Strong increase of foliar inulin occurs in transgenic lettuce plants (Lactuca sativa L.) overexpressing the Asparagine Synthetase A gene from Escherichia coli

Transgenic lettuce (Lactuca sativa L. cv. 'Cortina') lines expressing the asparagine synthetase A gene from Escherichia coli were produced to alter the plant nitrogen status and eventually enhance growth. The relative molecular abundance of water-soluble metabolites was measured by 1H NMR in transgenic and conventional plants at early developmental stages and grown under the same conditions. NMR metabolic profiles assessed that a transgenic line and the wild-type counterpart shared the same compounds, but it also revealed side effects on the carbon metabolism following genetic modification. Concerning the nitrogen status, the amino acid content did not vary significantly, except for glutamic acid and gamma-aminobutyric acid, which diminished in the transgenics. As for the carbon metabolism, in transgenic leaves the contents of sucrose, glucose, and fructose decreased, whereas that of inulin increased up to 30 times, accompanied by the alteration of most Krebs's cycle organic acids and the rise of tartaric acid compared to nontransformed controls. Lettuce leaf inulins consisted of short oligomeric chains made of one glucose unit bound to two/four fructose units. Inulins are beneficial for human health, and they are extracted from plants and commercialized as long-chain types, whereas the short forms are synthesized chemically. Hence, lettuce genotypes with high content of foliar short-chain inulin represent useful materials for breeding strategies and a potential source for low molecular weight inulin.     

Metabolite profiling of maize kernels--genetic modification versus environmental influence

A metabolite profiling approach based on gas chromatography-mass spectrometry (GC-MS) was applied to investigate the metabolite profiles of genetically modified (GM) Bt-maize (DKC78-15B, TXP 138F) and Roundup Ready-maize (DKC78-35R). For the comparative investigation of the impact of genetic modification versus environmental influence on the metabolite profiles, GM maize was grown together with the non-GM near-isogenic comparators under different environmental conditions, including several growing locations and seasons in Germany and South Africa. Analyses of variance (ANOVA) revealed significant differences between GM and non-GM maize grown in Germany and South Africa. For the factor genotype, 4 and 3%, respectively, of the total number of peaks detected by GC-MS showed statistically significant differences (p < 0.01) in peak heights as compared to the respective isogenic lines. However, ANOVA for the factor environment (growing location, season) revealed higher numbers of significant differences (p < 0.01) between the GM and the non-GM maize grown in Germany (42%) and South Africa (10%), respectively. This indicates that the majority of differences observed are related to natural variability rather than to the genetic modifications. In addition, multivariate data assessment by means of principal component analysis revealed that environmental factors, that is, growing locations and seasons, were dominant parameters driving the variability of the maize metabolite profiles.     

Proteomic analysis of four Brazilian MON810 maize varieties and their four non-genetically-modified isogenic varieties

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.     

Microbial and nematode communities associated with potatoes genetically modified to express the antimicrobial peptide magainin and unmodified potato cultivars

The antimicrobial peptide magainin II has activity against a range of micro-organisms. Tubers harvested from potatoes genetically modified (GM) to express a synthetic magainin gene show improved resistance to the bacterial pathogen Erwinia carotovora. The microbial and nematode communities associated with three magainin-expressing potato lines, their near-isogenic, unmodified parental cultivar (Iwa) and an unrelated cultivar (Karaka) were investigated on field-grown plants. Heterotrophic plate counts were used to enumerate aerobic culturable bacterial and fungal populations, while cultivation-independent analysis of bacterial communities was based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from community DNA from phyllosphere, rhizosphere and geocaulosphere (tuber surface) samples. Small but statistically significant differences in the population sizes of culturable bacteria, fungi and yeast were detected among some GM magainin-expressing lines and the unmodified control. However, these differences were typically smaller than the differences between the unmodified parental line control (Iwa) and the unrelated cultivar control (Karaka). Similarly, the difference in the proportion of the nematode population belonging to the fungal feeding trophic group between Iwa and Karaka was greater than that amongst Iwa and its near-isogenic GM lines, and was significantly so for the genus Aphelenchus. The nematode channel ratio (NCR) indicated a more fungal-dominated decomposition channel in soil beneath Karaka compared to Iwa at harvest. In general, eubacterial phylloplane communities were similar for all lines, while the rhizosphere communities associated with two of the three GM lines differed from communities associated with their unmodified parental line control. When roots were senescent, there was no significant difference among potato lines in rhizosphere eubacterial communities or individual trophic groups of the nematode community. Greater diversity was found in geocaulosphere; α-proteobacteria and actinomycete communities of two of the three GM lines differed significantly from their unmodified parental line control and the unrelated cultivar control, while the communities associated with the third GM line were more similar to those of the two control lines. This highlights the importance of testing several GM lines when assessing non-target effects. Results suggest that there is little likelihood of any major sustained non-target effect of genetic modification using a magainin II transgene on plant-associated and soil microflora and function.     

Unsupervised principal component analysis of NMR metabolic profiles for the assessment of substantial equivalence of transgenic grapes (Vitis vinifera)

Substantial equivalence is a key concept in the evaluation of unintended and potentially harmful metabolic impact consequent to a genetic modification of food. The application of unsupervised multivariate data analysis to the metabolic profiles is expected to improve the effectiveness of such evaluation. The present study uses NMR spectra of hydroalcoholic extracts, as holistic representations of the metabolic profiles of grapes, to evaluate the effect of the insertion of one or three copies of the DefH9-iaaM construct in plants of Silcora and Thompson Seedless cultivars. The comparison of the metabolic profiles of transgenic derivatives with respect to their corresponding natural lines pointed out that the overall metabolic changes occur in the same direction, independent of the host genotype, although the two cultivars are modified to different extents. A higher number of copies not only produces a larger effect but also modifies the whole pattern of perturbed metabolites.     

Identification and quantification of major steviol glycosides in Stevia rebaudiana purified extracts by 1H NMR spectroscopy

The use of (1)H NMR spectroscopy for the characterization of Stevia rebaudiana extracts is presented. The developed method allows qualitative and quantitative determination of the major steviol glycosides in purified extracts and fractions obtained from various stages of the purification process. Moreover, it proved to be a powerful tool to differentiate between glycosides which are naturally occurring in the stevia plant and artifacts formed in the course of the manufacturing process. Identification of steviol glycosides was achieved by the use of 2D NMR techniques, whereas quantification is based on qHNMR using anthracene as internal standard. The solvent mixture pyridine-d(5)-DMSO-d(6) (6:1) enabled satisfactory separation of the signals to be integrated. Validation of the method was performed in terms of specificity, precision, accuracy, linearity, robustness, and stability. Quantitative results were compared to those obtained with the JECFA HPLC-UV method and were found to be in reasonable agreement. NMR analysis does not rely on the use of reference compounds and enables significantly faster analysis compared to HPLC-UV. Thus, NMR represents a feasible alternative to HPLC-based methods for the quality control of Stevia rebaudiana extracts.     

Isolation and structural elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein from marigold flowers, kale, and human plasma

(13Z,13'Z,3R,3'R,6'R)-Lutein has been isolated and purified from extracts of marigold flowers, fresh raw kale (Brassica oleracea var. Acephala), and human plasma and fully characterized by (1)H and (13)C NMR, UV/vis, and MS. While the concentration of (13Z, 13'Z)-lutein in kale and human plasma compared to (all-E,3R,3'R, 6'R)-lutein was found to be quite low, this compound was readily isolated by fractional crystallization of lutein from marigold extracts. Thus, the mother liquors from two consecutive crystallizations of lutein from a saponified extract of marigold flowers were enriched in (13Z,13'Z)-lutein (8.7% of total carotenoids) and employed for the isolation of this compound by HPLC. The identity of the di-Z-lutein in kale and human plasma has been established by comparison of the HPLC-UV/vis-MS profiles of the purified compounds with those of a fully characterized sample, isolated from marigolds. 3-Hydroxy-beta,epsilon-caroten-3'-one and 3'-epilutein have also been identified in extracts from marigolds.     

Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.     

Assessment of the nutritional values of genetically modified wheat, corn, and tomato crops

The genetic modification in fruit and vegetables could lead to changes in metabolic pathways and, therefore, to the variation of the molecular pattern, with particular attention to antioxidant compounds not well-described in the literature. The aim of the present study was to compare the quality composition of transgenic wheat ( Triticum durum L.), corn ( Zea mays L.), and tomato ( Lycopersicum esculentum Mill.) to the nontransgenic control with a similar genetic background. In the first experiment, Ofanto wheat cultivar containing the tobacco rab1 gene and nontransgenic Ofanto were used. The second experiment compared two transgenic lines of corn containing Bacillus thuringiensis "Cry toxin" gene (PR33P67 and Pegaso Bt) to their nontransgenic forms. The third experiment was conducted on transgenic tomato ( Lycopersicum esculentum Mill.) containing the Agrobacterium rhizogenes rolD gene and its nontransgenic control (cv. Tondino). Conventional and genetically modified crops were compared in terms of fatty acids content, unsaponifiable fraction of antioxidants, total phenols, polyphenols, carotenoids, vitamin C, total antioxidant activity, and mineral composition. No significant differences were observed for qualitative traits analyzed in wheat and corn samples. In tomato samples, the total antioxidant activity (TAA), measured by FRAP assay, and the naringenin content showed a lower value in genetically modified organism (GMO) samples (0.35 mmol of Fe (2+) 100 g (-1) and 2.82 mg 100 g (-1), respectively), in comparison to its nontransgenic control (0.41 mmol of Fe (2+) 100 g (-1) and 4.17 mg 100 g (-1), respectively). On the basis of the principle of substantial equivalence, as articulated by the World Health Organization, the Organization for Economic Cooperation and Development, and the United Nations Food and Agriculture Organization, these data support the conclusion that GM events are nutritionally similar to conventional varieties of wheat, corn, and tomato on the market today.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.     

Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon)

Polyphenolic compounds in cranberries have been investigated to determine their role in protection against cardiovascular disease and some cancers. Extracts of whole fruit were assayed for radical-scavenging activity and tumor growth inhibition using seven tumor cell lines. Selective inhibition of K562 and HT-29 cells was observed from a methanolic extract in the range of 16-125 microg/mL. Radical-scavenging activity was greatest in an extract composed primarily of flavonol glycosides. Seven flavonol glycosides were isolated and purified from whole fruit for further evaluation; the anthocyanin cyanidin 3-galactoside was also purified for comparison with the flavonoids. Three flavonol monoglycosides were newly identified by (13)C NMR as myricetin 3-alpha-arabinofuranoside, quercetin 3-xyloside, and 3-methoxyquercetin 3-beta-galactoside (isorhamnetin); the other four isolated were the previously identified myricetin 3-beta-galactoside, quercetin 3-beta-galactoside, quercetin 3-alpha-arabinofuranoside, and quercetin 3-alpha-rhamnopyranoside. These compounds were evaluated for 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and ability to inhibit low-density lipoprotein oxidation in vitro. Most of the flavonol glycosides showed antioxidant activity comparable or superior to that of vitamin E; cyanidin 3-galactoside showed activity superior to that of the flavonoids as well as vitamin E or Trolox in both antioxidant assays.     

Analysis of glycosidically bound aroma precursors in tea leaves. 3. Change in the glycoside content of tea leaves during the oolong tea manufacturing process.

A direct qualitative and quantitative determination of the glycosides of tea aroma compounds at the four stages of the oolong tea manufacturing process (plucking, solar withering, indoor withering, and oolong tea product) was carried out by a capillary gas chromatographic-mass spectrometric analysis after trifluoroacetyl derivatization of the glycosidic fractions. Sixteen glucosides and primeverosides were identified and quantified in cv. Chin-shin-oolong and cv. Chinhsuan-oolong. A comparison of the glycosides in dried fresh leaves between the two cultivars showed significant differences. During the manufacturing process, the amounts of most of these glycosides increased from the solar-withering stage, reaching the highest level at the final stage of oolong tea production. It was noted that no glycoside decreased in its content during the manufacturing process, this being quite different from the manufacture of black tea. In addition, the contents of these alcoholic aroma compounds in the free aroma concentrate from each cultivar remained almost unchanged or slightly decreased, and they constituted only about 12 and 17% in amount of the whole oolong tea aroma compounds. However, jasmine lactone and indole were markedly higher in the final oolong tea products.     

Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes

Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.     

Analysis of phloem exudate collected from fruit-bearing stems of coconut palm: Palm trees as a source of molecules circulating in sieve tubes

Sieve tubes have been attracting widespread research interest because of their possible role in mediating physiological signals within the whole plant. However, progress in research into the function of sieve tubes has been limited by the low volume of sap available. To overcome this problem, we attempted to collect phloem exudate from tropical coconut palm trees (Cocos nucifera L. cv. Namhom). As much as 3 to 15 mL of exudate per hour was collected from the cut surface of the plant's fruit-bearing stem. Our analyses revealed that the characterized profiles of sugars (sucrose: 339 mM), amino acids (total concentration: 17.1 mM), cations (potassium: 48.3 mM), and proteins (total concentration: 0.1 /-lg /-lL-1) in the exudate were mostly consistent with those of phloem sap or phloem exudate collected from rice plants, castor bean plants, etc. This exudate was assumed to reflect the composition of the phloem sap from the source organs of coconut palm trees. The large volume of exudate collected contributed significantly to the analyses of the various compounds in the stream of sieve tubes.     

Lipid characterization in vegetative tissues of high saturated fatty acid sunflower mutants

Modifications of the fatty acid composition of plant vegetative tissues produce deficient plant growth. To determine the expression of the seed high-saturated sunflower (Helianthus annuus L.) mutant character during the vegetative cycle, five sunflower mutant lines (three high-stearic and two high-palmitic) have been studied during their germination and vegetative cycle. No significant variations with regard to the control lines were observed in the mutant vegetative tissue lipids; however, during seed germination important differences between lines were found. Although in the early steps of germination the palmitic and stearic acid levels in the respective mutants seedling cotyledons continued being higher than those of the control lines, they decreased and reached values similar to the controls, except in CAS-3. Variations in the cotyledon palmitic acid content with regard to the control line were also observed in high-stearic mutants, suggesting the expression of a modified acyl-ACP thioesterase or recycling of seed fatty acids during seedling development.     

转基因产品可追溯管理及溯源技术研究进展

家畜生产链中的转基因技术作为改善农业技术,提高农业经济效益的新技术,是21世纪不可回避的并需要积极应对的技术领域。鉴于转基因食品、动物饲料及其动物产品的安全与风险并存,本文阐述了转基因生物及转基因动物的概念内涵,总结了世界各国及其消费者对转基因技术及其产品的接受态度,其中欧盟对于GM食品、饲料及GM动物的开发持谨慎的态度,在相关标识及立法上起步早,并随着技术进步不断完善立法。对于食品和饲料的GM的标识阈值欧盟为0.9%,澳大利亚和新西兰则为1%,日本与韩国则对此标识未作要求,美国、加拿大和阿根廷等则对GM产品标识采取自愿的方法,而我国目前的相关立法着重于对转基因产品安全审批,但对终端产品管理和标识尚无界定。其次,本文总结了中国在转基因动物溯源技术方面所开展的工作,一是基本构建了基于基因改良的转基因动物数据库及溯源网络平台(www.gmanimal.com.cn);二是提出了转基因动物产品溯源的全程标识技术路线;三是使用Net及网络数据库技术开发了转基因动物(猪)及其产品全程溯源模式系统,可实现从是否使用转基因饲料和(或)使用转基因动物及其产品生产的正向跟踪及方向溯源,为满足政府的监管及消费者的知情权和选择权在技术上做好了储备。     

Universal primer-multiplex-polymerase chain reaction (UP-M-PCR) and capillary electrophoresis-laser-induced fluorescence analysis for the simultaneous detection of six genetically modified maize lines

To meet the labeling and traceability requirement of genetically modified (GM) maize and their products for trade and regulation, it is essential to develop a specific detection method for monitoring the presence of GM content. In this work, six GM maize lines, including GA21, Bt11, NK603, Bt176, Mir604, and Mon810, were simultaneously detected by universal primer-multiplex-polymerase chain reaction (UP-M-PCR), and the amplicons for the six event-specific genes as well as the endogenous Ivr gene were successfully separated by the method of capillary electrophoresis-laser-induced fluorescence (CE-LIF). The UP-M-PCR method overcame the disadvantages in conventional M-PCR, such as complex manipulation, lower sensitivity, amplification disparity resulting from different primers, etc., and in combination with CE-LIF, it obtained a high sensitivity of 0.1 ng for both single and mixed DNA samples. The established method can be widely used for the qualitative identification of the GM maize lines.     

Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes

Strawberry contains high levels of micronutrients and phytochemical compounds. These exhibit functional roles in plant growth and metabolism and are also essential for the nutritional and organoleptic qualities of the fruit. The aim of the present work was to better characterize the phytochemical and antioxidant profiles of the fruit of nine different genotypes of strawberry, by measuring the total flavonoid, anthocyanin, vitamin C, and folate contents. Cultivar effects on the total antioxidant capacities of strawberries were also tested. In addition, the individual contribution of the main antioxidant compounds was assessed by HPLC separation coupled to an online postcolumn antioxidant detection system. This study showed the important role played by the genetic background on the chemical and antioxidant profiles of strawberry fruits. Significant differences were found between genotypes for the total antioxidant capacity and for all tested classes of compounds. The HPLC analyses confirmed qualitative and quantitative variability in the antioxidant profiles. These studies show that differences exist among cultivars, applicable in dietary studies in human subjects.     

Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.     

Analysis of volatiles in porcine liver pâtés with added sage and rosemary essential oils by using SPME-GC-MS

The effect of the addition of two natural antioxidant extracts (sage and rosemary essential oils) and one synthetic (BHT) on the generation of volatile compounds in liver patés from Iberian and white pigs was analyzed using SPME-GC-MS. Lipid-derived volatiles such as aldehydes [hexanal, octanal, nonanal, hept-(Z)-4-enal, oct-(E)-2-enal, non-(Z)-2-enal, dec-(E)-2-enal, deca-(E,Z)-2,4-dienal] and alcohols (pentan-1-ol, hexan-1-ol, oct-1-en-3-ol) were the most abundant compounds in the headspace of porcine liver patés. Patés from different pig breeds presented different volatiles profiles due to their different oxidation susceptibilities as a probable result of their fatty acid profiles and vitamin E content. Regardless of the origin of the patés, the addition of BHT successfully reduced the amount of volatiles derived from PUFA oxidation. Added essential oils showed a different effect on the generation of volatiles whether they were added in patés from Iberian or white pigs because they inhibited lipid oxidation in the former and enhanced oxidative instability in the latter. SPME successfully allowed the isolation and analysis of 41 volatile terpenes from patés with added sage and rosemary essential oils including alpha-pinene, beta-myrcene, 1-limonene, (E)-caryophyllene, linalool, camphor, and 1,8-cineole, which might contribute to the aroma characteristics of liver patés.