Appl Microbiol Biotechnol
DOI 10.1007/s00253-016-7903-z
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Techno-functional differentiation of two vitamin B12 producing
Lactobacillus plantarum strains: an elucidation for diverse
future use
Bharat Bhushan 1,2 & S. K. Tomar 1 & Arun Chauhan 2
Received: 18 June 2016 / Revised: 17 September 2016 / Accepted: 27 September 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract An appropriate selection of Lactobacillus strain
(probiotic/starter/functional) on the basis of its techno-
functional characteristics is required before developing a nov-
el fermented functional food. We compared vitamin B12 (B12,
cobalamin) producing Lactobacillus plantarum isolates,
BHM10 and BCF20, for functional (vitamin over-production,
genomic insight to B12 structural genes, and probiotic attri-
butes) and technological [milks (skim and soy) fermentation
and B12 bio-fortification] characteristics. Addition of B12 pre-
cursors (5-amonolevulinate and dimethylbenzimidazole) to
cobalamin-free fermentation medium increased vitamin pro-
duction in BHM10, BCF20, and DSM20016 (a positive stan-
dard) by 3.4-, 4.4-, and 3.86-folds, respectively. Three impor-
tant B12 structural genes were detected in L. plantarum species
(strains BHM10 and BCF20) by PCR for the first time. The
gene sequences were submitted to NCBI GenBank and found
phylogenetically closer to respective sequences in B12 produc-
ing Lactobacillus reuteri strains. During comparative probiot-
ic testing, BCF20 showed significantly higher (p < 0.05 to
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-016-7903-z) contains supplementary material,
which is available to authorized users.
* S. K. Tomar
sudhirndri@gmail.com
Bharat Bhushan
bharatndri@gmail.com
Arun Chauhan
arun.chauhan@med.und.edu
1
Dairy1 Microbiology Division, ICAR-National Dairy Research
Institute, Karnal, Haryana 132001, India
2
Department of Biomedical Sciences, School of Medicine and Health
Sciences, University of North Dakota, Grand Forks, ND, USA
p < 0.001) gastrointestinal tolerance and cell surface hydro-
phobicity (p < 0.05) than BHM10. Moreover, only BCF20
was found positive for BSH activity and also exhibited com-
paratively better antagonistic potential against potent patho-
gens. Conversely, high acid and bile susceptible strain
BHM10 displayed significantly higher soy milk fermentation
and resultant B12 bio-fortification abilities during technologi-
cal testing. Two B12 quantification techniques, UFLC and
competitive immunoassay, confirmed the in vitro and in situ
bio-production of bio-available form of B12 after BHM10 fer-
mentation. Conclusively, techno-functional differentiation of
two B12 producing strains elucidates their diverse future use;
BCF20 either for B12 over-production (in vitro) or as a probi-
otic candidate, while BHM10 for cobalamin bio-fortification
(in situ) in soy milk.
Keywords L. plantarum . B12 production . Probiotic .
Bio-fortification . Soy milk . Bio-available
Introduction
The genus Lactobacillus, an important LAB, represents a
small but significant part of the human microbiota across dif-
ferent body sites and also found in a close association with
animals, plants, fermented foods, and beverages (Arena et al.
2014). Besides their well-established flavoring, texturing, pre-
serving (Hugenholtz and Smid, 2002), and bio-transformation
abilities (Saini et al. 2014), some Lactobacillus strains have
been exploited for the development of novel functional foods
by producing/ releasing/ increasing some macronutrients and
micronutrients (like vitamins) during fermentation (Capozzi
et al. 2012; Thakur et al. 2016). But, such novel strains first
have to be isolated from diverse ecological niches and then to

be selected for their extraordinary functional and technologi-
cal attributes.
On the same line, we have previously isolated two potent
vitamin B12 (B12 or cobalamin) producing Lactobacillus
plantarum strains, BCF20 (16S ribosomal RNA (rRNA) ac-
cession #KM396393) and BHM10 (16S rRNA accession
#KM396397), from infant’s fecal and mother’s milk samples,
respectively, by using a genotypic screening (cbiK gene de-
tection) strategy (Bhushan et al. 2016). A scope of over-
production has urged to extend our previous work in a way
to reveal the maximum production level in supplemented
growth conditions in the present study. Moreover, cbiK gene
detection in L. plantarum, a novel finding for this species,
encouraged us to go ahead for the detection of more B12 struc-
tural genes in both the strains.
Apart from production of bio-molecules, human-originated
lactobacilli are getting more attention because of their biolog-
ical origin, resistance against many physiological barriers, and
additional health-promoting effects (Walter 2008; van Baarlen
et al. 2013). This vitamin is produced intracellular by
lactobacilli (Taranto et al. 2003). Hence, a B12-producing
lactobacilli should either be susceptible to any of the stresses
(lysozyme, H2O2, acid, and bile) present in these organs (our
hypothesis) or should be autolytic to deliver this essential
vitamin in the B12 absorption corridor of human GI system
(Taranto et al. 2003). So, an analysis of B12-producing
lactobacilli for tolerance/ resilience/ susceptibility towards
GI conditions was found necessary before aspiring future
B12 supplementation outcomes. Moreover, safety and probi-
otic attributes of newly isolated LAB strains have consistently
been encouraged as important criteria in search of novel func-
tional and probiotic strains (Bove et al. 2012).
An appropriate selection of suitable microorganisms has
always been considered an important step before exploiting
them for the development of novel fermented functional foods
(LeBlanc et al. 2011; Capozzi et al. 2012). In addition, differ-
ent food bases (dairy/ soy/ cereal) should also be tested for
growth of such selected starter/ functional strains. Vitamin
B12-producing cultures are suggested to be used to decrease
the cobalamin losses of fermented dairy products during stor-
age (Adolfsson et al. 2004). Besides fermented dairy products,
soy milk has also been used for the development of B12 bio-
fortified functional food (Molina et al. 2012). Moreover, soy
beverages are also gaining ground for their high nutritional
values and low production cost (Molina et al. 2012). Hence,
we hypothesized that fermentation profile and resultant in situ
cobalamin production capabilities of both the strains in B12-
available (skim milk) and B12-free (soy milk) growth condi-
tions would give an overall idea about a perfect food base for
future B12 bio-fortification strategies.
L. plantarum has previously been reported for B12 produc-
tion potential in few studies (Madhu et al. 2009; Masuda et al.
2012), but with low production levels, negligible genomic
Appl Microbiol Biotechnol
data in support and scanty information for probiotic attributes
and milks bio-fortification profile.
Hence, the present study was designed to exploit B12 pro-
ducers, BHM10 and BCF20, for functional (vitamin over-pro-
duction, genomic insight to B12 structural genes. and probiotic
attributes) and technological [milks (skim and soy) fermenta-
tion, and B12 bio-fortification] characteristics in order to elu-
cidate the authenticated future use of both the strains.
Materials and methods
Chemicals and growth media
All chemicals were procured, if not mentioned separately,
from Sigma Aldrich Pvt. Ltd. (St. Louis, MO, USA). All
PCR reaction ingredients were purchased from New
England BioLabs (Hitchin, UK). Anaerobic and micro-
aerophilic conditions were provided by using Anaerocult gas
packs (Merck KGaA, Darmstadt, Germany). Different growth
media used in the study were MRS (for reviving and mainte-
nance), supplemented vitamin B12 assay medium (sVBAM,
for B12 over-production), tryptone glucose extract (TGE), and
brucella agar (for growth of indicator strains).
Biological materials
Two B 12-producing L. plantarum strains, BHM10 and
BCF20, were procured from the National Collection of
Dairy Cultures (NCDC771 and NCDC772, respectively),
which were previously isolated (Bhushan et al. 2016) from
human samples (milk and fecal, respectively) in Techno-
functional Starters Laboratory [TFSL, NDRI (India)]. The
standard B12 producer, Lactobacillus reuteri (L. reuteri)
DSM20016, was kindly gifted by Prof. Jean Guy Joseph
LeBlanc (CERELA, Argentina). Lactobacillus rhamnosus
strain GG was previously procured in the TFSL from
American Type Culture Collection (ATCC53103).
Pathogenic strains, Bacillus cereus (B. cereus) NCDC66 and
Staphylococcus aureus (S. aureus) NCDC109 were kindly
taken from NCDC, NDRI (India). Listeria monocytogenes
(L. monocytogenes) ATCC15313 and Salmonella typhi
(S. typhi) NCTC6017 were kindly obtained from Quality
Assurance Lab, NDRI (India). Helicobacter pylori
(H. pylori) DSM21031 was procured from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ)
culture collection.
Functional differentiation of B12-producing Lactobacillus
strains
For the B12 over-production by individual tested strain
(BCF20 or BHM10) in sVBAM [VBAM supplemented with
Appl Microbiol Biotechnol
dimethylbenzimidazole (DMBI, 15 mg/l) and 5-
amonolevulinate (ALA, 15 mg/l)], the fermentation condi-
tions were adapted from our previous work (Bhushan et al.
2016). Briefly, individual tested strain was serially sub-
cultured for 10 times in vitamin B12 - free growth conditions
and finally inoculated into sVBAM for sequential anaerobic
(initial, 10 h) and microaerophillic (4 h) fermentation condi-
tions. The concentration of DMBI was taken as suggested
previously (Hugenschmidt et al. 2010). DMBI (filter steril-
ized) was always added into medium after the first phase
(10 h) of anaerobic incubation, while ALA was added during
media preparation. The strategies for B12 extraction and its
precise quantification (using UFLC) have already been de-
scribed in our previous report (Bhushan et al. 2016) and sim-
ilarly followed in the present study. Both extraction and esti-
mation were carried out in minimum exposure of light so as to
reduce the vitamin loss and to determine the actual production
level. Microbiological assay procedure was followed as de-
scribed previously (Bhushan et al. 2016) for the bioavailabil-
ity testing of produced B12.
For detection of four B12 structural genes (Table S1), new
primers were designed (using Primer3 software), self stan-
dardized for PCR cycles (Table 1) and sequencing of partially
amplified gene products was outsourced from First Base
Laboratories (Sdn, Bhd, Malaysia). After checking the se-
quence similarity on BLAST program (http://blast.ncbi.nlm.
nih.gov), sequences were deposited in NCBI GenBank for
generation of accession numbers. To infer the evolutionary
relatedness within B12 - producing lactobacilli, phylogenetic
trees were constructed with the presently generated and pre-
viously reported GenBank sequences of four tested genes
(cobT, cbiB, cbiA, and cbiP) using the maximum likelihood
(ML) method.
Both strains were also tested for their in vitro survival ca-
pabilities against GI barriers (oral, gastric, and intestinal) and
for few other characteristics required to be a potent probiotic.
Survival percentages of tested isolates and L. rhamnosus GG
(a positive standard for GI tolerance) against tested GI barriers
were calculated for comparative assessment of survival with
the following formula:
Survival ð%Þ ¼ ðlogN t =logN 0 Þ  100
where Nt is the total count of viable bacterial cells in the
medium at time t (h), and N0 is the total count of viable
Table 1 Genetic profile of B12
producing L. plantarum strains Sr. no. Isolate
1 L. plantarum BHM10
2 L. plantarum BCF20
BHM Bharat human milk, BCF Bharat child fecal ( denotation adapted from Bhushan et al. (2016)
bacterial cells in the medium at time 0 h.
The ability to survive in oral conditions was assessed by
adding previously suggested lysozyme (100–300 μg/ml) and
hydrogen peroxide (H2O2, 10–30 μg/ml) concentrations
(Bosch et al. 2012) in to MRS medium with some modifica-
tions in the experimentation. Briefly, 7 ml of MRS medium
was inoculated with 1 % (7–8 log10 cfu/ml) of each bacterial
strain and incubated at 37 °C in micro-aerophillic conditions
for 60 and 20 min for lysozyme and hydrogen peroxide, re-
spectively. Differentiation of two B12 - producing isolates was
done by comparing survival percent of tested bacterial strains
at each concentration of lysozyme and H2O2.
Resistances to acid and bile stresses in liquid broths
were tested as suggested previously (Thakur and Tomar
2016) with some modifications. Each overnight-grown
culture (~9 log10 cfu/ml) was harvested by centrifugation
(5009×g at 4 °C) and re-suspended in equal volume of
PBS. As a modification, 1 % inoculum from PBS bacte-
rial suspension was tested for survival in to acidified (pH
3.5, 3.0, 2.5, 2.0, and 1.5) and ox bile [1 % (w/v] sup-
plemented MRS broth tubes. Moreover, survival percent
was evaluated by determining the residual viable counts
in MRS agar at 0, 1.5, and 3 h after inoculation in acidic
growth medium and at 0, 3, and 6 h after inoculation in
to bile supplemented medium. Serial dilutions for plating
were done in peptone water (0.1 %).
To observe whether bile-tolerant strains produced a func-
tional Bsh enzyme for the deconjugation of bile salts, the
isolates were streaked on MRS agar supplemented with high
concentration (1 %) of sodium taurodeoxycholate. A zone of
salt precipitation (partial or full) around the colonies of Bsh
positive strains were supposed to be observed on plates sup-
plemented with this bile salt.
Cell surface hydrophobicity (CSH) facilitates an organ-
ism to adhere to hydrocarbons present on the gut epithe-
lium. Overnight grown cultures (~9 log10 cfu/ml) were
harvested and re-suspended in 5 ml phosphate urea mag-
nesium (PUM) buffer (pH 6.5). Cell concentration was
standardized, in terms of OD595, of approx. 0.8–1.0 and
previously described procedure (Rosenberg et al. 1983)
was followed using n-hexadecane, n-octane, and xylene.
The measure of decreased absorbance (in percent) was
taken as a measure of the cell surface hydrophobicity
(%H) and calculated with the following equation.
Isolation source GenBank accession number
cobT cbiB cbiA cbiP
Human milk KU681025 KU681027 KU681029 KU681031
Child fecal KU681026 KU681028 KU681030 KU681032

ODinitial −ODfinal
%Hydrophobicity ¼  100
ODinitial
where ODinitial and ODfinal are the absorbances (at 595 nm)
before and after extraction.
Each culture was screened for its inhibitory spectra against
both Gram-positive and Gram-negative strains by agar well
diffusion assay (Chenoll et al. 2011) with some modifications.
Indicator organisms used were S. aureus, S. typhi,
L. monocytogenes, B. cereus, and H. pylori. Each
Lactobacillus spent culture supernatant (Lb-SCS) of log phase
culture (12–14-h-old) was prepared by centrifuging the tested
culture broth at 7826×g for 5–7 min at 4 °C. Fresh culture of
indicator bacteria was either poured or spread on preformed
agar plates. After 2 h of incubation, a volume of 150 μl of Lb-
SCS was put in to the wells (8 mm) precut in agar layer. The
plates were kept undisturbed for 2 h and subsequently incu-
bated at 37 °C as per requirement of tested organism. After
24–48 h of incubation, inhibition zones around the wells, in-
cluding well diameter, were measured.
For detection of any hemolytic activity, overnight-grown
tested culture was streaked on Columbia agar plates contain-
ing 5 % (w/v) buffalo blood (cattle yard, NDRI), and
anerobically incubated for 24–48 h at 37 °C. Blood agar plates
were examined for signs of β-hemolysis (clear zones around
colonies), α-hemolysis (green-hued zones around colonies),
or γ-hemolysis (no zones around colonies).
Antibiotic susceptibility test was performed by disk diffu-
sion method as done previously (Sharma et al. 2015). A total
of 20 antibiotics (Hi-Media Pvt. Ltd., Mumbai, India), named
Vancomycin, Cefuroxime, Ceftriaxone, Gentamicin,
Ciprofloxacin, Nalidixic acid, Cloxacillin, Cefaperazone,
Nitrofurantoin, Penicillin, Clindamycin, Norfloxacin,
Cotrimoxazole, Tobramycin, Amoxycillin, Amikacin,
Ceftazidime, Netillin, Ampicillin, and Erythromycin, were
tested against each tested strain. On the basis of inhibition
zone diameter (in mm), results were expressed in terms of
resistance (R) or susceptibility (S).
Technological differentiation of B12 producing
Lactobacillus strains
Growth and acidification of individual Lactobacillus strains
was separately tested in both skim and soy milks. Skim milk
was prepared by reconstituting skim milk powder [SMP,
Modern Dairy (Karnal)] in HPLC-grade water (concentration,
12 %), and autoclaving in standard conditions (121 °C for
10 min at 15 lbs). Soy milk preparation was adapted from
previous work (Hati et al. 2015) with some modifications to
attain B12-free conditions. Briefly, dry soybeans were soaked
and grinded in HPLC-grade water and milk obtained was
filtered through autoclaved (121 °C for 30 min at 15 lbs) mus-
lin cloth to rule out any exogenous B12 contamination.
Appl Microbiol Biotechnol
After inoculum preparation for each tested strain in VBAM
(Bhushan et al. 2016), washed cells were inoculated at the rate
of 1 % (v/v) into 2-screw capped milk vessels of each milk
type (total four tubes for each tested strain) to provide an initial
cell concentration of approx 107 cfu/ml. At 37 °C, one vessel
from each milk type was incubated for 14 h (10-h anaerobic +
4-h microaerophiliic), while other was undergone a total fer-
mentation of 24 h (16-h anaerobic + 8-h microaerophiliic).
After desired fermentation, lactobacilli counts were taken in
MRS agar and pH and titratable acidity were calculated using
pH meter (Thermo scientific, Waltham, MA, USA) and phe-
nolphthalein titration method, respectively.
Along with milk fermentation profile, B12 bio-fortification
status was assessed by vitamin estimation (pre and post-
fermentation) of fermented milks. ADVIA Centaur kit
(Seimens, Benedict Ave, Tarrytown, NY 10591, USA) was
used for B12 determination in autoclaved milks by competitive
immunoassay with direct chemiluminescent technology. This
method has previously been used for B12 determination in
human milk samples (Hampel et al. 2014).
Statistical analysis
All experiments were performed in triplicates on three differ-
ent days with values presented as mean ± SE. Microsoft office
(2007) was used for tabulation and descriptive representation
of data. B12 production data was comparatively analyzed
using one-way ANOVA with Tukey’s post hoc test. Data gen-
erated after probiotic testing was relatively evaluated by two-
way ANOVA with Bonferroni’s post hoc test. GraphPad Prism
(version 5) software was used for ANOVA. MEGA6 software
(Tamura et al. 2013) was used for analyzing phylogenetic
relatedness on the basis of maximum likelihood (ML) ap-
proach with bootstrap (500) using the Tamura-Nei model
(Tamura and Nei 1993).
GenBank accession numbers
GenBank accession numbers assigned for submitted gene se-
quences are given in Table 1.
Results
Functional differentiation of B12 producing Lactobacillus
strains
An enhanced B12 production in sVBAM (Fig. 1e) was ob-
served in cell extracts of BCF20 (102.549 ± 11.857 μg/l),
DSM20016 (77.512 ± 5.298 μg/l), and BHM10
(37.544 ± 3.185 μg/l) in comparison to our previous work
(Bhushan et al. 2016) where we reported BCF20
(23.90 ± 1.73 μg/l), DSM20016 (20.03 ± 4.17 μg/l), and
Appl Microbiol Biotechnol

Fig. 1 UFLC chromatograms for

B12 detection in lactobacilli cell
extracts (a–d) and comparative
GraphPad representation of data
(e). B12 peaks in chromatograms
are indicated by arrows in panels
[a cyanocobalamin standard
(40 ng/ml); b BCF20; c BHM10;
d DSM20016]. Retention time
(RT) of cyanocobalamin standard
is 13.925 min. RTs of tested cell
extracts [BHM10 (13.908);
BCF20 (13.933); DSM 20016
(13.917)]. All tested cell extracts
were diluted (2×) before analysis.
Comparative UFLC
quantification data (e). Data
(mean ± SE) was analyzed by one
way Anova using Tukey’s post
hoc test. Values with different
letters differ significantly
(p < 0.05)

BHM10 (10.91 ± 1.55 μg/l) as vitamin producers. UFLC
chromatograms for over-production are shown in figures;
Fig. 1a–d and Fig. S1. All the strains showed the same B12
production pattern (BCF20 > DSM20016 > BHM10) after
over-production as it was seen in un-supplemented conditions
(Bhushan et al. 2016). BCF20 produced a significantly higher
B12 (2.73-fold) than BHM10 in supplemented conditions with
a significance level of p < 0.01 (Fig. 1e). However, no signif-
icant difference (p > 0.05) in B12 production was observed
among BCF20 and DSM 20016.
Primer designing, PCR amplification, sequencing, and phylo-
genetic analysis were also completed with the interesting results.
Primers and PCR-related information are given in Table 2. The
use of a single PCR cycle for all the tested genes can be helpful to
other researchers using the same oligos for their work. Both B12
producers were found positive for all the tested genes and gel
electrophoretic images are given in Fig. 2. Only one out of four
tested genes (cbiP) has previously been reported in L. plantarum
species (LeBlanc et al. 2013) and rest three (cobT, cbiB, cbiA)
were detected for the first time on genomic DNA of this species.
During BLAST and phylogenetic analysis, all obtained se-
quences showed higher similarities to respective gene sequences
present in other B12 producing L. reuteri strains, including
L. reuteri DSM20016 (standard B12 producer) (Fig. 2).
Oral tolerance was checked as a primary requirement of
both, a probiotic strain and a non-probiotic B12 delivering
strain. The survival rates in lysozyme supplemented medium
for BCF20 (93–95 %) and LGG (97–101 %) were higher than
those of BHM10 (81–85 %), but a significant growth differ-
ence (p < 0.05) was observed between LGG and BHM10 only
(Fig. 3a). In case of H2O2, all strains showed very good sur-
vival (98–101 %) at all tested concentrations (10, 20, and
30 μg/ml) with no significant differences (Fig. 3b). Hence,
both tested strains can be considered as probable probiotic
candidates at first stage of testing.
The effects of different pH on the survivability of two B12
producing strains, BHM10 and BCF20, in the present study
are given in Fig. 4. At all pH ranges, BCF20 showed signifi-
cantly higher survival (p < 0.05 to p < 0.001) in comparison to
BHM10. After 1.5 h of incubation, BCF20 showed more than

Table 2 B12 structural genes
amplification by PCR Gene Primer sequences
name
cobT F: tgtgagcattgccttcactc
R:ccagcaatgtcgatagcaga
cbiB F:
taaggcacctgcaacaaca-
g
R: acttggtgaccctcattcgt
cbiA F: aatcgggaggcaaactcttc
R:ttgaaggcgttatgggactc
cbiP F: ctgctccatcctcacgattt
R:gaccagcaacgcattaaagg
60 and 74 % survival at pH 2.0 (Fig. 4a) and 2.5 (Fig. 4b),
respectively, in comparison to 0 and 31 % survival of BHM10
with a significant difference of p < 0.001. In addition, the
survival rates in bile supplemented growth conditions for
BCF20 (84–94 %) and standard strain LGG (93–94 %) were
significantly higher (p ≤ 0.05) than BHM10 (60–78 %)
(Fig. 5a). Furthermore, only BCF20 was found positive for
partial BSH activity (Fig. 5b). Hence, at second stage of GI
tolerance (acid and bile) testing, BHM10 can easily be
rejected as a probiotic candidate.
BCF20 showed significantly higher (p < 0.05) hydropho-
bicity (26 %) than BHM10 (17 %) and LGG (16 %) for n-
hexadecane, while LGG displayed highest hydrophobicity
(31 %) for remaining two hydrocarbons (xylene and octane)
as compared to BHM10 (27 and 28 %) and BCF20 (19 and
26 %), respectively (Fig. 6a).
Both of our L. plantarum isolates (BHM10 and BCF20)
inhibited most of the indicator pathogens tested with agar well
diffusion assay (Fig. 6b). They generated very large inhibition
zones (>20 mm) against three potent human pathogens
(S. aureus, H. pylori, and B. cereus). In contrast, comparatively
low zones (8–13 mm) were observed against S. typhi in accor-
dance to a previous study (5–10 mm) (Bosch et al. 2012).
Present results revealed both tested L. plantarum strains
negative for any zone of hemolysis (red cell lysis) around
colonies. Our results rendered negligible space for antibiotic
resistance transfer to pathogens as both strains exhibited sus-
ceptibility to all tested antibiotics, except Vancomycin,
Nalidixic acid Ciprofloxacin (Table S2) and hence can be
considered safe for human consumption.
Technological differentiation of B12-producing
Lactobacillus strains
Although tested cultures were not previously adapted before
for growth in skim milk, both strains grew and acidified skim
milk (Table 3). Being milk isolate, BHM10 thrived, unsurpris-
ingly, in a better way and started curdling the skim milk after
12 h; 8 h less than BCF20. As expected, the titratable acidity
Appl Microbiol Biotechnol
Product size Reference Common PCR cycle steps (1–6) for
(bp) all genes
597 Present 1. 94 °C for 4 min
study 2. 94 °C for 35 s
727 Present 3. 59 °C for 35 s
study
4. 68–72 °C for 45 s
5. Repeat step 2 to 4 (× 35 cycles)
652 Present 6. 72 °C for 7 min
study
709 Present
study
pursued inversely in comparison to pH; increased as the pH
decreased (Table 3). Soy milk, however, was satisfactorily
acidified and curdled only by BHM10 after 20–24 h of incu-
bation. BCF20 could not curdle this milk type even after 36 h
of incubation.
Although both isolates were pre-sensitized for B12 produc-
tion during VBAM growth phase, none could produce B12 in
skim milk, but used the already present vitamin in this milk
type (Table 4). Only BHM10 could increase the soy milk B12
level (~4 μg/l) (Table 4).
Discussion
In accordance with our report, extra addition of DMBI and
ALA has also been suggested previously as a requirement for
increased B12 production (Mohammed et al. 2014). ALA, one
of the primary precursors to B12, is required for synthesis of
uroporphyrinogen III, while DMBI is a late precursor required
for the lower ligand synthesis of vitamin B12 (Mohammed
et al. 2014). In comparison to low B12 production levels re-
ported for L. plantarum by other researchers [13 ng/g dry
biomass (Madhu et al. 2009); 0.2–1.8 μg/l (Masuda et al.
2012)], present study reports for a huge intracellular produc-
tion level (37–102 μg/l), in fact, highest ever for this species in
liquid broth medium. The B12 production level in BCF20
(>100 μg/l) was really an appreciable amount and this strain
can be proposed for future genetic engineering strategies and
further industrial bio-production setup.
The novel detection of some important B12 structural genes
[present report: cobT, cbiB, and cbiA; previous report: cbiK
gene (Bhushan et al. 2016)] in L. plantarum species (BHM10
and BCF20) and high phylogenetic relatedness (Fig. 2) of
detected sequences with the corresponding gene sequences
of L. reuteri strains (DSM20016 and IRT) support the previ-
ously supposed mechanism of probable transfer of B12 genetic
repertoire (LeBlanc et al. 2011) from well known B12-produc-
ing (like L. reuteri) to recently known B12-producing (like
L. plantarum) organisms. Interestingly, L. reuteri has already
Appl Microbiol Biotechnol
Fig. 2 B12 structural genes amplification in tested lactobacilli (a–d) and
underlying respective dendrograms showing molecular phylogenetic
relatedness with other vitamin producing strains on the basis of their
respective gene sequences. Agarose gel electrophoresis (AGE, 1.5 %)
of the PCR products in panels [a cobT (597 bp); b cbiB (727 bp); c
cbiA (652 bp); d cbiP (709 bp)] attained with primers designed against
respective gene sequences from standard B12 producer, L. reuteri
been reported as an autochthonous inhabitant of human GI
tract (Reuter 2001) and human milk (Sinkiewicz and
DSM20016. M molecular standards [100 bp (a–d)]; Lane 1 type strain
L. reuteri DSM20016; Lane 2 BCF20, Lane 3 BHM10, Lane 4 BIF77. In
dendrograms, bar indicates sequence divergence. Lactobacillus species
tested [L. plantarum (BHM10 and BCF20), L. reuteri (DSM20016, IRT,
SD2112, and CRL1098) and L. rossiae (DSM15814)]. Trees are
arbitrarily rooted and generated by MEGA6 tool
Ljunggren 2008), which were the coincident isolation sources
of presently investigated L. plantarum strains (BHM10 and

Fig. 3 Comparative oral tolerance (two-way ANOVA) of two B12-pro-
ducing strains in terms of survival (%) on a varied concentration range.
Oral tolerance among BCF20, BHM10 and LGG in panels [a lysozyme
tolerance; b hydrogen peroxide tolerance]. Values with different letters
differ significantly (p < 0.05)
BCF20) (Bhushan et al. 2016). Most likely, both B12-produc-
ing L. plantarum strains (BHM10 and BCF20) might have
acquired these genes from L. reuteri species through horizon-
tal gene transfer in their respective common ecological niches
(human milk and intestine). The cbiK, blub/cobT, and cbiB
genes have separately been reported in individual studies by
other researchers (Ferrer et al. 2013; Deptula et al. 2015; Yu
et al. 2015) as most important genes of the lengthy B12 bio-
production pathway.
Both high and low tolerance to oral stresses can be consid-
ered valuable for a B12-producing food grade organism. LAB
strain with high oral tolerance can be exploited further for
other probiotic attributes, while a susceptible strain would
deliver intracellular B12 at the initial site (oral cavity) of com-
plex cobalamin absorption pathway. Present results of a high
lysozyme and H2O2 tolerance of both tested L. plantarum
strains (BHM10 and BCF20) are in accordance with previous
report (Bosch et al. 2012) of the similar tolerance ranges of
lactobacilli for lysozyme (80–140 %) and H2O2 (88–110 %).
A probiotic candidate must be tolerant to lysozyme (salivary)
and H2O2 (toothpaste) for a transient or autochthonous stay in
mouth before going lower down in to GI tract (FAO/WHO
Appl Microbiol Biotechnol
2002). Hypothetically, Lactobacillus strains with high oral
tolerance/ survival could colonize (transiently/ autochtho-
nously) the oral cavity and be used to exclude the pathogenic
inhabitants (like H. pylori) in the niche. Hence, oral tolerance
results suggested both tested strains as better probiotic
candidates.
At the general pH range of the stomach (2.5 to 3.5)
(Holzapfel et al. 1998), L. plantarum BCF20 showed moder-
ate to higher survival rates (~63 to ~92 %) in comparison to
BHM10 which exhibited negligible to low acid tolerance pro-
file (0 to ~55 %). A minimum of 8–9 log cells per day are
suggested for any therapeutic effect to be generated by a pro-
biotic organism in the host (Kurmann and Rasic 1991) and a
higher tolerance to gastric acid is required for the delivery of
such numbers to lower gut. In that scenario, only BCF20 can
be considered as a potent acid resistant organism. Moreover,
probiotic bacteria with survival transit at pH 3.0 have been
considered as probable candidate for management of upper
GI tract diseases, including H. pylori infection (Berrada
et al. 1991). In contrast to BHM10, BCF20 not only showed
significantly higher survival in bile supplemented growth con-
ditions, but found BSH positive as well. The present results
are in accordance to previous report that bile tolerance differs
significantly among Lactobacillus strains within a species
(Mättö et al. 2004). The diversified acid and bile tolerance
profiles for vitamin (B2) producing Lactobacillus strains/ spe-
cies have also been reported in another recent study (Thakur
and Tomar 2016). Similar to our report, L. plantarum species
have previously been reported for in vitro taurocholate salt
deconjugation ability (Zhang et al. 2008). The second stage
of probiotic testing (acid and bile tolerance profile) rejected
the probiotic claim of BHM10.
BSH activity is one among the suggested probiotic features
(FAO/WHO 2002). Presence of BSH activity in fecal isolate
(BCF20) in the present study supported the previous report
regarding most probable finding of this activity in Intestinal
isolates (Bateup et al. 1995). Generally, the conjugated bile
acids enter into the cell, get protonated and deconjugated and
resultantly facilitate for lactobacilli its bile tolerance. In BSH
positive strains, this enzyme plays a supportive role and en-
sures the better survival of lactobacilli even in much higher
concentrations of bile salts (Bustos et al. 2012). Presence of
BSH activity strengthened the probiotic claim of strain
BCF20.
The human body is a diverse and complex ecological niche
where an organism producing antimicrobial substances can
get extra benefit of successful colonization and autochthonous
survival (Nagpal et al. 2012), hence advocated as an important
characteristic of probiotics (FAO/ WHO 2002). As a common
feature, strains BHM10 and BCF20 displayed a high antago-
nistic potential against all of the tested indicator strains.
Safety has been considered as one of the important attribute
of a probiotic organism (FAO/ WHO 2002). Moreover, a non-
Appl Microbiol Biotechnol
Fig. 4 Comparative acid tolerance (two-way ANOVA) among two B12-
producing Lactobacillus strains and LGG in terms of survival (%) on a varied
pH range at different times. Percent survival of three Lactobacillus strains
probiotic B12 producing organism (like BHM10) should also
exhibit a high range of safety after human consumption.
Present results of no red cell lysis are in accordance with
previous finding, where L. plantarum strains were found neg-
ative for any zone of hemolysis (Peres et al. 2014). Both tested
L. plantarum strains were found safe during preliminary safe-
ty testing.
Horizontal transfer of antibiotic resistance genes through
plasmids from probiotics to pathogens is assumed, as LAB is
known to have plasmids of different sizes (Sharma et al.
2014). Hence, we believe that a probiotic/starter/functional
Lactobacillus strain must be susceptible to most of the antibi-
otics. Present results are in accordance with a study carried out
on two L. plantarum strains and both were found susceptible
to all antibiotics tested (Bosch et al. 2012). Many researchers
have, however, reported for more antibiotic resistance profile
of tested LAB (Sharma et al. 2015).
Being a high lysozyme, H2O2, acid, and bile tolerant or-
ganism, BCF20 cannot be claimed, theoretically, to deliver a
significant amount of intracellular B12 in upper GI tract
(mouth, stomach, and small intestine), a corridor involved in
cobalamin absorption. However, it can definitely be further
exploited as a probiotic candidate to reach in to lower gut in
sufficient numbers, to colonize there and deliver strain-
specific benefits. Moreover, strain BCF20 with high acid re-
sistance and remarkable in vitro anti H. pylori potential offers
(BCF20, BHM10, and LGG) is shown in panels [a at pH 2.0, b at pH 2.5, c at
pH 3.0, d at pH 3.5]. Values with different letters differ significantly (p < 0.05)
itself as a potential candidate against this gastric pathogen.
Whereas, a high acid and bile susceptible B12-producing
Lactobacillus strain (BHM10), besides its elevated oral toler-
ance and remarkable antibacterial potential, could only be
better employed as a cobalamin delivery vehicle either to
fasting stomach (acid enriched area) or to proximal part of
small intestine (bile enriched area). And both of these two
delivery sites play an important role in complex B12 absorp-
tion mechanism (Nielsen et al. 2012). The upper GI tract
stresses (acid and bile) have been proposed for bacterial cell
death, cytoplasmic content liberation, and cell lysis. One such
experimentation on Lactococcus lactis reported for 14 and
91 % cell disruption/ lysis due to gastric and duodenal stress,
respectively (Drouault et al. 1999), while another report
showed the cell leakage from L. acidophilus strain
(Bezkorovainy 2001). Likewise, acid and bile mediated cell
leakage/ lysis of BHM10 can probably be hypothesized for
future in vivo B12 supplementation strategies in animal
models. Moreover, antibacterial potential, non-hemolytic na-
ture and high antibiotic susceptibility of both isolates addition-
ally support their safe use for human consumption.
Two human L. plantarum isolates from different sam-
ple sources (milk and fecal) showed diverse growth pat-
terns in two milk types and supported the previous find-
ings about versatile fermentation abilities of this species
(Kleerebezem et al. 2003).
 Appl Microbiol Biotechnol

Fig. 5 Comparative
differentiation of two B12-
producing strains and LGG for
bile tolerance (two-way ANOVA)
in terms of survival (%) (a) and
BSH activity in terms of salt
precipitation (b) at 1 % bile.
Percent survival of three
Lactobacillus strains (BCF20,
BHM10, and LGG) at different
times (a). Values with different
letters differ significantly
(p < 0.05). Growth potential and
bile salt deconjugation activity on
bile-supplemented MRS agar (b).
Bile salt precipitation (partial)
around colonies shows the
presence of BSH activity. + BSH
activity present; − BSH activity
absent

Present results, showing decreased cobalamin levels after
skim milk fermentation, supported the previously published
finding that exogenous B12 source in growth medium (skim
milk vitamin in our case) stopped bacterial endogenous cobal-
amin biosynthesis (Roth et al. 1996) as seen after 14 and 24 h
of fermentations in this study. Hence, present results reject the
possible use of skim milk as a food base for B 12 bio-
fortification strategies.
Soy products are highly nutritious and widely accepted
across the globe in recent times. Being a B12 deficient/ lacking
milk type, soymilk has been considered as a suitable food base
for in situ bio-fortification of this vitamin (Molina et al. 2012).
Only BHM10 could show a considerable growth in
soymilk and a resultant increase in the milk B12 level after
24 h of incubation. This indicates that in situ increase in
B12 level is a function of increased cell numbers of B12
producing lactobacilli. However, B12 level in BHM10-
fermented soymilk was far less (~9×) than in sVBAM.
Probable explanation behind this could be the little
growth potential of BHM10 in soymilk as compared to
sVBAM and possible partial inability of autoclaving in
recovery of B 12 molecules from dead bacterial cells.
L. plantarum has been known to grow in its own adapta-
tion style in different ecological niches (Kleerebezem
et al. 2003). Hence, higher concentrations of cell biomass
and resultant increase in B12 level would probably be
achieved after repeated sub-culturing of BHM10 in
soymilk. Another Lactobacillus strain, L. reuteri
CRL1098, has already been tested for the development
of B12 fortified functional soy-food (Molina et al. 2012).
But, vitamin form produced in that case was
pseudovitamin-B12 and showed different RT than cyano-
cobalamin, when detected with HPLC (Santos et al.
2007). While B12 form produced in present study showed
RT in accordance with standard cyanocobalamin when
tested with UFLC during over-production experiments.
Appl Microbiol Biotechnol

Table 4 B12 fortification profile (pre and post-fermentation) of milks

Isolate Milk type B12 concentration (μg/l)

0h 14 ha 24 hb

BHM10 Skim milk 0.617 0.041 <0.0001

Soy milk <0.0001 0.077 3.951
BCF20 Skim milk 0.617 0.065 <0.0001
Soy milk <0.0001 0.001 0.031
a
An initial anaerobic (10 h) and subsequent micro-aerophillic (4 h) incu-
bation at 37 °C
b
An initial anaerobic (16 h) and subsequent micro-aerophillic (8 h) incu-
bation at 37 °C

In terms of novelty, this is the first study reporting for

Fig. 6 Comparison of two B12-producing lactobacilli for hydrophobicity
in terms of adherence to hydrocarbons (a) and antibacterial potential in
terms of inhibition zone diameters against pathogens (b). Values with
different letters differ significantly (p < 0.05)
Moreover, ADVIA Centaur kit, used for B12 estimation in
milks, employs intrinsic factor for specific binding with
B12 molecule and renders no space for nonspecific detec-
tion of other forms of B12.
detection of three important genes (cobT, cbiB, cbiA) on ge-
nomic DNA of L. plantarum species. Present report is novel in
the form of highest (>100 μg/l) B 12 production from
L. plantarum species ever. We are first to report, to the best
of our knowledge, the use of L. plantarum for in situ soy milk
bio-fortification with biologically active form of B12. And
finally, we are innovative in using the techno-functional ap-
proach to elucidate the future use of two B12- producing
Lactobacillus isolates.
In conclusion, techno-functional differentiation displayed a
crystal clear picture for the future exploitation of two B12-
producing L. plantarum strains. Strain BCF20, the high B12
producer and the better probiotic, can be used for two separate
functionalities; either for in vitro B12 over-production in a lab/
industry setting or for its probiotic properties (like anti
H. pylori potential). Conversely, strain BHM10 (low B12 pro-
ducing, high acid and bile susceptible and good soymilk fer-
menter) can convincingly and only be exploited for vitamin
delivery to stomach or small intestine in the form of
biofortified functional soy food. An appropriate strain selec-
tion on the basis of such techno-functional testing is the most
important prerequisite step before going into in vivo animal
models experimentation and claiming any possible health

Table 3 Growth and

acidification profile of B12 Isolate Milk type Cell counts (cfu/ml) pH≠ Titratable acidity* (% lactic acid)
producing lactobacilli in milks
14 ha 24 hb 14 ha 24 hb 14 ha 24 hb

BHM10 Skim milk 2.4 × 108 3.5 × 109 5.3 4.41 0.55 0.78
Soy milk 5.0 × 107 3.0 × 108 6.1 5.5 0.24 0.54
BCF20 Skim milk 6.4 × 107 8.2 × 108 6.41 4.95 0.17 0.68
Soy milk 1.6 × 107 4.9 × 107 6.55 6.12 0.14 0.21

Initial cell concentration ~1 × 107 cfu/ml

≠
pH of the un-inoculated milks [skim, 6.73; soy, 6.82]
*The titratable acidity of un-inoculated milks [skim, 0.11; soy, 0.10]
a
An initial anaerobic (10 h) and subsequent micro-aerophillic (4 h) incubation at 37 °C
b
An initial anaerobic (16 h) and subsequent micro-aerophillic (8 h) incubation at 37 °C

improving effect. Interestingly, a B12-producing food grade
organism can anyways, whether probiotic or not, be of higher
importance.
Acknowledgments Bharat Bhushan was the PhD student supported by
ICAR-NDRI to carry out his research work. Authors would like to thank
Mrs. Kamna Saini, Dr. Alia Khan, and Dr. Namita Rokana from NDRI,
Karnal, India, for helping in the spectrophotometric work during routine
experimentation. The valuable support of Miss Rinky Gupta from NDRI,
Karnal, India, for antibacterial experiments was highly appreciable.
Authors are also thankful to Mr. Lalit Saluja and his team (SRL diagnos-
tics, Karnal) for their valuable help in B12 analysis of milk samples.
Compliance with ethical standards This article does not contain any
studies with human participants or animal performed by any of the
authors.
Conflict of interest The authors declare that they have no conflict of
interest.
References
Adolfsson O, Meydani SN, Russell RM (2004) Yogurt and gut function.
Am J Clinic Nut 80(2):245–256
Arena MP, Russo P, Capozzi V, López P, Fiocco D, Spano G (2014)
Probiotic abilities of riboflavin-overproducing Lactobacillus strains:
a novel promising application of probiotics. Appl Microbiol
Biotechnol 98(17):7569–7581. doi:10.1007/s00253-014-5837-x
Bateup JM, McConnell MA, Jenkinson HF, Tannock GW (1995)
Comparison of Lactobacillus strains with respect to bile salt hydro-
lase activity, colonization of the gastrointestinal tract, and growth
rate of the murine host. Appl Environ Microbiol 61(3):1147–1149
Berrada N, Lemeland JF, Laroche G, Thouvenot P, Piaia M (1991)
Bifidobacterium from fermented milks: survival during gastric tran-
sit. J Dairy Sci 74(2):409–413. doi:10.3168/jds.S0022-0302(91
)78183-6
Bezkorovainy A (2001) Probiotics: determinants of survival and growth
in the gut. The Am J Clinic Nutr 73(2):399s–405s
Bhushan B, Tomar SK, Mandal S (2016) Phenotypic and genotypic
screening of human-originated lactobacilli for vitamin B12 produc-
tion potential: process validation by micro-assay and UFLC. Appl
Microbiol Biotechnol 28:1–3. doi:10.1007/s00253-016-7639-9
Bosch M, Rodriguez M, Garcia F, Fernández E, Fuentes MC, Cuñé J
(2012) Probiotic properties of Lactobacillus plantarum CECT
7315 and CECT 7316 isolated from faeces of healthy children.
Lett Appl Microbiol 54(3):240–246. doi:10.1111/j.1472-765
X.2011.03199.x
Bove P, Gallone A, Russo P, Capozzi V, Albenzio M, Spano G, Fiocco D
(2012) Probiotic features of Lactobacillus plantarum mutant strains.
Appl Microbiol Biotechnol 96(2):431–441. doi:10.1007/s00253-
012-4031-2
Bustos AY, Saavedra L, de Valdez GF, Raya RR, Taranto MP (2012)
Relationship between bile salt hydrolase activity, changes in the
internal pH and tolerance to bile acids in lactic acid bacteria.
Biotechnol Lett 34(8):1511–1518. doi:10.1007/s10529-012-0932-5
Capozzi V, Russo P, Duenas M, Lopez P, Spano G (2012) Lactic acid
bacteria producing B-group vitamins: a great potential for functional
cereals products. Appl Microbiol Biotechnol 96(6):1383–1394.
doi:10.1007/s00253-012-4440-2
Chenoll E, Casinos B, Bataller E, Astals P, Echevarría J, Iglesias JR,
Balbarie P, Ramón D, Genovés S (2011) Novel probiotic
Bifidobacterium bifidum CECT 7366 strain active against the
Appl Microbiol Biotechnol
pathogenic bacterium Helicobacter pylori. Appl Environ
Microbiol 77(4):1335–1343. doi:10.1128/AEM.01820-10
Deptula P, Kylli P, Chamlagain B, Holm L, Kostiainen R, Piironen V,
Savijoki K, Varmanen P (2015) BluB/CobT2 fusion enzyme activity
reveals mechanisms responsible for production of active form of
vitamin B12 by Propionibacterium freudenreichii. Microb Cell
Fact 14(1):1. doi:10.1186/s12934–015–0363-9
Drouault S, Corthier G, Ehrlich SD, Renault P (1999) Survival, physiol-
ogy, and lysis of Lactococcus lactis in the digestive tract. App
Environ Microbiol 65(11):4881–4886
FAO/WHO J (2002) Working group report on drafting guidelines for the
evaluation of probiotics in food london. Ontario, Canada. Available
at: ftp://ftp.fao.org/es/esn/food/wgreport2.pdf
Ferrer M, Ruiz A, Lanza F, Haange SB, Oberbach A, Till H, Bargiela R,
Campoy C, Segura MT, Richter M, von Bergen M (2013)
Microbiota from the distal guts of lean and obese adolescents exhibit
partial functional redundancy besides clear differences in communi-
ty structure. Environ Microbiol 15(1):211–226. doi:10.1111/j.1462
2920.2012.02845.x
Hampel D, Shahab-Ferdows S, Domek JM, Siddiqua T, Raqib R, Allen
LH (2014) Competitive chemiluminescent enzyme immunoassay
for vitamin B12 analysis in human milk. Food Chem 153:60–65.
doi:10.1016/j.foodchem.2013.12.033
Hati S, Vij S, Singh BP, Mandal S (2015) β-Glucosidase activity and
bioconversion of isoflavones during fermentation of soymilk. J Sci
Food Agri 95(1):216–220. doi:10.1002/jsfa.6743
Holzapfel WH, Haberer P, Snel J, Schillinger U, Huis in't Veld JH (1998)
Overview of gut flora and probiotics. Int J Food Microbiol 41(2):
85–101. doi:10.1016/S0168-1605(98)00044-0
Hugenholtz J, Smid EJ (2002) Nutraceutical production with food-grade
microorganisms. Curr Opin Biotechnol 13(5):497–507. doi:10.1016
/S0958-1669(02)00367-1
Hugenschmidt S, Schwenninger S, Gnehm N, Lacroix C (2010)
Screening of a natural biodiversity of lactic and propionic acid bac-
teria for folate and vitamin B12 production in supplemented whey
permeate. Int Dairy J 20(12):852–857. doi:10.1016/j.
idairyj.2010.05.005
Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers
OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW,
Stiekema W (2003) Complete genome sequence of Lactobacillus
plantarum WCFS1. Proc Nat Acad Sci 100(4):1990–1995.
doi:10.1073/pnas.0337704100
Kurmann JA, Rašić JL (1991) The health potential of products containing
bifidobacteria. In: Robinson RK (ed) Therapeutic properties of
fermented milks. Elsevier, Applied Science Publishers Ltd,
London, pp. 117–158
LeBlanc JG, Laino JE, del Valle MJ, Vannini V, van Sinderen D, Taranto
MP, de Valdez G, de Giori GS, Sesma F (2011) B-group vitamin
production by lactic acid bacteria—current knowledge and potential
applications. J Appl Microbiol 111(6):1297–1309. doi:10.1111
/j.1365-2672.2011.05157.x
LeBlanc JG, Milani C, de Giori G, Sesma F, van Sinderen D, Ventura M
(2013) Bacteria as vitamin suppliers to their host: a gut microbiota
perspective. Curr Opin Biotechnol 24(2):160–168. doi:10.1016/j.
copbio.2012.08.005
Madhu A, Giribhattanavar P, Narayan M, Prapulla S (2009) Probiotic
lactic acid bacterium from kanjika as a potential source of vitamin
B12: evidence from LC-MS, immunological and microbiological
techniques. Biotechnol Lett 32(4):503–506. doi:10.1007/s10529-
009-0176-1
Masuda M, Ide M, Utsumi H, Niiro T, Shimamura Y, Murata M (2012)
Production potency of folate, vitamin B12 and thiamine by lactic
acid bacteria isolated from Japanese pickles. Biosci Biotechnol
Biochem 76(11):2061–2067. doi:10.1271/bbb.120414
Mättö J, Malinen E, Suihko ML, Alander M, Palva A, Saarela M (2004)
Genetic heterogeneity and functional properties of intestinal
Appl Microbiol Biotechnol
bifidobacteria. J Appl Microbiol 97(3):459–470. doi:10.1111
/j.1365-2672.2004.02340.x
Mohammed Y, Lee B, Kang Z, Du G (2014) Development of a two-step
cultivation strategy for the production of vitamin B12 by Bacillus
megaterium. Microb Cell Factories 13(1):102. doi:10.1186/s12934-
014-0102-7
Molina V, Médici M, de Valdez GF, Taranto MP (2012) Soybean-based
functional food with vitamin B12-producing lactic acid bacteria. J
Funct Foods 4(4):831–836. doi:10.1016/j.jff.2012.05.011
Nagpal R, Kumar A, Kumar M, Behare PV, Jain S, Yadav H (2012)
Probiotics, their health benefits and applications for developing
healthier foods: a review. FEMS Microbiol Lett 334(1):1–5.
doi:10.1111/j.1574-6968.2012.02593.x
Nielsen MJ, Rasmussen MR, Andersen CB, Nexø E, Moestrup SK
(2012) Vitamin B12 transport from food to the body’s cells—a so-
phisticated, multistep pathway. Nat Rev Gastroenterol Hepatol 9(6):
345–354. doi:10.1038/nrgastro.2012.76
Peres CM, Alves M, Hernandez-Mendoza A, Moreira L, Silva S, Bronze
MR, Vilas-Boas L, Peres C, Malcata FX (2014) Novel isolates of
lactobacilli from fermented Portuguese olive as potential probiotics.
LWT-Food Sci Technol 59(1):234–246. doi:10.1016/j.
lwt.2014.03.003
Reuter G (2001) The Lactobacillus and Bifidobacterium microflora of the
human intestine: composition and succession. Curr Iss Intest
Microbiol 2(2):43–53
Rosenberg M, Judes H, Weiss E (1983) Cell surface hydrophobicity of
dental plaque microorganisms in situ. Infect Immun 42(2):831–834
Roth JR, Lawrence JG, Bobik TA (1996) Cobalamin (coenzyme B12):
synthesis and biological significance. Ann Rev Microbiol 50(1):
137–181. doi:10.1146/annurev.micro.50.1.137
Saini K, Tomar SK, Sangwan V, Bhushan B (2014) Evaluation of
lactobacilli from human sources for uptake and accumulation of
selenium. Biol Trace Elem Res 160(3):433–436. doi:10.1007
/s12011-014-0065-x
Santos F, Vera JL, Lamosa P, de Valdez GF, de Vos WM, Santos
H, Sesma F, Hugenholtz J (2007) Pseudovitamin B12 is the
corrinoid produced by Lactobacillus reuteri CRL1098 under
anaerobic conditions. FEBS Lett 581(25):4865–4870.
doi:10.1016/j.febslet.2007.09.012
Sharma P, Tomar SK, Goswami P, Sangwan V, Singh R (2014) Antibiotic
resistance among commercially available probiotics. Food Res
Internat 57:176–195. doi:10.1016/j.foodres.2014.01.025
Sharma P, Tomar SK, Sangwan V, Goswami P, Singh R (2015)
Antibiotic resistance of Lactobacillus sp. isolated from com-
mercial probiotic preparations. J Food Safet 36(1):38–51.
doi:10.1111/jfs.12211
Sinkiewicz G, Ljunggren L (2008) Occurrence of Lactobacillus reuteri in
human breast milk. Microb Ecol Health Dis 20(3):122–126.
doi:10.1080/08910600802341007
Tamura K, Nei M (1993) Estimation of the number of nucleotide substi-
tutions in the control region of mitochondrial DNA in humans and
chimpanzees. Mol Biol Evol 10:512–526
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6:
molecular revolutionary genetics analysis version 6.0. Mol Biol
Evol 30:2725–2729. doi:10.1093/molbev/mst197
Taranto M, Vera J, Hugenholtz J, De Valdez G, Sesma F (2003)
Lactobacillus reuteri CRL1098 produces cobalamin. J Bacteriol
185(18):5643–5647. doi:10.1128/jb.185.18.5643-5647.2003
Thakur K, Tomar SK (2016) In vitro study of riboflavin producing
lactobacilli as potential probiotic. LWT-Food Sci Technol 68:570–
578. doi:10.1016/j.lwt.2015.12.059
Thakur K, Tomar SK, De S (2016) Lactic acid bacteria as a cell factory for
riboflavin production. Microb Biotechnol. doi:10.1111/1751-
7915.12335
van Baarlen P, Wells JM, Kleerebezem M (2013) Regulation of intestinal
homeostasis and immunity with probiotic lactobacilli. Trends
Immunol 34(5):208–215. doi:10.1016/j.it.2013.01.005
Walter J (2008) Ecological role of lactobacilli in the gastrointestinal tract:
implications for fundamental and biomedical research. Appl
Environ Microbiol 74(16):4985–4996. doi:10.1128/AEM.00753-08
Yu Y, Zhu X, Shen Y, Yao H, Wang P, Ye K, Wang X, Gu Q (2015)
Enhancing the vitamin B 1 2 production and growth of
Propionibacterium freudenreichii in tofu wastewater via a light-
induced vitamin B12 riboswitch. Appl Microbiol Biotechnol
99(24):10481–10488. doi:10.1007/s00253-015-6958-6
Zhang M, Hang X, Fan X, Li D, Yang H (2008) Characterization and
selection of Lactobacillus strains for their effect on bile tolerance,
taurocholate deconjugation and cholesterol removal. World J
Microbiol Biotechnol 24(1):7–14. doi:10.1007/s11274-007-9431-6