Browsing by Subject "Charakterisierung"

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Characterization and management of Jatropha curcas L. germplasm
(2018) Senger, Elisa; Melchinger, Albrecht E.
Jatropha curcas L. (jatropha) is a perennial plant of the Euphorbiaceae family that grows in the tropics and subtropics worldwide. Jatropha is targeted to be grown in marginal environments. The seeds are used mainly for production of food products and bioenergy, amongst others. Jatropha breeding is at an early stage. The first obstacle is to generate competitive cultivars for economically feasible cultivation. Mayor breeding objectives are to increase seed yield and yield stability, to decrease production costs, and to improve product quality adapted to specific markets. Jatropha breeding needs to be optimized in several research areas, such as methods and tools for germplasm characterization and breeding techniques, while considering requirements of the agronomic management and product processing. The germplasm can be separated into two naturally occurring germplasm pools that differ in the presence of phorbol esters (PE). These chemical compounds have antinutritional effects on humans and animals and cannot be inactivated or eliminated from the plant material on an industrial scale yet. Therefore, food production is based on cultivars lacking PE, while bioenergy production is less affected from PE presence. The germplasm needs to be characterized and grouped depending on breeding objectives and strategies. Tools for identification of plants that synthesize PE exist, but bear decisive disadvantages or need to be advanced. These tools are exploited for germplasm management and food safety strategies. The objectives of this study were to i) examine the variation of relevant traits among genotypes and between germplasm pools, ii) estimate phenotypic and genotypic trait correlations, iii) investigate location effects and genotype by environment interactions, iv) investigate parental and heterotic effects of genotypes from different germplasm pools as well as the effect of the mating type on expression of relevant traits, and v) develop recommendations for implementation of the findings in jatropha breeding programs. In the first two publications, stress response was investigated. Leaf chlorophyll content (SPAD) was used as a dynamic trait that can be influenced by e.g. water stress and nutrient deficiency. Different genotypes were screened at several locations and at different time points. High genetic diversity was found not only in stress response but also in SPAD value. The fast and non-destructive method is highly promising to be applied in further screenings or stress response studies. In the second publication, genotypic differences in aluminum tolerance were found among seedlings in a greenhouse trial. The rapid test method is applicable in further screenings. However, it needs to be proven that aluminum tolerance at the seedling stage observed under greenhouse conditions is expressed also at later plant developmental stages in the field. In the consecutive three publications, several traits were assessed on seeds and seedlings to detect significant differences between genotypes and/or between germplasm pools. Such traits would be highly valuable for germplasm management. We found that random variation is a disadvantage of quantitative traits and hinders clear assignment of each experimental unit to the respective germplasm pool. Thus, qualitative traits might be favored, such as the “silver shimmer inside the seed testa” that differentiated toxic from non-toxic seeds with a low error rate. However, these results need to be validated. Another application area of the investigated traits is the identification of self-fertilized material within hybrid progeny. In our study, self-fertilized seeds could be differentiated from cross-fertilized ones in specific genotype combinations. Similarly, many seedling traits showed heterotic effects. In the sixth publication, genotype by environment interactions were investigated and recommendations for breeding programs elaborated. A large set of genotypes was grown for four years at three different locations. We showed that selection at only one testing location is highly risky because cultivars with low yield stability could be selected. Therefore, it is indispensable for breeders to work in a network of testing locations that differ in edapho-climatic conditions and apply appropriate experimental designs and statistical tools. In the final publication, several parameters related to the nutritional value of kernels of non-toxic genotypes grown at two locations were assessed. The high nutritional value of this material was presented and compared to soybean, peanut, hazelnut, and corn. Furthermore, preliminary conclusions related to location effects and product processing were drawn. The findings of this thesis contribute to characterization of this novel crop with regard to stress tolerances, seed and seedling characteristics as well as food quality, and help to increase breeding efficiency by presenting simple methods for fast genotype screening as well as grouping of germplasm and by efficient exploitation of testing facilities.
Characterization and modulation of technofunctional properties of pea proteins
(2023) Moll, Pascal Bernd; Weiss, Jochen
Plant-derived ingredients for food formulation have gained increasing interest in recent years as animal products pose a higher burden on the environment. Among plant proteins, those from pea (Pisum sativum L.) are of particular interest because of their low allergenicity, low cost, high availability, and good reputation among consumers. However, the technofunctionality of pea proteins is often inferior to animal-derived proteins limiting a more widespread use in food products. These technofunctional properties include - among others - foaming, gelling, and binding of other ingredients and it depends on the food product, which functionality food scientists must utilize and optimize. Cost effective approaches to improve the technofunctionality of pea proteins are therefore desirable and would allow the industry to further implement the use of sustainable ingredients in foods. In line with these overall goals, the aim of the first section of this thesis was to characterize a commercial pea protein isolate and to modulate the physicochemical and technofunctional properties through homogenization for foaming application. The main goal of the second section was to mix pea proteins with pectin to obtain a suitable binder with desired properties for the application in meat alternatives. The mixing approach was based on previous research data that had shown that interacting protein-polysaccharide systems display a synergistic behaviour in terms of their functional properties. First section: Foams are two phase systems consisting of gas bubbles that are stabilized by surface-active ingredients such as proteins in the discontinuous, aqueous phase. The physico-chemical properties of proteins such as their solubility determines foaming performance. In Chapter I, a commercial pea protein isolate was fractionated into a water-soluble and a water-insoluble fraction for characterization. Although the two fractions were similar in protein composition, they showed distinct differences in physicochemical properties. For instance, the particle size of soluble pea proteins was around 40-50 µm at acidic pH (3-5), while no measurable particles were detected at neutral The insoluble pea proteins were large at pH 3 and 7 (> 80 µm) and ca. 40-50 µm close to their isoelectric point at pH 5. The results suggest that commercial pea protein isolates consisted of several fractions with differences in their physico-chemical properties. The yield of the water-insoluble fraction was higher and therefore used in Chapter II, where experimental results illustrated that dispersions of insoluble pea protein aggregates (5% w/w, pH 7) could be disrupted from 180 ± 40 µm (control) to 0.2 ± 0.0 µmm upon homogenization at pressures ≥ 125 MPa. This was attributed to a cleavage of intermolecular interactions such as disulphide bonds, hydrogen bonds, and hydrophobic interactions. The decrease in insoluble pea protein aggregate size was accompanied by an increase in solubility from 23 ± 1% to ≥ 80% that may be beneficial for its technofunctionality. Consequently, homogenization was applied to the same material at pH 3 and 5 with the aim of investigating its foaming performance in Chapter III. In general, unhomogenized dispersions of pea protein aggregates (5% w/w, pH 3 or 5) did not foam at both tested pH values due to large pea protein aggregates with low solubility and surface activity. At pH 3, the dissociation of pea protein aggregates into smaller, more soluble, and more surface-active proteins was responsible for a high foam capacity (FC = 360-520%) with medium foam stability as measured by drainage (FS = 19-30 min). Only a limited particle size reduction upon homogenization was observed at pH 5, which was close to the isoelectric point of the pea proteins. Nevertheless, the still large aggregates consisted of re-aggregated smaller protein particles that were able to form a smaller amount of rather stable foams with thick interfacial films (FC = 213-246%, FS = 32-42 min). Overall, homogenization of insoluble pea protein aggregates was shown to change its physicochemical properties thereby benefitting technofunctional properties such as foaming. Second section: Another technofunctionality of interest is binding of different structural elements in e.g., meat alternatives. For this, the binder must be i.) sticky to glue heterogeneous components together and ii.) able to readily solidify upon further processing thereby ensuring a coherent bulk matrix. In Chapter IV, the influence of pH (3.50, 4.75, 6.00) and biopolymer concentration (17.5-50.0% w/w) on the stickiness of a pea protein isolate – apple pectin mixture (mixing ratio r = 6:1) was investigated. It was found that biopolymer concentrations of 17.5-20.0% w/w led to low stickiness due to a lack of cohesive forces (WoA = 0.29-0.51 mJ). At high biopolymer concentrations of 40-50% w/w, the biopolymer mixtures were also not sticky because of adhesion being limited (WoA = 0.02-0.05 mJ). There was a good balance of adhesion and cohesion that facilitated a high stickiness (WoA = 0.48-0.65 mJ) at intermediate concentrations of 25-30% w/w, which was also indicated by a viscoelastic behavior (G’ ≈ G’’). At those concentrations, the mixtures at pH 6 were stickier due to increased swelling of the pea proteins. The importance of viscoelasticity for stickiness of biopolymer mixtures was confirmed in Chapter V, where pea protein isolate and apple pectin (25% w/w, pH 6) were mixed in different ratios r. Mixtures of pea protein and apple pectin and particularly the sample with r = 2:1 possessed high stickiness due to the development of a multiphase morphology that allowed for a good balance of adhesion and cohesion with distinct frequency dependency. Pea protein alone (r = 1:0, c = 25% w/w) had an elastic but soft texture with low stickiness due to limited viscous properties, whereas a sample solely consisting of apple pectin (r = 0:1, c = 25% w/w) was also not sticky because of its high cohesion and stiffness. The results of Chapter VI revealed that pea protein homogenization prior to mixing with apple pectin led to smaller protein particles in the blend that contributed to a higher cohesive strength. Interestingly, vacuum-dried pea proteins resulted in a higher network strength as this drying method prevented reaggregation of small protein particles to a higher extent as compared to freeze-drying. Overall, the mixture with homogenized and vacuum-dried pea proteins was nearly twice as sticky as the mixture with untreated pea proteins. In Chapter VII, sticky mixtures of different pea protein preparations (soluble, homogenized and unhomogenized pea proteins) and pectin (25% w/w, pH 6, r = 2:1) were tested for their ability to solidify upon different treatments, namely heating as well as the addition of transglutaminase, laccase, calcium, and combinations thereof. Calcium was found to facilitate crosslinking of pectin chains and thus induced solidification of the mixtures. For instance, the consistency coefficient K’ increased from 2800 ± 1000 Pasn for pea protein isolate – apple pectin mixtures to around 19000 Pasn when calcium was added. Heat treatment and transglutaminase did not lead to solidification indicating that pectin made up the continuous phase. Furthermore, laccase led to the highest degree of solidification when sugar beet pectin was used (K’ > 30000 Pasn) due to ferulic acid and pea protein tyrosine crosslinking. Consequently, the sticky mixture of pea protein and sugar beet pectin (25% w/w, pH 6, r = 2:1) with the addition of laccase for solidification was identified as the most suitable binder for a bacon type meat analogue, which was the object of the study carried out in Chapter VIII. This binder had the highest binding strength (W = 2.0-4.3 mJ) between textured protein, fat mimic, and both layers at 25 °C due to the introduction of covalent bonds by laccase within the binder and between the binder and the adherends. A control sample without laccase addition had lower binding properties (W = 0.7-1.0 mJ) and the binding strength of a methylcellulose hydrogel (6% w/w) serving as benchmark was only higher between two fat mimics at 70 °C (W = 1.8 ± 1.1 mJ) due to increased hydrophobic forces. Finally, the pea protein – sugar beet pectin binder (22.5% w/w, pH 6, r = 2:1) was tested in burger patty type meat analogues to glue textured vegetable protein and fat particles together (Chapter IX). The binder system did not influence the hardness of the burger patties suggesting that this property was governed by the structural elements and not the binder. However, the cohesiveness as determined by sensory analysis was found to be superior when the pea protein – sugar beet pectin binder was used (-0.7 ± 0.2) as compared to the methylcellulose benchmark (-2.9 ± 0.3). This was attributed to the sticky character of the biopolymer mixture that enabled improved binding of the different structural elements. Overall, this novel binder based on plant-derived ingredients was shown to be applicable in different meat alternatives. Last, Chapter X reviewed the functionality and binding mechanism of currently used binders in foods and showed that stickiness, hardening/solidification, and water holding capacity are of great importance. In many food products, the binder transitions from a sticky food glue to a solid matrix triggered by different process operations that depend on the characteristics of the applied binder. From the presented results, it can be concluded that pea proteins are useful functional ingredients in various application scenarios. The desired technofunctionality can be improved through different process operations such as fractionation, homogenization, or mixing with other plant-derived ingredients. For this, knowledge regarding structure-function relationship and other influential factors is needed. In some cases – such as in binders – process operations must be well orchestrated to induce structural transitions and therefore changes in functionality at the desired time during manufacturing. Overall, the results of this thesis contributed to a better understanding for a more widespread use of pea proteins to promote a more sustainable food system. The appended graphical abstract summarizes the key steps undertaken in this thesis to come to this conclusion.
Characterization of the rehydration behavior of food powders
(2019) Wangler, Julia; Kohlus, Reinhard
The rehydration behavior of food powders is of high importance in terms of powder processing and product quality. Rehydration of powders mainly depends on the physical powder characteristics particle size, porosity and wettability, the latter being expressed by the contact angle between solid and rehydrating liquid. With focus on food powders, it could be shown that the rehydration behavior is strongly influenced by dynamic changes of these physical characteristics. This includes the initiation of dissolution and swelling directly after powder-liquid contact. Especially in case of biopolymers, which were investigated in detail by the example of xanthan gum, guar gum and alginate, these processes are important to describe their rehydration behavior. Due to the special characteristics of these biopolymers dissolution and swelling result in an increase of viscosity as well as in a decrease of bulk porosity. The kinetics and interactions of these processes significantly affect the individual steps of rehydration and have to be considered in describing the process of food powder rehydration. For inert powder-liquid systems capillary liquid uptake into a powder bulk can be described by the Washburn equation which equates the capillary pressure and the hydrodynamic flow resistance. This approach was used as basic equation to describe capillary liquid uptake of food powders. The validity of the original approach is restricted to the case of constant powder and liquid properties. With regard to food powders, changes within the powder-liquid system were considered by a stepwise adaption of the variables of the Washburn equation. Thus, the first part of this thesis focused on establishing and defining methods to characterize the dynamics of the physical properties particle size, bulk porosity, viscosity and contact angle. This enabled a more detailed characterization of the interactions between food powder and liquid during rehydration. Wettability of food powders in contact with dist. water was assessed by contact angle measurements. Contact angles were 52° for alginate, 58.1° for xanthan gum and 70° for guar gum which confirmed their hydrophilic character. To describe the change of the bulk porosity a rheological measurement set-up was constructed to quantify the swelling behavior. Influence of viscosity on rehydration was determined by measuring the concentration dependent viscosity increase and the rate of viscosity increase over time. The change of viscosity as a consequence of dissolution allowed conclusions about the dissolution rate of biopolymers in highly concentrated situations. These results indicated that rehydration of guar gum is mainly influenced by viscosity effects whereas swelling has the highest impact on the rehydration behavior of xanthan gum and alginate. Further methods such as Nuclear Magnetic Resonance analysis enabled a more detailed characterization concerning the dynamics of powder-liquid interactions and the strength of water binding within these biopolymer gels. The strength of water binding was found to correlate with the stability of highly concentrated biopolymer aggregates. The aggregate stability was determined by rheological analyses and is of importance, particularly with regard to powder dispersability. To predict food powder rehydration, a model was established using a VoF approach. To simulate capillary liquid rise based on physical characteristics, dynamic changes were resolved both spatial and temporally. To describe particle and liquid properties more precisely, a model system consisting of biopolymer coated glass beads was developed by fluid bed technology. By the variation of the coating layer thickness and the coating material, dynamic changes within the system could be controlled which enabled a more differentiated description. A parameter variation study was conducted to simulate the influence and interaction of dynamic processes on capillary liquid uptake into such powder systems. Capillary liquid uptake into the coated glass beads was investigated experimentally. It could be shown that even with coating layers of 0.5 µm dynamic effects are sufficiently strong to cause a stop of capillary liquid uptake. It has been shown that viscosity development dominates guar gum rehydration whereas swelling is the prevalent mechanism in xanthan gum and alginate rehydration. Simulation of capillary liquid rise demonstrated that the influence of the coating layer thickness is not significant. This result could be explained by the slow dissolution rates of the biopolymer samples. Calculations indicated that even a coating layer of 0.5 µm could only be dissolved partially after a dissolution time of 250 s. This explains the little impact of coating layer thickness on viscosity development and thus on capillary liquid uptake. Further explanations focus on biopolymer swelling. Simulation showed that coating layers of 0.5 µm are sufficient to cause swelling-induced pore-blocking conditions.
Isolation, characterization and potential agro-pharmaceutical applications of phorbol esters from Jatropha curcas oil
(2012) Devappa, Rakshit K.; Becker, Klaus
Biodiesel is generally prepared from renewable biological sources such as vegetable oils by transesterification. Jatropha curcas seed oil is a promising feedstock for biodiesel production. During biodiesel production from Jatropha oil, many co-products such as glycerol, fatty acid dis-tillate and seed cake, among others, are obtained. The efficient use of these co-products would enhance the economic viability of the Jatropha based biofuel industry. However, the possible presence of phorbol esters (PEs) in these co-products restricts their efficient utilization. During biodiesel production, Jatropha oil is subjected to many treatments (stripping, degumming and esterification) wherein PEs present in the oil undergo partial or complete destruction depending on the treatment conditions. One of aims of this study was to develop and integrate methodolo-gies for using the PEs as a value added product instead of simply allowing them to be destroyed during biodiesel production. Potential uses of the phorbol ester enriched fraction (PEEF), ob-tained from Jatropha oil in agro-pharmaceutical applications were also investigated. The reason for choosing this group of compounds (PEs) was that they are highly bioactive both in vitro and in vivo, but they are currently considered to be merely toxic, unwanted biomaterial in the Jatro-pha biodiesel production chain. The recent increase in the cultivation of Jatropha cultivation means that there are potentially huge quantities of PEs that could be used for many purposes. This study revealed that a large proportion (85.7%) of PEs was localized in the endosperm portion of the Jatropha seed. Interestingly, the kernel coat contained PEs in high concentration. The endosperm portion of the kernel also contained antinutritional factors such as phytate (96.5%) and trypsin inhibitor (95.3%). The presence of high levels of antinutritional/toxic com-ponents in the kernel was presumed to be one of the factors that protect Jatropha seeds against predatory organisms during post harvest storage. Based on the presence or absence of PEs, a qualitative method was developed to differentiate between toxic/nontoxic Jatropha genotypes. In this method the methanol extract of seeds is passed through a solid phase extraction (SPE) column and the absorption (280 nm) of the result-ing eluate is measured. After screening Jatropha seeds collected from different parts of the world for toxic and non-toxic genotypes using the pre-established HPLC method for PEs, a cut off value of the absorbance was set up to differentiate toxic and nontoxic genotypes. Raw kernels whose SPE eluates had an absorbance ≥0.056 were considered as toxic and ≤0.032 as nontoxic. Corresponding absorbances for the SPE eluates of defatted kernel meal were ≥0.059 (toxic) and ≤0.043 (nontoxic). However, confirmation of the presence of PEs especially in Jatropha products for food applications should be carried out using the pre-established and validated HPLC method. The developed qualitative method could find its applications for screening the toxicity of products and co-products obtained from the Jatropha biodiesel industry. Conditions were optimized for the extraction of PEs as a phorbol ester enriched fraction (PEEF) from Jatropha oil using methanol as a solvent and a magnetic stirrer/Ultra-turrax as ex-traction tools. The extent of PE reduction in Jatropha oil was >99.4% using methanol as the sol-vent. The PEEF obtained (48.4 mg PEs/g) was 14 fold higher in PEs than in the original oil and this fraction was highly bioactive as determined by the most sensitive snail bioassay (LC100, 1 ppm) (see below). As the removal of PEs from oil took 60 min, which might be considered a long time in an industrial process, further conditions were optimized to extract maximum PEs in the shortest possible time with minimum solvent. The tools used for PE extraction (Ultra-turrax and magnetic stirrer) were effective with a treatment time of 2 and 5 min, resulting in 80 and 78% extraction of PEs, respectively. The biodiesel prepared from both the residual oils met European (EN 14214:2008) and American biodiesel standard (ASTM D6751-09) specifications. It was evident from the study that PEs could be easily extracted by either of the two methods with a high yield and the residual oil could be processed to produce high quality biodiesel. Also the residual oil with a lower PE content is expected neither to harm the environment nor the workers who had to handle it. The extracted PEEF was evaluated for its agricultural potential as a bio control agent. The PEEF had a high biological activity in aquatic bioassays using snails (Physa fontinalis), brine shrimp (Artemeia salina) and daphnia (Daphnia magna), when compared with microorganisms. The EC50 (48 h) of the PEEF was 0.33, 26.48 and 0.95 ppm PEs for snail, brine shrimp and daphnia respectively. High MIC (minimum inhibitory concentration) values (≥215 ppm) and EC50 values (≥58 ppm) were obtained for both the bacterial and fungal species. Among the bio-assays tested, the snail bioassay was the most sensitive, producing LC100 at 1 μg of PEs/ml. The snail bioassay could be used to monitor the presence of PEs in various Jatropha derived products, contaminated soil and other matrices in the ecosystem that might be involved in the production or use of Jatropha and its products. The study also demonstrated that the PEs exhibit molus-cicidal, antifungal and antibacterial activities. The shelf life of the PEEF was investigated. The PEEF was more susceptible to degradation when stored at room temperature (50% degradation after 132 days) than when stored at 4 °C or -80 °C (8% and 4% degradation respectively). Similarly, the PEEF lost biological activity (the snail bioassay) more rapidly at room temperature becoming ineffective after 260 days; while at 4 °C and -80 °C, only 27.5% and 32.5% activity was lost after 870 days. The degradation of PEs was due to auto-oxidation. Changes in fatty acid composition, increase in peroxide value and decrease in free radical scavenging activity of the PEEF reflected the auto-oxidation. Inclusion of antioxidants as additives (butylated hydroxyanisole (BHA), Baynox and α-tocopherol) pro-tected the PEs against degradation. The study demonstrated that the PEEF was susceptible to oxidation and addition of antioxidant stabilised the PEs during storage. In soil, PEs present in both the PEEF (2.6 mg/g soil mixture) (silica was used to adsorb PEs) and Jatropha seed cake (0.37 mg/g soil mixture) were completely degraded as the temperature and moisture content of the soil increased. PEs from silica-bound PEEF were completely de-graded after 19, 12, 12 days (at 13% moisture) and after 17, 9, 9 days (at 23% moisture) at room temperature (22 −23°C), 32 °C and 42 °C respectively. Similarly, at these temperatures, PEs from seed cake were degraded after 21, 17 and 17 days (at 13% moisture) and after 23, 17, and 15 days (at 23% moisture). The toxicity of PE-amended soil extracts when tested using the snail bioassay decreased with the decrease in PE concentration. The study demonstrated that PEs pre-sent in the PEEF or Jatropha seed cake are completely biodegradable in soil and the degraded products are innocuous. In preliminary studies, the PEEF exhibited potent insecticidal activity against Spodoptera frugiperda, which is a common pest in corn fields damaging maize crop across the tropi-cal/subtropical countries such as Mexico and Brazil. The PEEF produced contact toxicity with an LC50 of 0.83 mg/ml (w/v). The PEEF at higher concentration (0.25 mg/ml, w/v) also reduced food consumption, relative growth rate and food conversion efficiency (FCE) by 33%, 42% and 38% respectively. The study demonstrated that the PEEF has a potential to be used as a bio-control agent. Further in-depth field experiments on the effects of the PEEF on S. frugiperda will pave the way for its use under field conditions. The pharmaceutical potential of Jatropha PEs was also investigated. The PEs from Jatropha oil were purified. At least six purified PEs (designated as factors C1 to C6) were present in Jatropha oil. The identities of the purified PEs (factors C1 and C2) were confirmed by NMR. Whereas, factor C3 and factors (C4 + C5) were both obtained as mixtures. However, comparison of peak areas for phorbol 12-myristate 13-acetate (PMA) and Jatropha factor C1 in the HPLC method showed a difference in sensitivity of absorption at 280 nm of 41.3 fold. All the individual purified Jatropha PEs (factors C1, C2, C3mixture and (C4+C5)) and PEs-rich extract (factors C1 to (C4 + C5)) were biologically active when tested in the snail and brine shrimp bioassays. In ad-dition, all the Jatropha PEs produced platelet aggregation in vitro with an effective order of (based on ED50 (μM)): Jatropha factor C2 < factor C3mixture < factor C1 < factor (C4+C5). The PEs-rich extract (contains factor C1 to C6) was toxic to mice upon intra gastric administration, with an LD50 of 27.34 mg/kg body mass as PMA equivalent or 0.66 mg/kg body mass as factor C1 equivalent. The prominent histopathological symptoms were observed in lung and kidney. The Jatropha purified PEs-rich extract, purified PEs (factor C1, factor C2, factor C3mixture and factors (C4+C5)) and toxic Jatropha oil produced severe cellular alterations/disintegration of the epithelium and also increased the inflammatory response (interleukin-1α and prostaglandin E2 release) when applied topically to reconstituted human epithelium (RHE) and human corneal epithelium (HCE). In RHE, the nontoxic oil (equivalent to the volume used for toxic oil) pro-duced a lower cellular and inflammatory response than the toxic oil and the response increased with an increase in concentration of the PEs. In HCE, nontoxic oil (equivalent to the volume used for toxic oil) produced marked cellular alterations. The study demonstrated that the pres-ence of PEs in Jatropha oil increased the toxicity, both towards RHE and HCE. In addition, all the purified Jatropha PEs gave positive responses in the tumour promotion assay and negative responses in the tumour initiation assay in vitro (the assay was based on foci formation in Bhas 42 cells). In the tumour promotion assay, the order of transformed foci/well formation was: PEs-rich extract > factor (C4+C5) > factor C3mixture > factor C1 > factor C2. The tumour promotion ac-tivity was mediated by the hyper activation of protein kinase C (PKC). The aforementioned studies demonstrated that Jatropha PEs are toxic when administered orally or when applied topically to the skin or eye tissues. The data obtained should help in establishing safety measures for people working with Jatropha PEs. The potential of Jatropha PEs as a feedstock intermediate for the synthesis of Prostratin, a promising adjuvant in anti HIV therapy, was evaluated. The studies demonstrated that the Jatro-pha PEs could be synthesized sequentially by converting them first to crotophorbolone and then to prostratin. As analyzed by Nano-LC-ESI-MS/MSR, the prostratin synthesized from Jatropha PEs had similar mass and peak retention time to the reference prostratin (Sigma, St. Louis), The study showed that prostratin could be synthesized from Jatropha PEs. However, further optimi-zation studies are required to ascertain the synthesis reactions and yield of prostratin synthesized from Jatropha PEs. Some of the preliminary requirements for any successful bio-control agent are that it should have a high bioactivity on the target organism, a long shelf-life and a high biodegradability in soil. In addition, the bioactive phytochemical should be available in large quantities, it should be easily extractable and continuously available. The PEEF potentially satisfies these aforesaid re-quirements. The abundance and novelty of PEs present in Jatropha species could form a new ?stock? for the agro-pharmaceutical industries. Considering the projected oil yield of 26 million tons/annum by 2015 (GEXSI, 2008), huge amount of raw materials will be available for both biodiesel and pharmaceutical industries. PEs in the form of the PEEF could be used either as in-sect controlling agents in agricultural applications or as a ?stock? biomaterial for synthesizing prostratin in pharmaceutical applications.
Plasmopara viticola, the downy mildew of grapevine : phenotypic and molecular characterization of single sporangium strains infecting hosts with different resistance levels
(2015) Gómez Zeledón, José Javier; Spring, Otmar
The downy mildew of grapevine, Plasmopara viticola, is one of the most important pathogens in viticulture. Its genetic diversity had been assessed in some previous studies using molecular markers, but the diversity of the infection behavior has not yet been addressed adequately. Therefore, the development of a fast, reliable and uncomplicated assay to screen for pathogen phenotypes on host with different resistance levels was a major task of this work. A leaf disc test was proposed, evaluating sporulation and necrosis produced by the pathogen on Vitis plants with different susceptibility. Using this bioassay, interesting strains were assessed and kept for future studies. The urgent need to work with genetic homogeneous inoculum was shown, because the assays revealed a high phenotypic diversity in isolates collected from the field as a bulk sample. Hence, a cloning technique to obtain single sporangium strains was found useful to avoid working with mixed genotypes. The leaf disc bioassay also allowed screening for fungicide resistance in P. viticola populations. Isolates resistant to dimethomorph and metalaxyl, two important fungicides for oomycetes control, were detected. Higher resistance was associated with fields were the fungicide application was high as well. Some strains were even resistant to doses where the fungicide exhibits phytotoxic activity to grapevine. The approach of characterizing P. viticola pathotypes on different host plants of Vitis vinifera cultivars and Vitis species from North America and Asia revealed a broad spectrum of fully susceptible to completely resistant reactions. This information is of direct practical value in future plant breeding programs, but also provides the chance to select specific host-pathogen combinations to study the mechanisms of resistance or susceptibility. Fluorescence microscopy revealed how the infection progress of highly and lowly virulent strains advance in tolerant and susceptible hosts, and which points of the infection are interesting for future studies. On the molecular level, effectors were investigated to trace their possible involvement in the infection process. It was found that RXLR 1, NLP 1, Elicitin like 2, Glucanase inhibitor 2 and 4 , and 1,3-ß Glucanase 2 are candidates which are upregulated in the earliest infection stages. Following the here established methodology and suggested strategy it should be possible in the future to get a better insight in the mechanisms of infection and resistance of grapevine downy mildew.