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L. & Gripon, J.-C. (1989) Appl. Environ. Microbiol.
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23. Desmazeaud, M. J. & Zevaco, C. (1979) Milchwissenschaft 34,606-6 10
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Microbiol. 56,526-532
25. Exterkate, F. A. & de Veer, G. J. C. M. (1987) Appl.
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26. Niven, G. W. (1991) J. Gen. Microbiol. 137,
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27. Law, B. A. (1979)J. Appl. Bacteriol. 46, 455-463
(1981) Agric. Biol. Chem. 45, 159-165
29. van Boven, A,, Tan, P. S. T. & Konings. W. N. (1988)
Appl. Environ. Microbiol. 54, 43-49
30. Bosman. B. W., Tan, P. S. T. & Konings, W. N.
(1990) Appl. Environ. Microbiol. 56, 1839- 1843
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Received 12 April 1991
Micro-organisms as a novel source of flavour compounds
Mohamad 1. Farbood
International Flavours & Fragrances, Union Beach, N] 07735, U.S.A.
Consumer demand for natural flavour ingredients
for foods and beverages has resulted in substantial
research in production of these ingredients via processes which are considered to be natural. A
number of research papers and patents describing
these approaches have been published [ 1-31. In this
paper, the use of micro-organisms for commercial
production of natural flavour compounds is discussed.
Appropriate
micro-organisms
may
be
obtained from the many culture collections based
on information available in the literature or can be
isolated from nature using selective screening techniques. A survey of disclosed commercial processes
reveals that in most cases a natural precursor was
used which is closely related structurally to the
product. De nouo biosynthesis from substrates such
as carbohydrates, acetate, etc., in most cases has not
resulted in viable commercial processes. In other
words, it seems that controlled catabolic transformation of a precursor is preferred over de nouo biosynthetic pathways for production of natural flavour
compounds.
The historical development of industrial biotechnology may be divided into four eras [4]:(1)
production of foods, such as wine, beer, vinegar,
cheese, yogurt, bread, etc.; (2) large-scale production of specific materials under non-sterile conditions (e.g., organic acids, solvents, biomass, etc.), by
relatively simple technology; (3) production under
Volume 19
sterile conditions, of relatively expensive secondary
metabolites by more complex processes (e.g., penicillin); and (4) application of modern scientific
developments in biotechnology, e.g., enzyme
research, gene technology, molecular biology and
process engineering. Enzyme research has led to
processes involving immobilized enzymes and
whole cells. Application of developments in the field
of molecular biology and gene technology have led
to processes using micro-organisms with specifically altered gene structure. Process engineering has
led to the development of new reactors, optimization of processes, and better measurement and control of process parameters. In the near future
bioengineering advances may lead to computer
control of entire production processes from inoculation to product recovery.
There are several requisites for industrial
micro-organisms [ 51: purity, genetic stability,
amenability to change by mutagenic agents, facile
formation of reproductive units, acceptable growth
rate, and relatively short process time for product
accumulation. In addition, if possible, the strain
should protect itself against contamination, be capable of long-term culture maintenance, and ideally,
minimize or eliminate production of all toxic substances.
Such micro-organisms may either be obtained
from a culture collection or isolated from natural
sources by screening programmes. Screening may
Food Biotechnology
be defined as the application of highly selective
procedures to detect and isolate, from a large population, only those micro-organisms capable of producing the desired metabolite. The greatest
challenges are to establish a suitable assay system
and to determine the criteria for selection and isolation. Available information can be used as a guide,
but the selection, isolation, and improvement of cultures still rest mainly on empirical experience.
Identification of isolated micro-organisms allows a
comparison with those already described in the
literature. Such properties as production of desirable fermentation products, pathogenicity for plants,
animals, or humans, and special precautions for
handling such organisms must be considered.
Identification allows one, to a certain extent, to predict growth characteristics and other requirements
which need to be considered in the study of the isolated micro-organism.
These approaches to commercially successful
microbiological processes can be illustrated with a
few examples taken from the patent literature and
some of our own work. The information available in
published research can lead to development of a
commercial process in a relatively short period of
time. Production of methyl anthranilate is a good
example. Transformation of 4-(methylamino)benzaldehyde to 4-aminobenzaldehyde using Polyp o r n versicolor (Fig. l), had been described
previously [6]. From this work it was evident that
Polyporus species and related micro-organisms were
capable of N-demethylation reactions. This
property was used to advantage in the Ndemethylation of methyl N-methylanthranilate [7].
However, commercially acceptable yields could
only be achieved by optimization of growth conditions and substrate levels, and choice of appropriate
emulsifiers. Two disadvantages to this process are
the lengthy incubation time and the limited availability of methyl N-methylanthranilate. In another
approach to this compound via a bacterial process,
anthranilic acid is produced in high yield using I.tryptophan as the substrate [#]. The acid is subsequently esterified with methanol in a second
process. Disadvantages to this approach are the
availability of natural methanol and the need for the
additional esterification step.
Another example of the use of published
information to develop a commercial process for
flavour compounds in the production of esters by
Geotrichum fragrans [9]. This organism, originally
identified as Oospora suaveolans, was reported to
produce small amounts of ‘fruity’ esters [lo]. An
extensive strain development programme resulted
in substantial improvement in yield of esters. Selection of the appropriate amino acid precursor,
deduced from analysis of the esters produced and
knowledge of amino acid degradation pathways,
directed ester production to the most desirable
compounds. Ultimately, commercially feasible
yields of esters were achieved by continuously
sweeping volatile products from the fermentation in
the air stream and trapping them on activated
carbon. Obviously, this necessitated development of
an effective process for recovery of the product
from the carbon. Thus, one can see that in this case
commercial success resulted from a combination of
several factors most of which could not be foreseen
at the inception of the project. The possible formation of these esters from isoleucine was postulated
(Fig. 2).
In some cases, a soil screening programme
and published information may be combined to
Fig. 2
Postulated mechanism for formation of ethyl esters
via isoleucine degradation pathway
Fig. I
CH&H,$MX(NH&CKlH
CH,
N-demethylation activity of Polyporus versicolor
NH-CH,
Q-
CH3CHzOH
versicolor
CHO
CHO
4-(methylamino)benzaldehyde
4-aminobenzaldehyde
COOCH,
NH-CH,
0
H&H,G-OCH&H,
I
0
Polyporus
P
0
CH,CH=C-C-S-CoA
I
CH,CH&SCoA
Polyporus
versicolor
Methyl N-methylanthranilate
CH,-CCH-C-SCoA
Methylanthranilate
1991
69 I
Biochemical Society Transactions
692
develop a viable process [ 111. Numerous recently
published research and issued patents describe the
production of y-decalactone, an important component of many fruit flavours. In essence, most of
these processes are similar to that described in US.
Patent 4,560,656 [ 113. Obviously these patents
differ by employing different micro-organisms.
Although the first organism was isolated through
soil screening using castor oil or ricinoleic acid as
the sole carbon source, the underlying scientific
principle of this process was published in 1963
describing the metabolism of ricinoleic acid by
members of the Cundzifu genus [ 121. In this paper,
appearance and disappearance of 4-hydroxydecanoic acid during catabolism of ricinoleic acid
by Cundzifu guilliermondi was reported. The
patented process is very straightforward, using a
member of the Cundzifu genus and castor oil as substrate. However, the thought processes that went
into this work demonstrate the underlying complexity. An organism was needed with lipase activity, and one that preferably could tolerate high
levels of free fatty acid. A medium (‘chemical environment’) was needed that allowed the growth of
the micro-organism, but kept most of the released
fatty acids, including the product acid, in ionic form
to perhaps minimize their toxicity. Finally, using the
concept of co-oxidation, a small quantity of
decanoic acid added at the right time further
increased the accumulation of y-hydroxydecanoic
acid.
A similar approach using Cundia petrophilum
was devised to produce a mixture of saturated and
unsaturated y-decalactones at a very high yield
[ 131. In a recent patent application [ 141 a process is
disclosed for production of 6- and y-octalactone.
Members of the Mucor genus were employed and
octanoic acid or its esters was used as substrate.
Interestingly enough, these organisms differ from
Cundzifu species in their fatty acid metabolism.
Mucor species, besides possessing the normal poxidation pathway, hydroxylate the acid at the yposition. It seems in both cases that the bulkiness of
the hydroxyl group at the y-position slows down
the degradation of this acid resulting in its accumulation.
In the development of micro-organisms for
production of natural flavour compounds one may
use selective soil screening to isolate a parent strain
and then, via a combination of mutagenesis and
screening techniques, isolate a strain capable of
accumulating the desired product. A published process for the production of acetophenone is a good
example of this approach [15]. A parent strain was
Volume 19
isolated from soil by selective enrichment using
cinnamic acid as the primary carbon source in a
liquid minimal medium. Purified isolates were
screened simultaneously for adaptation to benzoic
acid, assuming that benzoic acid would be an intermediate in cinnamic acid degradation. Isolates were
mutated with N-methyl-N-nitro-N-nitrosoguanidine and mutants which grew well on benzoic acid
and poorly on cinnamic acid were selected and
purified. The mutant which accumulated the highest
levels of acetophenone was characterized by the
American Type Culture Collection as an unclassified Pseudomonus sp. (ATCC 53716). The parent
strain differs from the mutant in the distribution of
3-0x0-phenylpropionic acid between the two
alternative branches namely formation of acetophenone and degradation via benzaldehyde.
Transformation of sclareol (Z-ethenyldecahydro-2- hydroxy - 2 a , 5,5,8a-pentamethyl- 1-naphthalenepropanol) to related ambergris-like compounds has been of interest for a long time.
The target compound is Ambrox (dodecahydro3a,6,6,9a-tetramethyI-naphtho[2,1-b]furan), the
most important odour component of ambergris. In
a chemical process chromium or manganese oxidation is used to convert sclareol into sclareolide
(decahydro- 3a,6,6,9a-tetramethyl-naphtho [2,l- b] furan-2( lH)-one). The resulting sclareolide is
reduced to a diol (decahydro-2-hy droxy-2a,5,s,8atetramethyl-naphthalene-ethanol), and finally the
diol is cyclized to Ambrox using a reagent such as
p-toluene-sulphonyl chloride. The disadvantages of
the first step of this process are low yield (40-SO%),
disposal of spent heavy metals, and, above all, a
limited supply of starting material. Using selective
soil screening on a minimal medium and sclareol as
the sole source of carbon, two isolates were
obtained [ 161. Both isolates were classified by
Centralbureau voor Schimmel Cultures and designated to be Hyphozymu roseoniger, a new species.
The morphological characteristics of this microorganism are very unique; in liquid medium, growing on dextrose only a yeast-like form is present
during the first few days of growth. With sucrose as
the carbon source, a completely filamentous form
appears; the two forms are completely interchangeable by alternating between dextrose and sucrose.
The metabolic activity of the yeast form is also
slightly different from that of the filamentous form.
Optimization of conditions, use of the proper
emulsifier, and reducing the substrate particles to
optimum size, resulted in conversion of sclareol at a
2% level to the diol with a yield of 81%. The main
disadvantage of this process is its long fermentation
Food Biotechnology
period. Nonetheless, this process was the first
breakthrough
in sclareol biotransformation.
According to disclosed information, apparently, the
rate of degradation of epi-sclareol by H. roseoniger is
slower than that of sclareol. Various compounds
were synthesized and tested for possible conversion
by H roseon*er [16]. It is now evident that all of
these compounds were converted to the final
product.
Another patent describes a similar process
[ 171. A large scale selective soil screening programme using a minimal medium and sclareol as
sole carbon source resulted in four isolates, identified as Cryptococcus albidus (ATCC 20918 &
20921) (sclareolide producer), Cryptococcus luurentii
(ATCC 20920)
and
Bensingtonia
czlziztu
(ATCC 209 19) (diol producer). Cryptococcus ulbidus converts, quantitatively, as much as 150 g/l of
sclareol powder to sclareolide crystals. The product
is recovered by simple filtration and purified by
conventional crystallization procedures.
Several intermediates in the transformation of
sclareol by C albdus were isolated, purified, and
identified to determine the likely pathway. Rased on
our own research and published information [ 161, a
pathway for the transformation of sclareol to
sclareolide is proposed (Fig. 3). The steps in this
transformation are: allylic rearrangement, oxidation
of primary alcohol to corresponding acid, hydroxylation at B-position, two carbon cleavage, BaeyerVilliger oxidation, hydrolysis of ester, further oxidation of primary alcohol to carboxylic acid and cyclization in situ to form lactone. It should be
Fig. 3
Proposed pathway for transformation of sclareol to
sclareolide by Cryptococcus albidus
emphasized that identification of intermediates and
speculation on possible metabolic pathways are not
only of scientific interest, but also can be extremely
important for optimization of the process and biotransformation of related or unrelated molecules to
strengthen the protection of a patent. The initial
interest in sclareolide was for use as a starting
material for the fragrance material, Ambrox. Later,
is was discovered [ 181 that sclareolide imparts
mouthfeel, increases the saltiness of sodium
chloride, covers the bitterness of potassium chloride
and adds richness and creaminess to low fat ice
cream. Sclareolide has recently achieved GRAS
status (i.e. generally recognized as safe) for flavour
applications.
The processes decribed above are all based
upon the usage of wild-type organisms or ‘classical’
mutants thereof. It is likely that the introduction of
genetic engineering techniques will have a major
impact on this field. However, one must keep in
mind that cost limitation is an important factor in
the introduction of gene technology into this area.
The acceptable costs of many flavour and fragrance
compounds are, for the most part, far lower than
those for pharmaceutical products.
In conclusion, the specific examples presented
here describe commercially viable processes. These
approaches have been shown to be applicable to the
production of highly-valued natural flavour ingredients. Furthermore, the final example demonstrates
that fermentation processes can be developed as
viable alternatives to classical organic synthesis for
flavour, fragrance, and other fine chemicals.
1. Mironowicz, A. & Siewinski, A. (1906) Acta Biotechnol. 6, 141-146
2. Scharpf, L. G., Seitz, E. W., Morris, J. A. & Farbood,
M. I. (1986) ACS Symp. Ser. 317,323-346
3. Gatfield, I. I,. (1988) Food Technol. 42, 110-169
4. Rehm, H. J. & Reed, G . (1981) in Biotechnology
(Rehm, H. J. & Reed, G., eds.), vol. 1, pp. 2-3, Verlag
Chemie, Weinheim, Deerfield Beach, Florida & Basel
5. Hesseltine, C. W. & Haynes, W. C. (1973) Prog. Ind.
Microbiol. 12, 1-2
6. Schmidt, A. & May, R. (1965) Hoppe-Seyler’s Z .
Physiol. Chem. 340,283-286
7. Gregory, P., Scire, B. & Farbood, M. 1. (1989) PCT
Int. Appl. WO 89 00,203
8. Takasago Perfumery KK (1990) Japanese Pat.
J02135-093-A
9. Farbood, M. I., Morris, J. A. & Seitz, E. W. (1987)
U S . Pat. 4,686,307
10. Hatteri, S., Yamaguchi, T. & Kanisawa, T. (1974)
Proc. Int. Congr. Food Sci. Technol. 4th 1, 143-151
11. Farbood, M. I. & Willis, B. J. (1985) US. Pat.
4,560,656
1991
693
Biochemical Society Transactions
694
12. Okui, S., Uchiyama, M., Mizugaki, M. & Sugawara, A.
(1063) Hiochim. Hiophys. Acta 70, 348-35 1
13. Farbood. M. I., Morris, J. A., Sprecker, M. A,.
Hienkowski, I,. J., Miller, K. l’.? Vock, M. 11. &
Hagedorn, M. I,. ( 1 990) US. Pat. 4,960,597
14. Gregory, 1’. & Eilerman, K.G. (1989) PCT Int. Appl.
WO 8912-134A
15. Hilton, M. D. & Cain, W. J. (1990) Appl. Environ.
Microbiol. 56.623-627
16. Farbood, M. I., Willis, H. J. & Christenson, 1’. A.
(1086) S. African ZA 8.5 04,306
17. Farbood. M. I., Morris, J. A. & Lhwney, A. 1. (1000)
U S . Pat. 4,970,163
18. Huckholz, I,., Farbood, M. I., Kossiakoff, N. &
Scharpf, I,. G. (1990) 1J.S.Pat. 4,000,603
Keceived 12 April 1091
Anti-microbial substances produced by food associated micro-organisms
Hans Horn and Christina Msrtvedt
MATFORSK, Norwegian Food Research Institute, Osloveien I , N- I430 As, Norway
Introduction
Fermentative breakdown of carbohydrates results
in a range of small molecular mass organic molecules which exhibit anti-microbial action, including
lactate, propionate, acetate and ethanol. Conditions
favouring the growth of bacteria producing these
metabolic end products were discovered and used
thousands of years ago and are still exploited in the
production of a variety of products. They are
employed in different manners, either as food additives after production in industrial fermentors, or
produced in situ during fermentation of meat, vegetables and milk.
Today there is considerable renewed interest
in the use of naturally produced anti-microbial substances for food preservation and protection owing
to the low energy demand of the fermentation process and the discovery of an abundance of additional anti-microbial activities associated with the
fermentative microbes. Recombinant DNA technology has made it possible to identify and clone
genes encoding these anti-microbials and electrotransformation has enabled the transfer of the genes
to other bacteria, thus opening the era of tailormade starter cultures.
The present paper will survey the antimicrobials produced by food associated microorganisms with the main emphasis on the lactic acid
bacteria and related organisms.
Metabolic end products
Present knowledge suggests that the commonest
form of anti-microbial activity expressed in foods is
that associated with lactic, acetic and propionic
acids.
Lactic acid results from the metabolism of
many different types of bacteria, primarily LactoAbbreviation used: 3-HPA, 3-hydroxypropanal.
Volume 19
bacillus, Lactococcus, Leuconostoc and Pediococcus.
Lactic acid is produced from breakdown of hexoses
via the Embden-Meyerhof-Parnas pathway. Ideally,
in homofermentative breakdown one mole of
hexose gives rise to two moles of lactic acid and two
moles of ATP. The anti-microbial action of lactic
acid is only moderate. The acid produced lowers
the pH and the growth of most spoilage and pathogenic organisms is inhibited to some extent. Lactic
acid causes the leakage of hydrogen ions across the
cell membrane. This results in acidification of the
cell interior and inhibition of nutrient transport. The
energy-yielding metabolism is not influenced [ 11.
Lactic acid is the primary acid produced during fermentation of sausage, sauerkraut, olives, yoghurt,
etc.
In heterofermentative breakdown of sugars,
the dissimilation proceeds via the pentosephosphate shunt. One mole of hexose gives rise to
one mole of lactic acid, ethanol, CO, and ATP,
respectively. However, oxidation of NADH + H +
using alternative hydrogen acceptors (see below)
sometimes leads to the formation of acetic acid and
ATP instead of ethanol. Acetic acid is one of the
most used anti-microbials from micro-organisms.
Industrially it is produced through aerobic oxidation of ethanol by members of the genus Acetobacter. Acetic acid is mainly used as a food additive,
in the form of vinegar. It is added as a preservative
substance and a flavouring agent to many different
foods including mayonnaise, dressings, pickles and
mustard.
Acetic acid has a wide range of inhibitory
activity, inhibiting yeasts and moulds as well as
bacteria. Its action cannot be explained by pH
reduction alone. The undissociated form penetrates
the cell and hereby exerts its inhibitory action,
which is consistent with its anti-microbial activity
increasing with decreasing pH values.