Fungus

Details Fungus Fungus

1999-09-29

2001-08-07

Hendricks, Keith (Department: 1761)

Food or edible material: processes, compositions, and products

Dormant ferment containing product, or live microorganism...

C426S656000, C435S256500

Reexamination Certificate

active

06270816

ABSTRACT:

Human foodstuffs are produced commercially from Fusarium which is a haploid fungus. A particularly suitable strain of Fusarium A3/5, IMI 145425 is described in U.S. Pat. No. 4,347.
It is desirable in order to produce human foodstuffs of attractive mouth feel that the Fusarium should show little hyphal branching, and this is the case with Fusarium, IMI 145425. However, Fusarium in extended continuous fermentation invariably becomes more highly branched due to the appearance of variants in the fermenter culture. The appearance of highly branched variants appears to be a common feature of extended fungal fermentations. (Trinci, A. P. J, et al., (1990) Microbial Growth Dynamics, pp 17-38, IRL Press, Oxford U.K.)
It is therefore desirable that strains of Fusarium, of greater morphological stability should be produced.
In fungal strain breeding programmes the parasexual cycle has often been reported as a valuable means of improving commercially important production strains, since it enables the advantageous characteristics of two individuals to be combined via recombination, heterokaryosis or diploidy. For example, diploids have been claimed to give increased productivity of citric acid (Das & Roy, 1978), penicillin (Elander et al., 1973), and cephalosporin C (Takeda Chemical co., 1977). Reports of qualitative improvements include restoration of sporulation ability in
P. chrysogenum
(Calam et al., 1976), improved filtration characteristics in
A. niger
(Ball et al., 1978), generally improved vigour in
C. acremonium
(Hamlyn & Ball, 1979), reduced oxygen demand in
M. inyoensis
(Crueger, 1982), and elimination of p-hydroxypenicillin V production in
P. chrysogenum
(Chang et al., 1982). By contrast there appears to be no clear demonstration of parasexuality in Fusarium (Booth, 1984) although putative diploids of
F. oxysporum
have been described (Buxton, 1956). Attemps to demonstrate the production of diploids of
F. graminearum
NRRL 319 (Bu'Lock et al., 1986) were unsuccessful but heterokaryotes were reported.
According to Rowlands (1984) diploids are unstable and are likely to break down during fermentation with progressive loss of productivity unless some sort of selection pressure can be applied. In
S. cerevisiae
there is evidence that adaptive mutations occur at a higher (×1.6) frequency in an evolving diploid than in haploid populations (Paquin and Adams, 1983). This supports the view that diploid populations of fungi are unstable. We have however found that certain higher ploidy strains of
fungi imperfecti
show an increase in (morphological) stability under typical growth conditions relative to the parental haploids.
We have now made strains of Fusarium of higher ploidy and have devised means whereby other higher ploidy strains of
Fungi imperfecti
especially Fusarium may be produced. These have better morphological stability than haploid strains. It is thought possible that this may be at least partly due to reduced sensitivity to mutation arising from the fact that the mutant genes leading to higher branching may be recessive and that in this case corresponding mutations of both the duplicated genes in higher ploidy strains are necessary before a hereditary characteristic can be passed on by asexual reproduction.
Fungi imperfecti
differ from other fungi in that they do not normally pass through a sexual stage. Thus, it can be predicted that higher ploidy strains will generally show greater stability in inherited characteristics than haploid strains providing that they are cultured under conditions such that reversion or partial reversion of the haploid form is negligable.
By “higher ploidy” is meant higher ploidy than haploid including anenploid.
This invention comprises a human food product which comprises higher ploidy strain of a member of the
Fungi imperfecti
of which each parent has a genetic constraint on its ability to grow which is not shared by the other parent. The invention also comprises a higher ploidy strain of Fusarium. Preferably one parent is auxotrophic for two compounds selected from histidiine, arginine, leucine and adenine and the other is auxotrophic for the other two of the said compounds. Suitable strains are deposited by Marlow Foods Limited, Station Road, Stokesley, Middlesbrough TS9 7AB, as IMI 369190, 369191 and 369192 with The International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY on Nov. 6, 1995.
The human foodstuffs derived from
Fungi imperfecti
preferably have a reduced nucleic acid content of preferably less than 2% by weight.
This invention comprises a higher ploidy strain of
Fungi imperfecti
which is dead and of which the nucleic acid content is less than 2% by weight. The fungus may be killed and its nucleic acid content simultaneously reduced to less than 2% by treatment with water at a temperature of at least 60° C.; the water may be its own growth medium.
The genetic constraint may be that each parent is auxotrophic for a different metabolite or that each produces a gene product which is necessary for growth which has a temperature or pH sensitively greater than that of the corresponding gene product of the other parent. By growing the higher ploidy strain in an appropriate environment (in the above cases one lacking the nutrients for which the parents are auxotrophic or one which has a temperature of pH at which neither parent can grow without a supply of the other's gene product) one can select against reversion to haploid strains, thus maintaining the increased morphological stability of the fungus.
The invention also comprises human food which comprises a higher ploidy strain of
Fungi imperfecti
in which the fungus has been treated to remove nucleic acids and/or to modify its texture, taste or mouth feel.
The higher ploidy strains may be produced as follows. A corresponding haploid strain is cultured and subjected to mutagenesis, for example by ultra violet radiation until auxotrophic strains are produced which have lost the ability to make specific metabolites. The culture may be plated out for example onto agar plates and supplied with a complete medium which enables auxotrophs as well as unmutated organisms to grow. By replicate plating onto a minimal medium the unmutated strains are identified and the auxotrophs selected from the complete medium plate and their nutrient requirements identified. Two auxotrophs, for example, requiring different amino acids as nutrients may then be selected and used to produce a higher ploidy strain as subsequently described, or they may be cultured separately and the process repeated (except that in this case the minimal nutrient will contain the appropriate amino acid(s) required by each auxotroph). By this means two double auxotrophs may be derived each of which preferably requires two amino acids both of which differ from those required by the other. If desired triple or higher order auxotrophs may be prepared by corresponding means; they should preferably be auxotrophic for a group of metabolites which have no common member.
The invention comprises a process of preparing a higher ploidy strain of a member of the
fungi imperfecti
, for example Fusarium which comprises culturing two auxotrophs which require different auxotrophic nutrients together in a medium containing the nutrients necessary for both to grow (the joint culture stage) and then culturing organisms derived from such culture in a medium lacking the auxotrophic components (minimal medium) (the minimal medium culture stage) and recovering a higher ploidy strain from the minimal medium culture stage.
In the joint culture stage it is necessary to induce sporulation. This may be carried out by known means, suitably by subjecting the organisms to a nutritional challenge, for example by supplying only a complex carbon source as the main nutrient.
We have found that when transferred to a minimal nutrient medium, i.e. one lacking the auxotrophic components the higher ploidy strains derived from both auxotrophs multiply relative to the haploid strains and those higher ploidy strains

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