Health
Scientific Committees
Scientific Committee on Food
Outcome of discussions
Opinion on
Riboflavin as a colouring matter authorized for use in
foodstuffs produced by fermentation using genetically
modified
bacillus subtilis (expressed on 10 december
1998)
Terms of Reference
To advise on the safety in use of
riboflavin, produced by fermentation using genetically
modified
Bacillus subtilis, as a colouring matter authorized
for use in foodstuffs intended for human
consumption.
Introduction
The Committee has previously reported on
the safety in use of riboflavin and its metabolite
riboflavin-5'-phosphate as a colouring matter for
foodstuffs for human consumption (1). It has now been
requested to review the microbiological and toxicological
safety of riboflavin produced by fermentation as a
colouring matter for foodstuffs generally.
Background
Riboflavin (vitamin B
2) is a water-soluble vitamin, that is
synthesised by plants and many microorganisms but is not
produced by higher animals. It is an essential
micronutrient in the human diet, where it serves as
precursor to coenzymes e.g. flavine adenine dinucleotide
and flavin mononucleotide, which function as hydrogen
carriers in biological redox processes.
Riboflavin occurs naturally in peas,
beans, grains, yeast, milk, egg yolk and liver. Hitherto
riboflavin has been chemically synthesised for use in food
fortification and in small amounts as a colouring agent in
foods e.g. ice cream, processed meat, fish products, sauces
and soups. Riboflavin is also used in the fortification of
animal feedstuffs.
Although it has been known for many
years that riboflavin could be produced by bacteria using
fermentation technology, it was not until recently that a
very pure product could be produced using a genetically
modified strain of
Bacillus subtilis. The pathway of biosynthesis of
riboflavin in
B. subtilis involves 6 steps controlled by enzymes
and the precursors guanosine triphosphate and
ribulose-5-phosphate.
To evaluate the possible microbiological
risks for consumer health the microbiological status of the
production process requires consideration with regard to
the safety of the producer organism as well as the
fermentation process and product purification procedure.
Although riboflavin is not a Novel Food per se the safety
assessment can be performed following the strategy proposed
by the SCF in its Guide to the Assessment of Novel Foods
(2), particularly with reference to the section on Novel
Foods derived from genetically modified
microorganisms.
Producer organism
B. subtilis is an aerobic endospore-forming
bacterium commonly found in nature and generally not
considered to have a pathogenic or toxigenic potential.
There is a history of safe use of this bacterium in
large-scale fermentation production of specialty chemicals,
of enzymes used in food production processes, and of
several traditional relationships to food. It is used
traditionally in East Asia for the fermentative production
of Natto from wheat starch, a product also obtainable in
western countries. It is thus an organism with a tradition
in food use although the actual food product is little
known in the European Union. The organism is also involved
in many types of food spoilage.
The strain of
B. subtilis used in riboflavin production is
RB50::[pRF69]
n[pRF93]
m.Ade+ (3), a non-sporing derivative of
B.subtilis 168, which had been produced by classical
mutation and into which the two specific plasmids pRF69 and
pRF93 had been chromosomally, and thus stably, integrated
into the bacterial genome and amplified 20-25-fold and
10-15-fold respectively. The strain was identified by the
Deutsche Sammlung von Mikroorganismen (DSM Braunschweig,
Germany) as
Bacillus subtilis. The genetic modification includes
the chromosomal integration of the following
elements:
- pUC 19, a derivative of the common
E. coli cloning vector pBR 322, harbouring a DNA
fragment carrying the
rib operon and the
bla gene encoding ampicillin resistance, which
latter is not expressed in
B. subtilis.
- SPO1-15, the constitutive promoter
derived from a
B. subtilis bacteriophage which enables efficient
expression of the
rib operon.
- the marker gene
tet, encoding tetracycline resistance, originally
derived from
Bacillus cereus.
- the marker gene
cat, encoding chloramphenicol resistance,
originally derived from
Staphylococcus aureus.
The
tet as well as the
cat gene can be considered to be constituents of the
natural gene pool of a wide range of Gram-positive
organisms including
B. subtilis. The chromosomal organisation of the
gene inserts has been demonstrated by their functionality
as well as by southern blot analysis. These data were
supplemented by sequencing of specific fragments.
The fermentation process
The producer organism is grown by
controlled submerged growth in well-defined media
containing accepted components such as carbohydrates,
nitrogen sources, mineral salts, an antifoam agent and the
antibiotics chloramphenicol and/or tetracycline in the
inoculation media.
The production process uses inoculum
build-up followed by a feed-batch production fermentation.
It is carried out in contained vessels using axenic
large-scale microbiological techniques. The fermentation is
routinely tested to ensure cultural purity, the quality
control as described can be considered to be of the highest
standard to exclude contamination. The final fermentation
process will be performed in practice in compliance with
Good Industrial Large-Scale practice.
Down-stream processing
After fermentation the whole broth is
pasteurised to ensure that no viable cells of the
production organism are present in the final product. The
further processing is designed to remove cell debris, DNA
and contaminants. Hydrochloric acid is added as the only
purification agent. The concentration of acid and the
temperature conditions chosen were shown to degrade any DNA
still present, so that it can no longer serve as a
substrate in transformation or as a template in a
polymerase chain reaction. After removal of any residual
biomass the purity of the final product is 96% and this
material after washing and drying can then be used as such
in animal feed. Further processing with hot hydrochloric
acid produces a material of 98% purity (food grade). This
final purification step is identical with that currently
used for producing a chemically synthesised material of the
same 98% purity. Comparison of the HPLC chromatograms of
the chemically synthesised and the fermentation product
confirmed that no further impurities had resulted from the
fermentation process, the main degradation product
resulting from the acid treatment being
8-hydroxymethylriboflavin (4). The final product meets the
specification for riboflavin used as colouring matter for
foodstuffs for human consumption listed in Commission
Directive 95/45 EC (3).
Intake
The riboflavin produced by fermentation
is intended to be used for the same purposes as the
chemically synthesised product. No increased consumption is
expected as no new applications exclusive to the
fermentation-derived product are anticipated.
Nutritional aspects
As the synthetic product and the
fermentation product have identical specifications and
quality criteria and the same application levels, it is
unlikely that there will be any difference in
bioavailability, when one product is substituted for the
other. Hence a nutritional assessment is
unnecessary.
Toxicological aspects
The 96% fermentation product (feed
grade), the 98% fermentation product (food grade) and the
98% chemically synthesised product were examined in a
13-week oral feeding study in Wistar rats at doses of 20,
50 or 200 mg/kg b.w. There were no significant differences
between test and control groups regarding clinical signs,
feed consumption and water intake. There was a 6%
retardation of growth of females given 200 mg/kg b.w. of
the 98% fermentation product. No consistent or dose-related
changes in haematological parameters, urinalysis or
clinical chemistry were noted. Females given 200 mg/kg b.w.
of the 98% fermentation product showed a slight increase in
relative liver weight and males given 50 and 200 mg/kg b.w.
fermentation product showed a slight increase in relative
spleen weight, neither of these weight increases being
dose-related nor associated with any histopathological
findings. Gross and histopathology showed no significant
treatment-related lesions in any test group. The observed
changes were considered to be of no toxicological concern
(5)
A bacterial microsomal reverse mutation
test in
S. typhimurium was negative with and without S9 mix
over a dose range of 50-5000 µg/plate for the 96% and 98%
fermentation product (6,7)
Evaluation
The only genetic modifications of the
producer organism
B .subtilis concerned the introduction of the 2
genetic constructs containing the
rib operons, the
tet and
cat genes and the selected mutational changes for
deregulating the purine and riboflavin pathways and also
for preventing sporulation. PCR analysis, southern blot
analysis and sequencing of specific fragments showed that
the expected composition of the modified genome remained
stable under production conditions and that biologically
active DNA of the production strain was no longer present
in the fermentation-derived purified material. A
hypothetical genetic transfer to the human consumer or the
gut microorganisms of the novel genes in GM
B. subtilis following consumption of
fermentation-derived riboflavin could not occur since the
purification process destroyed any DNA that may have been
present.
There are no indications that the
impurities present in the fermentation-produced riboflavin
were different from those occurring in the chemically
synthesised riboflavin and were also shown with existing
analytical methods to be present at similar levels.
The toxicity study and the mutagenicity
assay showed no toxicologically relevant adverse effects
either in the 98% fermentation product or the 98% synthetic
material. On microbiological-hygienic grounds there are no
objections to the acceptance of the fermentation product.In
conclusion the Committee considered that riboflavin
produced by fermentation using genetically modified
Bacillus subtilis is acceptable as a food
colour.
References
1. SCF (1977) Fourth series of Reports,
European Commission, Luxembourg
2. SCF (1997) Fourtieth series of
Reports, European Commission, Luxembourg
3. Advisory Committee on Novel Foods and
Processes (1996) Annual Report, UK
4. Simon, Vogt (1989) Roche Research
Report BS/GCR 62589 submitted to the
EuropeanCommission
5. Buser, Hofmann et al. (1995) Roche
Research Report B 161165 submitted to the European
Commission
6. Albertini, S. (1995) Roche Research
Report B 164571 submitted to the European Commission
7. Albertini, S. (1995a) Roche Research
Report B 164572, submitted to the European
Commission
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