). Error bars represent the standard
deviation of folate recovery from triplicate determinations. Total folate content of bars with
same lowercase letters are not significantly different (P > 0.05).
Fig. 2. Comparison of the folate content (μg/100g FW) between soybean cultivars and
landraces. Different lowercase letters indicate statistical differences at P < 0.05.
Fig. 3. Folate vitamer distribution (μg/100 g FW) among the three major ecoregions in China;
Northern Region (NR), Huanghuaihai Region (HR) and Southern Region (SR). Different
lowercase letters indicate statistical differences at P < 0.05.
Fig. 4. Correlation between folate vitamers and other seed quality traits. THF, H4folate;
5MTHF, 5-CH3-H4folate; 5,10MTHF, 5,10-CH=H4folate; 10FFA, 10-CHO-PteGlu; 5FTHF,
5-CHO-H4folate; DHF, H2folate. *, **, and *** indicate significant differences at 5%, 1%
and 0.1%.
Fig. 5. Geographical distribution of 5-CHO-H4folate (5FTHF), 5-CH3-H4folate (5MTHF) and
total folate content (μg/100 g FW) across the three major soybean production areas in China.
Table 1. Calibration and sensitivity data for folate standards prepared in blank soybean
matrix (n = 3)
Folate
Limit of detection
(g/100 g)
Limit of
quantification
(g/100 g)
Slope (mean ±
SD n = 7 or 8)
Correlation
coefficient
R2
Linear range
(g/100 g)
Function
H4folate
0.098
0.328
1833.97±12.35
0.995
0.328–100
1/x
5-CH3-H4folate
0.207
0.627
3827.72±33.09
0.992
0.627–500
1/x
5,10-CH=H4folate
0.124
0.377
2176.55±34.88
0.997
0.377–100
1/x
MeFox
0.085
0.259
1263.45±11.82
0.998
0.259–1800
1/x
10-CHO-PteGlu
0.206
0.623
617.24±5.68
0.992
0.623–100
1/x
5-CHO-H4folate
0.366
1.109
1245.34±6.6
0.994
1.109–500
1/x
H2folate
0.080
0.232
176.29±1.49
0.993
0.232–100
1/x
PteGlu
0.148
0.447
376.78±1.43
0.991
0.447–200
1/x
MTX
0.226
0.685
4454.49±8.56
0.995
0.685–400
1/x
Author contributions
K.G.A-B, S.Z, S.I., R.G. and B.L. - Formal analysis, Investigation; Methodology, Software,
Writing - original draft, review & editing, Data curation. M.A., A.M.A., A.S., J.Q., S.G,
B.S.G., Y.F., J.L., Y.L - Investigation, Methodology. C.Z., L.Q.- Formal analysis, Resources,
Writing - review & editing. J.S., Q.L. and Z.L. - Conceptualization, Funding acquisition,
Project administration, Supervision, Resources, Writing - review & editing.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
Supplementary Figure S1. MRM transition of seven folate vitamers, MeFox and internal
standard, MTX.
Supplementary Figure S2. Folate distribution (μg/100g FW) among different seed coat
colors in soybean.
Different lowercase letters indicate statistical differences at P < 0.05.
Supplementary Figure S3. Folate distribution (μg/100g FW) among different seed weights
in soybean.
Different lowercase letters indicate statistical differences at P < 0.05.
Supplementary Table S1. Gradient program for folate analysis on HPLC MS/MS in
soybean seeds
Time (min)
Mobile phase A (%)
Mobile phase B (%)
Flow rate (mL/min)
0.00
95.00
5.00
0.30
2.00
91.00
9.00
0.30
7.90
90.50
9.50
0.30
8.20
80.00
20.00
0.30
11.20
80.00
20.00
0.60
11.40
95.00
5.00
0.60
14.40
95.00
5.00
0.60
14.50
95.00
5.00
0.30
16.50
95.00
5.00
0.30
Supplementary Table S2. Folates extracted (μg/100g FW) after using different pH conditions in soybean seeds
pH
H4folate
5-CH3H4folate
5,10-CH+H4folate
Mefox
10-CHOPteGlu
5-CHOH4folate
H2folate
PteGlu
Total Folates
4.5
4.30±0.16
7.52±0.05
1.74±0.23
84.07±9.18
16.20±1.15
75.97±3.38
1.55±0.05
12.59±0.22
119.88±4.38b
5.5
6.98±0.17
9.03±1.12
1.47±0.32
96.68±3.89
29.67±0.33
78.18±2.24
1.81±0.09
17.18±0.32
144.32±0.41a
6.5
5.59±0.79
7.18±0.99
1.05±0.17
82.8±11.35
31.27±0.70
56.45±2.82
1.43±0.61
13.15±0.13
116.13±5.94b
7
4.09±0.24
5.56±0.16
0.95±0.01
66.65±4.64
25.33±1.25
41.01±0.61
1.38±0.11
9.03±0.34
87.35±0.45d
7.5
4.74±0.25
5.47±0.51
0.90±0.14
67.83±2.91
29.78±1.50
47.49±0.74
1.16±0.04
8.85±0.81
98.40±1.41c
8.5
4.51±0.00
6.02±0.45
1.06±0.03
85.07±5.65
35.24±0.35
45.35±3.47
1.92±0.13
8.92±0.82
103.02±2.84c
9.0
4.10±0.02
5.78±0.59
1.07±0.07
99.54±6.14
36.32±1.62
31.62±1.81
1.72±0.08
9.12±0.07
89.74±2.77d
Folate vitamers μg/100g (mean±SD) according to pH treatment
Lowercase superscripts at the total folate values indicate significant differences at P < 0.05
Analysis of variance (ANOVA) of total folates by pH treatments
Treatment
Df
Sum Sq
Mean Sq
F value
Pr(>F)
6
4804.50
800.75
77.26
4.855e-06 ***
Supplementary Table S3. Folates extracted (μg/100g FW) after using different enzyme treatments and amounts in soybean seeds
Enzyme treatment
H4folate
5-CH3H4folate
5,10-CH+H4folate
Mefox
10-CHOPteGlu
5-CHOH4folate
H2folate
PteGlu
Total Folates
30 RS
9.54±0.32
10.51±1.34
1.09±0.16
64.78±1.51
9.41±0.160
78.63±0.84
0.62±0.10
3.78±0.09
113.58±0.20f
50 RS
12.39±0.04
13.05±1.92
1.37±0.28
85.90±2.77
11.60±0.26
104.45±0.36
0.74±0.12
5.07±0.19
148.66±1.98de
100 RS
13.90±0.60
13.45±0.75
1.79±0.76
82.79±10.8
13.71±0.33
114.62±4.11
0.83±0.14
5.55±0.23
163.85±6.65cd
150 RS
16.29±2.35
15.92±2.18
2.29±1.39
101.74±14.74
16.05±0.49
133.95±5.37
0.91±0.16
5.91±0.44
191.33±21.69b
100 CP
4.50±0.19
8.18±0.90
0.00±0.00
146.87±10.23
6.26±0.02
103.35±8.36
0.00±0.00
7.30±0.00
129.58±9.10ef
200 RS + 200 CP
23.88±0.16
21.15±4.86
2.98±0.17
105.81±4.10
15.97±0.49
149.49±0.25
1.25±0.35
5.83±0.22
220.54±4.75a
20 A, 15 P, 30 RS
9.85±1.54
9.88±2.04
0.00±0.00
64.78±1.51
32.01±7.45
89.95±0.26
1.58±0.01
12.53±3.65
155.8±14.43cde
20 A, 150 P, 100 RS
13.35±1.10
4.42±0.73
1.97±0.58
176.70±36.5
43.49±6.53
92.66±9.70
3.76±1.80
18.98±3.78
178.62±23.01bc
20 A, 150 P, 100 RS, 150 CP
16.25±1.48
7.25±1.23
1.45±0.10
185.98±15.51
52.05±1.97
100.97±7.45
3.41±0.08
19.58±1.91
200.95±10.24ab
Folate vitamers μg/100g (mean±SD) according to enzyme treatment
Lowercase superscripts at the total folate values indicate significant differences at P < 0.05; RS – Rat serum; CP – Chicken pancreas; A- -amylase; P- Protease.
Analysis of variance (ANOVA) of total folates by enzyme treatment
Treatment
Df
Sum Sq
Mean Sq
F value
Pr(>F)
8
19606
2450.71
16.24
4.558e-05 ***
Supplementary Table S4. Folates extracted (μg/100g FW) using different boiling times in soybean seeds
Boling time
H4folate
5-CH3H4folate
Mefox
5,10-CH+H4folate
10-CHOPteGlu
5-CHOH4folate
H2folate
PteGlu
Total Folates
5 mins
15.21±0.71
22.27±0.86
152.49±5.34
9.65±0.19
18.68±0.53
111.83±4.97
2.73±0.36
20.70±1.42
201.06±7.60c
10 mins
14.87±0.60
22.82±1.22
165.27±0.90
8.62±0.88
23.26±0.61
127.03±1.61
2.06±0.13
25.52±1.09
224.19±2.74b
15 mins
19.34±1.29
31.67±1.76
220.11±7.11
11.03±2.34
29.51±1.76
167.61±1.02
1.88±0.15
28.95±2.16
290.00±8.44a
20 mins
17.15±0.93
27.58±3.16
201.9±8.19
8.99±0.94
27.79±2.77
164.87±1.33
1.54±0.08
27.58±1.04
275.49±10.09a
Folate vitamers μg/100g (mean±SD) according to boiling time
Lowercase superscripts at the total folate values indicate significant differences at P < 0.05
Analysis of variance (ANOVA) of total folates by boiling time
Treatment
Df
Sum Sq
Mean Sq
F value
Pr(>F)
3
10580.10
3526.70
59.13
0.0009049 ***
Supplementary Table S5. Matrix effect and absolute recovery of folate vitamers
Compounds
Matrix Effect %
Absolute recovery %
Low
Medium
High
H4folate
94.97
38.48
37.39
35.27
5-CH3-H4folate
104.87
70.26
71.61
74.22
5,10-CH=H4folate
98.10
45.37
48.51
44.22
MeFox
97.99
97.4
95.85
94.34
10-CHO-PteGlu
90.60
74.85
80.95
80.03
5-CHO-H4folate
91.37
110.67
98.97
126.06
H2folate
81.86
15.18
19.90
11.43
PteGlu
91.40
56.95
57.83
59.17
MTX
93.36
81.44
85.93
75.33
MTX- Internal standard
Supplementary Table S6. Precision measurements of folates standards and BCR 485
Folates
Intra-day
precision %RSD
Inter-day
precision
RSD (%)
Intra-day
precision
(BCR
485) %RSD
Inter-day
precision (BCR
485) %RSD
Retention
time %RSD
H4folate
4.81
6.26
1.89
6.47
0.23
5-CH3-H4folate
5.79
6.79
3.20
3.03
0.14
5,10-CH=H4folate
3.81
7.95
5.92
7.44
0.21
MeFox
4.45
10.10
4.28
4.01
0.16
10-CHO-PteGlu
4.40
11.09
3.25
4.34
0.09
5-CHO-H4folate
5.36
7.04
4.92
5.57
0.25
H2folate
7.01
8.20
1.69
1.20
0.06
PteGlu
6.65
10.93
7.60
7.70
0.10
4.55
7.53
0.33
MTX
5.95
7.99
MTX: Internal standard; %RSD- Relative standard deviation
Supplementary Table S7. Comparison of the folate values (μg/100g) of BCR 485 detected in our study with previous studies and certified
value
Mean content
(Present Study)
Shohag et al., 2017
(LC-MS/MS)
Ringling et al.,
2013 (LC-MS/MS)
Vishnumohan et al.,
2011 (LC-MS/MS)
Finglas et al., 1999
(HPLC)
Certified value
(MA)
Indicative value
H4folate
3.66±0.01
28.78±1.86
8.00
ND
5
NA
NA
5-CH3-H4folate
332.79±0.05
249.00±11.13
320.90
375
202-294
NA
214.42
5,10-CH=H4folate
1.27±0.13
NA
0.10
NA
NA
NA
NA
MeFox
200.28±7.83
NA
NA
ND
NA
NA
NA
10-CHO-PteGlu
1.17±0.38
NA
1.10
NA
ND
NA
NA
5-CHO-H4folate
9.97±0.15
23.18±1.63
5.00
ND
ND
NA
NA
H2folate
0.35±0.10
NA
NA
NA
NA
NA
NA
PteGlu
0.87±0.06
NA
0.80
ND
ND
NA
NA
Total folates
(Without MeFox)
350.08±0.58
289.30±14.22
336.00
375.00±16.00
NA
315.00±28.00
NA
Extraction method
(conjugase)
Mono-enzyme
(CP+ RS)
Mono-enzyme
(CP+ RS)
Mono-enzyme
(CP+ RS)
Tri-enzyme (Human
plasma)
Mono-enzyme
(Hog kidney)
-
Mono-enzyme
(Hog kidney)
MA- Microbiological assay; HPLC- High-Performance liquid chromatography; LC-MS/MS- Liquid Chromatography Mass Spectrometry; NA- not analysed; ND- not
detected;”-“data not available
Supplementary Table S8. Descriptive statistics of 7 folate monoglutamates, total folates and MeFox of soybean accessions from Hainan 2018 extracted
using our optimized extraction protocol
Folates
Minimum (μg/100g)
Maximum (μg/100g)
Mean (μg/100g)
Standard Deviation
Coefficient of variation (%)
H4folate
0.68
66.49
20.53
9.92
48.32
5-CH3-H4folate
0.84
205.74
28.23
18.9
66.96
5,10-CH=H4folate
0.43
28.46
5.12
3.00
58.56
MeFox
110.00
1601.71
407.02
154.37
37.93
10-CHO-PteGlu
1.00
71.06
11.45
6.15
53.66
5-CHO-H4folate
33.02
590.59
162.25
64.65
39.84
H2folate
0.25
29.44
2.90
2.68
92.37
PteGlu
2.27
163.09
31.84
15.28
48.00
Total folates (Without MeFox)
64.51
691.24
262.01
84.29
32.17
Supplementary Table S9. List of soybean accessions containing > 400 g/100 g FW of total
folates
Identification
number/ name
ZDD14672
Accession type
Location/Ecoregion
Country
Total Folates
Landrace
SR
China
691.24
ZDD12910
Landrace
SR
China
680.87
ZDD12830
Landrace
SR
China
680.26
ZDD14683
Landrace
SR
China
634.05
ZDD02866
Landrace
HR
China
567.60
ZDD09581
Landrace
HR
China
549.53
ZDD13689
Landrace
SR
China
521.96
ZDD14783
Landrace
SR
China
516.82
WDD01594
Cultivar
USA
USA
515.93
ZDD06233
Landrace
SR
China
515.21
ZDD22642
Cultivar
NR
China
515.18
ZDD10734
Landrace
HR
China
506.48
ZDD06646
Landrace
SR
China
504.95
ZDD02277
Landrace
HR
China
504.13
ZDD02461
Landrace
HR
China
495.47
ZDD01818
Landrace
HR
China
487.19
ZDD23650
Cultivar
NR
China
487.19
ZDD01169
Landrace
NR
China
483.48
ZDD13815
Landrace
SR
China
478.77
Youbili
Cultivar
Russia
Russia
475.25
ZDD07197
Landrace
NR
China
471.24
Ha11-4519
Cultivar
NR
China
469.09
ZDD14729
Landrace
SR
China
465.38
WDD00476
Cultivar
USA
USA
463.04
Ls15
Cultivar
USA
USA
460.80
ZDD00163
Landrace
NR
China
453.740
ZDD22798
Cultivar
NR
China
448.00
ZDD23623
Cultivar
NR
China
445.81
ZDD10276
Landrace
HR
China
443.99
ZDD23632
Cultivar
NR
China
441.32
ZDD00393
Landrace
NR
China
440.13
ZDD01417
Landrace
NR
China
439.17
ZDD14052
Landrace
SR
China
438.21
ZDD06154
Landrace
HR
China
437.25
ZDD07088
Landrace
NR
China
430.83
ZDD00717
Landrace
NR
China
429.80
WDD01607
Cultivar
USA
USA
429.76
WDD00543
Cultivar
USA
USA
428.46
ZDD16874
Landrace
SR
China
426.70
ZDD06816
Cultivar
NR
China
426.36
ZDD04653
Landrace
SR
China
426.33
ZDD23615
Cultivar
NR
China
423.00
ZDD00269
Landrace
NR
China
419.12
ZDD00159
Landrace
NR
China
418.19
ZDD00127
Landrace
NR
China
417.92
ZDD02348
Landrace
HR
China
417.26
ZDD17542
Landrace
SR
China
416.22
WDD01253
Cultivar
Japan
Japan
415.94
ZDD13696
Landrace
SR
China
415.45
ZDD14780
Landrace
SR
China
410.62
WDD00631
Cultivar
USA
USA
410.05
WDD03008
Cultivar
USA
USA
409.61
16ZF310-5
Cultivar
HR
China
409.22
ZDD08013
Landrace
HR
China
407.83
ZDD00303
Landrace
NR
China
404.33
ZDD07024
Landrace
NR
China
404.22
ZDD00023
Cultivar
NR
China
404.11
ZDD24399
Cultivar
NR
China
401.70
WDD00984
Cultivar
USA
USA
401.69
Z13-653-1
Cultivar
HR
China
401.60
NR- Northern Region; HR- Huanghuaihai Region; SR- Southern Region
Supplementary Table S10. Correlations between the geographical factors and soybean
folates
Geographical
factors
Longitude
Latitude
Altitude
H4fol
ate
0.09
1*
0.09
6*
0.08
8*
5-CH3H4folate
5,10CH=H4fola
te
MeFo
x
10-CHOPteGlu
5-CHOH4folate
H2fola
te
Pte
Glu
0.165***
-0.065
0.183
***
0.14***
0.188***
0.133
***
-0.117**
0.041
0.081
*
0.126**
0.084*
-0.09*
-0.02
-0.091*
0.007
*
-0.08*
0.057
-0.014
0.04
2
0.00
8
0.06
1
Total
folates
0.086*
0.03
0.025

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Highlights
Conjugase treatment was sufficient for soybean folate extraction.
10-fold variation was observed for soybean folates, from 64.51 - 691.24 μg/100 g.
5-CHO-H4folate was the most dominant folate vitamer in soybean.
Folates are affected by accession type, ecoregion, and seed morphological traits.
Soybean is a key candidate for folate biofortification