Significance of volatile compounds produced by spoilage bacteria in vacuum-packed cold-smoked salmon (Salmo salar) analyzed by GC-MS and multivariate regression

Changes were studied in the concentration of 38 volatile compounds during chilled storage at 5 degrees C of six lots of commercially produced vacuum-packed cold-smoked salmon and sterile cold-smoked salmon. The majority of volatile compounds produced during spoilage of cold-smoked salmon were alcohols, which were produced by microbial activity. Partial least-squares regression of volatile compounds and sensory results allowed for a multiple compound quality index to be developed. This index was based on volatile bacterial metabolites, 1-propanol and 2-butanone, and 2-furancarboxaldehyde produced by autolytic activity. Only a few of the volatile compounds produced during spoilage of cold-smoked salmon had an aroma value high enough to indicate contribution to the spoilage off-flavor of cold-smoked salmon. These were trimethylamine, 3-methylbutanal, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-penten-3-ol, and 1-propanol. The potency and importance of these compounds was confirmed by gas chromatography-olfactometry. The present study provides valuable information on the bacterial reactions responsible for spoilage off-flavors of cold-smoked salmon, which can be used to develop biosensors for on-pack shelf-life determinations.     

以生物胺变化评价冷藏罗非鱼片腐败进程

为了探讨生物胺指标评价鱼肉腐败进程的可能性及其安全范围,应用反相高效液相色谱法测定冷藏罗非鱼片贮藏过程的生物胺变化,用以判断鱼片的腐败进程。结果表明:新鲜鱼片初始单胺和多胺总量较高,二胺总量很低。贮藏过程中单胺和多胺波动变化并呈下降趋势,尸胺、腐胺含量快速增加成为主要生物胺。含有尸胺+腐胺的二胺、生物胺指数、总生物胺等指标与其两者增长趋势相似。相关分析表明尸胺、腐胺、二胺、生物胺指数、总生物胺与贮藏时间、微生物数量和氨基态氮具有高度相关性。经回归分析表明假单胞菌、肠杆菌的增长与尸胺、腐胺具有重要的对应关系。尸胺、腐胺和二胺可简便有效评价鱼片腐败进程;但综合考虑组胺和酪胺的毒性,则生物胺指数(BAI)更适用,冷藏罗非鱼片生物胺指数的初步判别范围为:<20mg/kg,新鲜;20～40mg/kg,可接受;>40mg/kg,腐败。该研究为冷藏罗非鱼片生物胺的限量标准提供数据参考。     

Determination of biogenic amines in squid and white prawn by high-performance liquid chromatography with postcolumn derivatization

A simple method was developed for the determination of biogenic amines in aquatic food products using a reverse-phase high-performance liquid chromatography with postcolumn automatic o-phthalaldehyde derivatization and fluorescence detection. The linearity, repeatability, and recovery of the method for seven amines (tyramine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine) were evaluated. This optimized method was applied to detect biogenic amines in squid and white prawn during refrigerated storage. Sensory analysis, pH value, and total volatile base nitrogen value were combined to evaluate the freshness quality of these two raw materials. Agmatine and cadaverine in squid, cadaverine, and putrescine in white prawn were the most obviously changed amines during the storage at two different temperatures, and these biogenic amines could be the effective quality indicators for the freshness of the raw aquatic materials.     

贮藏温度对炝蟹品质的影响

为阐明不同贮藏温度下炝蟹品质的变化和食用安全性,本试验以三疣梭子蟹为材料制作炝蟹,探讨冻藏(-18℃)和冷藏(4℃)条件下炝蟹感官品质、菌落总数(TVC)、挥发性盐基氮(TVB-N)和生物胺的变化规律,并对其生物胺与其他品质指标进行相关性分析。结果表明,贮藏温度对炝蟹感官品质TVC、TVB-N等指标的劣变具有显著的影响。-18℃冻藏和4℃冷藏条件下,炝蟹感官品质分别于第10天和第6个月超出可接受限值;随着贮藏时间的延长,4℃冷藏条件下炝蟹TVC值显著增加,但至贮藏16 d时仍小于5 lg(CFU·g^-1),TVB-N值于贮藏12 d时超出一级鲜度临界值;-18℃冻藏过程中炝蟹TVC值略有增加,但变化不显著,TVB-N值于第6个月超出一级鲜度临界值。4℃冷藏过程中炝蟹中共检测出8种生物胺,而-18℃冻藏过程中共检测出5种生物胺。此外,色胺、腐胺和酪胺是炝蟹贮藏过程中产生的主要生物胺,且与其他品质均具有显著相关性。本研究结果为炝蟹在贮藏过程中的安全性和货架期预测提供了理论依据。     

降解亚硝酸盐和生物胺乳杆菌筛选及其改善鱼肉香肠品质效果

为了筛选出优良亚硝酸盐和生物胺的降解植物乳杆菌,并研究在鱼肉发酵香肠中的应用。该文主要考察了19株植物乳杆菌亚硝酸盐和生物胺的降解活性及相关基因的携带情况,从中筛选出优良菌株,并研究其对鱼肉香肠发酵过程中各种亚硝胺、生物胺、腐败微生物的抑制作用和对其他理化指标的影响。试验结果表明,L.plantarum CP3对亚硝酸盐和生物胺的降解能力最强,分别达到95%和77%,PCR结果显示此菌株中含有亚硝酸盐降解(假定蛋白(hypothetical protein,HP)、谷胱甘肽还原酶(glutathione reductase,GR)、一氧化还原酶(Oxidoreductase,ORD))和生物胺降解的相关基因(multicopper oxidases,suf I),且不含生物胺生成相关基因(hdc,tdc,odc)。GC-MS测定结果显示L.plantarum CP3对发酵香肠中4种亚硝胺-二乙基亚硝胺(N-nitrosodiethylamine,NDEA),二甲基亚硝胺(N-nitrosodimethylamine,NDMA),亚硝基吗啉(N-nitrosomorpholine,NMOR)和甲基乙基亚硝胺(N-nitrosomethylamine,NMEA)的生成都具有显著的抑制作用,联合接种L.saki M4可以协同提高亚硝胺的降解能力。并且,与对照组相比,接种L.plantarum CP3+L.saki M4的鱼肉香肠,发酵48 h后腐胺、尸胺和酪胺的含量分别降低了76.83%,93.33%和88%。另外,接种乳酸菌处理组香肠中的肠杆菌、假单胞菌等腐败菌及pH值、硫代巴比妥酸(thiobarbituric acid,TBA)和挥发性盐基氮(total volatile basic nitrogen,TVB-N)的含量显著低于对照组。该研究结果体现了L.plantarum CP3作为香肠等肉制品发酵剂的良好开发应用前景。     

Biogenic amines in wines from three Spanish regions

One hundred and sixty-three wines from La Rioja, Utiel-Requena, and Tarragona were analyzed to determine if there were any differences in the concentrations of six biogenic amines that are found in these three regions. The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was studied. Results show important differences in putrescine and histamine concentrations among regions, varieties of grape, and type of wine; differences were less appreciable for the remaining biogenic amines studied. Low pH prevented biogenic amine formation. Malolactic fermentation and short storage periods in bottle (3-6 months) showed increases in histamine concentration, whereas longer periods of storage led to a general decrease in histamine. Several strains of lactic acid bacteria were isolated in this work, and their ability to form biogenic amines was assayed in synthetic media, grape must, and wine. Grape varieties, different types of winemaking, pH, and lactic acid bacteria may be responsible for the differences observed in the biogenic amine concentrations of the wines analyzed.     

电子束辐照对真空包装章鱼生物胺的抑制作用研究

为分析电子束辐照处理对真空包装章鱼制品冷藏过程中生物胺形成的抑制作用,以真空包装的新鲜章鱼腕足段为研究对象,分别进行0、0.5、1.0、1.5、2.0和2.5 kGy剂量的电子束辐照处理,于4℃下冷藏63 d,每隔7 d进行感官品评和生物胺含量检测。结果表明,冷藏期间真空包装的章鱼中共检测出精胺、亚精胺、腐胺、尸胺、组胺和酪胺6种生物胺。电子束辐照处理能抑制章鱼冷藏过程中腐胺、尸胺、组胺和酪胺的生成,且抑制作用与辐照剂量成正比,其中对组胺生成的抑制效果最佳,仅在少数样品中被检出。此外,电子束辐照也抑制了章鱼中亚精胺和精胺在冷藏后期的分解。冷藏过程中的腐胺、尸胺和酪胺含量变化与样品感官品质表现出较好的一致性,高感官品质章鱼样品中的腐胺、尸胺和酪胺含量均≤10 mg·kg-1。综合电子束不同剂量辐照对章鱼感官品质和生物胺的抑制作用,1.0 kGy的电子束辐照剂量是真空包装章鱼4℃冷藏保鲜的最适前处理,其能延长高品质新鲜章鱼货架期至35 d。本研究结果为章鱼加工企业生产高品质章鱼产品提供了理论依据。     

Chemical, microbiological, and AromaScan evaluation of mahi-mahi fillets under various storage conditions

The quality for mahi-mahi stored at 1.7, 7.2, and 12.8 degrees C for 0, 1, 3, and 5 days was determined using biogenic amine analysis, microbial counts, and sensory evaluation (by a sensory test panel and an AromaScan). Biogenic amines in methanol extracts from mahi-mahi samples were analyzed using capillary electrophoresis (CE) with ultraviolet detection at 210 nm and a gas chromatography (GC) method that can simultaneously determine the contents of putrescine, cadaverine, histamine, spermidine, and spermine within 20 min after pentafluoropropionic anhydride derivatization. A good correlation R2= 0.99) was found between CE and GC methods for detecting histamine in mahi-mahi. Fish quality deteriorated and correlated with increasing microbial numbers. Biogenic amines may be useful indicators for mahi-mahi quality and safety. AromaScan was able to correlate quality changes for mahi-mahi in microbiological and sensory analyses.     

Freshness and quality criteria of iced farmed Senegalese sole (Solea senegalensis)

Senegalese sole (Solea senegalensis) is a high-value commercial species with increasing importance in aquaculture. The aim of this work was to study the quality changes of this species during chilled storage under refrigeration, through sensory and chemical methods. In particular, the optimization of a quality index method (QIM) scheme was proposed as well as the definition of sensory and chemical quality criteria. A shelf life of 15 days was reported, and a QIM scheme based on a total of 22 demerit points (dp) was proposed. Biogenic amines were never detected, and the usual spoilage indicators, such as TVB-N and TMA-N, were not significantly produced during the period of sensory-acceptable quality (15 days). On the basis of the significant correlations (p<0.001) between sensory data and Ki values, a quality index (QI) 19 dp and a Ki>40% indicate unacceptable quality of iced Senegalese sole.     

High-performance liquid chromatographic determination of biogenic amines in poultry carcasses

Biogenic amines, produced by bacterial decarboxylation of amino acids, have been associated with toxicological symptoms in broilers fed various poultry byproducts. A reversed-phase high-performance liquid chromatographic method is described for the quantitation of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in chicken carcasses. Amines were extracted with perchloric acid, derivatized with dansyl chloride, separated using gradient elution (methanol and water), and detected by fluorescence. Benzylamine was used as the internal standard. Linearity, repeatability, and recovery of the method were evaluated. The method was linear for all of the amines studied at concentrations ranging from 0.05 to 25 microg/mL. Average recoveries ranged from 92.6% to 96.8% for all amines except for histamine, which was 74.6%.     

Influence of the freshness grade of raw fish on the formation of volatile and biogenic amines during the manufacture and storage of vinegar-marinated anchovies

Volatile and biogenic amines from three batches of anchovies, marinated in vinegar, were studied. The anchovies had been vacuum-packed and kept in refrigerated storage for 3 months. Trimethylamine and total volatile basic nitrogen levels were very low and constant throughout marinating and storage process: less than 1 and 10 mg/100 g, respectively. Certain amine levels, mainly those of tyramine and serotonin, increased slightly, particularly during storage. However, even the highest recorded levels were much lower than those considered to be hazardous for human consumption. To study the influence of raw material freshness in the amine profile, two laboratory trials using fresh and spoiled anchovies and simulating the industry standard marinating process were carried out. Levels for both volatile and biogenic amines were dependent on raw material quality, proving consistently higher in those deriving from nonfresh fish. Vinegar marinating leads to a decrease in the accumulation of amines in anchovy while their concentration in the vinegar solution increases due to the vinegar effect as solvent extractor.     

High-performance liquid chromatographic method for determination of biogenic amines in feedstuffs, complete feeds, and animal tissues

Numerous methods to analyze biogenic amines in biological materials have been described. A versatile and rapid methodology to analyze these compounds in feedstuffs, complete feeds, and animal tissues, however, has not been reported. The current method was developed to address this need. Biogenic amines in feedstuffs, complete animal feeds, and animal tissues were extracted with 10% trichloroacetic acid, reacted with O-phthaladehyde using high-performance liquid chromatographic employing a cation exchange column. Detection limits were 50 pmol/mL for tyramine, histamine, putrescine, and spermine; 40 pmol/mL for cadaverine; and 25 pmol/mL for spermidine. Extraction efficiency of biogenic amines in feedstuffs, duodenum, liver, ileum + jejunum, and whole shrimp and shrimp hepatopancreas ranged between 99-105, 93-135, 80-85, 65-102, 88-98, and 88-97%, respectively. It can be concluded that the current method can be applied to individual feedstuffs, complete feeds, and animal tissues for the rapid and accurate determination of concentration of biogenic amines.     

Simultaneous HPLC analysis of biogenic amines, amino acids, and ammonium ion as aminoenone derivatives in wine and beer samples

A method has been developed for the simultaneous analysis of biogenic amines, amino acids, and the ammonium ion in wine and beer. Aminoenones formed by the reaction of amino acids, biogenic amines, and the ammonium ion with the derivatization reagent diethyl ethoxymethylenemalonate are separated by HPLC. Reaction takes place in methanolic alkaline medium for 30 min in an ultrasonic bath. Further heating at 70 degrees C for 2 h produces complete degradation of excess derivatization reagent and byproducts. Comparison of the results of ammonium analysis and enzymatic analysis showed a good correlation (r = 0.953). The proposed analytical method has the following advantages: easy derivatization of wines and beers; quantification of 24 amino acids, nine biogenic amines, and the ammonium ion in a single injection; use of the photodiode array detector; complete degradation of excess derivatization reagent during sample preparation; and detection limits below 0.40 mg/L for amino acids and below 0.06 mg/L for biogenic amines.     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.     

Effects of brine salting with regard to raw material variation of Atlantic salmon (Salmo salar) muscle investigated by Fourier transform infrared microspectroscopy

Atlantic salmon fillets differing with regard to raw material characteristics (prerigor, postrigor, frozen/thawed) and salt content were investigated by FT-IR microspectroscopy and light microscopy. Local variation within each salmon fillet was further taken into account by sampling from the head and tail part separately as they vary in fat and moisture content. The highest salt uptake was achieved for frozen/thawed quality during brine-salting with 16% NaCl for 4 h, while the uptake was least for prerigor fish. At the same time, salting caused muscle fiber swelling of about 10% for both frozen/thawed and postrigor qualities. Differences in the FT-IR amide I spectral region were observed implying a change in the muscle protein secondary structure. Prerigor was least affected by brine-salting, having a final salt concentration of 2.2%, while postrigor had a NaCl content of 3.0% and frozen/thawed of 4.1%. Local variation within the fillets had an effect on the amide I absorption characteristics before as well as after salting. Salt uptake of the samples was affected by raw material quality and at the same time the degree of swelling of the myofibers was influenced by raw material character.     

Relation of biogenic amines' formation with microbiological and sensory attributes in Lactobacillus-inoculated vacuum-packed rainbow trout (Oncorhynchus mykiss) fillets

The biogenic amine (BA) content of vacuum-packed filleted rainbow trout (Oncorhynchus mykiss) inoculated or not with two different Lactobacillus strains, individually or in combination, was monitored during refrigerated storage for 20 days and related to respective bacteriological and sensory changes occurring during the same period. Eight amines, namely putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, histamine, spermine, and spermidine, were determined, whereas agmatine was not detected in any of the samples. In all cases, BA concentration was higher (P < or = 0.05) in the controls compared to all inoculated treatments, whereas the trend with regard to the bacterial populations (Enterobacteriaceae, pseudomonads, and H2S-producing bacteria) and the off-odor scores was similar. Inoculation with Lactobacillus sakei CECT 4808 showed the best preservative effect among inoculated treatments. Concentrations of putrescine and cadaverine, the main BAs formed, correlated well with both spoilage bacterial counts and off-odor scores and can be useful indicators of shelf life. Spermine and spermidine contents decreased during storage, while levels of the other determined BAs remained below 10 mg/kg even after sensory rejection.     

Content of biogenic amines in a Chardonnay wine obtained through spontaneous and inoculated fermentations

This paper describes the content of biogenic amines in wines obtained from a Chardonnay must inoculated with different strains of Saccharomyces cerevisiae and in a wine fermented with the indigenous yeasts (control wine). The concentrations of nonvolatile amines and phenethylamine in the wines from the inoculated must were superior to those of the control wine. This was probably due to the fact that consumption of the precursor amino acids of these amines, during fermentation, was also greater in the inoculated samples than in the control sample. Furthermore, from the results obtained it may be said that, at least to some extent, the nonvolatile amines were formed by yeasts during fermentation. The concentrations of dimethylamine, ethylamine, and pyrrolidine (volatile amines) were different for the different wines, although they did not reach concentrations sufficiently high to have any effect on the aroma.     

Tolerance to Acid Water Among Strains and Life Stages of Atlantic Salmon (Salmo Salar L.)

Reintroduction of Atlantic salmon (Salmo salar L.) after liming of acidified barren salmon rivers could benefit by choosing acid tolerant strains. Testing different life history stages from fry to smolts of five salmon strains with different acidification history demonstrated strain-specific variation in tolerance to acid aluminum-rich waters for stages from fry to parr. Contrary to expectation, salmon from non-acidified rivers were more tolerant. Differences in sensitivity were found between life history stages. Within a specific life history stage, size-dependent sensitivity was found; small fish being more sensitive to pH while large fish were more sensitive to aluminum. Presmolts showed the same relative tolerance between strains as younger stages. These differences disappeared, however, when the smolt reached full smoltification, probably due to supersensitivity at this stage. Poor water quality during the last period of smoltification and outmigration can thus mask the genetic potential for tolerance to acidic rivers.     

Gas chromatography-mass spectrometry assessment of amines in Port wine and grape juice after fast chloroformate extraction/derivatization

A simple, reliable, and sensitive gas chromatography-mass spectrometry method for the quantification of volatile and nonvolatile biogenic amines in Port wines and grape juices was developed and evaluated. The method was based on a previously reported two-phase derivatization procedure with isobutyl chloroformate in a toluene medium, which provides a quantitative reaction in 10 min. Following the derivatization step, the excess of reagent was eliminated by treatment with alkaline methanol. The derivatization procedure was performed directly on 1 mL of sample, avoiding any fastidious and time-consuming cleanup extraction steps. The method allows the simultaneous quantification of 22 amines, which can be found in wines: methylamine, dimethylamine, ethylamine, diethylamine, propylamine, isopropylamine, butylamine, isobutylamine, amylamine, isoamylamine, 2-methylbutylamine, hexylamine, pyrrolidine, piperidine, morpholine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, 2-phenylethylamine, histamine, and tyramine. Because of the fact that histamine and tyramine derivatives are degraded during the isobutyl chloroformate elimination step, the corresponding determination was made after removal of the excess of derivatizing reagent by evaporating an aliquot of the toluene layer obtained after the reaction. The presented method showed excellent analytical characteristics in what linearity, recovery, repeatability, and limit of detections were respected. It was used to assess the concentration of biogenic amines in juice grapes and Tawny and Vintage Port wines with different aging times. On the whole, the total content of amines in Port wines was low. Most of the amines found in wines have their origin in the raw material used for their elaboration, so the Port winemaking process is not prone to the production of this kind of compounds. Total biogenic amine contents have shown a decrease with the aging of both types of Port wines.