Qualitätsanalytik im Dienste der Ölpflanzenzüchtung

Analytical Methods for Quality Traits in Plant Breeding In plant breeding programs selection for quality traits usually requires two kinds of methods, accurate quantitative methods for precise determination and rapid screening methods for mass selection. The demands on all methods are acceptable sensitivity and precision besides low costs and short times per analysis. In screening programs for fat and protein content NMR and NIR analysis are methods of choice. By own experiences, pyrolysis gaschromatography is a very useful tool for the determination of protein content. Rapid analysis of crude fibre content is still an unsolved problem, although there are many improvements of corresponding laboratory equipment. Paperchromatography is a common rapid method for fatty acid analysis. For quantitative determination gaschromatographic methods are very useful, especially in combination with automatic sample injection techniques. Some unsolved difficulties still exists with the analysis of glucosinolates. Screening-methods are based either on the colour formation of intact GSL molecules with palladium reagent or on the determination of enzymatically released glucose following myrosinase degradation of intact glucosinolates. Hitherto, quantitative determination of glucosinolates is based on gaschromatography as well. Unfortunately, these methods lack in determination of indole glucosinolates, due to their thermal instability. HPLC determination is well suited for the detection of both compounds, simultaneously. This is of great importance, because most of the breeding material low in total glucosinolate content shows remarkably increased indole glucosinolate proportions. For practical purposes, photometrical methods as well as X-ray fluorescence-spectrometry seems to be useful and rapid tools for the determination of total glucosinolate content.     

Recent advances in the analysis of glucosinolates

Occurrence and biochemistry of glucosinolates are briefly discussed. The chemistry of intact glucosinolates and their degradation products is considered in relation to the methods used for their determination. Different methods have been used, including ion- exchange chromatography, paper chromatography, high- voltage electrophoresis,¹H- and¹³C- NMR spectroscopy. The quantitative analysis of trimethylsilylated desulfoglucosinolates by gas chromatography and of intact glucosinolates by high performance liquid chromatography is discussed in relation to previously used methods based on the determination of glucosinolate degradation products.     

Gesamtkonzept zur Glucosinolatbestimmung — Möglichkeiten zur Erstellung von Bilanzen in Rapssamen und -schroten

A New Concept for the Determination of Total Glucosinolate Content — Possibilities to Draw up Balances in Rapeseed and Rapeseed Meal The indirect analysis of the original total glucosinolate content of rapeseed and rapeseed meal via the determination of released sulfate anion is an interesting alternative to the other methods. While the total sulfate content always represents the total glucosinolate content at the time of harvest, the amount of decomposed glucosinolates in the seeds or meals is represented by the amount of free sulfate. In this way a balance can be made between rapeseed harvest or rapeseed entering the oil mill and rapeseed meal leaving the oil mill. Oil milling processes always lead to a partial degradation of glucosinolates and the resulting meal will contain a variable portion of the original intact glucosinolates. Glucosinolates are hydrolysed during drying, analysing or processing either enzymatically by the action of myrosinase or thermally by the action of heat. In both cases one molecule sulfate is produced from one molecule glucosinolate. With the aid of the sulfate method it is therefore possible to draw up balances especially in rapeseed meal.     

Effect of dehulling on the composition of antinutritive compounds in various cultivars of rapeseed

Eight different rapeseed cultivars (Brassica napus L.) have been dehulled by hand. The contents of glucosinolates, sinapine, condensed tannins and inositol phosphates have been investigated in the resulting fractions of dehulled seeds and hulls as well as in the whole seeds by HPLC methods and a spectroscopical method for condensed tannins. After dehulling the content of glucosinolates as well as the content of sinapine and inositol phosphates increased significantly (P = 0.01) in the seeds of the different cultivars by 2.0 to 35.0%, 14.1 to 57.1% and 11.9 to 38.2%, respectively. The content of condensed tannins decreased by 26.0 to 81.2% after the dehulling process. In most of the cultivars the enrichment of antinutritive compounds corresponded to the share of hulls separated from the seeds. Only in the case of condensed tannins unproportional losses in the dehulled seeds were observed.     

Complex-Formation between Glucosinolates and Tetrachloropalladate(II) and its Utilization in Plant Breeding

A method is described which allows a fast determination of the content of glucosinolates in cruciferous seeds. Basically the reaction between glucosinolates and tetrachloropalladate (II) is used. For the quantitative measurement of the resulting coordination complexes an 8-channel photometer is recommended. Furthermore an analytical grist-mill has been constructed which allows a fast preparation of the samples.     

Untersuchungen über den Einsatz glucosinolatarmer Rapssaaten bzw. Extraktionsschrote als Futtermittel für monogastrische Tiere

Experiments on the Use of Seed and Meal of Rapeseed Low in Glucosinolate Content in Feeding Rations for Monogastric Animals Glucosinolates and their components in seed and meal of rapeseed may confine feed-uptake by animals. With broilers as experimental animals, the feed-uptake and consequently the growth rate were dependent on the amount of glucosinolates in the feed rations. Using short-term heat treated rapeseed meal with a low glucosinolate content ?Erglu”? the feed-uptake was normal; with heat treated products rich in glucosinolates the feed-uptake could be increased only partly. Heating inactivates those enzymes in rapeseed which split glucosinolates to nitrils; most of the glucosinolates, however, remain intact. There exists a good correlation between content of nitrils in rapeseed products and repression of feed-uptake with broilers.     

Determination of the Glucosinolate Content in Commercial Rapeseed Loads with a Pocket Reflectometer

A method is proposed which allows the determination of the glucosinolate content in commercial loads of rapeseed within 10 minutes. The procedure is based on the following steps: Homogenization of 10 g seeds, extraction of the glucosinolates with boiling water, enzymatic hydrolysis of the glucosinolates, removal of interfering substances with activated charcoal paper, detection of the liberated glucose using a glucose test paper, measurement of the colour intensity of the test paper with a pocket reflectometer and calculation of the glucosinolate content on basis of the measured values.     

The Use of Flash Chromatography for the Isolation and Purification of Glucosinolates (Mustard Oil Glycosides)

A rapid method for the isolation of several important glucosinolates in large (gram) amounts using flash chromatography is described. Emphasis has been paid to the major glucosinolates of the meal and green material of rape, Brassica napus, to enable further studies on their bioactivity and to supply standards for analytical purposes. The application of the method for the isolation of other glucosinolates is discussed.     

Total Glucosinolate Content in Rapeseed Using Reflectance

A method has been developed for the determination of total glucosinolate content in rapeseed. Following selective hydrolysis of glucosinolates by the endogeneous myrosinase in rapeseed, the glucose produced is detected with commercially available glucose test strips. The intensity of the colour on the strips is measured using a portable reflectometer. This method is suitable for use by plant breeders and for the determination of glucosinolate content in commercial rapeseed loads.     

Interaction Between Atmospheric CO<Subscript>2</Subscript> and Glucosinolates in Broccoli

Total and individual glucosinolate contents of broccoli cv Marathon were assessed at ambient CO₂ (430–480 ppm) and elevated atmospheric CO₂ (685–820 ppm) to determine the ecological relationship between changing atmospheric CO₂ concentrations and phytochemicals. Elevated atmospheric CO₂ concentration had a differing effect on individual glucosinolates and glucosinolate groups. Total glucosinolate content increased at elevated atmospheric CO₂ concentration as a result of a strong increase in both methylsulfinylalkyl glucosinolates glucoraphanin and glucoiberin. In contrast, indole glucosinolates simultaneously decreased, predominantly because of a reduction of glucobrassicin and 4-methoxy-glucobrassicin contents. We conclude that changes in N content and N/S ratios as well as alterations in photochemical processes at elevated atmospheric CO₂ concentration can influence total and individual glucosinolates contents of Brassicaceae, as demonstrated in the greenhouse, for broccoli.     

Differential Levels of Insect Herbivory in the Field Associated with Genotypic Variation in Glucosinolates in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glucosinolates are commonly found in Arabidopsis thaliana and its crucifer relatives, which are known for their role in defense against insect herbivory. In a common garden experiment, we assessed genotypic variation in glucosinolates in A. thaliana and evaluated the association between this chemistry and both plant damage and fitness. Specifically, glucosinolate concentrations were directly associated with damage levels and inversely associated with fitness. These results are contrary to the general expectation that enhanced chemical defense should result in decreased insect herbivory. As the measured insect community in this field trial was dominated by specialist herbivores, this positive relationship between glucosinolates and herbivory agrees with previous observations that glucosinolates (or their hydrolysis products) attract specialist insects. In addition, glucosinolate diversity in this common garden appeared to affect herbivore damage levels. For example, genotypes that contained alkenyl glucosinolates had higher mean damage levels than those that contained hydroxyalkyl glucosinolates. Results suggest that genotypic variation in glucosinolates may be a major factor in determining plant utilization patterns by insect herbivores in the field.     

Sequestration of Host Plant Glucosinolates in the Defensive Hemolymph of the Sawfly Athalia rosae

Interactions between insects and glucosinolate-containing plant species have been investigated for a long time. Although the glucosinolate–myrosinase system is believed to act as a defense mechanism against generalist herbivores and fungi, several specialist insects use these secondary metabolites for host plant finding and acceptance and can handle them physiologically. However, sequestration of glucosinolates in specialist herbivores has been less well studied. Larvae of the turnip sawfly Athalia rosae feed on several glucosinolate-containing plant species. When larvae are disturbed by antagonists, they release one or more small droplets of hemolymph from their integument. This reflex bleeding is used as a defense mechanism. Specific glucosinolate analysis, by conversion to desulfoglucosinolates and analysis of these by high-performance liquid chromatography coupled to diode array UV spectroscopy and mass spectrometry, revealed that larvae incorporate and concentrate the plant's characteristic glucosinolates from their hosts. Extracts of larvae that were reared on Sinapis alba contained sinalbin, even when the larvae were first starved for 22 hr and, thus, had empty guts. Hemolymph was analyzed from larvae that were reared on either S. alba, Brassica nigra, or Barbarea stricta. Leaves were analyzed from the same plants the larvae had fed on. Sinalbin (from S. alba), sinigrin (B. nigra), or glucobarbarin and glucobrassicin (B. stricta) were present in leaves in concentrations less than 1 mol/g fresh weight, while the same glucosinolates could be detected in the larvae's hemolymph in concentrations between 10 and 31 mol/g fresh weight, except that glucobrassicin was present only as a trace. In larval feces, only trace amounts of glucosinolates (sinalbin and sinigrin) could be detected. The glucosinolates were likewise found in freshly emerged adults, showing that the sequestered phytochemicals were transferred through the pupal stage.     

Antinutritive Compounds in Different Oilseeds

Different oilseeds, usable as renewable resources, are analyzed for their content of condensed tannins, glucosinolates, phytic acid and its degradation products as well as for sinapine, and the results are presented. Seeds of sunflower, flax, coriander, Euphorbia lagascae, iberic. dragon head, safflor, Camelina sativa, soy, mustard, hemp and rapeseeds are used. Apart from hemp, coriander and Euphorbia lagascae different cultivars or genotypes of each species are investigated. The variation of the results of each cultivar or genotype is shown. Tannins and phytic acid are found in different amounts in all oilseeds, whereas sinapine and glucosinolates are limited to seeds from species belonging to the plant family Brassicaceae.     

Schnelle und präzise Bestimmung des Gesamtglucosinolatgehaltes von Rapssamen-Vergleich der Röntgenfluoreszenzanalyse mit Gaschromatographie und Hochdruckflüssigkeitschromatographie

Rapid and Precise Determination of Total Glucosinolate Content in Rapeseed – Comparison of X-Ray Fluorescence Spectroscopy with Gaschromatography and High-Performance-Liquid-Chromatography The introduction of oilseed-rape cultivars poor in glucosinolates within the EG requires methods which allow a separation of the yield batches by the glucosinolate content. By means of the indirect determination of the glucosinolate content by X-ray-spectroscopy a method is introduced that produces a high correlation with conventional procedures (r² = 99% for GC-reference samples, r² = 92% for GC single data, r² = 96% for HPLC single data; deviations from average values: GC-reference samples 1.3%; single analyses of GC and HPLC 7.4 respectively 3.1%) and that needs only 4 minutes for one complete determination and is easily to perform.     

Herbivore-Mediated Effects of Glucosinolates on Different Natural Enemies of a Specialist Aphid

The cabbage aphid Brevicoryne brassicae is a specialist herbivore that sequesters glucosinolates from its host plant as a defense against its predators. It is unknown to what extent parasitoids are affected by this sequestration. We investigated herbivore-mediated effects of glucosinolates on the parasitoid wasp Diaeretiella rapae and the predator Episyrphus balteatus. We reared B. brassicae on three ecotypes of Arabidopsis thaliana that differ in glucosinolate content and on one genetically transformed line with modified concentrations of aliphatic glucosinolates. We tested aphid performance and the performance and behavior of both natural enemies. We correlated this with phloem and aphid glucosinolate concentrations and emission of volatiles. Brevicoryne brassicae performance correlated positively with concentrations of both aliphatic and indole glucosinolates in the phloem. Aphids selectively sequestered glucosinolates. Glucosinolate concentration in B. brassicae correlated negatively with performance of the predator, but positively with performance of the parasitoid, possibly because the aphids with the highest glucosinolate concentrations had a higher body weight. Both natural enemies showed a positive performance-preference correlation. The predator preferred the ecotype with the lowest emission of volatile glucosinolate breakdown products in each test combination, whereas the parasitoid wasp preferred the A. thaliana ecotype with the highest emission of these volatiles. The study shows that there are differential herbivore-mediated effects of glucosinolates on a predator and a parasitoid of a specialist aphid that selectively sequesters glucosinolates from its host plant.     

The Distribution of Glucosinolates in Different Phenotypes of Lepidium peruvianum and Their Role as Acetyl- and Butyrylcholinesterase Inhibitors—In Silico and In Vitro Studies

The aim of the study was to present the fingerprint of different Lepidium peruvianum tuber extracts showing glucosinolates-containing substances possibly playing an important role in preventinting dementia and other memory disorders. Different phenotypes of Lepidium peruvianum (Brassicaceae) tubers were analysed for their glucosinolate profile using a liquid chromatograph coupled with mass spectrometer (HPLC-ESI-QTOF-MS/MS platform). Qualitative analysis in 50% ethanolic extracts confirmed the presence of ten compounds: aliphatic, indolyl, and aromatic glucosinolates, with glucotropaeolin being the leading one, detected at levels between 0–1.57% depending on phenotype, size, processing, and collection site. The PCA analysis showed important variations in glucosinolate content between the samples and different ratios of the detected compounds. Applied in vitro activity tests confirmed inhibitory properties of extracts and single glucosinolates against acetylcholinesterase (AChE) (15.3–28.9% for the extracts and 55.95–57.60% for individual compounds) and butyrylcholinesterase (BuChE) (71.3–77.2% for the extracts and 36.2–39.9% for individual compounds). The molecular basis for the activity of glucosinolates was explained through molecular docking studies showing that the tested metabolites interacted with tryptophan and histidine residues of the enzymes, most likely blocking their active catalytic side. Based on the obtained results and described mechanism of action, it could be concluded that glucosinolates exhibit inhibitory properties against two cholinesterases present in the synaptic cleft, which indicates that selected phenotypes of L. peruvianum tubers cultivated under well-defined environmental and ecological conditions may present a valuable plant material to be considered for the development of therapeutic products with memory-stimulating properties.     

Determination of the total glucosinolate content in canola by reaction with thymol and sulfuric acid

Intact glucosinolates in seeds and meals of rapeseed and canola were isolated and purified on small DEA ion-exchange columns. After being eluted with potassium sulfate the glucosinolates were hydrolyzed with sulfuric acid to produce thioglucose which was determined as a complex with thymol. The method was compared to a gas liquid chromatography (GLC) procedure which determines aliphatic glucosinolates. The extra amount of glucosnilates found (ca. 14 ]gmmol/g oil-free) was equal to the sum of those not determined by GLC. The thymol method had a standard error of ±3 μmol/g compared with a standard error of ±1 μmol/g for the GLC procedure for the same set of 18 samples ranging from 10 μmol/g to 100 μmol/g (oil-free, 8.5% moisture basis) of aliphatic glucosinolates. This is paper No. 626 of the Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main St., Winnipeg, Manitoba, Canada R3C 3G8     

Möglichkeiten zum Nachweis einer Hitzeeinwirkung auf Rapssaat im Zusammenhang mit Glucosinolat-Bestimmungsmethoden

Ways to Detect Heat Exposure of Rapeseed in Relation to Glucosinolate Determination In wet summers rapeseed lots may have to be dried during harvesting by the action of hot air prior to storage in a silo. In the laboratory, samples for analysis must often be dried because rapeseed with a higher degree of humidity can be difficult to grind or mill. In some laboratory protocols, the ise of microwave ovens is recommended. When rapeseed is heated, components can be chemically transformed in such a way that they are no longer accessible to analysis methods designed for the original parent compound. This is also the case with glucosinolates and some of their analysis methods, particularly when microwave drying of samples is used. Discrepancies in glucosinolate results, particularly between the result of a sulfur or sulfate determination on the one hand, and GLC or HPLC of desulfoglucosinolates on the other hand, may have been caused by excessive drying if batches and by the fact that the GLC or HPLC methods in use do not determine decomposed glucosinolates. The known thermal decomposition of glucosinolates is particularly evident in 00-rapeseed because of the increased lability of 4-hydroxyglucobrassicin — a prominent 00-rapeseed glucosinolate. It was therefore of interest to find independent methods to detect heat exposure in rapeseed. A number of possibilities are discussed and investigations based on the pattern of chlorophyll degradation products are described. An analysis of the degree of formation of the pyropheophytins a and b permits to decide whether or not a sample of rapeseed had been heated excessively or dried by microwave absorption.     

Anforderungen der Tierernährung an die Rapszüchtung

Recommendations for Rape Breeding in regard to Animal Nutrition Full-fat rapeseed is one of the feedstuffs with the highest energy content. In rapeseed meal (RSM), protein and amino acids, however, also cell wall constituents and harmful substances mainly glucosinolates, are enriched. The content of lignin is 8 %. This “undigestible” part causes the low digestibility and energy content. In pigs dose-effect-relationships were investigated between dietary glucosinolates and growth, feed intake, weight as well as iodine content of thyroid, thyroid hormone concentration of blood serum and further criteria. The “no-observed-effect level” for pigs is in the range from 2 to 3 mmol glucosinolates/kg feed. In rapeseed the glucosinolate content should be lowered generally to < 30 mmol/kg defatted seed. The breeding of low hull varieties increases the energy content of RSM. With regard to produced pork the quality of rape fat will be better if it would contain more oleic acid instead of linoleic and linolenic acid.     

Extent of thermal decomposition of indole glucosinolates during the processing of canola seed

A series of samples representing various stages of canola seed processing were obtained from two commercial processing plants to assess the extent of thermal degradation of indole glucosinolates during the crushing process. The individual glucosinolate content of all samples was determined along with the content of indole glucosinolate thermal degradation products [indoleacetonitriles and thiocyanate ion (SCN)]. Only minor decomposition of indole glucosinolates was evident prior to the desolventization stage of seed processing. Major decomposition of indole glucosinolates occurred in the desolventization of seed processing with little or no effect during meal drying. Indoleacetonitriles (3-indoleacetonitrile and 4-hydroxy-3-indoleacetonitrile) and SCN together accounted for 45–60% of the degraded indoles in the samples studied.