Mridusmita Chaliha

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 degrees C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 degrees C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.

Key Role of Fe2+ in Epithiospecifier Protein Activity

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe(2+) is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe(2+) requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.

Effect of Packaging Materials and Storage on Major Volatile Compounds in Three Australian Native Herbs

Lemon myrtle, anise myrtle, and Tasmanian pepper leaf are commercial Australian native herbs with a high volatile or essential oil content. Packaging of the herbs in high- or low-density polyethylene (HDPE and LDPE) has proven to be ineffective in preventing a significant loss of volatile components on storage. This study investigates and compares the effectiveness of alternate high-barrier property packaging materials, namely, polyvinylidene chloride coated polyethylene terephthalate/casted polypropylene (PVDC coated PET/CPP) and polyethylene terephthalate/polyethylene terephthalate/aluminum foil/linear low-density polyethylene (PET/PET/Foil/LLDPE), in prevention of volatile compound loss from the three native herbs stored at ambient temperature for 6 months. Concentrations of major volatiles were monitored using gas chromatography-mass spectrometry (GC-MS) techniques. After 6 months of storage, the greatest loss of volatiles from lemon myrtle was observed in traditional LDPE packaging (87% loss) followed by storage in PVDC coated PET/CPP (58% loss) and PET/PET/Foil/LLDPE (loss of 23%). The volatile loss from anise myrtle and Tasmanian pepper leaf stored in PVDC coated PET/CPP and PET/PET/Foil/LLDPE packaging was <30%. This study clearly indicates the importance of selecting the correct packaging material to retain the quality of herbs with high volatile content.

Sensory quality of soymilk and tofu from soybeans lacking lipoxygenases

Abstract The oxidation of unsaturated lipids by lipoxygenases in soybeans causes undesirable flavors in soy foods. Using a traditional and a nontraditional soy food user group, we examined the cultural difference in perceiving the sensory characteristics of soymilk and tofu produced from soybeans with or without lipoxygenases (Lx123). The two groups described the samples using similar terms. The traditional users preferred the control soy milk and lipoxygenase‐free tofu while the nontraditional users preferred the lipoxygenase‐free soymilk with no preference for tofu. In a separate study, a trained descriptive taste panel compared the odor of soymilk and tofu from control soybeans or those lacking lipoxygenase‐1 and lipoxygenase‐2 (Lx12) or all three isomers (Lx123). The rancid/grassy odor was rated the lowest in Lx123 products, followed by Lx12 products with the control products given the highest rating. The Lx12 and Lx123 products were also sweeter and less bitter than the controls. Taken together, our results demonstrated that soybeans lacking lipoxygenases can produce soy foods with less undesirable aromas and are therefore likely more acceptable to the consumers.

Organic acids in Kakadu plum (Terminalia ferdinandiana): the good (ellagic), the bad (oxalic) and the uncertain (ascorbic)

The phenolic ellagic acid (EA) is receiving increasing attention for its nutritional and pharmacological potential as an antioxidant and antimicrobial agent. The Australian native Kakadu plum (Terminalia ferdinandiana) fruit is an abundant source of this phytochemical. The fruit also contains large amounts of vitamin C (mainly as ascorbic acid, AA) and possibly the undesirable oxalic acid (OA). Regular consumption of high oxalate foods poses a variety of health risks in humans including interference with calcium absorption and kidney stone formation. Oxalate is also the end-product of AA metabolism so that consumption of fruit with heightened AA content has the potential to elevate urinary oxalate levels. The aims of this study were to investigate the distribution of EA and the presence of other bioactives in other Kakadu plum tissues. Chemical analysis of Kakadu plum fruit and leaves for EA (free and total), OA (water-soluble and total), calcium (Ca) and AA indicated that EA and AA concentrations were high in the fruit while the leaves had significantly higher EA levels but little or no detectable AA. OA content in fruit and leaves was substantial with the fruit being placed in the high-Oxalate category. These findings suggest that there is potential to elevate oxalate levels in the urine of susceptible people and intake of fruit-derived products should be closely monitored. By measuring tissues collected from specific trees, high EA-producing or low OA-containing individuals were identified.

Bioactive rich extracts from Terminalia ferdinandiana by enzyme-assisted extraction: a simple food safe extraction method

A food grade compatible enzyme assisted extraction (EAE) technique for extracting bioactive compounds from freeze-dried Kakadu plum puree was evaluated. To optimise the extraction, a central composite rotatable design (CCRD) was conducted and effects of solvent concentration, enzyme concentration and time of reaction on extracted levels of free ellagic acid (fEA), ascorbic acid (AA) and total phenolic content (TPC) were determined. In the extracts, concentration of fEA ranged from 53.6 to 266.6 mg/100 g dry weight (DW) of Kakadu plum puree; AA 63.7 to 112.1 mg/100 g DW and TPC levels of 73.23 to 104.74 mg of gallic acid equivalent (GAE)/g. Extraction yield of fEA ranged from 10.3 to 51.3%. The model was found to be suitable for extraction of fEA - an important bioactive compound with documented antimicrobial properties from Kakadu plum fruit. A solvent (propylene glycol) concentration of 1.5% (w/w), enzyme (pectolytic enzymes) concentration of 300 mg/L and extraction time of 15 h was ascertained as optimum for the fEA extraction delivering a yield of 51.3%. The extraction method described here facilitates the provision of a simple, cost effective food-grade compatible extract that by-passes the need for organic solvents thereby obtaining an EA-rich aqueous extract with enhanced biological activities. This simple extraction method can also be applied to other EA rich plant material like pomegranate and peel of many common fruits which are generated as food processing by-products and can be easily adopted by numerous industries.

Physico-chemical characteristics and fungal profile of four Saudi fresh date (Phoenix dactylifera L.) cultivars

Physico-chemical and microbial analyses of four commercial fresh date cultivars (Sukari, Barhi, Khalas and Rothana) grown in Saudi Arabia were evaluated. Colorimetric assay indicated that Sukari had the highest total sugar content of 13.21g/100gFW while the lowest was in Rothana with 7.96g/100gFW. Total phenolic content (TPC) ranged from 76.74 to 122.20mgGAE/100gFW in Barhi and Rothana respectively, whereas antioxidant activity was highest in Sukari (105.99μgGAE/gFW) and lowest in Khalas (90.81μgGAE/gFW). The dominant fungal genera were Aspergillus sp., Rhizopus sp., Penicillium sp. and Sarocladium sp., occurring at 37, 18, 13 and 12% respectively. The highest fungal occurrence was in Barhi (30%) followed by Sukari (29%), Khalas (26%) and Rothana (15%). The TPC had a negative correlation with fungal occurrence whilst the total sugars had a positive correlation.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

ABSTRACT In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest. Graphical Abstract

Measuring free ellagic acid: influence of extraction conditions on recovery by studying solubility and UV-Visible spectra

Recent investigations into plant tissues have indicated that the free form of the natural polyphe-nolic antioxidant, ellagic acid (EA), is much more plentiful than first envisaged; consequently a re-assessment of solvent systems for the extraction of this water-insoluble form is needed. As EA solubility and its UV-Vis spectrum, commonly used for detection and quantification, are both governed by pH, an understanding of this dependence is vital if accurate EA measurements are to be achieved. After evaluating the pH effects on the solubility and UV-Vis spectra of commercial EA, an extraction protocol was devised that promoted similar pH conditions for both standard solutions and plant tissue extracts. The extraction so devised followed by HPLC with photodiode-array detection (DAD) provided a simple, sensitive and validated methodology that determined free EA in a variety of plant extracts. The use of 100 % methanol or a triethanolamine-based mixture as the standard dissolving solvents were the best choices, while these higher pH-generating solvents were more efficient in extracting EA from the plants tested with the final choice allied to the plants’ natural acidity. Two of the native Australian plants anise myrtle (Syzygium anisatum) and Kakadu plum (Terminalia ferdinandiana) exhibited high concentrations of free EA. Furthermore, the dual approach to measuring EA UV-Vis spectra made possible an assessment of the effect of acidified eluent on EA spectra when the DAD was employed.

Measuring the three forms of ellagic acid: Suitability of extraction solvents

Accurate quantification of ellagic acid and its derivatives, ellagic acid glycosides and ellagitannins, present in plant-based foods is a vital prerequisite for any study of their health-promoting properties. This goal is impeded by the lack of commercially available standards and the fact that these three forms differ widely in solubility. This disparity necessitates careful attention being paid to the choice of extraction solvents to ensure that precise and reproducible content measurements are achieved. This work sought to devise an extraction protocol that is effective for all ellagic acid forms whilst keeping the water-insoluble free ellagic acid solubilised during all analysis stages. To overcome this unavailability of commercial standards, the designated “targeted” ellagic acid derivatives identified in the selected fruit were monitored during the course of extraction that employed a number of commonly used solvents. Large variations in the extraction yield of the solvents tested for the ellagic acid and its derivatives were identified, extending even to the different fruit samples for the same form. It is regarded as unlikely that any selected extraction solvent could be universally employed to effectively extract all the ellagic acid compounds; however, the use of the solvent 50:50 vol. methanol-dimethylformamide satisfied most requirements.