Solid-Phase Extraction Embedded Dialysis (SPEED), an Innovative Procedure for the Investigation of Microbial Specialized Metabolites

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.     

Effects of Carbohydrate-free Diets on the Performance of Lactating Mink (Mustela vison) and the Growth Performance of Suckling Kits

The effects of carbohydrate-free diets on body weight changes, feed consumption, and plasma concentrations of nutrients, metabolites, and hormones in lactating mink and on growth performance in kits were investigated in a total of 108 litters during the lactation period of seven weeks. The litters were distributed in three experimental groups fed different ratios of metabolisable energy (ME) derived from protein and fat (61:38; 47:52; 33:66). The females and kits were weighed at parturition and 2, 3, 4, and 7 weeks after parturition. Blood samples were collected from females in the second, fourth, and sixth week of the lactation period. Carbohydrate-free diets fed to lactating mink can in principle support normal performance, health of the females, and growth performance of the kits. However, mink need a sufficient supply of available gluconeogenic precursors in the form of amino acids. In the present experiment, the diet with 33% of ME from protein contained insufficient amounts of gluconeogenic precursors for lactating mink, and three weeks post partum this group was excluded from the experiment. The differences in dietary protein level (61% and 47% of ME) and fat level (38% and 52% of ME) influenced the plasma concentrations of free fatty acids (FFA) and urea, but had minimal influence on plasma concentrations of other nutrients, metabolites, and hormones.     

Aniquinazolines A–D,Four New Quinazolinone Alkaloids from Marine-Derived Endophytic Fungus Aspergillus nidulans

Four new quinazolinone alkaloids, namely, aniquinazolines A–D (1–4), were isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined on the basis of chiral HPLC analysis of the acidic hydrolysates. The structure for 1 was confirmed by single-crystal X-ray diffraction analysis. All these compounds were examined for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.     

Effect of relative humidity at either acute or chronic moderate temperature on growth performance and droppings' corticosterone metabolites of broilers

The present study aimed to investigate the effect of relative humidity(RH) at either acute or chronic moderate ambient temperature(Ta) on growth performance and droppings' corticosterone metabolites of broilers.Two experiments were conducted: effect of RH(35,60 or 85%) on average daily feed intake(ADFI) and droppings' corticosterone metabolites at acute(1 d: 20–26 or 31–20°C,26 or 31°C for 6 h d–1 at 10:00–16:00) moderate Ta(experiment 1) and effect of RH(35,60 or 85%) on growth performance and droppings' corticosterone metabolites at chronic(step-wisely increasing temperature by 3°C every 3 d from 20 to 32°C within 15 d: 20–23–26–29–32°C) moderate Ta(experiment 2).Droppings were collected at the 2,4,6,8,and 22 h after Ta-RH controlled in experiment 1 and at the 2,4,6,and 22 h after Ta controlled to 32°C in experiment 2.The results showed that: 1) In experiment 1,85% RH increased(P0.05) the droppings' corticosterone metabolites at the 2,6,8,and 22 h and 35% RH increased(P0.05) it at the 2 and 22 h compared to the 60% RH.Moreover,85% RH further increased(P0.05) it compared to the 35% RH,however,no difference(P0.05) was found in ADFI among the three RH groups at acute moderate 26°C; 35 and 85% RH increased(P0.05) droppings' corticosterone metabolites at the 2,6,8 and 22 h and decreased(P0.05) ADFI compared to the 60% RH,moreover,85% RH further increased(P0.05) droppings' corticosterone metabolites and further decreased(P0.05) ADFI compared to the 35% RH at acute moderate 31°C; and the average of droppings' corticosterone metabolites in the whole period had a negative correlation(P0.02) with the ADFI.2) In experiment 2,85% RH increased(P0.01) droppings' corticosterone metabolites only at the 2 h and decreased(P0.02) ADFI and average daily gain(ADG) compared to the 60% RH,no difference(P0.05) in droppings' corticosterone metabolites was found between the 35 and 60% RH,however,35% RH decreased(P0.01) ADG compared to the 60% RH,and the average of droppings' corticosterone metabolites in the whole period also had a negative correlation(P0.02) with ADFI and ADG.In conclusion,droppings' corticosterone metabolites could be used as a RH stress index and low and high RH,especially high RH,reduced growth performance possibly through inducing RH stress at moderate temperature.     

武夷茶区茶园土壤养分状况及其对茶叶品质成分的影响

【目的】分析乌龙茶主产区武夷茶园中土壤养分状况及其对茶叶品质成分的影响,为改良茶园养分管理和提升茶叶品质提供理论依据。【方法】本研究在2008年福建省土壤普查数据的基础上,于2015年在武夷3大茶区,（桐木区、岩茶区和洲茶区）随机选取68个茶园,包括桐木区12个、岩茶区32个和洲茶区24个,分别采取茶园0—20 cm的土壤和一芽三叶的茶青样品。检测土壤pH,有机质、碱解氮、有效磷和速效钾含量等土壤养分指标,同时利用高效液相色谱系统（HPLC）定量测定茶青中的茶氨酸、咖啡碱、芦丁、ECG、EGCG和总儿茶素等6种次级代谢物含量做品质成分分析。【结果】通过对比2008年和2015年的土壤养分指标,发现近年来,武夷茶区土壤酸化严重,部分茶园土壤有效磷含量增加显著。三大茶区中,岩茶区茶园土壤养分状况变化最为明显,其土壤pH、有机质和碱解氮分别下降了0.65、45.29%和49.39%;土壤有效磷含量却大幅度上升,从5.21 mg?kg ^-1上升到平均值为245.70 mg?kg ^-1,上升幅度超过40倍。说明该区域茶园存在过度施肥的现象。土壤养分状况显著影响茶叶品质成分,并且不同土壤养分指标对不同品质成分的影响有所不同。通过边际效应分析,发现各次级代谢物的最高含量都有其对应的土壤养分范围。在此基础上,拟合了武夷茶区高品质茶园适宜的土壤养分范围：pH 4.5—5.0;有机质20—40g·kg ^-1;碱解氮60—100 mg·kg ^-1;有效磷10—100 mg·kg ^-1;速效钾100—150 mg·kg ^-1。 【结论】综合本研究结果,建议武夷岩茶区和洲茶区在养分管理方面,总体采取有机肥取代部分化肥,适量补氮和钾,严格控制磷肥施用等措施。     

2株高产蛋白微藻细胞代谢物的比较

以2株新近分离出来的链带藻(Desmodesmus sp. QL96和Desmodesmus sp. QJ74)为研究对象,检测胞内主要细胞代谢产物组成及含量变化,找出优势细胞代谢产物。收集相同时间间隔下培养的2株微藻,经过冻干、研磨、复溶,制备成细胞溶解液,再进行逐级萃取,分级检测每个萃取步骤中所得细胞代谢产物的含量。结果表明:在BG11培养基中生长30 d,链带藻Desmodesmus sp. QL96和Desmodesmus sp. QJ74的最高蛋白含量分别达到细胞干重的71.68%和62.14%,其他代谢产物,如碳水化合物、脂质和色素,在2株微藻中的含量都没有超过20%。对比其他微藻的文献报道,链带藻Desmodesmus sp. QL96是目前发现的蛋白含量最高的藻株,其蛋白的表达模式和蛋白相关产品的开发具有研究价值。     

人工饲粮对幼年工蜂生长发育及生理代谢的影响

本试验利用液相色谱-质谱联用技术(LC-MS)对幼年工蜂进行代谢组学分析,旨在探究不同人工饲粮对幼年工蜂生长发育及生理代谢的影响,为蜂王邮寄的人工饲粮配制提供一定理论依据。试验选取1日龄的意大利蜜蜂(Apis mellifera ligustica)300只,分为5组,分别饲喂5种人工饲粮,每组30只工蜂。对照组饲粮:蔗糖粉∶蜂蜜=3∶1;试验组A饲粮:蔗糖粉∶蜂粮∶蜂蜜=6∶5∶1;试验组B饲粮:蔗糖粉∶蜂粮∶蜂蜜=6∶3∶1;试验组C饲粮:蔗糖粉∶花粉∶蜂蜜=6∶5∶1;试验组D饲粮:蔗糖粉∶花粉∶蜂蜜=6∶3∶1。用5种人工饲粮进行室内喂养,记录蜜蜂7 d内的死亡数量;解剖试验组A和对照组第1、3、5、7天的工蜂咽下腺,测定其平均重量、颜色和饱满度;采用LC-MS方法检测试验组A和对照组蜜蜂饲喂7 d后的代谢物差异,对检测数据进行模式识别分析,筛选并鉴别差异代谢物。结果表明:1)对照组和试验组A蜜蜂第1~5天时均没有出现死亡,在第6和7天时试验组A蜜蜂死亡数量均显著低于对照组(P<0.05)。试验组B、C、D的蜜蜂在6 d内全部死亡。2)试验组A第7天时的蜜蜂咽下腺平均重量显著高于对照组(P<0.05),咽下腺颜色为全部乳白色,且咽下腺小体饱满。3)采用主成分分析(PCA)、偏最小二乘法判别方法(PLS-DA)和正交偏最小二乘方-判别分析(OPLS-DA)分析代谢组数据,结果显示试验组A和对照组明显分离,共鉴定出23个差异代谢物,包括氨基酸、脂类、糖类等,其中有10种代谢物上调,13种代谢物下调。差异代谢物通路分析发现,差异极显著的通路有牛磺酸和亚牛磺酸生物合成通路、赖氨酸降解通路、戊糖磷酸盐代谢通路、戊糖和葡萄糖醛酸相互转化通路(P<0.01),差异显著的通路有氨基酸的生物合成通路,色氨酸代谢通路,碳代谢通路,丁酸代谢通路,丙氨酸、天冬氨酸和谷氨酸代谢通路,丙酮酸代谢通路(P<0.05)。由此可见,试验组A的饲粮更适合蜜蜂工蜂的生长发育和生存,在邮寄环境中可代替常用的炼糖饲粮。     

凤梨草莓与黄毛草莓种间杂种果实香气成分的代谢谱分析

【目的】通过比较黄毛草莓和凤梨草莓的2个种间杂种PF（具黄毛草莓浓郁的蜜桃香气）和NF（无蜜桃香气）完熟期果实香气成分的代谢谱,为黄毛草莓蜜桃香气特征成分鉴定及野生草莓优异资源的开发利用提供参考。【方法】采用顶空固相微萃取和气相色谱/质谱联用技术（gas chromatograph tandem mass spectrometer technology,GC-MS）,对供试材料的香气成分进行检测。采用偏最小二乘法判别分析（partial least squares discrimination analysis,PLS-DA）模型第一主成分的变量投影重要度（variable importance in the projection,VIP）值和log2FC（Fold Change,FC）,结合T-test的P值来筛选差异性代谢物,PF相对于NF,设置阈值VIP>1.0, log2FC>1.0或log2FC<-1.0且P value<0.05,差异代谢物的相对含量采用峰面积归一化法计算,CAS号（Chemical Abstracts Service Registry Number）在https://pubchem.ncbi.nlm.nih.gov网站查阅。【结果】从检测到的383种总代谢物中筛选出67种差异代谢物,其中58种上调,9种下调,上调幅度较大的差异代谢物为内酯类物质,log2FC排在前3名的依次是(Z)-7-癸烯-5-酸（5.60）、丁位十一内酯（5.33）、δ-癸内酯（5.30）,下调幅度较大的差异代谢物为酯类物质,-log2FC排在前3名的依次是肉桂酸乙酯、亚硫酸（-7.19）,2-乙基己基异己酯（-6.65）和3-羟基丁酸乙酯（-4.14）。从相对含量来看,酯类在PF（37.69%）中大幅低于NF（57.20%）,内酯类在PF（20.91%）中大幅高于NF（6.12%）,酮类在PF（15.30%）中略高于NF（9.12%）,醇类、醛类、酸类、烯烃类和其他代谢物在PF和NF中的含量相当,NF中相对含量最大的酯是丁酸乙酯（17.92%）,PF中相对含量最大的内酯是δ-癸内酯（12.53%）,PF相对含量最大的酮与NF相同,均为2-庚酮。【结论】肉桂酸乙酯、丁酸乙酯和3-羟基丁酸乙酯等酯类可能是NF的关键香气成分,(Z)-7-癸烯-5-酸、丁位十一内酯和δ-癸内酯等内酯类可能是形成PF蜜桃香气的关键物质。     

两种可食用玫瑰生物碱代谢物差异分析

以云南滇红玫瑰(ZY)与墨红玫瑰(DH)两种云南主要的食用花卉品种为研究对象,采用超高效液相色谱-串联质谱(UPLC-MS/MS)方法比较两种玫瑰花次生代谢产物的差异性。结果表明,两种花瓣中共检测到82种代谢产物,包括55种氨基酸及其衍生物(67.07%)、16种生物碱(19.51%)、7种酚胺(8.54%)、4种吲哚类生物碱(4.88%),其中滇红玫瑰中谷胱甘肽含量是墨红玫瑰的26.35倍,具有较强的抗氧化能力;而L-天冬酰胺和S-(5'-腺苷)-L-高半胱氨酸却是墨红玫瑰中特有的物质,具有显著的降血压作用。与墨红玫瑰相比,滇红玫瑰15种差异代谢物显著下调,表明墨红玫瑰较滇红玫瑰有较高的营养保健功能,而滇红玫瑰高表达成分使其具有更强的抗氧化能力。     

GC-MS分析慢性氨气应激对肉鸡血清代谢物的影响

【目的】随着养殖业集约化和规模化的发展,舍内环境对畜禽生长的影响日益突出。在密闭舍饲条件下,不断产生有毒有害气体,其中氨气的危害最大,降低畜禽生产性能,威胁动物健康。为此开展慢性氨气应激对肉鸡血清代谢物影响的研究,从小分子物质和代谢途径方面探究氨气导致机体代谢发生的变化,为舍内氨气的合理调控提供数据支持。【方法】试验采用单因素完全随机设计,将96只21日龄健康AA肉鸡随机分成对照组和试验组,每组设4个重复,每个重复12只鸡。对照组氨气浓度为0 mg·kg ^-1,试验组氨气浓度为45 mg·kg ^-1。试验在人工模拟呼吸舱内进行,呼吸舱采用全自动化控制温湿度等养殖条件,采用网上平养,自由采食和饮水,24h 光照,试验期为21d。在肉鸡42日龄时,从每个重复中随机抽取2只,翅静脉采血,分离血清。将血清样品预处理后,采用气相色谱/质谱联用技术（gas chromatograph tandem mass spectrometer technology, GC-MS）检测代谢产物,利用质谱数据库对其进行鉴定。通过主成分分析、偏最小二乘法判别分析、正交偏最小二乘法判别分析、t-检验等,寻找差异代谢物,并利用生物信息学方法对差异代谢物进行通路富集分析。【结果】（1）利用 GC-MS 方法,结合质谱数据库对检测到的代谢物进行快速鉴定,在肉鸡血清中共检测到204种代谢物。将代谢组数据导入SIMCA-P软件进行多元统计分析,结合t-检验筛选出 23种差异代谢物,其中上调19个,下调4个。（2）差异代谢物主要涉及能量代谢（乳酸、α-酮戊二酸）、氨基酸代谢（L-别苏氨酸、L-高丝氨酸、烟酰甘氨酸）、脂肪酸代谢（硬脂酸、亚麻酸、亚油酸、胆固醇）以及核苷酸代谢（次黄嘌呤、尿嘧啶、胸腺嘧啶）等。（3）代谢通路富集分析表明,氨气应激主要影响了肉鸡血清的脂肪代谢通路,如亚油酸代谢、花生四烯酸代谢以及α-亚麻酸代谢。【结论】GC-MS较为全面地检测到血清的代谢物,能够准确地筛选出差异代谢物,同时慢性氨气应激显著影响肉鸡的血清代谢物含量,主要影响脂肪代谢通路,为阐明氨气应激影响营养代谢的机制提供参考。