Enzyme and Microbial Technology 35 (2004) 369376

Submerged culture conditions for the pridela f

Bu a, Taon Y

ungbukb Department of Microbial Engineering, Konkuk University, Seoul 143-701, Korea

c Department of Biotechnology, Daegu University, Kyungbuk 712-714, KoreaAccepted 1 December 2003

Abstract

Optimizatstudied. Therespectivelywere favoratank fermenagitation ratrate, and hydconditions, fwas a feathefermenter wFor a compawere lowerwere produc 2004 Else

Keywords: G

1. Introdu

For mancharides prmushroompharmacolostimulatingsupplemen

CorrespoE-mail add

0141-0229/$doi:10.1016/jion of submerged culture conditions for mycelial biomass and exopolysaccharide (EPS) production by Grifola frondosa wasoptimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 C and 5.5,

. Glucose (30 g/l) was the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract and polypeptoneble nitrogen sources for both mycelial biomass and EPS production. To find the optimal operational parameters in a 5 l stirred-ter, the self-directing optimization technique was used and the results were as follows: culture pH, 5.06; aeration rate, 1.16 vvm;e, 166 rpm. In the course of mycelial submerged culture, the mycelial morphology was significantly altered by culture pH, aerationrodynamic behavior, which subsequently affected the yield of EPS production. While compact pellets were formed at low aerationreely suspended mycelial growth was observed at high aeration conditions. The most desirable morphology for EPS productionr-like mycelial clump. Under optimal culture conditions, maximum biomass concentration and EPS production in a stirred-tankere 16.8 and 5.3 g/l, respectively, which were significantly greater than results prior to optimization (13 and 4 g/l, respectively).rative study, the fungus was further cultured in a 5 l airlift fermenter, but mycelial biomass yields (10 g/l) and EPS yields (4.53 g/l)than those in the stirred-tank fermenter. Eventually, three groups of polysaccharides of diverse molecular mass (4701100 kDa)ed either from the mycelial extract or from the culture filtrate precipitate.vier Inc. All rights reserved.

rifola frondosa; Exopolysaccharides; Self-directing optimization technique; Submerged culture

ction

y years, interest has concentrated on polysac-oduced by numerous microorganisms, especiallys, because of their various biological andgical activities [8,9,12]. These include immuno-, anti-tumor, hypoglycemic activities, as dietaryts for the enhancement of stamina, as a therapeutic

nding author. Tel.: +82 53 850 6556; fax: +82 53 850 6559.ress: jwyun@daegu.ac.kr (J.W. Yun).

for curing coughs and for blood circulatory problems or as atonic promoting longevity and improving the quality of life[11,25,28].

To obtain bioactive polysaccharides from mushrooms,most investigators have spent their efforts cultivating edi-ble or medicinal mushrooms on solid artificial media (forfruit body production) rather than in submerged cultures(for mycelial extract and/or exopolysaccharide (EPS) pro-duction). Submerged cultures obviously have the potential forhigher mycelial production in a compact space and in shortertime with fewer chances for contamination. In addition, EPSs

see front matter 2004 Elsevier Inc. All rights reserved..enzmictec.2003.12.015biomass and exopolysacchaBasidiomycete Grifo

m Chun Leea, Jun Tae Baea, Hyeong Bae PyoHye Jin Hwangc, Jong W

a R & D Center, Hanbul Cosmetics Co., Chroduction of mycelials by the ediblerondosae Boo Choeb, Sang Woo Kimc,unc,

369-830, Korea

370 B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376

which have synergistic effects with mycelia on biological ac-tivities, can be simultaneously produced [2,10,20,21].

Although many investigators have attempted to obtain op-timal submduction froon nutritionsubmergedrooms [1,5

Grifolaing to the[14]. Themushroompolysaccharides have[15,16].

Althougpolysacchatumor activoptimal substrated exte

In the prproductionfrondosa wtechnique.were carrielogical infldescribed.

2. Materia

2.1. Micro

A culturdistrict in Kdextrose aslants wereat 4 C. Thtaining 50 mextract, 2 p0.2MnSO4for 7 days.

2.2. Ferme

G. frondpetri dish,by punchinilized self-performedafter inocution experiminoculatedvated at 25Incheon, KLtd., Daeje

the fermentation medium (3 l) and was cultivated for 13 days.All fermentation experiments were performed in triplicate atleast.

Self-d

self-ding sims of vrimentlimita

e simpe sim

d andpoint

rimente of aion rables as undthe i

he expand r

ld refleminatspons

e a rulsfactoxperis they detet respoified ais conned:{2

e N ishe thrW is

Prepa

e fermin, an5m) andd vigode frapitates, whilcolleacchalial berged culture conditions for polysaccharide pro-m several mushrooms, currently available reportsal requirements and environmental conditions incultures are limited to only a few kinds of mush-

,24].frondosa is a Basidiomycete fungus belong-

order Aphyllopherales, and family Polyporaceaefruit body and liquid-cultured mycelium of thishave been reported to contain useful anti-tumorrides from various fractions. These polysaccha-been identified as glucans (e.g. -1,6- and -1,3-)

h several investigators have studied differentride fractions from G. frondosa and their anti-ities have been well characterized [17,22,26,29],merged culture conditions have not been demon-nsively so far.esent study, submerged culture conditions for theof mycelial biomass and EPS production by G.ere optimized using a self-directing optimizationFor a comparative study, a set of fermentationsd out in an airlift fermenter system, and morpho-uence affecting the EPS production has also been

ls and methods

organism and media

e of G. frondosa was isolated from a mountainousorea. The stock culture was maintained on potato

gar (PDA) slants. Unless otherwise mentioned,incubated at 25 C for 7 days and then stored

e seed cultures were grown in 250 ml flasks con-l of medium containing (g/l) 30 glucose, 6 yeast

olypeptone, 0.5MgSO47H2O, 0.5K2HPO4, and5H2O at 25 C on a rotary incubator at 120 rpm

ntations

osa was initially grown on PDA medium in aand then transferred to the seed culture mediumg out 5 mm of the agar plate culture with a ster-designed cutter. Flask culture experiments werein 500 ml flasks containing 100 ml of the medialating with 3% (v/v) of the seed. After optimiza-

ents were finished, the fermentation medium waswith 3% (v/v) of the seed culture and then culti-C in a 5 l stirred-tank fermenter (KoBioTech Co.,orea) or in a 5 l airlift fermenter (Best Korea Co.on, Korea). The seed culture was transferred to

2.3.

Arotatlevelexpementof thAs thlishemum

expeshapaeratvariacesse

Afterout, ttifiedshoudeterthe rehenca satinew e

minunewlwors

identdureobtai

N =

wherare truns;runs.

2.4.

Th20 ma 0.4USAstirrechariprecirideswere

polysmyceirecting optimization technique

irecting optimization technique (also called theplex method) was used to determine the optimal

ariables to maximize production processes whens have to be carried out sequentially due to equip-tion or operating feasibility [6]. The movementlex in the response plane monitors this process.plex moves, a self-correcting approach is estab-the simplex slowly converges towards the opti-. Initially, the design begins with a set of fours considering the fact that the simplex takes thetetrahedron composed of three variables (e.g. pH,te, and agitation intensity) [6]. The levels of there set up according to prior experience of the pro-er study, or from values reported in the literature.nitial four sets of experiments have been carriederiment which gave the worst response was iden-

eplaced by a new combination of variables whichct the worst point in the response plane. However,

ion of the reflection of a point of a tetrahedron ine plane is a complex mathematical procedure ande of thumb was applied which was found to givery approximation of the actual reflection [6]: Themental point is twice the average of the best pointsworst point (Eq. (1)). The experiment with thermined set of variables is than carried out and thense from the four remaining experiments are againnd replaced by a new set. This iterative proce-

tinued until no further improvement in response is

(B1 + B2 + B3)}3 W (1)

new experimental combination; B1, B2, and B3ee best points from the last four experimentalthe worst point from the last four experimental

ration of polysaccharides

entation broth was centrifuged at 8000 g ford the resulting supernatant was filtered throughmembrane filter (Millipore Co., Bedford, MA,mixed with four volumes of absolute ethanol,rously and left overnight at 4 C. The polysac-

ctions suspended in the upper parts of the ethanolwere pooled and designated as Fr-I polysaccha-

e the bottom fractions of the precipitated EPSscted after centrifugation and designated as Fr-IIrides. To extract intracellular polysaccharides, theiomass was submerged in hot water for 4 h at

B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376 371

100 C followed by extraction with ethanol for 24 h at 4 C,and the resulting polysaccharides were named Fr-III poly-saccharides.

2.5. Analytical methods

2.5.1. Estimation of mycelial growth and polysaccharideproduction

Samples collected at various intervals were centrifugedat 8000 g for 20 min, and the resulting supernatantwas filtered through a 0.45m membrane filter (Millipore).The resulting culture filtrate was mixed with four volumesof absolute ethanol, stirred vigorously, then left overnightat 4 C. The precipitated EPSs were centrifuged at 8000 g for 10 min, and the supernatant discarded. The totalweight of EPS was estimated by the summation of the topfraction of the filtrates and precipitates from the bottomfraction of EPS after lyophilization until constant weightwas confirmed. The dry weight of mycelia were measuredafter repeated washing of the mycelial pellets with dis-tilled water and drying overnight at 70 C to a constantweight.

2.5.2. Molecular weight determinationThe molecular weight of polysaccharides were estimated

on the basiters Co., Mcolumn (0.8using distil50 C; floweluate wastector (Alltwas standa(Polymer S

Fig. 1. Effect iomassexperimental

3. Results and discussion

3.1. Effect of carbon sources

Many kinds of mushrooms frequently require starch, su-crose, maltose, glucose, or galactose as carbon sources fortheir submerged cultures [1,2,10,20,21,28,29]. The influenceof carbon sources for mycelial biomass and EPS productionwas studied in media containing those carbohydrates, whereeach carbon source was added to the basal medium at 24 g/l,instead of potato dextrose in PMP medium. When the cellswere grown in the glucose medium, both mycelial biomassand EPS production reached their highest levels (Fig. 1a),and the maximum glucose concentration for both mycelialbiomass and EPS production were achieved at 30 g/l (Fig. 1b).

3.2. Effect of nitrogen sources

To investigate the effects of nitrogen sources on EPS pro-duction and mycelial growth, six kinds of nitrogen sourceswere examined (Fig. 2a). Maximum EPS production wasachieved when yeast extract (6 g/l, see Fig. 2b) was used.In comparison with organic nitrogen sources, inorganic ni-trogen sources gave rise to relatively lower mycelial biomassand EPS productions.

Effect

our prize wath ande fungm volum vs of the calibration curve obtained by HPLC (Wa-ilford, MA, USA) with Shodex OHpak KB-804cm 30 cm) (Showa Denko K.K., Tokyo, Japan)

led water as a mobile phase (column temperature,rate, 0.8 ml/min; injection volume, 20l). The

monitored by an evaporative light scattering de-ech Associates, Deerfield, IL, USA). The columnrdized with dextrans of diverse molecular masstandards Service Inc., Silver Spring, MD, USA).

of carbon sources (a) and glucose concentration (b) on the mycelial bdata are mean S.D. of triple determinations.

3.3.

Inlum sgrowof thoculuinocuand exopolysaccharide production in Grifola frondosa. All

of inoculum volume

evious investigations [20], it was found that inocu-s one of the important factors influencing mycelialpolysaccharide production during liquid cultureus, Cordyceps militaris. To find the optimal in-

lume, G. frondosa was cultivated under differentolumes ranging from 2 to 6% (v/v). Consequently,

372 B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376

Fig. 2. Effect of nitrogen sources (a) and yeast extract concentration (b) on the mycelial biomass and exopolysaccharide production in Grifola frondosa. Allexperimental data are mean S.D. of triple determinations.

the optimal inoculum volume for both mycelial biomass andEPS production by G. frondosa was found to be 3% (w/v) asshown in Fig. 3.

3.4. Effect

The effbiomass antial pHs (4.initial pH aproduction

Fig. 3. Effecttriple determi

b). These pH and temperature optima are similar to those forother mushrooms in submerged cultures [1,20].

3.5. Optimization results in a stirred-tank fermenter

e rotafor o

entatioable a

[23].ents wof initial pH and temperature

ect of initial pH and temperature on myceliald EPS production was studied under different ini-07.0) and temperatures (2030 C). The optimalnd temperature for both mycelial biomass and EPSwere pH 5.5 and 25 C, respectively (Fig. 4a and

Thniquefermavailouslyperimof inoculum volume on the mycelial biomass and exopolysaccharide production innations.ting simplex method is a simple and reliable tech-btaining suitable combinations of parameters forn when no prior experience about the process isnd experiments cannot be conducted simultane-In this study, a total of eight fermentation ex-ere necessary to obtain the best combination ofGrifola frondosa. All experimental data are mean S.D. of

B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376 373

Fig. 4. Effect de prodS.D. of triple

physical parate) for EPinitial fouration rate:knowledgeEPS by theup based oand generaliterature. IEPS produrate was hiruns after tment of themedium isis evident fincreased upH values.and it was ataining a hkeeping theproduction

Table 1Results of selffermenter

Run no.

12345678

ctionmes n

brean by aaxim

and 5and aepm (Tared, datais meng awsubsection

Morphof initial pH and temperature on the mycelial biomass and exopolysaccharideterminations.

rameters (e.g. pH, agitation intensity and aerationS production byG. frondosa (Table 1). During theexperiments, variable levels were pH: 36; aer-02.5 vvm; agitation rate: 50300 rpm. No priorwas available about the production conditions fororganism in bioreactor. The above levels were setn the previous experience in shake flask culturesl levels for fungal fermentation reported in thet was observed from the initial experiments thatction was low (Run nos. 2 and 7) when agitationgh and pH was low (Table 1). The fermentationhe initial set of experiments guided by the move-simplex revealed that the pH of the fermentationa key factor for optimum EPS production. Thisrom the observation that EPS production steadilyp to pH 5.06 and decreased in regions of higherAeration rate caused changes in EPS production

produenzyto theitatioThe m16.85.06166 rcomptivelyby thmoviwere

produ

3.6.pparent that a reasonably high aeration rate, main-igh dissolved oxygen level in the fermenter, andbroth well mixed was necessary for optimal EPS

. Most likely, agitation was unfavorable for EPS

-directing optimization for physical parameters during mycelial growth and exopoly

pH (controlled) Aeration rate (vvm) Agitationintensity (rpm)

6.0 0.5 1004.0 2.0 2506.0 2.0 1004.0 0.5 2506.6 0 505.06 1.16 1663.44 2.44 2946.0 0 50

Fungal mthe physicabehavior iscentrationsuction in Grifola frondosa. All experimental data are mean

, probably due to shear inactivation of some keyecessary for synthesizing the polysaccharides, ork-up of mycelial pellets or both. Hence, mild ag-irlift operation is sufficient for EPS production.um production of mycelial biomass and EPS were.26 g/l, respectively at a pH level (controlled) ofration of 1.16 vvm with an agitation intensity of

able 1), which significantly increased productionsto our preliminary results (13 and 4 g/l, respec-not shown). Further improvement in production

chanism was unlikely, as the simplex had starteday from the optimum combination. Further studiesquently performed to compare the polysaccharideusing an airlift bioreactor, as described later.

ological changes in a stirred-tank fermentersaccharide production byGrifola frondosa in a 5 l stirred-tank

Maximum mycelialdry weight (g/l)

Maximum exopolysaccharideconcentration (g/l)

11.38 2.536.5 0.8213.36 4.679.8 3.4310.5 2.1916.8 5.265.3 1.0310.2 2.20

orphology is an important parameter that affectsl properties of the fermentation broth. Rheologicalclosely related to morphology and biomass con-[3,4,18]. In order to identify the most productive

374 B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376

Fig. 5. Changes in mycelial morphology in response to the culture conditions: (a) pH 6.0, aeration rate 0.5 vvm, agitation speed 100 rpm; (b) pH 4.0, aerationrate 0.5 vvm, agitation speed 250 rpm; (c) pH 5.06, aeration rate 1.16 vvm, agitation speed 166 rpm (an optimized condition).

Fig. 6. The time profiles of mycelial biomass and exopolysaccharide production in a 5 l stirred-tank fermenter (a) and in an airlift fermenter (b). Stirred-tankfermenter was operated under the following conditions: controlled pH 5.06, aeration rate 1.16 vvm, and agitation speed 166 rpm. Airlift fermenter was operatedunder the following conditions: controlled pH 5.06, aeration rate 1.16 vvm.

B.C. Lee et al. / Enzyme and Microbial Technology 35 (2004) 369376 375

morphology of G. frondosa for EPS production, the mycelialmorphology was observed during optimization studies. Asshown in Fig. 5, it was observed that culture pH, aeration rate,and hydrodin mycelia:ditions (Figgrowth wanot shown)levels weremycelial clis not in acmentationscompact peet al. [19] pto that of tproduction

3.7. Ferme

It is obvagitation inof myceliagal fermenproduct yietween stirre[7,27]. Mohave not ustion by mu

Thus, itcan be sucproductionactor givesgus was cumycelial bical time prand polysain airlift feEPS produmenters duof the desirmycelial biairlift fermfermenter.cal featureswere grownmenter (feaogy observthroughout

3.8. Molec

The moltein for eaccharides ob(Fr-I) had htom fractio

Table 2Characterization of the three different polysaccharides produced from sub-merged culture of Grifola frondosa

ccharida

or nome

otal carbproteins

serum

cts (FacchareportolysacThereto proon of).

onclu

e preationof sub

PS pre pHnificaosa afermeefficilial bd areacchaer mu

rence

ae JT,ergedilomyceae JT,on souy subm001;91:elmar-Beiny MT, Thomas CR. Morphology and clavulinic acid pro-uction of Streptomyces clavuligerus: effect of stirrer speed in batchermentations. Biotechnol Bioeng 1991;37:45662.aniel O, Schonholzer F, Zeyer J. Quantification of fungal hyphae

leaves of Deciduous trees by automated image analysis. Applnviron Microbiol 1995;61:39108.ang QH, Zhong JJ. Two-stage culture process for improved pro-uction of ganoderic acid by liquid fermentation of higher fungusanoderma lucidum. Biotechnol Prog 2002;18:514.endrix C. Through the response surface with test tube and piperench. Chemtech 1980;10:48897.ynamic behavior affected morphological changecompact pellets were formed at low aeration con-. 5a and b), whereas freely suspended mycelial

s observed at high aeration conditions (figures. Maximum mycelial biomass and polysaccharideachieved when mycelial morphology was loose

ump with high hairiness (Fig. 5c). This findingcordance with results from other mushroom fer-: i.e., the most desirable morphology was oftenllets, in many cases [2,21,24]. In contrast, Parkointed out that feather-like morphology (similar

his work) was more favorable than pellets in theof arachidonic acid from Mortierella alpina.

ntation results in an airlift fermenter

ious that the mechanical shear raised by severestirred-tank reactors can cause decreased yields

l biomass and polysaccharide production in fun-tations. Comparative studies of fungal growth andld in highly viscous fermentation processes be-d-tank and airlift fermenter are scarcely availablereover, despite the feasibility, airlift fermentersually been employed for polysaccharide produc-shrooms in submerged cultures [13,14].is important to investigate whether G. frondosa

cessfully cultivated in an airlift fermenter for theof polysaccharides and whether this type of biore-advantages over stirred-tank fermenters. The fun-ltured in a 5 l airlift fermenter to compare yields ofomass and EPS production. Fig. 6 shows the typi-ofiles for substrate consumption, mycelial growthccharide production in stirred-tank (Fig. 6a) andrmenters (Fig. 6b), respectively. Cell growth andction were more favorable in the stirred-tank fer-e to efficient mixing and leading to the formationed morphology. The maximum concentrations ofomass (10 g/l) and EPS (4.53 g/l) obtained in theenter were lower than those in the stirred-tankIt was also of interest to compare morphologi-between the two fermenter types. Although cellsat the similar conditions as in the stirred-tank fer-

ther-like mycelial clump), the mycelial morphol-ed in the airlift fermenter was a compact pelletthe fermentation period.

ular properties of the polysaccharides

ecular mass and the ratios of carbohydrate to pro-h polysaccharide were investigated. The polysac-tained from the top fraction of culture filtratesigher molecular weights than those from the bot-n of culture filtrates (Fr-II) and from mycelium

Polysagroup

Fr-IFr-IIFr-III

a Fb T

Totalbovine

extrapolysdosathe phigh.dratefracti(27%

4. C

Thtimiztionand Ecultuto sigfronddosamore

mycetainepolysto oth

Refe

[1] Bm

c

[2] Bbb2

[3] Bdf

[4] DinE

[5] FdG

[6] Hwe Molecular weight(kDa)

Carbohydrateb(%, w/w)

Proteinb(%, w/w)

1100 87 13770 73 27470 82 18

nclatures for each polysaccharide, see Section 2.ohydrates were measured by the phenolsulfuric acid method.were measured by bicinchoninic acid (BCA) assay using

albumin as the standard.

r-III) (Table 2). In comparison to three groups ofrides obtained from mycelial extracts of G. fron-ed by Mizuno et al. [15], the molecular masses ofcharides obtained from this work were relativelywas a great difference in the ratios of carbohy-

tein among the three polysaccharides. The bottomthe EPS (Fr-II) had the highest protein content

sions

sent work demonstrated that the self-directing op-technique was an efficient tool for the optimiza-merged culture conditions for mycelial biomassroduction by G. frondosa. It was found that the, aeration rate, and hydrodynamic behavior lednt differences in the mycelial morphology of G.nd subsequently affected EPS yields. In G. fron-ntation, the stirred-tank fermenter was consideredent than airlift fermenter, in that higher yields ofiomass and EPS were achieved. The results ob-considered useful for the production of Grifolarides on a large scale and can be widely appliedshroom fermentations.

s

Sinha J, Park JP, Song CH, Yun JW. Optimization of sub-culture conditions for exo-biopolymer production by Pae-s japonica. J Microbiol Biotechnol 2000;10:4827.Park JP, Song CH, Yu CB, Park MK, Yun JW. Effect of car-rce on the mycelial growth and exo-biopolymer productionerged culture of Paecilomyces japonica. J Biosci Bioeng5224.

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[9] Kim DH, Yang BK, Jeong SC, Park JB, Cho SP, Das S, Yun JW,Song CH. Production of a hypoglycemic, extracellular polysaccha-ride from the submerged culture of the mushroom Phellinus linteus.Biotechnol Lett 2002;23:5137.

[10] Kim SW, Hwang HJ, Park JP, Cho YJ, Song CH, Yun JW. Mycelialgrowth and exo-biopolymer production by submerged culture of var-ious edible mushrooms under different media. Lett Appl Microbiol2002;34:5661.

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[17] Ohno NacterizatGrifola

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rager Mnd stirrioeng 1ang BKong CH

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[27] Ta

B[28] Y

StB

[29] ZFf, Oazi GN, Onken U, Chopra CL. Comparison of air-lifted reactor for fermentation with Aspergillus niger. J Ferment989;68:1126., Ha JY, Jeong SC, Das S, Yun JW, Lee YS, Choi JW,. Production of exo-polymers by submerged mycelial cul-ordyceps militaris and its hypolipidemic effect. J Microbiolol 2000;10:7848., Mizuno T, Ito H, Shimura K, Sumiya T, Kawade M.

ation and antitumor activity of polysaccharides from Grifolamycelium. Biosci Biotech Biochem 1994;58:1858.

Submerged culture conditions for the production of mycelial biomass and exopolysaccharides by the edible Basidiomycete {it{Grifola frondosa}}IntroductionMaterials and methodsMicroorganism and mediaFermentationsSelf-directing optimization techniquePreparation of polysaccharidesAnalytical methodsEstimation of mycelial growth and polysaccharide productionMolecular weight determination

Results and discussionEffect of carbon sourcesEffect of nitrogen sourcesEffect of inoculum volumeEffect of initial pH and temperatureOptimization results in a stirred-tank fermenterMorphological changes in a stirred-tank fermenterFermentation results in an airlift fermenterMolecular properties of the polysaccharides

ConclusionsReferences