Materials and Methods:

Materials and Methods: Study Area: Dharwad is selected as the study area for screening Spice mycoflora, the area is located at 15 0 44 North latitude 74 0 99 East longitude in Karnataka state (India), because of the rich vegetation and conducive atmosphere, harbors fungal growth. The sites selected for the present investigation is market area and different places of Dharwad. Local Market Area, Dharwad. Collection of samples: Sample collection: The five spice samples Viz; Elettaria cardamomum, Maton. (Cardamom), Coriandrum sativum, L. (Coriander), Cuminum cyminum, L. (Cumin), Cinnamomum tamala, Nees and Eberm (Indian cassia) and Piper nigrum, L. (Black pepper) were collected from Local market area Kirana merchant shops and spice sailors etc. on regularly monthly basis, from the chosen sites from Dharwad during the period of November 2009 to October 2012. The selected spice samples of unknown variety was collected in sterile polyethylene bags separately from the chosen sites, during the collection field data Viz; collection area, weight, type of sample, date of collection etc was carefully noted in the field note book. Collected samples were brought to the laboratory and used for further mycoflora analysis. 25 P a g e

Glassware and Chemicals: Petri plates, blotting paper with superior water holding capacity, conical flasks, pipettes, measuring cylinders and test tubes etc glassware from Borosil and HiMedia chemicals were used in the present study. Cleaning of Glassware: Glassware were first washed with a detergent and then placed in cleaning solution. The composition of the solution is Potassium dichromate 60gms, concentrated Sulphuric Acid 60ml and distilled Water 1000ml. The glassware were kept in the cleaning solution for 24 hours, later thoroughly washed with tap water and finally rinsed with distilled Water and dried. Sterilization of glassware: All the glassware were sterilized at 15 lbs pressure at 121 0 C for 15 min. in the autoclave and dried in the hot air oven. Preparation of cotton plugs: Cotton plugs are best filters of microbes. The required amount of nonabsorbent cotton should be rolled tightly to fit the mouth of the flask or tube. It must be possible to lift the flask or tube by holding the plug without the plug slipping off. Methods: There are various methods for screening of mycoflora of Spices, but here following three methods have been employed for fungal isolation which are suitable. Isolation of seed mycoflora: Screening mycoflora associated with five different Spice samples in the study were isolated by following methods. 1. Serial Dilution Method. 2. Agar Plate Method. 3. Standard Blotter Method. Serial Dilution Method (Waksman, 1927): Ten gram of Spice sample was weighed and this was then suspended in 250ml conical flask containing 90ml of sterile distilled water (leveled up to100ml) so as to get the stock solution. The flask was shaken for about 15min on a shaker. 26 P a g e

1ml of suspension was transferred to test tube containing 9ml of sterilized distilled Water with the help of sterilized 1ml pipette. This gives the dilution of 1:1000. This process was repeated to obtain serial dilution 1:10000, 1:100000, 1:1000000. All the dilutions were thoroughly shaken again and then 1ml from each dilution was poured on sterilized petriplates under laminar flow chamber, each plate was gently rotated to ensure uniform distribution of suspension. Sterilized media was poured in petriplates containing 1ml of suspension under aseptic condition; each plate was again gently rotated to ensure uniform distribution of suspension and media. After solidification of media in the plate, plates were incubated at 25±1 0 C incubation time 37 days and the plates were examined for mycoflora of Spices. Agar Plate Method (Muskett, 1948): Hundred samples of the selected Spices were taken to isolate the mycoflora of Spices. Sterilized media was poured in sterilized petriplates under aseptic condition. After solidification of media in plates, the plates were kept inverted for 12hrs, contaminated plates were rejected. Ten samples of each selected Spice were placed at equidistance in each petridish containing nutrient media. The plates were incubated by providing 12hrs. light and 12hrs. darkness alternatively. The plates were examined for the mycoflora under stereo binocular microscope and compound microscope. Standard Blotter Method (de Tempe, 1953): Sterile glass petriplates (9cm) were used in the study; their discs of blotter were moistened with sterilized water and were placed at the bottom of each petriplates. Excess water was drained from the blotters. Total 100 samples of the selected Spices were taken randomly from the lots for the study. Ten samples were placed at equidistance on blotter paper in each petriplates, for better growth and sporulation of fungal flora, 12hrs artificial light was provided by placing the plates below the Philips 40w tubes and alternated 12hrs darkness. After incubation of the plates, fungal growth was seen under stereo binocular microscope and compound microscope. Media used: In the present study following media were used isolate mycoflora of selected Spices from Dharwad. 27 P a g e

1. Potato Dextrose Agar (PDA) 2. Czapeks Dox Agar (CZA) 3. Malt Extract Agar (MEA) 4. Yeast Extract Agar (YEA) 5. Sabouraud Dextrose Agar (SDA), Table No. 1. Chemical composition of different media. Name of the Media chemicals PDA in CZA in MEA in YEA in SDA in gm gm gm gm gm Dextrose FeSO 4.7H 2 O 20 0.01 40 K 2 HPO 4 1.0 KCl 0.5 Malt Extract 30 MgSO 4.7H 2 O 0.5 NaNO 3 Potato Sucrose Yeast Extract Peptone Agar Distilled Water P H 200 15 1000ml 5.6±0.2 2.0 30 15 1000ml 7.3±0.2 5.0 15 1000ml 5.4±0.2 3.0 5.0 15 1000ml 7.2±0.2 10 15 1000ml 5.6±0.2 PDA Potato Dextrose Agar, MEA Malt Extract Agar, SDA Sabouraud Dextrose Agar. CZA Czapek Dox Agar, YEA Yeast Extract Agar. 28 P a g e

Sterilization of media: All the media were sterilized in an autoclave at 15 lbs pressure at 121 0 C for 15 min. To prevent growth of bacteria, the media must be either acidified or supplemented with antibiotics. Chloramphenicol Antibiotic was used to prevent the bacterial growth. Counting of Fungal Colonies: Fungal colonies start appearing on the inoculated plates which were incubated at 25±1 0 C, After 24 hrs white mycelium start to appear and in a couple of days, a full growth of the fungus takes place to form a colony. But in some cases, appearance of fungal colony takes many days as noticed in slow growing fungi. At the same time number of fungal colonies of a particular species or genera is noted or counted. At the same time colour of the colony from both the sides, textures of the colony were noted. The incubated plates were observed under stereobinocular microscope. To avoid loss of fungi which developed late and which might be covered by fast growing forms, all plates in a series were checked frequently under stereobinocular microscope for a period of two weeks. All plates observed for one month before discarding. Preparation of Slant and transferring of fungi: There is a competition for development of various fungi in the initial medium. Colonies appearing in this medium probably do not grow and develop in the full extent. Hence single colonies of different fungi were transferred from the petriplate to the slant by flame sterilized and cooled Nichrome wire or loop. All this process was done under laminar air flow chamber. This process is to be done quickly to minimize the contamination and to have the pure cultures under aseptic conditions. Preservation and Maintenance of Culture: There are different methods for preservation and maintenance of cultures. The pure cultures of most of the fungal forms isolated from seeds were maintained by repeated (once in three months) subcultures in an aseptic condition and were preserved in a refrigerator. 29 P a g e

Slide Preparations, Illustrations, PhotoMicrography: Lactophenol with cotton blue stain was used as mounting media for preparation of slides, which were sealed with DPX mountant. Illustrations of fungi given in the thesis were drawn with the help of Erma Camera Lucida at stages level using 5x, 10x, 40X, 43x objectives and 10x, 15x, eyepieces. The choice of magnification was according to the need. Photomicrographs of the slides were taken by Carl Zeiss Imager M2 Model Microscope with Jenoptic Prog. Res. C5 attached Camera using 5X, 10x, 20x, 40X objectives and 10x eyepieces. Selection of objectives and eyepieces were dependent upon the nature of the fungal structures. Identification: Identification of different fungi from five selected Spices was done with help of slides prepared by direct mount from the culture; prepared slides were made permanent and with help of Erma Camera Lucida drawings along with micrometric measurements, Photomicrography and by referring fungal monographs and manuals such as Thom and Raper, (1945), Raper and Thom, (1948), Tandon (1968), Subramanian, (1971), Barnett & Hunter, (1972), Domsch & Gams, (1972), Ainsworth, et al., (1973), Ellis (1971,1976), Ellis and Ellis, (1985), Raper & Fennell, (1965), Bilgrami, et al., (1981), Gilman, (2001), Nagamani, et al., (2006), Pande, (2008), Muthumary (2013) also research articles and other related literature fungal Identification and illustrations were made up to the Genera and Species level. All identified specimens are deposited, under the code number MASD (Mycoflora Analysis of Spices from Dharwad), in the Mycology Laboratory, Department of Botany, Karnatak University, Dharwad, Karnataka State, India. Data on maximum and minimum Temperature, Relative humidity and Rain fall taken from Meteorological, Department, University of Agricultural Sciences, Dharwad, Karnataka state, India. 30 P a g e

Presentation of the Data: The term Percentage of Fungal Occurrence Total number of individual fungal occurrence on all the samples in 36 months = X100 Total number of fungal occurrence on all the samples in 36 months The term Percentage of Seasonal occurrence = Total number of individual fungal occurrence on all the samples in particular Season of 36 months = X100 Total number of fungal occurrence on all the samples in particular Season of 36 months Analysis of Aflatoxins (B1, B2, G1, G2) and Ochratoxin A by High Performance Liquid Chromatography Analysis of Aflatoxins (B1, B2, G1, G2) Sampling: A total of 20 samples belonging to 5 different Spices viz., Elettaria cardamomum, Maton. (Cardamom), Coriandrum sativum, L. (Coriander), Cuminum cyminum, L. (Cumin), Cinnamomum tamala, Nees and Eberm (Indian cassia) and Piper nigrum, L. (Black pepper) 250 gram of each sample of unknown variety were randomly collected in polyethene bags from different places of Dharwad and stored at 4 0 C until the analysis. Apparatus: Blender jars, beakers, glass funnels, 10ml pipettes, 50ml graduated cylinders,10100 µl air displacement pipette, 10ml volumetric flask, parafilm, HPLC with fluorescence detector, Kobra cell pump stand etc. Reagents: Supelco Aflatoxin mix kit, HPLC grade Methanol, 80% Methanol+20% distilled Water, 20% tween 20, 1% Acetic acid in HPLC Water, HPLC Water, 31 P a g e

Aflatest p column, Sodium Chloride excelar grade, fluted filter paper, glass fiber filter paper etc. Procedure: ASTA, (1998) Sample preparation: Spices weigh 25gm in blender jar, Weigh 5gm salt into blender jar, Add 80% Methanol, cap blender jar with parafilm, blend at high speed for 1min, Filter the contents through fluted filter paper into a beaker, Pipet 10ml of filtrate into 50ml graduated cylinder, Filter contents of the graduated cylinder through a glass fiber filter into 250ml beaker, filtrate is made column clean up by aflatest P column by using 10ml HPLC water and 1ml of Methanol this Methanol eluent used for the injection. Instrumental conditions: Mobile phase: 63% DIwater 0.1g/L KBr 0.02 Nitric acid, 22% Methanol, 15% Acetonitrile For PHRED photochemical reactor 55% HPLC Water, 45% HPLC Methanol Flow rate 1.2ml/min, Column C18, Injection volume 50µl, Detection Excitation 365nm Emission 468nm, Kobra cell at 100µams/phred switched on Calculation: Aflatoxin (µg/kg) = pg (Conc)/25 LOQ0.5µg/Kg Average percentage of recovery value (Aflatoxin B190%, B285.6%, G181.8% and G282.3%) 32 P a g e

Analysis of Ochratoxin A Sampling: A total of 20 samples belonging to 5 different Spices viz., Elettaria cardamomum, Maton. (Cardamom), Coriandrum sativum, L. (Coriander), Cuminum cyminum, L. (Cumin), Cinnamomum tamala, Nees and Eberm (Indian cassia) and Piper nigrum, L. (Black pepper) 250 gram of each sample of unknown variety were randomly collected in polyethene bags from different places of Dharwad and stored at 4 0 C until the analysis. Apparatus: Blender jars, 12 position pump stand with syringes, HPLC with florescence detector and auto sampler, beakers 250ml, conical flask 250ml, graduated cylinder 50ml, Volumetric flask 50ml and 100ml and Parafilm etc. Reagents, Chemicals and other Accessories: Supelco OchratoxinA standard, HPLC grade Methanol, HPLC grade Acetonitril, HPLC grade Acetic Acid,1% Sodium Bicarbonate in distilled Water, 1X PBS buffer in HPLC Water, Tween 20, HPLC water, OTA immunoaffinity column, fluted filter paper (240 mm) etc. Procedure: AOAC, (2005) Preparation of 1X buffer solutions Preparation of 1X mycotoxin wash buffer: take 200 ml 5X buffers in a 1000 ml measuring Jar made up to 1000ml with HPLC water. Preparation of working standard 10ppb Take 1 ml of 500 ppb stock standard into 50 ml standard flask. Bring to volume with 50:50 Water: Methanol with 1% Acetic acid in 50ml Water. Extraction and quantification of Ochratoxin A for extraction of Ochratoxin A 10gms of spice sample put into blender Jar add 70 ml of methanol followed by 30 ml of 1% Sodium Bicarbonate into the jar mix thoroughly, blend at high speed for 1 min filter the extract through fluted filter paper (240mm) into a 250 ml beaker, after this pipette 10 ml of the filtrate into 50ml of graduated cylinder (if the sample is Nutmeg, Oregano or Black pepper add 40 ml of 20% tween 20 solution to the graduated cylinder, if the sample is not one of the product add 40ml 33 P a g e

of 1X buffer solution and mix thoroughly now transfer the content through microfiber filter paper, this will be used for clean up. Immune affinity column clean up: Connect OTA immunoaffinity column Take 10 ml of sample solution on the column and allow to absorb, rinse the column with 10 ml 1X buffer solution and then with 10 ml HPLC Water. Place 20ml stopper test tube under the tip of the column and add 1 ml 98:2 HPLC Methanol: Acetic acid to the column, Collect the Methanol eluent in the test tube Add 1ml HPLC water in to the column collect the water eluent also in the same test tube. Vortex the mixture for 1min the sample is now ready for injection into the HPLC. Instrumental conditions: Ochratoxin A was quantified by using following instrumental conditions HPLC with fluorescent detector Excitation 333nm and Emission 443nm, column C18 ; 4.6 X 50 mm (Part no.: 00B4252EO) at ambient temperature, 51% HPLC Water+48.8% HPLC grade Acetonitril + 0.2% HPLC grade Acetic acid was used as mobile phase; flow rate: 1ml/min injection volume: 50μl and average percentage of recovery value (mean) is 87%, Limit of Quantification is 5.0 μg/kg, Supelco OchratoxinA standard used as a standard for Ochratoxin A analysis, working standard 10 ppb. Calculation: Ochratoxin A (µg/kg) = pg (Conc)/10 (LOQ) is 5.0 μg/kg Average percentage of recovery value (mean) is 87% 34 P a g e