Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

This thesis is an investigation of the potential of Allyl Isothiocyanate (AITC) derived from oriental mustard meal (Brassica juncea meal) as a natural preservative suppression moulds growth on bread. Currently, clean labels and natural antimicrobial agents are interests of alternative preservatives. In this study, an antimicrobial sachet/patch containing B. juncea meal was developed to produce AITC vapour in situ; the efficacy of gaseous AITC/B. juncea meal on suppression of Penicillium spp. and other mould growth was investigated. The growth was completely inhibited for 28 days at 23˚C in the presence of 0.7-1.3 ppm AITC in the headspace (released from 50-100 mg B. juncea meal). Fifty mg mustard meal showed fungistatic activity, and ≥100 mg were fungicidal. The shelf life of sliced white bread (600 g) was prolonged for 14 days using 3g of B. juncea meal at 23˚C thereby illustrating the potential of AITC as an alternative to chemical preservatives. / Developing Innovation Agricultural Products (DIAP) program of AAFC and Mustard 21 (RBPI 2109)