1044 Chiang Mai J. Sci. 2014; 41(5.1)

Chiang Mai J. Sci. 2014; 41(5.1) : 1044-1057http://epg.science.cmu.ac.th/ejournal/index.phpContributed Paper

Growth, Glucoamylase, Pigments and Monacolin KProduction on Rice Solid Culture in Flask and KojiChamber Using Monascus sp. KB9Thunnaree Subsaendee, Vichein Kitpreechavanich and Busaba Yongsmith*Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.*Author for correspondence; e-mail: fscibus@ku.ac.th

Received: 17 September 2013Accepted: 29 October 2013

ABSTRACT

Monascus sp. KB9, with capability to promote high production of natural red

pigment and anti-cholesterol agent, monacolin K, was used for solid rice culture in staticflask and koji chambers. The effect of various factors on the optimal fermentationcondition for 15 days incubation was determined using 500ml-flask containing 100 grice substrate. The results indicated that incubation temperature of 30°C and 38% (w/w) initial moisture content of rice at neural pH gave the highest red pigments andmonacolin K production at 3,571.97 A420U/gdw, 2,697.71 A500U/gdw, and 13,536.61mg/kgdw, respectively. While the highest glucoamylase activity and glucosamine contentof 189,685.66 unit/gdw and 4.438 mg/gdw were obtained under 35°C incubation, with38% (w/w) initial moisture content of rice substrate at initial pH 5. The secondarymetabolites were drastically reduced (80-90%) at 41-43% (w/w) initial moisture contentalthough the biomass was similar in content. Remarkably, scaling up of fermentation inthe semi-automated koji chambers of 10 and 50 kg rice substrate by using informationfrom flask level showed that Monascus sp. KB9 could grow and produce higher pigmentsand monacolin K in a shorter time. Fermented rice of 10 kg and 50 kg koji chambersproduced 2 times higher pigments and 2 and 4 time higher monacolin K than thoseobtained from flask scale cultivation. Ready-to-use product of fermented red rice wasexamined for subsequent processing of storage stability. The results showed that theamount of pigments and monacolin K in red rice were stable by steaming at 100°C 30min for growth inactivation, drying at 50°C overnight and storing in the refrigerator(2-4°C) for 1.5 months.

Keywords: Monascus sp., growth, glucoamylase, monacolin K, red pigments, fermentedred rice, koji chamber

1. INTRODUCTIONRed yeast rice or Ang-kak, a

traditionally fermented product, wasproduced by fermenting Monascus sp. on

steamed rice and has been applied widelyin many countries. It is a dietary staple andis used as colorants, medicinal, and health

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supplements [1,2,3]. Monascus spp. couldproduce red pigment and monacolin Kcontent in solid state fermentation.Monacolin K, commercially known aslovastatin is a secondary metabolite whichis a regulatory and rate-limiting enzyme ofcholesterol biosynthesis [4]. Glucoamylaseis an exo-acting carbohydrolase whichliberates glucose units from the non-reducing end of starch. Glucose was thusused as precursor for growth and secondarymetabolites production. However, theconditions of fermentation were crucial incontrolling the quality of fermented redrice [5] and also affected the secondarymetabolites production. A wild type strainof M. kaoliang KB9, which was toxicitysafety proof by rat feeding trial, chromosomeaberration test on human lymphocyte andchicken embryo bioassay [6], was studiedon rice solid culture and found that initialmoisture content of rice affected glucoamylaseactivity [7]. Many studies reported thepigments and monacolins production bysolid state fermentation of Monascus sp. atlaboratory scale [8-10]. The understandingof relationship between growth, glucoamylaseactivity and secondary metabolites in ricesolid culture was necessary for scaling upfermented red rice production. Kojichamber is a simple bioreactor for scalingup rice solid culture in a tray with aperforated bed while the fermentationtemperature of rice was controlled by aself-compressor. Our fermented red ricehas been applied as supplement of feed inlaying hens and the results showed that0.5% (w/w) of fermented red rice couldsignificantly reduce 20% of serum and eggyolk cholesterol while enhance 20% of eggyolk pigmentation [11], The results couldlead to cholesterol and triglyceridereduction in human trials. Our aims are tofind the optimum conditions of these

functional rice products at flask scale andthus subsequently used in scaling up to 10kg and 50 kg rice solid culture in kojichamber to provide basic information forthe feasibility in industrial scale to higherproduction to meet the functional foodmarket demands.

2. MATERIALS AND METHODS2.1 Microorganism and Cultivation

A wild type strain, Monascus sp. KB9was used. The stock culture was maintainedon MY agar.

2.2 InoculumInoculum preparation: Monascus sp.

KB9 was cultured on C-medium slantculture at 30oC for 7 days. Conidia or asexualspores were collected and suspended insterilized 0.1% tween 80. The spores werescrapped under aseptic conditions. Thespore suspension was used (2% v/w) as theinoculums (106 spores/ml) in flask scale andthe fermented red rice powder (7 days age)(106 spores/ml) was used as the inoculumsin koji scale.

2.3 Solid-rice Fermentation of Monascussp. KB9

A local, non-sticky rice, Hom-malicultivar was used throughout the experiments.The preparation of rice substrate is as follow:broken-milled rice was soaked in tap waterfor 3 h. After the water was removed, thesoaked rice was drained for 10 min and thena 500-ml Erlenmeyer flask containing 100 grice was autoclaved at 121oC for 15 minand cooled to room temperature.

2.3.1 Effect of temperature on growth,glucoamylase, red pigment and monacolinK production

Two milliliters of inoculum wasinoculated in prepared flask. The pigments

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and monacolin K production were examinedat different temperatures 20, 25, 30, 35 and40oC for 15 days.

2.3.2 Effect of initial moisture on growth,glucoamylase, red pigment and monacolinK production

Samples were examined at differentmoisture by adding sterile water of 0, 5, 10,15 and 20 ml into the flasks to adjust initialrice moisture content. Levels of initial ricemoisture contents were 32%, 35%, 38%, 41%and 43%, respectively. The sample wasincubated at 30�C for 15 days. The pigmentsand monacolin K were investigated.

2.3.3 Effect of initial pH on growth,glucoamylase, red pigment and monacolinK production

To avoid any acid hydrolysis to the ricesubstrate, steamed rice was examined atdifferent pH adjusted water (pH 3 to 11)which was prior adjusted by 0.5 N HClor 0.5 N NaOH and then added about10 ml/flask to 100 g rice for proper moisturecontent of 38%. The sample was incubatedat 30� C for 15 days. Growth, glucoamylaseactivity, the pigments and monacolin Kcontent were investigated.

2.4 Koji PreparationThe size (w× l×h) of koji chamber 10

and 50 kg were (0.46×0.46×0.12 m) and(0.99 ×1.52×0.18 m), respectively. Thediagram of koji chamber was shown inFigure 5a. Polished rice (10 kg) and (50 kg)were washed and then soaked in water for3 h. After the water was drained 1 h, therice was steamed at 100-121oC for 60 minin 200 liters autoclave and subsequentlycooled to about 40oC. Red-koji moldstarter (2% w/w) was inoculated onto thesteamed rice, mixed in horizontallyrotating drum and incubated in koji

chamber for 48 h at 35oC. During thepreparation of koji, the temperature of theinoculated rice was measured with athermometer and temperature probe ofincubator at a central point. The fermentedrice was sprayed with water twice daily tokeep moisture content about 50% andcultivated for 15 days at a temperature of30-35oC.

2.5 Extraction of PigmentThe pigment of fermented rice was

extracted by the method described. Onegram of fermented rice was blended with39 ml of 70% ethanol in a 250 ml flask.Forty milliliters of sample was extractedon a rotary shaker at 300 rpm for 3 h. Theextract was filtered by No.4 of Whatmanfilter to remove suspended solids. Thepigment concentration was measured byspectrophotometry at 420 and 500 nm fororange and red pigments, respectively byusing a spectrophotometer (UV-240,Shimadzu, Kyoto) against a 70% ethanolblank. The moisture content of rice sampleswas determined by weight loss after dryingthe fermented rice in a hot air ovenovernight at 105oC

2.6 Extraction of Monacolin K [8]The fermented rice was steamed at

100oC for 30 min, dried at 50oC for 24 hand then ground by mortar and pestle topowder of which 0.5 g was extracted with25 ml of 70% ethanol at 50oC for 2 h,followed by filtration through a 0.2 μmfilter membrane and then measured by anHPLC system. The HPLC system consistedof a Shimadzu LC-10AT VP liquidchromatograph, a FCV-10AL VP pump, anLDC analytical spectromonitor 3100 detectorset at 238 nm and an LDC analytical CI-4100integrator. Chromatography columnspersuit C18, 5 μm, 250×4.6 mm were

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connected to a guard column (MetaGuardpursuit 4.6 mm 5μ C18) and 20 μl loopinjector. An isocratic mobile phase ofacetonitrile: water at the ratio of 65:35(v/v) was used. The flow rate was 1.0 ml/min at a temperature of 28oC. MonacolinK (Sigma) was dissolved in methanol toprepare the standard monacolin K (100-500ppm).

2.7 Glucoamylase Activity [7]The glucoamylase activity was routinely

assayed by the determination of reducingsugars liberated in the reaction mixture in20 min at 55oC. The reaction mixturecontained 0.5 ml of the enzyme solutionand 0.5 ml of boiled 1% soluble starch in0.05 M final concentration acetate bufferpH 5.5. The reducing sugar liberated wasdetermined using UV-240 spectrophoto-meter at 520 nm absorbance. Glucose wasused as a standard. One unit of theglucoamylase was defined as the amountof enzyme to liberate reducing sugarequivalent to 1 �g of glucose/ml in min at55oC.

2.8 Biomass Estimation [12]Fermented rice (1 g) was extracted with

5 ml of HCl at room temperature for 20 h.The mixture was cooled and filtrated. Twomilliliters of extract was mixed with onemilliliter of distilled water. The mixturewas boiled at 100oC for 2 hours andneutralized as pH7 with 30% NaOH. Themixture was filled to 50 ml volume withdistilled water and filtered with No.4 ofWhatman filter. The mixture was assayedfor glucosamine. One milliliter of acetylacetone reagent and boiled at 100oC for 20min. The mixture was added with 10 ml ofethanol and 1 ml of Erhlick’s reagent. Themixture was mixed well at static conditionfor 30 min. The glucosamine content was

determined using UV-1700 spectrophoto-meter at 530 nm absorbance. Glucosamineconcentration was estimated from astandards curve with 20-100 μg/mlglucosamine HCl.

3. RESULTS AND DISCUSSION3.1 Flask Cultivation

Effect of temperature on growth,glucoamylase, red pigments and monacolinK production.

Temperature is an important factor asit influences metabolic activities andmicrobial growth. The Monascus spp. couldgenerally grow between 18-45oC and theoptimum temperature between 30-38oC[13]. Our strain Monascus sp. KB9 could grow(0.013-0.047 gdw/ml) in the range of 17.5-47oC and produced red pigments (0.15-10.70A420U/ml and 0.12-12.30 A500U/ml)incubation for 7 days in MY broth usingthe temperature gradient techniques(machine). The temperature requirementsin growth and development (productionof cleistothecia and conidia) and otherculture characteristics on agar media havealready been reported by Chayawat et al.[10]. The Wort agar gave the best myceliumgrowth while good mycelium as well assexual structure were found on GYP agar.Moreover, multi amount of conidia as wellas ascospore within cleistothecia werefound under the light microscope. Thefermented rice of Monascus sp. KB9 with32% initial moisture content was incubatedat 20, 25, 30, 35 and 40oC for 15 dayscultivation. Fungal growth in this studywas actually detected as glucosamineconcentration presented in solid rice culture.Figure 1 indicated that the fermentation at20oC incubation gave the poorest growthand product formation. While at 30oCincubation was most favorable for secondarymetabolites formation such as pigments and

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anti-cholesterol at 2,229.15 A420U/gdw,1,119.95 A500U/gdw and 5,164.38 monacolinK mg/kgdw, respectively. The differentincubated temperature also affected theglucosamine content as well as glucoamylaseactivity. Result showed that both formationsincreased rapidly in the range of 30-35oCincubation, the highest growth andglucoamylase were 3.420 mg/gdw and45,998.02 U/gdw, respectively, at 35oCincubation. These results correspond to theprevious data showing that 35oC temperatureof fermentation effected on myceliumgrowth of Monascus sp. [2] while themaximum red pigment production of M.purpureus LBP97 was obtained at 30oC[14]. Teng and Feldheim [1] also found thatanka in flask fermentation at 30oC, theyield of pigments occurred between thefifth and fifteenth days and the fermentedrice should be least incubated for 10 daysand not longer than 15 days. The temperatureof fermented rice of KB9 strain shouldthen be maintained at 30-35oC in favor ofthe best propagation, mycelium growth

and red pigmentation. On the beginningdays of fermentation, the rice wouldappear a pink color and been stirred andshaken to distribute the moisture forgrowth and pigments production.A regular fermentation procedure took7 days to reach the deep red colored at thecenter of the rice [2]. Ganrong et al. [8] andGailing [15] reported the optimal temperaturefor monacolins production was 26oC whilethat of Tsukahara et al. [16] was 23oC.Chayawat et al. [10] showed that roomtemperature (28-32oC) was the besttemperature for monacolins production ofstrain KB9. Our study on this strain foundthat slow production of monacolinsoccurred at the 3-5 days of incubation withthe yield below 1,000 mg/kgdw. After that,the productivity increased rapidly till theend of 15 days incubation giving the yieldsof 5,164.38 mg/kgdw while that productionat 35oC gave only 4,613.85 mg/kgdw.Incubation temperature at 30oC was thusused in the subsequent experiments.

Figure 1. Effect of temperature (20-40oC) on growth, glucoamylase, pigments andmonacolin K production by Monascus sp. KB9 in flask scale. Experiments were carriedout with 32% (w/w) initial moisture of rice substrate for 15 days incubation.

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3.2 Effect of Initial Moisture onGrowth, Glucoamylase, Red Pigmentsand Monacolin K Production

Initial moisture content is a criticalfactor for growth and enzyme productionof solid state fermentation because it isnecessary for new cell synthesis [14]. Theeffect of initial rice moisture content at32-43% was found to be one of the keyfactors affecting glucoamylase activity aswell as pigmentation of Monascus [7]. In thisstudy, 32% initial moisture content of riceshowed low growth and low productformation with rather dry rice appearance.The glucosamine as well as glucoamylaseactivity increased at 35-38% initial moisturecontent while both reached their maximumat 3.448 mg/gdw and 182,947.00 U/gdw,respectively, from the 38% initial moisturecontent which could also accumulatehighest yields of secondary metabolites ofpigments and monacolin K at 3,571.97 A420,2,697.71 A500U/gdw and 13,536.41 mg/kgdw,respectively. For those having the higherinitial moisture content of 41-43%, the ricegrain looked wet but still promotedgrowth and glucoamylase activity whereaspigments and monacolin K were 80% and98% decreased remarkably (Figure 2).Following the fermentation time course indetails as shown in Figure 3, it is confirmedthat initial rice moisture content of 41-43%,the fungus grew well and reached its earlystationary phase at 5 days incubation whererice was hydrolyzed very fast and theamount of glucose liberation had highconcentration at 413.50 and 402.42 mg/gdwat 7 days of incubation. Pigments andmonacolins production could thus beinhibited to some extent by such highglucose content. Furthermore, highermoisture content (41% and 43%) not onlyresulted in lower pigments and monacolinK production but also gave the agglomeration

of the substrate which leads to reducedoxygen transfer and minimizes heatexchange. In addition, the air ventilationwas low, the CO2 accumulated and promoted0.6% ethanol synthesis [7]. It could thenbe a possible explanation for reduction inpigment and monacolin K production athigh initial moisture content. Generally,at 32-38% initial moisture content gave thehighest glucoamylase at 9 days incubationand the glucose was decreased after 7 daysincubation to be consumed up for secondarymetabolites where the pigments andmonacolin K were continuously increased.Surprisingly, the high initial moisturecontent (41-43%) of rice, Monascus sp. KB9rapidly grew and gave also earlier butlower enzyme activity. While the glucosewas highly liberation but it was slowly usedup with increasing moisture content up to71% would inhibit secondary metabolitesproduction. Occurred “air tight” phenomenaobtained from such high moisture contentas well as high glucose liberation importantlyaffected on the production of secondarymetabolites, pigments [17] or monacolins[18]. Lee et al. [5] reported that the initialmoisture content of approximately 50%was good for red pigment production ofM. purpureus ATCC 16362 but the initialmoisture content at 60% decreased the redpigment production drastically. However,Chen and Hu [9] concluded that cultivationof a mutant strain M. pilosus M12-69 yieldthe best monacolin K/citrinin ratio whenthe moisture contents was 55-75%. The redpigment and monacolin K production werehigh when the moisture about 50-70% andthis moisture was traditionally used for ricefermentation [19]. Other substrate such asjack fruit seed, Babitha et al. [14] reportedthat M. purpureus LPB 97 could producethe higher alpha amylase and glucoamylaseactivity at 50% initial moisture content.

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Figure 2. Effect of initial moisture content (32-43% w/w) on growth, glucoamylase, pigments andmonacolin K production by Monascus sp. KB9 in flask scale. Experiments were carried out at room temperature(28-30°C) for 15 days incubation.

Figure 3. Fermentation time course of growth, glucoamylase, pigments and monacolin K production byMonascus sp. KB9 on rice substrates with various initial moisture content (32-43% w/w) in flask scale.Experiments were carried out at room temperature (28-30°C) for 15 days incubation. (a) 32%,( b) 35%, (c)38%, (d) 41% and (e) 43%.

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In our study the 38% (w/w) initial moisturecontent at 30oC incubation were used inthe subsequent experiments.

3.3 Effect of Initial pH on Growth,Glucoamylase, Red Pigments andMonacolin K Production

Monascus sp. KB9 could grow in all initialpH adjusted water (pH 3, 5, 7, 9 and 11) tosolid rice culture, though the highestgrowth was obtained from the initial pHof 5.0. The initial pH also affected the redpigment formation. A pH range of 5 to7gave favorable red pigment productionsimilar to those observed by Lee et al. [5]who showed that the highest level of redpigment were obtained at a pH of 6.0.Pigment color tones of ethanol extractsolution changed correspond to pH variationindicated as: pH 3-4 orange yellow, pH 5orange red, pH 6 red and pH 7-10 purplered [20]. The red pigment was found morestable in neutral (pH 7) or alkaline (pH 9.5)than acidic condition (pH 3) [2]. However,the actual initial pH of rice, which sterile

pH adjusted water (pH 3, 5, 7, 9 and 11)addition after sterilization was changed as5.422, 5.942, 6.201, 6.358 and 7.128,respectively. The results from the solid-state flask culture performed at variousinitial pH adjusted water to steriled ricewere presented in Figure 4 and showed thatduring cultivation, the actual initial neutralpH (around 6.201) of rice affected the redpigments formation but the lower pH(around 5.942) was optimal condition forgrowth, glucoamylase activity and monacolinK content. The highest glucoamylaseactivity, monacolin K, and glucosamine at189,685.66 U/gdw, 8,557.62 mg/kgdw and4.438 mg/gdw respectively were obtainedat actual pH 5.942. The results of neutralactual pH (6.201) sample were best onpigments production of 2,991.34 A420U/gdwand 2,009.33 A500U/gdw at the 38% initialmoisture content. Initial actual pH of riceat 5-7 of this study played no influence toMonascus fermentation of primary orsecondary metabolite synthesis.

Figure 4. Effect of pH adjustment on growth, glucoamylase, pigments and monacolinK production by Monascus sp. KB9 in flask scale. Sterile water at pH (3, 5, 7, 9 and 11)was added to rice substrate of 38% (w/w) initial moisture content and incubated at 30°Cfor 15 days.

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3.4 Scaling Up of Red Rice Fermentationin Koji Chamber

In previous study, optimum conditionsin flask scale fermentation such as 30-35oCincubation temperature, 38% initial moisturecontent and initial pH 5-7 were applied in10, 50 koji scale for growth, glucoamylaseand secondary metabolites. Monascus spp. isgenerally quite slow-growing mold, whichits rice culture is easily contaminated undernon-fully aseptic conditions by fast-growingAspergillus or Rhizopus spp. Scaling up ofred rice fermentation to 10 kg and 50 kgcapacity were carried out in the koji chambers,shown in Figure 5. Steamed rice was thusprepared and then mixed with 2% red riceinoculums (106spores/g). Red rice powderinoculum was conveniently used at thehigher scale of 10-50 kg rice substrates forit could be steadily mixed with steamed ricewhile initial moisture content of rice in kojichamber of 10 and 50 kg were 43.65% and42.97%, respectively. Incubation in kojichamber was carried out with the lid closedfor 2 days incubation. The ability of Monascussp. KB9 to create volatile compounds suchas alcohol or esters was activated in kojichamber (10 kg and 50 kg) by controllingthe high initial temperature at 35oC. Duringfermentation, the rice grains were flippeddaily to release the heat generated frommetabolism so fermented rice might needan adequate amount of water to compensatefor the moisture lost during incubation.The fungus mycelium grew and coveredthe surface of the grains while the moisturecontent of rice was decreased. After 2 dayincubation, distilled water was added infermented rice (2 times/day) in order toincrease more moisture content to be within50% level and then mixed throughlyeveryday. Strain KB9 could grow andproduce red pigment within 5 day and thecolor gradually changed to dark red.

Monascus sp. produced volatile compoundssuch as alcohol, esters, or volatile fatty acidsto inhibit other fungi under increasedtemperature (not exceed 45oC) in 5 daysbut all was disappeared after 7 days ofincubation. Glucose in koji scale was highlyproduced more than that in flask scale butreadily consumed by the fungus. At the 9days, the fungal biomass had covered theentire rice surface and penetrated into therice. Fermented rice then became softwhile moisture content increased. At forglucoamylase in the capacities of 100 g,10 and 50 kg rice were produced at9,525.50, 83,979.07 and 296,625.19 U/gdwwhile glucose residues were 225.81, 354.08and 365.45 mg/gdw. The glucoamylaseactivity in 50 kg of fermented rice washigher than 10 kg of fermented rice butthe glucose residue was quite similar inamount. The catabolite repression was notoccurred due to the higher glucoamylaseshould accumulate more glucose but suchglucose content was subsequent used ascarbon source for growth and wastransformed in glycolysis to acetyl CoAand enter the TCA cycle and polyketidepathway for pigmentation and monacolinK production [21]. Monascus growth asglucosamine content was still graduallyincreased until the end of fermentation(15 days) while continuously accumulatedpigments and monacolin K content. Theoptimum temperature for maximumsecondary metabolites production isusually lower than that required for growth[22]. The temperature in koji was firstlyadjusted at 35oC for growth within 3 daysincubation and then the temperature wascontrolled to 30oC in order to release heatand promote pigment and monacolinsproduction. Temperature-shift cultivationhas been applied in koji scale [16]. Themonacolin K content was increased by

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Finally, glucoamylase activity offermented red rice in flask and koji scalesat 15 days incubation containing 100 g, 10,50 kg were 8,069.27, 104,850.72 and251,395.65 U/gdw while the glucosaminewere 2.935, 8.244 and 20.049 mg/gdw,respectively. The pigment productionachieved best results using the fermentationof 10 kg and 50 kg koji chambers and

produced 2 times greater pigments thanthose in flask. The pigments in 100 g riceflask scale were 1,820.48 A420 U/gdw and1,898.78 A500U/gdw while the pigments in10 kg rice koji scale 10 were 3,538.42 A420

U/gdw and 3,613.70 A500U/gdw and 50 kgrice koji scale were 3,942.86 A420U/gdw and4,206.48 A500U/gdw, respectively. MonacolinK in the similar 100 g rice flask and koji

shifting the temperature from 30 to 23oCat 4 days incubation. The monacolin Kcontent was nearly 20 times higher than

having the constant temperature ofcultivation at 30oC.

Figure 5. Fermented rice by Monascus sp. KB9 in koji chamber, (a) diagram of kojichamber, (b) 10 kg rice, (c) 50 kg rice for 15 days incubation.

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scales 10 and 50 kg rice were produced at7,183.75, 14,580.45 and 27,871.66 mg/kgdwrespectively (Figure 6). Remarkably, thescaling up of 10 and 50 kg rice in kojichambers succeeded in giving 2 and 4 timeshigher monacolin K than that in flask scale.Many researchers reported monacolin Kproduction of 0.53 mg/g by M. perpureusNTU601 [23] 2.52 mg/g by M. pilosus [9]and 2.584 mg/g by M. purpureus NTU301[24]. Strain of Monascus is an importantfactor for specification which couldproduce different amount of pigment andmonacolin K. We selected M. kaoliang KB9because it could rapidly grow and producehigh secondary metabolites. The aerationsystem influenced on pigments formationwhile the amount of pigment was directlyproportional to the biomass produced [25].Chiu et al. [26] reported temperature at 37-38oC was effective for reproduction and

growth but the final stage of fermentationhas to be kept at 34oC while we keptfermented rice at 30oC. After downstreamingprocess, only half biomass has beenobtained. The fermented rice was changedto dark red pigment while monacolin Kcontent was lower so we must chooseconditions for fungal inactivation, temperatureof drying process and preservative methodin order to keep most metabolites infermented rice.

3.5 Stability of Pigments and MonacolinK in Red Fermented Rice

The downsteaming process wasexamined for storage stability of fermentedrice. The resistance of pigments andmonacolin K content were found optimumusing steaming at 100oC for 30 min forfungal growth and metabolism inactivation.Pigments and monacolin K of fermented

Figure 6. Fermentation time course of growth, glucoamylase, pigments and monacolinK production by Monascus sp. KB9 on fermented rice (a) 100 g in flask; (b) 10 kg rice inkoji chamber; (c) 50 kg rice in koji chamber.

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rice was remained at 93%, 93% and 90%while the sterilization (121� C for 15 min)removed most pigments and monacolin K.Moreover, the pigments and monacolinK in fermented rice was further destroyedby dry heating (50oC overnight) andremained at 55%, 55% and 75%. Thefermented rice thus became dark red riceand giving typical odor red rice after

drying. Fermented rice was stable in therefrigerator (2-4oC) at least for 1.5 monthwhile the pigments and monacolin Kwere still remained at 37%, 38% and 41%(Figure 7). Li et al. [27] found that themonacolins decreased significantly underthe conditions of high humidity at hightemperature (75% RH, 60oC) and sunlight.

Figure 7. Percentage of remaining value of pigments and monacolin K in downstreamprocessing.

4. CONCLUSIONSThe results of solid state fermentation

of Monascus sp. KB9 in flask and kojichamber showed that both temperatureand initial moisture content significantlyaffected red pigment and monacolin Kproduction. The initial moisture contentwas the most influencing factor for growthand activity of enzyme of rice fermentationof this strain as well as for pigments andmonacolin K production. In flask, theinitial moisture content of rice at 38%(w/w) and incubated at 30oC was best forboth red pigment and monacolin Kproduction while at 35oC was good forgrowth and glucoamylase formation. Initialmoisture content (41-43% w/w) reducedpigment as well as monacolin K symthesisbecause it facilitated Monascus sp. KB9growth and glucoamylase activity so fast.

High glucose liberation under highmoisture content (70% w/w) accumulationcould give the rice substrate agglomerationwhich leads to decreasing oxygen transferand minimizes heat exchange, resulted inboth secondary metabolites reduction.Scaling up to 10 and 50 kg rice culture inkoji chamber could be time and area savingfor fermentation. In koji scale, the redpigments and monacolin K content werehigher than flask level. Water spraying wasimportant for maintaining moisture lossdue to fermentation heat generated fromgrowth and glucoamylase activity. Inaddition, temperature must be controlledin the range of 30-35oC for pigment andmonacolin K production. Glucose wascontrolled by metabolism of fungi whichaffected secondary metabolite production.It is not necessary to use aseptic technique

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in koji chambers scale because Monascus sp.KB9 could produce alcohol at the earlyperiod of fermentation which inhibitedother microorganisms. Fermented red ricewith good odor, deep red pigment andhigher monacolin K content in kojichambers had been obtained in a shortertime. Downstreaming of fermented Monascusrice was carried out by steaming at 100oCfor 30 min to inactivate fungal biomass, andthen drying at 50oC in hot air oven andkept in closed package in 2-4oC refrigerator.Further study was suggested to developmore automatic control system for facilatingthe higher quality consistency of red riceproduct to meet the needs of the functionalfood market.

ACKNOWLEDGEMENTThe authors would like to thank

Kasetsart University Research andDevelopment Institute (KURDI) and theGraduate School of Kasetsart Universityfor supporting the research.

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