Busaba Yongsmith a Kaew Kangsadalampai b and Nissana Harntrikanok b a Department of Microbiology, Faculty of Science, Kasetsart University. Bangkok, Thailand b Department of Food and Nutritional Toxicology. Institute of Nutrition. Salaya. Nakornpathom, Thailand Value-creation on New YELLOW Pigments Produced by a Unique Mutant Derived from Monascus kaoliang KB9

Presentation Outline Genealogy of the yellow isolates Cassava/soybean flour submerged culture Rice Solid Culture Downstreaming of the yellow pigment - crude extract - purified yellow pigment - direct utilization of fermented rice powder Antimutagen / Antioxidant of the yellow products Conclusion

Genealogy of the yellow isolates

Cassava/soybean flour submerged culture

Rice Solid Culture

Downstreaming of the yellow pigment

- crude extract

- purified yellow pigment

- direct utilization of fermented rice powder

Antimutagen / Antioxidant of the yellow products

Conclusion

Monascus spp. Project Raw Materials Downstreaming Technology Production Technology Social Interest Yes/No (1983)

Think Locally Act Globally

Monascus kaoliang (KB 9) uv Red mutant (KB 10M16) uv Yellow mutant (KB 20M10.2) SMF SSF Genealogy of the yellow producing isolates

Features of the yellow isolates

10 day CYA G25N Wort agar PDA GYP SS MYS Yellow mutant

SEM GYP medium 10 day

Main Characteristics of Monascus YELLOW mutant 1.) Producing high yellow pigmentation at single wavelength ( max 370) in spite of medium pH or formula 2.) UVA absorbing pigment producer 3.) High Ext. / Int. pigmentation 4.) Ade - mutant 

Why Yellow ? @ Yellow is one of three mother colors @ World market of the colors @ Functional properties - as antimutagens - as antioxidant - etc.

Dehulled rice Soaking in tap water 3 h water draining 5-10 min autoclaved cooked rice Fermentation SSF

SSF Fermented rice grinding 1 g 39 ml of 50% EtOH 15 min supernatant measured by spectrophotometer  max 370 nm Dilution Extraction 3 h on rotary shaker EXTRACTION AND DETECTION OF MONASCUS PIGMENTS

EtOH Crude extract antimutagenic lyophilization Brown powder AMES Test Fruit Fly Test

Nitrite treated 1-Aminopyrene or Nitrite treated Fish extract Effect during nitrosation of 1-aminopyrene or fish extract Nitrosated products (direct-acting mutagen) Effect on nitrosated products Adding nitrite Mutageneticity test Antiformation Antimutagenicity Nitrite treatment Mutagenicity Monascus colorants powder

(Thayat Sriyapai,2003) Salmonella tester strainsTA98 and TA100. Inhibit direct mutagen Monascus pigment

SMART test in Drosophila melanogaster was designed to detect the induction of genotoxic damage in a rapid and inexpensive way. Somatic Mutation and Recombination Test (SMART)

> 50% surviving flies

(Dalad Siriwan, 2005)  Non-toxic Oh ! Ha!Ha!

Monascus yellow rice EtOH Extract Concentrate Spray drying Mellow yellow powder Rice solid culture Crude yellow extract / Rice powder

Product Applications

Experimental Design 10, 1, 0.1% Phenolic content 10, 1, 0.1% 10, 1% 10% Nissana Harntrikanon, 2006) SMART Antioxidant %Survival Mutagenicity Antimutagenicity Co-administration study DPPH FRAP

Samples Preparation Rice flour Arrowroot starch Sugar Water M. powder 10, 1, 0.1% (w/w) rice flour Steaming, 10 min Eggs Sugar Lemon juice Cake flour Baking powder Vanilla M. powder 10, 1, 0.1% (w/w) cake flour Baking, 180 o C, 15-20 min Storage (Dessicator)

Co-administration SMART The survival of adult files and mutagenicity of samples 1 Modulating effect of samples on mutagenicity of urethane 2

Co-administration Study 2 Wing preparation Observation at 400X mwh/mwh ORR/flare Standard medium Trans -heterozygous larvae Exp. + URE & Control medium

Mutation Index MI = Spots per wing induced by URE administrated with sample Spots per wing induced by URE MI of URE = 1 Antimutagenicity > 0.8: negligible 0.6-0.8: weak 0.4-0.6: moderate < 0.4: strong

> 0.8: negligible

0.6-0.8: weak

0.4-0.6: moderate

< 0.4: strong

Discussion No information 1 prove Inhibition of CYP-450 Induction of GST Thai desserts made from Monascus rice powder could reduce the mutagenicity of urethane. Hypothesis 1 Scavenging of urethane

1 Mutagenicity of Samples

All samples were not mutagenic 1 Mutagenicity of Samples

2 Co-administration Study All s amples were antimutagenic against urethane Strong Moderate Weak

Co-administration Study 2 All s amples were antimutagenic against urethane Strong Moderate Weak

Discussion 4 Moist heat Steaming at 100 0 C, 10 min Dry heat Baking at 180 0 C, 15-20 min Khanom Nam Dok Mai had stronger protective effect than that of Khanom Khai . Hypothesis Effect of heat treatment

Discussion 1 All types of Monascus rice powder themselves had phenolic contents and antioxidant activity. Hypothesis Antioxidative potential

Discussion 2 prove Thai desserts made from Monascus rice powder remained total phenolic content and antioxidant activity. Hypothesis Some compounds of Monascus rice powder were heat stable

Discussion Hypothesis 2 Antioxidant may reduce O 2 ˙¯, NO˙ in urethane metabolism

Antioxidant Free radical scavenging activity (DPPH assay) Ferric reducing antioxidant power (FRAP assay) Antioxidant activity 1 Total phenolic content 2

Sample 10 g Sample/100 ml 80% MeOH 3 times Filtrated Sample conc.10 mg/ml 80% MeOH Filtrated Dissolved in 10 ml 80% MeOH DPPH assay FRAP assay Total phenolic content

Results & Discussion DPPH Assay 1

FRAP Assay 1

Total Phenolic Content 2

Discussion Yellow > Red > White 3 Different total phenolic contents and antioxidant activity among the three Monascus rice powders . Hypothesis Type of pigments or some others active ingredients of each color

Conclusion SMART Antioxidant The survival of adult files and mutagenicity of samples Antimutagenicity All samples were not toxic & mutagenic All s amples were antimutagenic against URE DPPH & FRAP Total phenolic content Samples had phenolic contents and antioxidant activity

Macerated in CH 2 Cl 2 (2 days) filtered Filtrate Concentrated to dryness CH 2 Cl 2 extract (16.5 g) Fr2 Fr1 Fr4 Fr3 Fr6 Fr5 Fr7 Fr8 Fr10 Fr9 Fr11 Fr12 Sephadex LH-20 MeOH as eluent Sephadex LH-20 MeOH as eluent Sephadex LH-20 MeOH as eluent Yellow Ung kak (yellow yeast rice) 1 kg

SiCC Acetone: CH 2 Cl 2 (1:100) Fr 67810125-(2-3),bFr 67810125- (4-5),…, Fr 67810125 (18), Fr 67810125 (19-24) Ung03 Ung02 Ung04 Ung01 Scheme2 Extraction of CH 2 Cl 2 extract of M. kaoliang KB20M10.2 grown on rice

Possible biosynthetic pathway of azaphilone pigments 1–4 by the fungus M. kaoliang .

Phytochemistry 65 (2004) 2569-2575

K.U. Ung kak C.U. novel bioactive -C 13 H 18 O 5 , C 17 H 18 O 5 M.U. antimutagens BIOTECH TRF Integration R & D on Monascus Project Food/Drink application Non-Food application KURDI

Our experiences showed that Monascus yellow rice…..not just a colorant Monascus KB 20M10.2 is a specific strain, pH independent to produce yellow pigments at single peak of 370 nm under SSF or SMF. Rice solid culture of this strain could give not only yellow pigment product but also a potential antimutagenic yellowish extract expressed by AMES or fruit fly tests. Crude ethanol yellow extract as well as its key monacusone A showed strong antioxidant analyzed by FRAP or DPPH assay. CONCLUSIONS

Our experiences showed that Monascus yellow rice…..not just a colorant

Monascus KB 20M10.2 is a specific strain, pH

independent to produce yellow pigments at single peak

of 370 nm under SSF or SMF.

Rice solid culture of this strain could give not only yellow

pigment product but also a potential antimutagenic

yellowish extract expressed by AMES or fruit fly tests.

Crude ethanol yellow extract as well as its key

monacusone A showed strong antioxidant analyzed by

FRAP or DPPH assay.

SAWADEE KA