The Effect of Water Activity and Storage Temperature on the Growth of Aspergillus flavus in Medicinal Herbs

• Abstract
• Introduction
• Materials and Methods
• Medicinal herbs
• Criteria for selection of these medicinal herbs
• Organisms
• Preparation of inoculum
• Sample peparation
• Water activity
• Inoculation of A. flavus in medicinal herbs
• Extraction and confirmation of aflatoxin in medicinal herbs
• Precautions and decontamination
• Results and Discussion
• Acknowledgements
• References

Abstract

The quality control of medicinal herbs post harvesting or after collection becomes very critical because of susceptibility to fungal invasion during storage depending on the temperature and humidity of the storage area. The information on moisture equilibrium is important on the process and storage of foods which can be extended to medicinal herbs. In the present study, the growth of Aspergillus flavus was observed on selected ten medicinal herbs with water activity a_w above 0.81 when stored at 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C except for Picrorhiza kurrooa and Alpinia galanga which were found to have anti-fungal properties. Aspergillus flavus did not grow in any samples of medicinal herbs with water activity a_w below 0.81 at temperatures of 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C. Also Aspergillus flavus did not grow in any samples of medicinal herbs with water activity a_w above 0.81 when stored below 10 ± 2 °C. Therefore it can be concluded that the contamination of medicinal herbs with aflatoxins can be minimized by controlling water activity and storage temperature. Sorption isotherms (desorption) can be interpreted to determine the optimum drying which can lower the water activity to the level required for preventing growth of Aspergillus flavus and also for ensuring quality of medicinal herbs which may get destroyed upon over drying. Furthermore, it also saves incremental cost in prolonged drying over the optimum drying.

Introduction

The use of medicinal herbs all over the world has increased significantly in recent times. A great number of national and international companies employ vegetal raw material in the elaboration of their drugs. Therefore the medicinal herbs turn the commercial culture and thereafter harvest improvement becomes an essential feature to serve market demands. The main aspect to be investigated is optimization of the production of medicinal herbs through techniques that allow the preservation of quality along the productive chain. This may include optimization of storage conditions of a medicinal herb through techniques that allow the preservation of quality before it is used in the finished formulation. Information on moisture equilibrium is important in the process and storage of foods which can be extended to medicinal herbs. A high frequency of the aflatoxins producing fungus A. flavus has been reported to be found in stored crude herbs. Degradation of alkaloids and medicinally valuable secondary metabolites of stored plant drugs due to fungal infestations has been reported [1], [2]. The World Health Organization (WHO) has also paid serious attention to mycotoxin contamination in herbal drugs, considering it as a global problem. However, its incidence is higher in tropical and subtropical countries as the harvesting practices and high temperature and moisture ( %) favor fungal invasion and mycotoxin elaboration. The fungal contamination will vary with geographic location, agricultural and agronomic practices and the susceptibility of commodities to fungal invasion during pre-harvest, storage and processing periods [2], [3].

It has been indicated in the literature that the main factors controlling invasion of stored food commodities (including grain, seeds) by fungi is moisture and temperature [4], [5], [6]. Published literature [7] indicates that good growth of A. flavus was observed at a relative humidity (RH) >85 % at 25 – 38 °C, whereas minimal growth was recorded at 80 % RH and 30 °C. The optimal time and temperature for aflatoxin production by A. flavus and A. parasiticus on sterilized peanut kernels and in liquid medium in culture flasks was found to be 7 – 15 days and 25 – 30 °C, respectively [8]. Furthermore, it has been demonstrated that water activity, a_w is a better measurement than moisture ( %). Water activity (or equilibrium relative humidity ERH) indicates the degree of freedom of the water absorbed in a material and shows dimensions, structure, cohesion, agglomeration properties as well as electrical and chemical properties better than moisture ( %) does. The effect of this water on physical properties such as differences in water vapor pressure and not moisture ( %) govern the interchange of water between a product and its surroundings. Two ingredients may have the same moisture ( %) but totally different water activity a_w. Water is a basic ingredient in foods or crude herbs and its control can be critical to guarantee microbial safety during shelf-life and consumption. Lowering the water activity is a necessary goal to preserve food or crude herb and guarantee shelf-life.

The knowledge from the published literature on the effect of water activity and temperature on the growth of A. flavus in foods has been extended to medicinal herbs (active botanical ingredients). The current study is an attempt to analyze the effect of water activity and temperature on growth of A. flavus in medicinal herbs and hence the risk of aflatoxin contamination. The study would also reveal the condition for minimum, optimum and maximum growth of A. flavus and determine appropriate conditions for storage of active botanicals. Also plots correlating food moisture ( %) (expressed as mass of moisture per unit mass of dry material) with a_w at constant temperature known as sorption isotherms (adsorption and desorption) can be extended to study the same in medicinal herbs [9]. These isotherms can be useful in assessing stability of medicinal herbs and delay the decay process. Thus this information would be useful in optimization of the drying process for assessing storage and packaging condition and for contributing to physical, chemical and microbiological stability of the medicinal herbs [10]. The sorption isotherms show the relation of equilibrium between the moisture ( %) of the food or crude herb and its water activity at constant pressures and temperatures.

Materials and Methods

Medicinal herbs

Medicinal herbs such as Tinospora cordifolia (Guduchi – stem), Adhatoda zeylanica (Vasaka – leaves), Swertia chirata (Chiraita – whole plant), Momordica charantia (Karela – fruit), Gymnema sylvestre (Gurmar – leaves), Bacopa monniera (Neer Brahami – whole plant), Andrographis paniculata (Kalmegh – whole plant), Picrorhiza kurrooa (Kutki – root, rhizome), Centella asiatica (Mandukparni – whole plant) and Alpinia galanga (Kulanjan – rhizome), were purchased from the local market Kharibawri, New Delhi, India under the local trade names. Authenticity of the purchased medicinal herbs was confirmed by the Taxonomist, Herbal Drug Research, Ranbaxy Laboratory Limited, Gurgaon and voucher specimens (registration number RAN/HDR/SSH/T/TC/01/06 for Tinospora cordifolia; RAN/HDR/SSH/T/AZ/01/06 for Adhatoda zeylanica; RAN/HDR/SSH/T/SC/01/06 for Swertia chirata; RAN/HDR/SSH/T/MC/01/06 for Momordica charantia; RAN/HDR/SSH/T/GS/01/06 for Gymnema sylvestre; RAN/HDR/SSH/T/BM/01/06 for Bacopa monniera; RAN/HDR/SSH/T/AP/01/06 for Andrographis paniculata; RAN/HDR/SSH/T/PK/01/06 for Picrorhiza kurrooa; RAN/HDR/SSH/T/CA/01/06 for Centella asiatica; RAN/HDR/SSH/T/AG/01/06 for Alpinia galanga) were deposited at the crude drug repository Herbal Drug Research, Ranbaxy Laboratories Limited, Gurgaon for future reference. The pharmacologically active parts as mentioned above for these medicinal herbs were taken and ground individually to make a coarse powder of 10-mesh size for further study.

Criteria for selection of these medicinal herbs

These medicinal herbs have been selected as they are widely used in different Ayurvedic and herbal formulations. These plants are found wild and so there is no control on exposure to environmental conditions. Furthermore, these are of varied habit and habitat. Some of these medicinal plants are shrubs; some are climbers. P. kurrooa grows at high altitudes whereas M. charantia grows in dry regions. B. monniera and C. asiatica are aquatic plants. Also different parts of these plants are pharmacologically active and used in medicines such as leaves, rhizome/roots, fruits, bark. The plants like S. chirata, A. paniculata, P. kurrooa, T. cordifolia have bitter principles and people in general have the perception that growth of A. flavus is not favored in such highly bitter plants.

Organisms

Filamentous fast-growing fungus A. flavus was procured from Prof. Kaushal K. Sinha, T. M. Bhagalpur University, Bhagalpur, Bihar, India. A. flavus was further identified in the laboratory through the mycological literature and maintained on potato dextrose agar (PDA) medium at 4 °C for further work.

Preparation of inoculum

Inoculum of A. flavus was prepared in the laboratory by multiplying the fungi on Pearl millet grains. The grains (150 g) were soaked in distilled water overnight and excess water was decanted, autoclaved twice (121 °C for 1 h) and inoculated with one agar disc (6 m diameter) cut from the margin of an actively growing 5 days old A. flavus on PDA plate. The flasks were incubated at 25 ± 2 °C in the dark for 15 days. Pearl millet seeds containing mycelial fragments plus spores served as inoculum. The population of A. flavus was recorded as 1.5 × 10⁷ colony forming unit (cfu) per Pearl millet seed by dilution plate technique.

Sample peparation

The selected ten medicinal herbs as coarse powder samples were examined for the determination of moisture ( %) using a 784 KFP Titrino Metrohm and water activity using an ERH meter, Rotronic Hygroskop DT as before and after autoclaved herbs with or without addition of water. The values are presented in [Table 1a] and [Table 1b].

For the evaluation of water activity and inoculation of fungi in medicinal herbs, twenty five conical flasks (150 mL) were taken individually and 10 g coarse powder of a medicinal herb were added to each one. These conical flasks were plugged with non-absorbent cotton and autoclaved at 121 °C for 20 min. Thereafter, sterilized distilled water was added sequentially 1 mL, 1.5 mL, 1.8 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL and 6.0 mL to each conical flask under aseptic condition, in duplicate except for one flask that served as control. The percentage of added moisture was determined by the formula X × 100/10 (where X is the volume of sterilized water added) to finally get 10 %, 15 %, 18 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, and 60 % of moisture ( %), respectively, in each conical flask for every medicinal herb. The content of each flask was mixed properly with a sterilized stainless steel spatula. Accordingly, the moisture ( %) in each conical flask for each medicinal herb will be the sum of the natural moisture present in the medicinal herb after autoclaving and the added moisture. For example, in the case of P. kurrooa, the moisture after autoclaving is 14.63 % and 10 % moisture is added, hence total moisture ( %) is 14.63 + 10 = 24.63 %. Hence for every medicinal herb at each temperature condition (10 ± 2 °C, 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C), the above needs to be repeated.

Water activity

Twelve conical flasks corresponding to different moisture ( %) as above were used for determination of water activity. Also the water activity was determined individually for each medicinal herb before autoclaving and before addition of water. The water activity was determined by using Equilibrium Relative Humidity (ERH) meter (Rotronic Hygroskop DT). The ERH meter was calibrated before use as it is pivotal to obtaining accurate and sensitive water activity a_w measurements [11]. The calibration was carried out using saturated salt solutions such as potassium sulfate, barium chloride, sodium chloride, magnesium nitrate and magnesium chloride which have known ERH ( %) or water activity, a_w at 25 ± 2 °C. For water activity determination approximately 3 – 5 g of each of the medicinal herb were taken and placed in the ERH meter cup and the reading was noted.

Inoculation of A. flavus in medicinal herbs

The remaining thirteen conical flasks containing medicinal herb (10 g) with different moisture ( %) such as 10 %, 15 %, 18 %, 20 %, 25 %, 30 % 35 %, 40 %, 45 %, 50 %, 55 %, and 60 % were used to inoculate the fungi A. flavus except for one flask that served as control. Pearl millet seeds (1.5 × 10⁷ cfu per seed of A. flavus) were inoculated individually in each conical flask under aseptic conditions. Conical flasks with different moisture ( %) as prepared above were incubated at different temperature conditions 10 ± 2 °C; 25 ± 2 °C; 30 ± 2 °C; 40 ± 2 °C for 5 – 15 days and observations were made in comparison to its corresponding control. After completion of the incubation period, fungal growth was visually observed and conical flasks were marked on the basis of their growth as no growth (absence of growth of A. flavus), low growth (<50 – 100 cfu/g), moderate growth (<10⁴ cfu/g) and rich growth (<10⁸ cfu/g). The colony forming unit of fungal colonies was enumerated in medicinal herbs by the dilution plate method on potato dextrose agar medium (PDA plate) after five days incubation at 22 ± 2 °C.

Extraction and confirmation of aflatoxin in medicinal herbs

The conical flasks with rich growth of A. flavus were selected for extraction and detection of aflatoxins. For extraction of aflatoxins, 75 mL of methanol were added into each conical flask, which was covered with aluminium foil and incubated in an incubator shaker at 12 ± 2 °C for 18 h at 115 rpm. Thereafter methanol was removed by filtration (Whatman No. 44) and the solvent was evaporated under a rotatory vacuum evaporator (Büchi). Concentrated material was collected in amber glass vials. Silica gel G TLC plates were developed with acetone : methanol 85 : 15 mobile phase. Bluish-fluorescent color spots were observed at the upper mid region of silica gel G plate under UV light at 365 nm. Trifluoroacetic acid (TFA) [12] was sprayed on the silica gel G plate and again observed under UV light at 365 nm. The bluish-fluorescent colored spots were changed to greenish-fluorescent colored spots confirming the presence of aflatoxins. Simultaneously aflatoxins standards were also run and R_f matched with that produced by the test solution.

Precautions and decontamination

Safety precautions were taken during sampling and extraction. This included wearing of N-95 respirator, goggles, rubber gloves and long laboratory coats. The work areas were mopped with 5 % sodium hypochlorite solution. Decontamination procedures for laboratory wastes of aflatoxin developed by the International Agency for Research of Cancer (IARC) were followed.

Results and Discussion

The minimal and optimal temperatures to support the growth of A. flavus and hence production of aflatoxins in the medicinal herbs were found to be 25 ± 2 °C and 30 ± 2 °C, respectively. Furthermore, A. flavus did not grow in any samples of medicinal herbs with water activity a_w below 0.81 at temperatures of 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C. Also A. flavus did not grow in any samples of medicinal herbs with water activity a_w above 0.81 when stored below 10 ± 2 °C. Growth was observed in medicinal herbs with water activity a_w above 0.84 when stored at 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C except for P. kurrooa and A. galanga ([Table 1a] and [Table 1b]). P. kurrooa was found to have anti-fungal properties as confirmed by zone of inhibition by crude herb and extract against A. flavus. An interesting observation that the growth of A. flavus was not favored in P. kurrooa under all test conditions, i.e, 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C. This is in agreement with the published literature [13], which reveals that picroliv present in P. kurrooa has hepatoprotective activity against aflatoxin B₁. It has been indicated in the literature that biochemical changes in rats induced by aflatoxin B₁ poisoning were significantly prevented by oral administration of Picrorhiza extract and this forms the basis for the hepatoprotective activity of this drug. Similar observations were made for A. galanga as it did not support the growth of A. flavus under all test conditions, i. e., 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C. This was further confirmed by a zone of inhibition observed by A. galanga crude herb and extract against A. flavus indicating its antifungal property.

The United States Pharmacopoeia [14] describes the application of water activity determination to non-sterile pharmaceutical products as a tool for the rationale of reducing the frequency of microbial limit testing and screening for objectionable microorganisms and emphasizes that reduced water activity will greatly assist in the prevention of microbial proliferation in pharmaceutical products.

The results of water activity and moisture ( %) were plotted (sorption isotherms) [15], [16], [17] and adjusted according to the following eleven mathematical models (best fit curve made):

 1) M = A*(W/(1 – W))**B Oswin

 2) M = A*(–LOG(W))**B Halsey

 3) M = A*(–LOG(1 – W))**B Henderson

 4) M = EXP(A*W + B) Miniowitsch

 5) M = A/(B – LOG(W)) Posnow

 6) M = A*W/(1 – B*W) Langmuir

 7) M = A*W/((1 – W)*(1 – B*W)) Brunauer et al. (BET)

 8) M = A*W/(1 – B*W)*(1 – C*W)) Anderson (GAB)

 9) M = A*(– LOG(W)**B + C Kuhn

10) M = A/(1 – B*W) + C Filononko-Chuprin

11) M = (A/(B – LOG(W)))**C Ferro-Fontan et al.

where: A, B, C: parameters; M: moisture; W: water activity.

The results for correlation coefficients (r) and the parameters (A, B and C) obtained with each of the eleven models for each of the ten medicinal herbs is presented ([Table 2a] and [Table 2b]). The calculations were made with the computer program, Statistical Analytical System (SAS) 8.2. Also the mean of correlation coefficient for each model for ten medicinal herbs was determined. It was observed that the Langmuir and Anderson (GAB) models were the best based on the greatest value of correlation coefficient (r) ([Table 3]).

Since no growth was observed in medicinal herbs with water activity below 0.81 so a perpendicular drawn on the best fit curve gives the corresponding moisture ( %) which would not support the growth of A. flavus and hence, the production of aflatoxins. The desired level of moisture ( %) can be obtained by drying the medicinal herb under vacuum at 45 ± 2 °C [18], [19]. The moisture ( %) of the various medicinal herbs which will not support the growth of microorganisms (water activity a_w below 0.81) and hence aflatoxins and also at the same time retain the quality is presented ([Table 4]). Thus sorption models have been used to demonstrate how the water activity changes with change in moisture ( %) and information on reduction of water activity will help to decide how much to dry. The optimum drying will reduce the cost involved in drying and also it will retain the quality of the medicinal herb.

The water activity in G. sylvestre and B. monniera was reduced to 0.43 – 0.45 by drying for 2 hours at 45 ± 2 °C under vacuum and no growth was observed at this water activity. This was further confirmed by inoculating A. flavus in dried B. monniera and G. sylvestre. It did not support the growth of A. flavus. Also at the same time, the TLC profiles of both the crude herbs before and after drying support the fact that the constituents including the marker compounds (bacosides for B. monniera and gymnemic acid for G. sylvestre) are retained after drying thus revealing that decreasing the moisture ( %) by drying at appropriate condition to a desired level will not support growth of A. flavus and would also retain the original properties of the medicinal herbs. From the sorption isotherms, it can be concluded that the moisture ( %) in B. monniera and G. sylvestre at levels 54 % and 32 % (water activity 0.81) respectively des not support growth of A. flavus, hence drying less than one hour at 45 ± 2 °C under vacuum to bring the moisture ( %) to the levels 54 % and 32 % in B. monniera and G. sylvestre, respectively, would be sufficient. Thus the incremental cost involved in extra drying which happened in the above study could be avoided. Alternatively, storage below 10 ± 2 °C will help to protect the crude herbs from aflatoxin contamination.

It can be concluded that determination of water activity gives more meaningful data than measurement of moisture ( %) in medicinal herbs. Therefore the specifications of medicinal herbs should include the test for water activity with its limit NMT 0.81 so that the growth of A. flavus is not favored. Any medicinal herb having a water activity more than this limit should be dried to moisture ( %) as obtained from the corresponding sorption isotherm at a water activity 0.81 and when the desired moisture level is achieved it can be stored at 25 ± 2 °C, 30 ± 2 °C and 40 ± 2 °C. In cases where the water activity cannot be reduced below 0.81, then the medicinal herbs should be stored in a storage area at below 10 ± 2 °C. In this way adequate control can be imposed on medicinal herbs so as not to allow conditions for growth of A. flavus and hence, production of aflatoxins.

Acknowledgements

The support and help rendered by Dr. Dusan Popovski, Faculty of Technical Sciences, University St. Clement Ohridski, Bitola, Macedonia in formulating the statistical models is gratefully acknowledged. Also we acknowledge the help rendered by Prof. Kaushal K. Sinha, T M Bhagalpur University, Bhagalpur, Bihar, India for providing the strain Aspergillus flavus.

References

• 1 Horie Y, Yamazaki M, Itokawa H, Kinoshita H. On the toxigenic fungi contaminating herbal drugs as raw materials in pharmaceutical industries.  Trans Mycol Soc Jpn. 1979;  23 435-7
  PubMedSearch in Google Scholar
• 2 Roy A K, Chaurasia H K. Aflatoxin problem in some medicinal plants under storage.  Int J Crude Res. 1989;  27 156-60
  PubMedSearch in Google Scholar
• 3 Roy A K. Mycological problems of crude herbal drugs overview and challenges.  Ind Phytopathol. 2003;  4 1-13
  PubMedSearch in Google Scholar
• 4 Urban-Diener L, Davis-Norman D. Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids by Aspergillus flavus in sterile peanuts.  J Am Chem Soc. 1967;  44 259-3
  PubMedSearch in Google Scholar
• 5 Christensen J AB. Water activity and the growth of microorganisms.  Biochem Biophys Food Res. 1957;  23 108-34
  PubMedSearch in Google Scholar
• 6 Semeniuk G. Storage of cereals grains and their products. Am Assoc Cereal Chemist St. Paul 1954: 77-151
  Search in Google Scholar
• 7 Austwick P KC, Ayerst G. Toxic products in groundnuts: groundnut microflora and toxicity.  Chem Ind (London). 1963;  2 55 -61
  PubMedSearch in Google Scholar
• 8 Diener U L, Davis N D. Aflatoxin production by isolates of Aspergillus flavus.  Phytopathology. 1966;  56 1390-3
  PubMedSearch in Google Scholar
• 9 Fennema O R. Water and ice in. Food chemistry, 3^rd ed. New York; Marcel Dekker Inc 1996: 1069
  Search in Google Scholar
• 10 Spices W EL, Wolf W R. The results of the cost 90. Physical properties of food London. Appl Sci 1983: 65-87
  Search in Google Scholar
• 11 Leonard S. Calibration of water activity measuring instruments and devices: collaborative study.  J Assoc Anal Chem. 1978;  61 1166-8
  PubMedSearch in Google Scholar
• 12 Przybylski W. Formation of aflatoxin derivatives on thin layer chromatographic plates.  J Assoc Anal Chem. 1975;  58 163-4
  PubMedSearch in Google Scholar
• 13 Rastogi R, Srivastava A K, Srivastava M, Rastogi A K. Hepatocurative effect of picroliv and silymarin against aflatoxin B₁ induced hepatotoxicity in rats.  Planta Med. 2000;  66 709-13
  Thieme ConnectPubMedSearch in Google Scholar
• 14 United States Pharmacopoeia. USP Commission 30 2007 1112: 586-8
  Search in Google Scholar
• 15 Roberta Z, Dasilva-Pedro M, Neves J O, Yamashita F, Santoro-Patricia H. Water sorption isotherms of Beauveria bassiana (Bals) Vuill. Conidia.  Neotrop Entomol. 2003;  32 347-50
  PubMedSearch in Google Scholar
• 16 Popovski D, Mitrevski V. Numerical experiments with the GAB models. Proceeding of the 30th international conference of SSCHE, Tatranske Matliare 26 – 30 May. 2003: 189
  Search in Google Scholar
• 17 Popovski D, Mitrevski V. A method for extension of the water sorption isotherm models.  Electron J Environ Agric Food Chem ISSN: 1579 – 4377. 2004;  3 799-3
  PubMedSearch in Google Scholar
• 18 Velezruiz J F, Lima-Carrera M E, Macedoy-Ramirez R C. Air drying of three aroma herbs (Basil, Dill and Tea herbs). Proceedings of the 14th International Drying Symposium (IDS 2004) Sao Paulo, Brazil; 2004
  Search in Google Scholar
• 19 Francelid S, Park K J, Magalhae P s, Melill Marina P. Desorption isotherms of Calendula officinalis L. Proceedings of the 14th International Drying Symposium (IDC 2004) Sao Paulo; 2004
  Search in Google Scholar

Rashmi Kulshrestha

Ranbaxy Laboratories Limited

Gurgaon (Haryana)

India

Phone: +91/124/419/4253

Phone: +91/124/427/8026

Fax: +91/124/234/3124

Email: rashmi.kulshreshtha@ranbaxy.com

References

• 1 Horie Y, Yamazaki M, Itokawa H, Kinoshita H. On the toxigenic fungi contaminating herbal drugs as raw materials in pharmaceutical industries.  Trans Mycol Soc Jpn. 1979;  23 435-7
  PubMedSearch in Google Scholar
• 2 Roy A K, Chaurasia H K. Aflatoxin problem in some medicinal plants under storage.  Int J Crude Res. 1989;  27 156-60
  PubMedSearch in Google Scholar
• 3 Roy A K. Mycological problems of crude herbal drugs overview and challenges.  Ind Phytopathol. 2003;  4 1-13
  PubMedSearch in Google Scholar
• 4 Urban-Diener L, Davis-Norman D. Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids by Aspergillus flavus in sterile peanuts.  J Am Chem Soc. 1967;  44 259-3
  PubMedSearch in Google Scholar
• 5 Christensen J AB. Water activity and the growth of microorganisms.  Biochem Biophys Food Res. 1957;  23 108-34
  PubMedSearch in Google Scholar
• 6 Semeniuk G. Storage of cereals grains and their products. Am Assoc Cereal Chemist St. Paul 1954: 77-151
  Search in Google Scholar
• 7 Austwick P KC, Ayerst G. Toxic products in groundnuts: groundnut microflora and toxicity.  Chem Ind (London). 1963;  2 55 -61
  PubMedSearch in Google Scholar
• 8 Diener U L, Davis N D. Aflatoxin production by isolates of Aspergillus flavus.  Phytopathology. 1966;  56 1390-3
  PubMedSearch in Google Scholar
• 9 Fennema O R. Water and ice in. Food chemistry, 3^rd ed. New York; Marcel Dekker Inc 1996: 1069
  Search in Google Scholar
• 10 Spices W EL, Wolf W R. The results of the cost 90. Physical properties of food London. Appl Sci 1983: 65-87
  Search in Google Scholar
• 11 Leonard S. Calibration of water activity measuring instruments and devices: collaborative study.  J Assoc Anal Chem. 1978;  61 1166-8
  PubMedSearch in Google Scholar
• 12 Przybylski W. Formation of aflatoxin derivatives on thin layer chromatographic plates.  J Assoc Anal Chem. 1975;  58 163-4
  PubMedSearch in Google Scholar
• 13 Rastogi R, Srivastava A K, Srivastava M, Rastogi A K. Hepatocurative effect of picroliv and silymarin against aflatoxin B₁ induced hepatotoxicity in rats.  Planta Med. 2000;  66 709-13
  Thieme ConnectPubMedSearch in Google Scholar
• 14 United States Pharmacopoeia. USP Commission 30 2007 1112: 586-8
  Search in Google Scholar
• 15 Roberta Z, Dasilva-Pedro M, Neves J O, Yamashita F, Santoro-Patricia H. Water sorption isotherms of Beauveria bassiana (Bals) Vuill. Conidia.  Neotrop Entomol. 2003;  32 347-50
  PubMedSearch in Google Scholar
• 16 Popovski D, Mitrevski V. Numerical experiments with the GAB models. Proceeding of the 30th international conference of SSCHE, Tatranske Matliare 26 – 30 May. 2003: 189
  Search in Google Scholar
• 17 Popovski D, Mitrevski V. A method for extension of the water sorption isotherm models.  Electron J Environ Agric Food Chem ISSN: 1579 – 4377. 2004;  3 799-3
  PubMedSearch in Google Scholar
• 18 Velezruiz J F, Lima-Carrera M E, Macedoy-Ramirez R C. Air drying of three aroma herbs (Basil, Dill and Tea herbs). Proceedings of the 14th International Drying Symposium (IDS 2004) Sao Paulo, Brazil; 2004
  Search in Google Scholar
• 19 Francelid S, Park K J, Magalhae P s, Melill Marina P. Desorption isotherms of Calendula officinalis L. Proceedings of the 14th International Drying Symposium (IDC 2004) Sao Paulo; 2004
  Search in Google Scholar

Rashmi Kulshrestha

Ranbaxy Laboratories Limited

Gurgaon (Haryana)

India

Phone: +91/124/419/4253

Phone: +91/124/427/8026

Fax: +91/124/234/3124

Email: rashmi.kulshreshtha@ranbaxy.com