Agr.Biol.Chem.,39 (5),959- 965,1975

On

the

Changein theCellulose

by

Mode

of the Aggregation

Chemical

Modification

Components

of Saketheof Sake Yeastsof theCell

withSurface

YeastsNobuo SUGANO,*Hiroichi AKIYAMA
and Kikuo NOSHIRO
ResearchInstituteof Brewing,Tax Administration
Agency,Kita-ku,Tokyo
ReceivedSeptember6, 1974
The changesin mode of the aggregationof Sakeyeastswithcelluloseby treatmentswith
variousreagentswhichextractthemannan-proteincomplexor modifyionizedgroupsontheyeast
cell surfacewas studied. The treatmentswiththe disinfectantssuchas 70%ethanoland 0.1% mercurichydrochloridehad no effecton the aggregation. In the treatmentwithdiluteNaOH,
trypsin,papain and ethylendiamine,whichhavegenerallybeenknownas the reagentswhich
extract the mannan-proteincomplexon the cell surfaceof yeasts,the aggregationcharacter
disappearedor lowered,thereforeit was supposedthat the mannan-protein
complexon the cellsurfaceparticipatedwith the aggregationcharacter. The modificationof phosphategroups
the cellsurfacewithuranylnitrate and carboxylgroupswith1,2-epoxypropane
and methanolicHCl had no effecton the aggregation. The modificationof aminogroupson the cellsurface
with HNO2and chloramineT eliminatedcompletelythe aggregationcharacter. From above
results, it was assumedthat the amino groups in the protein of cell surfaceparticipatedin
the aggregationof Sake yeastswith cellulose.
Two typesof theof surface

fermentation

appearanceof Sakeat the end

mash

were

ob-

in Sake mashes."
cells which aggregated

with

the

served.
One is the formationof a thick yeast
cover on the top of the mash and the other isthe smooth surface of the mash without yeast

cell walls free from the endospermof ricekernels, also aggregatedwith the fiber of filter

cover.

phenomenaof yeasts with cellulose were simi
lar to the flocculationof brewer's
yeasts.
It
has been consideredthat the nature of a bre

It was previously1) demonstrated that these
phenomena were influenced by the charactersof the yeast strains, whether or not cells ag
gregate with the cell walls free from the endo
sperm of rice kernels by the digestion withmold enzymes in Sake mash. That is to say,the yeast cells which aggregated with the cell
walls formed a thick yeast cover by floating
to the surface of the Sake mash with fermenting
CO, foams, and showed the slow fermentation in the mashes at the end because the cells
were separated from the fermentation system."On the other hand, the strains which did not
aggregate with the cell walls dispersed throughout the fermentation and indicated the normal*

fermentation
The yeast

Present
address:
Bureau, Sendai, Japan.

SendaiTax

Administrationpaper

or cellulose

powder."

The aggregation

wer's yeast to flocculate is due to the biochemi
cal propertyof the cell wall.
Therefore,theapproachof the studies on the mechanismofthe flocculationsome suggestionsof brewer's
yeasts may givefor the study on the aggregation of Sake yeasts with cellulose.
Eddy"
destroyed
flocculating
propertyofbrewer's
yeastsby treating
the cells withpapain.
He concluded
that the digested portion on the cell wall consists of the mannan
proteincomplex from analysis of the liberat
ed components
by the digestion.
Lindquist5)
has demonstrated
that the tendency
toward a
rapid flocculation
is attributable
to the presenceof ionized groups on the cell surface, and thatthe most importantin this conection
are the960

N. SUGANO, H. AKIYAMA and K. NOSHIRO

anionic
acid.

groupsJansenof eitherandnucleic

Mendlicks'

or carboxylic
have

shown

that
the
yeast
cell surface
is negatively
charged at pH levels above 2.3 such as in wort,
beer,buffersolution
and water.
Sumino7'and Momosee'
examined the electricalchargeof Sake yeasts by the methodof bounderymoving electrophoresis,
the sign of the electricalbetween 3 and 4.

and has shown thatcharge reserved at pH

Authers3)
indicatedthat the mechanismofthe aggregationof Sake yeasts with cellulose
was different from that of the flocculationofbrewer's yeasts, and speculated
that the proteinof yeast cell surface played a importantpart inthe aggregationof Sake yeasts with cellulose.
In

thepresent

paper,

furtherstudies

wereattempted
to know the change of the aggregation of Sake yeasts with cellulose when treatedwith the reagents which solubilized the mannanproteinchangecomplexin the cell surface,of the aggregationand thecharge when modified the ionizedcell surface of Sake yeasts.
MATERIALS

and theelectricalgroupsin theAND METHODS

1. Strain used and culture condition. Aggregating
Sake yeasts with cellulose: A-63 and S-23 strains.
Non-aggregating Sake yeast: Kyokai No. 7 strain.
These yeasts were cultured in koji extract (Balling 10)
for 10 days at 15°C.
2. Method of measuring aggregation.
After culture, yeast cells were washed three times with distilledwater. Four ml of distilled water was placed into a
10 ml test tube (to fit Shimazu spectrophotometer,
Spectronic 20), to which were added washed yeast cells
to the optical density at 660 urn of 0.4, followed by theaddition of 10 mg of the powdery cellulose. The mixture was shaken and then allowed to stand for 2 hr.
The test tube was shaken and after 5 min the opticaldensity (A) at 660 nm was measured. As a control,
the optical density (B) of the mixture without yeast cells
was measured in the same way as above.
The degree of aggregation strength was expressed asfollows
Aggregation rate (%) = (1-(A-B)/0.4)
3.

x 100

Method of treatment with various reagents

1) Sterilizers. Suspension of cells (5 mg dry weight)
in 5 ml of 75 % ethanol or 0.1 % mercuric hydrochloride

placed into the test tube were allowed to stand for
30 min.
2) NaOH.
Washed cells (25 mg dry weight) weresuspended in 100 ml of each solution in 0.05 N, 0.1 N,
0.2 N or 0.3 N NaOH, allowing to stand for 30 min.
3) Trypsin.
Suspension of cells (25 mg dry weight)
in 100 ml of 0.02 M Tris-HC1 buffer, pH 8.0 containing
20 mg or 200 mg trypsin (3 Crystalline, Seikagaku
Kogyo Co., Ltd.) were allowed to stand for chosen
period of time (30, 60, 120 and 240 min) at 30°C.
4) Papain.
Suspension of cells (25 mg dry weight)
in 100 ml of 0.02 M Tris-HCl buffer, pH 7.6 containing
50 mg papain (Tokyo Kasei Kogyo Co., Ltd.) wereallowed to stand for 20 min at 30°C.
5) Ethylendiamine.
Washed cells (20 mg dryweight) were suspended in 5 ml of anhydrous ethylendiamine for 3 days at 37°C as the method of Northcote.91
After treatment all cells were washed three times with
distilled water, and the degree of aggregation strength
was measured by the spectrophotometric method orobserved by the microscope.
4.

Method of chemical modification

1) Uranyl nitrate.
Washed cells (10 mg dry weight)
were suspended in 5 ml of 10-2 M uranyl nitrate solution
for 30 min at 30°C as the method of Lycette.10)2) 1,2-Epoxypropane.
Washed cells at the concentration of 5 x 107/ml were suspended in 5 ml of
0.05 M acetate buffer, pH 4.5 containing 5 % 1,2-epoxypropane over night at 30°C as described by Mill.11)
3) Methanolic-HCI.
Washed cells (10 mg dryweight) were suspended in 10 ml of methanol containing
0.1 N HCl for 50 min at 58°C. In control experiments
the cells were treated separately with methanol andwith 0.1 N HCI.
4) Nitrous acid.
Nitrous acid was prepared asfollows. 6 g of NaNO2 was added in 5 ml of acetic
acid, which was fieled up to 40 ml with distilled water.
Washed cells (10 mg dry weight) were suspended in 10ml nitrous acid for 3 hr at 5°C.
5) Chloramine T. Washed cells (10 mg dry weight)
were suspended in 10 ml of I % (w/v) chloramine T
aqueous solution for 6 hr at 37°C.
6) 12. Washed cells (10 mg dry weight) were suspended in 10 ml of 0.038 % I solution, which was adjusted pH to 3.2 by 0.01 N HCI, containing 0.033 % KI
for 4 hr at 25°C.
7) PCMB (p-chloromercuric-benzoic acid).
PCMB
solution was prepared as follows. Solution A: 2.2 gof sodium hexamethaphosphate was dissolved in 50 mlof distilled water, followed by the addition of 1 ml in
1 N HCI. Solution B: 20 mg of PCMB was dissolvedin 1 ml in I N NaOH.
Solutions of A and B weremixed and fieled up to 100 ml with distilled water .

Aggregationof Sake Yeasts

Washed cells (4 mg dry weight) were allowed to react in
30 ml of the mixed solution for 30 min at 20°C.
After treatment all cells were washed three times with
distilled water, and the degree of aggregation strength
was measured by the spectrophotometric method orobserved by a microscope.
5. Measurement of the electrophotoretic mobility
The electrophotoretic mobility of intact cells and cells
treated to modify the surface groups were measured
as described by Momose.8)
RESULTS

1.

Change in the mode of the aggregation ofSake yeasts with cellulose by the treatment ofvarious reagentsThe results are shown in Table I. Aggregation rate decreased slightly by the treatment
with 75 % ethanol and 0.1 % mercuric hydrochloride, but little change in the aggregation
was observed by microscope. Therefore, it
was recognized that life or death of yeast
cells had no relation to the aggregation withcellulose.
By the treatment of cells with 0.05 N NaOH,
aggregation rate decreased to some extent forwith Cellulose

both A-63 and S-23 strains, but it was foundby microscopic observation that the aggregation still remained. In the case of concentration over 0.1 N NaOH, both cells of A-63 andS-23 strains lost their ability to aggregate withcellulose.
The treatment of the cells of A-63 and S-23
strains with trypsin at the concentration of
0.02 % (w/v) for 240 min resulted in the decreaseof about 60 % in aggregation rate, but by micro
scopic observation the aggregation character
was seen to be maintained to some extent. In
the case of the treatment at the concentrationof 0.2 % (w/v) for 240 min, the aggregation
character was completely eliminated. In thetreatment with papain and ethylendiamine, thecells of A-63 and S-23 strains lost their ability
to aggregate with cellulose.
The treatment of the cells of Kyokai No. 7
strain with any reagent described above, had
no effect on the aggregation character withcellulose, remaining without aggregation.

TABLE I.THECHANGE
OFAGGREGATIONOF
SAKE
YEASTSWITHCELLULOSE
BYTHETREATMENTOFSAKEYEASTSWITH
VARIOUSREAGENTS
Numericalvaluesshowaggregationrate.

2.

The change of the electrophoretic mobility
and the aggregation of yeast cells by the treat
ment to modify the surface groups
TABLE II.

No aggregation (as observed by microscope)

THE CHANGE OF AGGREGATION OF

SAKE YEASTS WITH CELLULOSEBY THE CHEMICALMODIFICATION OF IONIZED GROUPSON CELL SURFACE
Numerical-:

961

values show aggregationrate.

+: Aggregation.
- : No aggregation (as observed by microscope)

962

N. SUGANO, H. AKIYAMA and K. NOSHIRO

After treatment
the change in the mode of theaggregationof Sake yeasts with cellulose areshown in Table II.
of A-63, S-23 anduranyl nitrate had

The treatmentof the cells
Kyokai No. 7 strains withno effect on the aggregation with cellulose.
After treatment
the changeof the pH-mobility
curves of cells are shown in
Fig. 1. There were little change in the pHmobility curves of Kyokai No. 7 strain, but themobility curve of treated cells of A-63 strain
decreased
as comparedwith that of normal
cells.

FIG.

1.

UranylThe

pH-Mobility

CurvesNitrated-treated, normal

Cells

KyokaiNo.

7; •¢•\•¢,

normalA-63; •¢•\•¢,

FIG.

2.

pH-Mobility

5%

1,2-Epoxypropane-treated

No.

No.

The

of

for
Sake

7; •›•\•›,

NormalYeastsmodified
modified

CurvesCells•›•\•›, normal

KyokaiNo.

7; •¢•\•¢,

normalA-63; •¢•\•¢,modified

7; •›•\•›,

forNormalofSake

modifiedand.•›•\•›The treatment of the cells of A-63 and S-23
strains with 5 % and 12 % 1, 2-epoxypropane
resulted in a some decrease of aggregation
rate, but under microscope no difference
observed between the aggregation of the treated cells and normal cells with cellulose. In thecase of Kyokai No. 7 strain also, after treat
ment there was no effect on the aggregation
character. The pH-mobility curves of 5 % 1,2-epoxypropane-treated cells and normal cellsof both strains are shown in Fig. 2. The
slope of pH-mobility curves of treated cells ofboth A-63 and Kyokai No. strains decreased ascompared with that of normal cells of respec
tive strains. The slope of pH-mobility curve
decreased at pH 4.0 - 5.6 more in Kyokai No.
7 strain than in A-63 strain.
The effect on the aggregation character due
to the treatment with methanolic-HC1 was thesame as the result of the treatment with 1, 2epoxypropane. In the control experiment of
the treatment with methanol and 0.1 N HCI,
0.1 N HCl did not influence aggregation rate
for any strain, but methanol lowered the ag
gregation rate of A-63 and S-23 strains at thesame extent as the treatment with methanolicHC1. Therefore, it may be assumed that thedecrease of the aggregation rate is due to theeffect of methanol.

KyokaiA-63.

andYeasts

FIG.
.

Kyokai
A-63.

3.

The

Chloramine, normalNo.

7; •¢•\•¢,

pH-Mobility

Curves

for

Cells

of

Yeasts. •›•\•›

KyokaiNo.

7; •›•\•›,

normalA-63; •¢•\•¢,

T-treated

Sake

NormalandmodifiedKyokaimodified

A-63.

Aggregationof Sake Yeasts with Cellulose

In the treatment with chloramine T, A-63
and S-23 strains lost completely their ability toaggregate with cellulose, but there was nochange on the aggregation character of Kyokai
No. 7 strain. Figure 3 shows the change ofthe electrophoretic mobilities of treated cells
and normal cells at various pH's. The increase of mobilities of the negative charge on
treated cells at pH 3.0-5.6 was recognized atboth A-63 and Kyokai No. 7 strains. The
mobility of treated cells of A-63 strain at pH
5.6 was 1.7 times as large as that of Kyokai No.
7 strain.
The change of pH-mobility curve of treatedcells with HNO3 was the similar to the resultof treated cells with chloramine T (Fig. 4).
The young-aged cells of A-63 strain cultured
for 4 days at 15°C, which do not aggregate withcellulose, were treated with chloramine T and
HNO2, and the electrophoretic mobilities oftreated cells at various pH values were compared with those of old-aged cells of A-63
strain cultured for 10 days at 15°C. The results
are shown in Figs. 5 and 6.
As to normal cells, there were no differences
between the electrophoretic mobilities of theyoung cells and old cells, but in the case of thetreated cells, the increase of amounts of thenegative charge was larger for the old cells
than for the young cells at pH 3.0 - 5.6.

FIG.

5.

The

pH-Mobility

HNO2-treated

Cells

of

A-63

(cultured•¢•\•¢, normalmodified

A-63

A-63

for

for

4 days).

6.

The

Chloramine

4for(culturedfor

4 days).

The

treatment

had

no

with

cellulose.

ofNo.

influence

10

of

and

Strain.

days);

x-x,

for
4

Normaldays);

x---x,

normalmodified

A-63

CurvesforNormal

and

Sake

Yeast

A-63

Strain.•¢•\•¢(cultured(cultured(cultured

Kyokaifor
10days);

Cells

A-63

A-63

and

A-63

pH-Mobility, normal

A-63

for

Yeast

for

T-treated

modified

CurvesSake(cultured(cultured(cultured

FIG.

963

for
10

10

days);days);days);

x-x,

the

cells

7

strains

with

on

the

aggregation

x---x,

normalmodified

A-63

of

A-63,

h

and

S-23
PCMB
character

DISCUSSION

In
FIG.

4.

The

pH-Mobility

CurvesforNormalthefrom
HNO2-treated

Cells

of Sake

KyokaiNo.

No.

7; •¢•\•¢,

normalA-63;

paper,3)itwaseffectofvariousaggregation

of

Sake

the

fractionstudiesontheassumedthat

conditions

Yeasts.•›•\•›
on, normalpreviousand7; •›•\•›,

modifiedKyokaimodifiedA-63.

lulose,theproteinyeastswithinmannan-

the

cel

964

N. SUGANO, H. AKIYAMA and K. NOSHIRO

protein complex present on the outer surfaceof the cell wall was related to the aggregation.
The mannan-protein complex on the brewer's
yeast cell surface was digested by the treatment
with papain4) and trypsin.12) Kijima13) indicated that flocculent brewer's yeasts became
non-flocculent when treated with 0.5 N NaOH
for 15 min. Northcote9) used anhydrous
ethylendiamine as milder extraction procedureof the mannan-protein complex than NaOH.
Lyons12) recognized that the removal of the
mannan-protein complex by the treatment
with anhydrous ethylendiamine destroyed theflocculation character of brewer's yeasts.
In the treatment of cells of the aggregating
Sake yeasts with cellulose, such as A-63 andS-23 with NaOH, papain, trypsin and anhy
drous ethylendiamine, their cells lost theability to aggregate with cellulose, nonaggregating Sake yeast as Kyokai No. 7 remained to be non-aggregating. Therefore, it isfurther speculated that the factor participatingin the aggregation of Sake yeasts with cellulose
exist in the mannan-protein complex of the cellsurface.
It has been generally considerd that there
were amino groups, carboxylic groups andphosphate groups as the surface ionogenic
groups of the cell. Rothstein14) reported thaturanyl ion was linked by the phosphate groups
on the cell surface. Lycette10)recognized thatthe flocculation character of brewer's yeasts
was lost by the treatment with uranyl nitrate.
Momose15) has speculated that the phosphategroups of the yeast cell surface are of importanceon the aggregation of yeast by lactbacilli
because the aggregation lowered remarkably
in the treatment of yeast with uranyl nitrate.
1, 2-Epoxypropane have been used for theesterification of protein carboxylic groups.
Mill"' observed that the flocculation characterof yeast cells was lost by the treatment with 5
1, 2-epoxypropane solution.
Timakova16)
carried out the methylation with methanolicHCl for modification of carboxylic groups inthe cell surface of E. coli, and Gittensl7) car
ried out the esterification with methanolic-HCI
for the cells of Aerobacter aerogenes.

HNO2 has been used for the modification ofamino groups.
It has been generally known
that chloramine T can be used for the modi
fication of amino groups, 1, was used for themodification of SH groups and PCMB wasused for the modification of S-S groups.
Haydon18) has shown that the measured
values by the electrophoretic mobility of cell
indicate charge density which exists at a depthof 10 A on the cell surface.
There was little
difference in the slope of the pH-mobility
curve of normal cells and uranyl nitratetreated cells of Kyokai No. 7 strain between pH
3.0 and 5.6. Therefore, it is not consideredthat free phosphate groups are present in the
superficial layer of cell walls. On the other
hand, in the case of A-63 strain, it is assumed
that free phosphate groups are located at thesuperficial layer of cell walls because the pHmobility curve of treated cells was different
from that of normal cells. However, as there
was no change in the aggregation character
after a treatment with uranyl nitrate, it is assumed that the phosphategroups have noconnection with the aggregation character.
The carboxylic groups in protein are those of
the side chain of glutamic acid and asparatic
acid residue groups or are those of the termina
tion of polypeptide chain,19) and therefore after
the modification of carboxylic groups with 5
1, 2-epoxypropane,
the larger decrease in theslope of the pH-mobility
curve of Kyokai
No. 7 strain than that of A-63 strain indicatesthe possibility that more acidic amino acids arepresent in the superficial layer of cell walls of
Kyokai No. 7 strain than in that of A-63
strain.
As the modification for the carboxylic
groups had no effect on the aggregation character, it is not considered that the carboxylic
groups in the surface layer of the cell walls
are related with the aggregation character ofSake yeasts with cellulose. The treatment withLand PCMB also had no effect on the aggregation character, therefore, both S-H and S-Sgroups in the surface layer of cell walls appear not to be related to the aggregation.
Though there are s-amino group in lysine
residue and end-amino group in polypeptide

Aggregationin protein,of Sake Yeasts with Cellulose

chain

as aminogroupsmonly

accepted

that

amino

groupsgroupsficationin thein lysine residue."'
By the modiof amino groups in cells, the increase
slope of the pH-mobility
curve wasthere

it is comis very

as comparedwithlittletheend-s-amino

3)
4)
5)
6)

larger for old-aged cells of A-63 strain, whichshowedthe aggregation
characterwith cellulose, than the young-agedcells of A-63 and7)

KyokaiNo. 7 strain.
It may be consideredthat the amountsof e-amino groups in lysine
residue are present more at the surface layer

9)in the old-agedcells of A-63
that in the young-agedcells
Kyokai No. 7 strain.
As the aggregation
with cellulose
wasstrain than atof A-63
andcharacterof A-63 strainmucheliminatedby themodification
for amino groups
in cells, it isspeculated
that the factor to participate
in theaggregationis c-amino groups of lysine residue
in the cell surface

1)
2)

8)

10)
11)
12)
13)
14)
15)

of A-63 strain.

16)
17)

REFERENCES

18)

N. Sugano, H. Akiyama and K. Noshiro, Nippon
Nogeikagaku Kaishi, 47, 763 (1973).
N. Sugano, Y. Goto, K. Kaya, H. Akiyama and

19)

965

K. Noshiro, J. Soc. Brew Japan, 67, 49 (1972).
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Nogeikagaku Kaishi, 47, 771 (1973).
A. A. Eddy, J. Inst. Brew., 64, 368 (1958).
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H. E. Jansen and F. Mendlick, Proc. Eur. Brew.
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K. Sumino, H. Kawase, Y. Tani and S. Fukui,
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H. Momose, K. Iwano and R. Tonoike, J. Gen.
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E. D. Korn and D. H. Northcote, Biochem. J., 75,
12 (1960).
R.M. Lycette and L.R. Hedrick, Appl. Microbiol.,
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Acta, 66, 237 (1963).
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