Studies on the Formation of the Cheese-like Flavor

Solution containing l-leucine and l-methionine cultured by Aspergillus flavus were found to develop cheese-like flavor.

α-Keto-isocaproic acid was isolated and identified from the culture of l-leucine and α-keto-β-methylmercaptobutyric acid from that of l-methionine. The flavor was also developed from the mixture of the synthetic sample of α-ketoisocaproic acid and α-keto-β-methylmercaptobutyric acid.     

Inhibitory effect of 5,8,11-eicosatrienoic acid on angiogenesis

IntroductionCartilage contains high levels of n-9 eicosatrienoic acid (20:3n-9) but no blood vessels. 20:3n-9 might inhibit angiogenesis.Materials and methodsAngiogenesis was measured in human umbilical vein endothelial cells and diploid fibroblasts. Co-culture was performed with vascular endothelial growth factor-A (VEGF-A, 10 ng/mL) and fatty acids (0.1–10 μmol/L). After 10 days of incubation and immunostaining for endothelial cells, vessel areas were calculated with image analyser software.ResultsAddition of 20:3n-9 and n-3 eicosatrienoic acid (20:3n-3) dose dependently inhibited VEGF-A-stimulated angiogenesis (more than the positive control suramin). Arachidonic, eicosapentaenoic, dihomo-γ-linolenic (20:3n-6) and oleic acids did not affect VEGF-A-stimulated angiogenesis even at 10 μmol/L. Arachidonic and dihomo-γ-linolenic acids enhanced angiogenesis without VEGF-A.Discussion and conclusionsWe suggest that the presence of 20:3n-9 in cartilage may be related to its vessel-free status and that 20:3n-9 may be useful for the treatment of disorders with excessive vasculature.AcknowledgementsThis work was partly supported by Polyene Project, Inc.     

黄酮类化合物对大豆脂肪氧合酶的抑制作用研究

研究了几种不同黄酮化合物(黄酮醇类化合物、异黄酮类化合物、查尔酮类化合物、二氢黄酮类化合物)对大豆脂肪氧合酶(LOX)的抑制作用,并根据实验结果初步探讨了不同结构黄酮类化合物对大豆脂肪氧合酶抑制作用的结构-活性抑制作用关系.结果表明:黄酮化合物均可对LOX有不同程度的抑制作用,除橙皮苷外,它们的抑制效果均与加入量成正比,黄酮醇类化合物的抑制效果最为明显,它们对LOX的半数抑制浓度(IC50)依次为芦丁>槲皮素>根皮素>大豆黄素>异甘草素>芒柄花素>甘草素,而橙皮苷的最大抑制率为0.47 μg/mL时的45.1%.     

Identification of Intermediates in the Formation of Onion Flavor

Headspace gas and ether extract of sliced or homogenized onion were analyzed by using GLC. Dialkyl disulfides, main components of characteristic flavor of sliced onion, were found to be very little when onion was homogenized. Addtion of NaBH₄ to the homogenate resulted in a marked production of disulfides while that of cysteine did not. SH compounds as cysteine and benzyl mercaptan were added to the homogenate to determine the intermediates which produced flavor with NaBH₄. The presence of propyl propanethiosulfinate, propanal and thiopropanal S-oxide was confirmed by isolation and identification of their sulfide derivatives.     

水稻种胚脂肪氧化酶Lox-1,Lox-2缺失对种子储藏特性的影响

用人工加速老化的方法对Lox-1,Lox-2缺失材料云恢290和Lox-1,Lox-2不缺失品种汕优63进行了储藏后生理和生化变化的研究,结果表明:云恢290有着突出的耐储藏特性,老化20天,汕优63的发芽率下降到5%,云恢290发芽率保持在42%;老化30天后云恢290电导率增加小于汕优63,丙二醛和游离脂肪酸的增加只有汕优63的1/2左右。脂肪氧化酶Lox-1、Lox-2缺失减轻了膜脂过氧化作用,保持细胞膜结构完整性,同时减少游离脂肪酸的积累,从而延长了云恢290种子的寿命。     

Effects of Lipoxygenase on the Activities of Photosynthetic Electron Transport in Cucumber Leaves

Photosynthesis and electron transport activity decreased with leaf aging, and however, lipoxygenase (Lox) activity increased correspondingly. Soybean Lox-1 inhibited significantly PSⅡ electron transport activity of chloroplasts isolated from cucumber (Cucumis sativus L. ) cotyledon. But the inhibition could be eliminated by the addition of propyl gallate (PG) or 3, 3, 4, 5, 7-pentahydroxyflavon (PF). The inhibition of PSⅠ activity by soybean Lox-1 was enhanced in the presence of 3, 4, dichlorophenyl-1, 1-dimethylurea (DCMU) or 2, 5-dibromothymoquinone (DBMIB), bfft could be restored to its original level when PG was added. Addition of 2, 2-diphenylcarbonic dihydrazide (DPC) to the mixture of isolated chloroplasts and Lox-1, PSⅡ activity resumed obviously. Chlorophyll a fluorescence study showed that Fm was decreased by Lox-1 and resumed slightly by DPC. Based on the above results, it was suggested that Lox might act at least on three sensitive sites located on Q, PQ and the oxidative side of PSⅠ . The bleaching of chlorophyll and carotenoid stimulated by Lox-l, and the inhibition of PSⅠ electron transport activity by active oxygen might be. one of the important reasons to explaine the effect of Lox on the function of photosynthetic membrane.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Contribution to Stale Flavor of 2-Furfuryl Ethyl Ether and Its Formation Mechanism in Beer

The effect of storing beer on the generation of stale flavor was investigated by a sensory evaluation and quantitative determination of the volatile components. The results of the sensory evaluation revealed 2-furfuryl ethyl ether (2-FEE) to be an important component responsible for the astringent stale flavor, and (E)-2-nonenal (E2N) for the cardboard-like stable flavor. However, the stale flavor could not be reproduced by adding 2-FEE or E2N alone to fresh beer, but was closely reproduced when 2-FEE and E2N were added together. The results of experiments on adding furfuryl acetate (FAC) and furfuryl alcohol (FAL) to a 5% ethanol solution or beer indicate that 2-FEE was generated by the reaction between ethanol and FAL, which was formed by the hydrolysis of FAC, during the storage of beer.     

Calcium-dependent oxidation of 5,8,11-icosatrienoic acid by the cyclooxygenase enzyme system

Mead (5,8,11-icosatrienoic) acid was found to be metabolized by the cyclooxygenase enzyme system of ram seminal vesicle microsomes in a calcium-dependent manner. Although the enzyme converted Mead acid to products more slowly and less completely than the isomeric 8,11,14-icosatrienoic acid, both oxidations were inhibitable by indomethacin. Experiments using purified cyclooxygenase confirmed the participation of this enzyme system in the calcium-dependent oxidation. The products of the oxidation were separated by high performance liquid chromatography and analyzed by ultraviolet and gas chromatography-mass spectrometry. The spectra obtained were consistent with the products having the structures 13-hydroxy-5,8,11-icosatrienoate (the major product), 11-hydroxy-5,8,12-icosatrienoate, 9-hydroxy-5,7,11-icosatrienoate, and two isomeric 8,11-dihydroxy-5,9,12-icosatrienoates. No prostaglandin-like, cyclized products could be identified. This report is only the second to illustrate a calcium-dependent oxidation of a polyunsaturated fatty acid by a cyclooxygenase enzyme system and further extends the metabolic potential of Mead acid.     

不同钙离子浓度对日本沼虾感光器细胞超微结构的影响

为了进一步研究细胞外钙离子浓度变化对甲壳动物感光细胞超微结构的影响,应用透射电子显微镜显示了日本沼虾感光细胞,在暗适应时高钙离子浓度中温育的感光器细胞的感杆束直径下降,微绒毛排列零乱;多囊体、板膜体数量增加;色素颗粒散布在细胞质中,呈现出光适应的结构特征。而温育在低钙离子溶液和生理溶液中的感光器细胞结构相同,呈现出暗适应的结构特征。另外,细胞器中储存的钙离子也受细胞外钙离子浓度的影响,在高钙离子溶液中温育后细胞器储存的钙离子量增加,膜下储泡囊、多囊体、线粒体、色素颗粒等细胞器中的焦锑酸钙结晶颗粒比温育在低钙溶液中的细胞明显增多。结果显示,细胞外钙离子浓度变化引起细胞内钙离子浓度变化,从而影响感光器细胞的结构而影响其生理功能。     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

Formation of Aldehyde Flavor (n-hexanal, 3Z-nonenal and 2E-nonenal) in the Brown Alga, Laminaria Angustata

2E-Nonenal and n-hexanal are the major and minor flavor compounds in the edible brown alga, Laminaria angustata, respectively. They are believed to characterize the flavor of this alga. However the metabolism of the two compounds is not precisely known. The pathways were clarified by elucidation of the intermediate structure through purification of the intermediate compounds from an enzymatic reaction and identification using HPLC and GC-MS techniques. Formation of n-hexanal, 3Z-nonenal and 2E-nonenal are proposed to be via two cascades from unsaturated fatty acids. They are C18:2(n-6), linoleic acid cascade and C20:4(n-6), arachidonic acid cascade through their hydroperoxides as intermediates by the lipoxygenase/fatty acid hydroperoxide lyase pathway.     

Effects of Salicylic Acid on Heavy Metal-Induced Membrane Deterioration Mediated by Lipoxygenase in Rice

Deterioration of membranes caused by lipoxygenase (LOX) activity under 10 μM PbCl₂ or 10 μM HgCl₂ was partially alleviated by the exogenous application of 100 μM salicylic acid (SA). In two cultivars of rice (Oryza sativa L. cvs. Ratna and IR 36), the presence of SA ameliorated the increased leakage of electrolytes, injury index, and the content of malondialdehyde caused by these heavy metals. Lead decreased H₂O₂ content whereas Hg increased it in both cultivars. Application of SA increased H₂O₂ in presence of Pb, while decreased it in presence of Hg. Both Pb and Hg decreased superoxide dismutase activity, while increased peroxidase activity. The activity of catalase was decreased by Hg but increased by Pb and SA reversed their effects. Thus, SA ameliorated the damaging effects of Pb and Hg on membranes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the biosynthesis of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) from 3-hydroxyindolin-2-one in Zea mays

The ring expansion of 3-hydroxyindolin-2-one to 2-hydroxy-1,4-benzoxazin-3-one (HBOA) was investigated by labelling experiments. Action of the cytochrome P450 enzyme BX4 from maize on 3-hydroxyindolin-2-one under an 18O2 atmosphere induced production of 2-hydroxy-1,4-benzoxazin-3-one in which the ring oxygen--but not the 2-hydroxy group of HBOA--is labelled. A mechanism for this transformation is proposed.     

盐碱地渗水钙镁离子对凡纳滨对虾酶活力的影响

研究了盐碱地渗水中不同钙镁离子总量、不同钙镁离子比例对凡纳滨对虾(Litopenaeus vannamei)的存活、生长和体内谷草转氨酶(GOT)、谷丙转氨酶(GPT)、Na -K -三磷酸腺苷酶(Na -K -ATPase)等3种酶活力的影响。实验用水盐度保持在5‰,钙镁总量分别为300、600、900mg/L,钙镁离子比例梯度1∶1、1∶3、1∶5、1∶7、1∶9、1∶11。经过20d的养殖实验,结果表明,当钙镁总量为600mg/L时,凡纳滨对虾的存活及生长、3种酶活力表现最佳;在此钙镁总量条件下,钙镁离子比为1∶5和1∶7时其存活率和体长、体重增加率较高,而钙镁离子比例为1∶3和1∶5时具有较高的酶活力。综合以上研究结果,在5‰的盐度下,使钙离子和镁离子浓度分别保持在100mg/L和500mg/L的养殖用水,能够保证凡纳滨对虾的存活和最佳生长,证明经适当调配的盐碱地渗水养殖凡纳滨对虾是可行的。     

Effect of Phenolic Antioxidants on Lipoxygenase Reaction

The effect of conventional antioxidants on soybean lipoxygenase reaction was examined. Inhibitory activities of o-diphenols such as pyrocatechol, homocatechol, propyl gallate and NDGA were higher than those of m- and p diphenols. The mode of inhibition by NDGA, one of the most effective inhibitors among the phenolic antioxidants tested, conformed to a competitive type and not to an induction period type. Under certain conditions, NDGA could be an irreversible inactivator for the enzyme. The effect of NDGA on the enzyme reaction could not be completely explained by the coupled oxidation theory. The inactivation by NDGA were effectively prevented by either of adding catalase, of incubating under anaerobic condition or in low pH medium or of adding borate. These facts showed that the inactivation of lipoxygenase took place in consonance with the autoxidation of NDGA.     

Effects of Seasoning and Heating Device on Mutagenicity and Heterocyclic Amines in Cooked Beef

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AαC (2-amino-9 H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AαC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.     

Stereoselective synthesis and glycosidase inhibitory activity of 3,4-dihydroxy-pyrrolidin-2-one, 3,4-dihydroxy-piperidin-2-one and 1,2-dihydroxy-pyrrolizidin-3-one

3,4-Dihydroxy-pyrrolidin-2-one, 3,4-dihydroxy-piperidin-2-one and 1,2-dihydroxy-pyrrolizidin-3-one have been synthesized, using a simple strategy based on the asymmetric dihydroxylation of vinylogous aminoesters and subsequent mild intramolecular cyclization. All these compounds show a partial inhibition of alpha-glucosidase, but were inactive towards other glycosidases.     

Structural Basis for the Enzymatic Formation of the Key Strawberry Flavor Compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone

The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of ≤1.6 Å, including those with HDMF as well as three distinct substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded in a Rossmann-fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF reduction by [4R-²H]NADH and chiral-phase analysis of the reaction product EHMF, our data show that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic achiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the plant secondary metabolism our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.