Journal of Food Composition and Analysis 109 (2022) 104464

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Biologically active or just “pseudo”-vitamin B12 as predominant form in

algae-based nutritional supplements?
Sabrina P. van den Oever , Helmut K. Mayer *
University of Natural Resources and Life Sciences, Vienna (BOKU), Department of Food Science and Technology, Institute of Food Science, Muthgasse 11/1, A-1190
Vienna, Austria

A R T I C L E I N F O A B S T R A C T

Keywords: In order to find out whether algae-based nutritional supplements contain both physiologically active vitamin B12
Vitamin B12 and its non-active pseudo-form, a simple and fast ultra-high performance liquid chromatography method with
Pseudo-vitamin B12 UV detection was developed to separate these two forms. A total of 57 commercially available microalgae
Microalgae
products based on Chlorella and Spirulina were analyzed. Results obtained showed a broad variety of the content
Chlorella
of total vitamin B12 ranging from not-detectable to 445.9 μg 100g− 1 dry matter for Chlorella samples, and be­
Spirulina
Nutritional supplements tween 92.8 and 164.1 μg 100g− 1 dry matter for Spirulina products. However, there was a big variation in the
UHPLC-PDA analysis concentration of pseudo-vitamin B12 within all samples analyzed. Chlorella products contained mainly physio­
logically active cobalamin, while pseudo-vitamin B12 was the prevailing form in Spirulina-labeled nutritional
supplements. The importance of being able to differentiate between cobalamin and its pseudo-form in algae is
not only relevant for food analysts, but also regarding consumer protection since they have to be able to rely on
the correct labeling of nutritional supplements.

1. Introduction
Among the B-vitamins, especially vitamin B12 (cobalamin) is of
utmost importance for a balanced diet and of great concern to vegans as
it is almost exclusively found in food of animal origin. Even though only
very small amounts are needed to meet the adequate intake (4 μg/day
for adults), a sufficient supply with vitamin B12 still appears challenging
nowadays, and deficiency symptoms are widespread (EFSA, 2015). The
biologically active cobalt-containing corrinoid is synthesized only by
certain bacteria and archaea – not by animals or plants – but is accu­
mulated in higher organisms in the natural food chain. Therefore,
common animal food sources of cobalamin are meat (especially liver),
milk, eggs, fish and shellfish (Bito et al., 2018; O’Leary and Samman,
2010; Watanabe, 2007). Microbial de novo biosynthesis of cobalamin
can occur via the aerobic or anaerobic pathway. Vitamin B12 is indus­
trially produced on a large scale via microbial fermentation (e.g., using
Pseudomonas denitrificans, Propionibacterium shermanii, or Sinorhizobium
meliloti, but also Escherichia coli has become important) (Fang et al.,
2017). In humans, three extracellular cobalamin-binding proteins are
involved in the uptake and transport of cobalamin (transcobalamin,
intrinsic factor, and haptocorrin). Different related native forms of
vitamin B12 (known as vitamers) exist in natural food samples having
different upper ligands such as methylcobalamin and 5′ -deoxy­
adenosylcobalamin acting as essential enzymatic cofactors for methio­
nine synthase and methylmalonyl CoA mutase within the human body,
respectively. Other vitamers are the naturally occurring hydrox­
ocobalamin or the artificial and more stable cyanocobalamin, which is
often used in dietary supplements. Both can be metabolized in order to
become useful for human beings, even though methylcobalamin is more
bioavailable than the commercially available cyanocobalamin. More­
over, the upper ligand might be replaced by water (aquocobalamin),
sulphur-containing groups (e.g., sulphitocobalamin), nitrite or halides
influencing the chemical and physical properties of cobalamin
(Kumudha et al., 2010; Madhubalaji et al., 2020; Nielsen et al., 2012).
Plant-based sources for vitamin B12 and numerous other valuable
nutrients (e.g., folate, vitamin D and E, potassium, calcium, magnesium,
iron and selenium as well as amino acids and fatty acids) have become
an important field in food sciences due to promoted health benefits as
well as trends towards an animal-free diet in many Western countries
(Raab et al., 2016). Important plant foods containing cobalamin in sig­
nificant amounts are edible macroalgae (e.g., Nori that is made from
species of the red algae genus Pyropia or Porphyra) (Kuda et al., 2005),

* Corresponding author.
E-mail address: helmut.mayer@boku.ac.at (H.K. Mayer).

https://doi.org/10.1016/j.jfca.2022.104464
Received 5 November 2021; Received in revised form 20 January 2022; Accepted 11 February 2022
Available online 12 February 2022
0889-1575/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S.P. van den Oever and H.K. Mayer
microalgae (e.g., Chlorella and Nostoc sp.) as well as the so-called
“blue-green algae” Spirulina (Arthrospira spp. from the order of Oscil­
lateriaceae). Spirulina as a single-celled organism actually belongs to
cyanobacteria but is considered as microalgae as well (Grosshagauer
et al., 2020; Gutiérrez-Salmeán et al., 2015; Jalilian et al., 2019). These
microalgae are widely used for nutritional supplements (mainly as
powder, capsules or tablets) nowadays. The largest producers of
microalgae in Europe are Germany, France, Spain and Italy (Araújo
et al., 2021; Madhubalaji et al., 2019; Ścieszka and Klewicka, 2019).
Especially Chlorella and Spirulina species are dominating the global
microalgae market as they are gaining popularity in health-food stores
(Koyande et al., 2019) since both (among others that are commercially
used in aquaculture and food supplements) do not produce any algal
toxin, and hence have “GRAS” status meaning that they are “generally
recognized as safe” by the United States Food and Drug Administration
(FDA) (Matos, 2017).
Chlorella – as unicellular alga – is commercially produced and
distributed worldwide, and has been reported to exhibit various phar­
macological effects (including immunomodulatory, antioxidant, anti­
diabetic, antihypertensive, and antihyperlipidemic activities). More
than 20 Chlorella species have been characterized, which can be divided
into three varieties: C. vulgaris, C. lobophora, and C. sorokiniana (that was
former called C. pyrenoidosa) (Bito et al., 2020; Krienitz et al., 2004).
Chlorella species and culture conditions are the main factors influencing
concentration of nutrients (especially vitamins) and bioactive com­
pounds. Chlorella cells can absorb and further accumulate exogenous
cobalamin, which stimulates their growth. Microalgae, which can not
synthesize the vitamin de novo but still require it for metabolic activities,
might obtain it via a symbiotic relationship with bacteria, since pro­
karyotes are seen as providers of trace amounts of vitamins for algal
growth in algal-bacterial symbiotic systems (Bito et al., 2016; Edelmann
et al., 2019; Helliwell et al., 2016; Ji et al., 2021; Santos and Reis, 2014).
Spirulina is the commercial name of mostly Arthrospira maxima or
Arthrospira platensis that have been promoted as “functional food” and
valuable source for high-quality protein, iron, γ-linolenic acid, vitamins
and minerals (Bai et al., 2021; Gutiérrez-Salmeán et al., 2015). More
than half of microalgal species require vitamin B12 for growth, which is
synthesized de novo by certain prokaryotes, including the majority of
cyanobacteria (Helliwell et al., 2016).
Both microalgae are rich in vitamin B12, but there might be a dif­
ference between nutritional supplements based on Chlorella or Spirulina
regarding the bioavailability (as a combination of bioactivity and bio­
accessibility) of vitamin B12 (Wells et al., 2017). Apart from the vitam­
ers, where the upper ligand differs, other variants (“analogues”) exist as
well, which are not necessarily functionally interchangeable. The lower
ligand of cobalamin is attached to the cobalt-coordinated corrin ring via
the nucleotide loop containing 5,6-dimethylbenzimidazole (DMB) as a
base. However, this lower ligand can vary as well resulting in analogues
of cobalamin. Especially cyanobacteria synthesize a form that is known
as “pseudo”-vitamin B12, where the lower ligand of the molecule is
replaced by adenine as a base (Grossmann, 2016; Kumudha et al., 2010;
Watanabe et al., 1999). Other analogues, where the lower ligand is
replaced, were identified as well over the past years such as cobinamide,
cresol, 2− CH3-adenine, 2− CH3-S-adenine, or 5− OH-benzimidazole
(Girard et al., 2009), but regarding (blue-green) algae, the lower ligand
reported is adenine. Cyanobacteria can use this pseudo-vitamin B12 as
cofactor, but it does not show any physiological activity in mammals.
However, certain microalgae seem to be able to restructure the
pseudo-form into cobalamin (Bito et al., 2018; Helliwell et al., 2016).
This change in chemical structure affects the affinity of the mammalian
B12-binding protein intrinsic factor for the compound, since intestinal
absorption of cobalamin strictly recognizes the structure of the vitamin
B12-molecule. However, it was reported that pseudo-cobalamin shows
moderate affinity to intrinsic factor (Taga and Walker, 2008; Watanabe
et al., 2002; Wells et al., 2017). Vitamin B12 synthesized by cyanobac­
teria is not seen as a reliable source for humans (Dagnelie et al., 1991;
2
Journal of Food Composition and Analysis 109 (2022) 104464
Kittaka-Katsura et al., 2002; Wells et al., 2017).
The fact that cyanobacteria produce a biologically non-active form of
vitamin B12 leads to major concerns in food science, as nutritional
supplements containing only pseudo-cobalamin could be considered
unsuitable regarding bioavailability for human beings. Since consumers
rely on the health-promoted effects of algae-based dietary supplements,
this important issue needs to be focused on in quality control as well.
Products with high contents of pseudo-cobalamin should not be adver­
tised to consumers as a labeled source of vitamin B12. However, there is
no standard procedure for the analysis of vitamin B12 and its analogues
making analytical selectivity essential in order to assess vitamin B12 in
any complex food matrix.
In general, cobalamin is considered a challenging compound for
analysis due to its complex structure as well as many possible vitamers
and different forms. Hence, false-positive results are likely caused by
pseudo-cobalamin. Microbiological assays were the first methods used
for the determination of vitamin B12 based on the requirement of
vitamin B12 for growth (e.g., using Lactobacillus delbrueckii subsp. lactis
(leichmannii)). Bacteria used in microbiological assays do not discrimi­
nate between vitamin B12 and pseudo-vitamin B12 leading to over­
estimated and false-positive results (Chaiet et al., 1954; Wells et al.,
2017), and it was reported that the AOAC-approved method may also
detect other corrinoids not bioavailable for humans (Castillejo et al.,
2017). Besides microbiological assays, spectrophotometric methods
were used. In UV and visible spectrophotometry, aqueous solutions of
cyanocobalamin exhibit maximum absorption in UV and visible region
at 278 nm, 361 nm, and 550 nm, but are influenced by solvent, tem­
perature, and pH making this method hardly suitable for accurate
determination. Other methods such as atomic absorption spectroscopy
or radioimmunoassay are also not selective for different forms of
cobalamin. Capillary electrophoresis can be used for cobalamin sepa­
ration, and differentiation between different forms of vitamin B12 was
reported as well (Karmi et al., 2011). Modern analytical methods are
often based on liquid chromatography coupled to mass spectrometry
(LC–MS or LC–MS/MS) offering very detailed information about the
actual form of vitamin B12 analyzed but requiring expensive and com­
plex devices. The correct identification of physiologically available
vitamin B12 in foods and especially in nutritional supplements has
become of major importance due to various non-bioactive analogues.
Hence, fast and reliable methods are urgently required in order to
monitor nutritional supplements to guarantee high-quality products. It
is crucial to avoid being misled by biologically non-active pseudo-forms,
and consumers have to be able to rely on the vitamin B12 concentration
labeled on dietary supplements, which highlights the need for rigorous
care in the analytical determination of the vitamin B12 content of algal
foods. Therefore, the aim of the present study was to develop a fast,
simple and reliable ultra-high performance liquid chromatography
(UHPLC) method based on UV detection to separate the physiologically
active form of vitamin B12 from the non-active pseudo-vitamin B12 that
might be present in numerous nutritional supplements. A single quad­
rupole mass spectrometer was used for correct identification of both
forms. Using the established method, the present research also aims to
extend knowledge about commercially available algae-based dietary
supplements for both food analysts and consumers.
2. Material and methods
2.1. Chemicals and standards
Reagents and chemicals were of analytical or LC–MS grade. Water
(Ultra-pure, ≥18.2 mΩ - UHQ) was provided by a SG Ultra Clear UC
System (SG-Water, Barsbüttel, Germany). Acetic acid glacial (Op­
tima® LC–MS grade) was acquired from Fischer Chemicals (Geel,
Belgium). LC–MS grade methanol was obtained from Chem-Lab NV
(Zedelgem, Belgium). Standard substances of hydroxocobalamin ≥98
from USP (Rockville, MD, USA), and cyanocobalamin ≥98 %,
S.P. van den Oever and H.K. Mayer Journal of Food Composition and Analysis 109 (2022) 104464

5′ -deoxyadenosylcobalamin ≥97 %, and methylcobalamin ≥97 %,
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Certified
reference material (ERM®-BD600, whole milk powder) for vitamin
B12 was obtained from the Joint Research Centre of the European
Commission (Geel, Belgium). Since there is no certified reference
material available yet for pseudo-cobalamin, quantification was per­
formed using the calibration curve for cyanocobalamin as suggested
by Edelmann et al. (2019). For calibration, a stock solution of
cyanocobalamin of 100 μM was completely dissolved in ultra-high
quality (UHQ) water in a light-protected flask, and used for dilution
of the standard calibration curve with the following levels: 2, 3, 4, 6,
8, 10, 12.5, 25, 50, 75, and 100 nM. The standard solution series were
measured in triplicate using the UHPLC method described in section
2.4 (calibration curve equation for cyanocobalamin: y = 121.67x
+20.146; R2 = 0.998).
2.2. Sample acquisition
A total of 57 algae-based nutritional supplements (n = 41 labeled as
Chlorella sp.; n = 14 labeled as Spirulina sp.; n = 1 labeled as containing

Table 1
1
Results of vitamin B12 and pseudo-vitamin B12 determined for all 57 algae-derived nutritional supplements analyzed (all results are given in μg 100 g− dry matter). If
indicated, the labeled vitamin B12 content is given. In addition, relevant ratios were calculated (a-d).
No Species Delivery form Pseudo-Vitamin B12 Vitamin B12 Sum B12 Labeled Ratioa Ratiob Ratioc Pseudod
content Sum/labeled B12/labeled B12/Pseudo (% of sum)
Mean ± SD Mean ± SD

1 Chlorella pyrenoidosa Capsules 4.6 0.1 115.5 0.1 120.1 77 1.6 1.5 25.1 4
2 Chlorella sp. Tablets 3.5 0.3 109.9 6.2 113.4 83 1.4 1.3 31.4 3
3 Chlorella pyrenoidosa Tablets 27.9 1.2 4.8 0.2 32.7 0.2 85
4 Chlorella vulgaris Powder n.d. 71.1 1.8 71.1
5 Chlorella sp. Tablets n.d. n.d.
6 Chlorella sp. Tablets 2.0 0.0 17.5 0.3 19.6 60 0.3 0.3 8.6 10
7 Chlorella vulgaris Tablets 3.8 0.1 121.5 2.9 125.4 31.8 3
8 Chlorella pyrenoidosa Tablets 3.9 0.2 99.4 1.2 103.3 3100 0.03 0.03 25.5 4
9 Chlorella vulgaris Tablets 34.1 1.2 367.2 9.0 401.3 420 1.0 0.9 10.8 8
10 Chlorella vulgaris Tablets 2.2 0.1 10.3 0.5 12.4 4.8 17
11 Chlorella sp. Tablets 3.0 0.2 70.9 4.1 73.8 24.0 4
12 Chlorella vulgaris Tablets 56.2 3.0 8.7 0.4 64.9 0.2 87
13 Chlorella sp. Tablets 77.1 5.3 76.4 4.8 153.5 50 3.1 1.5 1.0 50
14 Chlorella pyrenoidosa Tablets 44.2 2.9 101.8 5.7 146.0 2.3 30
15 Chlorella pyrenoidosa Tablets 28.1 1.4 406.4 10.0 434.6 14.4 6
16 Chlorella vulgaris Tablets 6.3 0.3 106.0 2.2 112.3 16.9 6
17 Chlorella vulgaris Tablets n.d. 111.4 2.6 111.4 300 0.4 0.4
18 Chlorella vulgaris Tablets 88.0 4.7 62.8 1.6 150.9 900 0.2 0.1 0.7 58
19 Chlorella vulgaris Tablets 6.9 0.1 87.1 0.2 94.1 12.6 7
20 Chlorella vulgaris Tablets 2.9 0.1 12.1 0.4 15.0 95 0.2 0.1 4.2 19
21 Chlorella sp. Tablets n.d. 15.1 0.3 15.1
22 Chlorella vulgaris Tablets 2.8 0.1 11.0 0.3 13.8 3.9 20
23 Chlorella sp. Tablets 2.1 0.0 84.5 2.0 86.5 38 2.3 2.2 41.1 2
24 Chlorella sp. Powder 25.8 0.3 96.3 1.4 122.2 3.7 21
25 Chlorella sp. Tablets n.d. 1.1 0.0 1.1
26 Chlorella sp. Powder n.d. n.d.
27 Chlorella pyrenoidosa Capsules 5.3 0.3 195.9 4.4 201.2 37.1 3
28 Chlorella sp. Powder n.d. 307.7 17.0 307.7
29 Chlorella sp. Powder 15.2 0.8 430.7 5.7 445.9 421 1.1 1.0 28.4 3
30 Chlorella sp. Tablets n.d. n.d.
31 Chlorella sp. Tablets n.d. 242.1 6.4 242.1
32 Chlorella vulgaris Powder 1.7 0.1 43.2 2.3 44.9 50 0.9 0.9 25.0 4
33 Chlorella vulgaris Tablets 52.9 2.0 144.7 4.4 197.6 50 4.0 2.9 2.7 27
34 Chlorella vulgaris Powder n.d. 124.3 4.8 124.3 100 1.2 1.2
35 Chlorella sp. Powder n.d. 96.6 3.7 96.6 150 0.6 0.6
36 Chlorella pyrenoidosa Tablets 4.4 0.2 156.6 5.5 161.0 35.2 3
37 Chlorella vulgaris Tablets 74.9 2.3 7.6 0.3 82.4 0.1 91
38 Chlorella pyrenoidosa Powder 2.4 0.1 4.3 0.2 6.7 1.8 35
39 Chlorella sp. Tablets 13.3 0.7 4.7 0.2 18.0 177 0.1 0.03 0.4 74
40 Chlorella sp. Powder n.d. 5.5 0.2 5.5 11 0.5 0.5
41 Chlorella vulgaris Tablets n.d. 74.7 2.2 74.7
42 Chlorella sp. Tonic (liquid) n.d. 2.8 0.0 2.8 17 0.2 0.2
43 Chlorella sp., Spirulina sp. Capsules 3.8 0.1 198.4 5.6 202.2 542 0.4 0.4 52.7 2
44 Arthrospira platensis Powder 142.3 5.3 8.4 0.4 150.7 0.1 94
45 Spirulina platentis Tablets 84.5 3.3 8.3 0.1 92.8 0.1 91
46 Spirulina platentis Tablets 142.6 8.9 11.0 0.9 153.7 0.1 93
47 Spirulina sp. Tablets 73.6 2.9 51.5 2.2 125.1 83 1.5 0.6 0.7 59
48 Arthrospira platensis Tablets 103.0 5.0 32.6 1.7 135.6 0.3 76
49 Arthrospira pacifica hawaii Capsules 93.3 5.1 35.1 0.4 128.4 79 1.6 0.4 0.4 73
50 Spirulina platensis Tablets 85.8 1.7 13.6 0.8 99.4 69 1.4 0.2 0.2 86
51 Spirulina platensis Powder 160.7 8.7 3.5 0.2 164.1 0.0 98
52 Spirulina platentis Tablets 117.0 5.3 6.4 0.3 123.5 0.1 95
53 Spirulina platentis Tablets 130.6 4.1 17.0 0.8 147.6 0.1 88
54 Spirulina platentis Powder 101.6 2.0 6.4 0.2 108.0 0.1 94
55 Spirulina sp. Powder 140.4 0.2 11.6 0.1 152.0 0.1 92
56 Spirulina platentis Tablets 134.7 3.2 19.1 0.3 153.8 0.1 88
57 Aphanizomenon flos-aquae Tablets 118.4 6.8 8.3 0.6 126.8 140 0.9 0.1 0.1 93
a
Ratio of sum (pseudo-vitamin B12 and vitamin B12 determined) to labeled B12-content; b Ratio of physiologically active vitamin B12 to labeled B12-content; c Ratio of
physiologically active vitamin B12 to its pseudo-form; d Relative percentage of pseudo-vitamin B12 (as % of the total content found); n.d. = not-detectable.

3
S.P. van den Oever and H.K. Mayer
Chlorella and Spirulina sp.; and n = 1 labeled as another cyanobacteria
sp.) were bought from local retail stores (Vienna, Austria) as well as
online (Table 1).
2.3. Sample preparation
All samples of various nutritional supplements were stored at room
temperature (dry, avoidance of light) as suggested on the packaging
until chemical analysis. Since vitamin B12 is sensitive to light, all oper­
ations were conducted using amber glassware, covered tubes using
aluminum foil or under subdued light. Sample preparation was per­
formed according to the manufacturer’s instructions of the purification
kit (EASI EXTRACT®, R-Biopharm; Glasgow, UK) as well as similar to
Nakos et al. (2017) with modifications necessary for these nutritional
supplements. In case of solid samples, a representative aliquot was
ground using a ceramic mortar, and homogenized prior to weighing in
0.5− 5 g (depending on the expected concentration of vitamin B12 or
adapted after preliminary tests) into a laboratory glass bottle, and mixed
with 60 mL of sodium acetate buffer (50 mM) adjusted to pH 4.0. In
order to free protein-bound vitamin B12, enzymatic digestion was per­
formed mixing pepsin (1 g) and taka-diastase (0.25 g) thoroughly into the
samples. To convert all possible vitamers into cyanocobalamin, 1 mL of
potassium cyanide solution (1% w/v) was added to the samples and
mixed. Samples were then incubated for 1 h at 37 ◦ C under continuous
shaking on a Heidolph Unimax 1000 (Walpersdorfer, Germany) for
enzymatic digestion as well as conversion of B12-vitamers into the stable
cyano-form. Enzymatic reaction was stopped by incubating samples for
30 min at 120 ◦ C in a Heraeus Oven (Hanau, Germany). After cooling to
room temperature, samples were transferred quantitatively into 100 mL
volumetric flasks, and brought to volume with 50 mM sodium acetate
buffer. Afterwards, samples were centrifuged at 8000 x g for 10 min. The
supernatant was filtered through a Whatman S&S 595 ½ filter paper
(Maidstone, UK). Clear sample solutions were stored (under light pro­
tection) between 3 and 6 days at room temperature prior to purification
procedure, since a slight increase in concentration of cyanocobalamin
was observed compared to no storage time of the extract.
Regarding nutritional supplements consisting of capsules with
powdered vitamin B12, a representative aliquot was homogenized prior
to weighing in, and for liquid samples, an aliquot of 2− 10 mL was used,
all other steps were conducted as described above.
In contrast to sample preparation by Nakos et al. (2017), the pH
value of sample solutions was not adjusted to pH 7 since no difference
between pH 4 and pH 7 could be determined for cyanocobalamin.
However, for pseudo-cyanocobalamin major differences were observed
showing far lower concentrations found at pH 7 compared to pH 4.
Therefore, no pH adjustment was performed since also manufacturer’s
instructions (R-Biopharm) do not suggest change in pH during sample
preparation.
Purification was performed using EASI EXTRACT® immunoaffinity
columns (R-Biopharm, Glasgow, UK), which were acclimated to ambient
conditions before use. The immunoaffinity column was attached to the
corresponding adapter, and column storage buffer was removed with a
flow rate of 2 droplets per second. Ten milliliters of filtered sample so­
lution were passed through immunoaffinity column with a flow rate of 1
droplet per second. A slow and steady flow rate is essential to ensure
adequate capture of the vitamin by the antibody. The column was
washed by passing through 10 mL of UHQ water (2 droplets per second)
and completely dried by using enhanced air pressure. For elution of
cyanocobalamin, 5 mL methanol (LC–MS grade) were used with a flow
rate of 1 droplet per second, and eluted vitamin was collected in an
appropriate tube with screw cap. Elution efficiency of 5 mL methanol is
above 96 % (Nakos et al., 2017). During elution, back-flushing was
performed several times as well as resting of 1 min to increase contact
time of solvent with the antibody to ensure complete elution of the
vitamin present in the sample. Afterwards, the eluate was dried over­
night at 40 ◦ C under reduced pressure using a SpeedVac Concentrator
4
Journal of Food Composition and Analysis 109 (2022) 104464
(SVC-200H, Savant Instruments Inc., Farmingdale, NY, USA). Dried
residue was reconstituted in 500 μL of solvent A (UHQ water modified
with 0.1 % acetic acid) containing 6% of methanol, and filtered through
a 0.20 μm syringe filter (Sartorius, Goettingen, Germany) prior to
chromatographic analysis.
2.4. UHPLC analysis
Chromatographic analysis of vitamin B12 was performed on a Waters
Acquity™ Ultra Performance LC (UPLC™) H-class system (Waters,
Milford, MA, USA) equipped with an Acquity™ photodiode array (PDA)
eλ detector for quantification as well as an Acquity™ QDa single
quadrupole mass detector for correct identification of different forms.
For separation of cyanocobalamin and pseudo-cyanocobalamin, an
Acquity UPLC™ column (BEH C18, 50 × 2.1 mm i.d., 1.7 μm) was used
with gradient elution. The applied solvent system consisted of UHQ
water modified with 0.1 % acetic acid (solvent A), and LC–MS grade
methanol with 0.1 % acetic acid (solvent B). Gradient elution was
operated at 45 ◦ C with a flow rate of 0.65 mL min− 1 as follows: ini­
tial–3.0 min/2–20 % B; 3.0–4.0 min/20–20 % B; 4.0–5.0 min/20–100 %
B; 5.0–7.0 min/100–100 % B; 7.0–7.5 min/100–2% B; and further re-
equilibration at initial conditions for another 3 min. Injection volume
was set to 50 μL. Column eluates were monitored using PDA detector at
361 nm for (pseudo-)cyanocobalamin at a resolution of 1.2 nm and a
sampling rate of 40 points/s. QDa parameters for correct identification
of different forms of vitamin B12 were as follows: sampling rate of 20
points/s, electro spray ionization (ESI) in positive ion mode, cone
voltage: 15 V, capillary voltage: 0.8 kV, probe temperature 600 ◦ C in
selected ion recording (SIR) with m/z 678.2914 for cyanocobalamin,
and m/z 672.7749 for pseudo-cyanocobalamin both as [M + 2 H]2+
(Tanioka et al., 2014). Chromatographic data were collected and pro­
cessed using Waters Empower™ software version 3.
2.5. Method validation
The developed method was validated following the EURACHEM
guide (Magnusson and Örnenmark, 2014). Precision was assessed as
repeatability (n = 6, over one day) and intermediate precision over four
days (n/d = 3). In addition, precision of total sample preparation pro­
cedure was evaluated by preparing the same sample six times over a
period of two days (n/d = 3). For assessment of limit of detection (LOD =
s’0*3) and limit of quantification (LOQ = s’0*10), low concentration
standards were used (n = 10). The estimated deviation (s’0) was calcu­
lated according to EURACHEM guide (s’0 = s0/√n). Method accuracy
was assessed using certified reference material (ERM-BD600, dried milk
powder) over 3 non-consecutive days (n/d = 2) using 2 operators and
changed solvents.
3. Results and discussion
3.1. Performance of the established UHPLC-PDA method
In order to separate the physiologically active form of vitamin B12
from the non-active pseudo-vitamin B12 that might be present in
numerous nutritional supplements, a reliable UHPLC method based on
UV detection was established (Fig. 1).
Precision of the chromatographic system was measured by evalu­
ating repeatability and intermediate precision. Repeatability was
assessed by consecutive injections of cyanocobalamin standard solutions
over one day, which was found to be ≤ 0.9 %. Intermediate precision
was assessed over four days (n/d = 3), and was found to be ≤ 1.4 %.
Precision of the total sample preparation procedure using selected
nutritional supplements (digestion of sample, conversion of B12-vitam­
ers into the cyano-form, purification by immunoaffinity columns, drying
using SpeedVac system, and reconstitution prior to injection) was
evaluated by preparing the same sample six times over a period of two
S.P. van den Oever and H.K. Mayer
Fig. 1. UHPLC-PDA chromatograms of three selected algae-derived nutritional supplements showing different ratios of cobalamin to pseudo-cobalamin in Chlorella
No 41 (a), Spirulina No 54 (b), and Chlorella No 18 (c).
days (n/d = 3), and resulted in ≤ 4.3 %. Determination of limit of
detection (LOD) and limit of quantification (LOQ) using PDA detection
signals were 0.36 nM for LOD, and 1.19 nM for LOQ. Standard cali­
bration curve was established between 2 and 100 nM having a value for
R2 of 0.99. Method accuracy was performed using certified reference
material (ERM-BD600, dried milk powder) over 3 non-consecutive days
(n/d = 2) using 2 operators and changed solvents showing values be­
tween 98.1 % and 101.4 %. Chromatographic separation of three
selected algae-derived nutritional supplements using the established
UHPLC-PDA method are shown in Fig. 1a-c. Chlorella No 41 (a) con­
tained only physiologically active cobalamin vitamin B12, whereas
Spirulina No 54 (b) had almost exclusively the pseudo-form (with only a
minor content of cobalamin). However, several algae-derived nutri­
tional supplements such as Chlorella No 18 (c) showed both forms in
different proportions.
3.2. Vitamin B12 in Chlorella- and Spirulina-based nutritional
supplements
Algae-based nutritional supplements can be a valuable source for
vitamin B12, but they do not need to contain cobalamin at all. In contrast
to the findings of Sandgruber et al. (2021), where vitamin B12 was below
LOQ in all 15 commercial microalgae powders, it was present in almost
all of the 57 samples in this study, even though no content was labeled
on several nutritional supplement since many algae-derived dietary
supplements are advertised for other essential nutrients such as amino
acids, fatty acids, minerals or other vitamins. Results obtained for all
algae-based nutritional supplements analyzed showed a broad variety of
different contents of total vitamin B12 ranging between not-detectable
(No 5) and 445.9 μg 100g− 1 (No 29). The results of all 57 algae-based
nutritional supplements analyzed within the present study are shown
in Table 1 given as μg 100 g− 1 dry weight. Furthermore, the analyzed
amounts of nutrients were compared to those provided by the infor­
mation label on the products.
Regarding Chlorella samples, 18 of the 41 nutritional supplements
analyzed had labeled contents for vitamin B12, while only 3 of the 13
Spirulina samples had an indicated value. Furthermore, one nutritional
supplement contained both Chlorella and Spirulina sp., and one other
cyanobacteria species (Aphanizomenon flos-aquae) was analyzed.
3.2.1. Comparison of the concentration found in selected Chlorella samples
with the labeled content of vitamin B12
Results obtained for Chlorella samples showed both lower and higher
5
Journal of Food Composition and Analysis 109 (2022) 104464
concentrations of vitamin B12 found compared to the content labeled.
However, several samples had comparable results as well showing the
big variation regarding the total content in the nutritional supplements
analyzed (Table 1).
Lower concentrations were found in some Chlorella samples with
values below half of the content compared to the total amount of vitamin
B12 labeled (Table 1, columna). In Chlorella No 39, only one-tenth of the
indicated value on the product were determined with 18 μg 100g− 1 of
total vitamin B12 compared to the labeled concentration of 177 μg
100g− 1. Approximately half of the labeled content could be determined
for sample No 40, where 5.5 μg 100g− 1 instead of 11 μg 100g− 1 were
found. A few other Chlorella-based nutritional supplements had values
between one-tenth and half of the labeled content (such as No 6, 17, 18,
20, 42, and 43). However, there was one exception, where only
approximately three-hundredths of the labeled value were determined,
since Chlorella No 8 should have an extremely high amount of total
vitamin B12 of 3100 μg 100g− 1, while only 103.3 μg 100g− 1 could be
determined. Regarding not only the ratio of total vitamin B12 found to
the content labeled, but also the ratio of only physiologically active
vitamin B12 to the indicated amount, similar results were obtained for
most of the samples described above, since these products contained
almost only or exclusively the biologically available form (Table 1,
columnb). Merely samples No 18 and No 39 showed values of only 0.07
and 0.03, respectively, when only physiologically active cobalamin is
compared to the total content labeled on the nutritional supplement
(due to higher amounts of the pseudo-form present in these two
samples).
In contrast, in some Chlorella samples a higher content of vitamin B12
was determined compared to the labeled value on the product (Table 1,
columna). For example, more than 1.3-times of the amount of total
vitamin B12 labeled was found for No 1 and No 2, while No 23 showed
more than double of the content labeled. A more than 3-times higher
concentration was determined for Chlorella products No 13 and No 33
with values of 153.5 μg 100g− 1 (instead of 50 μg 100g− 1 as labeled), and
197.6 μg 100g− 1 (instead of 50 μg 100g− 1 as labeled), respectively.
Interestingly, the ratio of only physiologically active cobalamin to the
content labeled was only 1.5-times higher for sample No 13 resulting in
comparable results, since 76.4 μg 100g− 1 of available vitamin B12 were
determined instead of the labeled 50 μg 100g− 1. In this sample, pseudo-
cyanocobalamin and cyanocobalamin were present to the same extent.
Other samples showed comparable results regarding the ratio of total
vitamin B12 found to the content labeled with values between 0.9 and
1.2 (e.g., No 9, 29, 32, and 34). Similar results were observed concerning
S.P. van den Oever and H.K. Mayer
the ratio of available vitamin B12 determined to the content labeled for
these Chlorella-based nutritional supplements (Table 1, columnb). For
example, No 29 showed a total content of vitamin B12 of 445.9 μg
100g− 1, whereof 430.7 μg 100g− 1 were determined to be physiologically
active cobalamin matching the labeled content of 421 μg 100g− 1.
3.2.2. Comparison of the concentration found in selected Spirulina samples
with the labeled content of vitamin B12
Results obtained for Spirulina samples showed a higher concentration
of vitamin B12 found for all three samples, where a content was labeled
on the nutritional supplement (No 47, 49, and 50) (Table 1). Regarding
these samples, between 1.4-times and 1.6-times higher concentrations
were analyzed with a total content of 125.1 μg 100g− 1 (instead of 83 μg
100g− 1 as labeled), 128.4 μg 100g− 1 (instead of 79 μg 100g− 1 as
labeled), and 99.4 μg 100g− 1 (instead of 69 μg 100g− 1 as labeled) for No
47, 49, and 50, respectively (Table 1, columna). However, regarding
only the ratio of physiologically active cobalamin to the content labeled,
only between 0.2 and 0.6 were found since far lower concentrations of
vitamin B12 were determined compared to its pseudo-form (Table 1,
columnb) meaning that Spirulina products showed significant higher
concentrations for pseudo-cyanocobalamin than for the respective value
of cyanocobalamin.
Apart from Spirulina, one other cyanobacteria species was analyzed
(Aphanizomenon flos-aquae). Sample No 57 had a ratio of total cobalamin
to the content labeled of 0.9, since 126.8 μg 100g− 1 were found coming
close to the indicated value on the product of 140 μg 100g− 1. However,
the ratio of physiologically active cobalamin to the content labeled was
<0.1, since only 8.3 μg 100g− 1 were determined.
3.2.3. Comparison of Chlorella and Spirulina samples
Interestingly, in only three Chlorella samples analyzed, no vitamin
B12 could be detected at all (No 5, 26 and 30) (Table 1). All three were
labeled as Chlorella, but no further species was stated on the original
product. In 11 Chlorella samples (No 4, 17, 21, 25, 28, 31, 34, 35, 40, 41
and 42), only physiologically active vitamin B12 was found. Among the
nutritional supplements labeled as Spirulina, all 13 samples analyzed
contained both cobalamin and pseudo-cobalamin showing a high con­
tent of total vitamin B12 as well. However, quite high concentrations of
the primarily present pseudo-form were determined. Besides the broad
variation of total vitamin B12 content in investigated algae-based
nutritional supplements ranging between not-detectable and 445.9 μg
100g− 1, there are also significant differences with regard to the distri­
bution and ratio of vitamin B12 to its pseudo-form.
Chlorella sample No 13 had a ratio of 1, because both forms had
approximately the same concentration. Samples No 1, 2, 7, 8, 11, 23, 27,
29, 32 and 36 showed a positive ratio of vitamin B12 to pseudo-vitamin
B12 with a more than 20- to even 40-fold higher content of the biolog­
ically available form (Table 1, columnc). The highest ratio of vitamin B12
to its pseudo-form was observed for the mixture of Chlorella and Spir­
ulina powder (No 43) with a more than 50-fold higher concentration of
available cobalamin. In contrast, some samples had a higher content of
pseudo-cobalamin, and hence a ratio <1 such as Chlorella No 3, 12 and
37 showing a significantly higher concentration of the pseudo-form.
Similar results were observed for samples No 18 and 39. These sam­
ples might not be a valuable source of vitamin B12 for humans.
Regarding Spirulina samples, the ratio of available vitamin B12 to
pseudo-vitamin B12 was between approx. 0.1 and 0.4 for samples
analyzed meaning that many Spirulina products (e.g., No 44, 45, 46, 51,
52, 54 and 55 as well as No 57) had a more than 10-fold higher con­
centration of pseudo-vitamin B12 to vitamin B12. One sample (No 47)
showed almost equal amounts having a ratio of available cobalamin to
the pseudo-form of 0.7 (Table 1, columnc).
Apart from the ratio of vitamin B12 to its pseudo-form, the relative
percentage of pseudo-vitamin B12 of the total content showed a big
variation for Chlorella-based nutritional supplements analyzed (Table 1,
columnd). In 19 products, the relative percentage of pseudo-cobalamin
6
Journal of Food Composition and Analysis 109 (2022) 104464
was <20 % (e.g., No 1, 2, 6–11, …), while 4 samples showed a high
concentration of the pseudo-form, where more than 70 % of the pseudo-
form were found with regard to the total content analyzed (No 3, 12, 37
and 39). The remaining Chlorella products had values between 20 % and
70 % of pseudo-cobalamin.
It has been reported that the major and minor analogues in Spirulina
were identified as pseudo-vitamin B12 and vitamin B12 with 83 % and 17
%, respectively (Watanabe et al., 1999). These findings are in accor­
dance with the results obtained in the present study although a broader
range was observed. The relative percentage of the pseudo-form was
between 72.6 % (No 49) and 97.9 % (No 51) of total cobalamin found,
with only one exception (No 47), where pseudo-cobalamin and cobal­
amin where almost equally distributed (Table 1, columnd).
There is a huge range regarding the total content of vitamin B12 in
commercially available Chlorella products between not-detectable and
500 μg 100g− 1 (Bito et al., 2020), which is consistent with the findings in
this study. For Spirulina, literature reported values between 127 and 244
μg 100g− 1 (Grosshagauer et al., 2020; Gutiérrez-Salmeán et al., 2015;
Watanabe, 1999), which is comparable to the results obtained as well.
Among Chlorella species, the vitamin B12 content is higher in
C. pyrenoidosa compared to C. vulgaris when grown under open culture
conditions (Bito et al., 2020). This was not necessarily the case in the
present study, but reliable information about exact conditions during
growth is rare and hard to gain. Also mislabeling of the actual Chlorella
species might be a problem since recent research has shown that
microalgae is far more diverse than supposed indicating that N-gly­
can-based characterization of microalgae might raise the attention of
taxonomists to overthink this topic (Mócsai et al., 2021).
A major problem in the production of homogeneous lots, and thus
algae-based nutritional supplements with a constant content of valuable
components labeled, is the seasonal variation and different coastal en­
vironments, as well as a variation in the nutritional and functional
composition of algae between species, which becomes important when
more than one species is added to the final product (Wells et al., 2017).
Strong deviations from the nutrients presented on the label information
of microalgae powder and the content analyzed were observed by Edel­
mann et al. (2019) and Sandgruber et al. (2021) as well. Variations in the
nutrient profile within the same species, but coming from different pro­
ducers, again highlight the dependency of nutrients on the available
external vitamin B12 in the cultivation process as well as further pro­
cessing of microalgae. Hence, consumers will probably not get the same
nutrients of the same microalgae species when purchasing the same
microalgae powder from a different producer. Possible additional rinsing
of the biomass might also affect the concentrations of certain nutrients in
the final product (Sandgruber et al., 2021). Moreover, further reduction
of certain heat-labile components might occur during the drying process,
which was also unknown for the studied microalgae powders. Especially
the physiologically active and naturally occurring vitamers of cobalamin
methylcobalamin and 5′ -deoxyadenosylcobalamin are far less heat-stable
compared to the cyano-form leading to a possible further reduction
during drying of the biomass. Another big problem with vitamin B12 is
the huge light sensitivity of methylcobalamin and 5′ -deoxyade­
nosylcobalamin due to chemical instability resulting in degradation of
valuable B12-vitamers, if this is not considered during processing steps as
well as during storage (Chamlagain et al., 2015). All these factors remain
unknown when purchasing commercially available Chlorella and Spir­
ulina products. Furthermore, overestimation of microbiological assays in
routine analysis occurs, since it was not able to discriminate the active
cobalamin form from non-active forms. Another aspect that needs to be
considered as well might be a possible “contamination” of microalgae
with (pseudo-)vitamin B12-producing bacteria (not only cyanobacteria
such as Spirulina sp.) resulting in a certain “impure” algal biomass or
extracts, especially when it comes to the presence of pseudo-forms
(Sandgruber et al., 2021).
Furthermore, there is no uniform regulation for commercially
available microalgae, and limitations regarding correct labeling of the
S.P. van den Oever and H.K. Mayer
content of vitamin B12 on algae-derived nutritional supplements come
from quantifying the bioavailability meaning that pseudo-forms need to
be quantified accurately as well (Wells et al., 2017). Even in Spirulina
samples, the presence of physiologically active methylcobalamin was
reported (Kumudha et al., 2010; Madhubalaji et al., 2019). Producers of
dietary supplements with high amounts of labeled vitamin B12 should
have their products analyzed for the presence of the pseudo-form. This
information should be provided on the product as well. It is crucial that
consumers are not misled by wrong information on nutritional supple­
ments since vitamin B12 is still a key nutrient in a healthy diet due to
modern trends regarding meat-less diets. Therefore, if pseudo-vitamin
B12 is present, it must not be counted for the total vitamin B12 content
labeled. Only the concentration of physiologically active cobalamin
should be declared on the final product. Due to huge variations
regarding the content of vitamin B12 in algae-derived nutritional sup­
plements, it is essential to have uniform rules for quality control, and to
establish new methods for routine analysis that can reliably differentiate
cobalamin from its pseudo-form in order to have high-quality products
as well as to protect consumers. However, no information about the
actual analytical method used to determine the total content of vitamin
B12 of these dietary supplements is available, which would be essential
for further investigation. Nevertheless, it is clear that there is substantial
evidence for algae as nutritional and functional foods (Wells et al.,
2017). Further studies focusing on the comparability of microbiological
assays and chromatographic data should be considered.
4. Conclusions
In order to find out whether commercially available algae-based
nutritional supplements contain the pseudo-form of vitamin B12, a
rapid UHPLC method has been developed for the chromatographic
separation of pseudo-cyanocobalamin and cyanocobalamin. Even
though, mass spectrometry was used for correct identification, quanti­
tation was done using UV signals resulting in a simple and reliable
UHPLC method for analysis of vitamin B12 and its pseudo-form.
Results obtained from 57 commercially available algae-based nutri­
tional supplements showed a broad variety of different contents of total
vitamin B12 ranging between not-detectable and 445.9 μg 100g− 1 for
Chlorella samples, and between 92.8 and 164.1 μg 100g− 1 for Spirulina
products. But besides the big range regarding the total content of
vitamin B12 analyzed, there was a big deviation in the concentration of
pseudo-vitamin B12 within all samples analyzed. Chlorella products
contained mainly and in higher quantities active vitamin B12 with some
exceptions, where a higher concentration of the pseudo-form was found.
In contrast, Spirulina-labeled nutritional supplements contained high
amounts of pseudo-vitamin B12 and only minor amounts of active
cobalamin based on the total content. The relative percentage of pseudo-
cobalamin was between 72.6 and 97.9 % with regard to Spirulina-
products analyzed.
This huge range regarding total content of vitamin B12 as well as
different concentration regarding pseudo-cobalamin highlight the
importance of further investigation since overestimation by microbio­
logical assays is very likely as reported by other authors as well. This
leads to the need for a consistent, reliable and accurate, but still also
simple and fast analytical method for the determination of cobalamin in
algae-derived nutritional supplements as well as other food samples that
can not only quantitate the total amount of vitamin B12, but also sepa­
rate different forms of vitamin B12. The importance of being able to
differentiate between cobalamin and its pseudo-form in algae as well as
other possible B12-analogues is not only relevant for food analysts but
also regarding consumer protection since they have to be able to rely on
the labeling of nutritional supplements. Unintentionally false claims
about excessive vitamin B12 levels should be avoided. Products with
high contents of pseudo-cobalamin should not be advertised to con­
sumers as a labeled source of vitamin B12. Hence, the present study
should offer a basis for further research as well as a review and possible
7
Journal of Food Composition and Analysis 109 (2022) 104464
change in routine analysis regarding the complexity of the analysis of
vitamin B12 especially regarding algae-based nutritional supplements.
CRediT authorship contribution statement
Sabrina P. van den Oever: Conceptualization, Methodology,
Formal analysis, Investigation, Validation, Data curation, Software,
Writing - original draft. Helmut K. Mayer: Conceptualization, Meth­
odology, Project administration, Supervision, Writing - review &
editing.
Declaration of Competing Interest
Both authors declare that they have no competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
Acknowledgements
This research project was partially funded by the “Hochschulju­
biläumsstiftung” Vienna (H-265139/2020) supporting young re­
searches. We want to thank Prof. Friedrich Altmann from the Institute of
Biochemistry (University of Natural Resources and Life Sciences, Vienna
- BOKU) for providing numerous Chlorella samples, which have
massively extended this research project. We are also very grateful to
supporting staff members of our Food Chemistry Laboratory (Iris Bie­
dermann, Nicole Schamberger and Sarah Sperrer) for their skillful
technical assistance.
References
Araújo, R., Vázquez Calderón, F., Sánchez López, J., Azevedo, I.C., Bruhn, A., Fluch, S.,
Garcia Tasende, M., Ghaderiardakani, F., Ilmjärv, T., Laurans, M., Mac Monagail, M.,
Mangini, S., Peteiro, C., Rebours, C., Stefansson, T., Ullmann, J., 2021. Current status
of the algae production industry in Europe: an emerging sector of the blue
bioeconomy. Front. Mar. Sci. 7, 626389.
Bai, R., Su, P., Zhen, G., Nguyen, T.T., Diao, Y., Heimann, K., Zhang, W., 2021. An
efficient protein isolation process for use in Limnospira maxima: a biorefinery
approach. J. Food Compos. Anal. 104, 104173.
Bito, T., Bito, M., Asai, Y., Takenaka, S., Yabuta, Y., Tago, K., Ohnishi, M., Mizoguchi, T.,
Watanabe, F., 2016. Characterization and quantitation of vitamin B12 compounds in
various Chlorella supplements. J. Agric. Food Chem. 64, 8516–8524.
Bito, T., Tanioka, Y., Watanabe, F., 2018. Characterization of vitamin B12 compounds
from marine foods. Fish. Sci. 84, 747–755.
Bito, T., Okumura, E., Fujishima, M., Watanabe, F., 2020. Potential of Chlorella as a
dietary supplement to promote human health. Nutrients 12, 2524–2545.
Castillejo, N., Martínez-Hernández, G.B., Goffi, V., Gómez, P.A., Aguayo, E., Artésa, F.,
Artés-Hernándeza, F., 2017. Natural vitamin B12 and fucose supplementation of
green smoothies with edible algae and related quality changes during their shelf life.
J. Sci. Food Agric. 98, 2411–2421.
Chaiet, L., Miller, T., Boley, E.A., 1954. Vitamin assay, determination of vitamin B12
content of feed supplements and effect of pseudo-vitamin B12. Agric. Food Chem. 2,
784–786.
Chamlagain, B., Edelmann, M., Kariluoto, S., Ollilainen, V., Piironen, V., 2015. Ultra-
high performance liquid chromatographic and mass spectrometric analysis of active
vitamin B12 in cells of Propionibacterium and fermented cereal matrices. Food Chem.
166, 630–638.
Dagnelie, P.C., van Staveren, W.A., van den Berg, H., 1991. Vitamin B-12 from algae
appears not to be bioavailable. Am. J. Clin. Nutr. 53, 695–697.
Edelmann, M., Aalto, S., Chamlagain, B., Kariluoto, S., Piironen, V., 2019. Riboflavin,
niacin, folate and vitamin B12 in commercial microalgae powders. J. Food Compos.
Anal. 82, 103226.
EFSA, 2015. European Food Safety Authority- panel on dietetic products, nutrition and
allergies. Scientific opinion on dietary reference values for cobalamin (vitamin B12).
EFSA J. 13, 4150.
Fang, H., Kang, J., Zhang, D., 2017. Microbial production of vitamin B12: a review and
future perspectives. Microb. Cell Fact. 16, 15–28.
Girard, C.L., Santschi, D.E., Stabler, S.P., Allen, R.H., 2009. Apparent ruminal synthesis
and intestinal disappearance of vitamin B12 and its analogs in dairy cows. J. Dairy
Sci. 92, 4524–4529.
Grosshagauer, S., Kraemer, K., Somoza, V., 2020. The true value of Spirulina. J. Agric.
Food Chem. 68, 4109–4115.
Grossmann, A., 2016. Nutrient Acquisition: the generation of bioactive vitamin B12 by
microalgae. Curr. Biol. 26, 319–337.
Gutiérrez-Salmeán, G., Fabila-Castillo, L., Chamorro-Cevallos, G., 2015. Nutritional and
toxicological aspects of Spirulina (Arthrospira). Nutr. Hosp. 32, 34–40.
S.P. van den Oever and H.K. Mayer
Helliwell, K.E., Lawrence, A.D., Holzer, A., Kudahl, U.J., Sasso, S., Kräutler, B.,
Scanlan, D.J., Warren, M.J., Smith, A.G., 2016. Cyanobacteria and eukaryotic algae
use different chemical variants of vitamin B12. Curr. Biol. 26, 999–1008.
Jalilian, N., Najafpour, G.D., Khajouei, 2019. Enhanced vitamin B12 production using
Chlorella vulgaris. Int. J. Eng. – Trans. A: Basics 32, 1–9.
Ji, X., Luo, X., Zhang, J., Huang, D., 2021. Effects of exogenous vitamin B12 on nutrient
removal and protein expression of algal-bacterial consortium. Environ. Sci. Pollut.
Res. - Int. 28, 15954–15965.
Karmi, O., Zayed, A., Baraghethi, S., Qadi, M., Ghanem, R., 2011. Measurement of
vitamin B12 concentration: a review on available methods. The IIOAB Journal 2,
23–32. ISSN: 0976-3104.
Kittaka-Katsura, H., Fujita, T., Watanabe, F., Nakano, Y., 2002. Purification and
characterization of a corrinoid compound from Chlorella tablets as an algal health
food. J. Agric. Food Chem. 50, 4994–4997.
Koyande, A.K., Chew, K.W., Rambabu, K., Tao, Y., Chu, D.-T., Show, P.-L., 2019.
Microalgae: a potential alternative to health supplementation for humans. Food Sci.
Hum. Wellness 8, 16–24.
Krienitz, L., Hegewald, E.H., Hepperle, D., Huss, V.A.R., Rohr, T., Wolf, M., 2004.
Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chloropyta,
Trebouxiophyceae). Phycologia 43, 529–542.
Kuda, T., Tsunekawa, T., Goto, H., Araki, Y., 2005. Antioxidant properties of four edible
algae harvested in the Noto Peninsula, Japan. J. Food Compos. Anal. 18, 625–633.
Kumudha, A., Kumar, S.S., Thakur, M.S., Ravishankar, G.A., Sarada, R., 2010.
Purification, identification, and characterization of methylcobalamin from Spirulina
platensis. J. Agric. Food Chem. 58, 9925–9930.
Madhubalaji, C.K., Rashmi, V., Chauhan, V.S., Shylaja, M.D., Sarada, R., 2019.
Improvement of vitamin B12 status with Spirulina supplementation in Wistar rats
validated through functional and circulatory markers. J. Food Biochem. 43, e13038.
Madhubalaji, C.K., Rashmi, V., Chauhan, V.S., Sarada, R., 2020. Improvement in vitamin
B12 status of Wistar rats by supplementing the diet with Chlorella vulgaris biomass.
J. Food Sci. Technol. 58, 4270–4281.
Magnusson, B., Örnenmark, U., 2014. Eurachem Guide: the Fitness for Purpose of
Analytical Methods – A Laboratory Guide to Method Validation and Related Topics
(2nd Ed.). ISBN: 978-91-87461-59-0.
Matos, A.P., 2017. The impact of microalgae in food science and technology. J. Am. Oil
Chem. Soc. 94, 1333–1350.
Journal of Food Composition and Analysis 109 (2022) 104464
Mócsai, R., Kaehlig, H., Blaukopf, M., Stadlmann, J., Kosma, P., Altmann, F., 2021. The
structural difference of isobaric N-glycans of two microalgae samples reveals
taxonomic distance. Front. Plant Sci. 12, 643249.
Nakos, M., Pepelanova, I., Beutel, S., Krings, U., Berger, R.G., Scheper, T., 2017. Isolation
and analysis of vitamin B12 from plant samples. Food Chem. 216, 301–308.
Nielsen, M.J., Rasmussen, M.R., Andersen, C.B.F., Nexø, E., Moestrup, S.K., 2012.
Vitamin B12 transport from food to the body’s cells – a sophisticated, multistep
pathway. Nat. Rev. Gastroenterol. Hepatol. 9, 345–354.
O’Leary, F., Samman, S., 2010. Vitamin B12 in health and disease. Nutrients 2, 299–316.
Raab, A., Stiboller, M., Gajdosechova, Z., Nelsonc, J., Feldmann, J., 2016. Element
content and daily intake from dietary supplements (nutraceuticals) based on algae,
garlic, yeast fish and krill oils – Should consumers be worried? J. Food Compos.
Anal. 53, 49–60.
Sandgruber, F., Gielsdorf, A., Baur, A.C., Schenz, B., Müller, S.M., Schwerdtle, T.,
Stangl, G.I., Griehl, C., Lorkowski, S., Dawczynski, C., 2021. Variability in macro-
and micronutrients of 15 commercially available microalgae powders. Mar. Drugs
19, 310–332.
Santos, C.A., Reis, A., 2014. Microalgal symbiosis in biotechnology. Appl. Microbiol.
Biotechnol. 98, 5839–5846.
Ścieszka, S., Klewicka, E., 2019. Algae in food: a general review. Crit. Rev. Food Sci.
Nutr. 59, 3538–3547.
Taga, M.E., Walker, G.C., 2008. Pseudo-B12 joins the cofactor family. J. Bacteriol. 190,
1157–1159.
Tanioka, Y., Takenaka, S., Furusho, T., Yabuta, Y., Nakano, Y., Watanabe, F., 2014.
Identification of vitamin B12 and pseudovitamin B12 from various edible shellfish
using liquid chromatography–electrospray ionization/tandem mass spectrometry.
Fish. Sci. 80, 1065–1071.
Watanabe, F., 2007. Vitamin B12 sources and bioavailability. Exp. Biol. Med. 232,
1266–1274.
Watanabe, F., Katsura, H., Takenaka, S., Fujita, T., Abe, K., Tamura, Y., Nakatsuka, T.,
Nakano, Y., 1999. Pseudovitamin B12 is the predominant cobamide of an algal health
food, Spirulina tablets. J. Agric. Food Chem. 47, 4736–4741.
Watanabe, F., Takenaka, S., Kittaka-Katsura, H., Ebara, S., Miyamoto, E., 2002.
Characterization and bioavailability of vitamin B12-compounds from edible algae.
J. Nutr. Sci. Vitaminol. 48, 325–331.
Wells, M.L., Potin, P., Craigie, J.S., Raven, J.A., Merchant, S.S., Helliwell, K.E., Smith, A.
G., Camire, M.E., Brawley, S.H., 2017. Algae as nutritional and functional food
sources: revisiting our understanding. J. Appl. Phycol. 29, 949–982.