Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

B-Group vitamin production by lactic acid bacteria – current

knowledge and potential applications
J.G. LeBlanc1, J.E. Laiño1, M. Juarez del Valle1, V. Vannini1, D. van Sinderen2, M.P. Taranto1, G. Font
de Valdez1,3, G. Savoy de Giori1,3 and F. Sesma1
1 Centro de Referencia para Lactobacilos (CERELA-CONICET) Chacabuco 145, Tucumán, Argentina
2 Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
3 Cátedra de Microbiologı́a Superior, Universidad Nacional de Tucumán (UNT), Tucumán, Argentina

Keywords Abstract
biotechnology, dairy, fermented foods,
Lactobacillus, Lactococcus. Although most vitamins are present in a variety of foods, human vitamin defi-
ciencies still occur in many countries, mainly because of malnutrition not only
Correspondence as a result of insufficient food intake but also because of unbalanced diets.
Jean Guy LeBlanc and Fernando Sesma, Even though most lactic acid bacteria (LAB) are auxotrophic for several vita-
CERELA-CONICET, Chacabuco 145, San
mins, it is now known that certain strains have the capability to synthesize
Miguel de Tucumán, Tucumán T4000ILC,
Argentina.
water-soluble vitamins such as those included in the B-group (folates, ribofla-
E-mails: leblanc@cerela.org.ar, vin and vitamin B12 amongst others). This review article will show the current
jeanguyleblanc@hotmail.com, knowledge of vitamin biosynthesis by LAB and show how the proper selection
fsesma@cerela.org.ar of starter cultures and probiotic strains could be useful in preventing clinical
and subclinical vitamin deficiencies. Here, several examples will be presented
2011 ⁄ 1144: received 8 July 2011, revised where vitamin-producing LAB led to the elaboration of novel fermented foods
31 August 2011 and accepted 11 September
with increased and bioavailable vitamins. In addition, the use of genetic engi-
2011
neering strategies to increase vitamin production or to create novel vitamin-
doi:10.1111/j.1365-2672.2011.05157.x producing strains will also be discussed. This review will show that the use of
vitamin-producing LAB could be a cost-effective alternative to current vitamin
fortification programmes and be useful in the elaboration of novel vitamin-
enriched products.

ization of the gut mucosal barrier, immune adjuvant

Introduction
properties, alleviation of intestinal bowel disease symp-
Lactic acid bacteria (LAB) represent a heterogeneous toms and improvement of the digestion of lactose in
group of micro-organisms that are naturally present in a intolerant hosts (Ouwehand et al. 2002; Deshpande et al.
wide range of ecological niches such as foods and in the 2011; Soccol et al. 2011). The probiotic and beneficial
gastrointestinal and urogenital tract of animals, including aspects of LAB have been extensively reviewed elsewhere
humans. In addition to their important technological and will not be the subject of this review.
properties in food production, several studies have shown Besides probiotic LAB, certain strains of LAB are able
that LAB can confer beneficial properties to their hosts, to produce ⁄ release and ⁄ or increase specific beneficial
in particular specific members of the genus Lactobacillus, compounds in foods. These functional ingredients are
reason for which these bacteria are the most commonly sometimes referred to as nutraceuticals a term that was
used probiotic micro-organisms. These latter can be first given by Stephen DeFelice in 1989 to describe ‘a food
defined as ‘live microorganisms which when administered (or part of a food) that provides medical or health bene-
in adequate amounts confer a health benefit on the host’ fits, including the prevention and ⁄ or treatment of a
(FAO ⁄ WHO 2001). Some of the health benefits ascribed disease.’ These ingredients can be macronutrients,
to probiotics include promotion of a normal microbiota, micronutrients (such as vitamins) or non-nutritive com-
prevention of infectious diseases and food allergies, reduc- pounds and can be naturally present in certain foods or
tion of serum cholesterol, anticarcinogenic activity, stabil- added during processing. The proper selection and

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Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology 1297
Vitamin production by LAB J.G. LeBlanc et al.

exploitation of nutraceutical-producing micro-organisms els of vitamin ingestion would be subject to excessive

is an interesting strategy to produce novel foods with intakes. In the case of folic acid fortification, excess intake
increased nutritional and ⁄ or health-promoting properties may in turn mask the early haematological manifestations
(Hugenholtz and Smid 2002). of vitamin B12 deficiency. This is important as it has been
estimated that 10–30% of people over 50 years have a
reduced ability to naturally absorb vitamin B12, and con-
Vitamins
sequently, 20% of the general population in industrialized
Vitamins are micronutrients that are essential for the countries are potentially deficient for this vitamin (Asrar
metabolism of all living organisms. They are found as and O’Connor 2005). As folate fortification levels are
precursors of intracellular coenzymes that are necessary to based on the requirements of the general population,
regulate vital biochemical reactions in the cell. Humans some groups could be exposed to extremely high levels of
are incapable of synthesizing most vitamins, and they folic acid such as children whose vitamin requirements
consequently have to be obtained exogenously (i.e. from are lower than adults or in pregnant women who take
their diet). Although most vitamins are present in a vari- folic acid supplements. It has even been suggested that
ety of foods, vitamin deficiencies still exist in many coun- the foetus may become exposed to excessive amounts of
tries including highly industrialized nations mainly folic acid because of prescribed supplementation of the
because of malnutrition, not only as a result of insuffi- mother during pregnancy in combination with the con-
cient food intake but also because of unbalanced diets. sumption of fortified foods, and this could favour the
The B-group (or B-complex) vitamins include thiamine selection of methylentetrahydrofolate polymorphism that
(B1), riboflavin (B2), niacin (B3), pyridoxine (B6), panto- is associated with a group of debilitating diseases (Lucock
thenic acid (B5), biotin (B7 or H), folate (B11–B9 or M) and Yates 2005). As natural folates’ such as 5-methyltetra-
and cobalamin (B12). Each B-group vitamin is chemically hydrofolate (5-MTHF) that is normally found in foods
different and acts in synergy to maintain the body’s and sometimes produced by micro-organisms do not
homeostasis by playing major roles in metabolic processes mask B12 deficiency, this folate form would be a more
such as energy production and red blood cell formation. efficient and secure alternative than supplementation with
B-group vitamins, normally present in many foods, are folic acid (Lamers et al. 2006).
easily removed or destroyed during cooking and food The use of vitamin-producing micro-organisms is thus
processing, so insufficient intake are common in many a more natural and economically viable alternative than
societies. For this reason, many countries have adopted fortification with chemically synthesized pseudo-vitamins,
laws to enforce the fortification of certain foods with spe- and it would allow the production of foods with elevated
cific vitamins and minerals. For example, in Argentina concentrations of vitamins that are less likely to cause
the food industry is obliged to fortify all wheat flour for undesirable side effects.
human consumption with iron, folic acid, thiamine, ribo-
flavin and niacin to reduce the incidence of anaemia and
Riboflavin
neural tube deformation. However, recent reports high-
lighted a lack of official enforcement of the fortification Riboflavin (vitamin B2) plays an essential role in cellular
requirement and insufficient fortification levels, and it is metabolism, being the precursor of the coenzymes flavin
therefore not surprising that serum vitamin levels in the mononucleotide (FMN) and flavin adenine dinucleotide
general population have only slightly improved and sub- (FAD) both acting as hydrogen carriers in biological redox
clinical deficiencies still persist (ENNyS 2007). reactions involving enzymes such as nicotinamide adenine
Although the beneficial effects of generalized fortifica- dinucleotide (NADH) dehydrogenase. Here, the term
tion programmes have been demonstrated, such as the ‘riboflavin’ is used to describe all biologically active forms
decreased incidence of neural tube defects (NTD) and of vitamin B2 flavins, including riboflavin (7,8-dimethyl-
neonatal mortality in countries such as Canada and the 10-(1¢-d-ribityl) isoalloxazine), riboflavin-5¢-phosphate
USA where folate fortification is mandatory since 1998 (FMN) and riboflavin-5¢-adenosyldiphosphate (FAD). The
(Blencowe et al. 2010), many countries have not adopted recommended riboflavin requirements for humans vary
a national fortification programme because of possible with respect to sex, ageand physiological state (pregnancy
unwanted side effects. The main concerns are based on and lactation). Normal adults need to consume between
the fact that vitamins are added at concentrations that 0Æ9 and 1Æ6 mg of this vitamin on a daily basis as the
allow persons with low vitamin intakes to reach their rec- human body cannot adequately store riboflavin (Institute
ommended daily allowance (RDA) so as to prevent of Medicine 1998). Although present in a wide variety of
pathologies associated with deficiencies. At these levels of foods, such as dairy products, meat, eggs and certain
fortification, however, those with normal or elevated lev- green vegetables, riboflavin deficiency (ariboflavinosis) still

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1298 Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology
J.G. LeBlanc et al. Vitamin production by LAB

occurs in both developing and industrialized countries 6 120

Vitamin B1/2/3/5/6/12 concentration

(O’Brien et al. 2001; Blanck et al. 2002). Symptoms of ari-

Vitamin B7/9 concentration

boflavinosis in humans include sore throat, hyperaemia, 5 100
oedema of oral and mucous membranes, cheilosis and
4 80
glossitis (Wilson 1983). Severe cases of ariboflavinosis are
not common in most societies; however, subclinical mani-
3 60
festations are frequent amongst all subpopulation groups.
Subclinical riboflavin deficiencies are only detectable by 2 40
measuring the vitamin concentration in body fluids such
as blood plasma and serum. Vitamin B2 status in humans 1 20
has usually been assessed by measuring the erythrocyte
glutathione reductase activation coefficient (EGRAC), 0 0
Milk Buttermilk Yogurt Kefir
which is the ratio between glutathione reductase activity
determined with and without the addition of the cofactor, Figure 1 Vitamin concentrations in dairy products (modified from
FAD (Glatzle et al. 1970). Glutathione reductase loses FAD Alm (1982) and LeBlanc et al. 2010b). Concentrations of thiamine
at an early stage in vitamin B2 deficiency, making EGRAC (B1, ), riboflavin (B2, h), niacin (B3, ), pyridoxine (B6, ) and panto-
a useful method for the diagnosis of vitamin B2 deficiency thenic acid (B5, ) are in mg l)1, whereas biotin (B7, ), folate
(B9 ⁄ B11, ) and cobalamin (B12, ) are in lg l)1.
(Bates 1993).
Although dairy products contain riboflavin, they are
not considered a good source of this essential vitamin. vin levels increased significantly (1Æ7 and 2Æ0 mg l)1)
Considering that milk contains c. 1Æ2 mg of riboflavin per compared with unfermented milk (1Æ2 mg l)1). It has
litre, an average adult person and a pregnant woman been shown that most yogurt starter cultures decreased
would need to consume, respectively, 1 and 1Æ6 l of milk riboflavin concentrations whereas others can increase the
per day to meet their daily requirement. This level of levels of this essential vitamin up to 60% of the initial
milk consumption far exceeds that of residents of indus- concentration present in unfermented milk (Kneifel et al.
trialized countries such as USA where the daily per capita 1992).
consumption of fresh milk is c. 200 ml (Putman and Alls- In a clinical trial, it was shown that daily consumption
house 2003). Increasing the levels of riboflavin in milk of 200 g of both a probiotic or conventional yoghurt for
would thus be very important to prevent ariboflavinosis 2 weeks can contribute to the total intake of vitamin B2,
in populations where milk consumption is low. as reflected by increased levels of plasma-free riboflavin in
Riboflavin biosynthesis has been described both in gram- healthy women (Fabian et al. 2008). However, as ribofla-
positive and gram-negative bacteria, with detailed studies vin levels returned to normal when the intake of fer-
performed for Bacillus subtilis (Perkins and Pero 2002) and mented milks was stopped (no long-term effect), the
Escherichia coli (Bacher et al. 1996). Microbial biosynthesis changes in plasma concentrations seem more likely the
of riboflavin from the precursors guanosine triphosphate result of regular yoghurt consumption as a fermented
(GTP) and d-ribulose 5-phosphate occurs through seven dairy product, rather than of the specific intake of the
enzymatic steps that have previously been reviewed in probiotic bacteria (Fabian et al. 2008). Previous results
detail (Bacher et al. 2000). The imidazole ring of GTP is from this group showed that unlike some yoghurt starter
hydrolytically opened, yielding a 4,5-diaminopyrimidine, cultures that are able to produce riboflavin, most probiot-
which is converted to 5-amino-6-ribitylamino-2,4(1H,3H)- ic strains of lactobacilli consume this vitamin and thus
pyrimidinedione by a sequence of deamination, side- decrease their bioavailability in fermented products
chain reduction and dephosphorylation. Condensation of (Elmadfa et al. 2001). Consequently, adequate selection
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with of strains is essential to increase the concentration and
3,4-dihydroxy-2-butanone 4-phosphate obtained from bioavailability of this essential vitamin in fermented
ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. foods.
Dismutation of the lumazine derivative yields riboflavin Tempeh, a traditional Indonesian fermented soybean
and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, food, was shown to contain elevated concentrations of B-
which is recycled in the biosynthetic pathway. group vitamins (such as riboflavin) because of microbial
Riboflavin concentrations can sometimes vary in cer- biosynthesis (Keuth and Bisping 1993). The latter article
tain dairy products (see Fig. 1) because of processing also reported on the isolation of LAB from tempeh,
technologies and through the action of micro-organisms which were shown to belong to the Streptococcus and
utilized during food processing(LeBlanc et al. 2010b). Enterococcus genera and able to significantly increase ribo-
This is the case for buttermilk and yogurt where ribofla- flavin concentrations in this fermented product.

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Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology 1299
Vitamin production by LAB J.G. LeBlanc et al.

Recently, a publication has described the screening of In another study, it was shown that the administration
riboflavin-producing strains from different fermented of a fermented milk that was produced with the roseofla-
milk products obtained in the Vellore region of India (Ja- vin-resistant and spontaneous riboflavin-overproducing
yashree et al. 2010). Half of the 48 isolates were able to strain P. freudenreichii B2336 was beneficial to riboflavin-
grow in a chemically defined medium (CDM) without depleted animals (LeBlanc et al. 2006). The fermented
riboflavin although just a single strain was identified as product containing P. freudenreichii B2336, with increased
being an efficient riboflavin-producing strain: Lactobacil- concentrations of riboflavin, eliminated most physiologi-
lus fermentum MTCC 8711 produced 2Æ29 mg l)1 of ribo- cal manifestations of ariboflavinosis, whereas a product
flavin after 24 h of growth in the CDM (Jayashree et al. fermented with a non–riboflavin-producing strain of
2010). These authors conclude that this strain could be P. freudenreichii did not show this beneficial effect (Le-
further exploited for the enhanced production of ribofla- Blanc et al. 2006).
vin using various strain improvement strategies to Another method to obtain riboflavin-producing strains
develop a better starter culture for the fermented food or to increase their production capacities is to use meta-
industry. They also propose that it could be used to bolic engineering strategies. A Corynebacterium ammoni-
replace the conventional strains that are being employed agenes strain harbouring a plasmid containing all of its
in these LAB-based fermented products. riboflavin biosynthetic genes was constructed through
The selection of spontaneous roseoflavin-resistant metabolic engineering using recombinant DNA tech-
mutants was found to be a reliable method to obtain nat- niques. This recombinant strain was shown to produce
ural riboflavin-overproducing strains of a number of spe- and accumulate riboflavin to levels that were 17-fold
cies commonly used in the food industry (Burgess et al. higher as compared to the plasmid-free parent strain
2006). The toxic riboflavin analogue roseoflavin was used (Koizumi et al. 2000).
to isolate natural riboflavin-overproducing variants of the By means of classical mutagenesis and gene technology,
food-grade micro-organisms Lactococcus lactis (Burgess the gram-positive bacterium B. subtilis was modified to
et al. 2004), Lactobacillus plantarum, Leuconosctoc mesen- become a suitable host for the commercial production of
teroides and Propionibacterium freudenreichii (Burgess riboflavin (Perkins et al. 1999). A sequential optimization
et al. 2006). In these studies, it was demonstrated that strategy, based on statistical experimental designs, was
spontaneous resistance to the toxic riboflavin analogue used to enhance the production of riboflavin by recombi-
roseoflavin frequently coincides with a riboflavin-overpro- nant B. subtilis RH44 (Wu et al. 2007). Recently,
ducing phenotype because of mutations in the regulatory enhanced riboflavin production was obtained by express-
region of the rib operon. ing heterologous riboflavin operon from Bacillus cereus
Recently, LAB were obtained from durum wheat flour ATCC14579 in B. subtilis (Yunxia et al. 2010).
samples and screened for roseoflavin-resistant variants to Previously, we described the genetic analysis of the ribo-
isolate natural riboflavin-overproducing strains (Capozzi flavin biosynthetic (rib) operon in the lactic acid bacterium
et al. 2011). Two riboflavin-overproducing strains of L. lactis ssp. cremoris strain NZ9000 (Burgess et al. 2004).
Lact. plantarum were isolated and used for the prepara- This strain was converted from a riboflavin consumer into
tion of bread (by means of sourdough fermentation) and a vitamin B2 ‘factory’ by overexpressing its riboflavin bio-
pasta (using a prefermentation step) to enhance their synthesis genes (Burgess et al. 2004). Substantial riboflavin
vitamin B2 content. The applied approaches resulted in a overproduction (24 mg l)1) was described when all four
considerable increase in vitamin B2 content (about a two biosynthetic genes (ribG, ribH, ribB and ribA) were overex-
and threefold increase in pasta and bread, respectively), pressed simultaneously (in L. lactis NZ9000 containing
thus representing a convenient and efficient food-grade pNZGBAH). It was demonstrated that milk fermented by
biotechnological application for the production of vita- this genetically modified riboflavin-producing strain was
min B2-enriched bread and pasta. effective in reversing ariboflavinosis in a riboflavin-defi-
The roseoflavin-resistant, riboflavin-producing strain ciency rat model (LeBlanc et al. 2005b). The manufacture
L. lactis CB010 was able to eliminate most physiological of a product of this nature would decrease the costs com-
manifestations of ariboflavinosis such as stunted growth, pared with current vitamin fortification programmes.
elevated EGRAC values and hepatomegalia that were A great advantage of genetic engineering strategies is
observed using a riboflavin depletion–repletion animal that more than one functional property can be conferred
model (LeBlanc et al. 2005a). The bioavailability of the to the host micro-organisms. By directed mutagenesis fol-
riboflavin produced by this strain was similar to that of lowed by selection and metabolic engineering, folate and
pure riboflavin demonstrating the usefulness of this strain riboflavin biosynthetic pathways were modified in L. lactis
for the development of riboflavin-enriched fermented resulting in simultaneous overproduction of both folate
foods. and riboflavin (Sybesma et al. 2004). According to these

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1300 Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology
J.G. LeBlanc et al. Vitamin production by LAB

authors, novel foods, enriched through fermentation LAB could potentially increase folate levels in milk (San-
using these multivitamin-producing starters, could com- tos et al. 2008b).
pensate the B-vitamin-deficiencies that are common even Another example of LAB producing folates is the com-
in highly developed countries. bination of Strep. thermophilus and Bifidobacterium ani-
malis that increased the levels of this vitamin sixfold
(Crittenden et al. 2003). It is well established that
Folates
Strep. thermophilus strains are dominant producers of
Because folate is involved in essential functions of cell folates in milk, principally producing 5-MTHF, what
metabolism such as DNA replication, repair and methyla- leads to yogurts with more than six times the 5-MTHF
tion and synthesis of nucleotides, vitamins and some content as compared to the control after 12 h of fermen-
amino acids, human life could not exist without it. Folate tation (Holasova et al. 2004). Also it was found that some
deficiency has been implicated in a wide variety of disor- strains of B. longum were moderate producers with a
ders from Alzheimer’s to coronary heart diseases: osteo- maximum increase of 73% in 5-MTHF after this fermen-
porosis, increased risk of breast and colorectal cancer, tation time. On the other hand, Propionibacterium
poor cognitive performance, hearing loss and NTDs (Le- freundenreichii ssp. shermanii strains did not modify folate
Blanc et al. 2007, 2010b; Laiño et al. 2011). In this review, levels. The maximum concentration of 5-MTHF was
the generic term folate will include the complete group of highest between 6 and 12 h of fermentation, then a
all natural folate derivatives, including 5-methyltetrahy- decrease was observed (Holasova et al. 2004).
drofolate (5-MTHF) and folylglutamates that are naturally Micro-organisms are also able to increase folate content
present in foods, but not folic acid, that is the synthetic in a wide variety of other foods. For example, fermentation
form of folate commonly used for food fortification and of rye dough to produce bread is frequently accompanied
nutritional supplements. by increases in folate concentrations (Kariluoto et al.
Considering that milk contains between 20 and 2006), but the increase in this vitamin during fermentation
50 lg l)1 of folate, an average adult person or a pregnant was shown to be mainly because of folate synthesis by
woman would need to consume 6–12 l of milk per day to yeasts, whereas LAB did not produce folate, they consumed
meet their daily requirement, and as this level of con- it. So replacing folate consumers for folate-producing LAB
sumption is unrealistic, it may be helpful to increase could significantly increase folate content in these breads.
folate intake using vitamin-producing micro-organisms to It has also been reported that it is possible to select
prevent the occurrence of folate deficiency. starter cultures of LAB that produce significant amounts
Many industrially important LAB such as L. lactis and of 5-MTHF (to almost twice the basal level) during vege-
Streptococcus thermophilus have the ability to synthesize table fermentation (Jägerstad et al. 2004). It is important
folate (Friend et al. 1983; Lin and Young 2000; Huge- to carefully check the folate concentration in raw vegeta-
nholtz and Smid 2002; Crittenden et al. 2003; Sybesma bles to optimize the entire process. Folate losses during
et al. 2003c; Papastoyiannidis et al. 2006). This explains processing must be limited as much as possible, and opti-
why some fermented dairy products, including yogurt, mizing the conditions to favour the microbiological bio-
contain higher amounts of folate compared with nonfer- synthesis of folates is essential to increase folate levels in
mented milks (see Fig. 1). It was shown that folate con- the final product.
centration in yogurt may be increased to values above Another example of the use of LAB to improve folate
200 lg l)1 (Wouters et al. 2002). However, the ability of content in fermented products is in the fermentation of
microbial cultures to produce or utilize folate varies con- corn flour where an increase in folate level of almost
siderably being a strain-dependent trait. Most authors threefold after 4 days of fermentation at 30C was
claim that Strep. thermophilus normally produce folates achieved (Murdock and Fields 1984).
whereas Lactobacillus delbrueckii subsp. bulgaricus is a Some studies performed with the aim to determine
folate consumer, so the selection of adequate combination whether the exogenous vitamin can affect folate synthesis
of strains is essential to develop fermented foods with by bacteria have shown that production is strain depen-
increased vitamin concentrations. dent; some bifidobacteria did not produce folate when
Not only Strep. thermophilus and L. lactis have the abil- this vitamin was already present, whereas others produced
ity to produce folates, but also other LAB like Lactobacil- it regardless of the vitamin concentration. This suggests
lus acidophilus and Lact. plantarum have been reported to that in some strains folate biosynthesis might not be reg-
produce folate in CDM (LeBlanc et al. 2010b) as have ulated; this was confirmed by the finding that the final
Leuconostoc lactis and Bifidobacterium longum. Also, Lacto- concentration of this vitamin was at least 50-fold higher
bacillus reuteri JCM1112, a well-known producer of vita- than the requirement after bacterial growth of the folate-
min B12, can produce high quantities of folates, so this producing strains (Pompei et al. 2007).

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Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology 1301
Vitamin production by LAB J.G. LeBlanc et al.

Different forms of folates are produced by LAB; some tase decreased (by 50%) the production of total folates.
even produce folates with more than three glutamyl resi- In addition, it was observed that the combined overex-
dues. In L. lactis, up to 90% of the total produced folate pression of folKE and folC favoured the accumulation of
remains in the cell as 5,10-methenyl-THF and presumably intracellular folate (Sybesma et al. 2003b). Furthermore,
10-formyl-THF, both with four, five or six glutamate resi- the overexpression of GTP cyclohydrolase I showed a very
dues (Sybesma et al. 2003c). In Strep. thermophilus, much promising potential to increase the flux through the folate
less was shown to remain in the cell, with this folate biosynthesis pathway. In consequence, the appropriate
being present as 5-formyl-THF and 5,10-methenyl-THF, combination of the overexpression of folKE with the
both with three glutamate residues. The differences in dis- increased or decreased expression of other folate biosyn-
tribution can probably be explained by the different thesis genes can significantly increase folate production
length of the polyglutamyl tail in the two micro-organ- (Sybesma et al. 2003b).
isms. One of the main functions of the polyglutamyl tail An example of that is the significantly improved folate
is thought to be the retention of folate within the cell. status in deficient rats that was shown upon supplementa-
The cell retention of folate can be a result of the negative tion with L. lactis overexpressing the folC, folKE or
charge of the carboxyl groups of (polyglutamyl) folate folC + folKE genes (LeBlanc et al. 2010a). The biosafety
(pKa of 4Æ6). Moreover, in Strep. thermophilus, the intra- assessment of these genetically modified LAB (GM-LAB)
and extracellular folate distribution was influenced by the was performed and demonstrated that there were as safe
pH. Cells that grew at low pH had a larger extracellular as the native strains from which they were derived (Le-
folate fraction than cells that were cultured at high pH. Blanc et al. 2010c).
The explanation can be that at low intracellular pH, folate Increases in folate production can be performed not
is protonated and so became electrically neutral, enhanc- only by overexpressing the genes involved in the biosyn-
ing transport across the membrane. In the case of L. lac- thesis, but also by overexpressing other genes involved in
tis, pH did not seem to affect intra- and extracellular the biosynthesis pathway of related metabolites. For
folate distribution (Sybesma et al. 2003c). example, the overproduction of pABA did not lead to ele-
The genes for folate biosynthesis have been identified vated folate pools on its own (Wegkamp et al. 2007).
in L. lactis (Sybesma et al. 2003a), in Lact. plantarum However, simultaneous overexpression of the pABA and
(Kleerebezem et al. 2003) and in Lact. delbrueckii ssp. bul- the folate biosynthesis gene clusters reached high folate
garicus (van de Guchte et al. 2006), but in the last one, levels (Wegkamp et al. 2007), which did not depend of
some of them are missing. Not every Lactobacillus is able pABA supplementation. The overproduction of pABA led
to produce folate because the genes involved in folate bio- to relatively low intracellular folate pools and a relatively
synthesis are lacking in the genome; this is the case for high secretion of folate. There exists a very tight correla-
Lactobacillus gasseri (Wegkamp et al. 2004), Lactobacillus tion between folate and pABA biosynthesis that was
salivarius (Claesson et al. 2006), Lact. acidophilus and Lac- shown through deletion of the pABA genes in L. lactis
tobacillus johnsonii (van de Guchte et al. 2006). where in consequence its ability to synthesize folate was
In cells, the polyglutamyl form is the main form as eliminated, causing a complete inability to grow in the
folate-dependent enzymes have increased affinity for poly- absence of purine nucleobases ⁄ nucleosides.
glutamyl folates compared with the monoglutamyl forms. In other trials, Lact. gasseri ATCC 33323 was converted
The enzyme responsible for polyglutamyl folate synthesis from being a folate consumer into a highly efficient
and the corresponding elongation of the chain is poly- folate-producing strain (Wegkamp et al. 2004). In this
glutamyl synthetase, encoded by the folC gen in L. lactis. strain, the folate biosynthesis genes are not present,
All sequenced microbial genomes (even those of strains except for folA and folC, which are involved in the regen-
not able to produce folate) possess folC or a homologous eration and retention of reduced folates absorbed from
gene (Sybesma et al. 2003b). the medium. When a plasmid containing the complete
Through metabolic engineering, it is possible to folate gene cluster (folA, folB, folKE, folP, ylgG and folC)
increase folate levels in L. lactis (Sybesma et al. 2003b; from L. lactis MG1363 was introduced into Lact. gasseri
Wegkamp et al. 2007), Lact. gasseri (Wegkamp et al. ATCC 33323, the resulting recombinant strain was con-
2004) and Lact. reuteri (Santos et al. 2008b). By control- verted into a folate-producing bacterium (Wegkamp et al.
ling the overexpression of folKE genes in L. lactis that 2004).
encode 6-hydroxymethil-dihydropterinpyrophosphokinase Although it is useful during technological applications
(folK) and GTP cyclohydrolase (folE) results in a tenfold to increase folate production, a recent study has been
increased production of extracellular folate and a three- shown that folate overproduction in Lact. plantarum
fold increased production of total folates; meanwhile, WCFS1 significantly reduced the growth rate of this
overexpression of folA that encode dihydrofolate reduc- micro-organism (Wegkamp et al. 2010). Even when folate

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1302 Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology
J.G. LeBlanc et al. Vitamin production by LAB

overproduction led to very little change in metabolite (especially liver and kidney), fish and eggs, or pharmaceu-
levels or overall transcript profile, the growth rate in tical products. Vitamin B12 deficiency can cause different
Lact. plantarum was reduced drastically, most likely pathological manifestations that affect the haematopoietic,
because the growth-related transcripts and proteins are neurological and cardiovascular system, amongst others.
diluted by the enormous amount of gratuitously produced One of the most extreme forms of B12 deficiency is known
folate-related transcripts and proteins (Wegkamp et al. as pernicious anaemia that is not normally associated with
2010). The results obtained in this study will be helpful in diet but rather with problems in the gastric system caused
designing future genetic engineering strategies taking into by a lack of production of a gastric glycoprotein called
account that transcript numbers can affect growth. intrinsic factor that facilitates the absorption of the vita-
In animal studies, it was shown that low-folate diets min in the small intestine (Beck 2001).
are associated with an elevated risk of colorectal cancer As indicated previously, only bacteria and archaea are
and that folic acid would suppress the growth of the can- able to synthesize vitamin B12, although relatively few can
cer (Giovannucci 2002). The use of folate-producing pro- synthesize it de novo. One of the first model organisms used
biotics have recently been proposed to efficiently confer for the study of B12 biosynthesis was P. freudenreichii that
protection against inflammation and cancer, both exerting is used in the industrial production of the vitamin. To cir-
the beneficial effects of probiotics and preventing the cumvent the instability of the biosynthetic intermediates,
folate deficiency that is associated with premalignant the aerobic B12-producing bacterium Pseudomonas denitrif-
changes in the colonic epithelia (Rossi et al. 2011). How- icans has been used for the isolation of various intermedi-
ever, large discrepancies exist between folate supplementa- ates and the characterization of the majority of the genes
tion and cancer prevention: some researchers have and corresponding products involved in the biosynthesis of
demonstrated a clear reduction in the risk of recurrence this vitamin (Battersby 1994; Thibaut et al. 1998).These
of adenomas with increased folate intakes, whereas others studies concluded that the biosynthesis of cobalamin could
observe the complete opposite (Carroll et al. 2010). It is be performed under either aerobic (oxygen dependent) or
all these conflicting results that have raised concerns the anaerobic (oxygen independent) conditions.
implementation of new fortification policies of many The anaerobic route was observed in the strains of
countries (Ulrich 2008). It is thus very important to P. freudenreicchii, Salmonella enterica and Bacillus megate-
establish the risk-benefit relationship of folate and folic rium (Warren et al. 2002; Warren 2006; Escalante-Seme-
acid supplementation before proposing their use as a rena 2007). The initial characterization problems were
chemopreventive agent. primarily due to the fact that the central Co2+ ion was
inserted into the corrinoid ring in an early step that
generated unstable intermediates that were difficult to
Vitamin B12
isolate. On the other hand, in the oxygen-dependent
The term vitamin B12 is generally used to describe a type route, cobalt is inserted in a later stage creating more sta-
of cobalt corrinoid, particularly of the cobalamin (cbl) ble intermediates.
group. In strict terms, vitamin B12 is the form of the vita- Because of the complexity of B12 biosynthesis and the
min obtained during industrial production and which does limitations of the scope of this chapter, interested readers
not exist naturally (Rucker et al. 2001). Cyanide stabilizes are invited to read excellent reviews that have been pub-
the molecule during the extraction procedure from micro- lished on this subject (Raux et al. 2000; Scott 2003;
bial cultures, forming cyanocobalamin. In its natural form, Roessner and Scott 2006; Escalante-Semerena 2007).
the vitamin is present principally as desoxyadenosilcobal- It was shown that Lact. reuteri CRL1098 was able to
amin (coenzyme B12), methylcobalamin or pseudocobal- metabolize glycerol in a B12-free medium; this being the
amin, amongst other forms. Animals, plants and fungi are first hint that a LAB might be able to produce cobalamin
incapable of producing cobalamin; it is the only vitamin (Taranto et al. 2003). The chromatographic analysis of
that is exclusively produced by micro-organisms, particu- the intracellular bacterial extract of Lact. reuteri CRL 1098
larly by anaerobes (Roth et al. 1996; Martens et al. 2002; confirmed that this strain was able to produce a cobala-
Smith et al. 2007). Furthermore, biochemical and genomic min-like compound with an absorption spectrum closely
data indicate that only a few bacteria and archaea possess resembling that of standard cobalamin but with a differ-
the ability to produce this vitamin (Roth et al. 1996; Ro- ent elution time, while cobalamin production was con-
dionov et al. 2003). Adult ruminant animals and strict veg- firmed using different bioassays (Taranto et al. 2003).
etarians can obtain the vitamin in specialized bacteria Genetic evidence of cobalamin biosynthesis by
present in the rumen. Humans, however, do not harbour Lact. reuteri CRL 1098 was then obtained through the use
such microbes in their small intestine and must absorb the of different molecular biology techniques, and it was
coenzyme from natural sources such as animal meats shown that at least 30 genes are involved in the de novo

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Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology 1303
Vitamin production by LAB J.G. LeBlanc et al.

synthesis of the vitamin. The genetic organization (cob from the end of the gestation period to weaning (Molina
and cbi genes) are very similar to those of Salm. enterica et al. 2008). In this experimental animal model, vitamin
and Listeria innocua (Santos et al. 2007). B12 deficiency caused a significant reduction in the hae-
One distinctive characteristic of the cob cluster of matological parameters (haemoglobin, haematocrit and
Lact. reuteri is the presence of hem genes in the middle of reticulocytes values) and anthropometric alterations in
the cluster. In the respiratory organisms Listeria and Sal- pregnant females compared with the control animals,
monella with similar cob clusters, the hem genes are which were fed a B12-sufficient diet. Moreover, the defi-
located at other positions of their genome. The presence cient females gave birth to smaller numbers of offspring,
of the hem genes in the cob cluster is a characteristic that which also showed growth retardation (smaller size) and
has only been observed in certain genomes of Clostridium a decrease in haematological values with associated histo-
(Rodionov et al. 2003). Recently, the transcription of a logical alterations in the small intestine and a decrease in
vast set of genes involved in cobalamin synthesis in sour- the number of IgA-producing cells of the females and in
dough prepared with strain Lact. reuteri ATCC 55730 was their offspring. This experimental model of murine
described (Hufner et al. 2008). females and their offspring allowed the assessment of the
In addition to CRL1098, other Lact. reuteri strains were incidence of maternal cobalamin deficiency in offspring,
shown to be capable of producing some corrinoids such as probably also representing a useful tool to evaluate the
Lact. reuteri DCM 20016 (Santos et al. 2008a), JCM1112 efficiency of functional foods containing B12-producing
(Santos et al. 2008b) and CRL 1324 and 1327, strains iso- micro-organisms to prevent the nutritional deficit of
lated from human vagina (Vannini et al. 2008). After the cobalamin. Using this model it was shown that Lact. reu-
detection of B12 production by Lact. reuteri CRL1098 and teri CRL 1098 was able to revert vitamin B12 deficiency,
the study of its cobalamin biosynthesis cluster, the geno- demonstrating the bioavailability of the vitamin produced
mic sequence of two strains of Lact. reuteri, with different by this strain (Molina et al. 2009). It is known that when
characteristics, was released by the Joint Genome Institute: Lact. reuteri CRL 1098 is grown in strict anaerobiosis, this
Lact. reuteri F275 (type stain DSM20016) isolated from micro-organism produces almost exclusively pseudocobal-
human faeces that is unable to colonize mice and amin (Santos et al. 2007), a variant of the vitamin that
Lact. reuteri 100-23 isolated from the mouse intestine. appears to be inactive in animals, though, relevant in the
Curiously, comparative genomic data revealed that the microbial studies. Salmonella enterica serovar typhimuri-
strain isolated from the human intestine (DSM 20016) um, a B12-producing facultative anerobic bacterium, syn-
contains the cobalamin biosynthesis cluster, which is asso- thesizes pseudocobalamin in anaerobiosis, but in
ciated with the anaerobic catabolism of glycerol (or 1,2- microaerophilic conditions the coenzyme B12 is also pro-
propanediol), whereas the mouse strain (100-23) neither duced (Keck et al. 1998). Preliminary studies from our
contained the cob nor the glycerol metabolism genes. Based laboratory indicate that an additional corrinoid com-
on the horizontal transfer hypothesis of the cob-pdu clus- pound (with an absorption spectrum similar to cobala-
ter, it would be expected that other strains of LAB would min) is produced when Lact. reuteri is grown in
also have received this genomic island by one of the many microaerophilic conditions (V. Vannini, G. Font de
mechanisms of genetic transfer. Notably, of the current Valdez, P. Taranto and F. Sesma, unpublished data).
sequenced genomes of LAB, only Lact. reuteri contain These results may explain the results from the biodispon-
the pdu-cob genes (DSM20016 ⁄ JCM1112 and the Biogaia ibility assays and emphasize the need for further meta-
strain Lact. reuteri ATCC55730). Recently, a reuterin- bolic studies to establish better conditions for the
producing strain of Lactobacillus coryniformis isolated from production of the active form of the vitamin.
goat milk was characterized and was shown to produce a A common method to improve B12 yields is random
cobalamin-type compound (Martin et al. 2005). Prelimin- mutagenesis and the use of genetic engineering (Martens
ary genetic and biochemical data from our laboratory et al. 2002; Burgess et al. 2009). Different metabolic engi-
(Vannini et al. 2008) indicate that the cob-pdu cluster is neering strategies have been applied to increase vitamin
indeed present in other lactobacilli (Lact. coryniformis and B12 production in P. freudenreichii (Piao et al. 2004a,b). A
Lactobacillus murinus). The possibility of various vitamin recombinant P. freudenreichii strain harbouring a plasmid
B12-producing strains and species of LAB is important for containing hemA, from Rhodobacter sphaeroides, and ho-
future studies on cobalamin production, not only in evolu- mologues of hemB and cobA showed 2Æ2-fold overproduc-
tionary studies to address how the cob-pdu genomic island tion of vitamin B12 (Piao et al. 2004b). These studies
was acquired, but also to explore its potential application show that multigene expression systems improve the vita-
in the development of products that contain B12. min B12 production levels in propionibacteria. On the
Recently, a vitamin B12-deficient murine experimental other hand, no similar studies have been conducted in
model was developed to evaluate maternal B12 deficiency lactobacilli. Knowledge of multiple genomic sequences of

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1304 Journal of Applied Microbiology 111, 1297–1309 ª 2011 The Society for Applied Microbiology
J.G. LeBlanc et al. Vitamin production by LAB

Lact. reuteri B12-producing strain will facilitate the design none-producing LAB strains identified by Morishita et al.
of strategies for constructing food-grade strains with could thus be useful to supplement vitamin K require-
enhanced capacity to produce this essential vitamin. ment for humans.

Other B-group vitamins Conclusions

Besides riboflavin, folate and vitamin B12, increased levels This review describes the possibility to increase vitamin
of other B-group vitamins such as niacin and pyridoxine concentrations in fermented foods through judicious
have also been reported as result of the LAB fermentation selection of the microbial species and cultivation condi-
in yoghurt, cheeses and other fermented products (Shah- tions. It is expected that the food industry will take the
ani and Chandan 1979; Alm 1982). The concentration of next step to use this information for selecting vitamin-
thiamine in milk was also positively influenced (11% producing strains as part of their starter cultures to pro-
increase) following 48 h of fermentation with B. longum duce fermented products with elevated levels of these
(Hou et al. 2000). Recently, it was shown that a slight essential compounds. Such products would provide
(but not statistically significant) increase in the thiamine economic benefits to food manufacturers as increased
and pyridoxine concentration occurred as a result of soy ‘natural’ vitamin concentrations would be an important
fermentation with Strep. thermophilus ST5 and Lactobacil- value-added effect without increasing production costs.
lus helveticus R0052 or B. longum R0175 (Champagne Consumers would obviously benefit from such products
et al. 2010). These authors state that their strains were as they could increase their vitamin intakes while con-
not as efficient, as those reported in literature, but clarify suming them as part of their normal diet.
that in these previous studies, fermentations had been With the increased availability of genome sequences
carried out in milk-based products, and results were not and the development of novel engineering tools for LAB,
subjected to statistical analyses. The authors then the manufacture of products using GM vitamin-produc-
conclude that this was the first study on thiamine and ing strains is now possible, and these are a viable cost-
pyridoxine amounts in soy beverages fermented by pure effective alternative to current vitamin fortification
cultures of Lact. helveticus and Strep. thermophilus. programmes. The ultimate use of such GM-LAB may rely
on the regulatory acceptance of genetically modified
organisms in nutrition and nutraceutical preparations.
Other vitamins
Undoubtedly, consumers will play a decisive role in such
Although B-group vitamins are the most commonly stud- a decision, and their position should be guided by the
ied because of their importance in nutrition and general safety status of such genetically modified organisms and
metabolism, a few reports have shown that other water- their associated, scientifically proven health benefits.
soluble vitamins can be produced by LAB. In one such
study, LAB were examined for their ability to produce
Acknowledgements
quinone compounds as vitamin K occurs naturally in two
forms, namely, K1 (phylloquinone) in green plants and The authors would like to thank the Consejo Nacional de
K2 (menaquinones) in animals and some bacteria (Mo- Investigaciones Cientı́ficas y Técnicas (CONICET), Agen-
rishita et al. 1999). Lactococcus lactis ssp cremoris (three cia Nacional de Promoción Cientı́fica y Tecnológica
strains), L. lactis ssp lactis (two strains) and Leuc. lactis (ANPCyT) and the Consejo de Investigaciones de la
were selected as high producers of quinone that synthe- Universidad Nacional de Tucumán (CIUNT) for their
sized more than 230 nmol of quinones g)1 of dried cells. financial support. Dr van Sinderen is a member of the
These strains, when grown either in reconstituted nonfat Alimentary Pharmabiotic Centre, which is a research cen-
dry milk or in a soymilk medium, produced a beneficial tre funded by Science Foundation Ireland (SFI; Grant
quantity for dietary supplement (i.e. 29–123 lg of men- no.s 02 ⁄ CE ⁄ B124 and 07 ⁄ CE ⁄ B1368), through the Irish
aquinones l)1 of the fermented medium) (Morishita et al. Government’s National Development Plan.
1999). Vitamin K is an essential cofactor for the forma-
tion of c-carboxyglutamic acid (Gla) residues in proteins,
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