Appeal No. 1997-2080
Application No. 08/159,939
d) selecting colonies by visual inspection from step c) which contain yeast-like cells
and exhibit reduced pigmentation compared to the other cells;
e) testing the selected colonies for pigment production; and
f) selecting the isolate which produces minimum pigment in submerged culture to
fermentation conditions in a fermentation broth and harvesting the substantially non-
pigmented pullulan from the broth; and then
g) subjecting the harvested pullulan to a heat treatment for a period of time and
temperature effective to deactivate any enzymes therein which decrease the molecular
weight of the pullulan.
31. A method for producing substantially non-pigmented pullulan having a M of at
W
6
least 6 x 10 comprising the steps of subjecting an isolated substantially pure microbial
culture selected from the group consisting of those having the following identifying
characteristics: A. pullulans ATCC 74100, A. pullulans ATCC 74101, A. pullulans 74102,
A. pullulans 74103, A. pullulans 74104, and A. pullulans 74105, and mutants thereof
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capable of producing substantially non-pigmented pullulan having a M of at least 6 x 10 ,
W
said cultures being capable of reproducing themselves and of producing pullulan in
isolatable amounts when cultured in a liquid growing medium containing assimilable
sources of carbon, nitrogen, and inorganic substances to fermentation conditions in a
fermentation broth and collecting the substantially non-pigmented pullulan having a
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molecular weight of at least 6 x 10 from the broth.
33. A method for producing substantially non-pigmented pullulan comprising the
steps of subjecting a pure culture strain of Aureobasidium pullulans which produces
minimal pigment when cultured, obtained by:
a) collecting at least one wild-type strain of Aureobasidium pullulans from natural
sources;
b) subjecting the collected strain to fermentation conditions so as to enrich for
organisms that grow as fungal yeast-like cells;
c) separating the yeast like cells thus produced;
d) growing colonies from the separated yeast-like cells;
e) selecting colonies by visual inspection from step d) which contain yeast-like cells
and exhibit reduced pigmentation compared to the other cells;
f) testing the selected colonies for pigment production; and
g) selecting the isolate which produces minimum pigment in submerged culture, to
fermentation conditions in a fermentation broth, adjusting the pH of the fermentation broth
to about 7.0 after the pH of the fermentation broth has stabilized, and thereafter, harvesting
the substantially non-pigmented pullulan from the broth; and then subjecting the harvested
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