SPE-UPLC-MS/MS测定葡萄酒中的高氯酸盐

为建立前处理简单、定量准确、灵敏度高的葡萄酒中高氯酸盐的快速测定和分析方法,本研究使用酸化甲醇提取葡萄酒样品,最终选择WAX固相萃取柱前处理净化,亲水性聚丙烯滤膜(GHP)过滤,经Phenyl-Hexyl色谱柱分离,以电喷雾离子源(ESI)在多反应监测(MRM)负离子模式下测定葡萄酒中高氯酸盐。结果表明,该方法的检测时间为12 min,检出限和定量限分别为0.15 μg·L^-1和0.5 μg·L^-1,在1、2、5、10和20 μg·L^-1水平的加标回收率介于94.5%~105%之间,相对标准偏差(RSD)小于3.17%。该方法的建立可为葡萄酒生产企业和相关监管部门提供一定的技术支撑。     

干白葡萄酒增香酿造工艺参数优化

试验以一种酿酒葡萄为原料,利用顶空固相微萃取/气相色谱-质谱联用技术(HS-SPME/GC-MS)检测了不同可同化氮含量(200、300、400 mg/L)、酵母多糖(150、250、350 mg/L)、发酵温度(14、18、22℃)、初始pH值(3.3、3.5、3.7)和SO2添加量(40、70、100 mg/L)处理发酵酒样中的挥发性香气化合物,探讨了复合酿造因子对贵人香干白葡萄酒主要香气物质含量的影响关系。结果表明,300 mg/L的可同化氮有利于高级醇、酯类、单萜化合物的积累;酵母多糖添加量为250 mg/L时,单萜化合物质量分数达到最大值(198.54μg/L);发酵温度从14℃升高到22℃时,高级醇含量显著升高,酯类和单萜含量显著降低;提高葡萄汁初始pH有利于单萜化合物的积累,不利于高级醇、酯类的生成;添加70 mg/L的SO2时,单萜化合物质量分数最高(181.73μg/L)。正交试验极差分析表明,发酵温度和SO2添加量对高级醇含量影响较大;发酵温度与可同化氮对酯类香气物质含量的影响较大,酵母多糖和pH值对单萜类香气物质含量影响较大。各处理组间的聚类分析可知,可同化氮和酵母多糖对主要香气化合物的影响关联度较高、葡萄汁初始pH值和SO2添加量关联度较高。较低的发酵温度有利于酒样中香叶醇、异戊醇、苯乙醇、辛酸乙酯的生成,添加中等浓度的可同化氮和酵母多糖可促进乙酸异戊酯、乙酸己酯和己酸乙酯的合成,较高的初始pH值有利于芳樟醇、香茅醇和香叶醇的积累。综合分析,发酵温度18℃、初始pH值3.5、70 mg/L SO2、300 mg/L可同化氮、250 mg/L酵母多糖酿造贵人香干白葡萄酒,可有效促进酒样中主要香气化合物的合成释放。     

超声波辅助液-液萃取结合GC检测单萜化合物工艺优化

为优化超声波辅助液-液萃取(LLE)结合气相色谱(GC)检测酿酒酵母菌株合成单萜化合物的工艺条件,本试验以模拟葡萄汁为原料,研究超声功率、超声时间、NaCl添加量和萃取剂体积对萃取效果的影响,在单因素试验的基础上,采用响应面试验优化超声辅助萃取模拟葡萄酒中单萜化合物的萃取工艺。结果表明,模拟葡萄汁单萜类化合物最佳萃取工艺参数为,超声功率405 W、超声时间15 min、NaCl添加量4.9 g、萃取剂体积15.5 mL,在此萃取条件下结合GC可从模拟葡萄汁发酵酒样中检测出芳樟醇、α-萜品醇、香茅醇、香叶醇4种单萜物质,总含量高达46.29 μg·L^-1,进一步证实了酿酒酵母可以从头合成单萜化合物。本研究结果为深入探究酿酒酵母菌株单萜化合物合成代谢机制提供了理论依据。     

超高效液相色谱-串联质谱法测定动物尿液中5种镇静剂类药物残留

为建立动物尿液中异丙嗪亚砜、异丙嗪、氯丙嗪、安眠酮和地西泮5种镇静剂类药物残留的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法,本研究以固相萃取为净化手段,采用HPLC-MS/MS检测猪、牛和羊尿液中5种镇静剂类药物的残留量。结果表明,尿液样品经离心,调节pH值,Oasis MCX固相萃取柱净化后,能够有效去除杂质,5种镇静剂在0.6~20.0 μg·L^-1范围内呈良好的线性关系,相关系数(R²)均大于0.993;5种镇静剂类药物的检出限和定量限分别为0.1和0.6 μg·L^-1,0.6、2.5和9.0 μg·L^-1 3个添加水平的回收率介于64.6%~110.2%之间,相对标准偏差(RSD)均小于6.0%。本研究结果为不同动物尿液中镇静剂类兽药残留的精准检测提供了技术支持。     

体外消化对油茶蒲提取物抑制α-葡萄糖苷酶活性的影响

为探索体外消化对油茶蒲活性成分的影响,本试验以油茶蒲为原料,分析消化前后油茶蒲提取物、水相、正丁醇相、乙酸乙酯相对α-葡萄糖苷酶抑制活性的变化,采用高效液相色谱(HPLC)、分子对接进一步探索油茶蒲中抑制α-葡萄糖苷酶的主要活性物质。结果表明,油茶蒲提取物、乙酸乙酯相、正丁醇相、水相对α-葡萄糖苷酶的IC₅₀为0.27~1.17 μg·mL^-1,经体外模拟消化后,IC₅₀上升至0.34~2.36 μg·mL^-1,其中乙酸乙酯相的活性最强。油茶蒲提取物成分分析结果显示,体外消化能影响各相萃取物的总酚含量,其中乙酸乙酯相的总酚含量最高(44.49 μg·mL ^-1)且在体外消化中具有良好的稳定性。相关性分析结果表明,油茶蒲提取物中有5个化合物与α-葡萄糖苷酶的IC₅₀呈显著相关,其中包括3-O-甲基鞣花酸-4'-O-β-D-吡喃葡萄糖苷(MEAG)。AutoDock分子对接结果也表明,MEAG具有良好的α-葡萄糖苷酶抑制活性,以及最佳的结合能(-7.99 kcal·mol^-1)和估计抑制常数(1.40 μmol·L ^-1), 其主要通过氢键和疏水作用与α-葡萄糖苷酶的氨基酸残基相互作用。本研究结果为开发新型α-葡萄糖苷酶抑制剂以及油茶蒲资源利用提供了数据参考。     

硒处理对薄壳山核桃幼苗生长的影响

为探究硒对薄壳山核桃幼苗生长的影响,利用不同浓度亚硒酸钠[0(CK)、0.5、5、10、20、40、80 μmol·L^-1]对薄壳山核桃幼苗进行处理,并测定相关生理指标。结果表明,低浓度硒对薄壳山核桃幼苗生长发育、生物量积累、酶活性均有促进作用,而高浓度硒抑制了幼苗生长、干物质的积累、酶活性、光合速率,且硒浓度越高,抑制作用越明显。80 μmol·L^-1 硒浓度处理后的根长较CK显著降低了55.36%(P<0.05),其他形态指标均无显著差异。硒处理显著提高了幼苗体内的硒含量,且根系>叶片。随着硒浓度的增加,叶、根内的硒含量呈先升高后降低的趋势,硒浓度≥0.5 μmol·L^-1时,其他处理均与CK存在显著差异。40 μmol·L^-1硒浓度处理下叶中的硒含量较CK显著增加了1 682倍,根中的硒含量提高了482倍。低硒浓度(≤10 μmol·L^-1)处理可以促进根中Mn²⁺、Mg²⁺含量和叶中的Zn²⁺含量的积累。当硒浓度≥5 μmol·L^-1时,会促进根中Zn²⁺含量的积累,硒浓度≥10 μmol·L^-1,促进茎中Mn²⁺含量的积累,其中在80 μmol·L^-1硒浓度处理下茎中Mn²⁺含量为CK的9.29倍。过氧化物酶(POD)、超氧化物歧化酶(SOD)活性随着硒浓度增加均呈先增后减的趋势,且在0.5 μmol·L^-1硒浓度处理下达到最高,明显高于CK,分别为1 698.63、1 912.28 U·mg prot^-1。综上,低浓度硒处理和施硒时间长短对幼苗光合作用影响不大,而高浓度硒处理会抑制光合作用。本研究结果为进一步揭示硒富集机理和硒的科学使用提供了理论依据。     

高亲和力大豆凝集素单抗制备及免疫学特性鉴定

为制备高亲和力的大豆凝集素(SBA)单克隆抗体,本试验以50 μg SBA蛋白/200 μL乳化液/只的剂量免疫BALB/c小鼠,间接ELISA和间接竞争ELISA鉴定多抗血清效价和灵敏性。选择血清效价和灵敏性较好的小鼠进行细胞融合。通过有限稀释亚克隆筛选出能稳定分泌SBA单克隆抗体的阳性杂交瘤细胞株。采用体内诱生腹水法批量制备SBA单克隆抗体并对其免疫学特性进行鉴定。结果表明,SBA免疫的4只小鼠中,1号小鼠效价最高且灵敏性最好,半数抑制浓度(IC₅₀)为571.4 μg·L^-1。选取该小鼠进行细胞融合,并最终得到1株杂交瘤细胞,命名为5B4E10。制备的SBA单克隆抗体的效价达到1∶409 600 以上,IC₅₀为82.04 μg·L^-1,亚型为IgG1型,亲和力常数(Ka)为1.83×10⁹ L·mol^-1。且该抗体与大豆球蛋白、β-伴大豆球蛋白和其他抗营养因子无交叉反应。本研究结果为建立大豆及其制品中SBA的免疫学检测方法奠定了良好的抗体基础。     

外源NO对镉胁迫下草地早熟禾种子萌发及幼苗生理特性的影响

为探究可缓解草地早熟禾镉胁迫的最适有效硝普钠(SNP)浓度,以草地早熟禾品种蓝月为试验材料,SNP为NO供体材料,研究不同浓度NO对镉胁迫下草地早熟禾叶片生理特性的影响。结果表明,镉离子浓度为200 μmol·L^-1时草地早熟禾的生长受到严重抑制,施加SNP(<100 μmol·L^-1)能够促进草地早熟禾种子发芽,增加草地早熟禾幼苗叶绿素含量,提高叶片干物质量、相对含水量,以及叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性,而其游离脯氨酸(Pro)和丙二醛(MDA)含量均有所降低。表明低浓度NO(<100 μmol·L^-1)可有效缓解草地早熟禾镉胁迫,高浓度SNP(>100 μmol·L^-1)则表现出抑制作用。本研究结果为草地早熟禾修复镉污染土壤提供了理论依据。     

苯并芘(BaP)对鲤鱼肝、肾抗氧化、非特异性免疫能力及组织结构的影响

为探究苯并芘(BaP)对鲤鱼的毒性机制,本研究检测了不同浓度BaP(0、0.1、0.5、1.0 μg·L^-1)暴露对鲤鱼肝、肾组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、丙二醛(MDA)及非特异性免疫酶溶菌酶(LZM)、酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性,以及硝基四氮唑兰(NBT)阳性细胞及肝、肾组织结构的影响。结果表明,1.0 μg·L^-1 BaP暴露30 d后,鲤鱼肝、肾组织SOD活性、MDA含量及肝组织CAT、肾组织GSH活性均显著低于对照组;1.0 μg·L^-1 BaP暴露的肝、肾组织LZM、AKP和PO活性以及肝组织ACP活性均显著低于对照组;不同浓度BaP暴露30 d后,鲤鱼NBT阳性细胞数均显著下降,且对肝、肾组织造成不同程度的损伤。相关性分析结果表明,BaP暴露浓度与肝、肾组织的SOD活性呈负相关,与MDA含量呈正相关,与肝、肾组织的AKP、ACP、PO活性呈负相关,与NBT阳性细胞数呈负相关。综上,水体中BaP暴露对鲤鱼肝、肾抗氧化和非特异性免疫能力及组织结构产生了一定程度的负面影响,本研究为水体中BaP的生态毒性效应研究提供了理论参考。     

高压脉冲电场法提取耐辐射奇球菌中类胡萝卜素的研究

为探讨高压脉冲电场(PEF)电学参数致耐辐射奇球菌破壁提取类胡萝卜素的影响及最佳工艺参数,采用正交试验法,分析了电场强度、处理时间、频率和脉宽4种电场参数对类胡萝卜素提取效果的影响。结果表明,PEF技术提取类胡萝卜素的最优提取水平组合为:电场强度30 kV·cm^-1,处理时间30 s,频率30 Hz,脉宽10 μs。该条件下,提取量为75.06 μg·g^-1,与未进行电场处理的样品提取量48.27 μg·g^-1相比,增加了55.5%。本研究结果为PEF技术的工业化大规模应用提供了理论依据。     

优选非酿酒酵母胞外酶增香酿造干白葡萄酒效果

为了研究非酿酒酵母胞外酶促进葡萄酒发酵产香的效果,该文在爱格丽葡萄汁酒精发酵12 h后,分别添加优选胶红酵母(Rhodotorula mucilaginosa,RM)胞外酶液,优选发酵毕赤酵母(Pichia fermentans,PF)胞外酶液和商业糖苷酶制剂(almondβ-glucosidase,AG)(10 mU/mL),以不添加酶制剂的酿酒酵母纯发酵为对照,发酵过程中每24 h取样,采用HS-SPME-GC/MS(headspace solid-phase microextraction-gas chromatography/mass spectrometry)监测挥发性成分的生成变化。葡萄酒含糖量低于2 g/L时终止发酵,低温满罐密封储存,次年4月,进行葡萄酒香气仪器和感官量化分析。胞外酶液共测得6种风味酶活性,其中RM酶液中的β-D-葡萄糖苷酶、α-L-鼠李糖苷酶、β-D-木糖苷酶活性均显著(P<0.05)高于PF中的,而α-L-阿拉伯糖苷酶和酯酶在PF酶液中活性更高(P<0.05)。发酵过程中,胞外酶处理显著促进了(P<0.05)品种香气和发酵香气物质的生成,其中RM酶液显著提高了萜烯类物质和苯乙基类化合物含量,其作用效果分别比商业酶处理高41.7%和31.8%,PF酶液显著促进了发酵香气化合物的生成,尤其是脂肪酸乙酯含量约为对照的2倍。酒样感官分析结果显示,两株酵母的胞外酶处理表现出各自增香酿造的优势,其中RM酶液促进温带酸果类香气的效果突出,PF酶液显著提升了柑橘类香气。研究结果为酵母风味酶应用于葡萄酒的增香酿造提供了理论和实践指导。     

霞多丽干白葡萄酒品种香和发酵香成分变化的比较研究

本研究对确立宁夏贺兰山东麓原产地葡萄酒质量的感官体系标准,以及对葡萄酒生产工艺优化和质量评价有着重要实用价值。采用溶液萃取法提取霞多丽品种果实与干白发酵香气成分,用气相色谱-质谱进行分离测定,结合计算机检索技术对分离化合物进行鉴定,应用色谱峰面积归一法测定各成分的相对含量。实验结果：果实和干白葡萄酒中分别分离出81和33个峰,鉴定出78和32个香气化学成分,共占其色谱流出组分总量的98.89%和98.97%;成熟果实的挥发性物质,主要是2-呋喃甲醛等脂肪醛、2-呋喃乙酮等脂肪酮和十六碳酸等脂肪羧酸类。经过发酵工艺处理原酒中挥发性物质,主要有3-甲基丁醇、丁二酸二乙酯、辛酸、苯乙醇等。相对含量较高的香气成分种类相似,而微量特征香气成分差异较为显著,从而构成不同产区同一品种葡萄酒相似和独特香气与风格。本结果将对不同原产区优质葡萄酒酿造工艺生产,最大限度的释放游离态芳香物质的能力,提高干白葡萄酒的果香,有重要指导意义。     

不同酶和酵母对干红葡萄酒香气影响的差异分析

为提高甘肃河西产区蛇龙珠干红葡萄酒的香气品质,优化酿酒工艺,该文采用顶空固相微萃取和气相色谱-质谱联用技术,分析比较了添加不同浸渍酶和酵母发酵的蛇龙珠干红葡萄酒的香气构成。结果显示:蛇龙珠干红葡萄酒中初步定性香气化合物75种,主要为酯、醇、酸、萜烯和酚类物质。比较酯类、萜烯类等香气成分质量浓度和香气物质释放总量,EX-V酒样明显高于EX和HC酒样,D254酒样明显高于BDX酒样。各组酒样主要香气成分构成相似,但微量香气成分差异显著。果香是香气强度最高的香气系列,植物香、脂肪香、花香次之,其香气强度EX-V酒样高于EX和HC酒样,D254酒样高于BDX酒样。浸渍酶和酵母对甘肃河西产区蛇龙珠干红葡萄酒香气品质的影响评价显示,浸渍酶EX-V优于EX和HC,酵母D254优于BDX。研究结果可为甘肃河西产区蛇龙珠干红葡萄酒香气品质的提高及酿造工艺研究提供科学数据参考。     

Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas.

Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes. Ruché was chosen as the model wine to study because of its unique fragrance. A yeast strain specific for Ruché was selected using a new and rapid isolation method for red wines. The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions. Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone. Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast. The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM). The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM. By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine. The individual primary aroma compounds, identified by GC-MS analysis in Ruché wine samples or in Ruché skin-yeast-SNM samples, could not explain the complexity of the typical Ruché wine odor. Only odors common to many wine varieties were identified by GC-olfactometry analysis.     

Effect of different winemaking technologies on phenolic composition in Tinta Miúda red wines.

The influence of different types of winemaking technology on the contents of catechins, proanthocyanidins, and anthocyanins in Tinta Miúda red wines was studied. The Tinta Miúda red wines were made by fermentation with carbonic maceration, fermentation with stem contact, and fermentation without stem contact, respectively. The analysis of individual catechins, procyanidins, and anthocyanins in these wines was performed by HPLC, and quantification of total catechins, total oligomeric proanthocyanidins, total polymeric proanthocyanidins, and total anthocyanins was carried out by spectrophotometric methods. The wine made by carbonic maceration contained the highest amounts of both catechins and oligomeric and polymeric proanthocyanidins, followed by the wine made by fermentation with stem contact, whereas the wine made by fermentation without stem contact contained the lowest of these compounds. On the other hand, the concentrations of total anthocyanins and nearly all individual anthocyanins in the carbonic maceration wine were lower than those in the wines made by fermentation with stem contact and fermentation without stem contact. These results indicated that, although the carbonic maceration technique could retain higher amounts of catechins and proanthocyanidins in wine, it did not favor retaining or stabilizing anthocyanins in wine.     

Use of dehydrated waste grape skins as a natural additive for producing rosé wines: study of extraction conditions and evolution

Dehydrated waste grape skins from the juice industry were used as an additive to produce rosé wines. Maceration time, particle size, dosage, alcoholic content, and maceration temperature were first studied in model wine solutions using two different dehydrated waste grape skins. Full factorial experimental designs together with Factor Analysis and Multifactor ANOVA allowed for the evaluation of each parameter according to the composition of color and phenolic and aroma compounds. Higher maceration time favored the extraction of anthocyanins; phenolic compound release was influenced by dosage independent from other factors studied. Rosé wines were produced by direct addition of dehydrated waste grape skins, according to selected parameters in two different white wines, achieving characteristics equivalent to commercial rosé wines. After three months of storage, rosé wine composition was stable.     

Fourier transform infrared spectroscopy and chemometric analysis of white wine polysaccharide extracts

Monovarietal white wines from Maria Gomes and Bical Portuguese Bairrada varieties were prepared according to different maceration and pectic enzyme clarification procedures. The polysaccharide-rich extracts, obtained by wine concentration, dialysis, and lyophilization, were fractionated by graded ethanol precipitation. A wide range of fractions rich in polysaccharides were obtained. Using the spectral region between 1200 and 800 cm(-)(1) of the FTIR spectra of the wine polysaccharide dry extracts, using PCA and CCA chemometric methods, it was possible to discriminate the extracts on the basis of their polysaccharide composition. Moreover, it was possible to identify the wine-making processes involved and their influence on the wine polysaccharides. Furthermore, a calibration model using a PLS1 was proposed for the quantification of mannose in the samples obtained by precipitation with 60% ethanol aqueous solutions. This information will allow an expeditious assessment and monitoring of the polysaccharide composition and modifications that occur during the wine-making processing and evolution.     

Effect of different lysis treatments on the characteristics of yeast derivatives for winemaking

The effects of three preparation techniques on the oenological properties of a yeast autolysate were investigated: enzymatic autolysis, thermolysis, and the combination of a slow freezing-defrosting and mechanical disruption were carried out on a commercial formulation of active dry yeasts (Saccharomyces cerevisiae). The powders obtained by freeze drying, were characterized: volatile compounds were analyzed by SPME-GC with mass spectrometric (MS) and olfactometric detection (O); the release of colloids in winelike solution was studied by SDS-PAGE and size exclusion chromatography (SEC). Finally, the effects of the powders addition on the aroma composition of a white wine were investigated by SPME-GC-MS, SPME-GC-O, and sensory evaluation. The products obtained were quite different from each other. In particular, enzymatic autolysis led to higher contents of nonglycosilated soluble proteins in the powders and determined a higher retention of wine aroma compounds. On the contrary, thermal autolysate was richer in glycoproteins, and it was able to increase the wine aroma intensity; nevertheless, in the wines treated with such preparation, a slight yeastlike olfactory note was perceived.