Beruflich Dokumente
Kultur Dokumente
Review
Department of Food Safety, Teagasc-Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland
NIZO food research, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands
c
TI Food & Nutrition. Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands
d
Department of Microbiology and Alimentary Pharmabiotic Centre, Biosciences Institute, National University of Ireland Cork, Western Road, Cork, Ireland
b
a r t i c l e
i n f o
Article history:
Received 25 December 2008
Received in revised form 12 April 2009
Accepted 14 April 2009
Keywords:
Riboavin
Folic acid
Cobalamin
Overproduction
a b s t r a c t
Consumers are becoming increasingly health conscious and therefore more discerning in their food choices.
The production of fermented food products with elevated levels of B-vitamins increase both their commercial
and nutritional value, and eliminate the need for subsequent fortication with these essential vitamins. Such
novel products could reduce the incidence of inadequate vitamin intake which is common in many parts of
the world, not only in developing countries, but also in many industrialised countries. Moreover, the concept
of in situ fortication by bacterial fermentation opens the way for development of food products targeted at
specic groups in society such as the elderly and adolescents. This review looks at how vitamin
overproduction strategies have been developed, some of which have successfully been tested in animal
models. Such innovative strategies could be relatively easily adapted by the food industry to develop novel
vitamin-enhanced functional foods with enhanced consumer appeal.
2009 Elsevier B.V. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . .
Riboavin . . . . . . . . . . . . . . . . . . . . .
2.1.
Riboavin biosynthesis and overproduction . .
3.
Folate . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Folate biosynthesis and overproduction . . . .
4.
Vitamin B12 (cobalamin) . . . . . . . . . . . . . .
4.1.
Vitamin B12 biosynthesis and overproduction .
5.
Concluding comments . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
In the developed world consumers are increasingly health conscious and aware of their nutritional requirements. Meanwhile, in
the developing world malnutrition can result in dietary deciencies, particularly in vitamins, which may cause a variety of health
disorders.
Vitamins are generally classied into two groups, the fat-soluble
vitamins, which include vitamins A, D, E and K, and the water-soluble
vitamins, which include vitamin C, biotin (vitamin H or B7) and a
series of B vitamins thiamin (B1), riboavin (B2), niacin (B3),
pantothenic acid (B5), pyridoxine (B6), folic acid (B11) and cobalamin
Corresponding author.
E-mail address: kaye.burgess@teagasc.ie (C.M. Burgess).
0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.04.012
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1
2
2
3
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4
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6
6
Fig. 1. Riboavin biosynthesis in prokaryotes. The E. coli genes encoding each enzymatic activity are indicated on the left while the corresponding B. subtilis genes are indicated to the
right. Adapted from Bacher et al. (1996) and Perkins and Pero (2002).
fortied with folic acid (Food & Drug Administration, 1996) and over
forty countries worldwide have introduced mandatory fortication
of foods. Despite the observed benecial effects of folic acid supplementation, fortication may have adverse affects in subpopulations,
such as masking vitamin B12 deciency, primarily in the elderly
(Cuskelly et al., 2007). Furthermore, there is conicting evidence in
the relationship of folate and cancer and for these reasons many
European countries have thus far not adopted mandatory folic acid
fortication (Bailey et al., 2003, Food Safety Authority of Ireland,
2008). The recommended daily intake of folic acid is 400 mg of dietary
folate equivalents (DFEs). DFEs adjust for the nearly 50% lower
bioavailability of naturally occurring food-derived folate compared
with that of synthetic folic acid (Food and Nutrition Board, 1998c).
A shortage of folate compromises DNA replication and cell division,
and therefore one of the noticeable effects of folate deciency is the
occurrence of abnormalities of cells which have the highest turnover
rates in tissues such as the intestinal mucosa, regenerating liver and
bone marrow. The most obvious manifestations of folate deciency
are seen in white and red blood cells. Dietary folate deciency is the
leading cause of megaloblastic anaemia in the world (Gallagher and
Ehrenkranz, 1995). The folic acid status of expectant mothers is also
known to affect the incidence of neural tube defects such as spina
bida and anencephaly in the unborn infant, where many studies have
shown consistent protective effects of folates (recently reviewed by
Pitkin (2007)). Folates have also been shown to reduce homocysteine
concentrations in the blood, thereby possibly playing a role in the
prevention of heart disease (Verhoef et al., 1998).
As mentioned above, unlike mammals many microorganisms and
plants possess the ability to synthesise folic acid derivatives de novo in
which the end product is tetrahydrofolate. Because of this exclusive
ability, the biosynthetic pathway has been used as a drug target in the
treatment of diseases such as malaria or Pneumocystis (Pn.) carinii
infections (Anderson, 2005; Fulton et al., 1995), although in the case of
malaria the causative agent, Plasmodium (Pl.) falciparum, has been
reported to develop resistance to these drugs (Gatton et al., 2004;
Sibley et al., 2001).
3.1. Folate biosynthesis and overproduction
3. Folate
Folate, also known as vitamin B11, is the generic descriptor for
folic acid (pteroylmonoglutamic acid) and related compounds
exhibiting the biological activity of folic acid. Folates can consist of
a mono- or polyglutamyl conjugate and these compounds are named
after the number of glutamyl residues (PteGlun), where n denotes
the total number of glutamyl residues. The folates function as enzyme
co-substrates in one-carbon (C1) metabolism of amino acids and
nucleotides where the fully reduced (tetrahydro-) form serves as an
acceptor or donor of a single carbon unit (Combs, 1992). The most
signicant role of folic acid appears to be its involvement in the synthesis of purines and pyrimidines, and therefore in DNA synthesis.
Methionine synthase uses 5-methyltetrahydrofolate in the conversion of l-homocysteine to l-methionine (Brody, 1991). Much of the
methionine formed is converted to S-adenosylmethionine, a universal donor of methyl groups for DNA, RNA, hormones, neurotransmitters, membrane lipids and proteins (For a comprehensive
overview see Selhub (2002)).
Folic acid is synthesised by both microorganisms and plants. Folate
biosynthesis requires the coupling of the pteridine molecule to paminobenzoic acid (pABA) to form pteroic acid. Mammals, which
synthesise large amounts of pteridines, cannot catalyse this coupling
and are therefore auxotrophic for the vitamin (Baku and Dickerson,
1996). The main dietary sources of folate are leafy, green vegetables,
dairy products and cereal products, especially fortied breads and
breakfast cereals (Finglas et al., 2003). In the USA the FDA ruled that it
is mandatory for all our and uncooked cereal grain products to be
Dihydrofolate is synthesised from the precursors GTP, p-aminobenzoate (pABA) and glutamate, in seven enzymatic steps (Fig. 2).
The ATP-consuming conversion step of 6-hydromethylneopterin
to its pyrophosphate ester is catalysed by FolK in B. subtilis and has
been shown to represent the rate-limiting step in folate (over)production (Perkins and Pero, 2002). In L. lactis this step is catalysed by a
bifunctional enzyme whose overexpression was shown to result in
a more than two fold increase in total folate production (Sybesma
et al., 2003a).
The second part of the folate biosynthetic pathway is the production of pABA. Exogenously added pABA can support growth of
certain folic acidauxotrophic strains (Green et al., 1996), while pABA
supplementation of the growth medium of L. lactis was shown to
result in a two-fold increase in folate production (Sybesma et al.,
2003b), pointing to an as of yet unidentied uptake system for this
compound. However, while overexpression of the pABA genes alone
does not increase folate production levels in L. lactis, folate overproduction is observed when pabBC overexpression is combined with
overexpression of the folate biosynthetic genes (Wegkamp et al.,
2007).
Industrially, folate is primarily synthesised via chemical synthesis
due to the low yields of folic acid produced by current bacterial strains.
However, there may be technological advantages to microbial
production of folate. These include the fact that microbially produced
folate does not require reduction prior to use by humans and the more
environmental friendly character of biological production as compared to chemical synthesis (Zhu et al., 2005).
Fig. 2. Folic acid biosynthetic pathway in prokaryotes. The genes known to encode the enzymes in E. coli are shown on the left while those in known to encode the enzymes in B.
subtilis are shown to the right. The enzyme names indicate that the activity has been shown but the corresponding gene encoding it has not been identied. The asterisk denotes that
the gene encoding this enzyme, folQ, has been identied in L. lactis. Adapted from Green et al. (1996), Perkins and Pero (2002) and Sybesma et al. (2004).
biosynthesis. Many excellent reviews describe the biosynthetic pathways for the vitamin (Heldt et al., 2005; Martens et al., 2002; Roessner
et al., 2001; Roessner and Scott, 2006; Scott and Roessner, 2002; Scott,
2003). Most of the work in characterising cobalamin biosynthesis has been performed in Salmonella (S.) typhimurium and Pseudomonas (Ps.) denitricans. Two different pathways exist for adenosylcobalamin (ado-cobalamin) biosynthesis: (1) an oxygen-dependent
pathway, which is found in Ps. denitricans, and (2) an anaerobic
pathway, which has been identied in, among others, S. typhimurium,
P. freudenreichii subsp. shermanii and B. megaterium. All genes
necessary for anaerobic cobalamin biosynthesis have been identied
on the genome of St. sanguinis (Xu et al., 2007). Genes encoding
enzymes contributing to the oxygen-dependent pathway have been
given the prex cob, while those involved in the oxygen-independent
pathway have the prex cbi (Martens et al., 2002). The genes involved
and the reactions which they catalyse in each pathway are listed in
Table 1. Due to the early insertion of cobalt in the anaerobic pathway
the remaining intermediates are cobalto-complexes and therefore
require enzymes with different substrate specicities than the
intermediates in the aerobic pathway although many of the reactions
catalysed are similar. It is clear from Table 1 that most steps of the two
pathways have been elucidated, although the genes encoding some
enzymes remain to be identied. A relatively recent nding is the
identication of CobZ in Rhodobacter (R.) capsulatus, which catalyses
a reaction similar to that promoted by CobG, although it appears that it
mediates this reaction in a different manner, as the two proteins
display no primary sequence similarity. CobZ has also been shown to
contain a avin in the form of a non-covalently bound FAD, two FeS
Table 1
Reactions and genes required for the biosynthesis of adenosylcobalamin.
The genes listed for the oxygen-dependent pathway are from P. denitricans. The genes indicated for the oxygen-independent pathway are from S. typhimurium. The ? indicates that
enzymatic activity has been identied, but that the corresponding gene is still unknown. The major differences in the pathway are highlighted in discriminating colours. Adapted
from Roessner et al. (2001) and Roessner and Scott (2006).
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