高温胁迫下硒硫互作调控西兰花芽苗生理及萝卜硫素代谢研究

为探讨高温胁迫下硒硫互作对西兰花芽苗生理及萝卜硫素代谢的影响,以西兰花籽粒为试验材料,经单独喷施硫酸锌(ZnSO₄)、亚硒酸钠(Na₂SeO₃)、二者联合喷施、以及结合高温胁迫下喷施处理,分析发芽期间西兰花芽苗主要生理生化指标并利用荧光定量PCR技术分析萝卜硫素代谢关键酶基因表达的变化。结果表明,单独施用Na₂SeO₃可显著增加芽长和芽苗单株鲜重(P<0.05),有效缓解高温及ZnSO₄对西兰花芽苗生长发育的抑制作用;发芽期间,相较单独喷施ZnSO₄处理,西兰花芽苗经高温后联合喷施ZnSO₄及Na₂SeO₃处理,其硒元素含量、总抗氧化能力、硫代葡萄糖苷含量、黑介子酶活性、异硫氰酸酯含量、萝卜硫素含量均显著提高(P<0.05),其中萝卜硫素含量相较单独喷施ZnSO₄处理增加了39%。高温联合ZnSO₄及Na₂SeO₃处理4 d的芽苗中MYB28、UGT74B1及ST5b基因表达量均较对照显著上调,而BoHMT1显著下调(P<0.05)。综上所述,西兰花芽苗经高温联合硒硫处理是富集萝卜硫素的有效方式,该研究结果为生产富含萝卜硫素的功能性芽苗菜提供了理论依据和技术支撑。     

曲酸处理对鲜切西兰花品质及生理变化的影响

为了探讨曲酸对鲜切西兰花的保鲜作用,为曲酸在鲜切西兰花保鲜研究中提供理论基础,本研究以西兰花为试材,采用2.5%曲酸浸泡鲜切西兰花15 min,并贮藏于4℃条件下12 d。研究曲酸处理对鲜切西兰花失重率、可滴定酸(TA)、维生素C(VC)、叶绿素、丙二醛(MDA)含量和多酚氧化酶(PPO)活性的影响。结果表明:曲酸处理可有效减少鲜切西兰花贮藏期间水分的散失,延缓TA、VC和叶绿素含量的下降,减缓MDA的积累,抑制PPO的活性。曲酸浸泡处理可显著提高鲜切西兰花的贮藏品质。     

氨基酸对甘蓝硫代葡萄糖苷的影响

以中甘21(ZG21)、06-115两个甘蓝品种为试验材料,研究叶面喷施6种氨基酸对甘蓝硫代葡萄糖苷组分及其含量的影响。结果表明,不同种类氨基酸处理甘蓝硫代葡萄糖苷组分相同,均含有9种硫苷。氨基酸对甘蓝硫苷含量有一定的影响,半胱氨酸和甲硫氨酸显著提高06-115硫苷的总含量、脂肪族硫苷的含量和4-甲基硫氧丁基硫苷含量,其中半胱氨酸处理含量最高,与对照相比分别提高63.0％、69.6％和69.1％;半胱氨酸处理提高了3-吲哚基甲基硫苷含量,达到725.6 μmol/kg, FW;半胱氨酸处理也显著提高了中甘21硫苷的总含量、脂肪族硫苷的含量、吲哚族硫苷含量和4-甲基硫氧丁基硫苷含量,与对照相比分别提高了27.2％、39.6％、15.3％和26.2％。半胱氨酸、苯丙氨酸、甘氨酸和谷氨酸处理都提高了3-吲哚基甲基硫苷的含量,其中半胱氨酸处理最高,达到292.7 μmol/kg, FW,其次是谷氨酸和甘氨酸处理。     

流态冰预冷近冰温贮藏对西兰花贮藏品质的影响

西兰花是深受人们喜爱的蔬菜,衰老黄化是其采后正常的生理代谢过程,也是引起西兰花品质劣变的主要因素。为此寻求开发有效、绿色安全的西兰花保鲜方法迫在眉睫。以甘肃特色高原蔬菜西兰花品种耐寒优秀为试材,研究流态冰预冷近冰温贮藏、0 ℃预冷4 ℃贮藏和对照不预冷4 ℃贮藏等3种处理方式对其贮藏品质的影响。结果表明,在整个贮藏期,流态冰预冷近冰温贮藏处理西兰花的叶绿素含量、萝卜硫素含量、Vc含量、氨基酸含量以及可溶性蛋白含量均显著高于0 ℃预冷4 ℃贮藏处理和不预冷4 ℃贮藏处理。与0 ℃预冷4 ℃贮藏和不预冷 4 ℃贮藏相比,流态冰预冷近冰温贮藏可以增加西兰花贮藏期萝卜硫素、总酚物质和多糖的含量。流态冰预冷近冰温贮藏可保持西兰花贮藏期营养品质,同时能够维持西兰花的功能成分,保持其商品价值。     

养分合理调控对西兰花生长、品质及土壤特性的影响

为避免不合理施肥及连作导致的西兰花产量低、品质差等问题,采用田间试验方法,设置农民习惯施肥(CK)、合理减量无机肥(MF)、合理减量无机肥配施生物肥(BF1、BF2)4个处理,研究了合理减量无机肥及配施生物肥对西兰花生长、产量、品质、养分吸收和土壤性状的影响。结果表明:每形成1 000kg西兰花花球,平均需要吸收N、P2O5、K2O分别为1.37kg,0.61kg,0.92kg。与CK相比,MF、BF1和BF2的花球单重和直径分别平均增加12.76%,12.11%;增产10.90%~14.16%;可溶糖含量平均提高10.40%,Vc含量平均增加22.83%;N、P2O5、K2O吸收量分别平均提高7.00%,26.83%和8.93%,地上部养分总吸收量平均提高11.32%;各处理土壤养分含量和微生物数量均有不同程度的变化,但差异不显著。与MF相比,BF1和BF2处理的蛋白质含量平均提高17.14%。合理减量无机肥及配施生物肥能促进西兰花生长,增强茎、叶、花球性状,促进同化产物的合成及其由茎叶向花球中的转移,促进养分吸收,提高产量;同时改善品质,提高可溶性糖和Vc含量,配施生物肥能提高蛋白质含量;对土壤养分含量和微生物数量无显著影响,但是由于其减少了氮肥用量,提高了西兰花氮素吸收量,降低了土壤中氮素残留过多污染环境的风险。综合考虑西兰花的生物学性状、养分吸收利用、产量、品质和土壤性状及实际生产需求等,以合理减量无机肥配施丰满屯生物肥效果最好。     

预处理加工对西兰花茎叶汁挥发性成分的影响

为研究热烫、冻融、超声3种预处理方式对西兰花茎叶汁挥发性成分的影响,本试验利用顶空固相微萃取-气相质谱联用仪对不同预处理加工后的西兰花茎叶制成的西兰花茎叶汁中挥发性成分进行测定。结果表明,西兰花茎叶汁的主要异味成分为己醛、庚醛、壬醛、(E)-2-己烯醛、(E,E)-2,4-庚二烯醛、1-戊醇、1-辛醇、1-己醇、(E)-3-己烯-1-醇、甲硫醇、2-乙基噻吩、硫氰酸甲酯、二甲基二硫醚、β-紫罗酮、6-甲基-5-庚烯-2-酮。与超声预处理相比,热烫和冻融处理可以较好地降低西兰花茎叶汁中挥发性成分的种类和含量。与未进行预处理的对照组相比,热烫处理的西兰花茎叶汁中虽然检出了甲硫醇和二甲基二硫醚,但醛类含量减少,酯类含量增加,此外,与冻融处理相比,热烫硫化物含量更低。本研究为西兰花茎叶的果蔬汁开发提供了理论依据。     

复合涂膜结合紫外处理对鲜切木瓜冷藏品质的影响

为了延长鲜切木瓜的保鲜时间,利用尼辛、那他霉素、薄荷油、氯化钙、香兰素、壳聚糖制成复合保鲜涂膜液,考察涂膜及紫外线处理对木瓜鲜切块贮藏品质(包括色泽、可溶性固形物含量、失水率、维生素C含量、可滴定酸、霉菌酵母数、菌落总数)的影响。由均匀设计试验优化涂膜液配方以及紫外线处理参数。研究结果表明:涂膜及紫外线处理可较好地保持鲜切木瓜的色泽,抑制褐变,保持较高的可溶性固形物含量,减少贮藏过程中的水分损失,抑制霉菌、酵母菌和细菌的增长。采用SAS软件建立各参数与菌落总数关系的回归方程。利用非线性规划法得到较佳的复合涂膜配方与紫外线处理参数为:壳聚糖质量分数1.0%,氯化钙质量分数0.6%,Nisin0.10g/kg,那他霉素14.0mg/kg,香兰素质量分数0.3%,薄荷油1.5mL/L,紫外照射时间22min,紫外照射距离范围44～66cm。此复合涂膜配方与紫外线处理可使货架期至少达到6天,为鲜切木瓜保鲜提供技术参考。     

甘蓝型油菜中黑芥子酶基因在大肠杆菌中重组质粒的构建和表达

实验通过RT-PCR程序从提取的油菜总RNA中扩增出硫代葡萄糖苷水解酶（又称黑芥子酶,EC3.2.1.147）基因,酶解后插入大肠杆菌表达载体（Escherichia coli）pGEX-4T-1,获得克隆菌株。基因测序在 GenBank中的登陆号为EF583560,翻译的氨基酸序列与已报道的黑芥子酶（GenBank中的登陆号为Q00326）中的一段有一个氨基酸不同,同源性达到99%。经过IPTG诱导表达,在91KDa左右处有表达量很高的一条带。     

6-苄基腺嘌呤对鲜莲子活性氧代谢及能量水平的影响

为探讨6-苄基腺嘌呤(6-BA)对鲜莲子活性氧代谢及能量水平的影响,以清水浸泡为对照,在25±1℃条件下,用20 mg·L-16-BA对莲蓬太空莲36号进行处理,研究其贮藏期间感官品质变化及呼吸作用强度,并分析莲子活性氧水平,抗氧化酶活性,丙二醛(MDA)、三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、磷酸腺苷(AMP)含量以及能荷水平的变化。结果表明,与对照相比,6-BA处理可有效抑制莲蓬呼吸作用、莲子活性氧水平上升及MDA的积累,且保持抗氧化酶活性的同时,维持了莲子较高的能荷水平,从而延缓了鲜莲蓬和莲子品质的下降,使其贮藏期延长了3~4 d。综上,6-BA处理对减缓莲蓬和莲子采后衰老具有显著效果,本研究为鲜莲蓬和莲子采后保鲜提供了理论及技术支持。     

6-Benzylaminopurine delays senescence and enhances health-promoting compounds of harvested broccoli

The effect of 6-benzylaminopurine (6-BA) on the color, antioxidant activity, and contents of total phenols, glucosinolate, and sulforaphane in broccoli florets was investigated. The results showed that 6-BA treatment markedly inhibited the increase of the L* value and malondialdehyde (MDA) content and retarded the decrease of the H value. 6-BA treatment reduced the rate of chlorophyll degradation by regulating the activities of chlorophyllase and Mg-dechelatase. When compared to control florets, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were enhanced in florets treated with 6-BA, whereas the activity of peroxidase (POD) was significantly reduced. The contents of total phenols, glucosinolate, and sulforaphane in broccoli florets were also profoundly increased after treatment with 6-BA. These results indicated that 6-BA could maintain the quality, delay senescence, and improve the nutritional value of broccoli.     

Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane

In some cruciferous plants, epithiospecifier protein (ESP) directs myrosinase (EC 3.2.3.1)-catalyzed hydrolysis of alkenyl glucosinolates toward epithionitrile formation. Here, for the first time, we show that ESP activity is negatively correlated with the extent of formation of the health-promoting phytochemical sulforaphane in broccoli (Brassica oleracea L. ssp. italica). A 43 kDa protein with ESP activity and sequence homology to the ESP of Arabidopsis thaliana was cloned from the broccoli cv. Packman and expressed in Escherichia coli. In a model system, the recombinant protein not only directed myrosinase-dependent metabolism of the alkenyl glucosinolate epi-progoitrin [(2S)-2-hydroxy-3-butenyl glucosinolate] toward formation of an epithionitrile but also directed myrosinase-dependent hydrolysis of the glucosinolate glucoraphanin [4-(methylsulfinyl)butyl glucosinolate] to form sulforaphane nitrile, in place of the isothiocyanate sulforaphane. The importance of this finding is that, whereas sulforaphane has been shown to have anticarcinogenic properties, sulforaphane nitrile has not. Genetic manipulation designed to attenuate or eliminate expression of ESP in broccoli could increase the fractional conversion of glucoraphanin to sulforaphane, enhancing potential health benefits.     

The metabolic fate of purified glucoraphanin in F344 rats

Dietary broccoli is commonly eaten cooked, exposing individuals to intact glucoraphanin rather than to its hydrolysis product, the anticarcinogenic isothiocyanate sulforaphane, since cooking destroys the hydrolyzing enzyme myrosinase. There is little information on the absorption and metabolism of glucoraphanin, due partly to the lack of purified compound. In this study, glucoraphanin was purified from broccoli seed and 150 mumol/kg was administered to male F344 rats. Glucoraphanin (5% of an oral dose) was recovered intact in urine, showing that it is absorbed intact, and no glucoraphanin or metabolites were found in feces. Total urinary products accounted for 20 and 45% of oral and intraperitonneal doses, respectively, including sulforaphane N-acetyl cysteine conjugate (12.5 and 2%), free sulforaphane (0.65 and 0.77%), sulforaphane nitrile (2 and 1.4%), and erucin (0.1 and 0.1%), respectively. Both glucoraphanin and its reduced form glucoerucin were identified in bile following intravenous glucoraphanin administration. We conclude that orally administered glucoraphanin is absorbed intact, undergoes enterohepatic circulation, and is hydrolyzed in the gut in F344 rats.     

Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement

Increased consumption of cruciferous vegetables such as broccoli may reduce the risk of various cancers. Myrosinase is required to convert dietary glucosinolates from broccoli into bioactive isothiocyanates. We evaluated isothiocyanate excretion profiles in healthy subjects who consumed broccoli sprouts or broccoli supplement (no myrosinase) with equivalent glucosinolate content. Urinary metabolites of two major isothiocyanates, sulforaphane and erucin, were measured by liquid chromatography coupled with tandem mass spectrometry. Peak excretion of sulforaphane and erucin was higher and occurred sooner in subjects who consumed broccoli sprouts as compared to subjects who consumed the supplement. A subject-dependent shift in the ratio of urinary sulforaphane to erucin metabolites was observed in both groups, indicating conversion of sulforaphane to erucin. Lower histone deacetylase activity was observed in the peripheral blood mononuclear cells only in subjects consuming sprouts. Fresh broccoli sprouts differ from broccoli supplements in regards to excretion of isothiocyanates and bioactivity in human subjects.     

Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli

The aim of this study was to determine the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, the hydrolysis product of glucoraphanin, from raw and cooked broccoli. Eight men consumed 200 g of crushed broccoli, raw or cooked, with a warm meal in a randomized, free-living, open cross-over trial. Higher amounts of sulforaphane were found in the blood and urine when broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%, p ) 0.002). Absorption of sulforaphane was delayed when cooked broccoli was consumed (peak plasma time ) 6 h) versus raw broccoli (1.6 h, p ) 0.001). Excretion half-lives were comparable, 2.6 and 2.4 h on average, for raw and cooked broccoli, respectively (p ) 0.5). This study gives complete kinetic data and shows that consumption of raw broccoli results in faster absorption, higher bioavailability, and higher peak plasma amounts of sulforaphane, compared to cooked broccoli.     

The effect of post-harvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica)

The effects of post-harvest and packaging treatments on glucoraphanin (4-methylsulfinylbutyl glucosinolate), the glucosinolate precursor of anticancer isothiocyanate sulforaphane [4-methylsulfinylbutyl isothiocyanate], were examined in broccoli (Brassica oleracea var. italica) during storage times. The results showed that at 20 degrees C, 55% loss of glucoraphanin concentration occurred in broccoli stored in open boxes during the first 3 days of the treatment and 56% loss was found in broccoli stored in plastic bags by day 7. Under both air and controlled atmosphere (CA) storage, glucoraphanin concentration appeared to fluctuate slightly during 25 days of storage and the concentrations under CA was significantly higher than those stored under air treatment. In modified atmosphere packaging (MAP) treatments, glucoraphanin concentration in air control packaging decreased significantly whereas there were no significant changes in glucoraphanin concentration in MAP with no holes at 4 degrees C and two microholes at 20 degrees C for up to 10 days. Decreases in glucoraphanin concentration occurred when the broccoli heads deteriorated. In the present study, the best method for preserving glucoraphanin concentration in broccoli heads after harvest was storage of broccoli in MAP and refrigeration at 4 degrees C. This condition maintained the glucoraphanin concentration for at least 10 days and also maintained the visual quality of the broccoli heads.     

Analysis of isothiocyanate mercapturic acids in urine: a biomarker for cruciferous vegetable intake

Cruciferous vegetables contain glucosinolates, which are degraded to isothiocyanates. These are easily absorbed, conjugated to glutathione, and excreted into the urine as their corresponding mercapturic acids. We have developed and validated a solid phase extraction-high-performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry method for the specific analysis of individual isothiocyanate mercapturic acids in urine. The range of reliable analysis was 1.0-310 microM in urine. Urine samples fortified with three different levels of isothiocyanate mercapturic acids were measured on six different days by three independent technicians. The relative standard deviation (RSD) of repeatability was 12, 6, and 3%; the RSD of reproducibility was 19, 14, and 8%, and spike recoveries were 103, 104, and 103%, respectively, for 1.04, 10.5, and 313 microM levels. In 24 h urine collected from two volunteers after they consumed broccoli and cauliflower, clearly sulforaphane mercapturic acid (133 micromol) and allyl isothiocyanate mercapturic acid (4.7 micromol) were found. This procedure demonstrates a reliable and efficient method to study the intake and mode of action of isothiocyanates in animal studies and clinical trials.     

Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile.

Epidemiological and laboratory studies suggest that dietary broccoli may prevent or delay a variety of cancers. Broccoli and other crucifers contain a relatively unique family of secondary metabolites called glucosinolates. Glucoraphanin, the major glucosinolate in broccoli, is hydrolyzed by an endogenous plant myrosinase to form either the potent anticarcinogen sulforaphane (SF) or sulforaphane nitrile (SF nitrile). The bioactivities of SF and SF nitrile were compared in rats and in mouse hepatoma cells. Male, 4-week-old, Fischer 344 rats were administered SF or SF nitrile (200, 500, or 1000 micromol/kg) by gavage daily for 5 days. Hepatic, colonic mucosal, and pancreatic quinone reductase and glutathione S-transferase activities were induced by high doses of SF, but not by SF nitrile. When Hepa 1c1c7 cells were exposed to increasing levels of each compound for 24 h, quinone reductase showed a 3-fold maximal induction over control at 2.5 microM SF and a 3.5-fold maximal induction over control at 2000 microM SF nitrile, the highest concentration tested. These results demonstrate that SF nitrile is substantially less potent than SF as an inducing agent of phase II detoxification enzymes. Therefore, glucoraphanin hydrolysis directed toward the production of SF rather than SF nitrile could increase the potential chemoprotective effects of broccoli.