Characterization of Biogenic Amines and Factors Influencing Their Formation in Traditional Chinese Sausages

Abstract: Biogenic amines in 42 traditional Chinese sausage samples obtained from different regions were determined by HPLC. The result showed that cadaverine was the major amine, followed by tyramine and putrescine. A total of 4 groups of samples were identified on the basis of total amines by cluster analysis. Group A included samples showing low amine contents (76.5 to 220 mg/kg) and accounted for 28.5% of the sausages examined. Group B included samples with moderate amine contents (220 to 600 mg/kg) and accounted for 45.2%. Group C included 11.9% of the samples showing high total biogenic amines contents (600 to 1000 mg/kg) and group D contained 14.28% of the samples showing very high levels of biogenic amines (higher than 1000 mg/kg). High correlation coefficients were found between the total counts of Enterobacteria and concentrations of total biogenic amines (r = 0.73). Sanitary quality of raw materials and the specific flora are import factors influencing biogenic amines formation in traditional Chinese sausages. Practical Application : Biogenic amines are considered potentially harmful substances to human health worldwide and are usually found in fermented sausage.Traditional Chinese sausage is one form of spontaneously fermented sausage and manufactured in small-scale plants following spontaneous fermentation. Little information, however, exists on the traditional Chinese sausage.     

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 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.     

Effectiveness of a Lactobacillus sakei starter culture in the reduction of biogenic amine accumulation as a function of the raw material quality

The effectiveness of an amine-negative starter culture (Lactobacillus sakei CTC494) in the reduction of biogenic amine production during the ripening of fermented sausages was examined. Four batches were manufactured in parallel: spontaneously fermented and starter-mediated sausages were manufactured from two lots of raw materials of different hygienic quality. Besides the biogenic amine contents, changes in the microbial counts, nitrogenous fractions, pH, and water content were measured at several sampling points during the ripening process. In sausages manufactured from good quality meat, the starter strain of L. sakei reduced and even inhibited biogenic amine accumulation during sausage fermentation, the end products showing extremely low biogenic amine contents (tyramine levels less than 15 mg/kg of dry matter and putrescine and cadaverine levels less than 5 mg/kg of dry matter). Nevertheless, starter-mediated sausages made from poorer-quality raw materials showed much higher amine contents (308, 223, and 36 mg/kg of dry matter of cadaverine, tyramine, and putrescine, respectively), which were only slightly lower than those of the spontaneously fermented sausages made from the same raw materials. The relatively high bacterial numbers of raw materials of poorer-hygienic quality diminished the beneficial effect of the starter strain. Therefore, the effectiveness of the starter was strongly dependent on the hygienic quality of the raw materials used.     

Levels of biogenic amines in retail market fermented meat products

The qualitative and quantitative profile of biogenic amines (BA) in 50 samples of dry fermented sausages sold in Greek retail markets were determined by HPLC. Putrescine, cadaverine, tryptamine, β-phenylethylamine spermidine, spermine were analysed by UV detection after pre-column derivatization with benzoyl chloride, whereas tyramine and histamine were analysed by fluorescence detection after post-column derivatization with o-phthaldialdehyde (OPA). With the exception of spermidine and spermine a wide variation of BA levels was observed among the samples. Of the BA examined, tyramine, putrescine, histamine and cadaverine showed high concentrations ranging from: 0 to 510 mg/kg (median: 197.7 mg/kg), 0 to 505 mg/kg (median: 96.5 mg/kg), 0 to 515 mg/kg (median: 7.0 mg/kg) and 0 to 690 mg/kg (median: 3.6 mg/kg), respectively. The histamine content of 28% of the samples exceeded the toxicity limit of 100 mg/kg set for histamine in some fish species. Levels of tryptamine and β-phenyl-ethylamine never exceeded 50 and 29 mg/kg, respectively. Results of the present study suggest that the amounts of BA in dry fermented sausages, sold in Greek retail markets, may pose a potential health risk for sensitive individuals or for those undergoing classical monoamine oxidase inhibiting (MAOI) drug therapy.     

Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic

The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25 mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewe’s milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200 mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumer’s health.     

Biogenic amine and microbiological profile of Serbian dry fermented sausages of artisanal and industrial origin and associated health risk

ABSTRACT

From the public health point of view, safety of dry fermented sausages is of concern due to possible presence of biogenic amines and some other food hazards. Taking into consideration that biogenic amines are chemical indicators of microbiological contamination, industrial and artisanal dry fermented sausages produced in Serbia were subjected to the microbiological and biogenic amine profiling. Microorganisms identified by MALDI-TOF MS included Cronobacter sakazakii and Listeria innocua, whereas the food pathogens were not detected. Biogenic amine content, determined by HPLC method in the range from 37.3 to 1186 mg/kg, was characterised as very low to low in 40% of the samples, moderate in 38%, high in 14% and very high in 8%. Risk assessment revealed that consumption of dry fermented sausages could cause histamine and tyramine intake up to 11.9% and 3.4% of threshold dose for healthy population, respectively. Adverse health effects would be rather unlikely for general population.     

Biogenic amines in Spanish fermented sausages as a function of diameter and artisanal or industrial origin

The distribution of biogenic amines in three types of fermented meat sausages (chorizo, fuet and salchichón) was examined with respect to the degree of acidification. The aim was to determine whether low‐acid sausages (artisanal/traditional) have a different biogenic amine profile than more acidic products (industrial). Despite wide variability, tyramine was always found and was generally the major amine, followed by putrescine. Their contents in both industrial and artisanal sausages were similar, but correlated with the diameter of the product. In contrast, industrial sausages showed a higher average content of cadaverine and histamine, especially in chorizo, which also showed the highest content of free amino acids. Moreover, a multiple analysis of variance confirmed that the processing plant had a significant influence on the overall biogenic amine composition of products, histamine being the most important amine accounting for this effect. Copyright © 2005 Society of Chemical Industry     

Evolution of amino acids and biogenic amines throughout storage in sausages made of horse,beef and turkey meats

The changes in concentration of free amino acids and biogenic amines, along 28 d of storage at 4 °C, were monitored in a wide range of European ripened sausages manufactured from horse, beef and turkey meats. Generally speaking, both chemical families became more concentrated with elapsing time – but rather distinct patterns were followed in each meat type: total free amino acids increased by 13-fold in the case of horse sausages, and 5-fold in the case of beef sausages, but decreased to one third in the case of turkey sausages; and total biogenic amines attained 730 mg/kg in turkey sausages, 500 mg/kg in beef sausages and 130 mg/kg in horse sausages by 28 d of refrigerated storage. For putrescine, maximum levels of 285 mg/kg were attained in turkey and 278 mg/kg in beef sausages; for cadaverine, maximum levels of 6 mg/kg in turkey and 9 mg/kg in beef; and for histamine, maximum levels of 263 mg/kg in turkey and 26 mg/kg in beef. Hence, public safety concerns may be raised in the case of turkey sausages.     

四川区域自然发酵香肠及人工接种牦牛肉发酵香肠中生物胺含量的研究

以四川省10 区域自然发酵香肠及人工接种发酵剂的牦牛肉香肠共21 个样品为研究对象,测定发酵香肠中的生物胺含量。结果发现,21 个发酵香肠样品中均检测到酪胺、亚精胺、精胺、尸胺、腐胺、色胺及组胺,β-苯乙胺均未检出;生物胺总量在57.34～411.12 mg/kg,除采自凉山州西昌的自然发酵牦牛肉香肠和广安的自然发酵猪肉香肠中酪胺含量超过了美国食品及药品管理局（Food and Drug Administration,FDA）规定标准（酪胺含量≤100 mg/kg）,其余均不存在生物胺安全问题。     

Mixed starter cultures to control biogenic amine production in dry fermented sausages

Several combinations of an amine-negative Lactobacillus sakei strain, along with proteolytic Staphylococcus carnosus or Staphylococcus xylosus strains, were used to study the influence of mixed starter cultures on biogenic amine production during the manufacture of dry fermented sausages. Changes in pH, water content, proteolysis, microbial counts, and biogenic amine contents were simultaneously examined in a spontaneously fermented batch and in three mixed starter-mediated batches. A double-controlled microbial charge initially inoculated as mixed starter culture of L. sakei and Staphylococcus spp. (all amine-negative strains) drastically reduced tyramine, cadaverine, and putrescine accumulation. No production of other aromatic amines such as histamine, phenylethylamine, or tryptamine was observed in any batch. The polyamines, spermine and spermidine, were found in raw materials and their levels decreased slightly in the spontaneously fermented batch. No correlation between proteolysis and biogenic amine production was observed. The use of proper technological conditions favoring starter development and the use of the raw materials with good hygienic quality make it possible to produce fermented sausages nearly free of biogenic amines.     

Impact of fermentation conditions and refrigerated storage on microbial quality and biogenic amine content of sauerkraut

White cabbage (Brassica oleracea L. var. capitata cv. Bronco) was fermented, at 0.5% and 1.5% NaCl, using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures and, subsequently, sauerkraut was stored at 4 °C for 3 months. Microbial populations and six biogenic amines (putrescine, cadaverine, histamine, tyramine, spermine and spermidine) were investigated. Fermentation and storage increased aerobic mesophilic bacteria and LAB populations in sauerkrauts, and this was accompanied by a rise in biogenic amine content. L. plantarum sauerkrauts produced with 0.5% NaCl had the highest microbial counts, whilst no differences between salt contents were found with L. mesenteroides. Total biogenic amine amount was lower at 0.5% NaCl than at 1.5% in both induced fermentations and L. mesenteroides produced a lower content than did L. plantarum. Spermidine was the major contributor to the total biogenic amine content, followed by putrescine, whilst histamine was present at the lowest level. The individual and total biogenic amine levels in the experimental sauerkrauts stored at 4 °C for 3 months were below the upper limits reported in the literature for fermented products, indicating good quality and safety of the sauerkrauts. L. mesenteroides starter and 0.5% NaCl were the optimal fermentation conditions for producing sauerkrauts with the lowest biogenic amine contents.     

Effect of fermentation temperature on the microbial and physicochemical properties of silver carp sausages inoculated with Pediococcus pentosaceus

The effect of fermentation with Pediococcus pentosaceus at different temperatures ranging from 15 to 37 °C on the quality characteristics of silver carp sausages was investigated. Higher temperature stimulated the rapid growth of lactic acid bacteria, resulting in a rapid decline in pH, and consequently suppressed the growth of Pseudomonas, Micrococcaceae and Enterobacteriaceae. However, increasing fermentation temperature gave a progressive increase in total volatile basic nitrogen and biogenic amines in fermented silver carp sausages. Histamine was the main biogenic amine, exceeding 100 mg/kg after 48 h of fermentation at temperatures above 30 °C. Higher content of non-protein nitrogen and α-amino nitrogen correlated with the electrophoretic studies, which showed that proteolysis of high molecular weight myofibrillar and sarcoplasmic proteins was more prominent at higher fermentation temperatures. Products fermented at 23–30 °C showed greatest consumer preference and most favourable textural properties.     

Changes in biogenic amine levels during storage of Mexican-style soft and Spanish-style dry-ripened sausages with different aw values under modified atmosphere

Two raw sausages were prepared: a soft and a dry-ripened one, both by local traditional and industrial manufacturing practices. Sausages were packaged under a CO₂/N₂ atmosphere at different targeted activity water (a_w) values: 0.96 and 0.92 (soft sausages) and 0.88 and 0.82 (dry-ripened sausages). Sausages were then stored at 5 °C for 42 days or at 12 °C for 240 days (soft and a dry-ripened sausages, respectively). The time-related changes in dominant microbiota, pH and biogenic amine contents during storage were determined. Tyramine was the most abundant biogenic amine in all the sausages. Biogenic amine levels were higher in dry-ripened sausages than in soft sausages at packaging. However, during refrigerated storage soft sausages were fermented and the levels of biogenic amines increased (P < 0.05). At the end of storage, traditional soft sausages with 0.96 a_w presented comparable levels of biogenic amines to traditional dry-ripened sausages.     

Biogenic amines in dry fermented sausages: a review

Biogenic amines are compounds commonly present in living organisms in which they are responsible for many essential functions. They can be naturally present in many foods such as fruits and vegetables, meat, fish, chocolate and milk, but they can also be produced in high amounts by microorganisms through the activity of amino acid decarboxylases. Excessive consumption of these amines can be of health concern because their not equilibrate assumption in human organism can generate different degrees of diseases determined by their action on nervous, gastric and intestinal systems and blood pressure. High microbial counts, which characterise fermented foods, often unavoidably lead to considerable accumulation of biogenic amines, especially tyramine, 2-phenylethylamine, tryptamine, cadaverine, putrescine and histamine. However, great fluctuations of amine content are reported in the same type of product. These differences depend on many variables: the quali-quantitative composition of microbial microflora, the chemico-physical variables, the hygienic procedure adopted during production, and the availability of precursors. Dry fermented sausages are worldwide diffused fermented meat products that can be a source of biogenic amines. Even in the absence of specific rules and regulations regarding the presence of these compounds in sausages and other fermented products, an increasing attention is given to biogenic amines, especially in relation to the higher number of consumers with enhanced sensitivity to biogenic amines determined by the inhibition of the action of amino oxidases, the enzymes involved in the detoxification of these substances. The aim of this paper is to give an overview on the presence of these compounds in dry fermented sausages and to discuss the most important factors influencing their accumulation. These include process and implicit factors as well as the role of starter and nonstarter microflora growing in the different steps of sausage production. Moreover, the role of microorganisms with amino oxidase activity as starter cultures to control or reduce the accumulation of biogenic amines during ripening and storage of sausages is discussed.     

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.     

Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening

The interaction between tyrosine-decarboxylase and proteolytic activities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.     

大蒜精油和发酵剂对熏马肠中生物胺及微生物分布的影响

为研究大蒜精油和发酵剂对熏马肠中生物胺的影响,采用高效液相色谱法测定熏马肠中生物胺含量,利用 宏基因组学技术分析熏马肠中微生物多样性和生物胺含量的变化情况。结果表明：将大蒜精油和发酵剂联合使用能 够很好地减少熏马肠中尸胺、腐胺、组胺、酪胺和苯乙胺的产生,对控制生物胺在熏马肠中的含量起到非常显著的 作用;熏马肠中含有丰富的微生物物种,通过样品科分类水平物种分析,得出乳酸菌在熏马肠整个加工过程都是优 势菌种,而葡萄球菌在成熟后期数量较多。     

原料肉贮藏时间对牛肉发酵香肠中微生物菌相变化和蛋白质降解的影响

采用传统微生物计数、高通量测序、凯氏定氮、高效液相色谱等方法,探究牛肉宰后0～4 ℃下不同贮藏时间（0、12、24、36 h）对发酵香肠加工过程中微生物菌相变化、蛋白质降解及其相关性的影响。结果显示,随着牛肉贮藏时间的延长,香肠中菌落总数、大肠菌群数、菌群多样性和丰富度以及蛋白质降解指标均呈上升趋势,总游离氨基酸含量呈下降趋势;香肠成熟阶段中总挥发性盐基氮含量与总生物胺含量之间呈极显著正相关（P＜0.01）。其中,贮藏36 h牛肉加工的香肠与未经贮藏牛肉加工的香肠对比,菌落总数平均值由2.40×107 CFU/g增加到4.03×107 CFU/g（P＜0.05）,反映菌群多样性的Shannon指数平均值由1.39增长至2.89,优势菌群由整个加工阶段均为葡萄球菌变成前期为魏斯氏菌,后期为葡萄球菌;总游离氨基酸含量平均值由7.09 mg/g减少至6.44 mg/g,总生物胺含量平均值由92.14 mg/kg增长为117.42 mg/kg（P＜0.05）。结论：控制原料牛肉贮藏时间可以有效减少香肠中微生物的总量,降低菌群多样性,抑制蛋白质的降解,减缓生物胺的积累,从而保障发酵香肠的安全性。     

Evaluation of N-Nitrosopiperidine Formation from Biogenic Amines During the Production of Dry Fermented Sausages

The aim of this study was to determine the role of the precursors cadaverine (CAD) and piperidine in N-nitrosopiperidine (NPIP) formation during the production of dry fermented sausages. The influences of pH (4.9 and 5.3), sodium nitrite (0 and 150 mg/kg), and ascorbate (0 and 500 mg/kg) were investigated by the use of a dry fermented sausage model. The biogenic amines and volatile N-nitrosamines were analyzed by HPLC-UV and GC-TEA. The major biogenic amines were tyramine, putrescine, and CAD, but their accumulation could be inhibited by NaNO₂. When no amine precursors were artificially added, no meaningful N-nitrosamine contamination was observed. The artificial addition of 500 mg/kg cadaverine dihydrochloride (CAD·2HCl) could not provoke increased N-nitrosamine formation. In contrast, the addition of PIP (10 and 100 mg/kg) resulted in higher NPIP concentrations. No influence of pH was observed, but the NPIP formation was more pronounced when NaNO₂ was added and sodium ascorbate was excluded from the formulation. The role of ascorbate as N-nitrosamine scavenger was only detectable during the early stage of production. In the end products, NPIP was degraded and the levels were no longer influenced by the initial addition of NaNO₂ or ascorbate.