A Selective Chromatographic Method to Determine the Dynamic of Biogenic Amines During Brewing Process

A highly selective method was established to study the dynamic of biogenic amine formation during the brewing process. The presence of histamine, tyramine, cadaverine, putrescine, phenylethylamine, spermidine, and spermine was determined in raw materials (barley, hop, yeast), intermediate products (sweet wort, hopped wort, green and matured beer), and beers. Dansylated amines were separated on a Zorbax-XDB C₁₈ column using a binary mobile phase composed of acetonitrile and water. Chromatographic conditions were optimized using an experimental design, and validation was performed following International Conference on Harmonization (ICH) recommendations. Calibration data fitted a linear regression model with R ²?>?0.99. Repeatability (n?=?6) and intermediate precision (n?=?3) in matrix showed relative standard deviation (RSD) values lower than 7.62 and 8.51 %, respectively. Recoveries, at three different concentrations, ranged from 73.97 to 114.94 %. The proposed method was applied to determine the dynamic of biogenic amine content during brewing process of three microbreweries, allowing the detection of the principal sources or stages of biogenic amine (BA) contribution/formation.     

Levels of biogenic amines as a measure of the quality of the beer fermentation process: Data from Belgian samples

Biogenic amines were determined by HPLC in four beer types. In spontaneously fermented beers (SF beers), the amounts of vasoactive amines reached high levels, namely a mean value (1) above 20 mg/l for tyramine and (2) close to 10 mg/l for histamine. Considering the bacterial origin of these amines, we established a calculation formula for a Beer biogenic amine index (Beer BAI), reflecting the microbiological quality of the fermentation process. Using this formula we determined a mean Beer BAI value lying between 0.84 ± 0.89 (high quality) in low fermented beers and 16.2 ± 13.9 in SF beers. BAI values ⩾10 (poor hygienic fermentation process) corresponded to beers showing values of vasoactive BA (>10 mg/l) that could cause health troubles in certain types of consumers (people under treatment with monoamine-oxidase medication or genetically more sensitive to food-born BA).     

A Direct and Sensitive Analysis Method for Biogenic Amines in Dairy Products by Capillary Electrophoresis Coupled with Contactless Conductivity Detection

A capillary electrophoresis method coupled to contactless conductivity detection was developed for the quantitative determination of biogenic amines. Complete resolution of five biogenic amines, which are mostly found in dairy products, were performed in less than 6 min. Separation electrolyte composition was selected as 500 mM α-hydroxyisobutyric acid (HIBA) at pH 2.05. The detection limits of the method for biogenic amines were found between 0.041 and 0.098 mg/L. The precisions for the corrected peak areas were calculated as between 2.35 and 4.25% RSD. The applicability of the method was demonstrated by analyzing biogenic amines in the cheese, yoghurt, and kefir samples. The method does not require any pre-concentration and derivatization steps and can be applied as a simple, rapid, and precise method for the direct determination of biogenic amines in food samples.     

Biogenic amines in commercially produced Yulu,a Chinese fermented fish sauce

Seven biogenic amines were determined in 35 commercially produced Yulu samples from three provinces of China by pre-column derivatisation with dansyl chloride (Dns-Cl) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Putrescine, cadaverine, histamine and tyramine were the major biogenic amines (more than 100 mg kg^?1), while tryptamine, spermidine and spermine were regarded as minor biogenic amines (less than 25 mg kg^?1). Twenty samples contained more than 50 mg kg^?1 histamine (the limit for histamine in seafood products as suggested by the Food and Drug Administration). Twenty-one samples contained more than 100 mg kg^?1 tyramine and 10 contained more than 1000 mg kg^?1 total biogenic amines. This study provided data on biogenic amine levels in Chinese fermented fish sauce. The results suggested that biogenic amine content should be monitored in commercially produced Yulu.     

Changes of yolk biogenic amine concentrations during storage of shell hen eggs

The RP-HPLC/UV method, using dabsyl derivatization, optimised for the determination of biogenic amines in egg yolk, was appropriate for quantification of putrescine, cadaverine, histamine, ethylamine, propylamine, ethanolamine, tyramine, tryptamine, spermine, spermidine, phenylethylamine. Detection limits ranged between 0.05 and 0.06 mg of biogenic amine/kg of egg yolk. Two experiments using, respectively, farm and avian eggs were conducted to evaluate yolk biogenic amine concentrations of fresh and stored eggs, and to explain the effect of temperature and time of storage in the levels of biogenic amines during egg shelf-life. Only five of the 11 biogenic amines under study were detected: putrescine, cadaverine, propylamine, ethylamine and ethanolamine. Storage time during shelf-life presented a significant effect on the levels of the five amines (p < 0.01). On the contrary, storage temperature did not presented a significant effect on the levels of the mentioned amines, p > 0.01. The significant reduction of biogenic amine concentration during the shelf-life justified the application of a multiple linear regression using stepwise method to estimate the storage time. The regression equation was applied with success to confirm the storage time of farm eggs and avian eggs that were stored at two different temperatures.     

不同啤酒生物胺含量的比较

使用反相高效液相色谱法,测定啤酒中8种常见生物胺含量。以苯甲酰氯为衍生试剂,Agilent EclipseXDB-C18为固定相,乙腈和乙酸铵溶液为流动相,紫外检测波长设置为254 nm,梯度洗脱。本文首先对国内市售啤酒生物胺种类及含量范围进行研究,检测结果表明国内啤酒中8种生物胺均有存在,但不同样品所含种类不同,且有个别胺含量偏低无法定量。不同厂家的产品在种类和含量上存在差别,但整体含量接近,生物胺总量为4.21～10.59 mg/L,不足以对人类健康产生危害。原麦汁浓度、酒精度与生物胺含量的关系显示,二者与生物胺有一定联系,但非主要因素,相对于生产厂家和发酵情况,原麦汁浓度、酒精度对生物胺含量的影响较小。     

Application of LC with Evaporative Light Scattering Detector for Biogenic Amines Determination in Fair Trade Cocoa-Based Products

In this study, the separation of eight biogenic amines (cadaverine, serotonin, histamine, spermidine, spermine, tyramine, putrescine and β-phenylethylamine) by a liquid chromatography (LC) method with evaporative light scattering detection (ELSD) was performed. The LC–ELSD method was validated by comparison of the results with those obtained through LC–ultraviolet (UV) determination, based on a pre-column dansyl chloride derivatisation step, and the recorded data showed as both analytical methods can be interchangeably used for biogenic amines determination. LC–ELSD methodology showed good precision and permitted to achieve, for standard solutions, limits of detection (LOD) ranging from 0.01 to 0.02 μg ml^?1 and limits of quantitation (LOQ) ranging from 0.03 to 0.05 μg ml^?1. The whole methodology, comprehensive of the homogenization–extraction process and LC–ELSD analysis, has been applied in the analysis of several samples of fair trade cocoa derivatives. The most abundant amine found was histamine for a total amount of biogenic amines in the range 5.81–38.82 μg g^?1. The highest amounts of biogenic amines (BAs) were found in the most processed products but never representing a possible risk for consumer health, according to the toxicity levels reported in literature and regarded as acceptable.     

Determination of Biogenic Amines in Sichuan-Style Spontaneously Fermented Sausages

The qualitative and quantitative profile of biogenic amines in 30 samples of Sichuan-style spontaneously fermented sausages sold in Sichuan retail markets was studied by HPLC. Tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine were analyzed by UV detection after pre-column derivatization with dansyl chloride. There were wide ranges of different biogenic amines in the samples, except spermidine. The results showed that the histamine was the major biogenic amines, followed by tyramine and cadaverine, which have a mean value of 196.06 mg/kg (ranging from 114.33 to 327.35 mg/kg), 164.67 mg/kg (ranging from 88.84 to 285.89 mg/kg), and 141.65 mg/kg (ranging from 19.14 to 376.46 mg/kg), respectively. The toxicity limit of histamine is 100 mg/kg, where all of the samples exceeded this limit. None of the samples of total biogenic amines content exceeded 1000 mg/kg. The results showed that the amounts of biogenic amines in Sichuan-style spontaneously fermented sausages sold in Sichuan retail markets may pose a potential health risk for sensitive individuals. This suggested that it is necessary to improve the production techniques and the raw materials’ hygienic quality, to ensure the quality and safety of this fermented sausages with respect to their biogenic amine content.     

Determination of biogenic amines in traditional Chinese fermented foods by reversed-phase high-performance liquid chromatography (RP-HPLC)

A survey of biogenic amine content of traditional Chinese fermented foods (douchi, sufu, fermented sausage, yulu, and shrimp paste) was carried out. Eight major biogenic amines including putrescine, cadaverine, histamine, phenylethylamine, tyramine, spermine, spermidine and tryptamine were separated by reversed-phase HPLC-DAD on Inertsil ODS-SP column after pre-column derivatisation with dansyl chloride. The results showed that spermine (1.65–3.96 mg 100 g^?1), putrescine (0.20–10.89 mg 100 g^?1), cadaverine (3.60–12.14 mg 100 g^?1), and histamine (0.57–20.24 mg 100 g^?1) were the most represented amines. All amines were detected in yulu, while sufu showed a much higher content of most amines. Shrimp paste showed the lowest level of total biogenic amines. Moreover, the composition and content of eight biogenic amines in the selected samples varied among different food types, origins and batches. Although the average content for each amine is within the range that may elicit direct adverse reactions, consumers should be aware of the potential synergistic effect among different amines and limit their consumption at each meal.     

Occurrence of ochratoxin A and biogenic amines in Asian beers sold in French markets

Fifty‐one samples of 17 Asian beer brands commercially available in France were analysed for the occurrence of ochratoxin A and biogenic amines, including ethanolamine, histamine, methylamine, ethylamine, tyramine, tryptamine, 2‐phenylethylamine, putrescine, isopenthylamine and cadaverine. The types and levels of biogenic amines varied from one brand to another. None of the ochratoxin A or biogenic amine contaminants in the Asian beer samples exceeded the recommended levels for safe consumption. Ochratoxin A was found to range from n.d. to 0.175 µg L^?1. Biogenic amines were detected in all samples at a level ranging from 6.15 to 47.30 mg L^?1. Isoamylamine was not detected in any of the beers. Ethanolamine, tyramine, putrescine, and cadaverine were the most abundant of the nine biogenic amines found in this study. The concentrations were 2.75–12.88 mg L^?1 for ethanolamine, 0.58–17.37 mg L^?1 for tyramine, 1.54‐9.78 mg L^?1 for putrescine and 0.31–11.39 mg L^?1 for cadaverine. The work revealed significant, strong correlations (p < 0.01) between concentrations of ochratoxin A and biogenic amines, especially tyramine, tryptamine and cadaverine. Copyright © 2013 The Institute of Brewing & Distilling     

Changes in biogenic amine and polyamine contents in smear-ripened cheeses during storage

The content of eight biologically active amines in two groups of smear-ripened cheeses (unwashed-rind and washed-rind) was analysed. The biogenic amine levels were determined after storage at 5 °C in three periods of shelf lifetime: at the minimum durability date; 1 and 2 weeks after this date. The analytical procedure consisted of amine extraction from sample matrix, derivatisation with dansyl chloride and ultra-performance liquid chromatography quantification. The content of biogenic amines and polyamines significantly differed according to the technology of ripening. The cheeses unwashed during ripening had much higher contents of all observed amines and polyamines in comparison with the washed-rind cheeses. The mean content of putrescine, cadaverine and tyramine exceeded 100 mg/kg in unwashed-rind cheeses, while the other amines occurred at lower levels. The content of all detected amines was very low in washed-rind cheeses; no tryptamine, phenylethylamine and histamine were found. The effect of storage on the amine formation was not confirmed.     

Biogenic Amine Content of Beers Consumed in Turkey and Influence of Storage Conditions on Biogenic Amine Formation

The biogenic amine content of beers available in Turkey, both domestic production and imported products, was investigated. A total of 17 domestic and 13 imported beers were evaluated in terms of 8 different biogenic amines (histamine, tyramine, spermine, spermidine, 2‐phenylethylamine, putrescine, tryptamine and cadaverine). HPLC methodology with pre‐column derivatization and photodiode array detection after derivatization with dansyl chloride was used for quantification. In addition, the evolution of important amines such as histamine, tyramine, putrescine and cadaverine were investigated during different storage conditions by response surface methodology. The results indicated that both storage temperature and storage time were important factors affecting biogenic amine content. Histamine and putrescine increased during storage, but then decreased after reaching a maximum level after six weeks. With the biogenic amines tyramine and cadaverine, the amounts increased during the entire storage period. At higher storage temperatures, the formation of biogenic amines increased.     

Validation of an HPLC Methodology for the Identification and Quantification of Biogenic Amines in Chicken Meat

This study validated a high performance liquid chromatography (HPLC) method to determine biogenic amines in chicken meat. For the identification of biogenic amines, an isocratic elution system coupled with a UV detector (254 nm) was used after a perchloric acid (5 %) extraction and benzoyl chloride derivatization of the samples. The standards of tyramine, putrescine, cadaverine, spermidine, and spermine were used for the following validation parameters: selectivity, linearity, accuracy, recovery, limit of detection and quantification, and robustness. Finally, chicken meat commercialized in two types of packaging was evaluated. The results showed an excellent selectivity and separation of all amines, r ²?>?0.99, relative standard deviation <5 %, recovery between 64 and 112 %, limits of detection and quantification between 0.03–1.25 and 0.15–5.00 μg?L^?1, respectively, and appropriate robustness for the proposed methodology. Moreover, both chicken meat commercial packages had similar values for all amines; only tyramine was significantly different (P?≤?0.05). The proposed method was suitable to detection and quantification of simultaneous five biogenic amines in chicken meat.     

Determination of Total Carbohydrate Content in Beer Using Its Pre-column Enzymatic Cleavage and HPLC-RI

Beside ethanol, carbohydrates are the main source of total energy in beer. While analyses of fermentable carbohydrates are important from the technological point of view, the total content of carbohydrates is relevant in terms of nutrition. A high-performance liquid chromatography (HPLC) method with refractometric (RI) detection was developed for determination of total carbohydrate content in beer. Using enzymatic reaction with amyloglucosidase, the carbohydrates were cleaved to yield glucose and short glucose oligomers of less than 10 units, and separated on HPLC ion exchanger Rezex RSO-Oligosaccharide column in Ag⁺ mode. Optimum parameters were established for the enzymatic sample treatment and for the HPLC separation of reaction products. Calibration curves of glucose, fructose, maltose and simultaneously analyzed glycerol ranged from 0.001 to 0.5 g/100 ml, correlation coefficients of all calibration curves were 0.9999. The instrumental limits of quantification were 0.001 g/100 ml and they were verified using repetitive injections, with coefficient of variation (CV) less than 10 % in five replicates. The method limit of quantification was 0.01 g/100 ml since it was necessary to dilute the beer samples before chromatographic analysis. Recovery of the method in non-alcoholic and alcoholic beer was 98.5 %, and 92.3 %, respectively. Finally, ten non-alcoholic and 15 alcoholic beers from Czech market were analyzed using the method, the average content of total carbohydrates in non-alcoholic and alcoholic beers being 4.21 and 3.70 g/100 ml, respectively. These results are in a good correlation with the real extract of beer, which is on average 4.58 and 4.27 g/100 ml.     

Occurrence of biogenic amines in Doubanjiang and Tofu

This study was performed to analyze biogenic amine contents and other parameters in doubanjiang and tofu. Through this study, it was found that doubanjiang contained considerably large amounts of most biogenic amines (>30 mg/kg of β-phenylethylamine in particular), and tofu had a relatively high level of spermidine (>20 mg/ kg). Therefore, the amounts of biogenic amines in the foods seem to be occasionally beyond the safe level for human consumption. Meanwhile, the biogenic amine contents in doubanjiang showed a good relationship with salt content (R²=0.89). The spermidine content in tofu samples was closely related to that in soybean, the raw material of tofu. There also appeared to be a good relationship (R²=0.82) between the biogenic amine contents and total plate counts in doubanjiang, but not in tofu. Most strains from the foods were capable of producing biogenic amines, and the identification revealed that bacterial ability to produce biogenic amines was determined at the level of strains rather than species. Taken together, it seems that biogenic amine contents in doubanjiang are mainly affected by fermentation processes, whereas those in tofu are primarily affected by raw materials.     

Biogenic amine content of kefir: a fermented dairy product

Kefir is a fermented dairy product. Ten samples of kefir supplied from different manufacturers in Turkey were analyzed for the first time to determine biogenic amine contents using HPLC with benzoyl derivatization. Of the 10 biogenic amines under study, putrescine, cadaverine and spermidine were detected in all samples. Tyramine was detected in all kefir samples except one. Tyramine was the prevailing biogenic amine. Tyramine concentrations of kefir samples changed from non detectable values to 12.8 mg/l. Total biogenic amine contents of kefir samples were between 2.4 and 35.2 mg/l. Concentrations of biogenic amines were far below the allowable limits. pH values of kefir samples were in the range from 4.11 to 4.53; acidities were in the range from 0.652 to 1.047% (as lactic acid, w/v); total dry matters were from 8.88 to 12.00% (w/w); total free amino acid contents were from 0.0070 to 0.0206% (as leucine, w/v). No significant correlations were detected between biogenic amine concentrations and pHs and total dry matter contents. Significant correlations were obtained between biogenic amine concentrations and acidities and total free amino acid contents of kefir samples.     

Determination of biogenic amines in beers and brewing-process samples by capillary electrophoresis coupled to laser-induced fluorescence detection

We have developed a sensitive capillary electrophoretic method for the simultaneous determination of 10 biogenic amine which usually appears in beer samples, using laser induced-fluorescence. Sample amines were first derivatized and filtered and then separated with an uncoated capillary tubing in the presence of 50 mM sodium borate and 20% acetone at pH 9.3 at 30 kV. It was possible to analyze biogenic amines in brewing-process samples and in beer samples in less than 30 min, obtaining detection limits between 0.3 μg l⁻¹ for ethylamine and 11.9 μg l⁻¹ for 1,6-hexanodiamine.     

Effects of heating milk and accelerating ripening of low fat Ras cheese on biogenic amines and free amino acids development

Two experiments were conducted to study the effects of heating milk and, consequently, incorporation of whey proteins into cheese curd, fat content, accelerating cheese ripening by attenuated lactobacilli, species of bacteria and method of attenuation on formation of biogenic amines and liberation of free amino acids. Total biogenic amines and total free amino acids increased as ripening period progressed in all cheese treatments. Total biogenic amines and total free amino acids decreased as the fat content was decreased. Heating of 3% fat milk up to 70°C caused a significant (p<0.05) increase in total biogenic amines and total free amino acid concentrations, while raising the temperature of heat treatment up to 75 and 80°C decreased them. However, heating of 2% fat milk up to 75°C caused a definite (p<0.05) increase in total biogenic arnines and total free amino acids; conversely raising heat treatment temperature to 80°C decreased them. These results indicate that there is a positive correlation between total biogenic amines and total free amino acids; moisture and salt contents affected the formation of biogenic amines, while incorporation of whey proteins had no significant effect on biogenic amines development. In a second experiment, addition of attenuated lactobacilli as adjunct bacteria caused a pronounced (p<0.05) increase in free amino acids and biogenic amines. Addition of either freeze- or heat-shocked Lactobacillus helviticus was more effective in promoting the build-up of biogenic amines and free amino acids in the resultant cheeses than in cheeses made with addition of freeze- or heat-shocked Lactobacillus casei, respectively. There was a positive correlation between free amino acids and biogenic amine contents. The type of bacteria and prolongation of ripening period significantly affected the development of biogenic amines. Tyramine was the highest biogenic amine followed by histamine, while spermidine was the lowest.     

Dynamic changes of the content of biogenic amines in Chinese rice wine during the brewing process

This study examined the biogenic amine content of the brewing raw material (wheat Qu, seed starter, mash or liquid) during fermentation and during the maturation and storage process. Pre‐column derivatization and high‐performance liquid chromatography were employed to analyse the dynamic changes in biogenic amines during the Chinese rice wine brewing process. Some aliphatic amines (putrescine, cadaverine, spermine) were already found to be present in the glutinous rice and wheat Qu, but the content was below 2.88 mg/kg. None of the amines were detected in the brewing water. The biogenic amine content in the seed starter exhibited a large variation range (16.43–87.72 mg/L), which could influence the total content of biogenic amines prior to fermentation. A much more obvious increase in biogenic amines stemmed from the fermentation process, with the presence of a biogenic amine‐producing bacteria and precursor amino amines. In the first 2 days, the biogenic amine content increased sharply, followed by a slow increase over the next 4 days, and the content reached a maximum value on day 6; subsequently there was a decrease or fluctuation in the concentration. After clarification and cooking, the biogenic amine content increased significantly; however, a slight decrease was observed during storage, especially for putrescine and histamine. Putrescine and tyramine were the most prevalent amines in all of the samples. Overall, the content of biogenic amines was found to be within the safe level for human health, ranging from 54.52 to 146. 35 mg/L. Copyright © 2013 The Institute of Brewing & Distilling     

Occurrence of biogenic amines in beers produced with malted organic Emmer wheat (Triticum dicoccum)

Because several groups of microorganisms are able to decarboxylate amino acids, the presence of biogenic amines (BA) can be seen as an index of the microbiological quality of the brewing process. BAs were quantified for the first time in the intermediate products and craft beers produced with malted organic Emmer wheat (Triticum dicoccum) in a small size brewery in order to assess the possible presence of critical control points related to biological hazard in the brewing process. BA levels in beers produced exclusively from malted organic Emmer wheat were between 15.4 and 25.2 mg l^–1 in the samples of light beer (Lt) and between 8.9 and 15.3 mg l^–1 in double malt beers (DM) ready for consumption (the beers stored for 90 days at 1–2°C). Cadaverine and tyramine were the main BAs in the Lt and DM beers, respectively. Increased concentrations of BAs seemed to be more related to the heat treatment of the processing product during mashing and wort boiling, rather than to the fermentation process. Much lower concentrations were found in finished beers obtained from 50% malted organic Emmer wheat and 50% malted barley (up to 3.2 mg l^–1) or from 30% malted Emmer wheat (up to 8.3 mg l^–1). Thus, Emmer wheat malt can be a useful alternative to wheat and spelt for the production of beer with a limited content of BA, if the processing technology is kept under control.