VNU Jo u m al of Science, N a tu ral Sciences a n d T echnology 23 (2007) 166-173 Application of fed-batch fermentation in high-gravity brewing Vu Tran Khanh Linh, Le Van Viet Man* Department o f Food Technology, Faculty o f Chemical Engineering, Ho Chi Minh City University o f Technology, Vietnam National University, 268 Ly Tỉiuong Kiet, District 10, Ho Chi Minh City, Vietnam R eceived 18 M ay 2006 A b s tra c t E conom ic d em an d s to intensify the brew ing p ro cess and increase the ferm enter p ro d u ctiv ity have stim ulated interest in high-gravity brevving Hovvever, in creasin g w ort sugar co n cen tration can h av e a d etrim ental effect o n íerm entation p erío rm a n ce, ad v ersely a íĩe c tin g yeast p hysiology and altering the physical and ílav o r properties o f th e b eer product M any m ethods such as: higher pitching rates, higher ferm entation tem peratures, m ore efficien t ae ratio n than in conventional brew ing, and im m obilised yeast w ere used to im prove this process T his study focused o n the ap plication o f fed-batch ferm en tatio n in h ig h -g v ity brew ing T w o fed-batch cu ltures w ith d iffe ren t specific g ravity o f the sup p lem en ted w orts w ere exam ined The sp eciíĩc gravity after the su pplem entatio n w as the sam e D esp ite the higher p rim a ry ferm entation tim e, b o th fed-batch cultures achieved higher co n cen atio n o f ethanol and lo w er diacety l content than those in batch ferm entation T hese resulted in the p o ssib ility o f in creasin g the fínal b eer volum e and o f shortening the m aturation tim e K eyw ords: F ed-batch íerm entation, high gravity brew ing, S a cch a ro m yces cerevisiae In tro d u c tio n T he traditional b rew in g is g en erally applied to ferm ent w orts w ith l-1 ° B x specific gravity to produce beers o f - % (v/v) ethanol W ith the need to increase pro d u ctiv ity and consequently co m p etitiv en ess on the m arket, m any brew eries are ch an g in g th eir m ethods and processes for p ro d u cin g beer A m ajor innovation in brew ing is high g ravity brew ing te ch n o lo g y -th e ferm entation o f w o rt containing 16 g or m ore o f đ issolved solids p er 0 g o f w ort T his technology h as b eco m e p o p u lar due to such advantages as in creasin g p lan t efficien cy an d cap acity , red u cin g energy, labour and Capital costs, as w ell as increasing in ethanol y ie ld p er u n it o f ferm entable extract M o reo v er, it can h elp im prove beer stability, pro d u ce sm o o th er taste an d g reater flexibility to the fm al pro d u ct [1-4] O n the other hand, the dravvbacks o f this p ro cess include decreased m aterial efficiency, as w ell as reduced foam stability an d ílav o u r m aích in g [5] In addition, d u rin g ferm en tatio n o f h igh-gravity w ort, the y east are exposed to ex trem e conditions: in creased osm otic p ressu re and increased toxicity o f p ro đ u ced ethanol [2,6,7], nutrient lim itatio n esp ecially c o n ce m in g oxygen and assim ilab le n itrogen [ Ị T h e arisin g p ro b lem s h ave been solved by * Corresponding author Tel.: 84-8-8646251 E-mail: Iwman@ hcmut.edu.vn usin g h ig h e r pitch in g rates, h ig h er ferm entation V.T.K Linh, L.v.v Man / V N U Ịoum al o f Science, Naturaỉ Sciences and Technology 23 (2007) 166-173 167 tem peratures [ ], m o re efficien t aeration than in F oster T ien G ian g Ltd C om pany T he pre- conventional brevving [ ] an d im m obilised y east [2] O ne o f th e p o ssib ilities to red u ce the detrim ental factors acting in hig h -g rav ity brew in g could be fed-batch ferm en tatio n This technique is com m o n ly u sed in m icrobial ferm entation b ecau se it co m b in es the advantages o f b o th batch an d co ntinuous ferm entations F ed-b atch íerm en tatio n is used to prev en t or decrease sub strate-asso ciated grovvth inhibition by co n o llin g n u trien t supply P rocess is starteđ as a batch p rocess w ith a sm all am ount o f biom ass an d substrate in inoculum the íerm entor T h en the substrate feed is applied w hen m ost o f the in itially ad d ed substrate has been consum ed T h is p ro ced u re h elp s to increase the total substrate content in the ferm entor, w hile alw ay s m ain tain in g a low substrate concentratio n d uring ferm en tatio n for reducing the n eg ativ e effect o f o sm o tic pressure on yeast T hus, fed-batch ferm entation allow s đecreasing the initial yeast co n cen tratio n and the cost o f inoculum pro p ag atio n T his study focused on the prim ary ferm entation in brew ing F ed -b atch cultures w ere ex am in ed in o rd er to increase ethanol content in the green becr w as ob tain ed from yeast cultures m ain tain ed on m alt-agar slants at 4°c The inoculum p ro p ag atio n w as carried out in the 10°Bx 10 X w ort The p itch in g rate w as about 106 v iab le cells m L '1 A ll íerm entations w ere p erfo rm ed in a b io reato r B IO ST A T B (B B raun B io tech In tern atio n al) at 17°c w ith 1,5 L o f 16°Bx w ort In fed-batch ferm entation, sam ples w ere exam ined: 1,5 L o f fennenting w ort w as su p p lem en ted b y 1,5 L o f 28°Bx w ort or 2,5 L o f 24°B x w ort In the fed-batch sam ples, th e sp eciíìc g ravity in the m edium aíte r sup p lem en tatio n w as the sam e (16°Bx) 2.3 Analytìcaỉ methods D uring the ferm entation, th e evolution o f pH , total n u m b e r o f yeast cell and viability, w ort sp eciĩic gravity, red u cin g sugar, íree am ino n itro g en , ethanol and diacetyl contents w ere ex am in ed T h e n u m b er o f yeast cell w as d eterm in ed b y usin g an Im proved N eubauer H aem o cy to m eter at x400 m agn iíĩcatio n w ith a light m icro sco p e d eterm in ed by Y east usin g th e viability w as m ethylene blue staining [9] A fter rem o v in g y east biom ass by cen trifu g atio n (6000 rpm , 15 m ins, 4°C), the M a te ria ls a n d m e th o d s specific g ravity w as m easured by a reử acto m eter C on cen tratio n o f reducing sugars w as q u an tified b y spectrophotom etric m ethod 2.1 Wort production usin g th e d in itro salicy lic acid reag en t [10] Free 16 °Bx w o rt w as p ro d u ced from 80% barley am ino n itro g en (F A N ) co n ten t w as m easured m alt (im p o rted ữ o m A u stralia) and 20% rice by sp ectro p h o to m etric m ethod, using ninhydrin (V iet N am ), by deco ctio n m ashing T h e 28 °Bx reagent and 24 °Bx w orts w ere p rep ared b y vacuum concentration at 60°c from the 16 °Bx w ort d eterm in ed b y a m eth o d based on distillation [11] E thanol concentration was and d en sity q u an tiíĩcatio n [12] C oncentration o f diacetyl w as determ ined by spectrophotometric m ethod u sin g o - p h en y len d iam in reagent [ 1 ] 2.2 Microorganừm andfermentation conditions Y east used in th is study w as a lager strain of Saccharomyces cerevisiae, supplied by T h e su g ar uptake rate (g/L h) w as calculated as the ratio o f the red u cin g su g ar content (g/L) consum ed b y y east to th e ferm entation tim e (h) 168 V.T.K Linh, L.v.v M an / V N U Ịoum al o f Science, Natural Sãences and Technology 23 (2007) 166-173 Results and discussion 3.2 Substrate assimilation 3.1 Yeast growth Y east grow th during th e íerm en tatio n is presented in Fig In the batch ferm entation, d u rin g the íĩrst 60 hours, yeast cell nu m b er in the cu ltu re increased quickly because the w ort w as rich in oxygen and nutrients T h e m ax im u m co n cen tratio n o f cells (approxim ately 120 obtained after 108 X hours 10 cells/m L ) w as A fter that, the biom ass decreased progressively T h u s the 108* íerm enting hour w as ch o sen as the m om ent for wort supplementation in the tw o fed-batch cultures In fed-batch íerm en tatio n , after fresh w ort feeding, the yeast co ncentration in sam ple w as low er than th at in sam ple T h is w as due to the higher volum e o f w o rt supplem entation O ur experim ental resu lts show ed that Tim e (h o u rs) Fig K inetics o f specific gravity during the fe rm e n ta tio n o f 1,5 L o f 16 °Bx w orts: ( * ) batch culture, ( ♦ ) fed-batch culture 1: supplem ented w ith L o f 28 °Bx w ort after 108* ferm enting hours, ( A ) fe d -b atch cu ltu re 2: supplem ented w ith 1,5 L o f 24 °Bx w ort the m axim um yeast co n cen tratio n reached in the tw o sam ples w as sim ilar; how ever, the grow ing tim e o f yeast in sam ple w as 24 hours longer than that in sam ple (Fig 1) Tim e (h o u rs) Fig K inetics o f yeast grovvth d u rin g the íerm entation o f 1,5 L o f 16 °Bx w orts: ( * ) batch culture, ( ♦ ) fed-batch culture 1: supplem ented w ith L o f 28 °Bx w ort after 108*1* ferm enting hours, ( A ) fed-batch culture 2: supplem ented w ith 1,5 L o f 24 °Bx w ort Fig sh o w s the ev olution o f the speciíĩc g rav ity d u rin g the batch and fed-batch íe rm en ta tio n s In th e b atch culture, after 108 ferm enting hours, th e sp eciíĩc g ravity did not change sig n iíĩca n tly It w as due to the low co n c e n ^ tio n of substrates and high co n ce n tratio n o f som e toxic m etabolites (ethanol an d o th er h ig h er alcohols) produced by y east in th e m edium It can be coníĩrm ed that the ch o se n m o m en t - the 108th ferm enting hour for fresh w o rt su p p lem en tatio n w as reasonable In th e fed-batch cultures, fresh w ort su p p lem en tatio n increaseđ the nutrient contents an d d ecre ase d ethanol concentration in the ferm en tin g m edium T herefore, the yeast p ro p ag atio n co n tinued (Fig 1) and the speciíìc g ravity d ecreased sig niíĩcantly, particularly d uring th e n ex t 48 hours after supplem enting F ro m th e 15 ó* hour, the speciíic gravity d ecre ase d w ith a slow er rate: the substrate assim ilatio n rate in fed-batch culture w as lo w er th a n th at in fed-batch culture At the end o f th e prim ary ferm entation, the speciíĩc V.T.K Linh, L.v.v M an / VNƯ ]oum al o f Science, Natural Sciences and Technology 23 (2007) 166-173 gravity o f green beer in b oth fed -b atch cultures w ere still rath er h ig h er than that in the batch culture P erhaps the increased co n cen tratio n o f ethanol in the m edium cau sed a h arm íu l effect on yeast cells, w hich led the ferm en tatio n to be sluggish o r even stuck In this study, the prim ary ferm en tatio n was considered to have co m p leted w hen the ethanol concentration increased less than 0,3 % (v/v) in th e successive 24 ferm enting h o u rs T he ỉerm entation tim e in fed-batch cu ltu res and w as 204 hours and 228 h o u rs resp ectiv ely T he fm al speciílc gravity in fed-batch cu ltu re w as low er than that in fed-batch cu ltu re (10,7 °Bx vs 11,5 °Bx) T hus, it can be co n clu d ed that in fed-batch culture , y east co n su m ed m ore n u tn e n ts than in fed-batch cu ltu re Y east grow th involves the u p tak e o f Free A m ino N itrogen (F A N ) for the sy n th esis o f cellular protein (Fig 3) Fig indicated that d uring th e íĩrst 84 hours, FA N concen tratio n d ecreased sharply in the cultures A fter that, it b eg an to increase slightly in b atch íerm en tatio n , th at m ay be due to the autolysis o f dead cells T h is phenom enon led to a release o f in tracellu lar n itro g en based com pounds Time (hours) Fig K inetics o f free am ino nitro g en assim ilation during the íerm en tatio n o f 1,5 L o f 16 °Bx worts: ( * ) b atch cu lture, ( ♦ ) fed-batch cu ltu re 1: supplem ented w ith L o f 28 °Bx w ort a íte r 108* ferm enting hours, (▲ ) fed-bũtch cu ltu re 2: su p p lem en ted w ith 1,5 L o f 24 °B x w ort 169 A fter w o rt supplem entation, the FA N co n cen tratio n in fed-batch culture w as higher than that in fed-batch culture D uring the next 48 hours, F A N co n ce n ữ a tio n decreased due to the cell grow th T h en it augm ented w hen the d eath p h ase o f yeast began (after the 108* ferm enting h o u r - Fig 3) and the autolysis took place H ig h ethanol co ncentration w as another harm ful factor to the y e a st’s viability, in creasin g the n u m b er o f dead cells D u rin g the last hours o f the prim ary ferm entation, th e F A N level in fed-batch culture increased m ore sứ o n g ly than that in fed-batch culture It could be reasoned that at the 156* h our, th e death phase o f yeast in fedbatch cu ltu re beg an w hile in fed-batch culture 2, the y e a st g row th still continued This ph en o m en o n affirm ed the results obtained in íĩg u re 2: afler 156 ferm enting hours, the yeast activity in fed-batch culture w as higher than that in fed-batch cu ltu re and the speciíĩc gravity in fed-batch cu ltu re w as low er than that in fed -b atch cu ltu re 3.3 Metaboỉiíe production Time (hours) Fig K inetics o f ethanol production during the ferm entation o f 1,5 L o f 16 °Bx w orts: ( * ) batch culture, ( ♦ ) fed-batch culture 1: supplem ented w ith L o f 28 °B x w ort afìer 108* ferm cnting hours, (▲ ) fed -b atch culture 2: supplem ented w ith 1,5 L o f 24 °Bx wort 170 V.T.K Linh, L.v.v M an / V N U Ịoum al o f Science, Natural Sríences and Technology 23 (2007) 166-173 Fig sh o w ed th a t in b atch ferm en tatio n , the final ethanol co n ce n tratio n co u ld re a c h o n ly 6,46 % (v/v) due to th e lo w n u trie n t co n ten t and h ig h ethanol level In fed -b atch ferm en tatio n , those h arm íu l e íĩe c ts co u ld be o v erco m e, resu ltin g in h ig h er íìn al eth an o l content T h e íìn al eth an o l co n ce n tratio n s w ere 8,52% (v/v) in b o th fed -b atch cu ltu res, alth o u g h the residual su g ar co n ten t w as still th e r high It can b e su p p o sed th a t th e activ ity o f the Saccharomyces cerevisiae strain u sed in this study decreased rem a rk ab ly at th a t ethanol concentration T h e o b tain ed resu lts in fíg u re co u ld explain for the đ ata in íĩg u re In fed -b atch cu ltu re 1, from the 18001 h o u r to the th hour, FA N co n cen tratio n in c re ased sh arp ly (F ig 3); during this p erio d , th e in crease in ethanol content from 7,27 °/o (v /v ) to 8,52 % (v /v ) w as observed (Fig 4) T h u s, it w as ev id en t th at the increase in ethanol co n ten t in th e cu ltu re h ad a b ad effect on y east v iab ility In fed -b atch cu ltu re , the sam e p h e n o m e n o n w as also observed F rom the 204 h o u r to th e 2 lh h our, the ethanol co n cen tratio n in creased from 7,79 % (v/v) to 8,52 % (v/v), the F A N co n cen tratio n also aug m en ted in the m e d iu m d u e to the autolysis o f dead cells D u rin g the alco h o lic ferm en tation, the d ecrease o f pH is related to the fo rm ation o f C Ơ an d organic aciđ s o f y east cells B esides, the in crease o f pH m ark s the b eg in n in g o f y east autolysis Fig p resen ted th e ev olution o f pH in b atch an d fed-batch ferm entations T h ere w as no sig n iíĩca n t d ifferen ce in pH v alu es o f the green b e e rs in the cultures Tim e (hours) Fig E volution o f d iacety l co n cen traú o n d u rin g the ferm en tatio n o f 1,5 L o f 16 °Bx worts: ( * ) batch culture, ( ♦ ) fed-batch cu ltu re 1: supplem en ted w ith L o f 28 °B x w ort after 108th íerm en tin g hours, ( A ) fed -b atch culture 2: sup p lem en ted w ith 1,5 L o f 24 °Bx w ort D iacety l is One o f th e m ost im portant by Products in alcoholic ferm entation A t low levels, it gives b e e r a slick m ou th íeel; at higher levels, th e ílav o r b eco m es buttery and that d ecreases the sen so ry p ro p erties o f the fmal product Fig Time (hours) Fig C h an g e in p H du rin g th e ferm en tatio n o f 1,5 L o f 16 °Bx w orts: ( + ) b atch cu ltu re , ( ♦ ) fed-b atch culture 1: su p p lem en ted w ith L o f 28 °Bx w ort after 108* ferm en tứ ig hours, ( A ) fe d -b atch culture 2: su p p lem en ted w ith 1,5 L o f 24 °B x w ort show ed th a t the d iacetyl level in creased during th e íĩrst 84 hours and reached the m ax im u m co n cen tratio n (approxim ately m g/L ) M an y au th o rs affiim ed that diacetyl ío rm atio n is clo sely related to the y cast grow th, p articu la rly to th e v alin e b io sy n th esis [13] In V.T.K Linh, L.v.v M an / V N U Ịournal oịScừnce, Natural Sciences and Technology 23 (2007) 166-173 com parison w ith the o b tain ed results in Fig 1, the intensive grow th o f y east w as observed d u rin g this period T h e n the diacety l co n ten t in the ĩerm enting m edia d ecreased by red u ctio n reaction catalyzed b y th e en zy m e system in yeast T his resu lt w as sim ilar to th o se in m any prev io u s studies [13,14] A fter fresh w ort su p p lem en tatio n , diacetyl concentrations in th e fed -b atch cultures increased again due to y e a st g row th an d then decreased It can be n o te d th a t the second peak o f diacetyl íò rm atio n in fed-batch cu ltu re w as m u ch low er than th a t in fed -b atch cu ltu re T h e explanation can b e th at th e F A N co n ten t in fed-batch culture w as m uch h ig h er than in fed-batch culture L o w d iacetyl level in green b e e r is an advantage in b rew in g becau se the m aturation tim e w ill be shorter 71 T ab le p resen te d th e p aram eters o f the p rim ary íe rm e n ta tio n in b e e r p ro d u ctio n using b atch an d fed -b atch cu ltu res In th e fed -b atch cu ltu res, in spite o f the lo n g er p rim a ry ferm en tatio n tim e, the sugar co n ten ts assim ila te d b y y e a st w ere higher than th a t in th e b atch cu ltu re B esid es, th e sugar u p tak e te in fed -b atch cu ltu re an d w ere 30,2 % an d ,6 % h ig h er than th at in the b atch culture T h e fin al eth an o l co n cen tratio n in the fedb atch ferm e n tatio n s w as 32 % h ig h er than that in th e b a tc h ferm en tatio n T h is is a very im p o rtan t ad v an tag e o f h igh g rav ity brevving b ecau se th e h ig h e r co n ce n trate d b e e r w e ach iev e, th e m o re v o lu m e o f fm al b eer w e p ro d u ce T h e resu lts from tab le shovved that, the v o lu m e o f final b e e r in the fed-batch fe n n e n ta tio n s w as m u ch h ig h e r than that in the b atch íerm en ta tio n T able P aram eters o f the p rim ary ferm en tatio n in b ee r p ro d u c tio n u sin g b a tc h and fed -b atch cultures 156 97,6 F ed -b a tc h íe rm e n ta tio n 1A 204 167,05 F ed-batch íerm e n ta tio n 2C 228 173,32 0,63 50,9 ,8 7,2 0,76 67,2 0,8 1,0 0,6 1,50 L o f 16 °Bx w ort -> 1.94 L o fb e e r 2,50 L o f 16 °Bx w ort 3,23 L o f beer L o f 16 °Bx w ort -> 3,88 L o f beer 1,50 L o f 16 °Bx w o rt and 1,00 L o f °Bx w ort -> ,2 L o f b eer 1.50L o f 16 °Bx w ort and 1,50 L o f 24 °B x w ort -> 5,11 L o íb e e r B atch ferm entation“ F erm en tatio n tim e (h) S ugar content assim ilated b y yeast (g /í) S u g ar uptake rate (g/L h) E thanol co n cen tratio n in green b ee r (g/L) D iacetyl co n cen tratio n in green b e e r (m g/L ) V olum e o f final b eer afte r b ein g d ilu ted to ethanol c o n c e n a tio n o f % (v /v ) *The initial volume of the wort was 1,50 L, the initial speciíic gravity was 16 °Bx bThe initial volumc of thc wort was 1,50 L, the initial speciíic gravity was 16 °Bx The culture was supplemented with 1L o f 28 °Bx wort The spccific gravity attcr supplementing was 16 °Bx c The initial volume of the wort was 1,50 L, the initia! speciíic gravity vvas 16 °Bx The culturc was supplemented with 1,50 L of 24 °Bx wort The spcciíic gravity aftcr supplcmcnting was 16 °Bx d It was supposed that the ethanol content in the green beer did not augmented during the secondary íermcntation V.T.K Linh, L.v.v M an / V N U Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 166-173 17 A lth o u g h the eth an o l co n te n t in th e tw o fedb atch cu ltu res w as sim ilar, fed -b atch cu ltu re w as co n sid ered to be m o re effic ien t th an fedb atch cu ltu re for tw o reaso n s F irstly , lo w er diacetyl o f green b e e r in fed -b atch cu ltu re m ade the m atu ratio n tim e b eco m e shorter S econdly, the p itch in g te in fed -b atch culture w as lo w er than in fed -b atch cu ltu re i f the volum es o f fínal b e e r in th e tw o cu ltu res w ere sim ilar [4] D.I Reilly, c ’Cleirigh, P.K VValsh, Laboratory-scale production o f high-gravity w ort suitable for a broad variety o f rescarch applications, J Am Soc Chem 62 (2004) 23 [5] G Dragone, D p Silva, J.B.A Silva, Factors inílucncing cthanol production rates at highgravity brcw ing, Lebensm -ỈViss u.- Technoỉ 37 (2004) 797 [6] M p B rosnan, D Donnelly, T c James, u B ond, The stress response is repressed during íerm entation in brew ery sừains o f yeast, J Appl M icrobio 8 (2 0 )7 C o n c lu s ỉo n F e d -b a tc h c u ltu re s h a v e s h o w n to b e m o re a d v a n ta g e o u s th a n b a tc h íe rm e n ta tio n in h ig h g v ity b re w in g In fe d -b a tc h íe rm e n ta tio n , th e g re e n b e e r c o n ta in e d h ig h e r e th a n o l c o n c e n tra tio n a n d lo w e r d ia c e ty l c o n te n t In th is s tu d y , fe d b a tc h c u ltu re s th a t, th e tio s u p p le m e n tin g íe rm e n to r w a s bcer production, Braz J Chem Eng 18 (2001) 59 w e re re a liz e d It w a s c o n c lu d e d : o f th e v o lu m e o f w o rt fo r to th e in itia l v o lu m e o f w o rt in th e m o re e ffic ie n t th a n th e tio : [8] E.J Lodolo, E ’C onnor-Cox, B Axcell, O ptim ization o f the dissolved oxygen supply for high-gravity brew ing MBAA Tech Quart 36 (1999) 139 [9] A S B C , M ethods o f anaỉysis, 8^ Edition, M innesota, The Am erican Society o f Brevving C hem ists, 1992 [10] G L M iller, U se o f dinitrosalicylic acid reagent for determ ination o f ređucing sugar Anal Chem 31 (1 9 )4 Reĩerences [1] G p Casey, C.A M agnus, W M Ingledew, H igh-G ravity Brew ing: Effects o f nutrition on yeast com position, ferm entative ability, and alcohol production, A p p i Environ M icrobiol 48 (19 )6 [2] J Pátková, D ẵm ogroviõová, z D ồm ẻny & p B aửncová, V ery high-gravity w ort ícrm entation by im m obilised yeast Biotechnol L e t t 22 (20 0 )1 [3] R.B A lm eida, J.B A lm cida c Silva, U.A Lima, D.p Silva, A.N A ssis, Evaluation of ferm entation [7] P.L Pratt, J.H Bryce, G.G Stewart, The Effects o f osm otic prcssure and ethanol on yeast viability and m orphology, J Inst Brew 109 (2 0 )2 param eters during high-gravity [11] European Brew ery Convention, Anaỉytica EBC , 5th Edition, Fachvcrlag Hans Carl publisher, N um berg, 1998 [12] A O A C , OjJìcial M ethods o f Analysis o f AOAC International, 15th Edition, AOAC International, M aryland, 1990 [13] w H anncm ann, R educing Beer Maturation T im e and Retaining Quality, MBAA Tech Ouart (2 ( ^ )1 [14] w Kunzc, Technology Brewing and M alíing, 4,h Edition, VBL, Bcrlin, 2004 ... s m ain tain in g a low substrate concentratio n d uring ferm en tatio n for reducing the n eg ativ e effect o f o sm o tic pressure on yeast T hus, fed-batch ferm entation allow s đecreasing... for wort supplementation in the tw o fed-batch cultures In fed-batch íerm en tatio n , after fresh w ort feeding, the yeast co ncentration in sam ple w as low er than th at in sam ple T h is w... enting F ro m th e 15 ó* hour, the speciíic gravity d ecre ase d w ith a slow er rate: the substrate assim ilatio n rate in fed-batch culture w as lo w er th a n th at in fed-batch culture At