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[J.Fermcnt TechnoL, VoL 60,No. 4,p. 311-319,!982]

Growth and Enzyme a Solid-State

CultureofProduction
Aspergillusin
oryzae

HmEKI NARAHARA,1 YosuKE KoyAMA,2 TosmoMl YosHmA, SuMALEE

PicHANaKuRA,S Ryuzo UEDA,4 and HisAHARu TAGucHl
intematienal ofCboperutiue
Clenter and Developmentinuarobial
R&searcii Ehgineering,
Jap`ut,
FlactttyofEagiheering,
Osdra Ciniverst'ty,
lhmada.ka, Sbeita-shi,
Osdea 5615;JbPan

An instrumented syst ¢ rn of a kofimaking process was developcd for a kineticstudy on

the solid-state cultivation ofAopergitlus eryzae var. bru,meusW03 on steamed rice.
Estimation of thegrowth ef the mieroorganism in the kofiwas successfu11y done by
monitoring CO! evolution.
The water activity of kojidecreased according to the growth of A. oryzae, mainly
becauseof an increase inreducing sugar.
Optimum conditions forgrowth were not always similarto those for enzyme
produc-
tion. An initialwater tontent of oo% was suitablc forgrowth the
proteaseactivity
while
was higherat 35% or less,and the optimum teTnperature was 3Bea forgTowth whercas
the proteascwas producod niorc at 30DC. At thestationary phase of growth, a-amylase
still increasedwhilc
activity proteaseas well as saccharifyirig activities were constant.
A supply of C02 at 2 to 5% stimulated the protcaseproduction.

After the 2nd World War, the method of which usually wil1 not be produced in
submerged culture became very popular in a subrnerged culture.
thcmicrebial industrybecauseof the successes Hesseltinei-3}
roviewed thes,e advantages
in antibiotic production. Despite the pro- and problems in detail emphasizing the
gress in the technology of submerged cultures, necessity of development of process work
solid-state cultivation, which was a common accompanied by monitoring devices to de-
mcthod in traditionalfermentationprocesses, termine moisture, pH, free oxygen and
has shown little progressand no significant carbon dioxide.
expansion of application. However, taking Kinetlcstudies on solid-state fermentations
into consideration the simplicity of the have scarcely been reported sincc a series of
cultivation equipment and the 1ffwer expense papersby Terui et al.4-e) One of the reasons
for operation, more application of this is the technical diMcultiesin chemical
traditional method is expected with the analysis of cell mass, substrate consumed,
advanced knowledge and approaches in productyields and so on as well as physical
biochemical engineering. As the micro- analysis.
organisms in a solid-state cultivation grow The water content of thc substrate, which
under conditions closer to their natural is a peculiar parameter to solid-state culti-
habitats,they may be rnore capable of vations, influencesthe productivitiesof cell
producing certain enzyrnes and metabolitos mass, enzymes, metabolites and so on･

Present address: Lindedelser and Cieglerio)investigated

i Higllchi MatsunosukeSheter) Co. Ltd.,Harimache, ochratoxin A production in the solid-state
Abenoku, Osaka 545, Japan fermentationof Asperll:'lltts ochraceus and con-
a
Ajinomoto ao. Ltd., Kaw4saki-shi 210,Japan
S Depariment of Micrebiology, Faculty of Science, firmed that the initial wheat moisture ievel
Chu]alongkornUniversity, Bangkok 5, Thailand was the most critical of all fermentation
4 Departmentof Fermentation Technology, Faculty of
Engineering, Osal[a University,Yamada-oka, Suita- conditions. However, previous studics were
shi, Osaka 565, Japan lessquantitative,so itisnecessary te investi-

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312 Nww et al. [J.Ferir!ent.TechnoL,

the between the water Culture e(luipment A schernatic diagrarnof

gate relationship
the ferrnentorused in this work isshown in
content and the productivities. In con- solid state

Fig.1. The .inlet firstpasse[1through a mem-

this, Nishie et al.M reccmtly
air was
nection with
- brane Mter. Thc humidityof the airwas increased
reported thatthe rate of rnaceratmg enzymes
to satumtion beforecntering the fanentor by thc
formation of Aslbergiaus niger was in proportion following procedure. The firststep of humidification
to the growth rate irrespeetiveof themoistuiz: was done with a humidifier (acy1inder of 20 crn dia-.
content of the subetrate. mctcr, 60 crn height, pacikeclwith smal1 Rasehig rings,
In this work, the water eontent of the was filled with hot water and maintained at around

7orC). Tltt bubbiing through the

substrate was talten intoconsideration passed
related air wai

bom bottom to tqp. In thc step, thc

activity (Aw),
next
to water which affects the cylindtr
alr was coolod by a heatec[chariging coil in
availability of water for mieroorganisms, to humidified
a water-beth to drainoff e]ccess water. The temper-
investigate the effects on growth and enzymc
ature- of this watcr-bath was controlled by ftcdbadlt
productionof knj'i ef the culture terripezature. The inlet air was passcd
A mcthod was investigated to estimate oell through thcfer"ientor bottom to tep and then exhaustod.
mass in the kofi processwith a strain ef The air from the fermer)tor was driedin co1umns of
AspergiUusezJLcas monitoring the carbon di- concentrated HtSO" and sllica
gel, and o:ganic

oxid ¢ evolution rate or thc oirygen uptake in thc air were rtmoved with ap activated
matcrials

could be detected directly. cbarcoal odlurrm. T!hetreated air was passed through
rate, which
an oxygeri witha zirconia cell, model LC-7ooE
Furthermore, the effects bf operationa1
analyscr
con-
(Toray Ltd., Tolryo) and a carbon dio)tidc analyser,
ditions such as cultivation temperatum,
rnodel ArA-21 {Horibe Lrd., Kyoto).
water content and partialpressureof COt Estim tion of cell mass Cellmass was tsti-
in the air on the cell growth were investigated, mated by the giu{)cmainine method.tS} rn this method
and a relationship between the operational the amount of mycelia on thc kofi was estimatetl frDm
conditions and yields of enzyrnes such as the glucosaminc content, based on the fbot that tbe

sacc.harifYing enzyme, a-amyiase, acld pro- eontent of glucosatnirre in the mycelia oM. eoroac was

tease and neutral proteasc produced by alrttost constant th:vugbout tlrcgrowth phasas and that
A. ecyzae was sought. itwas not present in the raw nct.
Wster oontent [ht watcr content of kofiwas
Adaterhlsand Medrods determined･ by measuring tbe weight lessof a sample
The rnicroorgani"n dried at 105"C for 24 h.
Microorganism used was

brw:neas WOS ffom Wster utivity (Aw) The samplc of kofiwas

AspergiUtis otrzas var. obtained
HigudhiMatsunesul:e Shotai Co. Lrd.,Osalca. imdiated with ultraviolet ightte stop thc grc,wth of
This irradiatien was donc witha
Medium Raw ricegrains were pelisliDdto B5% thc rnicroorganism.
of tlre eriginal weight and washec!. The waslted rice
was soalted in watetr overnight, and then stcamed for
SOmirL [he steatned rioe was (hied under vacuum
fortwo days at roem ternper-ture and stored. [fhis
'was with 2 of
treated rioe rnixed g a surface-active

agtnt (Emulsy,gly(erolestcr of carbox)rlic acid,

Talreda?harmaccutical Lrd.,Osal[a)per 1 ig of dry
matter to prevent adlicring of ricegrains when humidi-
fied. The rice was humldified to a duired water

contens and then sterilized at !21"C for 20 min.

Inoculum Conidia of A. ot7ew incubetecl on
ricekrains were suspended in stcrile water ceritaining
fer-
EA 1oo, Fig･ 1. Schematic
diagram ef the solid:substratc
O.Ol% of a surfaceactive agent (Noigeri merltor.
polytthylerieal]cylplienyi ether, Daiichi Kcigyo Phar- 1.Air pump, 2. Flow mcter, 3. Air filter,
4. Hu-
maceutieal Ltd., Kyoto). Tl)e conidia surpcnsion, midifier, 5. Water beth, 6. Ferrrientor, 7- Os an-

20ml, containing about 5x107 conldia per ml, was alyser, 8. COs analyser, 9. Ternperature controller,

added to1ig of the riec as the inoculum. Matured 10. COs tegulator, 1l.COt gas,12. Solenokl valve,

kofiresultecl afber cultivation for24 h or more. and 13. Ck)nstant terriperature box.

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Vol. 60, 1982] Enzyme Production in Solid-State Culture 313

15 W ultraviolet lamp for IO min, keepingthe sample at 40eC. One unit of enzymc activity was definedas
at a distance of 10 cm from the 1arnp. Then 5 g of the the amount of 1% soluble starch solution hydrolyseq
treated kofiwas put into a water activity detector by the enzyme in 30 min at 40"C.
<Implex Chemicals Ltd., West Gcrmany) and kept Reducing sugar of kofi Ten grams of koji
at 20eC forabout 3 h to measure Aw. sample were steeped in leO ml of O.5% NaCl so]utlon
Enzyme preparation The enzymes were for 3h at 15"C, and then fi1trated.The arnount of
extracted from 1Og of riec kojiwith 1oo ml of water for reducing sugar in the filtrate was determinedby
proteasasor an aqua solution of O.5% sodium chloricle Lane-Eynon's method.is)

fortheamylase assay by shaking for3h at 15eC, and Standard culture conditions Static culti-
the fi1trate
was used as theenzyme $olution. vations Were carried out with forcedaeration. The
Deterrninationof protease aetivity A modi- temperature and the moistur ¢ of the solid sutstrate
ficationof Anson'srnethodiS) was used forthedcterrni-were controlled by aeration throughout the cultivation.
nation of protease activities. Casein solution (2gof Thc standard culture conditions were as fo11ows:
casein and 5 ml of Mi1O lacticacid for the acid protease CuLtlvation temperature, 380C; temperature of th ¢
assay or 8 ml of N/1O sodium hydroxideforthe neutral outer box, 330C; acration ratc, 17l/min; and initial
protease assay in 1ooml), 1.5ml, Mcllvaine's citric- water content, 35%.
phosphate buffbr at pH 3.0 for acid pretease or at pH
6.0forneutral protease,1,O ml, and the enzyme solu- Results and Discussion
tion, O.5 mr, were mixed, and the mixture was incubated
Cell growth estimation Hesseltine
at 38eC for 60 min. The action of the protease was
i-s} discussed the advantages and･ dis-
stopped by adding 3 ml of O.4 M trichloroacetic acid,
advantages of solid substrate fermentation
and thc precipitate was rcmoved by filtration.
One
milliliter of the filtrate, 5 ml of O.5 M Na2COs and 1 cempared with liquid mcdium fermentation.
ml Folin'sreagenti4} were well mixed and kept for
of One of the ofdisadvantages
solid state

30 min at 380a. Th ¢ optical densitywas measured fermentation,that he pointed out, was the
at 660 nm. One unit of protease activity was defined technical diMculty in the chemical analysis
as the amount of enzymc thatdeveloped color equi- of the cell mass and the substrate consumed.
valent to one pmol of tyrosine per minute under The conventional methods employed to
these conditiens. estimate the mycelial weight of A. oc)tcae
Deter nination of amylase the
grains
activity
grown on steamed rice were
Sbcchartwng actibity 10 ml of a 2% soluble starch digestion
enzymatic methodi7) glucos-
and the
solution, 2.0ml ef O.1M acctate buffbr(pH 5) and amine method.i2} The second one is an
1.0rnlof the enzymc solution were mixed, and thc
mixture was incubatod at 40'C for 30Tnin. Thc indirectrnethod in which the glucosamine
content of moldy riceisused as an index of
reaction was stopped by adding 7 ml of NIrO NaOH
solution. Lane-Eynon's methodi5} was used to
the mycelial weight. This method is more
dctermine the amount of roduelng sugar in the three accurate than the first one, but ittakes timc

solutions: (1)reaction mixture; (2)enzyme solution; to purify glucosamine from moldy riccsam-
and (3)staTch solution. Thcsc values for reducing ples. In addition, Arima and UozumiiS)
sugar glucose were
as denQted as l,m and
n mg, respcc- and SakuraiiS)found that the glucosamine
tively. Thc arneunt of tota1 sugar (smg) in the starch content of mycelia increasedconsiderably
deterrnined as glucose after hydrelysis
phase. However, the
solution was
after the stationary
with HCI. The saccharifYing activity was calculated
glucosaminemethod was successfully applied
as fo11ows: to the estimation of the cell mass in this
Saccharifying
activity
research, because our interestwas mainly
l-m-n 100
focussedon the changes in the laterlogarith-
`=-
s-n
×
lo × 1oo (unitslg) (1)
a-Amplase ttctitrftv The activity of a-amylase was
mic growth phase. The oxygen uptake
rate and the carbon dioxideevolution rate
determined by a modification of Wohlgemuth's
methodiS) which isbasedon the estimation of thc time
are also used for prediction of metabolic
activities, becausethe present instrumentation
inwhich thg color of an iodine-tarchcomplex decreases
to a definiteextcnt. The reaction mixture containing technology allows automatic determination
1O ml of 1 % soluble starch, 2 ml ofO.1 M acetate buffer of the respiratory activity. The continuous

(pH 5) and O.5ml ef the enzyrne solution was kept determinationof the partialpressuresof

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314 NARAHARA et al. [J.Ferment. Technol.,

oxygen and carbon dioxidein the inletand rE1.2
E 1
is possible with an oxygen
)t
"1,O
outlet airlines
O.10ON-:Ho e
analyser and a crabon dioxideanalyser. eo.e

The relationship between the carbon

dioxide evolution rate and the growth rate
.g･o"
V.O.05oNsX-!

of the microorganism was investigatedto

developa method of monitoring c ¢ 11growth o
E
in the kc!iiprocess. For this purpose, several v

o
cultivation runs with differentinitialwater o 2o 4e 6o so
cQntents madc were
and the glucosamine
Cutture time (h}
Fig.3. Time courses of oxygen consumption rate
(Io,),
content and the respiration ratc were dioxide evolution
carbon rate (lbo,)
and respira-
measured. In this work, mycelial weight tory quotient(RQ)of the koji.
was calculated from the glucosaminecontent A. orvcae was incubat¢ d on steamed ricecontaining
35% of water at 380C.
with a conversion of 9.7.iB} ratio
-"'"", Ibt; -"-'L,
lbot;
Figure 2 shows the biomass time courses
,RQ.

by the glucosamincmethod
measured

the of diflbrentinitial
com- was found that the RQ (respiratory
quotient)
paring efTlects water value was maintained at 1.0 during
about
cDntents. Around 50h of cultivation, the the This
whole cultivation period. result
estimated value of cell mass increased again isin good agreement with those of Terui and
after a plateau of growth in some batch Morimotoi7)and Okazaki and Sugama.2o)
runs. As stated above, the reason for this Figure 4 shows the relationship between
increase is that the glucosamine content of the specfic the specihc
growth rate, p, and
the mycelia increasedafter the stationary
dioxideevo!ution, the
phase.
rate of carben Qco,
value was correlated to the value of p esti-
Figure 3 shows the time courses of matcdthefrom the glucosaminc content, in each
oxygen uptake rate and thc carbon dionide case at a diflerentinitialwater content.
evolution rate in the solid culturc at 38"C Nishioet at.ii) observed a linear relationship
with an initialwater content of 35%. It between oxygen uptake and growth rate in
solid ctiltivation ofA. nigen',' This result isin

good agreement with our data on considering

40
the RQ value mentioned above. Consequ-
-

No -

os:.E=eU
pt35 o,eto?)sege
:
[-
E.so
l'
) aoos-eescree

20

20 40 60 80 e
e o.os o.lo
C"Iture.tTine (h>
P{h-i)
Fig. 2. Time courses of biomass and water content Fig.4. Relationship between the specific growth rate
of substratc initia1
water eontents
under
ef koji.
conditions of varieus
(p)
and the specifi ¢ rate of earbon dioxideevolu-
tion (Qco,)
at different initialwater contents.
Conidia of A. o[reas were inoculatedonto stcarned
A. aybue was incuhatod on steamed rice containing
rice containing 25.6(e),29.5(o),
35.8(A)and 25.6(e),29.5(o),and 35.8(A)% of water at
4e.3 (A)% and incubated in the fer-
of water,
38"C. mcot: specific production rate of carbon
mentor 38"C. Clellmass was estimated from
at dioxidefor mainteriance (mol COEIg cellfh).
glucosamine content in kofi A: experimental constant (molCOslg cel1).

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Vol. 60, 1982] Enzyme Production in Solid-State

Culturc 31s

ently, p by measuring
itis possibleto estimate progress of kojimaking isdiscusscdbclow.
the rate dioxideevolution during
of carbon In this culture equipment, a portion of
growth. Okazaki et at.2i) proposeda mathe- the water in kofievaporated into the outlet
matical model based on the data of kofi gas-Iine, removing metabolig heat as usually
making with an automatic oxygen feeder observed in solid cultures. The sitarch of
and we dealwith this pointin the next report. ricekernelsin kofiwas enzymatically hydro-
Water activity (Aiv) in the kofi process lyzedto reducing sugar duringthe incubation
The water content of a solid substrate period. A part of the reducing sugar was
markedly influencedthe cell grewth in solid consumed but some still remained in the rice
state cultivation.iO,ii) In connection with kojias shown in the figure (Fig.5).
this, Narahara22) claimed that the true Rosss4} proposed ･an estimatidn method
eflbct of water on the growth of A. oryzae for the water activity of a moistured food;
isbetterindicatedby water activity23> which the overall water activity, Awo, isthe product
aflects the availability of water for micro- of the individual water activities of each
ingredient,as fo11ows,
.organlsms.

The growth rate of A. oryzae in the koji Awo-(Awi)(Arp2)(Aw3) (2)

processdecreasedwith a decreaseof Aw and
the growth ceased at an Aw of around
In
O.90 be
this work, tlie main solute isassumed to
reducing sugar and the water activities
under diflbrentculture conditions CFig.
5).
of ingredientsare lumped together.
other
The reason why Aw decreases with the
Hence, based on Eq. (2), water activity of
kofiisdefinedas follows,
l,
-.'h50
Awo=(Awr)(Aw") (3)
Where Atvr is the water activity forreducing
,O sugar and Awn isthat forother ingredients.
O3:
12o To estimate the water activity, Aw#,
E water activities of steamed rice with various

=eIO water contents were measured at 20"C.

O O
The amount of reducing sugar in steamed
rice was negligibly small, so the water activity
of steamed might coincide with Awn.
rice
A"oEYvo=-pEo=veor From the data shown in Table 1, the corre-
lationof Awn and the amount of ingredients
A was summarized in a regression line with
*
v55
-
c

! Table 1. Water activitics of stcamod rice with various

: watercontents.
:
s Water Water of ingredients*
eso
s content activity IIAwAmount
(%) Aw % onwet
basiskg/kgwater
40.S35. O.975O. 1.0261.0321.
59. 764. L481.
20 4Q 60 80

Cutture tjme(h) 93e.925.

969O. 169. 79Z
Fig. 5. Time courses of biomass,water activity, water 958O. 04al1. 174. 242.
content and reducing sugar at different
COx levels.
The data for three batches of incubations were 7 942 062 3 89
plotted at differentpartial pressures of COm of
O.5 (A,-------),
2 (D,-･---)and 5 (o, )%. * The amount ef ingtcdientserccluding glucosc was
Clesed symbols are usecl for growth and reducing estimated from the water content data assuming the
sugar and crpen symbols forAwo and water content. absence of reducing sugar in the steamed rice･

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316 NARiuiARA et al. [J.Ferment. Technol.,

a correlation coeMcient of O.999as fo11ows Table l which indicatethat any change in

'
water content very much lessaffected the
Al.-=o.o2ss(100.-X)+o.gs7 (4) watcr activity.

where x is the water content of steamed rice Effbct of environmental conditiDns on

(%). enzyme production in the kofi process

According to the water activities of kofi There are several environmenta1 parameters
samples rneasured with a water activity have to be taken into consideration
which

detectorand Awn values estimated by the for the growth of microorganisms on a solid

fbllowing equation, Eq. (5),which is derived substrate. Water content of the substrate,

from Eq. (4)taking account of the arnount cultivation temperature and the partial
of reducing sugar, S (%),to estimate the pressureof carbon dioxidein the atmosphere
amount ef other ingredients in kofi, in the fermentorseem to be very important
-.S-X) environmental factorsthat aflbct the growth
(100
-Olo2ss
+o.gs7 of microorganisrns and their subsequent
AS,
(5) production ef metabolites.

Awr values were calculated using Eq. (3)

Efact ofinitial water content Moistening
bringsa suitable Aw value for growth and
and are given in Fig.6. swelling of the substrate, and the latter
Figure 6 clearly that the decrease
shows
of water activity throughout the period of
the kofiprocess, shown in Fig. 5,was caused n04Y?eeE5l,vx'2.eEe:losE(eo
roo

mainly by accumulation of reducing sugar :tsoE

and less by a decrease of water content.
This is also supported by the resti1ts shown in tT60;4oJt

too
g2og
io

T095;( :-ohEPdtsbl[tgt's
t5
84g.

ts;io;eeox.pt-. ib
a'K56-

O.90 ..)--o2.d."
h=e

ve
5'E3･ e
'E1!=

O.85 :Yao
O 20 40 60 80 o
Culturetime(h) lnitialwater eentent (%)
Fig. 6. Time courses of estimatcd water activities for Fig. 7. Efllects
of initial
moisture Ievelon the activities
non-solute components (Aw.)and for roducing of amylascs and proteases in kofi,incubated till
sugar (Aw,)at differentCOs levels. the stationary phase of growth.
Aw. values were estimated by equation (5), and Incubations were carTied out at differentinitial
Awr values were calculatod by equation (3)using water contents, and enzyme activities were com-
the valueAwo deterrninedwith a water activity
of pared at 60 h incubation when growth reached
detector. The data for cstimation of Aw. and the stationary phasc in al1thc Tuns. Enzyrnc
Aw. were collected from three batches of incuba- activities were calculated as units per gram dry
tions at diflerentpartlal pressures of COs of O.5 matter (upperfigure) and units per gram cells
(A,------),2 (O,-'-'-)and 5 (o, )%, shown (Iowerfigure).
in Fig.5. Symbols: e, saccharifying activity; o, a-amylase
Open and solid symbols are used forAw# and Awr, activity; A, acid protease activity; A, neutral
respectivcly. proteascactivity.

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Vol.60, 1982] Enzymc ProductiQn

in Solid-StateCulture 317

causes easier utilization of the substrate by C02 pressure.E8,29)

A. orvzae because of easy penetrationof In this solid culture, the partial pressure
mycelia intothe substrate. However, Nara- of C02 could be controlled in the feedback
hara25} reported that the water content mode in the fermentoras shown in Fig. 1.
must be keptrelatively low inorder to preyent Severalpartialpressures, O.5,2, 5 and 10%,
bacterial contamination, were selected for the experiment to find the
Figure 7 shows the effect of initiai water optimum partial pressure of C02 for the
content on the yieldsofamylases and proteases production of the enzymes. The positive
at 60h after inoculation.The moisture eflbct of COa on the production ef each
levelin the substrate affected the enzyme
'
enzyme was recognized prominentlyat 2%
yields to a certain extent, as itwas observed in the early stage of the growth phase till
tha.t.a. high water content suppressed the 30 h and in the case of proteases the highest
activities of thc proteasesand the
raised yieldat the stationary phase of growth was
'11 activityofthea-amylaseconsiderably. How- observed under the condition of a 5% C02
ever, each specMc activity was decrcased supply irnplyingthat stimulation of pro-
at a high initial water content, though a high duction could be possibleby changing the
an increase
water content caused ef the eell panial pressureof C02 at the logarithmic
rnass CFig. 2). growth phase (Fig. 8). The infiuence of
Therefore, it may be possible to obtain C02 on the saccharifying enzyme and
higP enzyme in this solld state culti- neutral protease was same as for a-amylasc
.yielgs.
vatlon by malntammg the moisture levelof and acid protease,respectively.
I･ the subEtrate at an adcquate levelfor cell Kritzman et al.SO) reported that COz
growth in the growth phase and decreasing not only inhibited the tricarboxylic acid
it to a suitable levelfor productionof the cycle, but that the fungus S27terotium rogfkii
enzymes in the stationary phase. This overcame the partial inhibitionof the tri-
peint will be dealt in detail in the ncxt carboxylic acid cycie by using an alternative
report.
pathway, the glyoxylatepathway. Gandhi
Efact .ofpartiatPressure of' C02 Despite and K.jaergaad3i} showed that C02 increased
many reports26T29} dealing with enzymc the productivity of a-amylase by BasiUtcs
syntheses, no information is available on the snbtilis and suspected the role of C02 in the
phy?iological role of C02 on enzyme for- elimination of catabolite repression. How-
mation. In traditional kofi processes, the ever, itis necessary to investigate the eflbct
control of C02 isnot done, and there were of COx in detail.
several reports eflbct of a high
of a negative
Efact ofczaltivation temperature To find
the best temperature for the production of
? these enzymes, A. orvzae was grown at four

;.i/' i'
different temperatures, 30, S5, 38 and
Figure9 shows the time courses of a-amylase
.i
protease activities at difierent
410C.

i V. and aCid
tt2 temperatures. The temperature dependen-
g:o tgt cies of the saccharifying activity
'E - and neutral
-O
=
proteaseactivity were slmilarto those of a-
amylase and acid
Time Ch) nme (h) protcase activity, respective-

Fig.8. Effect
of COs concentration on the production
ly. The maximum a-amylase activity was
of a-amylase and acid protease. observed at 380C.On the contrary, a low
Four batches of kojiculture wcre carriecl out at temperature enhanced the production of
difltrentCOs concentrations of O.5(e),
2 (o),5
(i)and 10 (A)%. proteases.Although the activities of pro-
Other initial
conditions: water content, 35%; teases at 35eC increased rapidly in the early
incubation
temperature, 38"C. stage of cultures, increases of their activities

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318 NARAHARA et al. [J.Ferment.Technol.,

" activity and a slight decreaseof protease
9 =
lt ''y"S-"- s activity as shown in Fig. 10. 0n the con-
t .-. G100 trary, when the temperature was changed
i5 '
i
i from 38.to 300C, the protease activity in-
E4
fi･ X7N
･s-.--
vh'ee

lipt .t. tt- a creased remarkably, while the a-amylase

--iIi "60 activity decreased. From the resuks of the
,.-....
o
" .3 ':
e i-
itwas suggest-
jZ// I
1- i 34o temperature
shift experiments,
32 M
that the temperature should
ed be changed
crt
.i-r'e"n

: l'
;r'
s･2o
t/j'

from the suitable temperature for growth,

91
e g 380C, to a highertcmperature for a-amylase
Bo o 2o 4e 6o o 2o 4e 6o re :-
< t' '
< production, and a lower levelfor protease
Time (h) Time (h) production.
Fig.9. Effbct of cultivatien temperature on the pro- These data imply that combination of
duction of a-arnylase and acid protease. will lead to
thcse environmental conditions
Four batches Qf koficultures wcre carricd out at
differenttemperatures of 30 (e), 35 (A),38 (O) a wider range of variation in the balance
and 41 (i)OC.Inhial water conterit: 35%. and yieldsof enzymes.

30 h
the finalactivities of
and
Nomenclature
stopped after

proteases
at 35eC werc lower than those at Aw : wateractivity23)
30eC. Furthermore the figureindicates that Awo : overal1 water (cfl
activity Eqs. 2 and 3)
a-amylase activity increasedcontinuously in Aw" : water activity foringredientsexcluding
the stationary phase ofgrowth, while protease glucose of steamed rice and koji(c £
activity was constant. These data imply Eqs. 3 and 5)
that the change of the cultivation temperature forreducing sugar ofkofi
Awr : water activity
in the logarithmicgrowth phase may cause Fig.6) (cfl
Eq. 3 and
the increase of theactivity of each enzyme. ho, : carbon dioxideproduction rate, mol
To confirm the above suggestion, the C021kg dry matter/h
incubation temperature was shifted from
t : arnount ef glucose in the reaction mix-
380C to diflerentextents and the effbct of ture, mg (c £ Eq. 1)
shifting was investigatedon the enzyme m : amount of glucose in the enzyme solu-
activity. Shiftingthe temperature from 38 tion, mg (cf Eq. 1)
to 410C resulted in an increaseof a-amylase mco,:specinc production rate of CO! for
maintenance, mol C02!g cell/h (cfi
1ooeo
r.s .Fig. 4)
:t4 n : amount of glucose in the starch
.:t,,!g
solu-
t
org3
tion, mg (c£ Eq. 1)
Y. E, E-v$40 Qcot:specific productionrate of C02, mol
ls,
ti
-
th
C021g cell/h
oa

th20

:/o
;
,a l s :amount of total sugar in the starch

g- solution, mg (c Eq. 1)
£

%
vO

o
4e so
2a o 2o 4o 6o eo S : reducing sugar on a wet basisof koj'i,
Tirne (h] Tinne Ch)
x : water content on a wet basis, %
Fig.10. Effect of tcrnperature shifts from 380a to
differenttempcratures on the erizyme activities. A : experimental constant, mol a021g cell
Each sample of 50g was transferrod from the
fermenter to a 500ml conical flask after 30h
(c Fig.4)
£

pa : specific growth rate, h-i

preincubation and the enzyme activities were
measured after 19h incubation at varieus tcm- Aclmowledgements
peratures,25 (-), 30 (e), 35 (A),38 CO)and 41
(i)eC.Open squares indicatc incubation in the The authors are gratefUlto Mr. K. Tanaka forhis
fermentor throughout all the incubation enthusiastic cooperation in performing the expemments.
period as
a control. The authors alse thank the mernbers of Higuchi

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Vol.60,1982] Enzyme Production in Solid-StateCulture 3I9

Matsunosuke Shoten's laboratory for thc analysis of l5) Takahashi, M.: BtLU. Chem. Sbc. Jopan, 33, 178
cnzymeactavltles. (1960).
16) Wohlgemuth,J.: Biochem.Z.,9,1(1908).
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