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Wolffia globosa–Mankai Plant-Based Protein Contains Bioactive Vitamin B12 and Is Well Absorbed in Humans

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Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B12 (B12)) metabolism. We examined the presence of B12 in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B12 concentrations. Methods: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B12 experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B12 levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups. Results: The B12 content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B12); 5-deoxyadenosylcobalamin(Ado-B12); methylcobalamin(Me-B12); cyanocobalamin(CN-B12)) were identified in Mankai extracts, whereas no pseudo B12 was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B12 metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B12 = 420.5 ± 187.8 pg/mL), serum B12 increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups). Conclusions: Mankai plant contains bioactive B12 compounds and could serve as a B12 plant-based food source.
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nutrients
Article
Wola globosa–Mankai Plant-Based Protein Contains
Bioactive Vitamin B12 and Is Well Absorbed
in Humans
Ilan Sela 1, , Anat Yaskolka Meir 2,, Alexander Brandis 3, Rosa Krajmalnik-Brown 4,
Lydia Zeibich 5, Debbie Chang 5, Blake Dirks 5, Gal Tsaban 2, Alon Kaplan 2, Ehud Rinott 2,
Hila Zelicha 2, Shira Arinos 1, Uta Ceglarek 6, Berend Isermann 6, Miri Lapidot 1,
Ralph Green 7, * and Iris Shai 2, 8, *
1Research and Development Department, Hinoman Ltd., Rishon Lezion 7546302, Israel;
ilansal@gmail.com (I.S.); shirabella@gmail.com (S.A.); miri@hinoman.com (M.L.)
2Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel;
anatyas@post.bgu.ac.il (A.Y.M.); gtsaban@gmail.com (G.T.); alonkaplan47@gmail.com (A.K.);
ehudrinott@gmail.com (E.R.); hila.zelicha@gmail.com (H.Z.)
3Targeted Metabolomics Unit, Life Sciences Core Facilities Weizmann Institute of Science, Rehovot 76100,
Israel; Alexander.Brandis@weizmann.ac.il
4School of Sustainable Engineering and the Built Environment, Biodesign Center for Health Through
Microbiomes, Arizona State University, Tempe, AZ 85281, USA; Dr.Rosy@asu.edu
5
Biodesign Swette Center for Environmental Biotechnology, Arizona State University, Tempe, AZ 85287, USA;
lzeibich@asu.edu (L.Z.); dcchang@asu.edu (D.C.); bedirks@asu.edu (B.D.)
6Institute for Laboratory Medicine, University of Leipzig Medical Center, 04103 Leipzig, Germany;
Uta.Ceglarek@medizin.uni-leipzig.de (U.C.); berend.isermann@medizin.uni-leipzig.de (B.I.)
7Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine,
Sacramento, CA 95817, USA
8Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA
*Correspondence: rgreen@ucdavis.edu (R.G.); irish@bgu.ac.il (I.S.); Tel.: +916-734-8078 (R.G.);
+972-8-647-7449/3 (I.S.); Fax: +916-734-0299 (R.G.); +972-8-647-7637/8 (I.S.)
These authors contributed equally to this work.
Received: 13 September 2020; Accepted: 4 October 2020; Published: 8 October 2020


Abstract:
Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin
analogues, which may compete with cobalamin (vitamin B
12
(B
12
)) metabolism. We examined the
presence of B
12
in a cultivated strain of an aquatic plant: Wola globosa (Mankai), and predicted
functional pathways using gut-bioreactor, and the eects of long-term Mankai consumption as a
partial meat substitute, on serum B
12
concentrations. Methods: We used microbiological assay,
liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic
bioreactors for the B
12
experiments. We explored the eect of a green Mediterranean/low-meat
diet, containing 100 g of frozen Mankai shake/day, on serum B
12
levels during the 18-month
DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean
diet groups. Results: The B
12
content of Mankai was consistent at dierent seasons (p=0.76).
Several cobalamin congeners (Hydroxocobalamin(OH-B
12
); 5-deoxyadenosylcobalamin(Ado-B
12
);
methylcobalamin(Me-B
12
); cyanocobalamin(CN-B
12
)) were identified in Mankai extracts, whereas no
pseudo B
12
was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with
a genome containing a KEGG ortholog involved in microbial B
12
metabolism were observed, compared
with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n=294
participants; retention-rate =89%; baseline B
12
=420.5
±
187.8 pg/mL), serum B
12
increased by 5.2% in
control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets
(
p=0.025
between extreme groups). Conclusions: Mankai plant contains bioactive B
12
compounds
and could serve as a B12 plant-based food source.
Nutrients 2020,12, 3067; doi:10.3390/nu12103067 www.mdpi.com/journal/nutrients
Nutrients 2020,12, 3067 2 of 17
Keywords:
Wola globosa; vitamin B
12
; plant-based nutrition; flexitarians; weight loss; sustainability
1. Introduction
Cobalamin is an essential nutrient for humans. It has the largest molecular mass (1355.4 g/mol)
and the most complex structure of all vitamins [
1
]. The term “vitamin B
12
” is the name usually used for
cyanocobalamin (CN-B
12
), which is the most chemically stable form of cobalamin. In this study, vitamin
B
12
will be used to refer to all corrinoids exhibiting the qualitative biological activity of CN-B
12
[
2
],
including the following three natural forms: Hydroxocobalamin (OH-B
12
), 5-deoxyadenosylcobalamin
(Ado-B
12
), and methylcobalamin (Me-B
12
). CN-B
12
is the form used in most dietary supplements and
is readily converted to the coenzyme forms, Me-B
12
and Ado-B
12
in the body [
1
]. Me-B
12
functions
as a cofactor for the methionine synthase reaction involved in the conversion of homocysteine to
methionine through a transfer of a methyl group from methyltetrahydrofolate; Ado-B
12
functions as
a cofactor for methylmalonyl-CoA mutase in which methylmalonyl-CoA, a product of amino acid
and odd-chain fatty acid catabolism, is converted to succinyl-CoA [
1
]. At the cellular level, these
enzymes play an important role in several crucial functions, such as DNA synthesis, methylation,
and mitochondrial metabolism [3,4].
De novo synthesis of vitamin B
12
appears to be restricted to some bacteria and archaea [
2
,
5
].
The vitamin
is therefore found solely in foods fermented by B
12
-producing bacteria, or in those
derived from the tissues of animals that have ingested B
12
-containing foods or which have obtained
it from B
12
-producing microbiota of their commensal microflora [
2
]. Hence, animal-derived foods
(meat, milk, eggs, and shellfish) are considered to be the exclusive dietary source of B
12
vitamin in
humans [
5
,
6
]. However, a preference for diets that limit intake of animal products has arisen during
the past decade, largely from the belief that lower animal-source protein diets reduce risk factors
for cardiometabolic diseases, such as hypertension, dyslipidemia, hyperglycemia, type 2 diabetes,
and cardiovascular diseases [
7
10
]. On the other hand, since vitamin B
12
is not measurably present
in plant-based foods, individuals adhering to a vegan diet without vitamin B
12
supplementation
are at risk of developing vitamin B
12
deficiency with potentially serious and sometimes irreversible
consequences [
3
,
11
]. Indeed, various types of edible algae have been reported to contain vitamin
B
12
[
4
,
12
]. However, recent data indicate that pseudo B
12
forms, such as OH-pseudoB
12
, Ado-pseudoB
12
,
Me-pseudoB
12
, and CN-pseudoB
12
, which are considered inactive in humans, and might compete
with B12, are the predominant corrinoids present in the algae [4,12].
Wola globosa ‘Mankai’ is an aquatic plant of the duckweed family recently identified for its
nutritional value [
13
,
14
]. It has a unique nutritional composition profile, which includes about 45%
protein of its dry weight, with all nine essential amino acids in a ratio equivalent to that of egg
protein [
15
], a source of omega-3 fatty acids [
16
]; dietary fiber; polyphenols; iron; and several other
micronutrients that tend to have low abundance in animal-based foods diets (e.g., vitamin A as
beta-carotene, riboflavin, vitamin B
6,
and folate). One cup of Mankai shake, which is equivalent
to ~20 g of dry matter, provides the following proportions of recommended intakes: 18% whole
bioavailable protein [
15
], 75% bioavailable iron [
17
], 60% folic acid, and 21% vitamin B
12
. In our
previous bioavailability study, we found, unexpectedly, that the serum vitamin B
12
concentrations
increase and attain higher levels than the increase observed following other protein source meals [
15
].
To exclude the sporadic presence of B
12
and to evaluate the stability levels in Mankai biomass,
various Mankai samples, grown under dierent conditions, ranging from lab scale under artificial light
to commercial scale under sunlight, were examined for their B
12
content by two dierent methods.
In the DIRECT PLUS weight-loss trial, among 294 participants with abdominal obesity and normal
B
12
levels, we explored the eect of an 18-month intake of Mankai, consumed as an evening green
shake, as a partial protein plant-based substitute, on vitamin B
12
serum levels. Besides, we examined
changes in the gut microbiome when directly exposed to Mankai using anoxic bioreactors, to simulate
Nutrients 2020,12, 3067 3 of 17
the human colon environment/microbiota. We hypothesized that Mankai might serve as a consistent
vitamin B12 source, despite the reduction in red meat intake.
2. Materials and Methods
2.1. Mankai Laboratory Analyses
2.1.1. Plant Sources
Vitamin B12 Detection in Plants Cultivated Under Greenhouse Conditions
Cultivated plant samples: Mankai biomass is grown in closed controlled highly monitored aquatic
greenhouses using a proprietary precision agriculture cultivation system. We sampled the plant for
B
12
analysis at dierent seasons during the years 2014 to 2019. Plant biomass was sieve harvested,
washed with tap water for 2 min, and dried in a food dehydrator (Excalibur, Sacramento, USA) at
65
C for 16 h. Each dried plant sample was stored in a vacuum-sealed aluminum bag at 4
C, until
analysis was performed.
Vitamin B12 Detection in Axenic Culture
Generating axenic culture: Plant sterilization was achieved by submerging and agitating plants in
predetermined concentrations of sodium hypochlorite for 1–3 min. Treated fronds were transferred
to a 12-well plate containing sterile Hoagland solution (MgSO
4·
7H
2
O 0.246 g/L, Ca(NO
3
)
2·
4H
2
O
542 mg/L, KH
2
PO
4
68 mg/L, KNO
3
250 mg/L, FeNa
·
EDTA 37 mg/L, H
3
BO
3
1.5 mg/L, MnCl
2·
4H
2
O
9.1 mg/L, ZnSO
4·
7H
2
O 0.11 mg/L, Na
2
MoO
4·
2H
2
O 0.045 mg/L, CuSO
4·
5H
2
O 0.045 mg/L, and 1%
Sucrose (
All purchased
from Fisher Scientific, Leicestershire, UK). The Hoagland formulation does
not contain cobalt compounds. Furthermore, ICP-MS analysis performed by an accredited laboratory
was applied to this 10
×
concentrated Hoagland solution and revealed no cobalt traces (<0.01 ppm).
The plate
was covered with aluminum foil and kept at 25
C for 24 h. After the foil was removed,
the plants
were allowed to recover for an additional 6 days under a 24-h light regime at 120
µ
E. Bleached
mother fronds with green daughter fronds were transferred to a new sterile well to establish a sterile
Mankai culture. Three sterile cultures, derived from three independent treatments,
were continuously
grown in vitamin B
12
-free Hoagland medium that was replaced once a week. Culture sterility was
verified by incubation of whole and crushed fronds on PCA (plate count agar, Neogen, Michigan, USA)
at 30
C for at least 5 days. Vitamin B
12
analysis was performed on 5-month-old independent plant
cultures that were intensively washed with running tap water for two minutes and dried in a food
dehydrator as described above.
2.1.2. Vitamin B12 Analyses
Bioassay Method
Total vitamin B
12
in the plant samples was measured by the AOAC 952.20 microbiological analytical
method, utilizing the B
12
-requiring bacterium Lactobacillus Delbrueckii subsp. lactis ATCC7830, which
is the established vitamin B
12
determination method for foods [
18
]. The analysis was performed by
Eurofins Laboratories, Inc. (Des Moines, IA, USA) and by Bactochem Ltd. (Nes Ziona, Israel).
Some tests
were done by Hinoman Ltd., analyzing one gram of dried plant by the Vitafast B
12
microbiological
assay kit (R-Biopharm, AG, Darmstadt, Germany) according to the manufacturer’s instructions.
Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry (ESI LC-MS/MS) Assay
Extraction of Vitamin B
12
: The extraction of dried Mankai samples and two commercial Spirulina
powders that served as a reference for pseudo vitamin B12 are described in Supplementary File S1.
Purification of vitamin B
12
and LC-MS/MS: B
12
extracts were evaporated to dryness under
reduced pressure and then re-dissolved in 9 mL of double-distilled water. The obtained solutions
Nutrients 2020,12, 3067 4 of 17
were loaded onto an immunoanity column (EASI-EXTRACT vitamin B
12
immunoanity column
(AOAC 2014.02), R-Biopharm AG, Darmstadt, Germany) and purified according to the manufacturer’s
protocol. The recovery eciency of pseudo CN-B
12
was considered to be similar to that of authentic
CN-B
12
. Subsequently, 10-
µ
L aliquots of extracts were analyzed in optimized conditions determined
using individual B
12
standards. The concentrations based on standard curves were calculated using
TargetLynx (Waters, Milford, MA, USA). The LC-MS/MS assay was performed at the Life Sciences Core
Facilities of Weizmann Institute of Science. Further extraction and purification methods, as well as
retention times and Multiple Reaction Monitoring (MRM) parameters for the detection of corrinoids,
are given in Supplementary File S1 and Table S1.
2.2. The DIRECT PLUS Dietary Intervention Trial
2.2.1. Study Design
The 18-month DIRECT-PLUS (dietary intervention randomized controlled trial
polyphenols-unprocessed) trial (clinicaltrials.gov ID: NCT03020186) aimed to address the
residual beneficial eect of a green Mediterranean diet, richer in green plants and lower in meat,
compared with other healthy lifestyle strategies. The trial was initiated in May 2017 and was
conducted in an isolated workplace (Nuclear Research Center Negev (NRCN), Dimona, Israel), where
a monitored lunch was provided. This workplace includes a medical department where most of
the medical measurements were taken and where lifestyle intervention sessions were held. Of the
378 volunteers
, 294 met the inclusion criteria of age >30 years and characterized by abdominal obesity
(waist circumference (WC): men >102 cm, women >88 cm) or dyslipidemia (TG >150 mg/dL and
high-density lipoprotein cholesterol (HDL-c)
40 mg/dL for men,
50 mg/dL for women). Exclusion
criteria are detailed in Supplementary File S2.
All subjects gave their informed consent for inclusion before they participated in the study.
The study
was conducted in accordance with the Declaration of Helsinki, and the protocol was
approved by the Medical Ethics Board and Institutional Review Board at Soroka University Medical
Centre, Be’er Sheva, Israel (0280-16-SOR). All participants did not receive any financial compensation.
2.2.2. Randomization and Intervention
Randomization and intervention were described elsewhere [
17
,
19
]. Briefly, participants were
randomly assigned to one of three intervention groups, all combined with physical activity
recommendation (along with a free gym membership):
Healthy dietary guidelines (HDG) group: In addition to the workout program, the participants
received basic health-promoting guidelines for achieving a healthy diet.
Mediterranean (MED) group: In addition to the workout program, participants were instructed to
adopt a calorie-restricted Mediterranean diet as described in our previous trials: DIRECT [
20
] and
CENTRAL [21] trials, supplemented with 28 g/day of walnuts.
Green Mediterranean (green-MED) group: In addition to the Mediterranean intervention
(including the provided walnuts), the green Mediterranean dieters were further guided to avoid red
and processed meat, with the diet being richer in plants and polyphenols. The participants were guided
to further consume the two following provided items: 3–4 cups/day of 100 g frozen cubes of Mankai
(whole plant), replacing dinner and a potential source of protein, iron, and vitamin B
12
.
The MED
and
green-MED diets were equally calorie restricted (1500–1800 kcal/day for men and
1200–1400 kcal/day
for women). All the above (walnuts, green tea, and Mankai) were provided free of charge.
2.2.3. Outcomes
Blood samples were taken at 8:00 AM after a 12-h fast, at baseline and after 18 months of intervention.
The samples were centrifuged and stored at
80
C. Serum vitamin B
12
was analyzed with a competitive
Elektro Chemiluminescence-Immuno Assay “ECLIA” (Cobas 8000, Roche Diagnostics, Mannheim,
Nutrients 2020,12, 3067 5 of 17
Germany) using Intrinsic Factor as a binding protein. Serum folate was also measured by the ECLIA
competitive approach and was used as a marker for green leaf consumption [
22
]. All biochemical
analyses were performed at the laboratories of the University of Leipzig, Germany. Chemical and
hematological parameters in freshly drawn blood samples were assessed at the workplace clinic at
baseline and at the end of the intervention measurements (
±
1 month before/after initiating blood
draws). Additional outcomes measures (i.e., anthropometric, electronic questionnaires) are presented
in Supplementary File S3.
2.2.4. Statistical Analysis
The primary outcomes of the DIRECT PLUS study, as stated in clinicaltrials.gov, were 18-month
changes in adiposity parameters (a flow diagram for the study is presented in Figure S1). In this analysis,
we primarily aimed to assess serum vitamin B
12
change during the study period. Continuous variables
are presented as means
±
standard deviations for normally distributed variables and medians for
non-normally distributed variables, with the Kolmogorov–Smirnov test used to determine the variable’s
distribution. Nominal variables are expressed as numbers and percentages. Dierences between time
points were tested using the paired sample T-test or Wilcoxon test. Dierences between groups (i.e.,
intervention groups or tertiles) were tested using analysis of variance (ANOVA), Kruskal–Wallis test,
or Chi-square test. Ln transformations were applied when necessary to achieve normal distribution.
Kendal Tau correlation was used to examine pof trend. Multiple comparisons were addressed using
the Tukey post hoc test (for ANOVA), and Bonferroni correction (for Kruskal–Wallis). For adjustments,
we used general linear regression models, with the specific adjustments detailed with the results.
Sample size calculations were detailed elsewhere [
17
]. Statistical analysis was performed using SPSS
(version 25.0, IBM, Armonk, NY, USA). Statistical significance was set at 0.05 level, two-sided.
2.3. Anoxic Gut Microbiome Bioreactors Pilot Experiment
2.3.1. Microbiota Reactors (Human Fecal Mixture)
A mixture of human fecal samples obtained from 20 healthy male and female volunteers (age:
18–65 years) collected for a research study in 2017 (Krajmalnik-Brown Lab; IRB#STUDY00004850,
Arizona State University) was used to inoculate anoxic bioreactors. After donation, fecal samples
were kept at 4
C and 1 g of sample was supplemented with 500
µ
L of 40% (v/v) anaerobic glycerol
solution. The fecal mixtures, consisting of 20 homogenized fecal samples obtained from each donor,
were stored in anaerobic freezer bags at
80
C. Prior to use, 1 mL of fecal mixture was added to a
serum bottle filled with 70 mL of anoxic Base medium (see below). The bottle, containing the starter
culture,
was placed
in a shaking incubator for 24 h at 100 rpm and 37
C. Headspace gas quantification
was used to confirm microbial activity.
2.3.2. Media, Anoxic Bioreactor, Mankai Lysate, and Sampling
Two anoxic media were used to examine the potential eect of Mankai on human-derived gut
microbiota. Both media were based on the protocol described by McDonald et al. [
23
], with the
following modification to provide the same chemical oxygen demand (COD) amount (200 meq/L)
to all treatments. The final media consisted of an anoxic micronutrient-containing solution and an
anoxic macronutrient solution (Table S2). COD was measured to quantify the reducing equivalents
in both solutions. To obtain a (a) base medium for the bioreactors that lacked Mankai and for
the starter culture (see above), and (b) Mankai medium for the Mankai-supplemented bioreactors,
micronutrient-containing solution, and macronutrient solution were combined, accordingly (
Table S2
).
Before bioreactor inoculation (adding 1 mL of the starter culture (see above)), Mankai lysate was
prepared by blending 5 g of frozen Mankai biomass (Wola globosa ‘Mankai’) with 400 mL of deionized
(DI) water for 5 min and subsequently flushing with nitrogen for 5 min. After inoculation and before the
first fill and draw, the bioreactors were incubated for 48 h in the dark at 37
C and mixed continuously
Nutrients 2020,12, 3067 6 of 17
at 100 rpm. Full details regarding the media, anoxic bioreactor, Mankai lysate, and the sampling are
provided in Supplementary File S4.
2.3.3. Chemical and Molecular Analysis
Total COD was determined by adding 400
µ
L of solution, medium, or lysate to a HACH COD
vial (HACH, High Range 20–1500 mg COD/L) with 1600
µ
L of DI water followed by a 2-h incubation
at 150
C (HACH DRB200). The vials were then cooled and measured for COD concentration in
mgCOD/L using a spectrophotometer (HACH DR2800 Laboratory Spectrophotometer). For microbiome
composition analysis, we performed 16S rRNA gene amplicon sequencing using Illumina sequencing
technology and found core dierences as described [
24
,
25
]. Further detailing regarding the 16S rRNA
amplicon sequences is presented in Supplementary File S5.
3. Results
3.1. Mankai Plant Analyses
3.1.1. Content and Stability of Vitamin B12 Levels during Dierent Seasons
Overall, Mankai contained 2.8
±
0.5
µ
g B
12
/100 g dry weight (DW) and the concentration remained
relatively stable during the seasons (Figure 1), regardless of the water temperature (17
C–29
C)
or duration of light hours (10–14): autumn: 2.84
±
0.5
µ
g/100 g DW, n=5 (range: 2.34
µ
g/100 g to
3.62 µg/100 g DW
); winter: 2.83
±
0.6
µ
g/100 g DW, n=5 (range: 1.96
µ
g/100 g to 3.44
µ
g/100 g DW);
spring: 2.94
±
0.6
µ
g/100 g DW, n=4 (range: 2.19
µ
g/100 g to 3.52
µ
g/100 g DW); and summer:
2.6 ±0.5 µg/100 g DW, n=6 (range: 1.83 µg/100 g to 3.26 µg/100 g DW). (p=0.76 between seasons).
Nutrients 2020, 9, x FOR PEER REVIEW 6 of 17
in both solutions. To obtain a (a) base medium for the bioreactors that lacked Mankai and for the
starter culture (see above), and (b) Mankai medium for the Mankai-supplemented bioreactors,
micronutrient-containing solution, and macronutrient solution were combined, accordingly (Table
S2). Before bioreactor inoculation (adding 1 mL of the starter culture (see above)), Mankai lysate was
prepared by blending 5 g of frozen Mankai biomass (Wolffia globosa Mankai) with 400 mL of
deionized (DI) water for 5 min and subsequently flushing with nitrogen for 5 min. After inoculation
and before the first fill and draw, the bioreactors were incubated for 48 h in the dark at 37 °C and
mixed continuously at 100 rpm. Full details regarding the media, anoxic bioreactor, Mankai lysate,
and the sampling are provided in Supplementary File S4.
2.3.3. Chemical and Molecular Analysis
Total COD was determined by adding 400 µ L of solution, medium, or lysate to a HACH COD
vial (HACH, High Range 201500 mg COD/L) with 1600 μL of DI water followed by a 2-h incubation
at 150 °C (HACH DRB200). The vials were then cooled and measured for COD concentration in
mgCOD/L using a spectrophotometer (HACH DR2800 Laboratory Spectrophotometer). For
microbiome composition analysis, we performed 16S rRNA gene amplicon sequencing using
Illumina sequencing technology and found core differences as described [24,25]. Further detailing
regarding the 16S rRNA amplicon sequences is presented in Supplementary File S5.
3. Results
3.1. Mankai Plant Analyses
3.1.1. Content and Stability of Vitamin B12 Levels during Different Seasons
Overall, Mankai contained 2.8 ± 0.5 µg B12/100 g dry weight (DW) and the concentration
remained relatively stable during the seasons (Figure 1), regardless of the water temperature (17 °C
29 °C) or duration of light hours (1014): autumn: 2.84 ± 0.5 µg/100 g DW, n = 5 (range: 2.34 µg/100 g
to 3.62 µ g/100 g DW); winter: 2.83 ± 0.6 µg/100 g DW, n = 5 (range: 1.96 µg/100 g to 3.44 µg/100 g DW);
spring: 2.94 ± 0.6 µ g/100 g DW, n = 4 (range: 2.19 µ g /100 g to 3.52 µg/100 g DW); and summer: 2.6 ±
0.5 µg/100 g DW, n = 6 (range: 1.83 µg/100 g to 3.26 µg/100 g DW). (p = 0.76 between seasons).
Figure 1. Stability of vitamin B12 levels in Mankai™ along the year. Autumn refers to water temperatures of
2224.5 °C and 10:20-10:50 h of light. Winter refers to water temperatures of 1720 °C and 1010:20 h of light.
Spring refers to water temperatures of 2124 °C and 11:3013:30 h of light. Summer refers to water
temperatures of 2529 °C and 13:5014:15 h of light. For each season, the weekly average water temperatures
and daily light hours relate to the sampling date.
3.1.2. Inherent Presence of Vitamin B12 in Mankai Axenic Cultures
Figure 1.
Stability of vitamin B
12
levels in Mankai
along the year. “Autumn” refers to water
temperatures of 22–24.5
C and 10:20–10:50 h of light. “Winter” refers to water temperatures of
17–20 C
and 10–10:20 h of light. “Spring” refers to water temperatures of 21–24
C and
11:30–13:30 h
of light. “Summer” refers to water temperatures of 25–29
C and
13:50–14:15 h
of light. For each season,
the weekly average water temperatures and daily light hours relate to the sampling date.
3.1.2. Inherent Presence of Vitamin B12 in Mankai Axenic Cultures
B
12
concentrations in three independent axenic cultures, which were vegetatively propagated for
at least 5 months post establishment, were 2.08, 2.34, and 1.6 µg/100 g DW.
3.1.3. Identification of Vitamin B12 Purified from Mankai
To verify that the corrinoid detected by the bioassay was indeed a bioactive form of cobalamin,
we used LC-MS/MS. The presence of the active form was validated in all 10 tested samples: four plant
samples representing three dierent seasons (spring, summer, and autumn) and 6 samples grown
under indoor conditions. Representative data of a Mankai sample collected during mid-March 2019
from an outdoor basin are shown in Figure 2. Standard CN-B
12
was eluted as a peak with a retention
Nutrients 2020,12, 3067 7 of 17
time of 2.11 min (Figure 2A) and the plant extract sample showed a corresponding peak with the same
retention time (Figure 2B) for all MRM transitions. The intensity ratios between individual MRM
signals were kept similar in both standard and plant samples (Figure S2).
Nutrients 2020, 9, x FOR PEER REVIEW 7 of 17
B12 concentrations in three independent axenic cultures, which were vegetatively propagated for
at least 5 months post establishment, were 2.08, 2.34, and 1.6 µ g/100 g DW.3.1.3. Identification of
Vitamin B12 Purified from Mankai
3.1.3. Identification of Vitamin B12 Purified from Mankai
To verify that the corrinoid detected by the bioassay was indeed a bioactive form of cobalamin,
we used LC-MS/MS. The presence of the active form was validated in all 10 tested samples: four plant
samples representing three different seasons (spring, summer, and autumn) and 6 samples grown
under indoor conditions. Representative data of a Mankai sample collected during mid-March 2019
from an outdoor basin are shown in Figure 2. Standard CN-B12 was eluted as a peak with a retention
time of 2.11 min (Figure 2A) and the plant extract sample showed a corresponding peak with the
same retention time (Figure 2B) for all MRM transitions. The intensity ratios between individual
MRM signals were kept similar in both standard and plant samples (Figure S2).
Figure 2. Liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS)
chromatograms of CN-B12. (A). Retention time for CN-B12 standard (arrow). (B). Retention time for
CN-B12 extracted from Mankai sample (arrow). ES, electrospray; MRM, multiple reaction monitoring;
TIC, total ion current.
3.1.4. Quantification of Total Vitamin B12 Purified from Mankai
The extractions described above were performed in the presence of KCN, which converts the
naturally occurring forms of cobalamin to the stable CN-B12 form. Since this conversion is not always
complete [26], we analyzed all four vitamin B12 forms by LC-MS/MS, with the aim of determining the
total vitamin B12 content of Mankai. Commercial OH-B12, CN-B12, Ado-B12, and Me-B12 standards were
eluted as peaks with retention times of 1.87, 2.1, 2.25, and 2.31 min, respectively, and the plant extract
samples showed corresponding peaks with the same retention times (Figure S3). The intensity ratios
between individual MRM signals were kept similar in both standard and plant samples (data not
shown). These results indicate that all three natural forms were present in Mankai and that
incomplete conversion to CN-B12 had occurred. The identification of CN-B12, OH-B12, Ado-B12, and
Me-B12 was further validated by four, three, two, and four MRMs, respectively. In order to calculate
the total vitamin B12 in the plants, we measured the recovery rate of each form by analyzing the
standards, with or without immunoaffinity column purification. Namely, the solutions containing
the standard mix of four B12 forms in equal amounts were divided in two halves. One half was diluted
with acetate buffer and passed through a EASI-EXTRACT vitamin B12 immunoaffinity column
according to the manufacturer’s purification protocol. The obtained eluate was evaporated and re-
dissolved to the same volume as the second half. Samples thus obtained were analyzed by LC-
Figure 2.
Liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS)
chromatograms of CN-B
12
. (
A
) Retention time for CN-B
12
standard (arrow). (
B
) Retention time for
CN-B
12
extracted from Mankai sample (arrow). ES, electrospray; MRM, multiple reaction monitoring;
TIC, total ion current.
3.1.4. Quantification of Total Vitamin B12 Purified from Mankai
The extractions described above were performed in the presence of KCN, which converts the
naturally occurring forms of cobalamin to the stable CN-B
12
form. Since this conversion is not always
complete [
26
], we analyzed all four vitamin B
12
forms by LC-MS/MS, with the aim of determining the
total vitamin B
12
content of Mankai. Commercial OH-B
12
, CN-B
12
, Ado-B
12
, and Me-B
12
standards
were eluted as peaks with retention times of 1.87, 2.1, 2.25, and 2.31 min, respectively, and the plant
extract samples showed corresponding peaks with the same retention times (Figure S3). The intensity
ratios between individual MRM signals were kept similar in both standard and plant samples (data not
shown). These results indicate that all three natural forms were present in Mankai and that incomplete
conversion to CN-B12 had occurred. The identification of CN-B12, OH-B12 , Ado-B12, and Me-B12 was
further validated by four, three, two, and four MRMs, respectively. In order to calculate the total
vitamin B
12
in the plants, we measured the recovery rate of each form by analyzing the standards,
with or
without immunoanity column purification. Namely, the solutions containing the standard
mix of four B
12
forms in equal amounts were divided in two halves. One half was diluted with acetate
buer and passed through a EASI-EXTRACT vitamin B
12
immunoanity column according to the
manufacturer’s purification protocol. The obtained eluate was evaporated and re-dissolved to the same
volume as the second half. Samples thus obtained were analyzed by LC-MS/MS.
The results
showed
recovery rates of 55%, 37%, 16%, and 100% for CN-B
12
, OH-B
12
, Ado-B
12
, and Me-B
12
, respectively. The
analysis was performed on three plant samples that were obtained from greenhouse cultivation basins
during spring, summer, and autumn. The amount of each form was then measured in plant extracts
and the total B
12
level was calculated according to the recovery rates. The data showed that the average
total authentic vitamin B
12
concentrations in Mankai is 3.23
µ
g
±
0.5/100 g DW and stable during
dierent seasons: spring 2.86
µ
g, summer 3.84
µ
g, and autumn 2.99
µ
g/100 g DW. These concentrations
are in line with the results received by the bioassay method.
Nutrients 2020,12, 3067 8 of 17
3.1.5. Authentic CN-B12 and Pseudo CN-B12 in Mankai
To further study Mankai as a vitamin B
12
food source, we estimated the concentrations of pseudo
B
12
in the plant. To this end, we used LC-MS/MS to analyze samples of spirulina that are known to
produce large amounts of pseudo B
12
[
27
] and therefore can be used as a reference. This measurement
was performed assuming similar ionization products for both CN-B
12
and pseudo CN-B
12
, so the
standard CN-B
12
curve was used as a reference to quantify both compounds. Based on the dierent
molecular masses of CN-B
12
and pseudo CN-B
12
, the data revealed the presence of CN-B
12
and pseudo
CN-B
12
in a ratio 1:3 in two dierent spirulina samples, whereas no pseudo CN-B
12
was detected in
the Mankai samples (Figure 3and Figure S4).
Figure 3.
A comparison of chromatograms of TIC for authentic CN-B
12
and pseudo CN-B
12
in Mankai
and spirulina samples. (
A
C
): Active CN-B
12
; (
B
D
) Pseudo CN-B
12
in Mankai
(
A
,
B
) and spirulina
(
C
,
D
) samples. In panel B, a peak at 2.12 min does not represent pseudo CN-B
12
because pseudo
CN-B
12
should appear before the peak of CN-B
12
[
27
,
28
] as is observed with a peak from a spirulina
sample (at 2.09 min, panel D) and is present not just in one but in all 4 MRM transitions at measurable
levels (Figure S4). ES, electrospray; MRM, multiple reaction monitoring; TIC, total ion current.
3.2. DIRECT PLUS Trial
3.2.1. Baseline Characteristics
The baseline characteristics are presented in Table 1. The mean vitamin B
12
concentration was
420.4
±
187.8 pg/mL (range: 150–1500 pg/mL), with a mean of 414.3
±
182.5 pg/mL for men and
465.5 ±220.9 pg/mL
for women (p=0.21 between sexes). Triglyceride levels were lower in the highest
vitamin B
12
tertile compared with the lowest tertile (p=0.01). Details regarding baseline vitamin
supplementation are presented in Supplementary File S6.
Nutrients 2020,12, 3067 9 of 17
Table 1.
Baseline characteristics of the DIRECT PLUS participants across sex-specific vitamin B
12
tertiles.
Entire
n=294
Lowest
Tertile
n=99
Intermediate
Tertile
n=98
Highest
Tertile
n=97
pBetween
Tertiles 1
pBetween
Extreme
Tertiles 2
Vitamin B12, pg/mL 420.4 ±187 261.2 ±46.1 385.7 ±37.9 618.1 ±1 92.8 - -
Age, years 51.1 ±10.5 51.9 ±9.6 49.7 ±10.5 51.5 ±11.4 0.25 0.54
Men, number 259 87 86 86 0.98 -
BMI, kg/m231.3 ±4.0 31.3 ±4.3 31.3 ±3.9 31.2 ±3.7 0.84 0.87
WC, cm 109.7 ±9.5 110.1 ±9.7 109.6 ±10.5 109.4 ±8.1 0.67 0.93
Fasting glucose, mg/dL 101.9 ±17.1 104.0 ±19.5 101.2 ±14.7 100.6 ±16.5 0.62 0.35
Cholesterol, mg/dL 190.6 ±33.0 190.9 ±29.5 190.9 ±33.8 189.8 ±35.9 0.97 0.82
HDL-c, mg/dL 46.0 ±11.7 45.0 ±12.4 46.2 ±11.3 46.7 ±11.3 0.29 0.15
LDL-c, mg/dL 125.7 ±30.1 125.5 ±28.6 127.0 ±31.6 124.5 ±32.4 0.86 0.83
Triglycerides, mg/dL 146.3 ±66.8 159.4 ±66.9 139.8 ±60.0 139.5 ±68.8 0.02 0.01
ALT, U/L 34.9 ±16.8 34.3 ±14.4 35.4 ±20.4 34.9 ±15.0 0.79 0.71
AST, U/L 25.6 ±7.7 25.5 ±7.2 26.1 ±8.7 25.3 ±7.3 0.90 0.74
Continuous data presented as means
±
SD. Lowest tertile: Men: < = 322.49 pg/mL; Women: < = 318.43 pg/mL.
Intermediate tertile: Men: 322.50 pg/mL–439.02 pg/mL; Women: 318.44 pg/mL–478.32 pg/mL. Highest tertile: Men:
439.03 +pg/mL; women: 478.33 +pg/mL.
1
tested using ANOVA/Kruskal-Wallis.
2
tested using T-test/Mann-Whitney.
ALT, alanine transaminase; AST, aspartate transaminase. BMI, body mass index; HDL-c, high density lipoprotein
cholesterol; LDL-c, low density lipoprotein cholesterol; WC, waist circumference.
All chemical and hematological parameters (mean corpuscular volume (MCV), mean cell
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cells (RBCs)
hemoglobin and hematocrit; n=290 for hemoglobin; n=124 for other parameters) were similar and
within the normal range across intervention groups (data not shown).
3.2.2. The Eect of the Intervention on Serum B12 Levels
The trial’s 18-month subject retention rate was 89.8%. Higher and similar weight reductions were
observed, following a caloric deficit, in the two MED groups (MED:
2.9
±
5.2%; Green-MED/low-meat:
3.9
±
6.5%) compared with the HDG group (
0.6
±
5.1%, p<0.05 for both MEDs vs. HDG). Overall,
the green-MED/low-meat diet group significantly increased intake of fish, Mankai,
and green tea
,
and decreased
red meat and poultry compared with the two other groups (p<0.01 for all).
Both MED
groups increased egg and milk consumption compared with the HDG group [
16
]. Vitamin
supplementation usage at the end of the intervention did not dier between the intervention groups
(Supplementary File S6).
Dierences in serum vitamin B
12
concentrations between intervention groups are presented in
Figure 4. After 18 months, the HDG group had a non-significant 1.245
±
126.5 pg/mL (+5.2%) change in
serum vitamin B
12
levels (p=0.93 vs. baseline), while MED had a significant increase in serum vitamin
B
12
levels (32.6
±
76.2 pg/mL (+9.9%); p<0.001 vs. baseline) as well in group Green-MED/low-meat
(
48.8 ±124.9 pg/mL
(+15.4%); p<0.001 vs. baseline). P-of-trend was observed between the groups
(
p=0.02
), with a significant dierence between the HDG and the green-MED/low-meat groups
(
p=0.025
). When further adjusted for age, sex, and baseline B
12
concentrations, these significant
dierences remained (p=0.01).
Nutrients 2020,12, 3067 10 of 17
Nutrients 2020, 9, x FOR PEER REVIEW 10 of 17
groups (p = 0.02), with a significant difference between the HDG and the green-MED/low-meat
groups (p = 0.025). When further adjusted for age, sex, and baseline B12 concentrations, these
significant differences remained (p = 0.01).
Figure 4. The 18-month change in serum vitamin B12 across intervention groups.* Indicates within-
group change (baseline vs. T18) at the 0.05 level. Data presented as means and SEM. HDG, healthy
dietary guidelines; MED, Mediterranean.
3.2.3. Changes in chemical and hematological Parameters
After 18 months of intervention, among the sub-group of participants with available
hematological and chemical measurements (n = 71 for hemoglobin; n = 41 for other hematological
parameters), all groups demonstrated no significant changes in MCV, MCH, MCHC, RBC
hemoglobin, or hematocrit, and also did not differ between the groups (p > 0.05 for all comparisons).
3.2.4. Dietary Vitamin B12 Sources
Next, we examined red meat (reported as increased, decreased, or no change in consumption)
vs. Mankai frequency of intake tertiles, and change in serum folate (Green-MED/low-meat group
only). Those who decreased red meat intake throughout the intervention showed a significantly
increased serum folate associated with more frequent intake of Mankai (p of trend < 0.05; Figure S5a).
Across all intervention groups, among those who decreased red meat consumption, increased serum
folate was associated with increased serum vitamin B12 (p < 0.05) (Figure 5). The less red
meat/increased serum folate group had a comparable increase of serum vitamin B12 to the mor -red
meat/decreased serum folate group (86.0 ± 117.6 pg/mL vs. 77.9 ± 118.6 pg/mL, p = 0.88). In a similar
analysis, replacing red meat with fish, we observed that among participants who increased fish intake
throughout the intervention, an increase in vitamin B12 was observed, as well as serum folate (p of
trend <0.01 for both). Significant increases in serum folate and vitamin B12 were observed for
participants who both consumed more fish and Mankai, and demonstrated an increase in serum
folate levels, as compared with other groups (Figure S5b,c).
Figure 4.
The 18-month change in serum vitamin B
12
across intervention groups. * Indicates
within-group change (baseline vs. T18) at the 0.05 level. Data presented as means and SEM. HDG,
healthy dietary guidelines; MED, Mediterranean.
3.2.3. Changes in chemical and hematological Parameters
After 18 months of intervention, among the sub-group of participants with available hematological
and chemical measurements (n=71 for hemoglobin; n=41 for other hematological parameters),
all groups
demonstrated no significant changes in MCV, MCH, MCHC, RBC hemoglobin, or hematocrit,
and also did not dier between the groups (p>0.05 for all comparisons).
3.2.4. Dietary Vitamin B12 Sources
Next, we examined red meat (reported as increased, decreased, or no change in consumption) vs.
Mankai frequency of intake tertiles, and change in serum folate (Green-MED/low-meat group only).
Those who decreased red meat intake throughout the intervention showed a significantly increased
serum folate associated with more frequent intake of Mankai (pof trend <0.05; Figure S5a). Across all
intervention groups, among those who decreased red meat consumption, increased serum folate was
associated with increased serum vitamin B
12
(p<0.05) (Figure 5). The less red meat/increased serum
folate group had a comparable increase of serum vitamin B
12
to the mor -red meat/decreased serum
folate group (86.0
±
117.6 pg/mL vs. 77.9
±
118.6 pg/mL, p=0.88). In a similar analysis, replacing
red meat with fish, we observed that among participants who increased fish intake throughout the
intervention, an increase in vitamin B
12
was observed, as well as serum folate (pof trend <0.01 for
both). Significant increases in serum folate and vitamin B
12
were observed for participants who both
consumed more fish and Mankai, and demonstrated an increase in serum folate levels, as compared
with other groups (Figure S5b,c).
Nutrients 2020, 9, x FOR PEER REVIEW 11 of 17
Figure 5. Red meat consumption change at the end of the intervention (tertiles) vs. 18-month serum
folate change (tertiles) vs. 18-month change in vitamin B12. * indicated within-group significance
(baseline vs. T18) at the 0.05 level. Data presented as means and SEM.
No significant difference between extreme groups less red meat/most increase in serum folate
and more red meat/most reduction in serum folate was observed.
3.3. Anoxic Bioreactors Pilot Experiment
Predicted Functional Pathways-Gut Bioreactor
Based on 16S rRNA gene amplicon sequences obtained from all bioreactors at the end of
incubation (day 14), we predicted KEGG (Kyoto Encyclopedia of Genes and Genomes) present in the
genomes of the bacteria identified, using Predicted functional profile analysis via PICRUSt [29]. This
analysis, allowing us to predict KEGGS and the linear discriminant analysis effect size (LEFSE),
showed that Mankai-supplemented bioreactors displayed a significantly higher relative abundance
of 16S rRNA gene sequences associated with a genome containing a KEGG ortholog involved in
vitamin B12 uptake (btuB; KEGG identifier K16092) than control bioreactors that lacked Mankai.
Statistical analyses revealed a linear discriminant analysis (LDA) score of 2.19 (log10) and a relative
btuB abundance of 0.034 ± 0.008 and 0.00 ± 0.001 in Mankai-supplemented reactors and reactors that
lacked Mankai, respectively (p < 0.05 between reactors).
In total, 1180 of 5257 different 16S rRNA gene amplicon sequences were identified in the three
replicated Mankai-supplemented bioreactors that contributed to the increased predicted abundance
of microbes containing btuB. Six 16S rRNA gene amplicon sequences displayed a greater than 0.5%
relative 16S rRNA gene amplicon abundance, and three of these sequences (closely related to
Aeromonas hydrophila, Pelomonas aquatica, and Geobacter anodireducens) were present in all three
replicated Mankai-supplemented bioreactors (figure S3). In marked contrast, only nine different 16S
rRNA gene amplicon sequencesassociated with microbes potentially containing btuBwere
identified in the control reactors that lacked Mankai, of which (a) five of these nine were present in
all three replicates and (b) only one sequence (closely related to Escherichia coli) displayed a relative
abundance greater than 0.5% (Table S3).
4. Discussion
In the current study, we examined, using different methodologies, the presence of vitamin B12 in
a cultivated strain of Wolffia globosa (Mankai). We found that Mankai, cultured under closed-
controlled greenhouse conditions, contains a substantial amount of the known bioactive forms of
vitamin B12 and that its presence is stable throughout the year. In inoculated gut microbiome anoxic
bioreactors, a significantly higher relative abundance of 16S rRNA gene sequences associated with a
genome containing the KEGG ortholog involved in vitamin B12 uptake was observed, compared with
control bioreactors that lacked Mankai. In our human studies, results suggest that long-term
consumption of this plant, as part of a whole flexitarian diet, may increase rather than impair vitamin
Figure 5.
Red meat consumption change at the end of the intervention (tertiles) vs. 18-month serum
folate change (tertiles) vs. 18-month change in vitamin B
12.
* indicated within-group significance
(baseline vs. T18) at the 0.05 level. Data presented as means and SEM.
Nutrients 2020,12, 3067 11 of 17
No significant dierence between extreme groups less red meat/most increase in serum folate and
more red meat/most reduction in serum folate was observed.
3.3. Anoxic Bioreactors Pilot Experiment
Predicted Functional Pathways-Gut Bioreactor
Based on 16S rRNA gene amplicon sequences obtained from all bioreactors at the end of incubation
(day 14), we predicted KEGG (Kyoto Encyclopedia of Genes and Genomes) present in the genomes
of the bacteria identified, using Predicted functional profile analysis via PICRUSt [
29
]. This analysis,
allowing us to predict KEGGS and the linear discriminant analysis eect size (LEFSE), showed that
Mankai-supplemented bioreactors displayed a significantly higher relative abundance of 16S rRNA
gene sequences associated with a genome containing a KEGG ortholog involved in vitamin B
12
uptake
(btuB; KEGG identifier K16092) than control bioreactors that lacked Mankai. Statistical analyses
revealed a linear discriminant analysis (LDA) score of 2.19 (log10) and a relative btuB abundance of
0.034
±
0.008 and 0.00
±
0.001 in Mankai-supplemented reactors and reactors that lacked Mankai,
respectively (p<0.05 between reactors).
In total, 1180 of 5257 dierent 16S rRNA gene amplicon sequences were identified in the three
replicated Mankai-supplemented bioreactors that contributed to the increased predicted abundance
of microbes containing btuB. Six 16S rRNA gene amplicon sequences displayed a greater than
0.5% relative 16S rRNA gene amplicon abundance, and three of these sequences (closely related to
Aeromonas hydrophila,Pelomonas aquatica, and Geobacter anodireducens) were present in all three replicated
Mankai-supplemented bioreactors (Figure S3). In marked contrast, only nine dierent 16S rRNA gene
amplicon sequences—associated with microbes potentially containing btuB—were identified in the
control reactors that lacked Mankai, of which (a) five of these nine were present in all three replicates
and (b) only one sequence (closely related to Escherichia coli) displayed a relative abundance greater
than 0.5% (Table S3).
4. Discussion
In the current study, we examined, using dierent methodologies, the presence of vitamin B
12
in a
cultivated strain of Wola globosa (Mankai). We found that Mankai, cultured under closed-controlled
greenhouse conditions, contains a substantial amount of the known bioactive forms of vitamin B
12
and that its presence is stable throughout the year. In inoculated gut microbiome anoxic bioreactors,
a significantly higher relative abundance of 16S rRNA gene sequences associated with a genome
containing the KEGG ortholog involved in vitamin B
12
uptake was observed, compared with control
bioreactors that lacked Mankai. In our human studies, results suggest that long-term consumption
of this plant, as part of a whole flexitarian diet, may increase rather than impair vitamin B
12
levels,
without additional red meat intake. To our knowledge, this is the first reported study on the B
12
content and bioavailability in duckweed and specifically in Wola globosa.
Although some evidence for the presence of vitamin B
12
in Actinorhizal plants has been
reported [
28
], it is generally recognized that vitamin B
12
is absent from plant-derived food
sources [
1
,
2
,
5
,
30
]. Plants neither require nor synthesize vitamin B
12
because they contain no
cobalamin-dependent enzymes and instead encode a B
12
-independent form of methionine synthase [
31
].
To carefully examine our hypothesis regarding the presence of vitamin B
12
in Mankai, we analyzed,
over a period of 5 years, samples that were obtained from intensively grown plant cultures. Repeated
microbiological assay analyses revealed the presence of stable levels of vitamin B
12
in Mankai.
Furthermore, to exclude B
12
presence due to absorption from an external source, we tested vitamin
B
12
in axenic Mankai cultures, generated by propagating a green daughter frond that emerged from a
bleached mother frond, for several months under sterile conditions. We speculated that in Mankai
plants grown under these conditions, the level of any absorbed vitamin B
12
from an external source,
such as occasional bacteria or microalgal contamination, would be expected to decline and probably
Nutrients 2020,12, 3067 12 of 17
become undetectable in the axenic culture as the plants propagated for successive generations in the
sterile culture and as the biomass increased by several orders. However, B
12
analysis performed on
cultures that were propagated for at least 5 months, under sterile conditions in a B
12
-free medium,
revealed similar levels of the vitamin. Since the results described above were obtained by the
microbiological assay method, the reliability of which was recently put in question because lactic
bacterium, L. delbrueckii, was found to be able to utilize other corrinoids as well [
1
], we decided to
further study the B12 nature in the Mankai plant tissue.
The LC-MS/MS method is a reliable method to analyze and identify vitamin B
12
and its congeneric
forms. We analyzed the four major forms of the vitamin: OH-B
12
, Ado-B
12
, Me-B
12
, and CN-B
12
in
all Mankai samples. The results revealed the presence of all four B
12
forms in the Mankai samples.
It is well known that in animal cells, Me-B
12
serves as a cofactor for methionine synthase, while
Ado-B
12
is a cofactor of methylmalonyl-CoA mutase. However, plants contain no cobalamin-dependent
enzymes [
31
] and therefore, while one can assume that these metabolites do not play a biological role
in Mankai plants, it remains possible that the coenzyme forms of B
12
are produced in endophytic
bacteria, which are the presumed source of the B
12
. As the analysis was performed using the KCN
extraction method, we were unable to assess the original content of each of the three natural B
12
forms
in Mankai. However, we were able to determine the total level of B
12
in Mankai, and importantly,
these results were comparable to the microbiological assay method. Moreover, we further investigated
the presence of pseudo B
12
due to reports on the identification of large quantities of this compound
in non-animal food sources, such as algae [
27
]. Since pseudo CN-B
12
is not commercially available,
we used
spirulina extracts as a reference source of pseudo CN-B
12
and compared it with Mankai
extracts. Under the LC-MS/MS conditions used in this study, no pseudo CN-B
12
forms bearing identity
with the pseudo CN-B
12
seen in the spirulina extract were detected in any of the Mankai samples.
Therefore, the bioassay analysis is a reliable method to measure vitamin B12 levels in Mankai.
Although the anity of the gastric intrinsic factor binding protein for authentic B
12
is 500 times
greater than for pseudo B
12
[
32
,
33
], according to Herbert and Drivas [
34
], non-cobalamin vitamin
B
12
analogues, produced by algae and other organisms, may interfere with vitamin B
12
metabolism.
A recent study by Bito et al. demonstrated that pseudo B
12
can inhibit transcobalamin II-mediated
absorption in mammalian cultured COS-7 cells [35].
Functional microbial composition analysis based on genome prediction and sequence
matching of microbes in reactors that were inoculated with human fecal samples indicated that
Mankai-supplemented reactors displayed a significantly enhanced relative abundance of 16S rRNA gene
sequences of microorganisms that have the gene required to produce the vitamin B
12
transporter BtuB.
BtuB, located in the outer membrane of Gram-negative bacteria, is essential for the active uptake
of cobalamin across the outer membrane [
36
]. We could infer that the increased abundance in
gut microorganisms that produce the vitamin B
12
transporter is due to the increased abundance
of thisvitamin B
12
in the Mankai reactors. Vitamin B
12
is an essential cofactor in several microbial
anaerobic processes (e.g., propionate fermentation, butyrate fermentation via 3-methylaspartate,
methanogenesis), suggesting that this vitamin has the potential to stimulate fermentation and, thus,
the production of short-chain fatty acids [3741], which provide many benefits to the host [42].
The origin of the vitamin B
12
in Mankai was not determined in this study, but we speculate that it
is derived from an endophyte bacterial source. The fact that we did find B
12
in the axenic cultures does
not negate this hypothesis as axenic duckweed cultures, although often termed in the literature as
“sterile” cultures, may still contain a plant tissue that carries microbes, in its internal core, as described
by Gilbert et al. [
12
]. One may reasonably assume that a single or several such endophytic bacteria are
responsible for the production of B12 found in Mankai.
Collectively, these results indicate that the presence of B
12
in Mankai is not an occasional event
nor a result of uptake from the surrounding medium but is stably and consistently produced within
or in close association with the plant. Further studies should be conducted to identify the vitamin
B
12
-producing bacteria and characterize their interaction with the plant. These studies may lead
Nutrients 2020,12, 3067 13 of 17
to novel strategies for B
12
enrichment in Mankai and would contribute to its nutritional value as a
potential vitamin B
12
food source, particularly for individuals who prefer a vegetarian lifestyle or who
eschew any animal products in their diet.
The recommended dietary allowance of vitamin B
12
for adults is set at 2.4
µ
g/day [
43
]. The vitamin
B
12
content in Mankai, according to our repeated analyses, is about 0.5
µ
g/20 g DW (equivalent to
100 g
of frozen Mankai, as given to our participants as a green dinner shake), thus making it a desirable
plant substitute. Although advised to completely reduce red meat intake, we observed a significant
increase in vitamin B
12
levels among participants who were under a semi-vegetarian weight loss diet,
compared with participants who, although advised to adopt a healthy lifestyle, did not significantly
change their routine red meat intake. It has to be noted that a significant trend in vitamin B
12
increase
was observed between the intervention groups, even though the green-MED dieters were instructed
to avoid red/processed meat and their diet was further fortified with Mankai shake and green tea.
In addition
, participants who reduced red meat had an increase in serum folate (a marker for green
leafy vegetable consumption [
22
]), and in this study for Mankai consumption [
17
] had an increase in
vitamin B
12
comparable to participants who increased red meat and had a decrease in serum folate
levels. Reducing red meat consumption, especially processed meat products, has been a focus of
attention in recent years, due to increasing evidence of the association between meat consumption
and health risks [
44
]. However, reducing red meat, as vegan or some vegetarian eating patterns
suggest, might put one at risk of vitamin B
12
deficiency, which could result in megaloblastic anemia and
neurological damage [
45
,
46
]. Vegetarians and vegans in particular are at risk of developing vitamin
B
12
deficiency and infants born to mothers who follow such diets run a risk of neurodevelopmental
abnormalities and feeding diculties [
47
]. Therefore, the identification of a natural alternative vitamin
B12 source would be of major interest to nutrition professionals.
Natural sources of authentic vitamin B
12
include red meat and fish but also dairy and eggs [
31
,
46
].
However, it is well known that growing cattle for food requires a lot of land, water, and energy,
and generates
considerable waste [
48
,
49
]. In the search for a sustainable vitamin B
12
source, it has
been reported that some plant foods (e.g., mushrooms and edible Algae) are rich in corrinoids,
but those
foods either lack the bioactive form of vitamin B
12
, must be consumed in impractical amounts,
or because
of controversial data are a questionable source of bioavailable B
12
[
50
52
]. Alternatively,
insects have been proposed as a promising source of food for vitamin B
12
. Mealworms, grasshoppers,
crickets, and cockroaches were studied regarding their content of bioactive vitamin B
12
but exhibited
marked variations in their vitamin B
12
content [
53
]. Moreover, esthetic, religious, and psychological
barriers may further limit their use as a source of vitamin B12 replacement.
The limitations of this study include the inability to assess the origin of the vitamin B
12
in the plant,
as well as the bioavailability and specific digestibility pathway of vitamin B
12
directly among our human
participants. The bioassay method, based on the B
12
-requiring bacteria Lactobacillus delbrueckii, cannot
determine whether Mankai contains cobalamin or inactive corrinoids or both [
1
]. However,
the fact
that the LC-MS/MS method, which is a direct physico-chemical assay for B
12
, revealed comparable
levels to the bioassay method indicates that, in the case of Mankai, the bioassay results reflect solely
the concentrations of authentic B
12
forms and not analogues. We were also not able to isolate Mankai
as a sole source of vitamin B
12
from other dietary components rich in vitamin B
12
in the long-term
human trial. In order to overcome this limitation, we presented additional analyses from the electronic
questionnaires of other B
12
sources. We did not measure homocysteine or methylmalonic acid, which
might better reflect metabolic deficiencies of vitamin B
12
or serum folate [
54
], thus we cannot evaluate
the eect of the intervention in cases with low B
12
and high levels of these serum/plasma markers
indicative of biochemical B
12
deficiency. Furthermore, our participants had baseline serum B
12
levels
within the normal range, so, although we could observe significant increases, we could not demonstrate
ecacy for correction of B
12
deficiency status and further studies should be carried out to examine this
question. We also cannot point out the exact mechanism that explains the substantial B
12
content in the
Mankai plant, nor the way in which this may be controlled in the plant tissue. The data we showed
Nutrients 2020,12, 3067 14 of 17
for our bioreactors are from a small pilot study, and we consider them preliminary. Thus, an open
question remains concerning the possibility that the Mankai plant may modify the microbiota in the
intestinal tract with possible eects on the bioavailability of B
12
normally present in bile [
5
]. Strengths
of the data that we report here include the comprehensive multi-assessment of several aspects of B12,
including laboratory, gut-related, and a long-term human randomized controlled trial, with monitored
lunch and daily supply of Mankai to the participants.
5. Conclusions
The Mankai plant contains bioactive B
12
compounds and could potentially serve as a plant-based
food source of vitamin B
12
. Results from this study could provide additional insight regarding a
much-needed alternative healthy and sustainable B12 source.
Supplementary Materials:
The following are available: http://www.mdpi.com/2072-6643/12/10/3067/s1. File S1:
Further details on the extraction and purification of Vitamin B
12
, File S2: Exclusion criteria DIRECT PLUS
trial, File S3: Further outcome measurements of DIRECT PLUS trial, File S4: Full details regarding the media,
anoxic bioreactor, Mankai lysate and the sampling, File S5: Further details regarding the 16S rRNA amplicon
sequences, File S6: Supplementation usage, DIRECT PLUS trial, Table S1: LC and MS parameters for detection of
corrinoids, Table S2: Composition of micronutrient-containing solution and macronutrient solution that were
used to prepare Base medium and Mankai medium, Table S3: Relative 16S rRNA gene amplicon abundance and
taxonomy of phylotypes that (a) are predicted to contain btuB in their genome and (b) displayed a greater than
0.5% relative abundance in either the Mankai-supplemented or control reactor, Figure S1: The DIRECT PLUS trial
flow diagram, Figure S2: Comparison of chromatograms of dierent MRMs for CN-B
12
standard 0.1
µ
g/mL (A-D)
and plant sample (E-H), Figure S3: Liquid chromatography/electrospray ionization
tandem mass spectrometry
chromatograms of bioactive B
12
compounds, Figure S4: Comparison of chromatograms of dierent MRMs for
Pseudo CN-B12 in Mankai (A-D) and Spirulina (E-H) samples, Figure S5: Further nutritional analysis.
Author Contributions:
Conceptualization, I.S. (Ilan Sela), A.Y.M., R.K.-B., L.Z., G.T., A.K., E.R., H.Z., M.L., I.S.
(Iris Shai); Formal analysis, I.S. (Ilan Sela), A.Y.M., A.B., R.K.-B., L.Z., D.C., B.D., S.A., U.C., B.I., M.L., I.S. (Iris Shai);
Investigation, A.Y.M., G.T., A.K., E.R., H.Z., I.S. (Iris Shai); Resources, I.S. (Iris Shai), R.K.-B.; Supervision, I.S.
(Iris Shai), R.G.; Writing—Original draft, I.S. (Ilan Sela), A.Y.M., M.L., I.S. (Iris Sha); Writing—Review and editing,
I.S. (Ilan Sela), A.Y.M., A.B., R.K.-B., L.Z., G.T., A.K., E.R., H.Z., U.C., R.G., I.S. (Iris Shai). All authors have read
and agreed to the published version of the manuscript.
Funding:
DIRECT-PLUS was supported by the Deutsche Forschungsgemeinschaft (DFG—German Research
Foundation)—project no. 209933838, grant SFB1052; the Deutsche Forschungsgemeinschaft, Obesity Mechanisms;
Israel Ministry of Health grant 87472511 (to I Shai); Israel Ministry of Science and Technology grant 3-13604
(
to I Shai
); and the California Walnuts Commission (to I Shai). Gut microbiome study was supported by
Arizona-BGU collaborative grant (PIs: Rosa Krajmalnik-Brown, Iris Shai). Mankai plant B
12
analysis was funded
by Hinoman Ltd. and Weitzman Institute. None of the funding providers were involved in any stage of the design,
conduct, or analysis of the study and they had no access to the study results before publication.
Acknowledgments:
We thank the DIRECT PLUS participants for their valuable contribution. We thank the
California Walnut Commission, Wissotzky Tea Company, and Hinoman, Ltd. for kindly supplying food items
for this study. We thank Dov Brikner, Efrat Pupkin, Eyal Goshen, Avi Ben Shabat, Evyatar Cohen and Benjamin
Sarusi from the Nuclear Research Center Negev, Liz Shabtai and Yulia Kovshan from Ben-Gurion University of
the Negev, Monica Colt from Hinoman Ltd. and Janet King from UC Davis for their valuable contributions to
this study.
Conflicts of Interest:
Sela I., Arinos S. and Lapidot M. are employees of Hinoman Ltd.; Shai I. advises to the
Hinoman, Ltd. nutritional committee. All other authors declare no conflict of interest.
References
1.
Watanabe, F.; Bito, T. Determination of cobalamin and related compounds in foods. J. AOAC Int.
2018
,101,
1308–1313. [CrossRef] [PubMed]
2.
Burgess, C.M.; Smid, E.J.; van Sinderen, D. Bacterial vitamin B2, B11 and B12 overproduction: An overview.
Int. J. Food Microbiol. 2009,133, 1–7. [CrossRef] [PubMed]
3.
Green, R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood J. Am. Soc. Hematol.
2017,129, 2603–2611. [CrossRef] [PubMed]
4.
Watanabe, F.; Takenaka, S.; Kittaka-Katsura, H.; Ebara, S.; Miyamoto, E. Characterization and bioavailability
of vitamin B12-compounds from edible algae. J. Nutr. Sci. Vitaminol.
2002
,48, 325–331. [CrossRef] [PubMed]
Nutrients 2020,12, 3067 15 of 17
5.
Green, R.; Miller, J.W. Vitamin B12. In Handbook of Vitamins; Zempleni, J., Suttie, J.W., Gregory, J.F., III,
Stover, P.J., Eds.; CRC Press: Boca Raton, FL, USA, 2013; pp. 447–489.
6.
Rizzo, G.; Lagan
à
, A.S.; Rapisarda, A.M.C.; Ferrera, L.; Grazia, G.M.; Buscema, M.; Rossetti, P.; Nigro, A.;
Muscia, V.; Valenti, G.; et al. Vitamin B12 among vegetarians: Status, assessment and supplementation.
Nutrients 2016,8, 767. [CrossRef] [PubMed]
7.
Crowe, F.L.; Appleby, P.N.; Travis, R.C.; Key, T.J. Risk of hospitalization or death from ischemic heart disease
among British vegetarians and nonvegetarians: Results from the EPIC-Oxford cohort study. Am. J. Clin. Nutr.
2013,97, 597–603. [CrossRef] [PubMed]
8.
Yokoyama, Y.; Barnard, N.D.; Levin, S.M.; Watanabe, M. Vegetarian diets and glycemic control in diabetes:
A systematic review and meta-analysis. Cardiovasc. Diagn. Ther. 2014,4, 373.
9.
Wang, F.; Zheng, J.; Yang, B.; Jiang, J.; Fu, Y.; Li, D. Eects of vegetarian diets on blood lipids: A systematic
review and meta-analysis of randomized controlled trials. J. Am. Heart Assoc. 2015,4, e002408. [CrossRef]
10.
Yokoyama, Y.; Nishimura, K.; Barnard, N.D.; Miyamoto, Y. Blood pressure and vegetarian diets. In Vegetarian
and Plant-Based Diets in Health and Disease Prevention; Elsevier: Amsterdam, The Netherlands, 2017;
pp. 395–413.
11. Sukumar, N.; Saravanan, P. Investigating vitamin B12 deficiency. BMJ 2019,365, l1865. [CrossRef]
12.
Gilbert, S.; Xu, J.; Acosta, K.; Poulev, A.; Lebeis, S.; Lam, E. Bacterial production of indole related compounds
reveals their role in association between duckweeds and endophytes. Front. Chem. 2018,6, 265. [CrossRef]
13.
Appenroth, K.J.; Sree, K.S.; Bog, M.; Ecker, J.; Boehm, V.; Lorkowski, S.; Sommer, K.; Vetter, W.;
Tolzin-Banasch, K.; Kirmse, R.; et al. Nutritional value of the duckweed species of the genus Wola
(Lemnaceae) as human food. Front. Chem. 2018,6, 483. [CrossRef] [PubMed]
14.
Kawamata, Y.; Shibui, Y.; Takumi, A.; Seki, T.; Shimada, T.; Hashimoto, M.; Inoue, N.; Kobayashi, H.; Narita, T.
Genotoxicity and repeated-dose toxicity evaluation of dried Wola globosa Mankai. Toxicol. Rep.
2020
.
[CrossRef] [PubMed]
15.
Kaplan, A.; Zelicha, H.; Tsaban, G.; Yaskolka Meir, A.; Rinott, E.; Kovsan, J.; Novack, L.; Thiery, J.; Ceglarek, U.;
Burkhardt, R.; et al. Protein bioavailability of Wola globosa duckweed, a novel aquatic plant–A randomized
controlled trial. Clin. Nutr. 2019,38. [CrossRef]
16.
Yan, Y.; Candreva, J.; Shi, H.; Ernst, E.; Martienssen, R.; Schwender, J.; Shanklin, J. Survey of the total fatty
acid and triacylglycerol composition and content of 30 duckweed species and cloning of a
6-desaturase
responsible for the production of
γ
-linolenic and stearidonic acids in Lemna gibba. BMC Plant Biol.
2013
,
13, 201. [CrossRef]
17.
Yaskolka Meir, A.; Tsaban, G.; Zelicha, H.; Rinott, E.; Kaplan, A.; Youngster, I.; Rudich, A.; Shelef, I.;
Tirosh, A.; Brikner, D.; et al. A Green-mediterranean diet, supplemented with mankai duckweed, preserves
iron-homeostasis in humans and is ecient in reversal of anemia in rats. J. Nutr. 2019,149. [CrossRef]
18.
Ball, G.F.M. Microbiological methods for the determination of the B-group vitamins. In Water-Soluble Vitamin
Assays in Human Nutrition; Springer: Berlin, Germany, 1994; pp. 317–364.
19.
Rinott, E.; Youngster, I.; Meir, A.Y.; Tsaban, G.; Zelicha, H.; Kaplan, A.; Knights, D.; Tuohy, K.; Fava, F.;
Scholz, M.U.; et al. Eects of diet-modulated autologous fecal microbiota transplantation on weight regain.
Gastroenterology 2020. [CrossRef]
20.
Shai, I.; Schwarzfuchs, D.; Henkin, Y.; Shahar, D.R.; Witkow, S.; Greenberg, I.; Golan, R.; Fraser, D.; Bolotin, A.;
Vardi, H.; et al. Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet. N. Engl. J. Med.
2008
,
359, 229–241. [CrossRef]
21.
Gepner, Y.; Shelef, I.; Schwarzfuchs, D.; Zelicha, H.; Tene, L.; Meir, A.Y.; Tsaban, G.; Cohen, N.; Bril, N.;
Rein, M.; et al. Eect of distinct lifestyle interventions on mobilization of fat storage pools: Central magnetic
resonance imaging randomized controlled trial. Circulation 2018,137, 1143–1157. [CrossRef]
22. Moll, R.; Davis, B. Iron, vitamin B12 and folate. Medicine 2017,45, 198–203. [CrossRef]
23.
McDonald, J.A.K.; Schroeter, K.; Fuentes, S.; Heikamp-deJong, I.; Khursigara, C.M.; de Vos, W.M.;
Allen-Vercoe, E. Evaluation of microbial community reproducibility, stability and composition in a human
distal gut chemostat model. J. Microbiol. Methods 2013,95, 167–174. [CrossRef]
24.
Gutierrez, D.; Weinstock, A.; Antharam, V.C.; Gu, H.; Jasbi, P.; Shi, X.; Dirks, B.; Krajmalnik-Brown, R.;
Maldonado, J.; Guinan, J.; et al. Antibiotic-induced gut metabolome and microbiome alterations increase
the susceptibility to Candida albicans colonization in the gastrointestinal tract. FEMS Microbiol. Ecol.
2020
,
96, fiz187. [CrossRef] [PubMed]
Nutrients 2020,12, 3067 16 of 17
25.
Ilhan, Z.E.; di Baise, J.K.; Dautel, S.E.; Isern, N.G.; Kim, Y.M.; Hoyt, D.W.; Schepmoes, A.A.; Brewer, H.M.;
Weitz, K.K.; Metz, T.O.; et al. Temporospatial shifts in the human gut microbiome and metabolome after
gastric bypass surgery. NPJ Biofilms Microbiomes 2020,6, 1–12. [CrossRef] [PubMed]
26.
Jägerstad, M.; Arkbåge, K. Cobalamins properties and determination. In Encyclopedia of Food Sciences and
Nutrition; Caballero, B., Trugo, L.C., Finglas, P.M., Eds.; Academic: Cambridge, MA, USA, 2003; p. 1419.
27.
Watanabe, F.; Katsura, H.; Takenaka, S.; Fujita, T.; Abe, K.; Tamura, Y.; Nakatsuka, T.; Nakano, Y.
Pseudovitamin B12 is the predominant cobamide of an algal health food, spirulina tablets. J. Agric.
Food Chem. 1999,47, 4736–4741. [CrossRef] [PubMed]
28.
Chamlagain, B.; Edelmann, M.; Kariluoto, S.; Ollilainen, V.; Piironen, V. Ultra-high performance liquid
chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and
fermented cereal matrices. Food Chem. 2015,166, 630–638. [CrossRef] [PubMed]
29.
Langille, M.G.I.; Zaneveld, J.; Caporaso, J.G.; McDonald, D.; Knights, D.; Reyes, J.A.; Clemente, J.C.;
Burkepile, D.E.; Thurber, R.L.V.; Knight, R.; et al. Predictive functional profiling of microbial communities
using 16S rRNA marker gene sequences. Nat. Biotechnol. 2013,31, 814–821. [CrossRef] [PubMed]
30.
Watanabe, F.; Yabuta, Y.; Bito, T.; Teng, F. Vitamin B12-containing plant food sources for vegetarians. Nutrients
2014,6, 1861–1873. [CrossRef] [PubMed]
31. Watanabe, F. Vitamin B12 sources and bioavailability. Exp. Biol. Med. 2007,232, 1266–1274. [CrossRef]
32.
Fedosov, S.N.; Fedosova, N.U.; Kräutler, B.; Nexø, E.; Petersen, T.E. Mechanisms of discrimination between
cobalamins and their natural analogues during their binding to the specific B12-transporting proteins.
Biochemistry 2007,46, 6446–6458. [CrossRef]
33.
Stupperich, E.; Nexø, E. Eect of the cobalt-N coordination on the cobamide recognition by the human
vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin. Eur. J. Biochem.
1991
,199,
299–303. [CrossRef]
34. Herbert, V.; Drivas, G. Spirulina and vitamin B12. JAMA 1982,248, 3096–3097. [CrossRef]
35.
Bito, T.; Bito, M.; Hirooka, T.; Okamoto, N.; Harada, N.; Yamaji,R.; Nakano, Y.; Inui, H.; Watanabe, F. Biological
activity of pseudovitamin B12 on cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase
in mammalian cultured COS-7 Cells. Molecules 2020,25, 3268. [CrossRef] [PubMed]
36.
Schauer, K.; Rodionov, D.A.; de Reuse, H. New substrates for TonB-dependent transport: Do we only see the
‘tip of the iceberg’? Trends Biochem. Sci. 2008,33, 330–338. [CrossRef] [PubMed]
37.
Xu, Y.; Xiang, S.; Ye, K.; Zheng, Y.; Feng, X.; Zhu, X.; Chen, J.; Chen, Y. Cobalamin (vitamin B12) induced a
shift in microbial composition and metabolic activity in an
in vitro
colon simulation. Front. Microbiol.
2018
,
9, 2780. [CrossRef] [PubMed]
38.
Louis, P.; Flint, H.J. Formation of propionate and butyrate by the human colonic microbiota. Environ. Microbiol.
2017,19, 29–41. [CrossRef] [PubMed]
39.
Takahashi-Iñiguez, T.; Garc
í
a-Hernandez, E.; Arregu
í
n-Espinosa, R.; Flores, M.E. Role of vitamin B12 on
methylmalonyl-CoA mutase activity. J. Zhejiang Univ. Sci. B 2012,13, 423–437. [CrossRef] [PubMed]
40.
Banerjee, R.; Ragsdale, S.W. The many faces of vitamin B12: Catalysis by cobalamin-dependent enzymes.
Annu. Rev. Biochem. 2003,72, 209–247. [CrossRef]
41.
Buckel, W. Unusual enzymes involved in five pathways of glutamate fermentation. Appl. Microbiol. Biotechnol.
2001,57, 263–273. [CrossRef]
42.
Van Treuren, W.; Dodd, D. Microbial contribution to the human Metabolome: Implications for health and
disease. Annu. Rev. Pathol. Mech. Dis. 2020,15, 345–369. [CrossRef]
43.
Food and Nutrition Board, Institute of Medicine. Dietary Reference Intakes: Recommended Intakes for Individuals;
National Academy of Sciences: Washington, DC, USA, 2004.
44.
Micha, R.; Penalvo, J.L.; Cudhea, F.; Imamura, F.; Rehm, C.D.; Mozaarian, D. association between dietary
factors and mortality from heart disease, stroke, and Type 2 diabetes in the united states. JAMA
2017
,317,
912–924. [CrossRef]
45.
Obeid, R.; Heil, S.G.; Verhoeven, M.M.A.; van den Heuvel, E.G.H.M.; de Groot, L.C.; Eussen, S.J.P.M. Vitamin
B12 intake from animal foods, biomarkers, and health aspects. Front. Nutr. 2019,6, 93. [CrossRef]
46.
Green, R.; Allen, L.H.; Bjørke-Monsen, A.L.; Brito, A.; Gu
é
ant, J.L.; Miller, J.W.; Molloy, A.M.; Nexo, E.;
Stabler, S.; Toh, B.H.; et al. Vitamin B 12 deficiency. Nat. Rev. Dis. Prim. 2017,3, 17040. [CrossRef]
47.
Oussalah, A.; Levy, J.; Berthez
è
ne, C.; Alpers, D.H.; Gu
é
ant, J.L. Health outcomes associated with vegetarian
diets: An umbrella review of systematic reviews and meta-analyses. Clin. Nutr.
2020
. [CrossRef] [PubMed]
Nutrients 2020,12, 3067 17 of 17
48.
Lebacq, T.; Baret, P.V.; Stilmant, D. Sustainability indicators for livestock farming. A review. Agron. Sustain.
Dev. 2013,33, 311–327. [CrossRef]
49.
Van Zanten, H.H.E.; Herrero, M.; van Hal, O.; Röös, E.; Muller, A.; Garnett, T.; Gerber, P.J.; Schader, C.;
Boer, I.J.M. De Defining a land boundary for sustainable livestock consumption. Glob. Chang. Biol.
2018
,24,
4185–4194. [CrossRef] [PubMed]
50.
Watanabe, F.; Yabuta, Y.; Tanioka, Y.; Bito, T. Biologically active vitamin B12 compounds in foods for
preventing deficiency among vegetarians and elderly subjects. J. Agric. Food Chem.
2013
,61, 6769–6775.
[CrossRef] [PubMed]
51.
Suzuki, H. Serum vitamin B12 levels in young vegans who eat brown rice. J. Nutr. Sci. Vitaminol.
1995
,41,
587–594. [CrossRef] [PubMed]
52.
Dagnelie, P.C.; van Staveren, W.A.; van den Berg, H. Vitamin B-12 from algae appears not to be bioavailable.
Am. J. Clin. Nutr. 1991,53, 695–697. [CrossRef]
53.
Schmidt, A.; Call, L.M.; Macheiner, L.; Mayer, H.K. Determination of vitamin B12 in four edible insect species
by immunoanity and ultra-high performance liquid chromatography. Food Chem.
2019
,281, 124–129.
[CrossRef]
54.
Klee, G.G. Cobalamin and folate evaluation: Measurement of methylmalonic acid and homocysteine vs
vitamin B12 and folate. Clin. Chem. 2000,46, 1277–1283. [CrossRef]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... These diminutive plants have gone through an extreme reduction in body size, with some species less than 0.5 mm in size, thereby minimizing the need for non-photosynthetic organs and selecting for rapid multiplication through budding [3]. As a consequence of their fast growth rate, biomass production is high, providing practical applications to the duckweeds in food [4,5], feed [6], water treatment [7,8] and biotechnology [9,10]. ...
... A phylogenetic tree based on psbK-psbI and atpF-atpH sequences was constructed after multiple alignment of the sequences ( Figure 2) using a total of the 27 haplotypes collected in this study, as well as W. globosa "Mankai" (a domesticated duckweed haplotype that was isolated from the Golan Hights, Israel [5]) and additional reference sequences (S. polyrhiza 7498, W. arrhiza DW35, W. globosa 8789, L. minuta 5573, L. gibba 5504, L. minor 7123) taken from the NCBI database (one for each species). ...
... A phylogenetic tree based on psbK-psbI and atpF-atpH sequences was constructed after multiple alignment of the sequences (Figure 2) using a total of the 27 haplotypes collected in this study, as well as W. globosa "Mankai" (a domesticated duckweed haplotype that was isolated from the Golan Hights, Israel [5]) and additional reference sequences (S. polyrhiza 7498, W. arrhiza DW35, W. globosa 8789, L. minuta 5573, L. gibba 5504, L. minor 7123) taken from the NCBI database (one for each species). ...
Article
Full-text available
Duckweeds (Lemnaceae) are tiny plants that float on aquatic surfaces and are typically isolated from temperate and equatorial regions. Yet, duckweed diversity in Mediterranean and arid regions has been seldom explored. To address this gap in knowledge, we surveyed duckweed diversity in Israel, an ecological junction between Mediterranean and arid climates. We searched for duckweeds in the north and center of Israel on the surface of streams, ponds and waterholes. We collected and isolated 27 duckweeds and characterized their morphology, molecular barcodes (atpF-atpH and psbK-psbI) and biochemical features (protein content and fatty acids composition). Six species were identified—Lemna minor, L. gibba and Wolffia arrhiza dominated the duckweed populations, and together with past sightings, are suggested to be native to Israel. The fatty acid profiles and protein content further suggest that diverged functions have attributed to different haplotypes among the identified species. Spirodela polyrhiza, W. globosa and L. minuta were also identified but were rarer. S. polyrhiza was previously reported in our region, thus, its current low abundance should be revisited. However, L. minuta and W. globosa are native to America and Far East Asia, respectively, and are invasive in Europe. We hypothesize that they may be invasive species to our region as well, carried by migratory birds that disperse them through their migration routes. This study indicates that the duckweed population in Israel’s aquatic environments consists of both native and transient species.
... Vitamin B12 analysis can be based on two approaches: microbiological and chemical. The microbiological tests generally involve the use of Lactobacillus delbrueckii [14]; these methods were originally developed for the analysis of vitamin preparations but are also applied to fortified foods and food matrices in general. Regarding chemical-physical methods, spectrophotometry is applicable, but with a complex matrix, it is difficult to achieve good sensitivity [15]. ...
... ICP-AES (inductively coupled plasma atomic emission spectroscopy) was also used by Bartosiak et al. (2018) [24] for the determination of cobalt species in dietary supplements. In recent times, several LC-electrospray ionization (ESI)-mass spectrometry methods have been developed for the determination of vitamin B12 concentrations in food products [14,[25][26][27]; these techniques are more sensitive and selective, allowing researchers to quantify naturally occurring cobalamin in food at very low levels. However, most of the LC-electrospray ionization-mass spectrometry methods available in the literature for the determination of cobalamin have been developed on matrices with a medium-high water content or plant-based matrix. ...
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The analysis of natural cobalamins in dairy products still represents an analytical challenge. The matrix’s complexity, low concentration level, light sensitivity, and binding to proteins are just some of the aspects that make their quantification a difficult goal to achieve. Vitamin B12 plays a fundamental role in human nutrition, and its intake is closely linked to a diet that includes the consumption of food of animal origin. In the current literature, few studies have been carried out on the quantitation of cobalamin in ripened cheeses. A sensitive, selective, and robust ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was developed, validated, and applied on ripened cheeses from different species (cow, sheep, and goat) purchased from local Italian markets, highlighting species-dependent differences in vitamin B12 concentrations. The vitamin B12 extraction procedure was performed by converting all cobalamins to the cyanocobalamin form. Furthermore, solid-phase extraction was used for matrix clean-up and analyte preconcentration. The proposed method showed good performance in terms of linearity, sensitivity, reproducibility, and repeatability. The mean vitamin B12 content ranged from <LOQ to 38.9 ng/g. Sheep cheese showed the highest concentrations of vitamin B12, with a mean content of 29.0 ng/g.
... To optimize production of multiple essential human nutrients, a growing procedure would be desirable that includes growth in low/nonexcessive light combined with approaches that increase zeaxanthin content via either a sudden pre-harvest increase in growth PFD and/or via engineering of the xanthophyll cycle. Duckweeds provide an attractive mix of carotenoids and polyphenols [4,55,57,60] and duckweed consumption has benefits for human health [100][101][102][103][104][105][106][107]. Future studies of the impact of growth light environment on duckweed's nutritional quality should combine evaluation of carotenoid, antioxidant, vitamin, and phenolics production in consideration of their synergistic actions. ...
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Development of a nutritious, sustainable food source is essential to address worldwide deficiencies in human micronutrients. Aquatic floating plants (e.g., species in the family Lemnaceae, duckweeds) are uniquely suited for area-efficient productivity with exceptionally high rates of growth and nutritional quality. Here, we provide an overview of the role of dietary micronutrients (with a focus on carotenoids) in human health and the promise of Lemnaceae as sustainable crops. We examine the effect of growth light environment on plant biomass production and levels of the carotenoids zeaxanthin, lutein, and pro-vitamin A (β-carotene), as well as the antioxidant vitamin E (α-tocopherol), and protein. Data on each of these nutrients are reported on a plant dry biomass basis (as relevant for nutrition) as well as relative to the required input of light energy (as relevant to resource-use efficiency).
... One study showed that consumption of W. globosa might have beneficial postprandial glycemic effects [14]. Another showed that the presence of cyanocobalamin, iron, and folic acid in this plant improved the health of prediabetic patients when included in a standard Mediterranean style diet [15]. W. globosa was also found to help maintain iron and folic acid status in humans and completely reverse iron deficiency anemia in an experimental rat model [16]. ...
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Wolffia globosa, or watermeal, is an aquatic plant belonging to the Lemnaceae family that is consumed as food and sold in local markets of Thailand. The aim of this study was to quantify selected active compounds and minerals in W. globosa ethanolic extract and evaluate its antioxidant activity. Total phenolic, flavonoid, and anthocyanin contents were analyzed. High-performance liquid chromatography was used for the determination of beta-carotene, ferulic acid, luteolin-7-O-β-D-glucoside, and kaempferol. Mineral contents (iron, potassium, calcium, magnesium, zinc, and sodium) were determined by atomic absorption spectroscopy. Antioxidative activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azobis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays. The beta-carotene, ferulic acid, luteolin-7-O-β-D-glucoside, and kaempferol contents of the extract were 2.52 ± 0.10, 1.40 ± 0.10, 2.42 ± 0.50, and 1.57 ± 0.14 mg/g extract, respectively. The highest mineral content in the W. globosa extract was magnesium. The wet extract of W. globosa showed higher amounts of all minerals than the dry extract. Freshly prepared and boiled W. globosa extracts showed radical scavenging activity at 1000 µg/milliliter with 75.77 ± 0.93% and 67.10 ± 0.20% inhibition of DPPH and 70.40 ± 7.20% and 59.78 ± 3.16% inhibition of ABTS, respectively. This plant is a promising novel source of natural phytochemical constituents and antioxidants and has potential for development as a plant-based nutraceutical product for the treatment of diseases caused by free radicals.
... In the development of drug action, MALDI-TOF is frequently used to characterize metabolomic modifications in red blood cells [91,92]. MALDI-TOF and chromatography can be used to find specific molecules in plant sources and their targets, as Sela et al. evaluated the vitamin [12] content in aquatic plant Wolffia globosa (Mankai) by MALDI-TOF [93] and Wang et al. used drug carrier proteins coupled with mass spectrometry to recognize the high-affinity protein molecules targets of andrographolide (Andro), a natural p-agonist [94]. ...
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Compounds isolated from natural sources have been used for medicinal purposes for many centuries. Some metabolites of plants and microorganisms possess properties that would make them effective treatments against bacterial infection, inflammation, cancer, and an array of other medical conditions. In addition, natural compounds offer therapeutic approaches with lower toxicity compared to most synthetic analogues. However, it is challenging to identify and isolate potential drug candidates without specific information about structural specificity and limited knowledge of any specific physiological pathways in which they are involved. To solve this problem and find a way to efficiently utilize natural sources for the screening of compounds-candidates, technologies such as next-generation sequencing, bioinformatics techniques, and molecular analysis systems should be adapted for screening many chemical compounds. Molecular techniques capable of performing analysis of large datasets, such as whole-genome sequencing and cellular protein expression profile, have become essential tools in drug discovery. OMICs - genomics, proteomics, and metabolomics - are often used in targeted drug discovery, isolation, and characterization. This review summarizes technologies that are effective in natural source drug discovery and aid in a more precisely targeted pharmaceutical approach, including RNA interference or CRISPR technology. We strongly suggest that a multidisciplinary effort utilizing novel molecular tools to identify and isolate active compounds applicable for future drug discovery and production must be enhanced with all the available computational tools.
... B12 content analysis was in liquid chromatography-mass spectrometry,(Nakos et al. 2017) microbiological assay and LC-MS/MS.(Sela et al. 2020) Quality assessment14 All studies scored more than 9/10 of the suggested quality assessment questionnaire. Only one determined B12 content without replicate.(Nakos et al. 2017) Nakos et al. (2017) investigated sea buckthorn (Hippophae rhamnoides), a plant used as a herb and medicinal herb, using liquid chromatography-mass spectrometry. T ...
Article
Interest in plant-based diets and vegetarianism is increasing worldwide, however, a concern for total vegetarians is vitamin B12 (B12) deficiency. We conducted a systematic review to investigate non-animal food sources of B12. Databases were PubMed, LILACS, Cochrane, Embase and Google Scholar, up to September 9, 2020. Quality of the eligible studies were assessed. We identified 25 studies which assessed B12 content in seaweeds, mushrooms, plants and fermented foods. Initial studies were microbiological bioassay, ELISA and HPLC. In the last decade, more sensitive method for real B12 determination was used, the liquid chromatography-electrospray ionization tandem mass spectrometry chromatograms. Real B12 content varied from mean (SD) mcg/portion size of seaweed hijiki 3 × 10-3/7 g to nori 1.03 - 2.68/sheet; mushroom white button cap 2 × 10-3(7 × 10-4)/20 g dry weight (dw) to shiitake 0.79(0.67)-1.12 (0.78)/20 g dw; and fermented foods from soy yogurt 20/cup. It is possible that daily recommendations for B12 can be met by a varied diet containing non-animal B12 food sources. Future research should consider different methods of storage, preparation, fermented foods and standardization of the production of certain foods.
... Since the only known naturally source of vitamin B12 are animal food products (meat, poultry, fish, egg, milk, etc.) and the general absence of this vitamin in plant foods because there are no cobalamin-dependent enzymes in plants, strict vegetarians and vegans are advised to supplement vitamin B12 to avoid a deficiency [243][244][245]. Several B12 plant-based food sources were reported on over the last years, for example, Mankai plant [246], seaweed [247], Hippophae rhamnoides, Elymus, Inula helenium [248], some algal species [249] as well as next-generation nutritionally fortified plant-based milk substitutes [250]. One caveat that must be mentioned in this context, is that such natural sources often contain biological inactive vitamin B12 analogues. ...
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Alzheimer’s disease (AD) is the most common form of dementia in the elderly population, affecting over 55 million people worldwide. Histopathological hallmarks of this multifactorial disease are an increased plaque burden and tangles in the brains of affected individuals. Several lines of evidence indicate that B12 hypovitaminosis is linked to AD. In this review, the biochemical pathways involved in AD that are affected by vitamin B12, focusing on APP processing, Aβ fibrillization, Aβ-induced oxidative damage as well as tau hyperphosphorylation and tau aggregation, are summarized. Besides the mechanistic link, an overview of clinical studies utilizing vitamin B supplementation are given, and a potential link between diseases and medication resulting in a reduced vitamin B12 level and AD are discussed. Besides the disease-mediated B12 hypovitaminosis, the reduction in vitamin B12 levels caused by an increasing change in dietary preferences has been gaining in relevance. In particular, vegetarian and vegan diets are associated with vitamin B12 deficiency, and therefore might have potential implications for AD. In conclusion, our review emphasizes the important role of vitamin B12 in AD, which is particularly important, as even in industrialized countries a large proportion of the population might not be sufficiently supplied with vitamin B12.
... A successful example of genotype selection for a distinct application is the commercial work on the cultivation of Mankai, a high proteinstrain from Wolffia globosa. This strain was selected based on its nutritional value; it contains at least 45% protein on a dry-weight basis and boasts fatty acids, amino acids, iron, zinc, and other nutrients (Sela et al., 2020), and is grown in indoor, highly monitored, greenhouses. Thus, indoor bioreactors facilitate the pure culturing of duckweed species/strains whose characteristics align with the production goals of the bioreactors in a way that is not possible outdoors due to likely contamination risks. ...
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Lemnaceae, i.e., duckweed species, have gained considerable attention as a sustainable source of high-quality nutrition, biofuel, and pharmaceuticals, as well as effective organisms for phytoremediation of wastewaters. A protein content of up to 45% makes duckweed biomass nutritionally interesting as an ingredient for animal feeds or human food. Outdoor duckweed cultivation has become common in recent decades but can be difficult to optimise and to control operationally. Yet, duckweeds also represent a suitable crop for indoor farming, with most species due to their flat structure particularly suited for cultivation in multi-level (stacked) systems that use indoor floor space efficiently. Here we propose construction of stacked systems with up to 15 m² of duckweed per m² of floorspace. Such stacked systems are facilitated by limiting the water depth to about 5 cm. Indoor cultivation can maximise yields, and doubling times as short as 1.24 days have been reported under indoor conditions. Indoor cultivation also extends the scope for crop manipulation and enables cultivation under pest-free and even sterile conditions. Yet, the technical and operational parameters required for effective large-scale indoor cultivation of Lemnaceae have received scant attention in the literature. Here, it is concluded that technological advances in urban and/or vertical farming can be exploited to enable design and operation of novel duckweed cultivation systems. Recirculating, flow through technology can optimise nutrient supply and growth, while sensor support systems with artificial intelligence can facilitate autonomous cropping and even harvesting. Furthermore, advanced understanding of duckweed-biology can, amongst others, inform selection of (wastewater-based) media, flow-rates and media retention time, duckweed species and strains, and enhance performance through manipulation of the duckweed microbiome. Despite challenges and knowledge gaps, there are now realistic opportunities to develop and operate high capacity, autonomous, controlled cultivation of duckweed under indoor conditions, for a broad range of purposes.
... Dans sa zone d'origine, notamment au Laos, en Birmanie (Myanmar) et en Thaïlande, elle est appelée Khaï Nam et constitue une culture légumière consommée sous forme de soupe, légume ou omelette. Son intérêt diététique -et donc sa culture -se répandent dans le monde, car elle contient au moins 30% de son poids sec en protéines (autant que le soja) et également de la vitamine B12 dont la carence est cause d'anémie chez les végétaliens (Sela et al., 2020). Les Wolffia ont également des capacités d'épuration des eaux avec une bonne absorption des nitrates et l'absorption des nutriments en excès en particulier azote (N) et phosphate (PO4). ...
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In the south of France, around the Salagou lake (Hérault), the observation of three populations of Wolffia globosa (Roxb.) Hartog & Plas (Araceae), a small duckweed of paleotropical origin, is the first record of this species in France. This discovery brings the number of Wolffia present in France to three. Here we summarise the available data concerning the description of W. globosa and its differences with related species (via a key for the four species of the genus Wolffia present in Europe), its ecology, its use and its pathways of introduction.
... Remarkable differences have been noted in vitamin B12 levels in cohort studies of meat eaters, fish eaters, vegetarians, and vegans, highlighting the inadequate dietary intake among vegans and vegetarians [17,18]. Attempts have been made to identify naturally occurring cobalamin-rich, plant-derived foods, such as dried shiitake mushroom fruiting bodies, dried green laver (Enteromorpha species) and purple laver (Porphyra species), aquatic plant Wolffia globosa (Mankai), sea buckthorn (Hippophae rhamnoides) berries and granulate products, and sidea couch grass (Elymus repens) products (dry extract and ground) [19][20][21][22]. A clinical trial conducted in India to determine improvement in vitamin B12 status among deficient vegetarians with dairy cow's milk intake concluded that it can drastically improve blood cobalamin levels [23]. ...
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Metabolic syndrome (MS) is a collection of pathological metabolic conditions that includes insulin resistance, central or abdominal obesity, dyslipidemia, and hypertension. It affects large populations worldwide, and its prevalence is rising exponentially. There is no specific mechanism that leads to the development of MS. Proposed hypotheses range from visceral adiposity being a key factor to an increase in very-low-density lipoprotein and fatty acid synthesis as the primary cause of MS. Numerous pharmaceutical therapies are widely available in the market for the treatment of the individual components of MS. The relationship between MS and vitamin B complex supplementation, specifically folic acid and vitamin B12, has been a subject of investigation worldwide, with several trials reporting a positive impact with vitamin supplementation on MS. In this study, an all-language literature search was conducted on Medline, Cochrane, Embase, and Google Scholar till September 2021. The following search strings and Medical Subject Headings (MeSH) terms were used: "Vitamin B12," "Folate," "Metabolic Syndrome," and "Insulin Resistance." We explored the literature on MS for its epidemiology, pathophysiology, newer treatment options, with a special focus on the effectiveness of supplementation with vitamins B9 and B12. According to the literature, vitamin B12 and folate supplementation, along with a host of novel therapies, has a considerable positive impact on MS. These findings must be kept in mind while designing newer treatment protocols in the future.
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Wolffia is a genus of protein-rich aquatic plants. Mankai, a cultivated strain of Wolffia globosa, contains more than 40 % protein based on dry matter evaluation. Furthermore, Mankai is nutritionally excellent as a food material, and is expected to be applicable to various products as a substitute for animal protein. A battery of toxicological studies was conducted on the dried product of Mankai (Dry Mankai), with the expectation to utilize it as a raw material for food applications. Dry Mankai was not genotoxic in a bacterial reverse mutation test and in vitro micronucleus assay. In the subchronic toxicity study, rats were provided Dry Mankai in the diet at levels of 0 %, 5 %, 10 %, or 20 % (w/w), equivalent to 0, 3.18, 6.49, and 13.16 g/kg/day for males and 0, 3.58, 7.42, and 15.03 g/kg/day for females, respectively. No adverse effects that could be attributable to treatment were observed in clinical observations, body weight, food consumption, ophthalmology, hematology and blood chemistry, urinalysis, and macroscopic and microscopic findings. According to the repeated-dose study in rats, the no observed adverse effect level of Dry Mankai was 20 % (w/w) for both sexes (13.16 and 15.03 g/kg/day for males and females, respectively).
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Background & Aims We evaluated the efficacy and safety of diet-modulated autologous fecal microbiota transplantation (aFMT) for treatment of weight regain after the weight loss phase. Methods In the DIRECT-PLUS weight loss trial (May 2017 through July 2018), abdominally obese or dyslipidemic participants in Israel were randomly assigned to (1)healthy dietary guidelines, (2)Mediterranean diet, and (3)green-Mediterranean diet weight-loss groups. All groups received free gym membership and physical activity guidelines. Both iso-caloric Mediterranean groups consumed 28g/day walnuts (+440mg/d polyphenols provided). The green-Mediterranean dieters further consumed green tea (3-4 cups/day) and a Wolffia-globosa (Mankai strain;100g/day) green shake (+800mg/day polyphenols provided). After 6 months (weight-loss phase), 90 eligible participants (mean age, 52 years; mean weight loss, 8.3 kg) provided a fecal sample that was processed into aFMT by frozen, opaque and odorless capsules. The participants were then randomly assigned to groups that received 100 capsules containing their own fecal microbiota or placebo until month 14. The primary outcome was regain of the lost weight over the expected weight regain phase (months 6–14). Secondary outcomes were gastrointestinal symptoms, waist-circumference, glycemic status and changes in the gut microbiome, as measured by metagenomic sequencing and 16s-rRNA. We validated the results in a parallel in-vivo study of mice specifically fed with Mankai, as compared to control chow diet. Results Of the 90 participants in the aFMT trial, 96% ingested at least 80 of 100 oral aFMT or placebo frozen capsules over the transplantation period. No aFMT-related adverse events or symptoms were observed. For the primary outcome, although no significant differences in weight regain were observed among the participants in the different lifestyle interventions during months 6–14 (aFMT, 30.4% vs. placebo, 40.6%;P=.28), aFMT significantly attenuated weight regain in the green-Mediterranean group (aFMT, 17.1%, vs placebo, 50%; P=.02), but not in the dietary guidelines (P=.57) or Mediterranean diet (P=.64) groups (P for the interaction=.03). Accordingly, aFMT attenuated waist circumference gain (aFMT, 1.89cm vs placebo, 5.05cm;P=.01) and insulin rebound (aFMT, 1.46±3.6μIU/ml vs placebo, 1.64±4.7μIU/ml;P=.04) in the green Mediterranean group but not in the dietary guidelines or Mediterranean diet (P for the interaction=.04 and .03, respectively). The green-Mediterranean diet was the only intervention to induce a significant change in microbiome composition during the weight loss phase, and to prompt preservation of weight loss-associated specific bacteria and microbial metabolic pathways (mainly microbial sugar transport) following the aFMT. In mice, Mankai-modulated aFMT in the weight loss phase, compared with control diet aFMT, significantly prevented weight regain, and resulted in better glucose tolerance, during a high-fat-diet induced regain phase (P<.05 for all). Conclusions Autologous FMT, collected during the weight loss phase and administrated in the regain phase, might preserve weight loss and glycemic control and is associated with specific microbiome signatures. High-polyphenols, green plant-based or Mankai diet better optimizes the microbiome for an aFMT procedure. (ClinicalTrials.gov number, NCT03020186)
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Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.
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Although the etiology of obesity is not well-understood, genetic, environmental, and microbiome elements are recognized as contributors to this rising pandemic. It is well documented that Roux-en-Y gastric bypass (RYGB) surgery drastically alters the fecal microbiome, but data are sparse on temporal and spatial microbiome and metabolome changes, especially in human populations. We characterized the structure and function (through metabolites) of the microbial communities in the gut lumen and structure of microbial communities on mucosal surfaces in nine morbidly obese individuals before, 6 months, and 12 months after RYGB surgery. Moreover, using a comprehensive multi-omic approach, we compared this longitudinal cohort to a previously studied cross-sectional cohort (n = 24). In addition to the expected weight reduction and improvement in obesity-related comorbidities after RYGB surgery, we observed that the impact of surgery was much greater on fecal communities in comparison to mucosal ones. The changes in the fecal microbiome were linked to increased concentrations of branched-chain fatty acids and an overall decrease in secondary bile acid concentrations. The microbiome and metabolome data sets for this longitudinal cohort strengthen our understanding of the persistent impact of RYGB on the gut microbiome and its metabolism. Our findings highlight the importance of changes in mucosal and fecal microbiomes after RYGB surgery. The spatial modifications in the microbiome after RYGB surgery corresponded to persistent changes in fecal fermentation and bile acid metabolism, both of which are associated with improved metabolic outcomes.
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Surface superhydrophobicity makes bacterial biofilms very difficult to fight, and it is a combination of their matrix composition and complex surface roughness which synergistically protects these biomaterials from wetting. Although trying to eradicate biofilms with aqueous (antibiotic) solutions is common practice, this can be a futile approach if the biofilms have superhydrophobic properties. To date, there are not many options available to reduce the liquid repellency of biofilms or to prevent this material property from developing. Here, we present a solution to this challenge. We demonstrate how the addition of metal ions such as copper and zinc during or after biofilm formation can render the surface of otherwise superhydrophobic B. subtilis NCIB 3610 biofilms completely wettable. As a result of this procedure, these smoother, hydrophilic biofilms are more susceptible to aqueous antibiotics solutions. Our strategy proposes a scalable and widely applicable step in a multi-faceted approach to eradicate biofilms.
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Antibiotic-induced alterations in the gut ecosystem increases the susceptibility to Candida albicans, yet the mechanisms involved remains poorly understood. Here we show that mice treated with the broad-spectrum antibiotic cefoperazone promoted the growth, morphogenesis and gastrointestinal (GI) colonization of C. albicans. Using metabolomics, we revealed that the cecal metabolic environment of the mice treated with cefoperazone showed a significant alteration in intestinal metabolites. Levels of carbohydrates, sugar alcohols, and primary bile acids increased, whereas carboxylic acids and secondary bile acids decreased in antibiotic treated mice susceptible to C. albicans. Further, using in vitro assays, we confirmed that carbohydrates, sugar alcohols and primary bile acids promote, whereas carboxylic acids and secondary bile acids inhibit the growth and morphogenesis of C. albicans. In addition, in this study we report changes in the levels of gut metabolites correlated with shifts in the gut microbiota. Taken together, our in-vivo and in-vitro results indicate that cefoperazone -induced metabolome and microbiome alterations favor the growth and morphogenesis of C. albicans, and potentially play an important role in the gastrointestinal colonization of C. albicans.
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The EAT-Lancet commission recently suggested that transformation to healthy diets by 2050 will require a reduction of at least 50% in consumption of foods such as red meat and sugar, and a doubling in the global consumption of fruits, vegetables, nuts, and legumes. A diet rich in plant-based foods and with fewer animal source foods confers both improved health and environmental benefits. Notably, the risk of vitamin B12 deficiency increases when consuming a diet low in animal products. Humans are dependent on animal foods such as dairy products, meat, fish and eggs. Vitamin B12 deficiency is common worldwide, especially in populations with low consumption of animal foods because of low socioeconomic status, ethical reasons, or because of their lifestyle (i.e., vegans). According to the European Food Safety Authoroty, the recommended adequate intake of vitamin B12 is 4.0 μg/d for adults, and vitamin B12 requirements are higher during pregnancy and lactation. Infants and children from deficient mothers and elderly people are at risk for vitamin B12 deficiency. Diagnosis of vitamin B12 deficiency is hampered by low specificity of available biomarkers, and there is no consensus yet regarding the optimal definition of low vitamin B12 status. In general, a combination of at least two biomarkers is recommended. Therefore, this review presents an overview of vitamin B12 biochemistry and its biomarkers. We further summarize current recommendations of vitamin B12 intake, and evidence on the associations of vitamin B12 intake from different nutrient-dense animal foods with vitamin B12 status markers. Finally, potential consequences of low vitamin B12 status on different health outcomes for pregnant women, infants and elderly are presented.
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Background: Decreased dietary meat may deplete iron stores, as plant-derived iron bioavailability is typically limited. Objectives: We explored the effect of a low-meat Mediterranean (green-MED) diet, supplemented with Wolffia globosa duckweed (Mankai: rich in protein and iron) as a food source for humans, on iron status. We further examined the iron bioavailability of Mankai in rats. Methods: Two hundred and ninety-four abdominally obese/dyslipidemic [mean age = 51.1 y; body mass index (kg/m2) = 31.3; 88% men] nonanemic participants were randomly assigned to physical activity (PA), PA + MED diet, or PA + green-MED diet. Both isocaloric MED groups consumed 28 g walnuts/d and the low-meat green-MED group further consumed green tea (800 mL/d) and Mankai (100 g green shake/d). In a complementary animal experiment, after 44 d of an iron deficiency anemia-inducing diet, 50 female rats (age = 3 wk; Sprague Dawley strain) were randomly assigned into: iron-deficient diet (vehicle), or vehicle + iso-iron: ferrous gluconate (FG) 14, Mankai 50, and Mankai 80 versions (1.7 mg · kg-1 · d-1 elemental iron), or FG9.5 and Mankai 50-C version (1.15 mg · kg-1 · d-1 elemental iron). The specific primary aim for both studies was changes in iron homeostasis parameters. Results: After 6 mo of intervention, iron status trajectory did not differ between the PA and PA + MED groups. Hemoglobin modestly increased in the PA + green-MED group (0.23 g/dL) compared with PA (-0.1 g/dL; P < 0.001) and PA + MED (-0.1 g/dL; P < 0.001). Serum iron and serum transferrin saturation increased in the PA + green-MED group compared with the PA group (8.21 μg/dL compared with -5.23 μg/dL and 2.39% compared with -1.15%, respectively; P < 0.05 for both comparisons), as did folic acid (P = 0.011). In rats, hemoglobin decreased from 15.7 to 9.4 mg/dL after 44 d of diet-induced anemia. After depletion treatment, the vehicle-treated group had a further decrease of 1.3 mg/dL, whereas hemoglobin concentrations in both FG and Mankai iso-iron treatments similarly rebounded (FG14: +10.8 mg/dL, Mankai 50: +6.4 mg/dL, Mankai 80: +7.3 mg/dL; FG9.5: +5.1 mg/dL, Mankai 50-C: +7.1 mg/dL; P < 0.05 for all vs. the vehicle group). Conclusions: In humans, a green-MED low-meat diet does not impair iron homeostasis. In rats, iron derived from Mankai (a green-plant protein source) is bioavailable and efficient in reversal of anemia. This trial was registered at clinicaltrials.gov as NCT03020186.
Article
Background Several meta-analyses evaluated the association between vegetarian diets and health outcomes. To integrate the large amount of the available evidence, we performed an umbrella review of published meta-analyses that investigated the association between vegetarian diets and health outcomes. Methods We performed an umbrella review of the evidence across meta-analyses of observational and interventional studies. PubMed, Embase, Cochrane Database of Systematic Reviews, and ISI Web of Knowledge. Additional articles were retrieved from primary search references. Meta-analyses of observational or interventional studies that assessed at least one health outcome in association with vegetarian diets. We estimated pooled effect sizes (ESs) using four different random-effect models: DerSimonian and Laird, maximum likelihood, empirical Bayes, and restricted maximum likelihood. We assessed heterogeneity using I² statistics and publication bias using funnel plots, radial plots, normal Q-Q plots, and the Rosenthal’s fail-safe N test. Results The umbrella review identified 20 meta-analyses of observational and interventional research with 34 health outcomes. The majority of the meta-analyses (80%) were classified as moderate or high-quality reviews, based on the AMSTAR2 criteria. By comparison with omnivorous diets, vegetarian diets were associated with a significantly lower concentration of blood total cholesterol (pooled ES = -0.549 mmol/L; 95% CI: -0.773 to -0.325; P < 0.001), LDL-cholesterol (pooled ES = -0.467 mmol/L; 95% CI: -0.600 to -0.335); P < 0.001), and HDL-cholesterol (pooled ES = -0.082 mmol/L; 95% CI: -0.095 to -0.069; P < 0.001). In comparison to omnivorous diets, vegetarian diets were associated with a reduced risk of negative health outcomes with a pooled ES of 0.886 (95% CI: 0.848 to 0.926; P < 0.001). In comparison to omnivores, Seventh-day Adventists (SDA) vegetarians had a significantly reduced risk of negative health outcomes with a pooled ES of 0.721 (95% CI: 0.625 to 0.832; P < 0.001). Non-SDA vegetarians had no significant reduction of negative health outcomes when compared to omnivores (pooled ES = 0.973; 95% CI: 0.873 to 1.083; P = 0.51). Vegetarian diets were associated with harmful outcomes on one-carbon metabolism markers (lower concentrations of vitamin B12 and higher concentrations of homocysteine), in comparison to omnivorous diets. Conclusions Vegetarian diets are associated with beneficial effects on the blood lipid profile and a reduced risk of negative health outcomes, including diabetes, ischemic heart disease, and cancer risk. Among vegetarians, SDA vegetarians could represent a subgroup with a further reduced risk of negative health outcomes. Vegetarian diets have adverse outcomes on one-carbon metabolism. The effect of vegetarian diets among pregnant and lactating women requires specific attention. Well-designed prospective studies are warranted to evaluate the consequences of the increased prevalence of vitamin B12 deficiency during pregnancy and infancy on later life and of trace element deficits on cancer risks. Prospero Registration Number CRD42018092470.
Article
The human gastrointestinal tract is home to an incredibly dense population of microbes. These microbes employ unique strategies to capture energy in this largely anaerobic environment. In the process of breaking down dietary- and host-derived substrates, the gut microbiota produce a broad range of metabolic products that accumulate to high levels in the gut. Increasingly, studies are revealing that these chemicals impact host biology, either by acting on cells within the gastrointestinal tract or entering circulation and exerting their effects at distal sites within the body. Given the high level of functional diversity in the gut microbiome and the varied diets that we consume, the repertoire of microbiota-derived molecules within our bodies varies dramatically across individuals. Thus, the microbes in our gut and the metabolic end products they produce represent a phenotypic lever that we can potentially control to develop new therapeutics for personalized medicine. Here, we review current understanding of how microbes in the gastrointestinal tract contribute to the molecules within our gut and those that circulate within our bodies. We also highlight examples of how these molecules affect host physiology and discuss potential strategies for controlling their production to promote human health and to treat disease. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 15 is January 24, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.