CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
GLUTINOUS RICE FERt1ENTATION:
,,
ISOLATION AND CHARACTERIZATION OF f~ICROORGANISMS
RESPONSIBLE FOR THE FERMENTATION ACTIVITY
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Home Economics
by
Karen Lambert Mays
August, 1974
The thesis of Karen Lambert Mays is approved:
Committee Chairman
California State University, Northridge
August, 1974
ii
To my parents
iii
ACKNOWLEDGEMENTS
Strictly Germ-proof
The Antiseptic Baby and the Prophylactic Pup
Were playing in the garden when the Bunny gamboled up;
They looked upon the Creature with a loathing undisguised;-It wasn't Disinfected and it wasn't Sterilized.
They said it was a Microbe and a Hotbed of Di sea.se;
They steamed it in a vapor of a thousand-odd degrees;
They froze it in a freezer that was cold as Banished Hope
And washed it in permanganate with carbolated soap.
In sulphureted hydrogen they steeped its wiggly ears;
They trimmed its frisky whiskers with a pair of hard-boiled shears;
They donned their rubber mittens and they took it by the hand
And 'l ec ted it a member of the Fumigated Band.
There'.s not a Micrococcus in the garden where they play;
They bathe in pure iodoform a dozen times a day;
And each imbibes his rations from a Hygienic Cup-The Bunny and the Baby and the Prophylactic Pup.
Arthur Guiterman
With deep appreciation to Dr. Marjory Joseph, Dr. Donald
Bianchi, and especially Dr. Charles R.
~~eston,
my advisors, either officially or unofficially.
thanks to Dr.
Tung~Shan
all of whom served as
I give very special
Chen for his patience and advice during this
work and for the knowledge I've gained by working with him, extraneous
to this study.
iv
TABLE OF CONTENTS
Page
LIST OF TABLES .
vi
LIST OF FIGURES
vii
ABSTRACT . .
viii
INTRODUCTION
1
LITERATURE REVIEH
3
Fermentation . . . . . . . .
Oriental Food Fermentations
Lao-chao . . . , . . . . . . .
The Role of Starters . . . . . . . .
Microbial Composition of the Starter .
MATERIALS AND METHODS
3
4
5
7
8
10
Materials
Methods
10
11
RESULTS AND DISCUSSION • .
23
Identification of SY Isolate . .
Identification of the SF Isolate
Chemical Changes in Inoculated Rice
23
40
45
BIBLIOGRAPHY .
53
APPENDICES .
57
I.
II.
Media for Yeast Identification .
Standard Description of Endomycopsi.s bv;r>tonii
v
58
61
LIST OF TABLES
Table
1.
Page
Fermentation and assimilation of carbon and
nitrogen compounds by SY isolate . . . .
29
2.
Summary of identification test results--SY isolate
35
3.
Chemical changes in glutinous rice fermented with
test cultures incubated at 2JOC for three days .
4·8
vi
LIST OF FIGURES
Page
Figure
1.
Pseudomycelium of SY isolate on cornmeal agar slide . . .
36
2.
SY isolate grown on potato plugs for seven weeks,
stained with modified Kufferath carbol-fuchsin stain
38
3.
Sporangium of SF isolate, grown on YM agar
43
4.
Sporangium of RCW culture, grown on YM agar
46
vii
ABSTRACT
GLUTINOUS RICE FERMENTATION:
ISOLATION AND CHARACTERIZATION OF tHCROORGANISMS
RESPONSIBLE FOR THE
FERI'~ENTATION
ACTIVITY
by
Karen Lambert Mays
Master of Science in Home Economics
August, 1974
Lao-chao is a fermented rice product traditionally prepared
in Chinese households from glutinous rice inoculated with a commercial
starter.
The microbial composition of a commercial lao-chao starter
from Taiwan was studied.
A yeast and a mold were isolated from
lao~
chao prepared from the starter and from the starter, respectively.
The yeast was identified as Endorrrucopsis burtonii, according
to morphological, cultural, sexual, and physiological characteristics,
and the mold was identified as a species ofRhizopus on the basis of
morphological characteristics:
The isolated microorganisms were tested for their ability to
ferment glutinous rice.
Samples of glutinous rice were inoculated
with either the isolated yeast, the mold, or a combination. The
samples were analyzed for pH, reducing sugar content, and ethanol
viii
content.
The results indicated that both the mold and the yeast have
the ability to saccharify the starch ·in glutinous rice and produce
ethanol.
Data indicate that the saccharifying and ethanol producing
abilities of the mold are superior to those of the yeast)
however~
satisfactory lao-chao product cannot be produced by the mold alone.
ix
a
INTRODUCTION
Fermentation is one of the oldest methods of food preparation
employed by man.
Fennented foods are indigenous to 'all parts of the
world, a.lthough the raw mate\··ials used vary with the area.
Occidental
fermentations center on grapes, dairy products, breads, and sausages,
whereas Oriental fermentations, 'irt particular Chinese and Japanese,
utilize soybeans and rfce to the greatest extent (Pederson, 1971).
In general, Oriental fermentations have not been as extensively
studies as Occidental fermentations.
The basic functions of fermenta-
tion in the Oriental culture is to render some foodstuffs, such as
soybeans,more digestible and to add interesting flavors to an
wise bland diet of vegetables and rice.
Until the 1950's, very little
research had been done on the Oriental fermented foods.
example of these foods that
\~estern
other~
The only
people had much exposure to was
In the last two decades, research has been con-
soy sauce or shoyu.
ducted on several of the better known fermented foods, such as
miso, and ontjom.
tempeh~
There is still quite a lot of research to be done
on the lesser known fermented products, most of which are produced in
the home (Hesseltine and Wang, 1967; Wang and Hesseltine, 1970).
Lao-chao is one example of these lesser known fermented
products. Traditionally, it is domestically produced from glutinous
rice using a starter.
purposes:
The wine-like end product is used for various
as a food; as a seasoning; and as a starter for shao-shing
wine fermentation.
1
2
The pur·pose of this study was to isolate and identify the
major fermentative microorganisms in a Chinese commerc·ial starter
used
to make lao-chao, a slightly alcoholic sweet fermented food produced
from glutinous rice.
Chemical changes in the rice as the result of
microbial action were also studied.
LITERATURE REVIEW
Fermentat·ion
Fermentation is the metabolism of organic compoundss such as
the carbohydrates in
food~
by microorganisms, predomi nant'ly yeasts and
bacteria, although a few molds a1e capable of this process (Stanier
et al . , 1970).
The products of fermentations are usually ethanol and
carbon dioxide, other a·lcohols, or various organic acids.
of
fermentation~
lactic acid and
nected with food products.
alcoholic~
Two types
are most commonly con-
The history of food fermentations predates
written records and the development of such foods rests more with
trial and error on the part of early man than on scientific knowledge
(Pederson, 1971).
The bulk of food fermentation research deals with
the better known products such as cheeses, pickles, and sausages.
Fermented food products of Oriental countries have been largely neglected in the literature, perhaps because most of the foods are not
generally used in Occidental countries, with the exception of soy
sauce (Pederson, 1971).
Fermentation of a food serves several purposes:
it introduces
new flavors and colors into the diet; results in the formation of
preservatives; improves nutritional value; improves the digestibility;
and adds color (Hesseltine and Wang, 1967).
Fermented products in the
Oriental diet serve all these purposes, particularly the addition of
new flavors.
3
4
Oriental Food Fermentations
The two
majm~
substrates used for food fermentation in As·ian
countr·ies are soybeans and cereals.
Several studies on soybean
products such as tempeh (Steinkraus et al., 1960; Roelofsen and Talens,
1964; Smith et al., 1964; Wang and Hesseltine, 1966), sufu (Hang and
Jackson, 1967a; Hang and Jackson, 1967b; Wang and Hesseltine, 1970),
shoyu (Teramoto et al., 1965), and some rice products such as sake
(Teramoto et al., 1965) and lao-chao (Wang and Hesseltine, 1970; Liu
et al., 1959) have been published.
Reviews covering some of these
same products, tempeh (van Veen and Steinkraus, 1970; Hesseltine and
Wang, 1967; Gray, 1970) sufu (Hesseltine, 1965; Hesseltine and Wang,
1967; Gray, 1970), shoyu (Hesseltine, 1965; Hesseltine and Wang, 1967;
Gray, 1970) and lao-chao (van Veen, 1972), as well as some other
products such as miso (Hesseltine, 1965; Hesseltine and Wang, 1967;
Shibasaki and Hesseltine, 1962; Gray, 1970), ang-kak (Hesseltine, 1965;
Gray, 1970), and ragi (Hesseltine, 1965) have been published.
Accord-
ing to Hesseltine (1965), untreated soybeans do not soften well with
cooking and are difficult to digest.
Fermentation of soybeans over-
comes both of these problems (Hesseltine and Wang, 1967).
Fermented
rice products are more often used an inocula for other fermentations
or as coloring agents, than as actual foods in themselves.
Ang-kak,
or red rice, is used to impart color to fish, Chinese cheese, and
wines .(Hesseltine,.l965).
Koji (Hesseltine and Wang, 1967; Ito, 1929),
ragi, and Chinese yeast (Hesseltine, 1965) are all used as inocula for
further fermentations of various substrates.
These inocula are
obtained by seeding rice or rice flour with appropriate microorganisms
5
and allowing them to ferment unti"l adequate enzymes or numbers of
microorganisms are produced to proceed with the second fermentation
(Pederson) 1971).
Two exceptions are sake and lao-chao fermented rice
products which are complete in themselves.
Although both sake and 1ao-chao are a·! cohol ic fermentation
products~
they diffel'' in several respects.
Sake is produced from
ordinary polished white rice of a variety indigenous to Japan (Arima,
1957) which has been inoculated with koji.
The rice is fermented from
10-14 dayss after which it is separated from the liquor by filtration.
The resulting wine has an alcohol content of 14-17% (Teramota et al .,
1965; Frazier, 1967).
Lao-chao is produced from glutinous rice inocu-
lated with Chinese yeast ball or a similar starter.
The fermentation
process takes 2-3 days, after which the product, both rice and liquor,
is consumed.
The alcohol content is 2-6%.
Lao-chao is also used as a
koji for the production of shao-shing wine, the most popular rice
~\fine
in China.
Lao-chao
Lao-chao also known as sweet rice wine, chiu-niang, or tienchiu-niang, is a fermented rice product indigenous to China.
Lao-chao
has never been produced commercially, but has been prepared in Chinese
households for thousands of years.
The process consists of steaming
and cooling glutinous rice, then mixing it with a small amount of
commercial starter.
This mixture is lightly packed into a bowl,
covered and incubated for 2-3 days at room temperature (Wang and
Hesseltine, 1970).
alcoholic.
The resultant product is juicy, soft, sweet and
Both the solid and liquid-portions of the wine are consumed
...
together as iss or in combination with eggs, fruits or seafood.
Traditionally, lao-chao is not consumed as a part of the daily diet,
but is reserved for special occasions.
For example, lao-chao may be
found in the diet of a new mother because it is believed the product
will help her regain her strength (Wang and Hesseltine, 1970).
The rice used to produce lao-chao is the glutinous or waxy
type.
Glutinous rice is differentiated from ordinary rice
starch content.
by
its
Whereas the ordinary r·ice starch is composed of
approximately 84% amylose and 16% amylopectin, glutinous rice starch
is almost 100% amylopectin (Houston and Kohler, 1970; Juliano, 1965).
The high amylopectin content gives glutinous rice its high degree of
stickiness.
Wang and Hesseltine (1970) postulate that it is this
stickiness which creates the semi-anaerobic conditions conducive to
fungal activity during the lao-chao fermentation.
Ordinary rice
inoculated with starter will not produce a satisfactory product (Wang
and Hesseltine, 1970).
The commercial starter used to inoculate glutinous rice for
the lao-chao fermentation is a mixed culture of saccharifying mold and
yeast (Wang and Hesseltine, 1970).
It is a solid cake referred to as
Chinese yeast ball (chiu-yueh or peh-yueh) (Wang and Hesseltine, 1970)
or Chinese yeast (Banks et al., 1967). The identity of the major
microorganisms in the starter varies with the starter.
Wang and
Hesseltine (1970) examined several samples of .commercial starter from
Taiwan and found several types of mold, all of which belonged to the
genus Mucor, and a yeast identified as a species of Endomycopsis.
The Role of Starters
Fermentation of foodstuffs originated several thousand years
before the deve·lopment of the microbiological techniques.
To r-epro-
duce a fermented product man could not rely on pure culture techniques,
but had to use some other method of transferring the fermentative
microorganis1ns to a fresh batch of food.
Starters serve this purpose.
In the general sense, a starter is an uncooked portion of the
fermented food which is used to seed a fresh batch of food.
l.Jith the
advance of science, pur_e culture techniques have been developed and
have been used for wines, vinegars, and most breads and cheeses
(Frazier~
1967), however, many foods are still best produced with
original raw material starter, such as sauerkraut, green olives,
fermented pickles (Frazier, 1967), and sourdough bread (Ng, 1972).
Pure culture techniques with which the fermentative microorganisms
are kept in a pure and active state until ready for use, allows the
food manufacturer to produce a consistently good product and has led
to the development of new fermented products.
Oriental fermented foods are normally inoculated with a
natural starter in the form of koji, raji, Chinese yeast ball, or
previously fermented raw material (Frazier, 1967; Hesseltine, 1965).
Centuries of using various inocula to prepare foods in the home has
led to the development of reliable natural starters which produce good,
'but not necessarily consistent products.
When the production of foods
such as soy sauce and miso became industrialized, standardization of
the processes and inocula occurred, resulting in a consistently good
product (Pederson, 1971).
Not all fermented foods are produced
8
commercially, hence the same food may vary slightly in flavor or
texture from household to household.
This may be due to the methods
followed or to differences in the microbial composition of the starter
used (Pederson, 1971 ).
Microbial
Co~Qositio~
of the Starter
A preponderance of Oriental fermented products originate from
foods high in starch, e.g., soybeans and rice, thus a saccharifying
mold is many times used to start the fermentation due to its ability
to produce amylolytic er:Jzymes, as in lao-chao, soy sauce, and miso
(Arima, 1957).
The inoculum used to introduce the mold may also con-
tain the yeast or bacteria v1hich will eventually ferment the available
sugars, or these microorganisms may be added after the action of the
mold is complete.
Mold
The purpose of employing the mold is to hydrolyze the starch
which it does by producing extracellular amylases (Banks et
al.~
1967).
Several genera of molds have been found in fermented foods, particularly Aspergillus_, Rhizopus_, Mucor_, Actinomucor_, and Penicillium
(Hesseltine and Wang, 1967).
Amylolytic enzyme production by molds
in Chinese coiTUllercial starter was first demonstrated by Calrnette in
1894 when he isolated a. species of Mucor (Banks et al., 1967; Thaysen
and Galloway, 1930).
He showed that members of the genus Rhizopus had
particularly good amylolytic properties (Banks et al., 1967; Liu and
Chen, 1962a; Liu and Chen, 1962b; Yue et al ., 1966b).
With the isola-
tion of amylolytic molds came the development of the Amylo process
9
which
\tas~ essentially~
the substitution of mold amylases for malt in
Occidental brewing processes. Molds from the genera Mucor or Rhizopus
are grown on the grain mash in order to convert the starch to sugar
before inoculation with the yeast (Hao et al
·~
1943).
Yeast and bacteria
Yeast and
bacteria~
either alone or in combination, may be
added to the food to be fermented.
This may be done either in the
starter or after saccharification by the mold.
The yeasts founq in traditional fermented foods are primar·ily
from the genera Monascus and Saccharomyces (Hesseltine and
1967).
Wang~
Under anaerobic conditions, the yeast catabolize haxoses to
alcohol and carbon dioxide (de
Becze~
1960).
The hexoses catabolized are those made available by the action
of fungal amylases on starch, in particular, glucose and maltose.
However, other sugars, such as sucrose are fermentable, due to further
breakdown by enzymes produces by the yeasts (de Becze, 1960).
Fermentation by bacteria leads to the formation of various
organic
acids~
notably lactic acid.
These acids give distinctive
flavors to such products as kimchi (Pederson, 1971), while acting as
preservatives.
Genera of bacteria corrmonly found in Oriental fer-
mented foods include Acetobacter Bacillusj and Lactobacillus
3
{Hesseltine and
Wang~
1967; Hahn, 1968).
MATERIALS AND METHODS
Materials
Glutinous Rice
Glutinous
rice~
also known as svJeet rice or mochigome is
produced and mal'keted by Bill
&
Ed Koda5 South Dos Palos, Ca1ifotnia.
The rice for this study was purchased in a local market.
Co~nercia1
Starter
Commercia·! lao-chao starter was obtained from a source in
Taiwan in the form of Chinese yeast balls.
The starter was crushed
into a powder form before use.
Media
Cultures were maintained on yeast-malt extract (YM) agar.
Acidified YM agar was used to isolate the microorganisms.
The agar
was acidified by adding 1 N HCl aseptically after the agar was autoclaved.
Final pH of the agar was 3.7 at 45°C as determined with a pH
meter.
The exact amount of HCl necessary to adjust the pH was deter-
mined using a duplicate quantity of non-sterile agar.
Other media
used in identification tests are listed in Appendix I.
Organisms
For comparative purposes, a known species of mold, Rhizopus
/
chinensis NRRL #367l,was obtained from Northern Regional Research
10
Laboratory, Peoria, Illinois, courtesy of Dr. Hwa L. Wang.
culture is designated
RCl~
(Rhizopus chinensis
This
~lang).
~1ethods
Preparation of Lao-chao
A 500 g portion of glutinous rice was washed and soaked in
cool tap water overnight.
The rice was thoroughly drained in a
colander and evenly spread approximately 3/4 inch thick in a stainless
steel steamer pan.
One hundred milliliters of tap water were sprin-
kled over the rice and this was steamed for 15 minutes at 15 psi
(121°C) in a pressure cooker (Steam-it, model ST-AG).
The steamed
rice was immediately cooled and the grains separated under cold running
tap water.
This was then drained in a colander and inoculated with a
mixture of l g commercial starter dispersed in 10 g of all-purpose
white wheat flour.
The starter mixture was thoroughly mixed with the
rice and the entire mass was lightly packed into a 2000 ml glass
beaker, leaving a center well for aeration, which is conducive to mold
growth and production of amylolytic enzymes.
The beaker was lightly
covered with plastic wrap and incubated at 33°C for 24 hours, after
which the plastic wrap was secured with a rubber band to create a
semi-anaerobic condition, which is conducive to alcohol production by
both the yeast and mold.
Incubation continued until the center well
was almost filled with liquid (2.5-3 days). At this point incubation
was terminated and the lao-chao refrigerated.
12
Isolation of Yeast from the Lao-chao
Acidified YM agar plates were used for easier isolation of
the yeast from the mixed culture in the lao-chao (Ladder, 1970).
The plates were streaked with one loopful each of lao-chao liquor
(Collins and Lyne, 1970).
three days.
The plates were incubated at 27°C for
Successive plates were inoculated with a loopful of
growth from a solitary yeast-like colony until a pure culture was
obtained.
YM agar slants were then inoculated, incubated and stored
at approximately 4°C in a refrigerator.
The first few subcultures
were maintained on acidified YM agar slants as a deterrent against
possible bacterial contamination.
This culture is designated as SY
(starter yeast).
Isolation of Maid from the Commercial Starter
The results from preliminary tests indicated that acidified
YM agar prevents bacterial contamination of the mold culture without
inhibiting growth.
Thus, the acidified YM agar was also used for
isolation of the mold from the commercial starter.
A starter suspension was prepared with 0.5 g of commercial
starter and 20 ml of distilled water.
A 0.1 ml aliquot of this sus-
pension was pipetted onto each acidified YM agar plate and then spread
over the surface with an alcohol-sterilized bent glass rod.
plates were incubated at 27°C for three days.
The
Growth was successively
streaked on plates until a pure culture was obtained.
YM slants were
then inoculated, incubated and stored at approximately 4°C in a
refrigerator.
This culture is designated SF (starter fungus).
.13
Maintenance of Isolated Cultures
Both isolated cultures were maintained on loosely capped YM
agar slants.
Isolates were subcultured every two months and were
incubated at 27°C for three days.
Maintenance of the RCW Culture
The RCW culture was revived on potato dextrose agar according
to instructions supplied with the culture.
RCW was maintained on YM
agar and subcultured as stated above.
preparation of Microbial Inocula for
Identification Tests
Yt·1 agar slants were inoculated from fresh subcultures of SF,
SY, and RCW and incubated at 27°C for three days until the culture
was in a state of active growth.
Growth from each of the slants was
loosened with a sterile loop and suspended in 5 ml of sterile distiiled
water. The suspensions were pooled and the cell counts in a known
volume were determined with a counting chamber .. ~0 Bright-Line).
The
suspensions were stored at 4°C until used as inocula for identification tests.
Yeast Identification Tests
The isolated yeast was identified according to methods
described by Ladder (1970).
Tests cover morphological, cultural,
.sexual, and physiological characteristics.
14
Morphological Tests
Characteristics of asexual reproduction and vegetative cells.-Two percent glucose-yeast extract-peptone (GYP) water flasks and sol-id
GYP plates were inoculated with a loopful of inoculum.
Flasks and
plates were incubated at 25°C for three days after which length and
width of 20 cells were measured.
The cultures were also examined for
budding or other types of asexual reproduction.
Both the flasks and
plates were then incubated at room temperature for four weeks and the
length and width of 20 cells were again measured.
Formation of pseudomycelium or mycelium.--A Petri dish containing a glass slide resting on aU-shaped glass rod was sterilized
by dry heat at 160-180°C for two hours.
The slide was removed from
the Petri dish with flame-sterilized tweezers, quickly dipped into
sterile cornmeal agar and replaced in the dish.
The solidified agar
was inoculated lightly in three lines along the length of the slide.
A sterile coverslip was placed over the center section of the slide.
Sterile water was poured into the bottom of the dish so the agar would
not dry out.
The dishes were incubated at 25°C for five days.
To
observe, the agar was wiped off the back of the slide and the area
under and around the coverslip was examined for mycelium or pseudomycelium .
. Cultural Characteristics
Cultural characteristics were studied in the same liquid and
solid 2% GYP cultures used for the vegetative cell tests.
characteristics in both media are recorded.
Growth
Sexual Characteristics
Ascospore formation, shape, and number per ascus were determined.
Potato plugs, malt extract agar slants, and cornmeal agar
slants were used as sporulation media.
Each slant was streaked with
a loopful of inoculum, incubated at 25°C for three days, and observed
microscopically for sporulation.
If sporulation had not occurred in three days, the cultures
were incubated at room temperature for four to six weeks and observed
periodically.
Staining with a modified Kufferath carbol-fuchsin
stain was used to verify ascospore formation.
A loopful of growth was
heat--fixed in two drops of buffered formal in sucrose fixative on a
slide.
This preparation was then flooded with Ziehl-Neelsen carbol-
fuchsin and steamed for two minutes without allowing the slide to dry.
The slide was decolorized with acid-ethanol (solution of 95% ethanol
and 1% HCl) for ten seconds and then rinsed with cool running tap
water.
This was counterstained with 1% methylene blue for three-five
minutes and then rinsed with running water and dried in bibulous
paper.
Mature ascospores stain red and vegetative cells blue.
Physiological Characteristics
Fermentative use of carbon compounds.--Glucose, galactose,
maltose, sucrose, lactose, and raffinose were used as carbon sources.
Sugar solutions and the basal medium, yeast nitrogen base (YNB), were
prepared and sterilized separately.
Six percent (w/v) solutions of
all the sugars except raffinose (12% w/v) were prepared and filter-
sterilized with a Gelman 47 mm glass (Seitz) filter funnel using a
...
membrane with
0.45~
mean flow pore size.
The basal medium was
16
prepared \'lith the addition of bromothymol blue to be used as an indicator dur·ing the fermentation.
6.0-7.6, yellow to blue.}
(Bl"omothymol blue has a pH range of
Five milliliters of the basal medium were
pipetted into Durham tubes and autoclaved for 15 minutes at 15 psi.
One milliliter of the sterilized sugar solutions was added aseptically
to the cooled Durham tubes to make the final medium.
also prepareds containing basal medium only.
with 0.1 ml of inoculum and incubated at 25°C.
A blank tube was
Each tube was inoculated
The tubes were shaken
gently every other day and the relative gas evolution in the insert
tube and the color change of the medium were observed.
Oxidative use of carbon comoounds.--Glucose, galactose,
maltose, sucrose, lactose, and raffinose were used as carbon sources.
A tenfold concentrated medium was prepared by dissolving 6.7 g of YNB
and .an amount of carbohydrate containing carbon equivalent to that in
5 g of glucose, in 100 ml of distilled water.
The YNB-raffinose
medium was prepared so that the carbon content was twice that of
glucose.
medium.
A blank was prepared by omitting the carbon source from the
These media were sterilized by filtration.
Sterile water
blanks were prepared with 4.5 ml of distilled water and autoclaved for
15 minutes at 15 psi.
To prepare the final medium, 0.5 ml of the YNB-
carbon solutions was aseptically pipetted into each of the sterile
water blanks and was mixed.
The tubes were inoculated with one drop
of inoculum and incubated at 25°C, shaken every two days and observed
weekly for four weeks.
Incubation took place in a separate incubator
from the fermentation tests to prevent interference by any ethanol
.......
1!
formed.
At the end of four weeks, relative growth was determined
with cell counts using an AO Bright-Line counting chamber.
Hydrolysis of arbutin.--Arbutin agar slants were streaked with
a loopful of inoculum and incubated at 25°C.
served as control.
An uninoculated tube
The tubes were examined daily from the second day
for the development of dark brown color which indicates the hydrolysis
of arbut·i n.
Utilization of nitrogen compounds.--Nitrogen compounds used
were potassium nitrate, sodium nitrite, and ethylamine hydrochloride.
A tenfold concentrated medium was prepared by dissolving 11.7 g of
Bacto-yeast carbon base (YCB) and either 0.78 g potassium nitrate,
0.26 g sodium nitrite, or 0.64 g ethylamine hydrochloride in 100 ml of
distilled water.
A blank was prepared by omitting the nitrogen source.
These media were filter-sterilized.
Sterile water blanks were pre-
pared with 4.5 ml of distilled water and autoclaved 15 minutes at
15 psi.
The final medium was prepared by aseptically pipetting 0.5 ml
of the YCB-nitrogen media into the sterile water blanks and shaking to
mix.
The tubes were inoculated with one loopful of inoculum and incu-
bated at 25°C.
After one week, one loopful of growth from each of the
tubes was transferred to a second, fresh tube and incubation was continued for another week.
\
Cell counts were used to establish relative
utilization of the nitrogenous compounds.
Growth on media of high osmotic pressure.--The medium was prepared by dissolving 16.7 g Bacto-vitamin-free yeast base in 100 ml of
distilled water, warming if necessary, and filter-sterilizing.
0.5 ml.
18
of this sterile medium was aseptically pipetted into 4.5 ml sterile
water blanks and shaken to mix.
These tubes were inoculated with one
drop of inoculum and incubated at 25°C.
After one
week~
one loopfu1
of growth from each tube was transferred to a fresh tube of medium
and incubation was continued for one more week.
Cell counts were used
to establish whether growth had occurred.
§rowth on media of high osmotic pressure.--Glucose-yeast
extract agar slants were prepared containing 50% and 60% (w/w) glucose.
The slants were streaked with a loopful of inoculum and incubated at
25°C for four
weeks~
when the growth was examined.
Growth at high temperatures.--YM agar plates were point inoculated.
The control plates were incubated at 25°C and the test plates
at 37°C for two days.
The growth was compared on the basis of colony
diameter.
Acid production.--Slants of glucose-chalk agar were inoculated
and incubated at 25°C for two weeks.
The cultures were periodically
checked for clarification of the agar around the colonies.
Ester production.--The control plate (25°C) from the high
temperature growth test was used.
hours, the plate was checked, by
After an incubation time of 24
smell~
for the presence of a fruity
odor, indicating ester production.
Mold Identification
Keys to the genera of fungi published by Clements and Shear
(1931), Gilman (1957), and Ainsworth (1973) were used as guides to
19
the characterization of the isolated mold, along with morphological
comparison to the RCW culture.
Both mold cultures were grown on YM agar plates and slants
incubated at 25°C for three days.
foliowing characteristics:
The cultures were examined for the
septation of mycelium; presence of
sporangia; shape and co ·1 Ot' of sporangia; presence of zygotes; presence
of
apophyses~
stolons and rhizoids; shape and size of spores.
Rapidity
of growth was measured by colony size on a point inoculated plate.
Optimum temperature was determ·ined by measurement of colony size on
point inoculated YM agar plates incubated at 25°C and 37°C.
Chemical Changes in Glutinous Rice as the
Result of Inoculation with Microorganisms
Inoculation and incubation of rice samples
One hundred gram portions of steamed glutinous rice in 400 ml
glass beakers were inoculated with cell suspensions of
alone or in combination.
SY~
SF, or RCW,
The SY and SF samples were inoculated with
one milliliter of suspension per beaker, the RCW samples were inoculated with two milliliters of suspensions, the RCW-SY samples received
one milliliter of SY and two milliliters of the RCW inocula, the SF-SY
samples were inoculated with one milliliter of each suspension.
Uninoculated samples and samples prepared with the commercial starter
served as controls.
All samples, except the uninoculated rice were
incubated for three days at 27°C. The uninoculated rice was not incubated.
The inocula were prepared in
the identification tests.
th~
same manner as those used for
20
After inoculation, the
ri~e
was mixed thoroughly, packed
lightly, and covered with plastic wrap.
Where the inoculum was a
mold, or the starter, the beakers were lightly covered with the
plastic wrap; with SY as the inoculum, the wrap was secured with a
rubberband.
When the yeast and either SF or RCW were used in combi-
nations, a koji method of incubation was used, i.e., the rice was
inoculated with the mold, lightly covered, and allowed to incubate
for 24 hours before inoculation with the yeast.
At this time, the
plastic wrap was secured with a rubberband.
Preparat·ion of samples for chemical analysis
The incubated rice samples were prepared for chemical testing
by the following method:
the rice was transferred from the beaker to
a 250 ml Erlenmeyer flask, 50 ml of distilled water was added and the
flask was shaken by hand for one minute.
This was filtered through
cheesecloth and the residue washed with 25 ml portions of distilled
water.
The filtrate was made up to 150 ml and was used for all chemi-
cal tests.
Chemical analysis
~
determination.--The pH value of the filtrates was deter-
mined with a pH meter.
Reducing sugar determination.--The Lane-Eynon titrimetric
method for reducing sugar as described by the Association of Official
Analytical Chemists (1970) was adapted.
The sample was prepared by
.pipetting 50 ml of the rice filtrate into a 100 ml volumetric flask
and adding 5 ml of saturated lead acetate solution and two drops of
glacial acetic acid.
minutes~
This solution was allowed to stand for ten
made to volume with distilled water, and then filtered
through Whatman #1 filter paper into a 250 ml Erlenmeyer flask containing 2 g of sodium phosphate.
When the filtration was
complete~
a small amount of additional sodium phosphate was added to the solution to insure that all lead had precipitated out.
This solution was
allowed to settle and the test sample was taken from the clear upper
layer.
The test sample was analyzed in the following manner:
10 ml
of the sample solution and 12.5 ml of Soxhlet reagent were placed in
a 250 ml Erlenmeyer
flask~
heated to boiling, and titrated with a
0.5% invert sugar solution until a faint blue color remained.
Three
drops of 1% methylene blue were added and titration continued, dropwise, to completion as indicated by the brick red color of the foam.
The titration had to be completed in three minutes after boiling began.
The amount of reducing sugar in the original filtrate is calculated
using the formula:
(ml 0. 5~; sugar
for Soxhlet)
(ml 0.5% sugar x 0 . 005
for sample)
10 ml
x dilution
= grams reducing sugar/100 ml sample
Ethanol determination.--The gas chromatography method described
by Trachman (1969) was adapted.
A Varian Aerograph model 1200 gas
chromatograph with.flame ionization detector was used.
The column
used was stainless steel, five feet in length with an outside diameter
of l/8 inch and packed with Poropak Q, mesh size 50/80.
the carrier gas.
Gas flow rates were:
Nitrogen was
n1trogen, 60 ml/minute;
22
hydrogen, 30 ml/minute; air, 300-400 ml/minute.
Temperatures were:
injector, 100°C; detector, 295°C; column l00-200°C with a ptogram
rate of l0°C/minute.
The rice filtrates were used directly.
was 1 microliter.
Injected sample size
Standards were prepared by diluting aliquots of
absolute ethanol to volume in a 100 ml volumetric flask.
Quantitative ethanol concentrations are calculated using
triangulation (peak area) and the formula:
Area
= 1/2 base x height,
and the peak areas of samples were compared to those of standards.
RESULTS AND DISCUSSION
Identification of SY Isolate
Morphological tests
Characteristics of asexual reproduction
Asexual reproduction in yeast may occur as budding, fission,
or a combination of the two.
A variation of budding which is not very
common is by formation of sterigmata upon which the new bud forms.
Budding is the development of a small protrubence from a
1arger parent cell.
The mature bud may either detach itself from the
parent cell or remain and give rise to clusters of other small cells
or chains of cells.
Some buds may extend to a pseudomycelium.
Bipolar budding occurs when buds form at two distal poles of the
parent cell.
Multipolar budding occurs when buds form at different
points on the surface of the parent cell; a classical form of reproduction among yeasts {Ladder, 1970).
Blastospores may be formed by
budding, usually at the junction of two cells in a pseudomycelium
(Kreger~van
Rij, 1969).
Fission occurs when a septum forms inward from the cell wall
along the length of the cell, dividing the cell in two.
Fissionis
typical of the Endomyeetaeeae and Sehizosaeeharomyeoideae (Ladder,
1970).
23
The SY isolate grown in 2% GYP water and on 2% GYP agar showed
primarily unipolar budding, i.e., at one end of the cell only, and
some bipolar budding.
No ster·igmata wer-e formed.
Characteristics of vegetative cells
Size and shape of vegetative cells serve as useful taxonomic
criterion when they are distinctive enough to differentiate between
genera, such as the triangular cells of.Trigonopsis (Ladder, 1970).
Normally~
this is not the case.
In liquid medium (2% GYP water) cells of the SY culture
measured 5-7 11m x 3-6 ·11m.
The cells appeared slightly smaller on
solid 2% GYP, measuring 4-6 11m x 3-5 11m. t1ost cells are slightly oval
in shape.
Formation of pseudomycelium or mycelium
A true hypha grows at the tip and crosswalls (septa) may be
formed.
The terminal unit on a hypha may often be longer than the
preceding cell because septation lags somewhat.
A pseudomycel ium is formed by budding cells which remain
attached, elongate, and thus form a long filament.
Septa are charac-
teristically missing and the terminal cell is often shorter than the
other cells, because growth does not occur at that end.
Formation of mycelium and pseudomycelium may occur concurrently, as in Endomyeopsis and Trichosporon (Ladder, 1970). This
appears to be the case with the SY isolate.
Cornmeal agar slides
observed under the microscope showed the presence of mycelium with
definite septation and profuse blastospore formation.
The septate
25
mycelia have many side branches arranged at approximate 90° angles
to the main hyphae.
The presence of pseudomycelium was also observed.
Cultural characteristics
Cultural characteristics of yeast both in liquid and on solid
media which may be observed with the naked eye are somewhat limited
in taxonomic value (Ladder, 1970).
Formation of a sediment, a ring, islets, or a pellicle, a dull,
dry-looking scum (de Becze, 1962), may result from yeast growth in
1 iquid
medium.
Yeasts .v1hich require oxygen to carry on metabolic
processes tend to form a pellicle.
Yeasts which form true mycelium
may produce a thick surface
later turning into a mucoid mass.
growth~
The SY culture forms a thick sediment at the bottom of the
2% GYP water flask.
After approx·imately four weeks at room tempera-
ture, the sediment \'las even thicker and almost mucous-like.
Micro-
scopic examination showed that a great deal of mycelium had formed.
No pellicle, ring, or islets was formed.
On solid 2% GYP agar, the SY formed
white colonies.
compact~
round, powdery,
Formation of colonies of this type is generally asso-
ciated with the production of pseudomycel ium or mycelium (Ladder,
1970).
Sexual characteristics
Sexual reproduction or ascospore formation is an important
taxonomic criterion because it serves to classify the microorganism
as either a Zygomycotina, Ascomycotina, or Basidiomycotina. The shape
of the spores, number of spores per ascus, and mode of germination may
all facilitate differentiation of one genus from another.
Ascospores
form under adverse conditions, when the nutrient content of the
support medium is inadequate.
It is sometimes difficult to induce a
yeast to sporulate, so several different types of sporulation media
must be tf'i ed.
Ascospore formation in haploid homothallic yeasts which reproduce exclusively by budding is thought to be preceded
diploidization.
by
karyogamy and
Diploidization can occur in either of two ways.
the first, a vegetative cell undergoes mitosis and forms a bud.
two haploid nuclei of the mother cell and bud fuse.
In
The
The diploid
nucleus undergoes division and one to four ascospores are discernible
in the mother cell.
jugate.
In the second method, two independent cells con-
Diploidization may be accomplished either by direct fusion
of the two cells, or by fusion of copulatory protubences formed by the
cells.
The latter gives the cells the appearance of a dumb-bell,
within which the
ascospore~
are delimited.
Diploid homothallic yeasts may form ascospores in unconjugated
asci.
In the case of heterothallism, cells of opposite mating types
are required for sporulation (Kreger-van Rij, 1969).
The number of ascospores in the ascus may vary from one to
many.
This is not a highly important taxonomic criterion.
However,
the shape of the ascospores does provide a good criterion for differentiation (Kreger-van Rij, 1969).
reniform, cylindrical,
Round, oval, sickle-shaped,
crescent-shaped~
and hat- orsaturn-shaped
• ascospores occur (Ladder, 1970). The cell wall of mature ascospores
may be acid-fast, thus staining with an acid-fast dye may help detect
_sporulated cells.
27
Potato
plugs~
malt extract agar, and cornmeal agar slants
were used for this test.
After three days of
the cultures were heat-fixed and stained.
incubation~
growth from
No sporulation had occurred.
The cultures were allowed to stand at room temperature for seven weeks,
during which time growth was stained and examined several times.
Sporulation was finally detected on the potato medium during the
seventh week.
Both single cells and dumb-bell shaped cells, indicating
conjugation, were found.
A few hat-shaped ascospores were seen.
Physioloqical character.istics
Utilization of carbon· compounds
Yeasts utilize carbohydrates in either of two ways; fermentation or assimilation.
Fermentation is the term used to describe
anaerobic utilization of carbon compounds, notably simple sugars.
The
Embden-Meyerhof-Parnas (EMP) pathway is the most common system for the
anaerobic metabolism of carbohydrates.
use of carbohydrates.
Assimilation is the aerobic
The most common oxidative pathway used by
yeasts is the tricarboxylic acid (TCA) cycle, although other pathways
are also used.
Oxidative use of carbohydrates is much more efficient
than anaerobic utilization.
Fermentative use of carbon compounds
The ability of a yeast to ferment carbon compounds depends
on the presence of an adequate transport system to allow utilization
of the sugar in the presence of little or no oxygen and on the presence
of appropriate enzymes for the anaerobic glycolytic breakdown to
ethanol and carbon dioxide.
Simple fermentation tests do not establish
the definite presence or absence of either of these factors, but merely
indicate that one or the other of the factors is in effect.
Simple fermentation tests utilize the presence or absence of
carbon dioxide evolution as an indication that fermentation has
occurred.
duction.
The Durham tube is specially designed to measure gas proConcurrent use of an indicator in the medium also aids in
assessment of carbon dioxide production.
How fast fermentation proceeds is determined by the time
required for formation of the inducible enzyme systems necessary for
the process and by gas ·evolution.
If a sugar is vigorously fermented,
a relatively large amount of gas may be observed in the insert tube
within a day or two.
If fermentation does occur, glucose wi11 always
be fermented.
Data as presented in Table 1
indi~ate.that
ferments glucose, sucrose, and maltose.
the test culture
Maltose fermentation was
particularly vigorous, but this may be due .to,the fact that the SY was
maintained on YM agar.
According to Ladder (1970), prolonged mainte-
nance on malt agar may result in the yeast acquiring the ability to
utilize maltose.
The sugars which were fermented did so quite vigor-
ously, as indicated by a change ·in the bromothymol blue indicator from
green to yellow or slightly yellow (indicating carbon dioxide in
solution) by the second or thfrd day of fermentation.
Raffinose was
fermented, but not as vigorously as the other fermented sugars.
Tests
to discern which part of the triose was being fermented were not performed.
29
Table 1.
Fermentation and assimilation of carbon and
nitrogen compounds by SY isolate
Carbon compound
Fermentation
glucose
Assimilation
++
++
+*
galactose
sucrose
++
++
maltose
++
++
+*
1actose
raffinose
+*
+*
Nitrogen compound
nitrate
+
nitrite
+*
amino alkane
++
++-utilized vigorously
+* - indicates a weak or slow reaction
. 30
Oxidative use of carbon compounds
The ability of a carbon compound to permeate the cell wall and
the presence of appropriate enzyme systems determine whether or not
the compound will be utilized
(Kreger~van
Rij, 1969). Generally, the
assimilation tests are more sensitive for detecting enzyme systems
than the fermentation tests (Ladder, 1970).
However, it is not possi-
ble by simple growth tests to determine wh·ich factor may account for
the ability or inability of a compound to be utilized nor is it possible to differentiate enzyme systems.
The list of carbon compounds which can be used for the assimilation tests is far more extensive than that for the
tests.
fet~mentation
Wickerham and Burton (1948) introduced a list of approximately
30 compounds which may be used.
Assimilation of the compounds in
liquid medium may be measured visually as relative turbidity (cell
mass} or by cell counts, although the latter is not considered as
reliable (Thorne, 1957).
Since all carbon compounds cannot be assimi-
lated by all yeasts, it is possible to differentiate between genera or
species on the basis of assimilation tests.
Yeasts will, however,
assimilate any sugar they ferment (de Becze, 1960). As in the fermentation tests, it is possible for a yeast to utilize a carbon com• pound in testing which it might not normally utilize due to the effect
of the maintenance medium.
Cell counts were used to determine assimilation.
As data in
Table 1 illustrate, maltose, sucrose, glucose, lactose, galactose, and
raffinose are assimilated by SY, in that order, with the latter three
being very slight.
31
'
Hydrolysis of arbutin
Confirmation of S-glucosidase activity in yeasts is achieved
with this test.
If the yeast is capable of splitting the S-glucoside,
the hydroxyquinone liberated reacts with any soluble ferric salts in
the medium, giving a brown color.
The SY culture showed a positive reaction to this test after
three to four weeks.
Utilization of nitrogen compounds
Si nee nitrogen .is one of the basic growth factors, the ability
or inability to assimilate different sources can be used to identify
yeasts.
The ability to utilize nitrate-nitrogen is mediated by the
sequential action of a series of "reductase systems
11
(Ladder, 1970)
which aid in the breakdown of the nitrate to other reduced compounds.
Utilization of nitrate is highly valued in differentiating yeasts
(Kreger-van
nitrite.
Rij~
1969).
Yeasts which utilize nitrate can utilize
However, those which use nitrite do not invariably have the
capacity to use nitrate.
Nitrate cannot be used in certain situations
where it might normally be used as a nitrogen source, because it may
be reduced to nitrite, which has a highly toxic effect on many microorganisms (Thorne, 1957; Ladder, 1970).
Aliphatic amine nitrogen
sources are used by many yeasts as a source of nitrogen.
Nitrogen utilization in liquid media was measured as an
increase in cell count.
The SY culture used the amino alkane form,
ethylamine hydrochloride, of nitrogen most readily.
Nitrate and
nitrite were also used, with the latter being only weakly utilized.
l
Gr·owth in vitamin-free medium
Some yeasts are capable of surviving in a vitamin-free
environment, while others have an absolute requirement for one or more
vitamins.
Well-nourished yeast cells may continue to proliferate in
the test medium due to carry over of needed growth factors.
Trans-
ferring growth from one tube of vitamin-free medium to another is a
method designed to eliminate this interfering factor by depleting the
stored growth factors.
The medium is complete except for vitamins,
par·ticularly of the B-complex, which have been omitted.
Growth is deter·mined by cell count, an increase of which
indicates the ability to proliferate in the medium.
Increase of cell
count with the SY indicates the ability to grow in the vitamin-free
medium.
Growth on media of high osmotic pressure
Most yeasts are capable of growing on media with a glucose
concentration of up to 40% (w/w) however, the osmophilic species may
grow on media with concentrations from 40-70%.
This characteristic is
litnited in usage, except in certain genera, such as
Saccharomyces~
where it might help delineate varieties of a species (Lodder, 1970).
The SY isolate is capable of fairly heavy growth on 50% glucose
agar, but no growth on 60% (w/w) glucose agar was observed.
Growth at high temperatures
Optimal temperature for growth of most yeasts is in the range
of 20-28°C.
Yeasts which normally grow in association with warm-
blooded animals are capable of growing at temperatures of approximately
37°C.
Generally, the ability to grow at 37°C or above is used only
to confirm a species.
Growth of the SY culture at 37°C was compared to growth at
25°C on the basis of colony diameter.
The diameter of the colony
incubated at 37°C increased more rapidly than the diameter of the
culture at 25°C, indicating the ability of this yeast to grow at
elevated temperatures.
Acid production
This test is used to determine excessive production of acetic
acid by the yeast.
Almost all yeasts produce acids, either volatile
or nonvolatile, as byproducts of their· metabolic processes.
Only
excessive acetic acid production serves as a taxonomic criterion.
Solid medium containing calcium carbonate, which causes a
slightly milky appearing agar, is used.
If the yeast produces exces-
sive acid, the calcium carbonate will act to neutralize it, as indicated by clarification of the agar around the colonies.
The SY isolate
reacted negatively to this test, indicating that it does not produce
substantial amounts of acetic acid.
Ester production
Many yeasts form esters during metabolism.
It is only when
ester production is substantial enough to be detected by odor that
this ability becomes of taxonomic importance.
Ethyl acetate is the
principle ester formed (de Becze, 1960). The ester gives a somewhat
fruity odor to the medium on which the yeast is growing.
The isolated
34
yeast (SY) gave off a definite fruity odor on
Y~1
agar, therefore it
may be considered to produce esters.
Summary of identification of the SY isolate
Using the characteristics elucidated by the various identification tests, it was possible to trace the SY isolate through the
identification keys presented by Ladder (1970) to a final genus choice
of Hansenula or Endomycopsis.
found in Table 2.
A summary of these characteristics is
The culture was traced through both genera, however,
the SY description did .not fit descriptions of the various species of
HansenuZa indicated by the key for several reasons.
Characteristics
of the HansenuZa genus which were not in keeping with those of the
isolate include the absence of true mycelium, pellicle formation in
a liquid culture, and cell measurements which did not coincide with
those of the SY.
One trait which the SY isolate and HansenuZa genus
have in common is the assimilation of nitrate, but since the SY utilizes an amino alkane form of nitrogen much more readily than .the
nitrate form, undue importance should not be placed on this factor.
Therefore, it may be assumed that the SY isolate is most likely a
species of the genus Endomycopsis.
The characteristics of the SY most
closely resemble those cif Endomycopsis bUl.'tonii (see Appendix II),
except in vigor of sugar fermentations, assimilation of nitrate~. and
growth at 37°C.
E. bUl.'tonii ferments the same sugars as the SY culture,
glucose, maltose, sucrose, and raffinose, but generally does so less
vigorously than the SY.
E. bUl.'tonii does not assimilate nitrate and
growth at 37°C is only weakly positive, whereas the SY isolate does
assimilate nitrate and growth at 37°C is definitely positive.
The
Table 2.
Summary of identification test results--SY isolate
Asexual reproduction: · by budding
Vegetative cells: Slightly~val in shape, 4-7 pm x 3-6 pm in
liquid medium, 4-6 pm x 3-5 pm on solid medium.
Formation of pseudomycelium and mycelium: Both formed on cornmeal
agar. Buds formed all over the hyphae. Side branches on
true hyphae at ca. 90° angle to hyphae.
Cultural characteristics: Formed a sediment in liquid medium, and
compact, powdery colonies on solid medium.
Sexual reproduction: Formed ascospores, preceded by conjugation,
hat-shaped, one to four per ascus, on potato plugs after
seven weeks. ·
Fermentation of carbon compounds:
glucose galactose
maltose lactose sucrose raffinose
positive
- negative
positive
negative
positive
- positive
Assimilation of carbon compounds:
glucose galactose
maltose lactose sucrose raffinose
positive
- weakly positive
positive
weakly positive
positive
- weakly positive
Hydrolysis of arbutin:
positive
Utilization of nitrogen compounds:
Growth in vitamin-free medium:
potassium nitrate - positive
sodium nitrite- weakly positive
ethylamine hydrochloride positive
positive
Growth on high osmotic pressure medium:
Growth at high temperatures:
Acid production:
Ester production:
neqative
positive
37°C:
50% (w/w) glucose- positive
60% (w/w) glucose- negative
positive
37
38
Figure 2.
SY isolate grown on potato plugs for seven weeks, stained
with modified Kufferath carbol-fuchsin stain
39
•
;~.o
same sugars are assimilated
by
both, except that the SY very weakly
assimilate lactose and E. burtonii does not.
Both have positive
reactions to the hydrolysis of arbutin, growth on vitamin-free medium,
and growth on 50% (w/w) glucose-yeast extract agar tests.
Identification of the SF Isolate
Taxonomy
Taxonomic classification of fungi rests heavily on morphological characteristics, although it is possible to differentiate
species with supportive nutritional and biochemical tests.
At present,
all fungi are classified as plants, a fact which causes controversy.
Ainsworth (1973) suggests treating the fungi as a separate kingdom
and constructs his keys accordingly.
Traditionally, taxonomic arrangements of the fungi begin by
distinguishing those which form mycelium from those which do not
{plasmoidal fungi).
Myxomycota.
The former are termed Eumycota and the latter
Eumycota are divided into five subdivisions based on the
types of sexual spores produced.
Mastigomycotina form.motile spores,
Deuteromycotina form no spores, Zygomycotina form zygospores,
Ascomycotina form ascospores, and Basidiomycotina form basidiospores.
In previous family keys, the type of mycelium formed also served to
differentiate between Zygomycotina (Phycomycetes) and Ascomycotina
(Ascomycetes), the former having aseptate mycelium and the lattet•
septate mycelium (Clements and Shear, 1931).
This was not totally
tenable because some Zygomycotina developed septate mycelium in older
cultures.
The subdivision Zygomycotina was previously 1.a catch:...all, a
situation which is now being corrected by separating the subdivision
36
Figure 1.
Pseodomycelium of SY isolate on cornmeal agar slide
41
·into at least two classes; Zygomycetes and Trichomycetes (Ainsworth!>
1973), and separating all other types into classes under other subdivisions.
Zygomycetes and Trichomycetes are differentiated by whether
or not they are saprobic and their mode of attachment to their host
or substrate.
Trichomycetes live in close connection w'ith arthnopods,
whereas Zygomycetes are differentiated by their manner of reproduction,
as well as other characteristics.
The Zygomycetes are represented
by three orders; Mucorales, Entomophthorales, and Zoopagales.
Ainsworth (1973), and Hesseltine and Ellis (1973), present comprehensive keys to the classes and genera of each of these orders, however,
they do not present keys to the species in the genera.
Zycha (1935)
has compiled what is still considered a most comprehensive key and
description of the genus Rhizopus in his book Mucorineae.
11
11
Description of the SF isolate grown on YM agar
Colonies on YM agar were first white changing to pale or dark
gray after two days of incubation at 27°C.
slightly more rapid at 37°C.
at 37°C.
Growth of the colony was
There was no change in mycelial growth
Sporangiophores are standing upright or bending slightly.
They appear to originate on stolons.
is globose and dark brown in color.
Slight apophysis is present.
slightly oval in shape.
Mycelium is aseptate.
Sporangium
Young sporangia are buff colored.
Sporangiospores are round or very
Size of spores:
4-6
~m
x 3-5
~m.
Zygospores
are present, globose in shape or slightly compressed between syspensores, and dark brown in color.
warty.
Rhizoids present.
Surface of zygospore appears slightly
42
De~cription
of RCW culture on YM agar
Colonies on YM agar were first wh·ite changing to dark gary
after two days of incubation at 27°C.
at 37°C.
Growth of colony was more rapid
Shorter mycelia were foY'rned at 37°C.
Sporangiophores are
standing upright or bending slightly and appear to originate on
stolons.
Mycelium is aseptate.
Sporangium is globose, with the lower
side flat, and dark brown in color.
spores are round in shape.
Apophysis is present.
Size of spores:
Sporangia-
4-6 ]lm x 4-6 ]lm.
Zygo-
spores are present, globose in shape or slightly compressed between
suspensors, and dark brown in color.
slightly warty.
Surface of zygospore appears
Rhizoids present.
Characterization of the SF isolate
Based on the presence of mycelium, zygospores, and sporangia,
the SF isolate was traced through identification keys presented by
Ainsworth (1973), Clements and Shear (1931), and Gilman (1957) to the
order Mucorales.
It was further possible to trace the culture through
the Mucorales keys presented by Zycha (1935) and Hesseltine and Ellis
(1973} to the genus Rhizopus, based on the presence of an
stolons, and rhizoids.
apophysis~
It was not possible to trace the culture
further due to information which was not obtained, such as zygospore
measurements, sporangia measurements, and height of sporangiophores,
all of which help pinpoint the species.
Cornparison:with the RCW culture revealed enough divergent
characteristics to conclude that the SF is not Rhizopus chinensis.
These include differences in sporangiospore size, degree of apophysis,
shape of sporangium and growth at 37°C.
The similarities between the
43
Figure 3.
Sporangium of SF isolate, grown on YM agar
44
two cultures are strongenough, however, to conclude that the SF
isolate is a member of the genus Rhizopus.
rapidity of
gr~owth,
Similarities include
appearance of myce1 ium and stolons, general
appearance of .zygospore and sporangium.
Chemica"! Changes in Glutinous
---rnocuTa.1ecr\~-1Jr!1ift1 croor·gan i
Rice
srns-
The changes in chemical composition of rice samples inoculated
with either SF, SY, RCW, or in various combinations were determined in
order to assess and compare the saccharifying and fermenting ability
of these microorganisms.
Data on the pH value, reducing sugar content,
an~ ethanol content of the rice sample are shown in Table 3.
pH value
At three days, all samples have a pH value below 4.7, down
from the average of 6.5 at the time the samples were prepared.
.
ge~era 1,
In
a lower pH favors development of fermentative m-fcroorganTS.ifis;
either acidic or
may interfere.
alco~olic,
by excluding other microorganisms which
However, the pH must be in the range suitable for
growth of the fermentative microorganism.
The lowering of the pH in
the rice samples is largely due to the presence of organic acids
produced by the microorganisms (Rhodes et al., 1959; Pederson, 1971).
Reducing sugar content
The average reducing sugar value measured for the SF is twice
as high as for SY, however, considering the differences within the
samples, this may not be significant.
When the two isolates are used
together, the reducing sugar value is somewhat higher than of either
46
Figure 4.
Sporangium of RCW culture, grown on YM agar
48
Table 3.
Chemical changes in glutinous rice fermented with test
cultures incubated at 27°C for three days
·
Chemical changes in rice
--------
Test .culture
Reducing sugar
(gil 00 rrd)
pH
-*.
-*
6.77
6.34
Average
Ethanol
content (%)
0.4
0
0
0.3
6.55
0.3
0
-----SF
SF
4.20
4.45
Average
4.42
4.69
Average
RCW
RCW
RCW-SY
RCW-SY
RCW-SY
4. 41.7
3.54
3.84
3.74
Average
starter
starter
starter
3.60
3.54
3.54
Average
0.2
0.5
0.2
79.3
60.3
3.57
3.33
3.35
3.36
0.3
1.5
0.6
69.8
3.35
1.1
0.6
1.3
0.8
56.7
72.5
55.6
61.6
0.9
0.8
1.7
2.0
11.8
11.6
12.0
3.56
0.1
6.6
3.71
Average
0
0.2
4.1
9.2
6.5
3.57
3.57
0.1
3.0
4.56
Average
0.1
0.1
6. i
4.33
SY
SY!
SF-SY
SF-SY
SF-SY
6.1
6.0
11.8
1.2
*The uninoculated rice samples were tested directly without
incubation at 27°C.
-·
----- -
- ----
.
. 49
alone,
which~
if significant, may be the result of a greater number
of organisms in the combined inoculum.
In contrast, the RCW culture
alone or in combination with the starter yeast produces a great deal
more reducing sugar than the starter culture isolates alone or in
combination.
decreases~
When RCW is combined with SY, the reducing sugar
but is still higher than any combination which did not
include RCW.
The very low reducing sugar value in the starter sample
in comparison with either of the RCW
low values for both SF and SY.
samples~
is consistent with the
These data indicate that RCW is a very
powerful saccharifying agent relative to the SF and SY isolates which
possess only modest saccharifying ability.
Several researchers have discussed the amylolytic properties
of Rhizopus (Yue et al ., 1966a; Vue et al., 1966b; Thaysen and
Galloway, 1930; Banks et al ., 1967; Hesseltine and Wang, 1967).
Various members of the genus have been isolated from Chinese yeast
balls (Thaysen and Galloway, 1930) and Oriental fermented food
products such as tempeh, ragi, and lao-chao (Wang and Hesseltine,
1970; Hesseltine and Ellis, 1973), and have been shown to possess
strong saccharifying ability.
Species of Rhizopus have also been
used commercially in the Amylo process in Occidental countries
(Hanson et al., 1955).
The SY isolate shows the ability to produce enzymes capable
. of breaking down starch into smaller components, including reducing
sugars, although to a lesser degree than either of the molds.
Endomyaopsis is one of the few yeasts which produce amylases and are
thus capable of utilizing starch (Wang and Hesseltine, 1970).
5()
Ethanol content
As shown in Table 3, ethanol was detected in all samples,
except the uninoculated rice and one SY sample.
This latter result
was unexpected and cannot be explained, as preliminary tests showed
that SY-inoculated rice contained ethanol.
Average ethanol production
is approximately the same for both the SF and SV isolates.
When used
in combination, the ethanol concentration increases slightly.
The
higher number of microorganisms in the combined inoculum may account
for this slight increase.
Ethanol production by the RCW culture alone
is 3.5-11 times higher than that of the SF, SV, of SF-SY combination.
When combined with the isolate, the RCW culture produced slightly less
. ethanol than by itself.
This is an interesting discrepancy in that
yeasts are usually expected to produce more ethanol than a mold.
More
extensive testing must be done before any conclusion concerning this
decrease in ethanol can be dt·awn.
Ethanol production by both ROJ and
RCW-SV is up to 11 times higher than the SF-SV combination.
The
starter sample contains slightly more ethanol, but still approximately
11 times more than the SF-SY combination.
The cell concentration in
the starter is unknown, so it is possible that it contains a higher
number of microorganisms than any of the other inocula used.
It is not unusual that the mold cultures both produced some
ethanol, since they produce the amylases necessary to form fermentable
sugars and the ability to produce ethanol is not uncharacteristic of
the Rh-izopus genus.
In studies conducted on fermenting microorganisms,
Verachtert (1970) proved that Rhizopus oryzae was capable of producing
ethanol.
Zycha (1935), in fact, used ethanol production as a
d~ffer-
entiating characteristic for solile of the Rhizopus species.
Comparison of SF, SY, and SF-SY
In general, the SY isolate produces less reducing sugar than,
but equal amount of ethanol to that produced by the SF isolate.
The
SY culture does not appear to have much impact on the fermentation,
based on the average reducing sugar and ethanol values for SF as compared with the SF·-SY combination.
This implies that the SF and SY
isolates differ little jn their abilities to break down starch to
re<iucing sugars and to produce ethanol.
It further implies that the
mold alone could carry out the fermentation of glutinous rice.
How-
evers preliminary test results indicate that this is not the case.
Without the yeast present, an unsatisfactory fermented product occurs;
no liquid is formed, sporulating mycelia grow on the surface, and the
flavor is "moldy,
product.
11
although the odor is comparable to a satisfactory
Wang and Hesseltine (1970) made the same observation and
postulated that the yeast seemed to promote utilization of sugar and
stimulate the fermentation.
When the yeast alone is used to ferment
the rice, liquid is formed and ethanol is produced, but the flavor
lacks the sweetness characteristic of a satisfactory fermented glutinous rice product.
These results indicate that both the mold and the yeast isolated from the starter have the ability to saccharify starch and to
produce ethanol, but that both organisms are necessary to give the
liquefaction, aroma, and flavor of lao-chao.
Future work on this
subject might more deeply explore the relative roles of the yeast and
52
the mold in the fermentation.
Exact determination of the amylolytic
enzymes produced by both the mold and yeast would also expand the
scant literature on this particular type of fermentation.
More definitive identification of the mold and the yeast
present in the commercial starter could be accomplished by further
study of morphological characteristics of the former and further
testing of nitrogen and carbon utilization for the latter.
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Association of Official Analytical Chemists. 1970. Lane-Eynon
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Arima, K. 1957. Sake and similar yeasts.
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In Yeasts:' W. Roman, ed.,
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Banks3 G. T., F. Binns, and R. I. Cutcliffe. 1967. Recent developments in the production and industrial applications of amylolytic
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Gilman, J. C. 1957. A Manual of Soil Fungi,
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Gray, W. D. 1970. The Use of Fungi as Food and in Food Processing,
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Hahn, P. A. 1968. "Chemicals from Fermentation,
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Hang, Y. D., and H~ Jackson. 1967a. Preparation of soybean cheese
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Hang, Y. D., and H. Jackson. l967b. Prepar·ation of soybean cheese
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Hanson, A.M., T. A. Bailey, R. C. Malzahn, and J. Corman. 1955.
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Hao, L. C.~ E. I. Fulmer, and L.A. Underkofler. 1943. Fungal
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Hesseltine, C. W. 1965. A millennium of fungi, food, and fermentation. Mycologia. 57(2):149.
, and H. L. Wang. 1967.
Biotechnol. Bioeng. 9(3):275.
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Traditional fermented foods.
, and J. J. Ellis. 1973. t~ucorales. In "The Fungi: An
Advanced Treatise, G. C. Ainsworth, F. K. Sparrow, and A. S.
Sussman, eds., chapt 11. New York: Academic Press.
11
Houston, D. F., and G. 0. Kohler. 1970. Nutritional properties of
rice, p. 8. Washington, D.C.: National Academy of Sciences.
Ito, M. 1929. Studies on enzymic substance contained in koji made
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Juliano, B. 0. 1965.
pines. 1965:5.
Our daily rice.
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Kreger-van Rij, N. J. W. 1969. Taxonomy and systematics of yeasts.
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Harrison, eds., chapt 2. London: Academic Press.
11
Liu, P. W., S. H. Chen, T. H. Chiu, and C. C. Chen. 1959. Studies
on shao-shing wine I. Taxonomical studies of saccharifying mold
from chiu-chu of shao-shing wine. Chemistry (Taipei) 3:181.
Lius P. Wq and S. 1-1. Chen. 1962a. Studies on the amylases of
amylomyces from Chinese chiu-chu (a starter of Chinese wine) I.
Isolation~ purification. and properties of the amylases from
Rhizopus tienehiuUensis. Chemistry (Taipei) 3:111.
. 1962b. Studies on the amylases from amylomyces of
chiu-chU v. On the activities of isoamylase and isomaltase
in the amylases of molds. Chemistry (Taipei) 11:21.
---.~C~h~in-e-se
Ladder, J ed. 1970. The Yeasts--A Taxonomic Study," pp. 1-120,
166-208. Amsterdam: North-Holland Publishing Co.
11
03
Ng, H. 1972. Factors affecting orgarric acid production by sourdough
(San Francisco) bacteria. Appl. Microbial. 23:1153.
"Microbiology of Food Fermentations~~~ chapts
Westport, Conn.: Avi Publishing Co.
C. S. 1971.
1 2, 3, 5, 6, 10.
Pederson~
5
Rhodes, R. A., A. J. Moyer, M. L. Smith, and S. E. Kelley. 1959.
Production of fumaric acid by Rhizopus arrhizus. /l.ppl. Microbiol.
7:74.
Roelofsen, P. A., and A. Talens. 1964. Changes in some B vitamins
during molding of soybeans by Rhizopus oryzae in the preparation
of tempeh kedelee. J. Food Sci. 29:224.
Shibasaki, K., and C. W. Hesseltine.
Bot. 16:180.
1962.
Miso fermentation.
Econ.
Smith, A. K., J. J. Rackis, C. W. Hesseltine, M. Smith, D. J. Robbins,
and A. N. Booth. 1964. Tempeh: Nutritive value in relation to
processing. Cereal Chern. 41:173.
Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. 1970. 11 The
Microbial World, 11 p. 176. Englewood Cliffs~ N.J.: PrenticeHall, Inc.
Steinkraus, K. H., Y. B. Hwa, J. P. van Buren, M. I. Provvidenti, and
D. B. Hand. 1960. Studies on tempeh--An Indonesian fermented
soybean food. Food Res. 25:777.
Teramoto, S., H. Taguchi, and R. Ueda. 1965. The organic acid constituents of Japanese fermented food products and some aspects
of their methods of production and qualities. In 11 Proceedings
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Thaysen, A. C., and L. D. Galloway. 1930.
Starch and Sugat'S, pp. 22-39. London:
11
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Thorne, R. S. W. 1957.
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In
The Microbiology of
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11
Yeasts~u
W. Roman, ed.,
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·
van Veen, A. G. 1972. Fermented rice foods. In 11 Rice: Chemistry
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·
Wang, H. L., and C. W. Hesseltine.
43:563.
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Sufu and
1966.
lao~chao.
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11
pp. 4-49, 106-118.
Leipzig:
Verlag
APPENDICES
57
Appendix I
Media for Yeast Identification
The following dehydrated media were rehydrated and sterilized according
to instructions on the product label.
1.
Bacto-YM agar - Difco 0712-01
2.
Bacto-potato dextrose agar - Difco 0013-01
3.
Bacto-malt extract agar - Difco 0186-01
4.
Bacto-yeast nitrogen base - Difco 0392-15
5.
Bacto-yeast carbon base - Difco 0391-15
6.
Bacto-vitamin-free yeast base - Difco 0394-15
The following media were prepared in the laboratory according to
Ladder (1970).
1.
Acidified YM agar- 41 g of YM agar (Difco 0712-01) were
suspended in 1000 ml of distilled water, heated to boiling,
and autoclaved at 15 psi for 15 minutes.
Cool agar to 45°C
and acidify to pH 3.7 with sterile 1 N HCl (ca. 24.5 ml
1 N HCL/500 ml agar).
2.
2% (w/v) glucose-yeast extract-peptone water - Dissolve 20 g
glucose, 10 g Bacto-peptone (0118-02), 5 g Bacto-yeast extract
(Difco 0127-0l) in 1000 ml distilled water.
autoclave for 15 minutes at 15 psi.
58
Dispense and
3.
2% (w/v) glucose-yeast extract-peptone agar - Add 2% (w/v) agar
to 2% glucose-yeast extract-peptone water, heat to boiling, and
autoclave 15 minutes at 15 psi.
4.
Potato plugs - Vegetables were peeled and cut into long cylinders about 1 em in diameter.
to obtain wedges.
The cylinders were cut obliquely
Wedges were rinsed with cold water and
placed in culture tubes containing ca. 5 ml distilled water
to prevent desiccation.
The tubes were sterilized by auto-
claving for 15 minutes at 15 psi.
5.
Fermentation basal medium - 4.5 g Bacto-yeast extract (Difco
0127-01) and 7.5 g Bacto-peptone (Difco 0118-02) were dissolved
in 1 liter of distilled water.
Sufficient bromothymol blue
was added to give a deep green color (ca. 0.25 g).
5 ml
aliquots were placed in culture tubes containing an inverted
insert tube.
Tubes were sterilized by autoclaving at 15 psi
for 15 minutes, during which time the insert tubes filled with
medium.
These tubes were cooled and 1 ml aliquots of filter-
sterilized 6% sugar solutions were aseptically added for the
final fermentation media.
6.
50% and 60% (w/w) glucose-yeast extract agar - 50 and 60 g of
. glucose were dissolved in 50 and 40 ml, respectively, of 1%
yeast autolysate (Bacto-yeast extract, Difco 0127-01).
The
volume was noted and an amount of agar (Bacto-agar, Difco 014001) to make the final agar concentrafion 3% (w/v) was added and
dissolved in a water bath.
The medium was dispensed in 5 ml
. 60
aliquots in culture tubes, autoclaved for 15 minutes at
15 psi, and slanted.
7.
Arbutin agar - 0.5% (w/v) arbutin and 2% agar were dissolved
in 10% yeast autolysate by heating.
The medium was dispensed
in 5 ml aliquots in culture tubes and autoclaved for 15 minutes
at 15 psi.
Immediately after sterilization, 2-3 drops of a
sterile "1% ferric ammonium citrate solution was aseptically
added to the liquefied agar in each tube.
The tubes were
shaken carefully to avoid frothing and then slanted.
8.
Corn meal agar
~
12.5 g of yellow corn meal were mixed with
300 ml of distilled water, heated in a 60°C water bath for
1 hour, and filtered through paper.
The filtrate was made up
to 300 ml and 3.8 g of agar were added.
The medium was auto-
claved for 15 minutes at 15 psi, filtered through absorbent
cotton wool while hot, dispensed, and sterilized at 15 psi for
15 minutes.
9.
Glucose-chalk agar- 50 g glucose, 5 g calcium carbonate, and
20 g agar were added to 1 liter of 0.5% yeast extract and
sterilized by autoclaving for 15 minutes at 15 psi.
The
medium was cooled to ca. 50°C, swirled to suspend the chalk
evenly, and distributed into Petri dishes on a cold surface
·-
to avoid settling of the chalk.
Appendix II
Standard Description of E'ndomycopsis but?tonii
(Ladder, 1970)
Growth in malt extract:
After 2 days at 25°C budding cells are oval,
3-51-!X 5,..:9.511. In addition, mycelial hyphae with cross \'Jalls are
present.
In the mycelium dichotomous ramifactions occur.
Buds
are formed all over the threads, occasionally on small elevations.
Side branches are generally perpendicular to the threads.
drical arthrospores of variable length may be present.
Cylin-
A floccu-
lent sediment, and occasionally, a ring and islets are formed.
After one month at l7°C a thick, yellow-brown mass and a broad ring
are present.
Growth on malt agar:
After 2 days at 25°C the same elements as men-
tioned for malt extract may be present.
The strains differ in the
proport·ion of budding cells to mycelial hyphae.
After one month at l7°C the streak culture is yellowish-white, tough,
raised, wrinkled and hairy.
, Slide cultures on potato- and corn meal agar:
with blastospores.
branched chains.
threads.
True mycelium is formed
They are occasionally arranged in small
Side branches often perpendicular to the mycelial
The mycelium may split up into arthrospores.
blasto- and arthrospores occur outside of the coverslip.
mycelium may be found.
61
Most
Aerial
62
Formation of ascospores:
Conjugation of cells of opposite mating type
may precede ascus formation. ·Diploid, bixesual strains occur.
spores are hat-shaped; two are formed per ascus.
liberated from the ascus.
The
They are easily
In one of the strains which is diploid,
spores were observed on Gorodkowa- VB- and acetate agar.
Seven of
the 15 strains studied sporulated after mixing with the appropriate
mating type on V8 agar; two belonged to one type, five to the other.
Fermentation:
glucose + (often slow)
lactose -
galactose + (weak) or -
melibiose-
sucrose + (often slow)
raffinose +
maltose + (often slow or weak)
raffinose +
Assimilation of carbon compounds:
glucose +
D-ribose +
galactose +
L-rhamnose -
L-sorbose + or -
ethanol +
sucrose +
glycerol +
maltose +
erythritol +
cellobiose +
ribitol +
trehalose +
galactitol -
lactose -
D-mannitol +
melibiose -
D-glucitol +
raffinose +
a-methyl-D-glucoside +
melezitose + or -
salicin
inulin -
DL-lactic acid -
soluble starch +
succinic acid +
63
D-xylose +
citric acid +
L-arabinose + or -
inositol -
D-arabinose Splitting of arbutin:
positive
Assimilation of potassium nitrate:
Growth in vitamin-free medium:
negative
positive
Growth on 50% (w/w) glucose-yeast extract agar:
Growth at 37°C:
weakly positive or negative
-·· ..
positive