The Significance of Iso‐α‐Acids for Beer Quality Cambridge Prize Paper

The iso‐α‐acids and their chemically‐modified variants play a disproportionately large role in the final quality of beer. Here, fundamental aspects of two of these quality issues — foam and bitterness — are discussed. A common feature of both issues is the dependence on the hydrophobic character of the hop compounds on both bitterness potency and ability to stabilise foams. Thus the isocohumulones appear significantly less bitter than the other, more hydrophobic hop compounds. Also apparent were the differences in bitterness between the cis‐ and the trans‐isomers, with the former being the more potent. Also described are the differences in the partitioning of the cis‐ and trans‐iso‐α‐acids into beer foam. The trans‐isomers are enriched in foam relative to their cis‐counterparts and may account for the observed enrichment of cis‐isomers in the final beer relative to the common ratios observed upstream.     

Alterations of the Profiles of Iso‐α‐Acids During Beer Ageing,Marked Instability of Trans‐Iso‐α‐Acids and Implications for Beer Bitterness Consistency in Relation to Tetrahydroiso‐α‐Acids

Age‐induced decomposition of iso‐α‐acids, the main bittering principles of beer, determines the consistency of the beer bitter taste. In this study, the profiles of iso‐α‐acids in selected high‐quality top‐fermented and lager beers were monitored by quantitative high‐performance liquid chromatography at various time intervals during ageing. The degradation of the iso‐α‐acids as a function of time is represented by the ratio, in percentage, of the sum of the concentrations of trans‐isocohumulone and trans‐isohumulone to the sum of the concentrations of cis‐isocohumulone and cis‐isohumulone. This parameter is relevant with respect to the evaluation of bitterness deterioration in aged beers. Trans‐iso‐α‐acids having a shelf half‐life of less than one year proved to be significantly less stable than cis‐iso‐α‐acids, but it appears feasible to counteract degradation if a suitable beer matrix is available. The fate of the trans‐iso‐α‐acids in particular adversely affects beer bitterness consistency. In addition to using hop products containing low amounts of trans‐iso‐α‐acids, brewers may profit of the remarkable stability of tetrahydroiso‐α‐acids, even on prolonged storage, for the production of consistently bitter beers.     

Degradation of Iso‐α‐Acids During Wort Boiling

A detailed study on the degradation of iso‐α‐acids was conducted. Because of the complexity of the wort matrix and interfering interactions during real wort boiling, the investigation of degradation kinetics was performed in an aqueous solution. Degradation was investigated as a function of time (0–90 min), temperature (80–110°C), pH value (4–7), original gravity (10–18°P) and ion content of the water (0–500 ppm Ca²⁺ and Mg²⁺). After 90 min of boiling, over 20% of the dosed iso‐α‐acids could no longer be detected. A strong dependence of degradation could be shown due to high temperature, low pH, high original gravity and a high Mg²⁺ content. The cis:trans ratio and co‐iso‐α‐acid content did not change significantly. Losses of isohumulones could be lowered by reducing the temperature and original gravity, as well as by heightening the pH value. High amounts of Ca²⁺ and Mg²⁺ salts also led to an increase in degradation products. Solutions to decrease degradation and thereby possible improvements in sensory bitter quality are discussed.     

Relationships of Sensory Bitterness in Lager Beers to Iso‐α‐Acid Contents

Iso‐α‐acids and their chemically modified variants play a large role in evoking the bitter sensory attributes of lager character, but individual consumers may vary in their perception of bitterness. Sixteen lagers were scored in rank‐rating for bitterness by 14 trained assessors and the concentrations of the six bitter components in these beers were determined by high performance liquid chromatography. Relationships between bitterness intensity and the bitter components were modelled well using partial least square regression with a correlation value of 0.92. When 8 assessors carried out time‐intensity scoring of bitterness, profiles for single products were very different. However, single assessor profiles for multiple products showed qualitative similarities but quantitative differences. That individual assessors perceived bitter characters differently in relation to time has implications for new product development.     

Gas Chromatography‐Combustion‐Isotope Ratio Mass Spectrometry for Traceability and Authenticity in Foods and Beverages

As consumers demand more certainty over where their food and beverages originate from and the genuineness of ingredients, there is a need for analytical techniques that are able to provide data on issues such as traceability, authenticity, and origin of foods and beverages. One such technique that shows enormous promise in this area is gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS). As will be demonstrated in this review, GC‐C‐IRMS is able to be applied to a wide array of foods and beverages generating data on key food components such as aroma compounds, sugars, amino acids, and carbon dioxide (in carbonated beverages). Such data can be used to determine synthetic and natural ingredients; substitution of 1 ingredient for another (such as apple for pear); the use of synthetic or organic fertilizers; and origin of foods and food ingredients, including carbon dioxide. Therefore, GC‐C‐IRMS is one of the most powerful techniques available to detect fraudulent, illegal, or unsafe practices in the food and beverages industries and its increasing use will ensure that consumers may have confidence in buying authentic products of known origin.     

Simple and Fast Sample Preparation Followed by Gas Chromatography‐Tandem Mass Spectrometry (GC‐MS/MS) for the Analysis of 2‐ and 4‐Methylimidazole in Cola and Dark Beer

We have developed a simple and fast sample preparation technique in combination with a gas chromatography‐tandem mass spectrometry (GC‐MS/MS) for the quantification of 2‐methylimidazole (2‐MeI) and 4‐methylimidazole (4‐MeI) in colas and dark beers. Conventional sample preparation technique for GC‐MS requires laborious and time‐consuming steps consisting of sample concentration, pH adjustment, ion pair extraction, centrifugation, back‐extraction, centrifugation, derivatization, and extraction. Our sample preparation technique consists of only 2 steps (in situ derivation and extraction) which requires less than 3 min. This method provided high linearity, low limit of detection and limit of quantification, high recovery, and high intra‐ and interday repeatability. It was found that internal standard method with diluted stable isotope (4‐MeI‐d₆) and 2‐ethylimidazole (2‐EI) could not correctly compensate the matrix effects. Thus, standard addition technique was used for the quantification of 2‐ and 4‐MeI. The established method was successfully applied to colas and dark beers for the determination of 2‐MeI and 4‐MeI. The 4‐MeI contents in colas and dark beers ranged from 8 to 319 μg/L and from trace to 417 μg/L, respectively. Small quantity (0 to 8 μg/L) of 2‐MeI was found only in dark beers. The contents of 4‐MeI (22 μg/L) in colas obtained from fast food restaurants were significantly lower than those (177 μg/L) in canned or bottled colas.     

Microwave‐Assisted Extraction and Determination of Citrus Red 2 Dye in Oranges and Orange Juice by Liquid Chromatography‐Tandem Mass Spectrometry

Abstract: A fast, simple, low cost, and high‐throughput method has been developed for the determination of citrus red 2 dye in orange and orange juice samples. The procedure is based on microwave‐assisted extraction followed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. The method was optimized, and the analyte was efficiently extracted from the samples in 30 min using hexane/acetone (v/v, 3 : 1). The method was validated and showed good linearity and selectivity. The limits of quantification (LOQs) were 5 μg/kg (sample size of 2 g) for both orange and orange juice samples. The average recoveries, measured at 3 concentration levels (5, 10, and 20 μg/kg), were in the range 77.5% to 87.6% for the compound tested with relative standard deviations below 7.3%. The proposed method is rapid, accurate, and could be utilized for the routine analysis of citrus red 2 dye in orange and orange juice samples.     

Determination of Xylazine and 2,6‐Xylidine in Animal Tissues by Liquid Chromatography—Tandem Mass Spectrometry

Xylazine is a potent α2‐adrenergic agonist used in veterinary medicine for sedation, analgesia, muscle relaxation, and so on. Its residue in animal‐derived food may cause the food safety problem. Moreover, the metabolite 2,6‐xylidine was reported to be a genotoxic and carcinogenic compound. Therefore, it is necessary to develop a high sensitive method for analyzing xylazine and metabolite residue in animal products. Here, we described a LC‐MS/MS method for simultaneous determination of xylazine and 2,6‐xylidine in 4 animal tissues: liver, meat, kidney, and fat. The samples were extracted by acetonitrile, and further clean up by hexane. The analysis was performed on a C18 reversed‐phase column and API 5000 Triple Quadrupole mass spectrometry with positive electrospray ionization interface operating in the multiple‐reaction monitoring mode. For all of the investigated sample matrix, the limit of detection (limit of quantitation) for xylazine and 2,6‐xylidine were 0.06 (0.2) and 1.5 (5) μg/kg, respectively, the recoveries were between 63.5% and 90.8%. The precision was within the range of required criteria for method development. The presented method is sensitive and reproducible, and thus suitable for accurate quantification of xylazine and metabolite residue in animal‐derived food products.     

Classification of Barley Shochu Samples Produced Using Submerged Culture and Solid‐state Culture of Koji Mold by Solid‐phase Microextraction and Gas Chromatography‐Mass Spectrometry

An easy and fast method of analyzing volatile components contained in shochu, by headspace solid‐phase microextraction and gas chromatography‐mass spectrometry, was established to determine the difference in the content of the various components of barley shochu produced using a submerged culture of koji mold (submerged‐culture shochu) and a solid‐state culture of koji mold (solid‐culture shochu). The contents of the volatile components between the two types of shochu were compared. The results of the comparison revealed that the content of ethyl lactate, ethyl benzoate, 3‐methyl‐1‐pentanol, diethyl succinate, citronellol, and 2‐phenethyl acetate differed between the submerged‐culture and the solid‐culture shochu samples. In addition, the classification of barley shochu samples, including those on the market, into submerged‐culture shochu and solid‐culture shochu, was carried out by multivariate analysis using the quantitative values of the above‐mentioned six compounds, and distinct discrimination was found possible.     

Effects of β‐Glucan,Shearing and Environmental Factors on the Turbidity of Wort and Beer

The presence of added β‐glucan in wort caused increased turbidity levels, which increased at higher molecular weights and concentrations of the polymer. Levels of pH, maltose and ethanol, and shear experienced in a brewery also influenced the turbidity of wort and beer. Haze levels of beer after 0.45 μm membrane filtration were found to decrease due to the removal of non‐β‐glucan particles. Cold storage at 4°C for two weeks was found not to lower the turbidity caused by high concentrations of high molecular weight β‐glucan polymers.     

Effects of β‐Glucans and Environmental Factors on the Viscosities of Wort and Beer

This paper reports on the influence of molecular weight and concentration of barley β‐glucans on the rheological properties of wort and beer. Environmental conditions such as pH, maltose level in wort, ethanol content of beer, shearing and shearing temperature were also examined for their effects on wort and beer viscosities. In the range of 50–1000 mg/L, β‐glucans increased solution viscosity linearly with both molecular weights (MW) of 31, 137, 250, 327, and 443 kDa and concentration. The influence of MW on the intrinsic viscosity of β‐glucans followed the Mark‐Houwink relationship. Shearing wort and beer at approximately 13,000 s^?1for 35 s was found to increase the wort viscosity but reduce beer viscosity. Shearing wort at 20°C influenced β‐glucan viscosity more than shearing at 48°C and 76°C whereas the shearing temperature (0, 5 and 10°C) did not effect the viscosity of beer. At lower pHs, shearing was found to reduce the viscosity caused by β‐glucans in wort but had no effect in beer. Higher concentrations of maltose in wort and ethanol in beer also increased the viscosity of β‐glucan polymers. It was found that β‐glucans had higher intrinsic viscosities in beer than in wort (5°C), and lower critical overlap concentrations (C^*) in beer than in wort.     

同位素内标-高效液相色谱-串联质谱法同时测定小麦和玉米中19种真菌毒素

建立高效液相色谱-串联质谱法测定小麦和玉米中19种真菌毒素的检测方法。样品经乙腈∶水∶甲酸(80∶18∶2)溶液提取、离心、浓缩后,以C18色谱柱分离,甲醇-甲酸水溶液为流动相梯度洗脱,采用电喷雾正负离子(ESI+、ESI-)同时电离,多反应监测模式检测,同位素内标法定量。19种真菌毒素在一定含量范围内均具有良好的线性关系(R²≥0.992),定量限范围为0.12～30μg/kg,在低、中、高3个添加浓度水平下的回收率范围为61%～117%,相对标准偏差小于15%(n=6),满足日常检测方法性能要求。本方法操作简便、灵敏度高、重现性好,适用于小麦、玉米等粮食中多种真菌毒素的同时测定。     

Simultaneous Analysis of Nε‐(Carboxymethyl)Lysine and Nε‐(Carboxyethyl)Lysine in Foods by Ultra‐Performance Liquid Chromatography‐Mass Spectrometry with Derivatization by 9‐Fluorenylmethyl Chloroformate

Isotope dilution ultra‐performance liquid chromatography–electrospray tandem mass spectrometry with derivatization by 9‐fluorenylmethyl chloroformate was successfully applied to quantify N^ε‐(carboxymethyl)lysine (CML) and N^ε‐(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end‐products, which may pose a risk to human health.     

Ser71 of αS1‐Casein is Phosphorylated in Breast Milk—Evidence from Targeted Mass Analysis

Scope

The casein phosphoproteins in mother's milk supply calcium and phosphate ions and make them biologically available to the newborn. Human αS1‐casein is of particular interest being also an autoantigen and proinflammatory cytokine. Phosphorylation of αS1‐casein by casein kinase 2 completely abolishes binding to toll‐like receptor 4 and proinflammatory effects. It is, however, not known, which amino acids are affected. Therefore, breast milk samples were analyzed in an effort to detect the phosphorylation sites of αS1‐casein.

Methods and results

Breast milk samples were tryptically digested. Target tandem MS analysis confirmed the known phosphorylation sites S33 and S41; evidence for pS89 was found in some samples. Experimental support for the presence of pS31 and pS34 was weak. Phosphorylation of a new site in αS1‐casein, S71, was reproducibly measured in all samples, albeit at much lower intensity than pS33 and pS41.

Conclusion

Phospho‐occupancy rates varied greatly and could not be confidently correlated to other parameters within the cohort of 20 donors. The new phosphosite S71 is located in the neighborhood of the serine‐rich region and may contribute to the cluster of high charge density at normal milk pH, likely exerting an influence on protein tertiary structure and thus function.

     

A Comparison of Polyvinylpolypyrrolidone (PVPP), Silica Xerogel and a Polyvinylpyrrolidone (PVP)–Silica Co‐Product for Their Ability to Remove Polyphenols from Beer

Proanthocyanidins have been characterized and levels compared in an ale and a lager using normal‐phase liquid chromatography– mass spectroscopy (LC/MS) after a pre‐concentration step employing Sephadex LH‐20 chromatography. Twenty proanthocyanidins were identified including both non‐galloylated and galloylated forms of the monomers, dimers and trimers. Oligomers greater than trimer were not detected. A proportion of all classes of proanthocyanidin adsorbed non‐specifically to a silica xerogel product, but polyvinylpolypyrrolidone (PVPP) and a product that comprises polyvinylpyrrolidone (PVP) on a surface of amorphous silica displayed greater capability (i.e. specificity) for binding these materials. Of the two specific adsorbents, it was the PVPP that had the greater binding capability compared to the PVP‐silica co‐product.     

高效液相色谱-串联质谱法测定动物肌肉组织中氨基甲酸酯类杀虫剂及其代谢物残留

建立动物组织中氨基甲酸酯类杀虫剂及其代谢物(共16种)残留的高效液相色谱-串联质谱分析方法。样品经乙腈提取、浓缩、净化,液相色谱串联质谱测定,内标法定量。16种杀虫剂在1.0～100μg/L范围内线性关系良好(r＞0.9959);方法定量限为0.5～2.5μg/kg;样品添加5.0、10.0、20μg/kg时,加标回收率为71.4%～105.5%;相对标准偏差为3.2%～13.7%。该方法具有简便快捷、灵敏度高的特点,适用于动物肌肉中氨基甲酸酯类杀虫剂及其代谢物残留量的检测。     

The Effects of Hop‐α‐Acids and Proline‐Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop α‐acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.     

高压液相色谱-质谱法和高效液相色谱法分析籽瓜中环磷酸腺苷

采用高压液相色谱-质谱法鉴定籽瓜中的环磷酸腺苷,并建立高效液相色谱法测定籽瓜中环磷酸腺苷的含量,对样品提取条件进行优化。结果表明：环磷酸腺苷在0.1～100 mg/L质量浓度范围内线性关系良好,相关系数为0.999 9;平均加标回收率为98.40%,相对标准偏差为3.22%。经单因素试验和正交试验,确定优化的提取条件为料液比1∶12（鲜样）和1∶60（干样）水提取,提取温度90 ℃、提取时间40 min、提取次数3 次。样品前处理和分析方法准确、简便、可靠、重复性好,可用于籽瓜中环磷酸腺苷含量的分析测定。