International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 1 Physiological Studies on the Monascus ruber Red Pigment and GC/MS Analysis Eman Mostafa Mohamed Botany and Microbiology Department, Faculty of Science, Assiut University, PO Box 71516, Assiut, EGYPTAvailable online at: www.isca.in, www.isca.me Received 21th July 2015, revised 7th August 2015, accepted 16th September 2015 Abstract Physiological studies and effect of mono-sodium-glutamate (Na-G) and agitation on the two M. ruber strains are studied. The GC/MS analysis recorded that the different effects of Na-G and agitation on the red pigment, spectrophotometeric colour intensity, number and kinds of the antioxidant, flavouring metabolites. Visual estimation of the soluble pigment by tested M. ruber strains AUMC 4066 and 5705 were cultivated on four kinds of media including glucose, malt, malt+Na–glutamate and glucose+Na–glutamate, incubated statically and submerged at 120 rpm for 10 days at 30°C are studied. Also the colour intensity estimated at A 500nm by spectrophotometer are studied. The results clearing that the deepest red colour of the soluble pigment are recorded in malt, and malt+Na–glutamate of the tested of M. ruber strain AUMC 4066. The media fortified by glutamate are inhibited the red soluble colours and gives the red or brown-red colour. The highest colour intensity of the soluble pigment: are detected in submerged malt and malt+Na–glutamate of M. ruber 4066 are 3.528 and 3.477. Also all the reading clearing that the strain No.4066 have the highest colour intensity on the all tested media. Numbers and kinds of the metabolites detected in the ethanolic extracts of the selected strain AUMC 4066 has been studied by using GC/MS analysis. 140 flavouring and antioxidant metabolites are detected in the extracts of the four tested culture media incubated at 120rpm, 30°C for ten days. Malt medium recorded the highest numbers of metabolites followed by Malt+Na-glutamate, glucose+Na-glutamate and glucose media are 76, 25, 20 and 11, respectively. The highest numbers of the classified recorded metabolic products includes 48 phenolic compounds, 38 esters, 23 alkanes and alkenes, 10 ketons, 5 indolic compounds, 4 from (azole and terpenoids), 2 for each of (amides and amines), one for each of (aldehyde, free fatty acids, pyran and cyclo-arabitol) are detected at 1 mass fractions in the ethanolic extracts of the tested medium. Keywords: Monascus ruber strains, spectrophotometer, GC/MS, antioxidants, flavouring metabolites, Na-Glutamate, agitations. Introduction Monascus is a commercial and applied fungus used in different places around the world specially the Asian countries.Monascus is highly red pigmented ascomycetous filamentous fungusable to produced three colouring groups "red, yellow and orange" colours. Monascus pigment has many 2ry metabolites for protective their cells against lethal (photo-oxidations, UV radiation, extremes heat and cold stress and other microbes), source of nutrients, such as iron and source of energy. It also acting as cofactors in enzyme catalysis, sexual and a sexual spores productions for completed the fungal developments and natural life cycle are helpful the fungal development and act as virulence factors for enhance the survivability of spores2,3. Monascus natural pigments act as a source of wide available and diverse metabolites which using as the food colorants. It have high ranks in annual consumption and sales around the world. Their globule production increased and was valued at $12 million4,5Monascuspigments are natural products have many advantages and ideal characters referred for cocustomer due to it producing from fungi including independence from weather conditions, wonderful colors with different shades, produced by simple and non expensive methods by fermentation on the grains, inexpensive substrates "specially agro-industrial residues", through few days (7-12 days), easily extracted, high (safety, stability, solubility in water and alcohol. It also have high "nutritional, flavoring or aromatic, bioactive or medicinal" values without any bad impact on human health and environment. Since human civilization Monascus pigment is appreciated for flavor and aroma and is widely accepted as flavoring material in many industrial applications around the world includes food additive, coloring agents in "foodstuffs, tissue texture, leathers, cosmetics, perfume and soups" for centuries, recently it used in "pharmacology and medicine", preservative and agricultural importance. Monascus pigment have high medicinal value due to the presences of numerous bioactive metabolites, they have beneficial health effects without any negative side-effects. It also possess antioxidant metabolites which used in prevention of many dangerous disease such asanticancer, immunomodulatory, hepatoprotective, antidiabites, antimicrobial, anti inflammatory, anticholesterol, anti-cardiovascular diseases agents, indigestion, Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 2 muscle bruises, dysentery, anthrax, immune enhancer and support human health agents1,6. We designed this investigation for study the red pigment production by two M. ruber strains and compeer between them. Also investigate the effect of Na-glutamate, agitations on the capability of red pigment production on four kinds of media [malt, glucose, malt+sodium-glutamate and glucose+sodium-glutamate]. Also study, compeer between the four tested media, determine and select the best media based on the soluble pigment and colour intensity estimation. Also select the best and highest pigment producing strain for further investigation. By using the GC/Ms analysis known the different kinds of the 2ry metabolites found in the ethanolic extracts of the M. ruber AUMC 4066 selected strain.Known the common and IUPAC name, chemical "structural, formula and groups" of the each recorded flavouring metabolites. Material and MethodsSource of the Used Materials: Ethanol HLPC grade and mono-sodium-glutamate are obtained from Sigma Aldrich International Company. Fungal Tested Strainsand Inoculums Preparation: According to methods described by de Carvalho J.C. et. al. and Moharram et al.Fermentation Media and Cultivation: Moharram et al.,were recorded that the malt medium gave the pest results for pigment and highest number of detected metabolites. Many literatures also recommended the using glucose medium supplemented by monosodium glutamate for enhancement the red pigment and their bioactive metabolites from Monascus strains4,5. According to the results in the literature we selected the tested medium in this investigation for present study. Malt Yeast Extract Medium: Semi-synthetic fermentation medium contained (malt extract 5, yeast extract 3, glucose 5) g/ L distilled water8,9Malt Yeast Extract Supplemented by Mono-Sodium-Glutamate Medium: Contained g/L (malt extract 5, yeast extract 3, glucose 5 and 5 monosodium glutamate) g/ L distilled water. Glucose Mono-Sodium Glutamate Medium: Contained g/L (20 glucose, 5 Na-glutamate, 5 KHPO, 5 KHPO, 0.1 CaCl, 0.5 MgSO.7HO, 0.01 FeSO.7HO, 0.01 ZnSO.7HO and 0.03 MnSO4.O) and distilled water 1L at pH=6.5. 100 mL aliquots from each medium type were dispensed each in 500 mLErlenmeyer flask and sterilized at 121°C for 20 min, each flask was inoculated with 2 mL of Monascus spore suspension and incubated at 30°C for 10 days statically and in fermentor shaker 120 rpm as a submerged and solid state fermentation1,4,5,8-10. Extraction of the Red Pigment: Monascus ruber cultures were extracted with 95 % ethanol (by volume), 50 mL/flask, while agitating at 10 000 rpm for 24 h. The mixture was filtered through Whatman filter paper No. 2 and dried over anhydrous NaSO1,5,8. Spectrophotometric Analysis: Colour intensity of the ethanolic extracts of red pigments of the four tested medium were estimated by spectrophotometer. The absorbance of the ethanolic extract was measured against pure solvent (ethanol) as a blank at 500 nm, near the absorbance peak of red pigments using spectrophotometer (Spectronic 2000 colourimeter, Bausch and Lomb) according to researchers1,7,8 Gas Chromatography/Mass Spectrometric (GC/MS) Analysis: Flavouring metabolites in ethanolic extracts of crude red pigment are analyzed by GC/MS analysis.Ethanolic extract of the cultures of the four tested medium cultivated by two tested M. ruber strains were analyzed by GC/MS analysis. Result and Discussion Table-1 and figure-1 are clearly shows that the M. ruber strain AUMC5705 had a red to brown colour soluble pigment and recorded the lowest spoctrophotometric reading of the colour intensity are 0.244, 0.382, 0.801 and 1.717 were recorded on glucose and malt (fortified by Na-glutamate), malt by strain No. 4066 and malt by AUMC5705 strain, respectively. The colour intensity are increased when incubated under submerged than static condition and the deep red colour were recorded 3.528 and 3.477 on malt and malt fortified by Na-glutamate by strain, AUMC 4066 which selected for further investigation. In this respect, the optimization of the environmental and nutritional growth conditions and maximization of the red pigment yield by different Monascus species are studied by many Authors hose found the following:Lee et al,18 recorded that the pigment good yield when pH 6.5, agitation 700 rpm, using 30 g/L of glucose, 1.5 g/L of monosodium glutamate, Fe2+ and Mn2+ trace elements are the strongest stimulates. Monascus pigments can be obtained by solid-state and submerged fermentations. However, the solid-state fermentation is a preferable technique than submerged because it is simple, requires less capital investment, features lower levels of catabolic repression and end-product inhibition, produces lower amount of waste water output, allows for better product recovery, and yield a high-quality product. Rasheva et al.,26recorded that maximum pigment production was obtained on Malt Agar (MEA) plates supplemented with ammonium nitrate. The presence of the glucose with the Na-glutamate inhibited red colour pigment production by the two tested strains. Chatterjee et al.,11 recorded that the M. purpureus, maximum pigment yield formed on 20g/L glucose and 0.3% MSG at 6 pH, 30°C. Research Journal of Biological Sciences _ _____________________ Vol. 4(10), 1-14, October (2015) International Science Congress Association All the following tables are clearing that the 4066 cultivated on the four tested media (Glucose, Malt, Glucose + Na- glutamate and Malt + Na incubated as submerged cul tures at 30°C for 10 days. The ethanolic extracts of each tested media are analyzed by GC/MC Visual estimation of the colour of the soluble pigment of the two tested strains of cultivated on four kinds 0f media including glucose, malt, malt + Na statically and submerged at 120 rpm for 10 days at 30°C and the colour intensity estimated by spectrophotometer at Media Colour Malt deep red Malt + Na-glutamate deep red Glucose+ Na-glutamate deep red Malt + Na-glutamate deep brown Glucose + Na-glutamate deep brown Glucose+ Na-glutamate deep brown Malt red to brown Malt Red Malt +Na-glutamate Brown Glucose+Na-glutamate Brown Colour intensity at A 500nm of the soluble pigment of the two tested strains of M. ruber AUMC (4066 and 5705) cultivated _____________________ _________________________ ____________ International Science Congress Association All the following tables are clearing that the M. ruber AUAC 4066 cultivated on the four tested media (Glucose, Malt, glutamate and Malt + Na -glutamate) and tures at 30°C for 10 days. The ethanolic extracts of each tested media are analyzed by GC/MC analysis. The ethanolic extracts of M. ruber AUMC 4066 strain cultivated on the malt static cultures, submerged [malt, Na (malt or glucose)] at 30°C for ten days t 11. Table-1 Visual estimation of the colour of the soluble pigment of the two tested strains of M. ruber AUMC (4066 and 5705) cultivated on four kinds 0f media including glucose, malt, malt + Na – glutamate and glucose+ Na statically and submerged at 120 rpm for 10 days at 30°C and the colour intensity estimated by spectrophotometer at 500nmStrain No. Colour intensity at A500nm 4066 3.528 4066 3.477 4066 1.601 deep brown 4066 1.442 deep brown 4066 1.401 deep brown 5705 1.400 red to brown 5705 0.936 4066 0.801 5705 0.382 5705 0.244 Figure-1 Colour intensity at A 500nm of the soluble pigment of the two tested strains of M. ruber AUMC (4066 and 5705) cultivated ____________ ISSN 2278-3202 Int. Res. J. Biological Sci. 3 AUMC 4066 strain cultivated submerged [malt, Na -glutamate+ ten days t able 1-7 and figures 2- AUMC (4066 and 5705) glutamate and glucose+ Na –glutamate, incubated statically and submerged at 120 rpm for 10 days at 30°C and the colour intensity estimated by spectrophotometer at A Incubated as Submerged culture Submerged culture Submerged culture Static culture Static culture Static culture Static culture Static culture Submerged culture Submerged culture Colour intensity at A 500nm of the soluble pigment of the two tested strains of M. ruber AUMC (4066 and 5705) cultivated Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 4 on four kinds of media and incubated static and submerged culturesTable-2 The metabolites recorded in the both glucose and malt mediaMass fractions Detected Metabolites Glucose Malt 93 94 hexadecanoic acid, ethyl ester or palmtic acid, ethyl ester 87 90 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester 87 88 1,2-benzenedicarboxylic acid, diisooecyl ester 83 87 1,2-benzenedicarboxylic acid, dioctyl ester 62 68 2-butenedioic acid (E)-,bis(2-ethylhexyl) ester 50 59 phenacyl-thiocyanate,2-oxo-2-phenylethyl-thiocyanate 25 26 2-ethylacridinePh Table-3 One metabolite was recorded in the malt and malt+ Na-glutamate mediaDetected Metabolites acetamide -2-(2,5-dioxo-pyrroidin-1-yl)-N-(5-propyl[1,3,4]thiadiazol-2-yl) Az Table-4 Detected metabolites in the ethanolic extracts of the glucose tested mediumMF Detected Metabolites 90 dodecanoic acid, ethyl ester 52 benzeneacetic acid,-oxo-,methyl ester 52 3,5(2H,4H)-dione,6-benzoylthio 1,2,4-triazinePh 35 3,4-dimethyl- heptaneAlka 25 3-[2,2-bi(methoxycarbonyl)-2-formamidoethyl-1]-4-(4-hydroxy-3-methylbut-2-en-1-yl)-,methyl- indoleIn Gas Chromatography/Mass Spectrometric analysis was detected 140 flavoring and antioxidant metabolites are detected in the ethanolic extracts of the four tested media. Figure-7 clearing that the main nucleus of the functional groups which classified the detected metabolites into 13 different chemical groups table 2-7 and figures 2-6. The detected metabolites including seven metabolites are detected in both extract of the glucose and malt includes six esters 94-50 and one phenol 25, 26 mass fractions. All the metabolites on malt have the highest mass fractions than the metabolites on glucose. The most detected metabolites are detected by many literatures12-26. Five flavoring metabolites are detected in the ethanolic extracts of the glucose tested medium including 2 esters, one of each phenol, alkaline, 35 and indole are 90 and 52, 52, 35 and 25 mass fractions, respectively. Twenty flavoring metabolites were detected in the ethanolic extracts of the Glucose+Na-glutamate medium at mass fractions 1 including 10 phenols, 4 esters, 3 alkans, 2 ketons and one indole. Table-5 Detected metabolites in the ethanolic extracts Glucose + Na-glutamate tested mediumDetected Metabolites (E)-9-octadecaneAlka pentacosane Alka 4-ethenyl-1,4-dimethyl- cyclohexane Alka ethyl-oleate 1,2-benzenedicarboxylic acid, bis(2-methylpropyl)ester 4,5-bis-dimethoxymethyl-octanedioic acid, dimethyl ester decanoic acid,5-ethyl-3,5,5,9-trimethyl-,methyl ester 2-carboxlic acid,6-(4-ethoxyphenyl)-3-methyl-4-oxo-4,5,6,7-tetrhydro-isopropyl-ester-1-H-indole Ind 4-(2-methylpropyl)acetophenone 2-hydroxy-3,5,5-trimethyl-cyclohex-2-enone 2-cyano-4-methylbiphenylPh 5-(p-aminophenyl)-4-(o-tolyl)-2-thiazolamine Ph 3-methylisothiazolo[4,5-b]pyridine Ph (1-pentylheptyl)-benzene Ph 2-(acetoxymethyl)-3-(methoxycarbonyl)biphenylene Ph 4-methox-3,5-dihydroxybenzenzoic acid Ph 1,1-[oxybi(2,1-ethanediyloxy)]bis- ethylene Ph 2-(1H-imidazo[4,5-b]pyridine-2-yl)-1-(4-morpholyl)-ethanone Ph 1-[4-[4-(2-hydroxyethyl)-1-piperazinylsultonyl]phenyl]- ethanone Ph Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 5 2(2-oxo-2-phenyl-ethyl)-malononitrile Ph Table-6 Detected metabolites in the ethanolic extracts of the Malt tested mediumMF Detected Metabolites 70 1,4-dione,hexahydro-pyrrolo[1,2-a]pyrazine Ph 58 1,4-diaza-2,5-dioxobicyclo[4,3,0]nonae Alka 50 2-phenyl-4-ethylidene-2-oxazolin-5-one,(4Z)-4-ethyldiene-2-phenyl-1,3-oxazol-5(4H) -one K 38 1-ethyl-2-hexene Alka 35 9-methyl-1-undecene Alke 35 phathalic acid,hexyl-isopropyl-ester E 35 1,2-benzenedicarboxylic acid, bi(2-methylpropyl)-ester 35 methylall-trans-9-(4-hydroxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonate E 1-methyl-2-phenybenzenimidazole Az 2-(E-4,4-dicyano-3-N-methylanlino-1,3-butadien-1-yl)-4-(methox ycarbonyl)-1,2,3-triazol Az 5-(1-cyclohexenyl)-5-ethyl-1,3-diazinane-2,4,6-trione k 2,6-di-T-butyl-4-methylene-2,5cyclohexadiene-1-one k octahydro-1,2,4-methenocyclobut[cd]inden-3(1H)-one k 4,4a,9,10-tetrahydro-4a-methyl-2(3H)-phenanthernone k 3,5-dihydroxy-4,4-dimethyl- cyclohexadien1-one k 2-hydroxy-3,5,5-trimethyl-cyclohex-2-enone k 1,1-(2-methyl-1,3-propanedyl)bis-cyclohexane Alka 2,4-dimethyl- docosane Alka 5,14-dibutyl- octadecane Alka 5,5-dimethyl- cyclohexane-1,3-dione-2-alylaminomethylene Alke 1-nonadecene Alke 5-methyl-decane Alka 1,2-5,6-dipyrainocyclooctane Alka heneicosane Alka 2,6,10-trimethyl- tetradecane Alka 2-undecene Alka 4-undecene Alke cyclopentyl acetylene Alke 2-methyl- hexadecane Alka 1-H-indole,1-methyl-2-phenyl-2-methyl-7-phenylindole Ind 3-[2,2-bi(methoxycarbonyl)-2-formamidoethyl-1]-4-(4-OH-3-methylbut-2-en-1-yl)-,methyl- indoleInd 1-methyl-2-phenyl-1-H-indole Ind 7-hexadecenal Ald [1-pentene,1,3-diphenyl-1-ylmethyl)amino] Am etoloxamine Am N-(2-dimethylamino-3-oxo-1-butenyl)- acetamide Amid nonanamide Amid Detected Metabolites 2-hydrazino-4-methyl-6-methyl-thiopyrimidine Ph propiontrile,3-[(isochroman-1-methyl)amino] Ph 1,4-dione,hexahydro-3-(2-methylpropyl)- pyrrolo [1,2-a]pyrazine Ph 1,2-dicarboxylic acid 2-(4-tert-butybenzylamide)1-phenylamid-pyrrolidine Ph 1,3,4,5,6,7-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalene Ph 4,4a,5,6,7,8-hexahydro-4,4a-dimethyl-6-(1-methyletenyl)-2(3H)-naphthalenone Ph 2-hydrazino-4-methyl-6-methylthiopyrimidine Ph 2-benzoyl-3-formyl-6-methyl-4,5-diphenyl-o-diacylbenzole Ph 2-propyn-1-one,1-(2-thienyl)-,(2,4-dinitrophenyl) hydrazone Ph 3,5-bis(1,1-dimethylethyl)-1,2-benzenediol Ph 3-hydroxy-5-methoxy- benzenemethanol Ph ethyldibenzothiophene Ph 2,4-dimethyl- benzo[1]quinoline Ph (3,3-dimethyldecyl)- benzene Ph 3,5-dimethoxy- phenol Ph 2,6-disopropylnaphthalene Ph 1-acetyl-3-(6-methyl-3-pyridyl)-pyrazoline Ph 4(3H)-one,5-ethyl-2-methylthio-pyrimidin Ph 2-methyl-2-propyl- oxazolidine Ph O-(P-tolyl)1-azetidinecarbothioate E di-(2-ethylhexyl) phthalate E L-proline, N-(hexanoyl)-decyl ester E L-proline,N-vleryl-,nonyl ester E hexyl-N-valerate E stearic acid,3-(octadecyloxy) propyl ester E acetic acid,2-acetoxymethyl-1,2.3-trimethyl butylester E (6,11-bicyclolides: bridged biaryl macrolide)L-proline,N-butoxycabonyl-heptyl-ester E 1,2,4-benzenetricarboxylic acid, 1,2-dimethyl-nonyl ester E sulfurous acid, octadecyl 2-propyl ester E octadecanoic -acid ethyl ester E methyl(methyl 4-O-methyl--D-mannopyranoside)urinate E a.a-dipyridyl,Fe(2.2-bipyrid)Fe,monocarbonyl-(1,3-butadien-1,4-dicarbonic acid,diethyl ester) formic acid,1-(4,7-dihydro-2-methyl-7-oxopyrazolo[1,5-a)pyrimidin-5-yl)-,methyl ester E hexadecanoic acid or palmtic acid FFA 2-(2-heptadecynyloxy)tetrahydro-2H-pyran Py cyclobarbital CSAL vouacapenic acid,phenanthro[3,2-b]furen-4-carboxylic acid,1,2,3,4,4a,5,6, 6a,7,11, 11a,11b-dodecahyd T acetate-megaesterol T Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 6 di-TMS hexasterol T Table-7 Summarized results of table 2-6Chemical group Total No. Glucose GNaG Malt MNaG Gl and M M andMNaG No. MF No. MF No. MF No. MF No. MF No. MF 1 1 1 1 1 1 1 1 1 1 Phenol 48 2 52,25 - 10 - 10 20 1= 70 19 15 - 15 1 25-26 - - Esters 38 7 90-52 - 4 - 4 17 3=58, 38, 35 15 4 - 4 6 93-50 - - Alkane and alkenes 23 1 35 - 3 - 3 16 3= 35 13 3 - 3 - - - - Ketons 10 - - - 2 - 2 7 1=50 6 1 - 1 - - - - Indole compounds 05 1 25 - 1 - 1 3 - 3 - - - - - - - Azole compounds 04 - - - - - - 2 - 2 1 - 1 - - 1 1 Terpenoids 04 - - - - - - 3 - 3 1 - 1 - - - - Amides 02 - - - - - - 2 - 2 - - - - - - - Amines 02 - - - - - - 2 - 2 - - - - - - - Aldehyde 01 - - - - - - 1 - 1 - - - - - - - Cyclo-sugar-alcohol 01 - - - - - - 1 - 1 - - - - - - - Free fatty acid 01 - - - - - - 1 - 1 - - - - - - - Pyran 01 - - - - - - 1 - 1 - - - - - - - Total No. 140 11 90-25 - 20 - 20 76 8 69 25 - 25 7 - 1 - MF= Mass fractions; G= Glucose, M= Malt, MNaG=Malt +Na-glutamate, GNaG= Glucose + Na-glutamate, Ph=Phenol, E=Esters, Alka and Alke = lkane and alkene, K=Ketons, Az=Azole c, Ind=Indole, T=Terpenoids, Al=Alcohol, FFA= Free fatty acids, Ald=Aldehyde, Amid=Amide, Am= Amine, Py= Pyran, CSAL=Cyclo-sugar-alcohols. Notes: 8 metabolites are detected in the both malt and glucose tested media Table 2, Malt=76+7+1=84, MNaG=25+1=26, GNaG= 20, Glucose=11+7=18, Total No.of the detected metabolites in all tested media= 140, [Malt= 76, Malt + Na glutamate= 25, Glucose + Na glutamate= 20, Glucose= 11, Both Glucose and Malt= 7 and Both Malt and Malt+ Na glutamate= 1] Figure 2-6 recorded the total No. of the detected metabolites in the four tested media including G=Glucose; GSG= glucose + Na glutamate; M=Malt; MSG= Malt + Na glutamate, GandM= Glucose and Malt and M and MSG= Malt and Malt + Na glutamate media classified into "phenols, esters, alkane and alkene, ketons, azole, indole, terpenoids, amine, amide, aldehyde, alcohol. Figure 7-10 recorded the detected metabolites in the four ethanolic extracts of the G=Glucose; GSG= glucose + Na glutamate; M=Malt; MSG= Malt + Na glutamate, G and M= Glucose and Malt and M and MSG= Malt and Malt + Na glutamate media by GC/MS analysis. Three [alkanes 38, 35 and 35 and esters 58, 35 and 35] and one from the each phenols 70 and ketons 50 mass fractions. Also seventy metabolites are recorded only in the ethanolic extracts of the maltmedium at mass fractions 1 including [19 phenols, 15 esters, 13 alkaine, 6 ketones, three metabolites from each of azole, indole and terpenoids and two from each of amine and amide and one from each of aldehed, cyclo-sugar alcohol and fatty acids]. Eighty flavoring metabolites are detected in the ethanolic extracts of the Malt tested medium including [17 Research Journal of Biological Sciences _ _____________________ Vol. 4(10), 1-14, October (2015) International Science Congress Association Phenol "One has 70 mass fraction and 16 less fraction", 24 includes 8 "90 – 35" and 16 less fraction. Esters, 19 alkane and alkene, 3 (58- 35) + 16 less mass fraction, 8 Ketons "one metabolites has 50 and 7 metabolites has 1 mass fraction", 3 metabolites for (azole Comparison between all the tested media in total No. _____________________ _________________________ ____________ International Science Congress Association Phenol "One has 70 mass fraction and 16 less than 1 mass 16 less than 1 mass 35) + 16 less than 1 mass fraction, 8 Ketons "one metabolites has 50 and 7 1 mass fraction", 3 metabolites for (azole compounds, indole and terpenoids), two (amide fatty acids), one metabolites related to (al cyclo-sugar- alcohols). Notes: + means all the metabolites recorded at less than one mass fraction. Figure-2 Comparison between all the tested media in total No. of the detected chemical groups Figure-3 Glucose media ____________ ISSN 2278-3202 Int. Res. J. Biological Sci. 7 compounds, indole and terpenoids), two (amide , amine and free fatty acids), one metabolites related to (al dehyde, pyran and alcohols). Notes: + means all the metabolites recorded at less than one mass fraction. detected chemical groups Research Journal of Biological Sciences _ _____________________ Vol. 4(10), 1-14, October (2015) International Science Congress Association Twenty five flavoring metabolites were detected in the ethanolic extracts of the Malt +Na- glutamate tested media including [15 phenol, 4 esters, 2 alkane, 2 azole and one of each (Keton, aldehed, thiols and terpenoid)] all the metabolites are recorded at with mass fractions 1.3,5(2H, 4H)- dione,6 1,2,4- triazine is thiophenolic compounds which act as _____________________ _________________________ ____________ International Science Congress Association Figure-4 Glucose Na glutamate media Figure-5 Malt media Figure-6 Malt + Na glutamate media Twenty five flavoring metabolites were detected in the ethanolic glutamate tested media including [15 alkane, 2 azole and one of each (Keton, aldehed, thiols and terpenoid)] all the metabolites are recorded dione,6 -benzoylthio- triazine is thiophenolic compounds which act as herbicide and dyes. Table-7: Summarized results of table 2 11 are recorded 140 flavoring and antioxidant metabolites are detected in the ethanolic extracts of the four tested media includes: Malt medium recorded the highest numbers of ____________ ISSN 2278-3202 Int. Res. J. Biological Sci. 8 table 2 -6. Table and figures 2- recorded 140 flavoring and antioxidant metabolites are detected in the ethanolic extracts of the four tested media includes: Malt medium recorded the highest numbers of Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 9 metabolites followed by Malt+Na- glutamate, Glucose+Na-glutamate, glucose are 76, 25,20 and 11, respectively. The highest numbers of the recorded metabolic chemical groups includes 48 phenolic compounds, 38 esters, 23 alkanes and alkenes, 10 ketons, 5 indolic compounds, 4 (azole and terpenoids), 2 for each of amides and amines, one for each of aldehyde, free fatty acids, pyran and cyclo-aribitol are detected at 1 mass fractions in the ethanolic extracts of the tested medium table 1-7 and figures 2-7. Figure-7 Malt  \n Figure-8 Malt Na- Glutamate  \r Research Journal of Biological Sciences _ _____________________ Vol. 4(10), 1-14, October (2015) International Science Congress Association Palmitic acids (free fatty acid) Ester Alkane Ketone Alcohol Aldehyde Primary Secondary Normal Sulfonamide Phosphor Imidazole Isoxazole Oxazo The main nucleus of the functional groups of the classified the detected metabolites into 14 chemical groups Many litterers recorded that the Monascus and other numerous fungi specially mushrooms are play an important role in the enhancement of aroma compounds in many fermented foods as indicated by Moharram et al.. Eighty eight flavouring compounds are detected by GC/MS analysis in ethanolic extract of the red pigments of two _____________________ _________________________ ____________ International Science Congress Association Figure-9 Glucose  \r  \n Figure 10 Glucose Na- Glutamate Palmitic acids (free fatty acid) Arabitol Terpenoids Ester Alkane Ketone Alcohol Aldehyde Primary Secondary Amines Normal Sulfonamide Phosphor - amide Phenol Pyran Indole Amides Imidazole Isoxazole Oxazo le Pyrazole Azole compounds Figure-11 The main nucleus of the functional groups of the classified the detected metabolites into 14 chemical groups and other numerous fungi specially mushrooms are play an important role in the enhancement of aroma compounds in many fermented foods as Eighty eight flavouring compounds are detected by GC/MS of the red pigments of two Monascus ruber strains grown on four tested medium substrate includes alcohols, benzaldehydes, esters, lactones, phenol, terpenoid, thiols and mercapto compounds Table 2 and3, figures 2- 11 recorded 48 phenolic found in all extract of the four tested media. Fungal phenols are the hydroxylated aroma tic hydrocarbon ____________ ISSN 2278-3202 Int. Res. J. Biological Sci. 10 Arabitol Terpenoids Ester Alkane Ketone Alcohol Aldehyde Primary Secondary Tertiary amide Phenol Pyran Indole Thiazole The main nucleus of the functional groups of the classified the detected metabolites into 14 chemical groups strains grown on four tested medium substrate includes alcohols, benzaldehydes, esters, lactones, phenol, and mercapto compounds 1,8,18,19. 11 recorded 48 phenolic compounds found in all extract of the four tested media. Fungal phenols are tic hydrocarbon s act as antioxidant Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 11 agents and as act as antioxidants, antimicrobial, and anticancer agents22. The ethanolic extract of the red pigments of two M. ruber has 4-diethyl aminomethyl-2,5-dimethylphenol23. Thirty five esters includes six esters are repeated in the both Glucose and Malt tested media (palmtic acid, ethylester; 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)ester; 1,2-benzenedicarboxylic acid, diisooecyl ester; 1,2-benzene dicar boxylic acid, dioctyl ester; 2-butenedioic acid (E)-,bis(2-ethylhexyl)ester; phenacyl-thiocyanate, 2-oxo-2-phenylethyl-thiocyanate "93,94"; "87,90"; "87,88"; "83,87"; "62,68" and 50,59" in Glucose and Malt media, respectively". The esters in Malt medium extract recorded the highest mass fractions than the glucose medium extract. Table- 2 and 3, Figures 2-11. Esters are responsible for the fruiting fragrance. The ethanolic extract of the red pigments have 27 esters including eight butyric acid esters (2-ethylbutyric acid, hexadecyl ester) Different fruity fragrance compound such as methyl butyrate (applefragrance), methyl butanoate (pineapple), ethyl butyrate (orange), ethyl butanoate (pineapple), pentyl butyrate (pear), pentyl butanoate (apricot). Five other esters including oxalic acid, monoamide, N-propyl, decyl ester; thiocyanic acid, 2-(2-butoxyethoxy)ethyl ester; benzoic acid, 4-methoxy-,2-(2-furoythydrazono methyl) phenyl ester; 1,2-benzene-dicarboxylic acid, disoctyl-ester and 1,2-benzenedicarboxylic acid, mono-(2-ethylhexyl)-ester1,8,18,19. Twenty three alkane and alkene includes: one alkane in glucose tested medium 3,4-dimethyl- heptanes "35 mass fractions". Three alkanes in Glucose+Na-glutamate medium (4-ethenyl-1,4-dimethyl-cyclohexane; pentacosane and (E)-9-octadecane (at 1 mass fractions). Also three alkane and alkene in Malt tested medium (1,4-diaza-2,5-dioxobicyclo[4,3,0]nonae; 1-ethyl-2-hexene; 9-methyl-1-undecene58; 38 and 35 mass fractions, respectively). Fourteen alkane and alkene in Malt+Na-glutamate tested medium (at 1mass fractions) and includes heneicosane; 2,6,10-trimethyl-tetradecane; 2-undecene; 1,1-(2-methyl-1,3-propanedyl)bis-cyclohexane; 5-methyl-decane; 2,4-dimethyl-docosane; 5,14-dibutyl-octadecane; 1,2-5,6dipyrainocyclo octane; 2-methyl-hexadecane; 4-undecene; cyclopentylacetylene; 5,5-dimethyl-cyclohexane -1,3-dione-2-alylaminomethylene; 1-nonadecene; 2,6-disopropylnaphthalene (at 1 mass fractions) and two (at 1 mass fractions) in Malt+Na-glutamate 4,5-dimethyl-nonane and 2-thione,3,3-dimethyl-bicyclo [2.2.2.1] heptanes table 2 and 3 figures 2-7. Alkane is a saturated hydrocarbon and play a role in fungi as a source of carbon and energy. Zhang et al.,20 study the volatile compositional characteristics of mushrooms by using GC/MS analysis and recorded that seventeen volatile compounds included alkene, alkane, alcohol, aldehyde, ketone, and other organic compounds. C volatiles consisted of the major volatile compositions of straw and oyster mushrooms. 1-Octen-3-ol and 3-octanone are the main volatile compounds. 1-octen-3-ol and 3-octanone could be considered as biologically active compounds, which are not only an important factor in the fungal flavor but also contain crucial bio-information related to antioxidant activity. Ten ketonic compounds are detected in all extracts of all tested medium. In Glucose+Na-glutamate medium two ketones "4-(2-methylpropyl)acetophenone and 2-hydroxy-3,5,5-trimethyl-cyclohex-2-enone ( 1 mass fraction). In Malt medium eight ketons were recorded includes 2-phenyl-4-ethylidene-2-oxazolin-5-one,(4Z)-4-ethyldiene-2-phenyl-1,3-oxazol-5(4H)-one (50 mass fraction); 5-(1-cyclohexenyl)-5-ethyl-1,3-diazinane-2,4,6-trione; 2-hydroxy-3,5,5-trimethyl-cyclohex-2-enone; 3,5-dihydroxy-4,4-dimethyl-cyclohexadien1-one; 4,4a,9,10-tetrahydro-4a-methyl-2(3H)-phenanther none; 2,6-di--butyl-4-methylene-2,5-cyclohexadiene-1-one; octahydro-1,2,4-methenocyclobut[cd]inde-3(1H)-one; 2-propyn-1-one,1-(2-thienyl)-,(2,4-dinitro phenyl)hydrazone ( 1 mass fraction)Table 2 and 7 Figures 2-11.Butyrolactone(at 1 mass fraction) was recorded in Malt+Na-glutamateTable 2 and3 Figures 2-11. Butyrolactone is a (methyl-4-hydroxy-2-[[4-hydroxy-3-(3-methylbut-2-enyl)-phenyl] methyl] -3-(4-hydroxyphenyl)-5-oxofuran-2-carboxy late). Butyrolactone I is inhibit CDK1 and CDK2 kinases, exhibiting antiproliferative activity against diverse tumour cell lines, e.g., lung cancer cells, pancreatic cancer cells and prostate carcinoma cells. Butyrolactone I was produced and recently discovered in Candida albicans, Aspergillus flavus,A. terreus, bacteria and Streptomyces. Butyrolactone Known as autoregulatory or quorum sensing molecules. Quorum sensing molecules include acyl-homoserine lactones, -butyrolactones and small peptides in bacteria and oxylipins and farnesol in fungi. Quorum sensing-molecule increase in 2ry metabolism of lovastatin and sulochrin production and switch on morphological development. It also increase the fungal growth, cell density, pre courser of the 2ry metabolites antibiotics, anticholesterols, synthesis of virulence factors, sporulation and bioluminescence for population and act as co-ordinately for adapt to the environmental conditions21. Three lactones are detected the ethanolic extract of the red pigments of two M. ruber strains in including 3-hydroxy-4,4-dimethyl--butanolactone-pantolactone; -lutalactone and 2,4-dihydroxy-3,3-dimethyl butyric acid -lactone8,21. Five indole compounds includes "3-[2,2-bi(methoxycarbonyl)-2-formamido ethyl-1]-4-(4-hydroxy-3-methylbut-2-en-1-yl), methyl-indole (25 mass fractions) in Glucose medium, 2-carboxlic acid, 6-(4-ethoxyphenyl)-3-methyl-4-oxo-4,5,6,7-tetrhydro-isopropylester-1-H-indole in Glucose+ Na-glutamate medium, two indole metabolites in Malt medium includes (1-methyl-2-phenyl-1-H-indole; 1--indole,1-methyl-2-phenyl-2-methyl-7-phenylindoleand 3-[2,2-bi(methoxycarbonyl )-2-formamid ethyl-1]-4-(4-hydroxy-3-methylbut-2-en-1-yl), methyl-indole all the forward metabolites are detected (at 1 mass fractions) in the ethanolic extracts of the tested medium table 2-7 and figures 2-11. Indole is an aromatic amino acid is Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 12 widely produced by fungi for regulates much physiological process such as spores formation and spores virulence. Monascushas high flavoring value duo to the presence of alkene, alkane, alcohol, esters, lactones, benzaldehyde, bezene acetaldehyde, ketones nonanal, diethyl disulfide, and other. Linoleic acid act as precursor of 1-octen-3-ol which responsible about flavor. Also aromatic fatty acid phenylalanine could decompose to benzene acetaldehyde. It often has the scent of anise due to the presence of benzaldehyde (almonds like taste)19,22. Azole or "heterocyclic nitrogenous five members ring" metabolites are detected includes acetamide-2-(2,5-dioxo-pyrroidin-1-yl)-N-(5-propyl-[1,3,4]thiadiazol-2-yl) was detected in both extracts of malt and malt+ Na-glutamate. But the methyl-2-phenybenzenimidazole and 2-(E-4,4-dicyano-3-N-methylanlino-1,3-butadien-1-yl)-4-(methoxycarbonyl)-1,2,3-triazol were detected in Malt medium only. Many azoles are used as antifungal drugs, inhibiting the enzyme 14demethylase which produces ergosterol an important component of the fungal plasma membrane table-2 and 3 figures 2-11. Three sulphour containing metabolites or thiols are detected in M. ruber strains extracts included 2,3-butanedithiol; 1.4-bis (methylthio)-butane and, 4-methylthio-butyric acid and one acetate-3-mercapto butyric acid (grapefruit fragrance) were determined by Moharram et al.,Three terpenoids includes vouacapenic acid, phenanthro [3,2-b] furen-4-carboxylic acid, 1, 2, 3, 4, 4a, 5, 6, 6a, 7, 11, 11a, 11b-dodecahyd and di-TMS hexasterol at ( 1 mass fraction) are detected in the ethanolic extracts of the Malt tested medium and limonene was detected in Malt+Na-glutamate tested medium.Table-2 and 3, figures 2-7. Terpenoids are derived from five-carbon isoprene units. It have aromatic characters and act as antibacterial, antineoplastic, other pharmaceutical functions and also act as a steroids and sterols precursors. Limonene is a cyclicterpene gives the orange fragrance detected in the ethanolic extract of the red pigments of M. ruber. Terpenoids are includes steroids and sterols “ergosterols and ergothioneine” have antibacterial, antineoplastic agents. Specially lanostane is act as anti-alzheimer, antidiabetic, anticholesterol, immunemodulatory, antioxidant, anticancer and antimicrobial. Phenols includes tocopherol, ascorbic acid, flavonoid, phenolic acids, phenylpropanoids, melanins, tannins which have antioxidant, anticancer and antimicrobial activity22-26. Two metabolites for each aldehydes [3-(1-dimethylamino naphthalene-5-sulfonyloxy) benzaldehyde detected in Malt+Na-Glutamate" and 7-hexadecenal], amide (-(2-dimethyl amino-3-oxo-1-butenyl)-acetamide and nonanamide) and amine (1-pentene,1,3-diphenyl-1-ylmethyl) amino and etoloxamine) at 1 mass fraction in Malt medium.Table 2 and 3 figures 2-7. Aldehyde (R-CHO) give M. ruber red pigment ethanolic extract their fragrances. Also aromatic fatty acid phenylalanine could decompose to benzeneacetaldehyde. It often has the scent of anise due to the presence of benzaldehyde which has almond smells26 One metabolites detected for each of free fatty acids "hexadecanoic or palmtic acid", pyran "2-(2-heptadecy nyloxy) tetrahydro-2H-pyran" and cyclo-sugar-alcohols "cyclobarbital" are detected in Malt1,8. Moharram et al., detected 22 pyran derivatives in ethanolic extracts of the tested M. ruber strains. Many references are reported that the pyran act as anticancer agents.1,,8,19. Alcohol is a saturated straight chain alcohols, the general formula for which is C2n+1OH. Four alcohols were detected in the ethanolic extract of the red pigments of M. ruber includes 1,2,3,4-butanetertrol, 1,2,3,4-butanetertrol,[S-(R,R)], tetrahydro-4H-pyran-4-ol, and glycerol. Cyclo-sugar-alcohols arabitol support the overgrowth of intestinal microbes such as Candida albicans or other yeast and it may cause gastrointestinal upset in some peopleEman Abbady reported that the Monascus pigments have numerous bioactive 2ry metabolites which act as antimicrobial agents. Also whose reported that the fatty acids palmitic acid and oleic acid which has antioxidant activity and act as amedicine. The most detected metabolites are recorded by many literatures as fungal bioactive and antioxidant agents includes the following metabolites amines, catechols, cerebrosides, glycans, homoglucans, heteroglucans, glycopeptides, glycoproteins, isoflavones, quinones, protein, RNA-Protein complexes sesquiterpenes, steroids, selenium and triacylglycerols which act as anticancer agents1,8,22-26Cheng et al.,23 who suggested that the contents of -coumaric, ferulic, and sinapic acids in adzuki bean were highly increased by fermentation with M. pilosus, and enhanced various antioxidant activity. Conclusion Malt and malt+Na–glutamate media and strain No. 4066 gives the best red pigment productions. 142 flavouring and antioxidants metabolites are detected in the extracts of the four tested media. Malt medium recorded the highest No. of metabolites followed by Malt+Na-glutamate, Glucose+Na-glutamate and Glucose media are 86, 26, 20 and 19, respectively. The classified metabolic products includes 48 phenolic compounds, 39 esters, 23 alkanes and alkenes, 10 ketons, 5 from (azole and indolic compounds), 4 terpenoids, 2 for each of (amides and amines), one for each of (aldehyde, free fatty acids, pyran and cyclo-sugar-alcohols) are detected at 1 Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 13 mass fractions. The flvouring, nutritional and medicinal values of Monascus pigments and their 2ry metabolites formed with the pigments depend up on the Monascus species, substrates or growing media, environmental conditions and methods of extraction and detections. We needed more and more investigations to obtain many of therapeutic substances for outweigh diseases. Also make full use of the advantages of this fungus like that fast food, Ctejat object, simple, cheap and modest growth. Acknowledgements We thank Prof A. H. Moubasher and all members of Assiut Univ. Mycological Center for give me the M. ruber strains. Also much acknowledged Prof N. Abo-El. Mally and all members of Analytical Chemistry Unit, Assiut University, Egypt, for good help. References 1.Eman M.M. and Abbady M.S., Secondary metabolites and bioactivity of the Monascus pigments, Review, Global J. Biot. Bioch., 9(1), 01-13 (2014)doi:10.5829/idosi.gjbb.2014.9.1.8268. 2.Calvo A.M., Wilson R.A., Bok J.W. and Keller N.P., Relationship between secondary metabolism and fungal development, Microbiol. Molec. Biol. Rev., 66(3), 447–459 (2002) www.ncbi.nlm.nih.gov/pmc/articles/ PMC120793/ 3.Liu G.Y. and Nizet V., Color me bad: microbial pigments as virulence factors, Review, Trends Micro., 17(9), 406-413 (2009) doi:10.1016/j.tim.2009.06.006. 4.Lee Y., Chen D., Chauvatcharin S., Seki T. and Yoshida T., Production of Monascus pigments by a solid-liquid state culture method, J. Fer. Bioeng., 79, 516-518 (1995).../0d0400e8c2360b389b875594. 5.Lee B.K., Park N.H., Piao H.Y. and Chung W.J., Production of red pigments by Monascus purpureus in submerged culture, Biotech. Biop. Eng., ,341-346 (2001) Sscielo.br/scielo.php?script=sci_arttextandpid = S0101. 6.Arunachalam C. and Narmadhapriya D., Monascusfermented rice and its beneficial aspects, Review, Asian J .Pharm. Clin. Res.4, 0974-2441 (2011)www.ajpcr.com/Vol4Issue1/230. 7.de Carvalho J.C., Oishi B.O., Woiciechowski A.L., Pandey A., Babitha S. and Soccol C.R., Effect of substrates on the production of Monascus biopigments by solid-state fermentation and pigment extraction using different solvents. Indian J. Biotech., 6,194–199 (2007)nopr.niscair.res.in/.../1/IJBT%206(2)%20194-1998.Moharram A.M., Eman M.M. and Ismail M.A. Chemical profile of Monascus ruber, Food Technol. Biotech., 50(4), 490–499 (2012) search.ebscohost.com/login.aspx? direct=trueandprofile 9.Lian X., Wang C. and Guo K., Identification of new red pigments produced by Monascus ruber, Dyes Pigments, 73,121–125 (2007)10.Hajjaj H., Klaebe A., Loret M.O., Tzedakis T., Goma G. and Blanc P.J., Production and identification of N-glucosyl rubropunctamine and N-glucosylmonascorubramine from Monascus ruber and the occurrence of electron donor-acceptor complexes in these red pigments, App. En. Microbio., 63, 2671–2678 (1997)11.Rasheva T., Hallet J.N. and Kujumdzieva A., Taxonomic investigation of Monascus purpureus 94-25 strain, J. Cult. Collect., 2,51–59 (1998) 12.Chatterjee S., Maity S., Chattopadhyay P., Sarkar A., Laskar S. and Sen S.K., Characterization of red pigment from Monascus in submerged culture red pigment from Monascus purpureus, J. App. Sci. Res., , 2102-2108 (2009)13.Vidyalakshmi R., Paranthaman J.R., Murugesh S. and Singaravadivel K., Stimulation of Monascus pigments by intervention of different nitrogen sources, Global J. Biotech. Bioch., 4,25–28 (200914.Dikshit R. and Tallapragada P., Monascus purpureus: A potential source for natural pigment production, J. Micr. Biotech. Res.1(4), 164-174 (2011)15.Shazwani N.S. The optimization of red pigment by Monascus purpureus FTC 5356 in solid state fermentation. MsC thesis Faculty of Universiti Putra Malaysia Institutional (2012)psasir.upm.edu.my /view/year/ 2009.default.html16.Rashmi D. and Padmavathi T., Exploring Monascus sanguineus as a potential natural source for pigment production, Int. Res. J. Biolog. Sci., 2(5), 59-67 (2013)17.Srianta I. and Harijono, Monascus-fermented sorghum: pigments and monacolin K produced by Monascus purpureus on whole grain, dehulled grain and bran substrates, Int. Food Res. J., 22(1), 377-382 (2015)18.Daigle P., Gelinas P., Leblanc D. and Morin A., Production of aroma compounds by Geotrichum candidum on waste bread crumb, Food Microbiol., 16,517–522 (1999)19.Eman M.M. and Farghaly F.A., Bioactive compounds of fresh and dried Pleurotus ostreatus mushroom, Inter. J. Biot. W. Ind., 3, 4-14 (2014) doi: www.lifescienceglobal.com/pms/index.../1822 20.Zhang Z.M., Wu W.W. and Li G.K., A Gas Chromatographic Mass Spoctrophotometric study of the volatile organic composition of straw and oyster mushrooms during maturity and its relation to antioxidant Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 1-14, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 14 activity, J. Chromat. Sci., 46, 690-699 (2008) doi: www.ncbi.nlm.nih.gov/pmc/articles/PMC2228291/ 21.Palonen E.K., Neffling M., Raina S., Brandt A., Keshavarz T., Meriluoto J. and Soini J., Butyrolactone I quantification from lovastatin producing Aspergillus terreus using Tandem Mass Spectrometry evidence of signalling functions, Microorg,2,111-127 (2014)doi:10.3390 /microorganisms2020111. 22.Barros L., Ferreira M.J., Queiros B., Ferreira I.C.F. and Baptista P., Total phenols, ascorbic acid, -carotene and lycopene in portuguese wild edible mushrooms and their antioxidant activities, Food Chem.103, 413–419 (2007)doi:10.1016/j.foodchem.2006.07.038 23.Cheng J., Lee S.K., Palaniyandi S.A., Suh J.W. and Yang S.H. Effect of fermentation with Monascus pilosus on the antioxidant activities and phenolic acid contents of adzuki bean Vigna angularis, J. Coas. Life Medic., 3(4),276-283 (2015)ifrj.upm.edu.my/.../1%20IFRJ%2021%20(01)%202014 24.Su Y.C., Wang J.J., Lin T.T. and Pan T.M., Production of the secondary metabolites c-aminobutyric acid and monacolin K by Monascus, J. Ind. Microbiol. Biotechnol., 30,41–46 (2003) DOI: 10.1007/s10295-002-0001-5 25.Smith H.A., Production of antimicrobials and antioxidants from filamentous fungi. PhD thesis in National University of Ireland, (2014)26.Rees A.M., Austin M.P. and Parker G., Role of w-3 fatty acids as a treatment for depression in the perinatal period, Aust. N.Z.J. Psychiatry, 39, 274–280 (2005)