Food Borne Pathogen Inhibition by Citrinin from Monascus purpureus

Kanokwan Sroykesorn1, Arpa Wanleeluk2 and Sasithorn Kongruang3 Department of Biotechnology, Faculty of Applied Science,

King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand 1518 Piboonsongkram Road, Bangsue, Bangkok, Thailand 10800

e-mail : pae_amon@hotmail.com1 , candy_syndrome@msn.com2 , stk@kmutnb.ac.th3

Abstract—Citrinin is one of the secondary metabolites of the Monascus speices and exhibits a wide range of activities in the biological systems such as antimicrobial, phytoxic, cytotoxic and enzyme inhibitory effects. The citrinin contents in the extracellular metabolic mixtures from 3 generations (G1-G3) from ultrasonic induced treatment of M. purpureus TISTR 3541 were quantified by HPLC technique and studied for an antibacterial activity toward food-borne pathogen. Results showed that the citrinin concentrations in G2 and G3 were 12.72 and 45.11 µg/ml. The 50 µl of 1 mg/ml pure citrinin inhibited Bacillus subtilis, Pseudomonas aeroginosa, Salmonella typhimurium and Staphylococcus aureus more than that of at 30 and 40 µl. For the fungal citrinin extract, the concentration of citrinin at 4 mg/ml was the best antibacterial activity when compared with 1 and 2 mg/ml pure citrinin. The strain of Gram (+) bacteria belonging to Bacillus subtilis has been more sensitive to the action of the extract containing Monascus citrinin whereas the Pseudomonas aeroginosa, gram (-) strain has been less influenced by the citrinin presence. For G3, results revealed that both pure and extracted citrinin inhibited the gram-positive pathogens better than those of the gram negative.

Keywords-Food Pathogen, Citrinin, Monascus purpureus, Inhibition zone

I. INTRODUCTION Citrinin (C13H14O5) is a toxic secondary metabolite of the

Monascus species which has a negative impact on the acceptability of red mold rice product. This substance causes nephrotoxic in mammalian system which it act as the mycotoxin found in many fungi for examples, Penicillium citrinum, Aspergillus and Fusarium. This metabolite exhibits a wide range of activities in diverse biological systems, including antimicrobial, phytotoxic, cytotoxic, hypochloresterolemic, and enzyme inhibitory effects [1]. Thus, there are strong economic and safety demands for suppressing citrinin production in commercially useful Monascus strains. The Monascus species actually produced other benefit substances for food additives such as a group of pigments (yellow pigment, ankaflavin and monascin; orange pigment, monascorubrin and rubropunctanin; and red pigment, monascorubramine and rubropuctamine) [2], a group of antihypercholesterolemic agents including monacolin K and a hypotensive agent, γ-aminobutyric acid (GABA) [3] , antioxidant compounds including dimerumic

acid [4] and 3-hydoxy-4-methoxy-benzoic acid [5]. Attempts to obtain the mutant strains with low citrinin production have been investigated from the modification of Monascus culturing conditions, induce mutation to gene insertion [6,7,8]

As focusing in the molecular level, this microtoxin belongs to the group of polyketides synthesized by the iterative type I polyketide synthase (PKS). The biosynthesis of citrinin in Monascus originates from a tetraketide arising from the condensation of one acetyl-CoA molecule with three malonyl-CoA molecules [9]. Its biosynthesis gene cluster has revealed that citrinin is synthesized by polyketide synthase in M. purpureus BCRC 33325 [10]. Further report of identification and in vivo functional analysis by gene disruption of ctnA, an activator gene involved in citrinin was found in M. purpureus and M. kaoliang, but was absent in M.sanguineus [11]. The serious consequences of citrinin contamination create a significant impact on the heath of man and animal; however, the benefit side of using a citrinin as the antibacterial substance is getting a future application in pharmaceutical application. Therefore, we investigated the antibacterial activity of citrinin extracts from M.purpureus TISTR 3541 toward food pathogenic bacteria. Comparison of the effectiveness of citrinin extracts from ultrasonic induced mutation in each generation of M. purpureus with the pure citrinin isolated from Penicillium citrinum was also reported.

II. MATERIAL AND METHODS

A. Chemicals Citrinin was purchased from Sigma Chemical Co. (St.

Louis, MO). LC grade acetonitrite was purchased from Merck Co (Darmstadt, Germany). Potato Dextrose Agar, yeast extract and malt extract were purchased from Himedia Laboratories (PVt., Ltd, India).

B. Organism used M.purpureus TISTR 3541 which was induced by

ultrasonic treatment for higher pigment productivity was used for the study. The mutant strains (G1-G3) were cultivated in tubes on potato-dextrose-agar at 30 0C for a week before used as producer organism. Bacillus subtilis TISTR 001, Pseudomonas auroginosa TISTR 781, Escherichai coli TISTR 780, Salmonella typhimurium TISTR 292 and Staphylococcus aureus TISTR 517 were purchased from Thailand Institute of Scientific and

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2011 International Conference on Bioscience, Biochemistry and Bioinformatics IPCBEE vol.5 (2011) © (2011) IACSIT Press, Singapore

Technological Research (Bangkok,Thailand) and used as test bacteria maintained in nutrient agar medium.

C. Medium, Inoculation and Condition for Pigment Production The spore suspensions at 106 spores/ml of 0.1% tween 80

were used as the inoculum after obtained from the stock culture of M.purpureus TISTR 3541 grown on PDA at 28 0C for 7 days by using cork borer. The seed cultures were transferred to a Modified YM broth (MYB) consisting of 5% peptone, 3% yeast extract powder, 3% monosodium glutamate, 10% cassava starch in distilled water. Media pH was adjusted to 4.5 prior to sterilization. Cultivations were carried out in 100 ml of sterilized modified YM on a reciprocal shaking incubator for 7 days at 28 0C and 200 rpm. The fermentation broths were then filtrated with the filter paper (Whatman no.1) through vacuum pump. The mycelium was then collected for the ultrasonic treatment.

D. Radial growth determination Radial growth of the strains was measured by using one

point inoculation of spore suspension at the center of PDA (Difco, Detroit, Mich., USA) on plates in triplicate. During incubation at 28 0C, the cultures were measured the radius of the colonies at interval for 12 days. Three perpendicular axes were measured and the average values were reported.

E. Ultrasonic treatment The filtrate were then suspended in 7% saline before

treated by ultrasonic wave at a frequency of 45 kHz, a power of 200W for 2 min, then was diluted 10 times. 0.1mL of mycelia suspension was culture by one point inoculation on the plate at 28 0C for 7 days. The growth mycelium was called generation 1 (G1). This 7 days culture was then transfer to MYB and cultivated in the same condition as previously described. The mycelium of G1 was then filtrated and resuspended in 7% saline before subjected to ultrasonic treatment. The experiment was repeated in the same manner until generation 3 (G3).

F. Preparation of mold extract and detection of citrinin The filtrate samples from wild type, G1,G2 and G3 were

centrifuged at 10000g for 10 min at room temperature. The upper layer was passed through a 0.20 µm filter and measured by an HPLC (Waters chromatography division. Milford, MA, USA) .A C18 column, 25cm x 4.6 mm, i.d.5µm was used as the analytical column. Injector volume: 40 µl was used. The mobile phase contained acetonitrile 55%, nano water 45 %and trifluoroacetate 0.1%. The flow rate was set at 1 ml/min and citrinin was detected using a UV detector set at 238 nm. Pure citrinin (Sigma Co.,Ltd) was used to construct the standard curve. Chromatographic profiles and the linear regression of the standard curve were reported [12].

G. Determination of Antimicrobial Activity by Disc Diffusion Assay Pure citrinin (Sigma Co., Ltd) at 1 mg/ml was used to test

the pathogenic inhibition. Samples of citrinin extract from

each generation were prepared to the final tested concentration at 1, 2 and 4 mg/ml using the spectrophotometer at A 280 nm. Paper discs impregnated with solution with different volumes of extracts from 20 µl were prepared with the solvents as standard for comparison. Antibacterial activity was determined by measurement of inhibition zone around each paper disc. The test samples were then incubated at 30°C for 24 h. All experiments were carried out in triplicate.

III. RESULT AND DISCUSSION

A. Morphological characterization of M.purpureus strain On potato-dextrose-agar medium in the culture plates of

M.purpureus of both wild type and mutants (G1, G2 and G3) formed fluffy, yellow colored with an orange spot in the middle of colony during the 8 days of cultivation. The middle part of all colonies turn to red as mycelia became matured during 10-12 days and then all mycelium turned to reddish after 12 days of cultivation as illustrated in Table1.The average growth rate of wild type was highest followed by G3, G1 and G2 as shown in Table 2. Wild type showed the highest rate of growth, 0.9563, over the ultrasonic mutant of G1 G2 and G3 with the ratios of 1.19 , 1.31 and 1.06, respectively (Table2).Our results showed that all M.purpureus TISTR 3541 and ultrasonic induce multant produced yellowish red and red.;however,it is known that M. aurantiacus, M. pilosus, M.purpureus, M. ruber and M. sanguineus produce red orange soluble pigments, but M. floridanus, M. lunisporasand M. pallens are non-red or orange pigment producing species.[13,14]

B. Citrinin The secreted fermentation broths were quantitative

analysis by HPLC to determine citrinin.Chromatograms of standard and citrinin samples were previously reported [15].Results showed that the effect of citrinin concentration was strongly dependent on the ultrasonic induction. As depicted in Figure 1, the concentration of citrinin in the filtrated sample increased as the generation increasing.The significantly increase in citrinin concentration was detected as and approximately 70 times found in the G3 when it is compared with the wild type. Normally, cultured M.purpureus in the liquid fermentation produces the citrinin as growth-related product and is biosynthesized as a primary metabolite. However, under oxygen-excess condition it would produce as a secondary metabolite during the stationary phase.The mycotoxin could also be detected in 12 different commercial Monascus samples at concentrationsvarying between 0.2 and 17.1 mg/g [15]

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TABLE I. PHYSICAL CHARACTERISTICS OF MONASCUS PURPUREUS GROWTH ON POTATO DEXTROSE AGAR (PDA)

Incubation time (day)

Monascus purpureus TISTR 3541

Wild type G1 G2 G3

2

4

6

8

10

12

TABLE II. GROWTH RATE OF MONASCUS PURPUREUS TISTR 3541

M.purpureus TISTR 3541 Equation R2 Rate of growth

(cm/day)

Wild type y = 0.9563x + 0.0347 0.9561 0.9563 G1 y = 0.8069x + 0.5327 0.9760 0.8069 G2 y = 0.7314x + 0.500 0.9776 0.7314 G3 y = 0.9014x + 0.1033 0.9835 0.9014

C. Antibacterial Activity The values obtained for the inhibition zone diameters

demonstrated that the pure citrinin had an antibacterial activity toward food pathogen. The antibacterial activity increased as the amount of citrinin was increased from 30 to 50 µl as shown in Table III. For the extracts, most of fermented red Monascus of G1 presented a lower antibacterial activity than G2 and G3 , but different as

function of tested strain type. In G1 citrinin extract, the percentages of antibacterial activity were found toward B.subtilis, E.coli, P.aeroginosa, S.typhimurium and S.aureus as 30%, 33%, -25%, -29% and -29%, respectively. These inhibition results also found in G2 and G3 citrinin extracts with the percentages of 50%, 50%, -100%, 26% and 39% for G2 and 47%, 41%, 39%, -29% and 0%, respectively. The strain of Gram (+) bacteria belonging to B.subtilis has been more sensitive to the action of the extract containing Monascus citrinin whereas the P.aeroginosa, gram (-) strain has been less influenced by the citrinin presence.The results from this experiment was similar to the report presented the study of the antimicrobial activity of red yeast rice obtained from a M.purpureus strain[16].They showed that The two strains of Gram (+) bacteria belonging to Bacillus genus has been more sensitive to the action of the extract containing Monascus pigments whereas the Pseudomonas Gram (-) strains has been less influenced by the extract presence.Previous studies showed that the antibacterial substance as citrinin was produced by Monascus species has a strong bacterial effect against gram-positive bacteria [17]. Our results are in agreement with the investigation of the M. purpureus NTU 601 strain was proven to produce citrinin in earlier study [18] which postulated that the antibacterial effect of citrinin on B. subtilis was related to the size of the inhibition zone.

Although the detailed molecular mechanism of the toxicity of citrinin is not well documented, it has been identified and reported that citrinin mainly effects on mitochondria in cells. This substance permeated into the mitochondria , alters Ca2+ homeostasis [19] , and interfered the electron transport system. All this mechanism would have negative effects resulting in the reduction of bacterial cell or leading to lethal death of the cell.This study pointed out that ultrasonic induced mutation method may be used to produce a strain that has higher citrinin product. If citrinin could be increased in red Monascus strains although in most of the food application to use the secreted pigment from Monascus strains has a strongly requirement to a minimum amount of citrinin, it may become an alternative important medicinal source to human being.

0

20

40

60

80

100

120

140

160

G0 G1 G2 G3

Citr

inin

con

cent

ratio

n (μ

g/m

l)

Generation

Figure 1. Citrinin concentration in Monascus purpureus TISTR 3541 of Wild type and mutants (G1-G3)

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TABLE III. SAMPLES OF INHIBITION ZONE CROSSING BY CITRININ UNDER 2 DAYS INCUBATION AT 37OC

Microorganism Nutrient agar plate

Bacillus subtilis TISTR 001

Escherichia coli TISTR780

Pseudomonas aeroginosa TISTR 781

Salmonella typhimurium TISTR 292

Staphylococcus aureus

TISTR517

TABLE IV. TANTIBACTERIAL ACTIVITY OF THE PURE CITRININ

Microorganism Amount of citrinin (µl)

Diameter of Inhibition zone(mm)

Bacillus subtilis TISTR 001

30 2.0±0.0

40 3.1±0.2

50 3.8±0.5

Escherichia coli TISTR780

30 1.0±0.0

40 1.7±0.6

50 1.7±0.6

Pseudomonas aeroginosa TISTR 781

30 2.0±0.0

40 3.1±0.2

50 3.8±0.5

TABLE V. ANTIBACTERIAL ACTIVITY OF THE PURE CITRININ (CONT.)

Microorganism Amount of citrinin (µl)

Diameter of Inhibition zone(mm)

Salmonella typhimurium TISTR 292

30 1.4±0.2 40 1.8±0.7 50 2.0±0.3

Staphylococcus aureusTISTR517

30 1.7±0.0

40 2.0±0.3

50 3.4±0.2

TABLE VI. ANTIBACTERIAL ACTIVITY OF THE MYCOTOXIN CITRININ DETECTED FROM MONASCUS EXTRACTS

Microorganism Generation Concentration (mg/ml)

Inhibition zone

diameter

in mm

Bacillus subtilis TISTR 001

G1

1 2.3 ± 0.6

2 2.9 ±0.4

4 2.6± 0.7

G2

1 3.7 ± 0.3

2 4.9 ±0.7

4 4.0± 0.9

G3

1 2.6 ± 0.2

2 1.9 ±0.5

4 3.8± 0.4

Eschericia coli TISTR 780

G1

1 1.5 ± 0.5

2 ND

4 1.3± 0.3

G2

1 2.0 ± 0.7

2 4.7 ±0.6

4 1.0± 0.0

G3 1 1.7 ± 0.3

2 5.0 ±0.1

4 1.0± 0.0

Pseudomonas aeroginosa TISTR

781

G1

1 1.5 ± 0.5

2 ND

4 1.0± 0.0

G2

1 ND

2 1.2 ±0.3

4 3.7± 1.2

G3 1 3.3 ± 0.6

2 ND 4 ND

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TABLE VII. ANTIBACTERIAL ACTIVITY OF THE MYCOTOXIN CITRININ DETECTED FROM MONASCUS EXTRACTS (CONT.)

Microorganism Generation Concentration (mg/ml)

Inhibition zone

diameter

in mm

Staphylococcus aureus TISTR 517

G1

1 1.2 ± 0.3

2 1.7 ±0.3

4 1.2± 0.3

G2

1 2.3 ± 0.0

2 1.3 ±0.3

4 1.7± 0.3

G3

1 1.0 ± 0.0

2 1.4 ±0.4

4 1.3± 0.3

Salmonella typhimurium TISTR

292

G1

1 1.0 ± 0.0

2 2.0 ± 1.7

4 1.7± 0.6

G2

1 2.3 ± 0.3

2 1.5 ±0.5

4 1.4± 0.2

G3

1 1.4 ± 0.2

2 3.8 ±1.9

4 3.7± 0.6

*ND = not detectable

ACKNOWLEDGMENT The author is grateful for financial support provided by

King Mongkut’s University of Technology North Bangkok, Thailand through the research grant. The author also wishes to express appreciation to Dr.Sittiruk Roytrakul from BIOTEC Central Research Laboratory, Thailand Science Park for his experiment assistance.

REFERENCES [1] H. Hajjaj, A. Klaebe, M.O. Loret, G. .; Goma, P.J. Blanc, and

J.Francois.”Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance”. Appl. EnViron. Microbiol.vol 65,1999, pp.311–314.

[2] H.C. Wong, and P.E. Koehler, “Production and isolation of an antibiotic from Monascus purpureus and its relationship to pigment production”. J. Food Sci. vol 46,1981, pp. 589-592.

[3] Y. Aniya , I.I. Ohtani, T. Higa, C. Miyagi, and H. Gibo, “Shimabukuro, M.; Nakanish, H.; Taira, J. Dimerumic acid as an antioxidant of the mold, Monascus anka”. Free Radical Biol. Med. Vol 286,1999, pp. 999-1004.

[4] G.F. Wu, and X.C. Wu, “Screening DPPH radical scavengers from Monascus sp”. Acta Microbiol. Sin. Vol 40,2000, pp.394-399.

[5] J-J Wang, C-L Lee, and T-M Pan, “Modified Mutation Method for Screening Low Citrinin-Producing Strains of Monascus purpureus on Rice Culture”. J. Agric. Food Chem. Vol 52,2004, pp. 6977-6982.

[6] S.F. Orozco, and B.V. Kilikian, “Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802”. World J. Microbiol. Biotechnol. Vol 24,2008, pp. 263–268.

[7] M-J Xu, Z-L Yang, Z-Z Liang, and S-N Zhou, “Construction of a Monascus purpureus Mutant Showing Lower Citrinin and Higher Pigment Production by Replacement of ctnA with pks1 without Using Vector and Resistance Gene”.J Agric Food Chem. Vol 20,2009,pp. 9764-9768.

[8] K. Suktham, O. Suwansungsa, B. Wonganu, S. Kongruang, and S. Roytrakul “Identification of Citrinin Biosynthesis Gene under the Ultrasonic Induction of Monascus purpureus”.2010 International Conference on Chemistry and Chemical Engineering (ICCCE2010). 2010,pp.35-39.

[9] T. Shimizu, H. Kinoshita, S. Ishihara, K. Sakai, S. Nagai, and T. Nihira, “Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus.” Appl. EnViron. Microbiol. vol 71,2005, pp. 3453–3457.

[10] T. Shimizu, H. Kinoshita , and T. Nihira, “Identification and in vivo functional analysis by gene disruption of ctnA, an activator gene involved in citrinin biosynthesis in Monascus purpureus”. Appl. EnViron. Microbiol. Vol 73,2007, pp. 5097–5103.

[11] C.L. Lee, J.J. Wang, and T.M. Pan “Synchronous analysis method for detection of citrinin and the lactone and acid forms of monacolin K in red mold rice”. JAOAC Int .vol 89,2006,pp.669-667.

[12] GM. Chagas , MBN. Oliveira,AP. Campello ,and MLW. Kluppel. “Mechanism of citrinin-induced dysfunction of mitohondria”.4. Effect on Ca2+ transport. Cell Biochem Funct.vol 13,1995,pp.53-59

[13] Sabater-Vilar, M., Maas, R.F.M., Fink-Gremmels, J., 1999. Mutagenicityof commercial Monascus fermentation products and therole of citrinin contamination. Mutat. Res. 444, 7–16.

[14] Udagawa, S., Baba, H., 1998. Monascus lunisporas, a new species isolated from mouldy feeds. Cryptogamie Mycologie. 19,269–276. [15] U. Camelia ,and F. Mariana, “Antibacterial and antifungal activity of

red rice obtained from Monascus purpureus”.IBIC 2010-Industrial Biotechnology 2 nd International Conference /webpapers/ 70Ungureanu.pdf/ 30-Mar-2010

[16] M-J Xu, Z-L Yang, Z-Z Liang, and S-N Zhou, “Construction of a Monascus purpureus Mutant Showing Lower Citrinin and Higher Pigment Production by Replacement of ctnA with pks1 without Using Vector and Resistance Gene”.J Agric Food Chem. Vol 20,2009,pp. 9764-9768.

[17] Y.C. Su, J.J. Wang, T.T. Lin, and T.M. Pan, “Production of the secondary metabolites ç-aminobutyric acid and monacolin K by Monascus”. J. Ind. Microbiol. Biotechnol. Vol 30,2003, pp. 40-46.

[18] SMR. Ribeiro , GM. Chagas , AP. Campello , and MLW. Kluppel. “Mechanism of citrinin-induced dysfunction of mitohondria”.5 Effect on the homeostasis of the reactive oxygen species.Cell Biochem Funct.vol 15 , 1997 , pp.203-209

[19] Li, Z., 1982. A new species of the genus Monascus. Acta Microbiol.Sin. 22, 118–122.

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