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nutrients
Article
Wolffia 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: 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 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 effect 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 different 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:
Wolffia 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].
Wolffia 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 different conditions, ranging from lab scale under artificial light
to commercial scale under sunlight, were examined for their B
12
content by two different methods.
In the DIRECT PLUS weight-loss trial, among 294 participants with abdominal obesity and normal
B
12
levels, we explored the effect 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 different 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 immunoaffinity column (EASI-EXTRACT vitamin B
12
immunoaffinity column
(AOAC 2014.02), R-Biopharm AG, Darmstadt, Germany) and purified according to the manufacturer’s
protocol. The recovery efficiency 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 effect 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. Differences between time
points were tested using the paired sample T-test or Wilcoxon test. Differences 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 effect 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 (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
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 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
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 20–1500 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 (10–14): 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
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
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 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-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 immunoaffinity 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
buffer and passed through a EASI-EXTRACT vitamin B
12
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-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
different 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 different
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 different spirulina samples, whereas no pseudo CN-B
12
was detected in
the Mankai samples (Figure 3and Figure S4).
Nutrients 2020, 9, x FOR PEER REVIEW 8 of 17
MS/MS. The results showed recovery rates of 55%, 37%, 16%, and 100% for CN-B12, OH-B12, Ado-B12,
and Me-B12, 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 B12 level was calculated according to the recovery rates.
The data showed that the average total authentic vitamin B12 concentrations in Mankai is 3.23µg ±
0.5/100 g DW and stable during different seasons: spring 2.86 µg, summer 3.84 µg, and autumn 2.99
µg/100 g DW. These concentrations are in line with the results received by the bioassay method.
3.1.5. Authentic CN-B12 and Pseudo CN-B12 in Mankai
To further study Mankai as a vitamin B12 food source, we estimated the concentrations of pseudo
B12 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 B12 [27] and therefore can be used as a reference. This measurement
was performed assuming similar ionization products for both CN-B12 and pseudo CN-B12, so the
standard CN-B12 curve was used as a reference to quantify both compounds. Based on the different
molecular masses of CN-B12 and pseudo CN-B12, the data revealed the presence of CN-B12 and pseudo
CN-B12 in a ratio 1:3 in two different spirulina samples, whereas no pseudo CN-B12 was detected in
the Mankai samples (Figure 3 and Figure S4).
Figure 3. A comparison of chromatograms of TIC for authentic CN-B12 and pseudo CN-B12 in Mankai and
spirulina samples. (A–C): Active CN-B12; (B–D) Pseudo CN-B12 in Mankai™ (A,B) and spirulina (C,D) samples.
In panel B, a peak at 2.12 min does not represent pseudo CN-B12 because pseudo CN-B12 should appear before
the peak of CN-B12 [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. DIRECT PLUS Trial 334
3.2.1. Baseline Characteristics
The baseline characteristics are presented in Table 1. The mean vitamin B12 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 B12 tertile compared with the lowest tertile (p = 0.01). Details regarding baseline vitamin
supplementation are presented in Supplementary File S6.
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 Effect 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 differ between the intervention groups
(Supplementary File S6).
Differences 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 difference 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
differences 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 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 (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 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 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 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 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 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 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 different methodologies, the presence of vitamin B
12
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 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 Wolffia 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 affinity 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 [37–41], 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 difficulties [
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 effect 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
efficacy 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 effects 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 different 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 different 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.
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