In vitro synthesis of biogenic amines by Brochothrix thermosphacta isolates from meat and meat products and the influence of other microorganisms

Twenty Brochothrix thermosphacta strains tested for biogenic amines (BAs) production, formed histamine (6.6-16.2 mg/kg) and tyramine (18.7-35.4 mg/kg) but neither putrescine nor cadaverine. Six of the twenty strains were also investigated in respect of their influence on the synthesis of BAs by Escherichia coli, Pseudomonas sp., Proteus mirabilis and Lactobacillus sakei. In pure culture Escherichia coli produced all of the studied amines (histamine, tyramine, putrescine and cadaverine) with a total concentration of 167.7 mg/kg, P. mirabilis produced a total of 56.7 mg/kg histamine, tyramine and cadaverine, while Lactobacillus sakei and Pseudomonas sp. produced histamine and tyramine, totaling 37.9 and 35.2 mg/kg respectively. All B. thermosphacta promoted cadaverine production by Escherichia coli which increased by 12-68%, and some of them contributed to the appearance of this amine among the metabolites of Pseudomonas. The presence of B. thermosphacta decreased the potential ability of P. mirabilis to produce BAs.     

我国市售不同类型及品牌腐乳中生物胺含量的分析与比较

为了揭示我国商业腐乳中生物胺存在形式和水平,采用丹磺酰氯柱前衍生结合高效液相色谱法（HPLC）对我国市售的20个品牌白方腐乳和20个品牌红方腐乳中的生物胺含量进行了分析。结果表明,我国市售的腐乳中存在的主要生物胺为酪胺、组胺、腐胺、色胺、β-苯乙胺和尸胺,其平均含量分别为73.7 mg/kg、43.7 mg/kg、38.5 mg/kg、27.7 mg/kg、5.4 mg/kg和5.4 mg/kg;每种生物胺在白方腐乳中的含量均显著高于红方腐乳（P＜0.05）,前者的酪胺、组胺、腐胺、色胺、β-苯乙胺和尸胺的平均含量分别为后者的1.6、1.9、3.4、3.3、2.3和3.0倍;且每种生物胺的水平在同一类型不同品牌腐乳之间均呈现高度差异化。     

BIOGENIC AMINES IN FINNISH DRY SAUSAGES

A study was conducted to determine the biogenic amine levels of Finnish dry sausages and to compare the results with those of other surveys of different types of fermented sausages. Sausages with high amine concentration were analyzed microbiologically in order to identify amine-producing microorganisms. Tyramine was the most common amine: the mean of 68 samples was 82 mg/kg and the mode 110 mg/kg. Other vasoactive amines found were histamine (<1–200 mg/kg), phenylethylamine (<1–48 mg/kg) and tryptamine (<10–91 mg/kg). Increased amounts of the spoilage indicator amines putrescine and cadaverine were found from most of the samples containing vasoactive amines. The formation of amines can be linked with problem(s) in hygienic conditions of raw materials and manufacturing practices and it is thus proposed that the sum of tyramine, histamine, putrescine and cadaverine could serve as an indicator of quality in fermented meat products.     

Production of Biogenic Amines and Their Potential Use as Quality Control Indices for Hake (Merluccius merluccius, L.) Stored in Ice

ABSTRACT: The production of tyramine, histamine, putrescine, cadaverine, agmatine, and spermidine was studied for use as quality indices in muscle of gutted hake stored in ice for 25 d. The changes in biogenic amine concentrations were correlated with trimethylamine nitrogen levels. All concentrations except for spermidine, increased progressively throughout storage. During storage, cadaverine reached 72.14 mg/kg while there were no significant increases in histamine and tyramine. The levels of free amino acids, which may be metabolized into biogenic amines, were not correlated to respective biogenic amines levels. The production of cadaverine and agmatine occurred prior to spoilage and these biogenic amines may indicate freshness of hake stored in ice.     

姜辣素对培根风干成熟过程中微生物及生物胺形成的抑制效应

以五花肉为原料,腌制时添加不同含量（100、300、500 mg/kg）的姜辣素进行风干成熟,测定样品理化指标、微生物及生物胺含量变化情况,研究姜辣素对培根风干成熟过程中微生物及生物胺形成的抑制效应。结果表明：与对照组相比,姜辣素能显著抑制菌落总数和肠杆菌（P＜0.05）;在风干培根样品中共检测到腐胺、尸胺、组胺、酪胺、精胺5 种生物胺;生物胺总量随着风干时间的延长呈显著增加趋势（P＜0.05）,100、300、500 mg/kg处理组与对照组总生物胺含量存在显著性差异（P＜0.05）。300 mg/kg处理组总生物胺含量最低,为94.13 mg/kg,相对于对照组降低了34.56%,且此时腐胺、尸胺、酪胺、组胺和精胺含量也得到了很好的抑制,相对于对照组分别下降了26.60%、40.59%、37.08%、72.19%和23.59%。添加姜辣素能显著降低样品中挥发性盐基氮含量（P＜0.05）,对于提高培根的品质及安全性具有较好的效果。     

不同NaCl添加量对牦牛乳硬质干酪成熟过程中生物胺产生的影响

以不同盐添加量（1.0%、1.3%、1.8%和2.3%）牦牛乳硬质干酪为研究对象,分析牦牛乳硬质干酪0～6 个月成熟过程中生物胺的动态变化,并对干酪中产胺微生物进行筛选。结果显示：不同盐添加量牦牛乳硬质干酪中生物胺主要为色胺、β-苯乙胺、尸胺、酪胺和腐胺,未检测到组胺,生物胺积累阶段主要集中在成熟后期。不同盐添加量干酪中色胺含量最低,且在成熟后期含量差异较小。当盐添加量从2.3%减少到1.0%时,干酪中β-苯乙胺含量减少。盐添加量分别为1.0%和1.3%时,干酪中尸胺含量较低,且未检测出腐胺。当盐添加量在1.8%～2.3%时,随着盐添加量的增加,干酪中尸胺和腐胺含量整体呈现增加趋势。不同盐添加量干酪成熟过程中,其酪胺含量范围为3.13～49.81 mg/kg,且盐添加量为1.3%干酪中酪胺含量较高。不同盐添加量干酪中生物胺总量最高为304.18 mg/kg。采用显色培养基筛选出一株产胺微生物,经分子生物学鉴定为Enterococcus durans。     

利用高效液相色谱法测定不同发酵时长郫县豆瓣酱中的8种生物胺

本研究基于高效液相色谱(HPLC)技术,测定了不同发酵时长(1~96个月)郫县豆瓣酱中色胺、β-苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺和精胺8种生物胺的含量。结果显示,HPLC分析中,8种生物胺的检出限在0.17~0.35 mg/L之间,定量限在0.55~0.92 mg/L之间,各生物胺标品在1~60 mg/L的质量浓度范围内线性关系良好,可被准确定量;各生物胺回收率在73.10%~104.13%之间,说明本方法具有较高的重复性和灵敏度。腐胺、尸胺、酪胺和组胺是郫县豆瓣酱中的主要生物胺;随着发酵时间的延长,腐胺含量快速上升,尸胺含量在发酵前54个月迅速上升,之后明显下降,酪胺和组胺在发酵54个月后含量有所增加;其他生物胺含量较低,且发酵期间变化不明显;郫县豆瓣酱中总生物胺含量随发酵时间的延长不断积累,从(37.86±6.69) mg/kg增加到(155.09±8.53) mg/kg,其含量在发酵前54个月快速上升,之后趋于稳定。目前食品中的多数生物胺尚没有限量标准,但参考现有的毒理学数据来看,本研究中郫县豆瓣酱的生物胺含量水平相对安全。     

温度和姜精油对金枪鱼品质影响及生物胺相关性

将分别贮藏于不同温度(0、4、15℃)条件下的金枪鱼样品,以及4℃条件下用姜精油作为生物抗氧化剂处理过的样品作为研究对象,定期测定金枪鱼生物胺(腐胺Put、尸胺Cad、组胺His、酪胺Tyr)、反式尿刊酸的含量、挥发性盐基氮(total volatile basic nitrogen,TVB-N)值、K值和菌落总数,研究温度和姜精油对金枪鱼生物胺含量的影响及组胺与反式尿刊酸的关系。结果表明,温度对金枪鱼生物胺生成有较大影响,0℃条件下生物胺含量显著低于其他贮藏温度,到第6天时组胺、酪胺、腐胺和尸胺含量分别为28.25、5.47、14.84 mg/kg和17.05 mg/kg,而此时15℃条件下金枪鱼组胺、酪胺、腐胺和尸胺含量分别为135.4、14.63、29.49 mg/kg和41.55 mg/kg;生物胺中组胺含量最高,而酪胺含量始终处于低水平状态,即使在15℃条件下到第7天时其含量仍为16.24 mg/kg;结果同时表明,生物抗氧化剂对生物胺的产生和微生物生长有一定的抑制作用,并能有效地延缓蛋白质和ATP降解。相关分析和回归分析表明,组胺、酪胺、尸胺、与TVB-N值、K值和菌落总数高度相关,其相关系数r在0.804~0.981之间,且生物胺和菌落总数、TVB-N值之间存在重要对应关系,反式尿刊酸对组胺的产生影响不大,两者之间相关性较弱,在4℃时其相关系数r为0.630。     

发酵剂对牦牛乳硬质干酪成熟过程中生物胺的影响

乳酸菌产生物胺的能力具有菌株特异性,因此,为了探究不同种类发酵剂对牦牛乳硬质干酪中生物胺形成的影响,该试验利用高效液相色谱对3种不同发酵剂制作的硬质干酪成熟过程中生物胺进行了测定和分析。结果表明,嗜热和嗜温发酵剂牦牛乳硬质干酪中检测出2-苯乙胺、腐胺、尸胺、组胺和酪胺,混合发酵剂干酪中检测出腐胺、2-苯乙胺、尸胺和酪胺。各生物胺之间呈现正相关性。3种不同发酵剂干酪在1～6个月成熟过程中,其各生物胺整体呈现增加趋势,嗜热、嗜温和混合发酵剂干酪中总生物胺最高含量分别为(448.3±9.6)、(456.8±58.4)、(293±24.5)mg/kg。组胺和酪胺是2种毒性相对高的生物胺,嗜热发酵剂干酪中组胺和嗜温发酵剂干酪中酪胺最高,其最高含量分别为(20.8±7.9)、(92.9±6.7)mg/kg,混合发酵干酪中未检测出组胺,酪胺含量次之,3种不同发酵剂干酪中组胺、酪胺含量均低于推荐安全剂量50 mg/kg和100 mg/kg。这为合理选择发酵剂和控制干酪中生物胺形成提供了依据。     

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.     

CHANGES IN BIOGENIC AMINE CONTENTS AND SENSORY QUALITY OF SARDINE (SARDINA PILCHARDUS) STORED AT 4C AND 20C

Biogenic amines (tyramine, putrescine, cadaverine, histamine, spermidine and spermine) in sardine during the storage at two temperatures (4C and 20C) were investigated. Changes in sensory scores were also recorded. Significant differences between storage times were found. The levels of tyramine, putrescine, cadaverine and histamine increased as decomposition of sardine progressed at both 4C and 20C. Spermidine and spermine did not change throughout storage. Putrescine was the main biogenic amine formed, followed by tyramine, cadaverine and histamine. Histamine remained lower than the toxic level of 100 mg/kg in the samples stored at 4C whereas it exceeded the toxic level after 16 h at 20C. The increase in histamine concentration was lower than tyramine and putrescine at both temperatures. Sensory scores gradually decreased during the storage.     

我国传统干酪中生物胺含量检测分析

通过反相高效液相色谱法对来自内蒙古、新疆、西藏、云南、北京市场共17种中国传统干酪样品生物胺含量进行了分析。结果表明,反相高效液相色谱法能准确地测定干酪中生物胺含量;在全部干酪样品中,8种生物胺含量大小不一,生物胺总量在1.30～41.27 mg/kg范围内,均低于国家标准规定要求,其中有14个样品的生物胺总量低于10 mg/kg,第10号干酪生物胺含量为41.27 mg/kg,为所有样品中含量最高,其次为第1号样品和第9号样品,分别为18.48 mg/kg和10.35 mg/kg。相关性分析结果显示,色胺与苯乙胺,腐胺与尸胺、酪胺、亚精胺,组胺与精胺,酪胺与精胺之间在0.01水平上具有显著相关性。     

Influence of pasteurised milk, raw milk and different ripening cultures on biogenic amine concentrations in semi-soft cheeses during ripening

In semi-soft cheeses, produced with pasteurised milk, raw milk and different starter cultures, the concentrations of cadaverine, histamine, phenylethylamine, putrescine and tyramine were investigated. The cultures (pasteurised milk cultures, raw milk cultures and starter cultures) strongly influenced the biogenic amine concentrations in the cheeses ripened for 5 months. Two cheeses made with identical pasteurised milk, but different ripening cultures, differed greatly in their total biogenic amine concentrations (51 vs 371?mg/kg). In general, the biogenic amine concentrations increased markedly between month 2 and month 3 of cheese ripening. The high content of enterococci and Enterobacteriaceae yielded the biogenic amine concentrations. In contrast, Lactobacilli did not seem to be important. However, unspecified bacteria have to be considered, since cheeses with comparable microbiological profiles differed enormously in their biogenic amine concentrations. Semi-soft cheeses produced from pasteurised milk showed remarkably lower total biogenic amine concentrations compared to semi-soft cheeses produced from raw milk (51–1096?mg/kg vs 1011–3133?mg/kg, depending also on the ripening cultures). The highest total biogenic amine concentration (4817?mg/kg) was detected in a cheese produced from raw milk that had been stored for 36?h. In this cheese, the concentrations of cadaverine, phenylethylamine, putrescine and tyramine were higher than in all other cheeses. The highest histamine concentration was found to be in another raw milk cheese (573?mg/kg).     

Influence of hygienic quality of raw materials on biogenic amine production during ripening and storage of dry fermented sausages

The effect of the hygienic status of raw materials on biogenic amine production during ripening and storage (at 4 and 15 degrees C) of fermented sausages was studied. Two portions of fresh lean and back fat pork were stored for 5 days at -20 degrees C (treatment A) and at 4 degrees C (treatment B), respectively. Raw materials of treatment A maintained their hygienic quality high and low amine content. Raw materials of treatment B showed from 1 to 3 log (CFU/g) higher microbial counts and a biogenic amine index near 50 mg/kg, indicating poorer hygienic quality. The quality of raw materials influenced the composition and the concentration of biogenic amines produced during the ripening sausages. Sausages of treatment A (A-sausages) showed a large accumulation of tyramine (up to 100 mg/kg dm) followed by putrescine and cadaverine (<15 mg/kg). In contrast, B-sausages resulted in earlier and much greater amine production, and cadaverine, tyramine, and putrescine levels were 50-, 2.6-, and 6.5-fold higher than those of A-sausages. Other biogenic amines, such as octopamine, tryptamine, phenylethylamine, and histamine were also produced in B-sausages. The higher proteolysis and the lower pH of B-sausages might have favored the decarboxylase activity of microorganisms. Biogenic amine contents of sausages during storage depended on the raw materials used and storage temperature. No significant modification on the amine contents was observed during the storage of A-sausages at either temperature. Greater changes occurred in B-sausages stored at 15 degrees C than in those stored at 4 degrees C. Higher temperatures favored proteolytic and decarboxylase reactions, resulting in increased amine concentrations after storage.     

六类鱼制品中生物胺的HPLC法测定

针对不同类型的鱼制品建立同时检测腐胺、尸胺、精胺、亚精胺、酪胺、苯乙胺、组胺和色胺8种生物胺含量的高效液相色谱(HPLC)法。应用该方法,测定了干燥制品、油炸制品、烘烤制品、发酵制品、混揉制品以及萃取制品6类鱼干制品中的生物胺含量。结果显示,萃取制品-鱼油中未检测出生物胺,其余5类产品中生物胺含量最高的为发酵制品(73.42~70.59 mg/kg)和混揉制品(55.48~49.88 mg/kg),其次为油炸制品(17.72~14.32 mg/kg),而鱼干制品(5.28~5.17 mg/kg)和烘烤制品(4.75~4.69 mg/kg)中的生物胺含量相对较少;另外,尸胺、组胺、精胺和亚精胺四种生物胺为除萃取制品之外的5类鱼制品中普遍存在的4种生物胺成分;其中尸胺是鱼制品中变化最大、影响最为明显的单体生物胺,检测产品中尸胺含量有利于监控、评判鱼干制品的质量。     

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.     

BIOGENIC AMINES FORMATION IN SARDINE MARINADE DURING REFRIGERATED STORAGE

Biogenic amines (tyramine, putrescine, cadaverine, histamine, spermidine, spermine) in sardine marinade were investigated. Sardine were beheaded, gutted, filleted, washed and marinated by immersing the fish into solutions containing 2 and 4% acetic acid and 10% NaCl for 24 h. Biogenic amine content, sensory scores and pH values of marinated sardine were recorded during the storage at 4C for up to 5 months. Biogenic amine content in marinated sardine was higher than in raw material. There were significant (P<0.05) differences in biogenic amine content between acetic acid concentrations of 2% and 4% just after marinating. Initial values of biogenic amines in marinade with 4% acetic acid were significantly (P<0.05) lower than those in marinade with 2% acetic acid. The contents of tyramine***, putrescine and histamine in sardine marinade decreased in the first 2–3 months of storage, followed by continuous increase up to 5 months of the storage. After decrease in the first month, cadaverine level did not significantly change. According to results, the marination process influences biogenic amine accumulation.     

牦牛乳硬质干酪成熟过程中生物胺与细菌群落结构分析

成熟干酪易产生生物胺,生物胺对人体健康具有影响,因此,为了探究牦牛乳硬质干酪的质量安全性,本实验利用高效液相色谱和高通量测序技术对干酪成熟过程中生物胺和细菌群落结构进行测定和分析。结果表明：牦牛乳硬质干酪中主要生物胺为腐胺、2-苯乙胺、酪胺、组胺和尸胺,在1～6 个月成熟过程中,干酪中各生物胺含量呈现增加趋势。干酪中生物胺含量最高阶段均出现在成熟期5～6 个月。干酪中组胺、酪胺和总生物胺含量分别低于推荐安全剂量50、100 mg/kg和1 000 mg/kg。干酪中游离氨基酸含量与除组胺之外的各生物胺含量、总生物胺含量、成熟时间之间具有显著正相关性（P＜0.01,P＜0.05）,各生物胺含量之间也存在正相关性。不同成熟期干酪包含相同属的微生物,其中链球菌属（Streptococcus）为优势菌属,平均相对丰度为84.63%,明串珠菌属（Leuconostoc）次之,平均相对丰度为6.91%,其他分别为乳杆菌属（Lactobacillus）、拉乌尔菌属（Raoultella）、不动杆菌属（Acinetobacter）、假单胞菌属（Pseudomonas）、肠杆菌属（Enterobacter）和无分类的肠杆菌科（unclassified_Eunnterobacteriaceae）。不同成熟期干酪中各菌属的相对丰度具有差异。本研究可为评估牦牛乳硬质干酪的质量安全性和生物胺形成机理提供理论依据。     

白鱼腐败菌产生物胺能力分析

为了探讨白鱼（Anabarilius）腐败菌产生物胺的能力,釆用聚合酶链式反应技术检测腐败菌的组氨酸、赖氨酸、鸟氨酸和酪氨酸的脱羧酶基因,应用反相高效液相色谱技术测定接种腐败菌培养基和无菌白鱼鱼块中生物胺含量,并对主要腐败菌的生物胺产量因子进行分析。结果表明：8 种腐败菌均含有至少1 种氨基酸脱羧酶基因;接种不同腐败菌的液体培养基和无菌白鱼鱼块均检测到多种生物胺,其中Morganella sp.wf-1和Morganella sp.wf-2具有很强的产腐胺、尸胺和组胺能力,使得接种这两种腐败菌的无菌白鱼鱼块中腐胺、尸胺和组胺含量、单胺、二胺、生物胺指数和总生物胺含量等指标在腐败过程中均显著高于对照组和接种其他腐败菌的无菌白鱼鱼块。同时,Morganella sp.wf-1和Morganella sp.wf-2的腐胺、尸胺、组胺、单胺、二胺、生物胺指数和总生物胺的产量因子均高于其他腐败菌。结果表明,Morganella sp.wf-1和Morganella sp.wf-2是白鱼腐败过程中产生物胺的主要腐败菌。     

高效液相法测定草鱼糜发酵过程中生物胺变化

本文采用纳豆芽孢杆菌和戊糖片球菌组成的混合发酵剂对草鱼鱼糜进行发酵,建立了发酵鱼制品中六种生物胺(色胺、腐胺、尸胺、组胺、酪胺、亚精胺)的高效液相色谱分析方法,研究了鱼糜发酵过程中生物胺含量的变化以及微生物数量的变化。结果发现,在30 min内各种生物胺得到很好的分离,且线性关系良好(R2>0.999),回收率在97.35%~103.00%,精密度、重现性RSD均小于10%。添加混合发酵剂可以明显抑制金黄色葡萄球菌、假单胞菌和大肠杆菌等杂菌的生长,降低发酵过程中腐胺、尸胺、组胺和酪胺含量的积累;利用纳豆芽孢杆菌,戊糖片球菌组成的混合发酵剂以及一系列负控脱羧酶活动能有效抑制鱼糜发酵过程中生物胺的形成,抑制腐败菌和致病菌的生长,提高鱼糜发酵食品的品质。