新的啤酒蛋白质组图谱的构建及在啤酒质量控制方面的应用

本研究通过凝胶双向电泳(2-DE)对啤酒中的蛋白质组进行分析,进一步通过质谱技术结合数据库检索方法对凝胶上的主要蛋白点进行鉴定归类,构建出了一个全面的啤酒蛋白质组图谱。结果显示,检测到的199个蛋白质点中有85个能被成功鉴定,此85个蛋白点可归为12类蛋白质。为了证明不同的大麦品种和麦芽溶解度对啤酒蛋白质组成和啤酒品质特征的影响,本文研究了11个啤酒样品,它们由蛋白质溶解度不同的8个品种的大麦麦芽酿造而成。通过2-DE分析啤酒蛋白质组并进行对比,发现大麦品种和麦芽溶解度均会影响啤酒中几种蛋白质的浓度,而啤酒中蛋白质的浓度与啤酒的品质特性,如泡沫稳定性有关。此外,观察到源于酵母的蛋白质也可能影响啤酒的品质。啤酒蛋白质组图谱的应用可为啤酒品质相关蛋白质的检测和研究提供一个强有力的平台。     

啤酒大麦种子蛋白质组的分级制备及初步分析

大麦种子蛋白主要分为清蛋白、球蛋白、醇溶蛋白和谷蛋白,它们在大麦种子中起到不同的作用,因此对每个组分的研究具有重要的理论和实际意义。根据4种蛋白质组分的溶解性不同,可利用分级提取的方法获得4种组分的蛋白质溶液;进而利用TCA沉淀和TCA-丙酮沉淀法对4种蛋白质进行除杂和浓缩,获得适用于双向电泳的蛋白质提取液。利用SDS-PAGE和双向电泳检测提取效果,发现所得SDS-PAGE凝胶条带清晰,双向电泳图谱分辨率高,蛋白质点清晰;经PDQuest分析,谱图结果显示检测到450个水溶蛋白质点和100个醇溶蛋白质点。大麦种子中各组分蛋白质组的分级提取方法的构建,为后续对各组分蛋白组进行质谱鉴定及功能分析奠定了基础。     

酿酒酵母蛋白质双向电泳条件优化及图谱建立

利用双向电泳技术分离酿酒酵母全蛋白质,并比较了不同样品制备方法、不同凝胶参数,以及不同染色方法对电泳结果的影响。经过各个环节的条件优化,最终获得了无明显横纵条纹,背景清晰,分辨率较高,重复性较好的酿酒酵母全蛋白质双向电泳图谱。经PD-Quest软件分析,所得图谱约分离到1 000个蛋白质点,该双向电泳图谱的构建为后续氮代谢研究奠定了基础。     

麦芽溶解度对啤酒泡沫稳定性酌影响及其与大麦二聚α-淀粉酶抑制剂(BDAI-I)的关系

啤酒泡沫稳定性是评价啤酒质量的一个重要指标.本文旨在通过双向凝胶电泳来研究啤酒中的发泡蛋白而阐明麦芽溶解度(蛋白质、淀粉等的降解)与啤酒泡沫稳定性的关系.研究发现用大麦品种B、C制成的麦芽生产的啤酒,其泡沫稳定性随着大麦溶解度的提高而下降;而用大麦品种A麦芽所酿的啤酒,其泡沫稳定性却保持不变.为了研究大麦品种A所制得啤酒具有良好泡沫稳定性的原因,采用双向凝胶电泳分析了啤酒中的全蛋白、盐析蛋白和泡沫蛋白质.结果表明:在A样品中,随着溶解度的升高,某特定区域的蛋白质点在这三部分蛋白中的含量均没有改变;然而,B和C样品中的这种蛋白质点都在减少.进一步采用基质辅助激光解吸离子化飞行时间一质谱方法(MALDI-TOF-MS)确定的肽质量指纹图谱(PMF)对这些蛋白质点进行鉴定,结果显示,这些蛋白质点均为大麦二聚α-淀粉酶抑制剂(BDAI-I).上述结果表明BDAI-I对啤酒泡沫的稳定性有重要的贡献.     

黄酒麦曲浸提液中宏蛋白质组的制备

麦曲在黄酒酿造中具有重要作用,它能为黄酒酿造提供各种酶类(如水解酶等)及各种风味物质前体。该研究基于宏蛋白质组学理论,分别采用TCA-丙酮沉淀法、丙酮沉淀法、2-D Cleanup Kit试剂盒法提取了黄酒麦曲浸提液中的宏蛋白质组,比较了采用3种不同方法制备样品的双向电泳结果。研究发现,用2-D Cleanup Kit试剂盒制备的样品在溶解性、蛋白纯度和双向电泳分离效果等方面都优于另外2种方法,图谱分辨率高,蛋白点清晰。初步建立了适用于黄酒麦曲浸提液的宏蛋白质组制备方法,获得了较为理想的双向电泳图谱,为后续利用质谱分析手段对宏蛋白质组中的蛋白组成进行全面、准确鉴定奠定了基础。     

啤酒行业蛋白质组学的研究进展

介绍了蛋白质组学的概念,以及从样品中蛋白质组的制备、双向电泳分离到质谱分析结合数据库搜索鉴定的主要技术路线。综述了蛋白质组学在啤酒行业中应用的研究进展,主要运用于区分大麦品种、揭示蛋白质组与麦芽和啤酒某些品质之间的关系。对啤酒及原料中蛋白质组今后的研究方向进行了展望,蛋白质组学的研究策略将在啤酒及其原料品质相关的研究中,日益发挥重要的作用。     

蕨菜总蛋白质双向电泳技术体系的建立

为了建立蕨菜中总蛋白质的双向电泳技术体系,作者以新鲜蕨菜为实验材料,分别比较了不同蛋白提取方法,不同上样量以及不同pH范围IPG胶条对2-DE图谱的影响。结果表明,采用TCA-丙酮结合酚抽法提取蕨菜中的蛋白质,采用IPG胶条pH5-8、蛋白质的上样量为1 200μg、考马斯亮蓝G-250胶体考染法进行染色。在此条件下进行蕨菜全蛋白质的双向电泳所得到的图谱蛋白质点多达719个,且蛋白质点分布均匀、背景清晰、分辨率较高。     

利用聚丙烯酰胺凝胶电泳和高效液相色谱法对Scarlett和Prestige大麦(大麦属vulgare L.)的麦芽、麦汁以及啤酒中蛋白组分的比较研究

本研究利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和反相高效液相色谱法(RP-HPLC),对Scarlett仕和Prestige大麦麦芽的蛋白组分(大麦醇溶蛋白、清蛋白和其他可溶性蛋白)进行了研究,同时跟踪分析了由这两种麦芽制成的麦芽汁以及啤酒中的蛋白质变化情况.此外,对这两个大麦品种进行了工业酿造规模的研究.通过对反相高效液相色谱数据进行统计学分析,发现大麦醇溶蛋白在发芽期间存在一个水解过程,导致其含量降低,同时产生较少的疏水性缩氨酸.相比之下,清蛋白和其他可溶性蛋白质在发芽过程中含量会增加.一些麦芽水溶性蛋白会由大麦醇溶蛋白质水解产生.我们还观察分析了这两个品种麦芽蛋白组分数量上的差别.聚丙烯酰胺凝胶电泳图显示出麦芽汁中的绝大部分组分在成品啤酒中也可以观测到.然而,高效液相色谱数据的化学计量分析显示Scarlett和Prestige大麦麦汁蛋白组分间存在数量上的差异.Scarlett大麦麦汁比Prestige麦汁中含有更多的蛋白质.与两种麦芽汁相比,两种成品啤酒样品中的蛋白相似性更强.试验将最优方法成功用于两种大麦麦芽蛋白质组分的研究中,同时跟踪分析了发芽期间蛋白质组分的变化情况以及麦芽汁和成品啤酒中蛋白质组分的变化.     

啤酒大麦、麦芽中多酚类化合物HPLC指纹图谱分析技术研究

以10个进口和8个国产啤酒大麦品种及其相对应的麦芽为样本,采用高效液相色谱（HPLC）建立大麦和麦芽中14种多酚类物质的指纹图谱,并分别进行相似度分析、聚类分析（CA）和主成分分析（PCA）。结果表明,进口大麦样品的相似度（0.938～0.989）高于国产大麦样品（0.911～0.937）,而进口大麦麦芽的相似度（0.892～0.967）普遍低于国产大麦麦芽的相似度（0.956～0.981）;CA（判别距离＜5）结果和PCA结果一致,8个国产大麦和1个进口大麦样品B2聚为一类,8个国产麦芽和3个进口麦芽样品M2、M3、M4聚为一类,说明通过大麦、麦芽多酚类物质的HPLC指纹图谱技术能基本区分国产和进口大麦品质的差异。     

四种大麦间泡沫蛋白质含量差异的比较

本实验建立了从麦芽中提取泡沫蛋白的便捷方法,并利用考马斯亮蓝法(Bradford 法)和SDS-PAGE 电泳技术进行定量分析,直观地检测和比较了四种大麦和麦芽中泡沫蛋白质的含量、组分和各组分的相对含量及其变化情况,可为评估大麦和麦芽质量以及预测啤酒泡沫的品质提供借鉴。     

茭白总蛋白质双向电泳技术体系的建立

为建立茭白总蛋白质双向电泳技术体系,研究了TCA/丙酮沉淀法和改良酚抽法两种不同总蛋白质提取方法、蛋白质上样量、pH值范围及凝胶质量浓度等条件对2-DE的影响。结果表明,改良酚抽法更适合茭白总蛋白质的提取,采用17 cm、pH 4~7的胶条、1.0 mg的蛋白质上样量、12 g/dL的凝胶浓度、考马斯亮蓝G-250胶体考染法染色,最终可获得蛋白质点较多,背景清晰,分辨率较高的2-DE图谱,为进一步开展茭白差异蛋白质组学研究奠定了基础。     

猪肝脏蛋白质组双向电泳体系的建立及优化

建立猪肝脏蛋白质组的双向电泳体系,并从样品处理、电泳参数、染色方法等方面对该体系进行优化。样品处理采用超速离心法,Cleanup试剂盒处理,17cm pH5～8的双向电泳预制干胶条,蛋白上样量120μL,得出匹配率为76%,蛋白点有1412±8个。参照全盲法,对各种不同的猪肝脏样品进行准确性和重复性实验,样品匹配率均在70%以上,且图谱中点的相对迁移率都在实验室的控制标准之内,可以筛选出既具有统计学意义又具有量分析意义的蛋白点,也可用于区分不同的猪肝脏样品。该体系能满足不同猪肝脏样品的双向电泳分析。     

Construction of a novel beer proteome map and its use in beer quality control

Beer proteins were analysed by two-dimensional gel electrophoresis (2DE). The protein species associated with major spots on 2DE gels were identified by mass spectrometry followed by a database search to construct a comprehensive beer proteome map. As a result, 85 out of 199 protein spots examined were positively identified and categorised into 12 protein species. A total of 11 beer samples were brewed from the malt of eight cultivars having different levels of protein modification. This experiment was designed to demonstrate the influences of barley cultivar and malt modification on beer protein composition and beer quality characters. The beers produced from these brewing trails were subsequently analysed by 2DE and their proteomes were compared. Cultivars and malt modification affected the concentration of several proteins in beer. Beer protein concentration was associated with differences in the desirable beer quality trait, foam stability. In addition, expression of yeast derived proteins were observed that may also influence beer quality. Overall, the application of a comprehensive beer proteome map provides a strong platform for detection and potential manipulation of beer quality related proteins.     

Effect of varying starch properties and mashing conditions on wort sugar profiles

The aim of this research was to investigate the relationship between starch composition in barley and its malted counterpart alongside malt enzyme activity and determine how these factors contribute to the fermentable sugar profile of wort. Two Australian malting barley varieties, Commander and Gairdner, were sourced from eight growing locations alongside a commercial sample of each. For barley and malt, total starch and gelatinisation temperature were taken, and for malt, α‐ and β‐amylase activities were measured. Samples were mashed using two mashing profiles (infusion and Congress) and the subsequent wort sugar composition and other quality measures (colour, original gravity, soluble nitrogen) were tested. Variety had no significant (<0.05) effect on any barley, malt, enzyme or wort characteristics. However, growing location impacted gelatinisation temperature, colour, malt protein content and original gravity. The gelatinisation temperature in malt samples was higher, by ~0.8°C, than in the equivalent barley sample. Several malt samples, even with protein contents <12.0%, had gelatinisation temperature >65°C. The fermentable sugars measured in the malt prior to mashing showed a higher proportion of maltose than glucose or maltotriose. In addition, there were significant differences in the amount of sugar produced by each mashing method with the high temperature infusion producing a higher amount of sugar and proportionally more maltose. There is scope for further research on the effect of genetics and growing environment on gelatinisation temperature, mash performance and fermentable sugar development. Routinely measuring gelatinisation temperature and providing this information on malt specification sheets could help brewers optimise performance. © 2019 The Institute of Brewing & Distilling     

焦麦芽烤甜香关键成分的确定及制备工艺优化

为开发能够增加卷烟烟气烤甜香香韵的烟用香料,以焦麦芽为原料,采用卷烟感官作用导向分析和气质联用（GC/MS）方法确定了焦麦芽提取物中的烤甜香关键成分,并以这些成分的含量为指标优化了焦麦芽烤甜香香料的制备工艺。结果表明,①经凝胶渗透色谱（GPC）分离和感官评价确定的焦麦芽烤甜香特征组分中含有麦芽酚、4-羟基-2,5-二甲基-3(2H)-呋喃酮、甲基环戊烯醇酮等15种关键成分。②优化的加热提取工艺参数为95%乙醇、料液质量比1：6、提取温度80℃、提取时间2.0 h、提取2次;脱蜡工艺参数为回溶乙醇浓度95%、回溶液中干物质含量20%和冷冻温度-20℃。③制备的焦麦芽烤甜香特征香料中烤甜香关键成分含量明显高于市售麦芽提取物和焦麦芽粗提物,且添加该烤甜香特征香料的样品卷烟烟气烤甜香更加突出。     

Quality control of beer using high-resolution nuclear magnetic resonance spectroscopy and multivariate analysis

High-resolution nuclear magnetic resonance (NMR) spectroscopy is introduced for the quality control and authenticity assessment of beer in official food control. Measurements were performed using a 400-MHz NMR spectrometer using flow injection technology for automatic sample changing. Only degassing and addition of buffer (pH 5.6 in D₂O for locking and 0.1% TSP for referencing) is required to prepare the beer samples. Differences in the spectral profiles of beers varying in type and origin were studied by principal component analysis (PCA), considering the spectrum to be a characteristic fingerprint. For the first time, the high throughput of a Flow-Injection NMR system allowed a comprehensive database of beer spectra for PCA classification to be established efficiently. Beers made with barley malt could be distinguished from those made with wheat malt. Clustering of beers from the same brewing sites was observed, as well as significant discrimination of beers with deteriorated quality. Using the partial least squares (PLS) method to correlate NMR spectra with results from reference methods, models for calculating the original gravity, ethanol and lactic acid were established. The results obtained suggest that NMR is a useful tool in the quality control of beer samples, since quantitative determination of essential compounds as well as chemometric classification are simultaneously possible. Compared to conventional methods, ¹H NMR spectroscopy is faster and requires simpler sample preparation.     

Protein changes during malting and brewing with focus on haze and foam formation: a review

Beer is a complex mixture of over 450 constituents and, in addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides, and lipids. In beer, several different protein groups, originating from barley, barley malt, and yeast, are known to influence beer quality. Some of them play a role in foam formation and mouthfeel, and others are known to form haze and have to be precipitated to guarantee haze stability, since turbidity gives a first visual impression of the quality of beer to the consumer. These proteins are derived from the malt used and are influenced, modified, and aggregated throughout the whole malting and brewing process. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality wort and beer. During mashing, proteins are solubilized and transferred into the produced wort. Throughout wort boiling proteins are glycated and coagulated being possible to separate those coagulated proteins from the wort as hot trub. In fermentation and maturation process, proteins aggregate as well, because of low pH, and can be separated. The understanding of beer protein also requires knowledge about the barley cultivar characteristics on barley/malt proteins, hordeins, protein Z, and LTP1. This review summarizes the protein composition and functions and the changes of malt proteins in beer during the malting and brewing process. Also methods for protein identification are described.     

Relationship of ergosterol content and fungal contamination and assessment of technological quality of malting barley preserved in a metal silo using the near-ambient method

Environmental microbiologists frequently use ergosterol, a fungal-specific membrane lipid, as an indicator of fungal infection in grain and other plant materials. Microbiological loading and technological quality of barley was determined directly after harvest, after post-harvest drying, and during storage. The conventional plate count method was used to measure fungal contamination (CFU). Ergosterol concentration (ERG) was determined by extraction, saponification and quantification using high-performance liquid chromatography (HPLC) with UV detection. The laboratory malting method was used to determine technological quality of the malt. Results showed a significant correlation between ERG and CFU (the coefficient of correlation was 0.92). Analyses also indicated that the high germinative energy and technological quality of the malt produced from dried barley was retained.     

麦芽糊精基因组DNA不同提取方法的比较

采用不同方法对麦芽糊精基因组DNA进行提取,从中选取满足麦芽糊精材料进行分子标记检测的最好的DNA提取方法。以市售麦芽糊精为材料,分别采用CTAB、SDS和异硫氰酸胍3种方法提取基因组DNA,比较不同方法的提取效果。光密度检测结果显示,异硫氰酸胍法的提取量和纯度均优于SDS法和CTAB法;琼脂糖凝胶电泳检测结果显示异硫氰酸胍法提取的基因组DNA谱带清晰、重复性好,SDS法和CTAB法提取的基因DNA观察不到谱带;3种方法提取的麦芽糊精基因组DNA都能够满足PCR分析的需要。结果表明,3种方法均能有效地从麦芽糊精中获得DNA,然而异硫氰酸胍法优于SDS法和CTAB法。