应用超高效液相色谱结合荧光检测法测定酿造原料和啤酒中的赭曲霉毒素A

在2008～2009年,使用超高效液相色谱（UPLC）结合荧光检测法（FLD）,对237种样品的啤酒大麦、麦芽、啤酒花、麦汁和啤酒进行了赭曲霉毒素A（OTA）污染的分析。相比于其他常用的方法,UPLC法是一种具有低检测限和定量限（LOD和LOQ）的快速检测技术。啤酒的LOD和LOQ值分别为0．0003nWmL和0．001ng／mL,大麦或麦芽为0．05μg／kg和0．2μg／kg,啤酒花为0．16μg／kg和0．5μg／kg。赭曲霉毒素A在其中一种大麦样品（0．3μg／kg）,一种麦芽样品（0．7μg／kg）和一种啤酒花样品（0．6μg／kg）中被检测到,对啤酒酿造过程中的OTA含量也做了检测。此外,对从当地商店购买的国内外啤酒样品也进行了分析,OTA在其中的39％啤酒样品中被检测到,水平介于0．001～0．0544ng／mL之间,只有一个啤酒样品中OTA含量达到了0．2438ng／mL。     

污染物赭曲霉素在大麦、麦芽、啤酒中的测量

利用ELISA(RIDASCREEN)方法研究大麦、麦芽、啤酒样品中存在的赭曲霉素A(OTA)。OTA的检测限很低，啤酒是0．08μg／L、大麦和麦芽是0．4μg／L。在29种大麦样品中有26种OTA含量是在0．53～12μg／kg之间。在24种麦芽样品中有23种OTA含量在0．5～6．6μg／kg之间，仅有一种麦芽样品没有检出OTA。在150种啤酒样品中有42种OTA含量在0．1～8．10μg／L之间(占28％)，108种啤酒样品(占72％)没有发现OTA，仅有一种啤酒样品OTA含量高于5μg／L。     

制麦和酿造过程中脱氧雪腐镰刀菌烯醇的初步研究

2008年江苏某农场的赤霉病感染相对严重的KA-4B大麦中,脱氧雪腐镰刀茵烯醇（DON）的含量为1．91mg/kg。以此大麦为原料,实验室规模下进行制麦和酿造实验,结果表明,浸麦可以洗去大麦本身含有的绝大部分DON;而大麦内部没有被洗掉的镰孢霉属真菌孢子在发芽阶段重新萌发、生长代谢,形成并积累大量的DON;焙燥阶段不能破坏DON,成品麦芽和麦根中DON的含量分别是原大麦中DON含量的51％和89％。麦芽中的DON可以经过糖化和发酵过程流入到啤酒中,啤酒中DON的总含量是麦芽粉中DON总量的86％。而同一年份相邻农场的基本未感染赤霉病的KA-4B大麦中,DON含量低于0．1mg/kg,绿麦芽和麦根中均检测到低于0．1mg／kg的DON,而成品麦芽、麦汁和啤酒中均未检测到DON。制麦及酿造实验表明,与基本未感染赤霉病的KA-4B大麦相比,赤霉病感染程度严重的KA-4B大麦微生物污染严重,DON含量相对高,大麦品质较差,发芽率较低,麦芽浸出率低,所制得麦汁的过滤速度快,麦汁和啤酒的色度均较高。     

羽LC／MS／MS分析麦芽和啤酒中的单端孢菌毒素（Trichothecene）

建立了一种简单、灵敏的用液相色谱电子喷射离子化联用质谱(LC／MS／MS-ESI)分析五种单端孢茵毒素(Trichothecene)的方法。这五种单端孢茵毒素分别为：HT-2毒素(HT-2)、T-2毒素(T-2)、脱氧雪腐镰刀茵烯醇(DON)、瓜萎镰茵醇(NIV)和3-乙酰基脱氧雪腐镰刀茵烯醇(3-AcDON)。分析了7种进口麦芽和17种国产啤酒(日本)，仅从一种麦芽中检测出了5ng／g的HT-2和23ng／g的NIV，啤酒中仅检测出了0．5～1．4ng／g的DON。该方法可应用在食品质量检测方面。     

制麦和酿造过程中脱氧腐镰刀菌烯醇的初步研究

2008年江苏某农场的赤霉病感染相对严重的KA-4B大麦中,脱氧雪腐镰刀菌烯醇(DON)的含量为1.91mg/kg.以此大麦为原料,实验室规模下进行制麦和酿造实验,结果表明,浸麦可以洗去大麦本身含有的绝大部分DON;而大麦内部没有被洗掉的镰孢霉属真菌孢子在发芽阶段重新萌发、生长代谢,形成并积累大量的DON;焙燥阶段不能破坏DON,成品麦芽和麦根中DON的含量分别是原大麦中DON含量的51%和89%.麦芽中的DON可以经过糖化和发酵过程流入到啤酒中,啤酒中DON的总含量是麦芽粉中DON总量的86%.而同一年份相邻农场的基本未感染赤霉病的KA-4B大麦中,DON含量低于0.1 mg/kg,绿麦芽和麦根中均检测到低于0.1mg/kg的DON,而成品麦芽、麦汁和啤酒中均未检测到DON.制麦及酿造实验表明,与基本未感染赤霉病的KA-4B大麦相比,赤霉病感染程度严重的KA-4B大麦微生物污染严重,DON含量相对高,大麦品质较差,发芽率较低,麦芽浸出率低,所制得麦汁的过滤速度快,麦汁和啤酒的色度均较高.     

大麦和麦芽萃取物抗氧化力的比较——采用高效液相色谱-库仑陈列检测器测定其游离酚类化合物含量

分析了10种大麦及其麦芽的热水浸出物的抗氧化能力。三价铁还原抗氧化能力（FRAP）和自由基消除能力（ABST）的值分别为，麦芽0．23-0．45mgGAE／gdw，大麦0．12-0．25mgGAE／gdw。无壳麦芽KM1910的抗氧化能力最强，而抗氧化力最高的大麦品种是Merlin。FRAP，ABST和ITT值之间存在着明显的正相关性（p〈0．01）。研究了施肥（20公斤N／公顷）对大麦抗氧化力的影响，结果表明施肥的影响并不明显，而基因类型对大麦抗氧化力影响明显。使用Folin—Ciocalteu‘s的方法测定总多酚含量，其范围在0．6-2．9mgGAE／gdw，与所有使用的测定抗氧化力的方法之间存在正相关性（p〈O．01）。采用配库仑阵列检测器的HPLC方法测定游离酚，含量最多的是阿魏酸，其含量范围是，大麦12．5～21．9μgGAE／gdw，麦芽7．8～56．1μgGAE／gdw，大麦及麦芽中儿茶酸的含量范围分别为11．0-17．0μgGAE／gdw，0．9-12．1μgGAE／gdw。     

正丁基-3-甲基咪唑四氟硼酸离子液体介导在呕吐毒素酶联免疫检测中的应用研究

呕吐毒素是食品中常见的一种真菌毒素,目前最常用的检测方法为酶联免疫检测。本文研究合成了一种水溶性离子液体正丁基-3-甲基咪唑四氟硼酸,并将其应用于呕吐毒素（DON）酶联免疫检测。结果表明:当呕吐毒素酶联免疫（ELISA）体系中含有0.014g/mL的该离子液体时,其检测灵敏度提高了约33.92%。半数抑制浓度分别为54.95μg/kg和36.31μg/kg（前者不含离子液体）。重复测定多次,其半数抑制浓度平均值为36.85μg/kg,表明重现性较好。另外,该方法已用于啤酒和麦芽汁中DON加标测定回收率。      

正丁基-3-甲基咪唑四氟硼酸离子液体介导在呕吐毒素酶联免疫检测中的应用研究

呕吐毒素是食品中常见的一种真菌毒素,目前最常用的检测方法为酶联免疫检测.本文研究合成了一种水溶性离子液体正丁基-3-甲基咪唑四氟硼酸,并将其应用于呕吐毒素(DON)酶联免疫检测.结果表明:当呕吐毒素酶联免疫(ELISA)体系中含有0.014g/mL的该离子液体时,其检测灵敏度提高了约33.92%.半数抑制浓度分别为54.95μg/kg和36.31μg/kg(前者不舍离子液体).重复测定多次,其半数抑制浓度平均值为36.85μg/kg,表明重现性较好.另外,该方法已用于啤酒和麦芽汁中DON加标测定回收率.     

北京地区部分市售食用植物油中玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的污染状况分析

目的分析北京市售植物油中玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的污染状况。方法实验采用同位素稀释-超高效液相色谱-串联质谱法,在多反应监测模式下对所采集玉米油、花生油、调和油等食用油样中的玉米赤霉烯酮(ZEN)、脱氧雪腐镰刀菌烯醇(DON)进行了检测,并对其污染现状进行分析。结果 ZEN和DON在3~500 ng/mL浓度范围内线性良好,相关系数为0.999,加标回收率为95.05%~107.10%,ZEN和DON方法的检出限分别为0.03μg/kg和0.9μg/kg;ZEN和DON方法的定量限分别为0.1μg/kg和3μg/kg。对北京市售30个油样检测结果表明,DON为未检出,ZEN检出率为100%,最高含量为333μg/kg,最低含量为1.95μg/kg,平均含量为67.7μg/kg,低于欧盟规定的限量标准400μg/kg。结论通过分析食用油中真菌毒素的含量状况,初步了解了北京市售植物油中玉米赤霉烯酮和脱氧雪腐镰刀碱烯醇的污染状况。     

检测大麦、麦芽和啤酒中反式-2-王烯醛的现代分析方法SPME和SPDE的优化

反式-2-壬烯醛是导致贮存啤酒中不协调异味和不新鲜黄油味的一种醛类物质。本研究优化和介绍了自动固相微萃取（SPME）与气相色谱（GC）联用技术及固相动态萃取（SPDE）与气相色谱（GC）联用技术检测大麦、麦芽和啤酒中反式-2-壬烯醛的方法。比较了SPME的五种不同位置涂有固定涂层的萃取纤维头（100μmPDMS,65μmPDMS／DVB,85μmCAR／PDMS,50／30μmDVB／CAR／PDMS,85μmPA）和两个针（501μmPDMS,50μmPDMS／AC）顶空（HS）SPME和SPDE萃取大麦、麦芽和啤酒中反式-2-壬烯醛的萃取率。当NaCl的添加量为1．5g,萃取时间20min,温度60℃的条件下,PDMS／DVB的HS—SPME能够达到最大的萃取率;当温度60℃,NaCl的添加量1．5g,萃取次数15次,PDMS针的HS—SPDE达到最大萃取率。研究证实了HS—SPME与气相色谱-质谱分析（GC—MS）联用技术能够检测到反式-2-壬烯醛;HS—SPME—GC与气相色谱火焰电离探测器（GC—FID）联用技术能够对样品进行分析。     

运用免疫亲和柱和高效液相色谱(HPLC)检测啤酒大麦中的脱氧雪腐镰刀菌烯醇

采用免疫亲和柱净化、高效液相色谱定量等方法对啤酒大麦中的脱氧雪腐镰刀菌烯醇(DON)进行检测。向啤酒大麦中加入标准品,加标的范围在0.10～2.00μg/g,加标回收率为92.04%～97.29%,相对标准偏差是0.92%～2.67%。检出限是0.01μg/g。所测样品中DON的含量范围在0.0123～0.0926μg/g,其中江浙、云南和东北产区的啤酒大麦中DON含量较高。     

Determination of ochratoxin A in brewing materials and beer by ultra performance liquid chromatography with fluorescence detection

In 2008–2009 the total set of 237 samples of malting barley, malt, hop, wort, and beer was analysed for ochratoxin A (OTA) contamination using the ultra performance liquid chromatography (UPLC) coupled to fluorescence detection (FLD). The UPLC method is a fast technique with low limits of detection and quantification (LOD and LOQ) compared to other methods used routinely. LOD and LOQ values were 0.0003 and 0.001 ng/ml for beer, 0.05 and 0.2 μg/kg for barley and malt, 0.16 and 0.5 μg/kg for hop, respectively.     

Natural occurrence of Fusarium mycotoxins of the 1990 barley crop in Korea.

During the barley harvest in June 1990, there was a great deal of rainfall and high humidity in the southern part of Korea, and natural occurrence of Fusarium mycotoxins was suspected in barley samples. The samples of undergrade barley were obtained from four provinces and analysed for the presence of deoxynivalenol (DON) and nivalenol (NIV) by gas chromatography and zearalenone (ZEN) by high performance liquid chromatography. Of 37 samples, 33, 37 and 10 were positive for DON, NIV and ZEN, respectively. The husked barley contained 29-677 ng/g for DON, 114-1546 ng/g for NIV and 183-1416 ng/g for ZEN. The naked barley contained 38-645 ng/g for DON, 85-4569 ng/g for NIV and 40-1081 ng/g for ZEN. The average concentration of NIV in naked barley was higher than that in husked barley, but the average concentration of DON in husked barley was higher than that in naked barley. The survey indicates that the 1990 barley crop in Korea was heavily contaminated with Fusarium mycotoxins.     

Further survey on the Fusarium mycotoxins in Korean cereals

Fifty-one samples of cereals from the 1984 harvest from Korea were analyzed for nivalenol (NIV), fusarenon-X (FX), deoxynivalenol (DON) and 3-acetyl-DON by gas chromatography (GC) utilizing a 63Ni electron capture detector (ECD), and were quantitated for zearalenone (ZEN) by high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Trichothecenes and ZEN in the positive samples were confirmed by GC-mass spectrometry (MS). Out of 51 samples, 51, 46 and 42 were positive for NIV, DON and ZEN, respectively, and one malt sample was heavily contaminated with NIV (2675 ng/g) and DON (246 ng/g), and one wheat sample was heavily contaminated with NIV (3169 ng/g). Neither FX nor 3-acetyl-DON was detected in any of the samples. The data reported here indicates that Korean cereals harvested in 1984 are simultaneously contaminated with NIV, DON and ZEN, and the incidences and levels are similar to those observed in the cereals harvested in 1983.     

Effect of ozone treatment on the safety and quality of malting barley

Molds and their mycotoxins are an expensive problem for the malting and brewing industries. Deoxynivalenol (DON) is a mycotoxin that is associated with Fusarium spp. These fungi frequently cause Fusarium head blight in wheat and barley in the midwestern region of the United States; Manitoba, Canada; Europe; and China. Barley growers and malt producers would benefit from a postharvest control method for mold growth and DON production. We evaluated the use of gaseous ozone (O(3)) for preventing Fusarium growth and mycotoxin production while maintaining malt quality characteristics. Micromalting was performed in three replications under standard conditions. Ozone treatment was applied to malting barley during steeping via a submerged gas sparger. Ozone treatment conditions were 26 mg/cm(3) for 120 min after 2 and 6 h of steeping. The effects of gaseous ozone on DON, aerobic plate counts, Fusarium infection, and mold and yeast counts of barley throughout the malting process were measured. Various quality parameters of the malt were measured after kilning. Statistical tools were used to determine the significance of all results. Ozonation of malting barley during steeping did not lead to significant reductions in aerobic plate counts but did lead to a 1.5-log reduction in mold and yeast counts in the final malt. The influence of gaseous ozone on DON concentration was inconclusive because of the low initial concentrations of DON in the barley. Ozone significantly reduced Fusarium infection in germinated barley. Gaseous ozone did not negatively influence any aspect of malt quality and may have subtle beneficial effects on diastatic power and β-glucan concentrations.     

Fundamental study on the influence of Fusarium infection on quality and ultrastructure of barley malt

Barley infection with Fusarium species has been a long standing problem for the malting and brewing industries. In this study, we evaluate the impact of Fusarium culmorum infected raw barley on the final malt quality. Barley grains were infected for 5 days at optimum fungal growth conditions. Grains were fully characterized and compared to standard barley grains. Due to fungal infection, germinative energy of infected barley grains decreased by 45%; its water sensitivity increased dramatically, and grains accumulated 199 μg/kg of deoxynivalenol (DON). Barley grains were subsequently malted for 8 days, fully characterized and compared to standard malt grains. Fungal growth behavior was evaluated during malting using a PCR-based assay and mycotoxins were measured using HPLC. Fungal biomass increased in grains, during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Kernel ultrastructure was evaluated using scanning electron and confocal laser scanning microscopy. Infected malt grains were characterized by extreme structural proteolytic, (hemi)-cellulolytic and starch deterioration with increased friability and fragmentation. Infected grains had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt in comparison to the control. The results of this study revealed that 20% F. culmorum infected barley kernels lead to a significant reduction in malt quality as well as mycotoxin formation.     

Effect of electron-beam irradiation on the safety and quality of Fusarium-infected malting barley

Utilization of Fusarium-infected barley for malting may lead to mycotoxin production during malting and decreased malt quality. Electron-beam irradiation may prevent safety and quality defects and allow use of otherwise good quality barley. We evaluated electron-beam irradiation for preventing Fusarium growth and mycotoxin production while maintaining barley-malt quality characteristics. Four barley lots with varying deoxynivalenol (DON) concentrations were irradiated at 0, 2, 4, 6, 8, and 10 kGy. Treated barley was malted in a pilot-scale malting unit. Barley and malt were analyzed for Fusarium infection (FI), germinative energy (GE), aerobic plate counts (APC), mold and yeast counts (MYC), and DON. Malt quality parameters included malt extract, soluble protein, wort color, wort viscosity, free amino nitrogen, alpha-amylase, and diastatic power. FI, APC, and MYC decreased in barley with an increase in dosage. The APC and MYC for malts from barley exposed to 8–10 kGy were slightly higher than in other malted samples indicating that irradiation-resistant microflora could flourish during malting. Barley GE significantly decreased (3–15%) at 8–10 kGy. Although irradiation had no effect on DON in raw barley, DON decreased significantly (60–100%) in finished malts prepared from treated barley (6–10 kGy). Malt quality parameters were slightly affected by electron-beam radiation. The results suggest 6–8 kGy may be effective for reducing FI in barley and DON in malt with minimal effects on malt quality.     

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg^?1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).     

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF BARLEY PROTEINS: RELATIVE QUANTITIES OF HORDEIN FRACTIONS CORRELATE WITH MALT EXTRACT

Several high performance liquid chromatography (HPLC) approaches have been used to study the relationships between amounts of particular groups of grain storage proteins (hordeins) and malt extract, using two sets of barley samples. The areas of several chromatogram peaks varied between samples in a manner that correlated with malt extract. Size-exclusion HPLC of protein aggregates extracted by sonication gave negative correlations of the relative areas of the three largest chromatogram peaks with malt extract in a set of 39 samples of the one variety, but not in a set of samples of 8 varieties grown at 4–5 sites. Under reducing conditions, disulphide-bonded hordeins were quantitatively extracted and the proportion of hordein in the largest peak, comprised of D- and B-hordeins, correlated well with malt extract in both sets of samples. The hordeins sequentially extracted into 50% (v/v) 1-propanol and 50% (v/v) 1-propanol-50 mM dithiothreitol (DTT) were also analysed by reversed-phase HPLC, and could be resolved into several peaks of B-, C- and D- hordeins. Relationships between the amount of certain C- hordeins in the propanol extract, and certain B- hordeins in the propanol-DTT extract and malt extract were seen. As well as providing a better understanding of the relationship between barley grain protein composition and malting quality, these HPLC methods are useful additional methods to enable partial prediction of malt extract using un-malted barley.