1032 Chiang Mai J. Sci. 2014; 41(5.1)

Chiang Mai J. Sci. 2014; 41(5.1) : 1032-1043http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Effect of Ammonium Salts on Pigments Productionby Monascus anka Mutant in 5L BioreactorBo Zhou* [a,b,c], Yan Wang [a,b,c], Huamin Lu [a,b,c] and Yanqing Zhou [a,b,c][a] School of Food Science and Engineering, Central South University of Forestry and Technology,

Changsha, 410004, P. R China.[b] National Engineering Laboratory for Rice and By-products Processing, Changsha, 410004, P. R China.[c] Hunan Province Key Laboratory of Grain, Oil Processing and Quality Control, Changsha, 410004,

P. R China.*Author for correspondence; e-mail: zhbo78@hotmail.com

Received: 4 July 2013Accepted: 6 September 2013

ABSTRACTThis work characterized the fermentation and Monascus pigments production of

Monascus anka mutant in a 5L bioreactor under the different ammonium salt conditions:ammonium chloride, ammonium sulfate or ammonium nitrate. All the experimental resultsobtained in this work suggest that not the pH environment established by ammonium saltsbut ammonium salts itself played a critical role on the production of Monascus pigments andthe growth of Monascus anka mutant. ammonium ion could inhibits the activity of some keyenzyme for Monascus pigments synthesis and the inhibition effect on some key enzyme forMonascus red pigments synthesis is stronger than on some key enzyme for Monascus yellowpigments synthesis. Key enzyme for inhibited Monascus pigments production would beresearched clearly by experiments in our future researching.

Keywords: Monascus anka mutant, ammonium salts, pH, yellow pigments, red pigments

1. INTRODUCTIONIn food industrial nowadays, yellow

pigments production has two mainapproaches: chemical synthesis and plantextraction. The former, with products suchas tartrazine [1], due to its safety risk, haswithered in use. Meanwhile, the latter, withextracts such as gardenia yellow[2]ÿsafflower yellow [3], is natural and lowin toxicity, but is subject to the seasonchange, resulting in an unstable industrialproduction. Compared with these twoapproaches, microbiological fermentation

for yellow pigments production, becauseof its few safety hazards, low cost, and widevariety of resource choices [4-7], hasreceived increasing attention as a promisingalternative. As far as industrial productionis concerned, Monascus is one of themicrobial resources now in use to producepigments.

Thousands years before ancientChinese had been using Monascus-fermented red rice as a food colorant tomake red rice wine, red soybean cheese,

Chiang Mai J. Sci. 2014; 41(5.1) 1033

meat and fish products and so on [8-10].The genus Monascus produces a mixture ofthree categories of pigments, orange, red,and yellow pigments. These three pigmentshave two components of polyketide originin common, which are secondarymetabolites possessing an azaphiloneskeleton [8, 11-13]. The red pigmentsproduced by Monascus are applied widelydue to their safety with the characteristicsof high protein adhesion, thermal stability,and wide-pH stability [14]. But withcitrinin production by Monascus wasdiscovered [15], which is nephrotoxic (kidneydamaging) and possibly carcinogenic[16-18] to human beings, the safety ofMonascus products has been paid more andmore attention to.

The production of Monascus redpigments has been researched a lot. Someparameter factors affecting the growth ofMonascus and production of red pigments,such as gas environment [19], agitation andaeration [20], carbon and nitrogen sources[21] and pH [22-24] were reported, and theperstractive fermentastion of intracellularMonascus pigments in nonionic surfactantTriton X-100 micelle aqueous solutionwas also reported [25-27]. However, due tounavailability of microbial species, researcheson production of Monascus yellow pigments(Monascusone A and B) is very limited[28, 29]. One Monascus anka mutant ableto produce high yield of yellow pigments(Ankaflavin and monascin) screened byphysical and chemical combinationmutagenesis was obtained in ourlaboratory, the yellow pigments producedby Monascus anka mutant have maximumabsorption at 410 nm [30]. After incubatingfor 3 days on MEA medium (Per Litercontains malt extract 20 g, peptone 1 g,glucose 20 g, and agar 15 g), the colonies ofMonascus anka mutant showed yellowish

in color with the size of 25-30 mm. Afterincubating for 5 days, the aerial myceliumwas white; the colonies showed yelloworange in color with the size of 29-30 mm,and the air-borne mycelium was white.Both conidiospores and ascocarpes werecolorless. The conidiospores were 40-50 μmin size and the ascospores were oval-shapedwith the size of 30-35 μm.

As the use of available microbial sourceof nitrogen, it was key inhibition effectfactors for antibiotics formation by highconcentrations of nitrogen sources, suchas 14-membered macrolide antibioticerythromycin [31]and the 16-memberedmacrolide antibiotics leucomycin [32].But in our previous study, ammonium saltsplayed a critical role on Monascus yellowpigments production by Monascus ankamutant in the shaker cultivation [30].

In present study, we characterized thefermentation and Monascus pigmentsproduction of Monascus anka mutant in thebioreactor under different ammoniumsalt conditions, ammonium chloride,ammonium sulfate or ammonium nitrate,and then made an analysis about ammoniumsalts affects on Monascus yellow pigmentsproduction by Monascus anka mutant.

2. MATERIALS AND METHODS2.1 Organism and Cultivation

The microorganism used in this work isMonascus anka mutant able to producecomparatively high-production of yellowpigments, which was screened bycombination of physical and chemicalmutagenesis in our laboratory. The originalstrain of Monascus anka obtained fromChina Center of Industrial CultureCollection(CICC 5013).

Stock cultures of the mutant weremaintained and periodically subcultivated

1034 Chiang Mai J. Sci. 2014; 41(5.1)

on wort agar slants, which contains 15°wort (provided by Guangzhou Zhujiang BeerCo., Ltd, China) and 20g/L agar (DifcoLabatory, Loveton Circle, USA). Cultureswere reactivated by being transferred ontofresh wort agar slants. After cultivation of2-3d at 31 °C, spores were collected with5 mL of sterilized water, and the sporesuspension corrected was used as inoculumpreparation. 1 mL of spores suspensionwas inoculated in 100 mL of seed medium(30g/L corn powder, 3g/L NaNO3, 0.01g/L FeSO4.7H2O, and 4g/L KH2PO4, pH6.0),and cultured at 31 °C, 200rpm for 2d in arotatory shaker as the seeds cultures.

The seed cultures were inoculated inthe submerged culture medium at volumeratio of 1:9, and fermented in a 5Lbioreactor (BIOFLO 3000 Batch/ContinuousBioreactor, New Brunswick Scientific Edison,N.J., USA) at 300 rpm, 31 � � and aeration of0.15 m3.h-1 for 6-7d. Submerged culturemedium contains ammonium salts, 12g/Lcorn steep liquor, 5g/L KH2PO4 , 20g/Lglucose, 40g/L starch and 0.1g/L CaCl2,pH4.0. The type of ammonium salts wasvarious in different experiments, and thedosage of ammonium salts was determinedby NH4

+, 0.3moL/L of which was used foreach fermentation. The 5L bioreactor loaded3 L of submerged culture medium.2.2 Measurement of Pigments

5 mL of the culture broth wasextracted with 5 mL of 70% (v/v) ethanolat room temperature for 1h, and thencentrifuged at 4000rpm for 20min. Thesupernatant obtained was filtered with filterpaper. The filtrate contained two pigments:yellow pigments and red pigments, whoseconcentrations were determined by measuringthe optical density of the filtrate using a2802SUV/VIS spectrophotometer (UnicoshScientific Instrument Co., Ltd, Shanghai,China) at 410nm and 510nm respectively

(Yoshimura et al. 1975). Results wereexpressed with OD units per mL offermented broth. The linearity equationbetween absorbance and dilutingproportions is y = -0.0054x + 1.5462 (R2

= 0.9907). In where, y is absorbance and xis dilution proportions (in the range from100 to 300).

2.3 Measurement of Dry Cell Weight(DCW)

5mL of cultures broth was filteredthrough fibre-glass membranes (WhatmanGF/C), the retained biomass was rinsed twicewith distilled water, vacuum dried completelyat 60!, and weighed.

2.4 Data Calculation and AnalysisThe specific growth rate, m(h-1), was

calculated with the equation: whereX is the cell concentration (g/L) at time t (h).The specific production rate of yellowpigments, qY(OD units /h), was calculatedwith the equation: where Y isthe yellow pigments value (OD units) at timet (h). The specific production rate of redpigments, qR(OD units /h), was calculatedwith the equation: where R is thered pigments value(OD units) at time t (h).

2.5 Data AnalysisAll the data shown in Figures were

expressed as mean ± standard deviation ofthe three replicates. The statisticalevaluation of all data was done byOriginPro8.0.

3. RESULTS AND DISCUSSION3.1 Effect of Ammonium Salts on pH ofCultures in Fermentation of Monascusanka Mutant

When using ammonium chlorideor ammonium sulfate as nitrogen sourcefor production of pigments, the pH

Chiang Mai J. Sci. 2014; 41(5.1) 1035

value of cultures changed in downtrendduring the whole fermentation and thefinal pH value of cultures was 1.90 and2.30, respectively (Figure 1). When usingammonium nitrate for productionof pigments, pH value of the cultureschanged in downtrend within first54hrs and pH value reached 2.97 at 54h,however, afterwards pH value ofthe cultures increased gradually and gotto 6.00 in the end of fermentation(Figure 1).

Three kinds of ammonium salts usedin this work are all strong-acidity andweak-alkalescence salts. Ammoniumchloride is the strongest in acidity, andfollowed by ammonium sulfate andammonium nitrate successively. Along withNH4

+ consumed as nitrogen sources forMonascus growth, the acidity offermentation broths rises and the pH ofcultures declines. The lowest pH (pH<2)environment was established because of

Figure 1. Effect of ammonium salts on culture pH by Monascus anka mutant fermentation ina 5L bioreactor.

strong acidity of ammonium chloride.When using ammonium sulfate forproduction of Monascus pigments, the finalpH value could maintain within the rangefrom 2 to 2.5. But the different situationoccurred while using ammonium nitratefor production of Monascus pigments,because Monascus can not only use NH4

+

for Monascus growth but also use NO3- for

production of Monascus pigments [21]. Inthe beginning, NH4

+ was used for Monascusgrowth and the pH of cultures declined,but 54hrs after fermentation, the pH ofcultures rose due to a great deal of NO3

-

consumption for production of redpigments.

3.2 Effect of Ammonium Salts on Growthof Monascus anka Mutant

Figure 2 indicates that ammonium saltsused in this work have no significant effectson the maximum dry cell weight (DCW), butbear significant relationship with the growthprocess. The trend of growth is similarbetween the fermentations using ammoniumchloride and ammonium sulfate, but differentfrom the fermentation using ammoniumnitrate. The time to reach maximum DCW inthe fermentation using ammonium nitrate isearlier than those using ammonium chlorideand ammonium sulfate. The maximumDCW could arrive at 27.86 g/L, 27.46 g/Land 27.78 g/L at 84h, 102h and 102h,

1036 Chiang Mai J. Sci. 2014; 41(5.1)

respectively (Figure 2).However, ammonium salts have effects

on specific growth rate (μ) (Figure 2).The trend of is similar between thefermentations using ammonium chlorideand ammonium sulfate. When using

ammonium nitrate, however, the value ishigher than that using ammonium chlorideand ammonium sulfate before 40h. Theirmaximum value can get to 0.048 h-1 at 24h,0.041 h-1 at 12h and 0.045 h-1 at 12h,respectively.

Figure 2. Effect of ammonium salts on dry cell weight (DCW) and specific growth rate (μ)of Manascus anka mutant.

In a way, ammonium salts littleinfluence the yield but affect the specificgrowth rate of Monascus anka mutant.

Effect of ammonium salts on Monascuspigments production by Monascus anka mutant

As far as yield of yellow pigments, themaximum amounts of yellow pigments hasno significant difference betweenammonium chloride, ammonium sulfateand ammonium nitrate, the maximumamounts of yellow pigments got to 54.87OD units at 96h, 51.12 OD units at 96h,49.56 OD units at 120h, respectively(Figure 3). But it has obviously differencefor yield of red pigments, the maximumamounts of red pigments got to 11.26 ODunits at 80h, 34.08 OD units at 96h, 42.06

OD units at 132h, respectively (Figure 3).When using ammonium chloride as

nitrogen source for production of Monascuspigments, compared to yellow pigments,the production of red pigments wasremaining at a very low level during thewhole fermentation. (Figure 3). Before48hrs, the trend of production of yellowpigments was similar to that of red pigmentswhen using ammonium sulfate forproduction of pigments, but the productionof yellow pigments was obviously higherthan that of red pigments after 48hrs, andthe pH value of cultures was 2.56 at thistime (Figure 1). The pH of culturesdeclined to 2.97 after 54hrs of fermentationwhen using ammonium nitrate as nitrogen

Chiang Mai J. Sci. 2014; 41(5.1) 1037

source for production of Monascuspigments (Figure 1). Compared to yellowpigments, the production of red pigmentswas obviously retarded before 54hrs andthen accelerated, and the yield of yellowpigments was 4.5 times as much as that ofred pigments before 54hrs. In the end offermentation, the yield was not ofsignificant difference between yellow andred pigments (Figure 3). The maximum

production of red pigments usingammonium chloride or ammonium sulfate isjust 26.77% and 81.10% as much as thatusing ammonium nitrate, respectively(Figure 3). The maximum yield of yellowpigments using ammonium chloride,ammonium sulfate, ammonium nitrate, was4.87 times, 1.50 times, 1.18 times,respectively, as much as that of redpigments (Figure 3).

Figure 3. Effect of ammonium salts on red and yellow pigments production by Monascusanka mutant in a 5L bioreactor.

The specific production rate of yellowpigments (qY) can maintain at a low level(the maximum qY can get to 0.032 ODunits /h at 72h) when using ammoniumnitrate for fermentation of Monascuspigments. When using ammonium chloride

and ammonium sulfate as nitrogen sources,from 36h to 60h, the value of qY can increaseto 0.08 OD units /h from 0.05 OD units /h. The maximum value of qY can reach 0.076OD units /h and 0.072 OD units /h at 54hand 36h, respectively (Figure 4).

1038 Chiang Mai J. Sci. 2014; 41(5.1)

Figure 4. Effect of ammonium salts on specific production rate of red pigments(qR) andyellow pigments (qY) of Manascus anka mutant.

Compared with using ammonium nitrateand ammonium sulfate, the production ofred pigments was inhibited more obviouslywhen ammonium chloride was used, but thetrend is similar to that when using ammoniumsulfate and only the inhibitory degree isdifferent, the specific production rate ofred pigments (qR) can be up to 0.037 ODunits/h for ammonium chloride and 0.048OD units /h for ammonium sulfate at 36h.But when using ammonium nitrate as nitrogensource, at 84h, the can get to 0.043 ODunits/h, and the can be kept at this value orup for longer than that when using ammoniumchloride and ammonium sulfate (Figure 4).

Despite the trend of production of redpigments using ammonium chloride asnitrogen source is similar to that usingammonium sulfate, the descending speed ofusing ammonium chloride is faster thanthat using ammonium sulfate. Thus, the yieldof red pigments using ammonium sulfateis higher than that using ammoniumchloride (Figure 3). When ammonium nitrate

was used for production of red pigments,not only the can keep at a low value (low to0.016 OD units/h ) for a long time, but alsothe time to reach maximum is later, andthe dropping range is narrower than those usingammonium chloride and ammonium sulfate(Figure 4). The trend of production ofyellow pigments is similar to that usingammonium chloride or ammonium sulfate.In the case of ammonium chloride, both therising and dropping slopes of are steep. As aresult, the yield of yellow pigments was of nosignificant difference between using ammoniumchloride and ammonium sulfate (Figure 3).When using ammonium nitrate for productionof yellow pigments, not only the can keepat a low level (from 0.01 OD units/h to0.03 OD units/h) for a long time, but also thetime to reach maximum is later than thatusing ammonium chloride and ammoniumsulfate(Figure 4). So, the yield of yellowpigments using ammonium nitrate was of nosignificant difference from using ammoniumchloride and ammonium sulfate (Figure 3).

Chiang Mai J. Sci. 2014; 41(5.1) 1039

Kurono et al. reported that theammonium salts were bad for productionof yellow pigments and also had nosignificant influence on production of redpigments [14], Chen et al. observed thatlow pH was good for production of yellowpigments [24]. Our results obtained in thepresent work indicate that althoughammonium salts did obviously influencenot only yellow pigments production butalso red pigments production by Monascusanka mutant, they did influence the specificproduction rate of yellow pigments (Fig.4). Ammonium salts influenced the yieldas well as specific production rate of redpigments (Fig. 4). Taken together with theresults that ammonium salts did notinfluence the yield but affected the specificgrowth rate of Monascus anka mutant andwith the results that ammonium saltsaffected the pH of cultures (Figs. 1-2).Three kinds of ammonium salts used in thiswork are all strong-acidity and weak-alkalescence salts. Ammonium chloride isthe strongest, ammonium sulfate the secondand ammonium nitrate the third in acidity.Along with NH4

+ consumed as nitrogensources for Monascus growth, the acidityof fermentation broths rises and the pHof cultures declines. There is still onedifferent point, however, in the case ofammonium nitrate, it is clear as Pastranaet al. reported that NH4

+ as one nitrogensource is mainly used for the growth ofMonascus [21], NO3

- is a good nitrogensource able to be used not only for thegrowth of Monascus but also for productionof red pigments. The pH of cultures usingammonium nitrate first declines thenincreases gradually along with theconsumption of NO3

-. In the end offermentation, the pH of cultures usingammonium chloride, ammonium sulfate orammonium nitrate was 1.90, 2.3 and 6.0,

respectively (Figure 1). Low pH of cultures(<2) like using ammonium chloride greatlyinhibits the production of red pigments andhigh pH of cultures like using ammoniumnitrate is beneficial for the production ofred pigments. Lin et al. also reported thesimilar results [22]. The maximumproduction of red pigments usingammonium chloride is just 26.77% as muchas that using ammonium nitrate in ourexperiments. Taken together with theresults, we made an assumption that pHof cultures of Monascus anka mutantdirectly influenced the production ofMonascus pigments, and pH of culturesinfluences the whole process of theproduction of Monascus pigments includingyellow and red pigments.

In order to verified our assumption,A two-stage pH control strategy wascarried out in an attempt to maximizeyellow pigment formation in this paper.Firstly, an initial pH of 6.5 was keptwith 0.1 mol/ L NaOH at 31oC for 2 daysin order to obtain maximum biomass. Inthe second stage, the pH was adjustedto 2.5 with 0.1 mol/L H2SO4 to stimulatethe production of yellow pigment forthe next 4 days. However, yellow pigmentyield was no greater than with the singlestage fermentation (data not shown), theresults was the same as described byYongsmith [33]. Our results furtherdemonstrated that not the pH environmentsestablished by ammonium salts but theammonium ion itself played a critical roleon the production of Monascus pigmentsand the growth of Monascus in the Monascusanka mutant fermentation.

As the use of available microbial sourceof nitrogen, it was key inhibition effectfactors for antibiotics formation by highconcentrations of nitrogen sources [34, 35],such as 14-membered macrolide antibiotic

1040 Chiang Mai J. Sci. 2014; 41(5.1)

erythromycin [31] and the 16-memberedmacrolide antibiotics leucomycin [32]and tylosin [36] is inhibited by highconcentrations of ammonium. Theproduction of polysaccharides byAureobasidium pullulans was also suppressedunder high nitrogen levels [37]. Theinhibition effect was considered that thekey enzyme activity for antibioticssynthesis has been inhibited, such as valinedehydrogenase [38] and threoninedehydratase [39] as key enzyme providingthe precursors for tylosin formation arerepressed by ammonium ion in batchculture.

According to the results in this paperand previously reported results,ammonium ion maybe inhibits the activityof some key enzyme for Monascus pigmentssynthesis, and the inhibition effect on somekey enzyme for Monascus red pigmentssynthesis is stronger than on some keyenzyme for Monascus yellow pigmentssynthesis. This hypothesis should beverified by experiments in our futureresearching.

The production of mycotoxin citrininof Monascus is affected not only by thegrowth conditions [39, 40] but also bygenus itself [15, 41]. Sandra et al. reportedthat the high pH (about 6) was not goodfor but low pH was good for the citrininproduction by Monascus [42]. In this study,the citrinin was unable to be detected byHPLC method as described by Xu [43],namely, the Monascus anka mutant mayproduce no or just a little citrinin (thedetection limit is 0.1mg/L).

ACKNOWLEDGEMENTSThis work was supported by the National

Natural Science Foundation of China(Nos.: 31301550), the youth Scientific Research

Fundation of Central South University ofForestry & Technology (Nos: QJ2010009A).

REFERENCES

[1] Simone P.A., Daniel M.B., dira C.B.A.and Annibal D.P.N., Determinationof synthetic dyes in selectedfoodstuffs by high performance liquidchromatography with UV-DADdetection, Food Chem., 2008; 107(1):489-496.

[2] Sato S., Kitamura H., Chino M.,Takei Y., Hiruma M. and Nomura M.,A 13-week oral dose subchronictoxicity study of gardenia yellowcontaining geniposide in rats, FoodChem. Toxicol., 2007; 45(8): 1537-1544.

[3] Chen L.G., Ding L., Zhang H.R.,Li J., Wang Y.T., Wang X.P.,Qu C.L. and Zhang H.Q., Dynamicmicrowave-assisted extraction coupledwith on-line spectrophotometricdetermination of safflower yellow inFlos cartham, Anal. Chim. Acta,2006; 580(1): 75-82.

[4] Takaichi S., Maoka T., Akimoto N.,Sorokin DY, Banciu H. andKuenen J.G., Two novel yellowpigments natronochrome andchloronatronochrome from thenatrono(alkali)philic sulfur-oxidizingbacterium Thioalkalivibrio versutusALJ 15, Tetrahedron Lett., 2004; 45:8303-8305.

[5] Sophie E. and Bert S., Structure andbiosynthesis of chrysophysarin A, aplasmodial pigment from the slimemould Physarum polycephalum(myxomycetes), Tetrahedron, 2000;56: 363-365.

[6] Shier W.T., Lao Y.B., Terry W.J.S.and Hamed K.A., Yellow pigmentsused in rapid identification of axatoxin

Chiang Mai J. Sci. 2014; 41(5.1) 1041

producing Aspergillus strains areanthraquinones associated with theaxatoxin biosynthetic pathway, Bioorg.Chem., 2005; 33: 426-438.

[7] Yuka M., Akira I., Jun M., ShigeruS., Kentaro Y. and Masami I.,Physarigins A-C, three new yellowpigments from a cultured myxomycetePhysarum rigidum, Tetrahedron Lett.,2003; 44: 4479–4481.

[8] Blanc P.J., Loret M.O., Santerre A.L.,Pareilleux A., Promel D., Laussac J.C.and Goma G., Pigments of Monascus,J. Food Sci., 1994; 9: 862-865.

[9] Ma J., Li Y., Ye Q., Li J., Hua Y., JuD., Zhang D., Cooper R. and ChangM., Constituents of red yeast rice, atraditional Chinese food and medicine,J. Agric. Food Chem., 2000; 48: 5220-5225.

[10] Wild D., Toth G. and Humpf H.U.,New Monascus metabolite isolatedfrom red yeast rice, J. Agric. FoodChem., 2002; 50: 3999-4002.

[11] Juzlova′ P., Martinkova′ L. and KrenV., Secondary metabolites of thefungus Monascus, J. Ind. Microbiol.,1996; 16: 163-170.

[12] Sato K., Goda Y., Sakamoto S.S.,Shibata H., Maitani T. and YamadaT., Identification of major pigmentscontaining D-amino acid units incommercial Monascus pigments, Chem.Pharm. Bull., 1997; 45: 227-229.

[13] Suchada J., Prasat K. and Busaba Y.,The fermentation of rice for ankapigment production, J. Ind. Microbiol.Biotechnol., 2000; 25: 141-146.

[14] Kurono M., Nakanish K., Shindo K.and Tada M., Biosynthesis ofmonascorubrin and monascofla, J.Chem. Pham. Bull., 1963; 11: 359-362.

[15] Blanc P.J., Loret M.O. and Goma G.,Production of citrinin by variousspecies of Monascus, Biotechnol. Lett.,1995; 17: 291-294.

[16] Ciegler A., Production of patulin andcitrinin from Penicillium expansum,Appl. Environ. Microbiol., 1977; 33:1004-1006.

[17] Wu M.T., Production of citrinin byPenicillium viridicatum on countrycured ham, Appl. Microbiol., 1974; 27:427-428.

[18] Franco C.M., Fente C.A. and VazquezB., A Simple and sensitive high-performance liquid chromatography-fluorescence method for thedetermination of citrinin, applicationto the analysis of fungal cultures andcheese extracts, J. Chromatogr. A.,1996; 723: 69-75.

[19] Hamdi M., Blanc P.J. and Goma G.,Effect of aerations conditions on theproduction of red pigments byMonascus purpureus growth on pricklypear juice, Process Biochem., 1996; 31:543-547.

[20] Hajjaj H., Blanc P., Groussac E.,Uribelarrea J.L., Goma G. andLoubiere P., Kinetics analysis of redpigment and citrinin production byMonascus ruber as a function of organicacid accumulation, Enz. Microb.Technol., 2000; 27: 619-625.

[21] Pastrana L., Blane P.J., Santerre A.L.,Loret M.O. and Goma G., Productionof red pigments by Monascus ruber insynthetic media with a strictlycontrolled nitrogen source, ProcessBiochem., 1995; 30: 333-341.

[22] Lin C.F., Isolation and culturalconditions of Monascus sp. forproduction of pigment in a submergedculture, J. Ferment. Technol., 1973; 51:407-414.

1042 Chiang Mai J. Sci. 2014; 41(5.1)

[23] Yoshimura M., Yamanada S., MitsugiK. and Hirose Y., Production ofMonascus pigment in a submergedculture, Agric. Biol. Chem., 1975; 39:1789-1795.

[24] Chen M.H. and Johns M.R., Effectof pH and nitrogen source on pigmentproduction by Monascus purpureus,Appl. Microbiol. Biotechnol., 1993; 40:132-138.

[25] Hu Z.Q., Zhang X.H., Wu Z.Q., QiH.S. and Wang Z.L., Perstraction ofintracellular pigments by submergedcultivation of Monascus in nonionicsurfactant micelle aqueous solution,Appl. Microbiol. Biotechnol., 2012; 94:81-89.

[26] Hu Z.Q., Zhang X.H., Wu Z.Q., QiH.S. and Wang Z.L., Export ofintracellular Monascus pigments bytwo-stage microbial fermentation innonionic surfactant micelle aqueoussolution, J. Biotechnol., 2012; 162: 202-209.

[27] Kang B.Y., Zhang X.H., Wu Z.Q.,Qi H.S. and Wang Z.L., Effect ofpH and nonionic surfactant on profileof intracellular and extracellularMonascus pigments, Process Biochem.,2013; 4: 759-767.

[28] Somboon T., Nattapat L. andYodhathai T., Azaphilone pigmentsfrom a yellow mutant of the fungusMonascus kaoliang, Phytochem., 2004;65: 2569-2575.

[29] Yongsmith B., Krairak S. andBavavoda R., Production of yellowpigments in submerged culture of amutant of Monascus spp, J. FermentBioeng., 1994; 78: 223-228.

[30] Zhou B., Wang J.F., Pu Y.W., ZhuM.J., Liu S.M. and Liang S.Z.,Optimization of culture medium foryellow pigments production with

Monascus anka mutant using responsesurface methodology, Eur. Food Res.Technol., 2009; 6: 895-901.

[31] Flores M.E. and Sanchez S., Nitrogenregulation of erythromycin formationin Streptomyces erythreus, FEMSMicrobiol. Lett., 1985; 26: 191-194.

[32] Omura S., Tanaka Y., Kitao C.,Tanaka H. and Iwai Y., Stimulationof leucomycin production bymagnesium phosphate and itsrelevance to nitrogen cataboliteregulation, Antimicrob. AgentsChemother., 1980; 18: 691-695.

[33] Yongsmith B., Tabloka W.,Yongmanitchai W. and Bavavoda R.,Culture conditions for yellowpigment formation by Monascus sp.KBIO grown on cassava medium,World J. Microbiol. Biotechnol., 1993;9: 85-90.

[34] Shen Y.Q., Heim J., Solomon N.A.,Wolfe S. and Demain A.L.,Repression of β-lactam production inCephalosporium acremonium bynitrogen sources, J. Antibiot., 1984; 37:503-511.

[35] Castro J.M., Liras P., Cortes J.and Martin J.F., Regulation ofα-aminoadipylcyste~nyl-valine,isopenicillin N synthetase, isopenicillinisomerase and deacetoxycephalosporinC synthetase by nitrogen sources inStreptomyces lactamdurans, Appl.Microbiol. Biotechnol., 1985; 22: 32-40.

[36] Omura S., Tanaka Y., Mamada H. andMasuma R., Effect of ammonium ion,inorganic phosphate and amino acidson the biosynthesis of protylonolide,a precursor of tylosin aglycone, J.Antibiot., 1984; 37: 494-502.

[37] Seviour R.J. and Kristiansen B., Effectof ammonium ion concentrationon polysaccharide production by

Chiang Mai J. Sci. 2014; 41(5.1) 1043

Aureobasidium pullulans in batchculture, Eur. J. Appl. Microbiol.Biotechnol., 1983; 17: 178-181.

[38] Omura S., Taki A., Matsuda K. andTanaka Y., Ammonium ions suppressthe amino acid metabolism involved inth biosynthesis of protylonolide in amutant of S. fradiae, J. Antibiot., 1994;37: 1362-1369.

[39] Lee S.H. and Lee K.J., Aspartateaminotransferase and tylosinbiosynthesis in Streptomyces fradiae,App. Environ. Microbiol., 1993; 59:822-827.

[40] Pisareva E., Savov V. and KujumdzievaA., Pigments and citrinin biosynthesisby fungi belonging to genus Monascus,J. Biosci., 2005; 60: 116-120.

[41] Wang Y.Z., Ju X.L. and Zhou Y.G.,The variability of citrinin productionin Monascus type cultures, FoodMicrobiol., 2005; 22: 145-148.

[42] Sandra F.B., Orozco B. and Vahan K.,Effect of pH on citrinin and redpigments production by Monascuspurpureus CCT3802, World J. Microbiol.Biotechnol., 2008; 24: 263-268.

[43] Xu G., Chen Y. and Yu H., HPLCfluorescence method for determina-tion of citrinin from Monascuscultures, Archiv. Lebensmittelhy.,2003; 4: 82-84.