猪肉自然发酵香肠中细菌和乳酸菌对生物胺积累的作用

在猪肉香肠的自然发酵时，检测了选择的污染菌对形成的生物胺的性质和产量的作用。本文主要研究的是在肉腐败过程中胺的变化和微生物生长的变化，而且重点研究的是加入产酪胺的短乳杆菌（L．brevis）和产尸胺的阴沟肠杆菌（E．cloacae）的猪肉发酵香肠在发酵时生物胺的积累。在腐败和香肠发酵时相关微生物和胺的量较多。尸胺的高度积累和大量的不理想肠细菌有关，在香肠发酵时，酪胺的产生并不完全是乳酸菌的作用。另一方面，也涉及到必要的生产香肠的自然发酵菌，污染菌对于腐胺和其它生物胺的作用，现在还不清楚。     

不同发酵方式对发酵马肉肠贮藏期品质劣变及食用安全性的影响

为有效确保发酵马肉肠贮藏期间的品质及安全性,研究添加汉逊德巴利酵母菌1808（Debaryomyces hansenii 1808）、木糖葡萄球菌21445（Staphylococcus xylose 21445）、植物乳杆菌E11（Lactobacillus plantarum E11）的复合发酵剂发酵组（FP）和单一乳酸菌发酵组（DZ）、自然发酵组（ZR）马肉肠贮藏过程中生物胺的变化,考察不同发酵方式对发酵马肉肠pH值、水分活度（aw）、色差、总挥发性盐基氮（total volatile basic nitrogen,TVB-N）含量、硫代巴比妥酸反应物（thiobarbituric acid reactive substances,TBARs）值及菌落总数的影响。结果表明：在贮藏期间,相比于自然发酵组和单一乳酸菌发酵组,添加复配发酵剂可有效减缓发酵马肉肠TVB-N含量、TBARs值、pH值的增幅,抑制贮藏后期有害杂菌的生长,防止产品过早发生腐败变质;此外,复配发酵剂可显著降低贮藏过程中马肉肠的水分损失（P＜0.05）,维持色泽的稳定,抑制生物胺（尸胺、腐胺、酪胺、组胺）的积累。综上,随着贮藏时间的延长,复配发酵剂可显著抑制发酵马肉肠品质的劣变及不良生物胺的过多积累。     

Species diversity and metabolic impact of the microbiota are low in spontaneously acidified Belgian sausages with an added starter culture of Staphylococcus carnosus

Quality of fermented sausages is affected by acidifying lactic acid bacteria (LAB) and colour- and flavour-promoting coagulase-negative staphylococci (CNS), whether or not used as starter culture. Artisan fermented sausages are often perceived as superior to industrial variants, partially because of the specific microbiota due to spontaneous acidification, which may be considered as an artisan characteristic. Therefore, two kinds of spontaneously acidified Belgian sausages were prepared (Belgian-type salami and Boulogne sausage), but with addition of a Staphylococcus carnosus culture. The Belgian-type salami was made from pork and beef, whereas the Boulogne sausage contained pork and horse meat. In all cases, Lactobacillus sakei was the dominant LAB species present on the raw materials and during fermentation, whereas enterococci remained present in the background. Enterobacteriaceae vanished after fermentation. The CNS species diversity on the raw materials was large and differed between the pork, beef, and horse meat. Nevertheless, this species diversity was annihilated during fermentation by the added S. carnosus culture. The volatiles fraction was mainly composed of aldehydes that originated from lipid oxidation and spices-derived compounds. Aromatic compounds that are typically associated to CNS activity, such as end-products from the metabolism of branched-chain amino acids, were not present in the Belgian-type salami and only marginally present in the Boulogne sausage. In conclusion, spontaneous acidification of Belgian-type fermented sausages leads to dominance of L. sakei and is no guarantee for bacterial contribution to the aroma profile when S. carnosus is added as a starter culture.     

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.     

Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for fermentation of Fuet sausages

The ability of Lactobacillus sakei CTC494, a negative amino acid-decarboxylase starter culture, to reduce biogenic amine accumulation during sausage fermentation and storage at 4 and 19 degrees C was studied. The effect on the amine formation of the tyramine producer Lactobacillus curvatus CTC371, as a positive strain, was also examined in comparison to a spontaneous fermentation process without starter culture (control batch). The polyamines spermine, spermidine, and diaminopropane were not influenced by the ripening, and their levels slightly decreased in all the batches throughout the storage. Tyramine, cadaverine, and putrescine were the main amines formed during the ripening. The addition of starter culture resulted in a decrease on the biogenic amine formation, depending on the strain inoculated. A great reduction in tyramine content was achieved when L. sakei CTC494 was inoculated, whereas L. curvatus CTC371 only attenuated tyramine accumulation compared with the control batch. Both starters were able to significantly limit the production of putrescine and cadaverine, and they inhibited tryptamine and phenylethylamine formation by the wild microbial flora. Tyramine levels of the control sausages rose during the storage at both temperatures, whereas those of cadaverine only increased at 19 degrees C. On the contrary, sausages manufactured through the starter controlled fermentation did not show changes of amine contents during the storage. The addition of a proper selected starter culture is advisable to produce safer sausages with low contents of biogenic amines.     

Biogenic amine formation in Nham,a Thai fermented sausage,and the reduction by commercial starter culture, Lactobacillus plantarum BCC 9546

Biogenic amines are of concern for sausage due to their toxicological effects on nervous, blood pressure, gastric and intestinal systems. In this study, the influence of raw pork meat quality and starter culture inoculation on biogenic amines accumulation in Nham, a Thai traditional fermented pork, were studied. Before Nham processing, pork meat was stored at 30 °C for 6 h, and at 4 and −20 °C for 2 days. Formation of biogenic amines (cadaverine, putrescine, histamine and tyramine) was significantly higher in Nham processed from stored meat. Accumulation of these biogenic amines in Nham reduced significantly by the addition of Lactobacillus plantarum BCC 9546, a commercial Nham starter culture. The results highlight the importance of using fresh meat products and the inclusion of an appropriate starter culture to minimise the formation of biogenic amines during the process of Nham fermentation.     

冷却猪肉及托盘表面细菌生物被膜分析和肉源荧光假单胞菌的鉴定与被膜研究

为研究冷却猪肉及其接触面中细菌生物被膜能力并探讨肉类特定腐败菌的生物被膜特征,分析了腐败的冷却肉和销售托盘表面的细菌生物被膜形成能力;利用形态学观察、16S r DNA分析和VITEK2微生物鉴定系统鉴定了冷却肉中一株生物被膜能力较强的荧光假单胞菌(Pseudomonas fluorescens);测定了该菌在培养基和猪肉浸提液中的生物被膜能力;用激光共聚焦和扫描电子显微镜观测了其生物被膜的结构特征。结果发现:冷却肉中及其销售托盘表面的细菌能够形成生物被膜的比例较高,37%和45%的细菌具有较强生物被膜形成能力。肉源性荧光假单胞菌能够在培养基和肉液中形成大量生物被膜,具有较强的生物被膜能力。显微成像结果表明荧光假单胞菌在培养6 h后有明显黏附,18 h后多糖分泌增多、菌体堆积并形成具有生物功能的立体生物被膜。这些特点可能有利于荧光假单胞菌在冷却肉表面黏附并成为优势腐败菌。     

Evaluating the Influence of Malolactic Fermentation Inoculation Practices and Ageing on Lees on Biogenic Amine Production in Wine

Biogenic amines are compounds, produced primarily by lactic acid bacteria (LAB) that negatively affect the wholesomeness of wine. Standard winemaking practices can greatly influence the levels of biogenic amines in wine. The aim of this study was to determine the relative contribution of different malolactic fermentation (MLF) practices and ageing of wines on fermentation lees to the final levels of biogenic amines. Wines were made on small scale over two harvest seasons with two red grape cultivars. Treatments included spontaneous MLF, co-inoculated MLF, MLF inoculated after alcoholic fermentation (conventional inoculation) and 4 months of ageing in the presence and absence of fermentation lees of all MLF treatments. Biogenic amine concentrations were measured by high-performance liquid chromatography at key winemaking stages and statistically analysed for the effects of MLF treatment and winemaking stage. Results indicate that the presence of indigenous LAB increased the risk of biogenic amine formation. Inoculation proved to reduce biogenic amine production over time compared to spontaneous MLF and co-inoculation even more than conventional inoculation. The presence of yeast lees during ageing generally led to higher final concentrations of biogenic amines in wines than the absence of lees. This study confirms other works that conclude that spontaneous MLF and uncontrolled ageing on yeast lees are generally unpredictable and pose a risk of biogenic amine contamination in finished wines.     

不同储藏时期香肠菌群结构的变化及优势腐败菌研究

以天然肠衣的熏煮香肠为研究对象,将新鲜香肠和腐败香肠分别取样,提取DNA,采用高通量测序技术对两种香肠的菌群结构进行比较研究,并确定优势腐败菌。结果表明:腐败香肠菌群丰度和均匀度较新鲜香肠降低;在属的水平上,新鲜香肠中丰度最高的是Staphylococcus(葡萄球菌属90.64%),而腐败香肠中丰度最高的是Weissella(魏斯氏菌属65.93%),其次是Staphylocococcus(葡萄球菌属33.79%);在种的分类水平上,新鲜香肠中Staphylococcus equorum(马胃葡萄球菌25.59%)和Staphylococcus vitulinus(小牛葡萄球菌18.01%)丰度最高,腐败香肠中丰度最高的是Weissella viridesecens(绿色魏斯氏菌46.04%),其次是Weissella cibaria(食窦魏斯氏菌19.68%)。结果提示:与新鲜香肠的菌群组成相比较,腐败香肠的菌群结构发生了显著的变化;腐败香肠中的优势腐败菌是Weissella viridesecens,该种微生物在香肠储藏过程中大量增殖,主导了产品的腐败;在生产过程中需消除Weissella viridesecens,以延长产品货架期,提高产品品质。     

Freezing of meat raw materials affects tyramine and diamine accumulation in spontaneously fermented sausages

Biogenic amine accumulation was studied in spontaneously fermented sausages (Fuet) manufactured from unfrozen-fresh meat (U-sausages) and frozen-thawed meat (F-sausages). The aim was to investigate whether the frozen storage of raw materials affects the microbial composition and its aminogenic activity during sausage fermentation. Tyramine was the major amine in all sausages. Although the final levels were similar, tyramine accumulated more rapidly in F-sausages, which contained putrescine as the second amine. By contrast, U-sausages accumulated much more cadaverine than putrescine. F-sausages showed a slightly lower pH and free amino acid content as well as higher counts of technological flora (lactic acid and gram positive catalase positive bacteria) and lower counts of enterobacteria. Therefore, to freeze the meat raw materials for few days before sausage manufacture could be a useful practice, especially for the artisan fermented sausages (without starter), because it helps to reduce enterobacteria development and cadaverine production.     

发酵肉制品中生物胺及其产生菌株研究进展

生物胺是一类具有生物活性的含氨基小分子有机化合物,其广泛存在于蛋白丰富的食品中。生物体内适量生物胺具有增强代谢、促进生长等功能,但当含量达到临界值时,会产生诸多不良影响。存在大量微生物的食品中,生物胺的含量较高,同类发酵肉制品中生物胺的种类和含量也有很大不同,如发酵时间、贮藏时间和包装材料等都会对此产生影响。本文主要介绍生物胺的常见种类、生理作用,发酵肉制品中产生物胺的微生物种类、形成途径、菌株筛选方法及检测方法,旨在为今后发酵肉制品中生物胺的进一步研究提供帮助,以期为发酵肉制品的质量保障提供参考。     

微生物亚硝化抑制剂对风干肠发酵成熟期间微生物菌落变化的影响及其感官评价分析

为研究微生物亚硝化抑制剂（microbial nitrosation inhibitor,MNI）对风干肠发酵和成熟过程中微生物群落动态变化及感官特性的影响,该试验设计4组风干肠,MNI组：添加MNI;MNIP组：添加MNI并接入PRO-MIX5商业发酵剂（木糖葡萄球菌、清酒乳杆菌、类植物乳杆菌）;FBFAP组：发酵牛骨调味基料和复配抗氧化剂（fermented beef flavorings and compound antioxidant,FBFA）并接入PRO-MIX5;CK组。结果表明：在发酵阶段,4组风干肠乳酸菌数逐渐增多,MNI组和MNIP肉馅分别在发酵的15 h、12.5 h达到发酵终点即pH降到5.4~5.6,乳酸菌数分别为8.99 lg cfu/g、8.91 lg cfu/g,显著高于CK组发酵终点（22 h）的乳酸菌数8.88 lg cfu/g（p<0.05）。到成熟终点时,革兰氏阴性腐败微生物在MNI组的相对丰度占比最低（19.35%）,对肠杆菌科和假单胞菌的抑制率分别为30.43%、27.22%,仅次于MNIP组37.00%、33.79%,优于CK组和FBFAP组。综合分析,MNI能作为促生长因子,促进肉馅中乳酸杆菌属菌的生长,在接种有发酵剂PRO-MIX5的风干肠中加入MNI对腐败微生物的抑制效果优于FBFAP。单独添加MNI或MNI与PRO-MIX5协同作用均能提高风干肠微生物安全性和感官性能,说明MNI在提高风干肠安全品质方面具有广阔的应用前景。     

发酵香肠干制过程中理化及微生物指标的变化

采用传统中式方法制作猪肉香肠,定期检测发酵香肠干制过程中理化及微生物指标的变化。通过对水分、pH值、水分活度、酸值和挥发性盐基总氮等理化指标以及细菌总数的测定,确定发酵香肠干制过程中各项指标变化范围,并对比了发酵组和对照组各项指标的差异,为发酵香肠干制提供理论依据。     

Effect of delayed gutting on biogenic amine contents during ripening of European anchovies

The effect of delayed gutting on biogenic amine formation during ripening of European anchovies (Engraulis encrasicholus) was studied. The main difference between the two processes was the use of gutted or ungutted fish in the first step of ripening. Biogenic amines and microbial counts were studied in seven batches of samples taken during manufacturing from fresh anchovies and from anchovies throughout ripening. The formation of amines commonly related to fish spoilage (histamine, tyramine, cadaverine, putrescine and agmatine) was significantly higher in the first step of ripening of batches in ungutted fish. In contrast, no differences were observed between the content of the natural polyamines spermine and spermidine.     

不同乳酸菌发酵剂对发酵红肠品质的影响

为研究乳酸菌发酵对红肠品质的影响,将发酵技术应用于本无发酵工艺的红肠制品中,筛选出能够提高红肠品质的乳酸菌发酵剂。分别将常应用于发酵肉制品的7 种商业乳酸菌发酵剂（木糖葡萄球菌＋戊糖片球菌（THM-17）、木糖葡萄球菌＋清酒乳杆菌＋类植物乳杆菌（PRO-MIX5）、木糖葡萄球菌＋肉葡萄球菌＋清酒乳杆菌（WBL-45）、木糖葡萄球菌＋戊糖片球菌＋植物乳杆菌（VHI-41）、木糖葡萄球菌＋戊糖片球菌＋植物乳杆菌（SHI-59）、肉葡萄球菌＋木糖葡萄球菌（WBX-43）和戊糖片球菌＋木糖葡萄球菌＋肉葡萄球菌＋乳酸片球菌（VBM-60））及8 种单菌（弯曲乳杆菌、戊糖乳杆菌、清酒乳杆菌-1、戊糖片球菌、木糖葡萄球菌、肉葡萄球菌、清酒乳杆菌-2、植物乳杆菌）以107 CFU/g的接种量接种至腌制后的肉馅中,拌馅灌肠后于35 ℃、80%湿度条件下发酵12 h,取样测定发酵后样品的乳酸菌数和细菌总数,再经干燥、蒸煮、烟熏、烘烤制得成品,测定其感官、pH值、色差、质构、亚硝酸盐、硝酸盐、生物胺及N-亚硝胺含量等指标。结果表明：15 种发酵剂中以木糖葡萄球菌和植物乳杆菌2 种乳酸菌发酵剂应用效果较好,所制得产品pH值分别为5.26和5.04,色泽美观,弹性适中,亚硝酸盐残留量（10.84、10.13 mg/kg）低,可显著抑制N-亚硝胺的形成（N-二甲基亚硝胺含量分别为1.29、2.51 μg/kg）,生物胺总量较低。由此说明,木糖葡萄球菌和植物乳杆菌能够显著提高红肠产品的安全品质。     

生物胺荧光响应膜在肉及肉制品新鲜度检测中的应用研究进展

肉和肉制品的新鲜度可以通过产品腐败过程中产生的代谢物含量进行评价。生物胺是肉及肉制品腐败过程中产生的物质之一,是监测食品新鲜度的重要指标。生物胺的传统检测方法繁琐、耗时、费力。建立快速、便捷的检测方法具有重要的现实意义和科研价值。生物胺荧光响应膜是一种由荧光材料与传感薄膜组成的固体光学传感器,能够通过传感薄膜的荧光颜色变化对生物胺进行实时检测,近年来因其灵敏度高、成本低、操作简单、响应速度快等优点被广泛应用于肉及肉制品中新鲜度的检测。本文简要概述肉及肉制品中生物胺的形成与分类以及生物胺对人体的危害,重点综述基于不同荧光响应原理构建的荧光响应膜在肉及肉制品新鲜度监测中的应用。     

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.     

Effect of different starter cultures on the biogenic amines production as a critical control point in fresh fermented sausages

The selection of critical control points is one of the most important steps in the design of a hazard analysis and critical control points system. In this study, the influence of starter culture inoculation on biogenic amine accumulation in fresh fermented sausage as critical control point was studied. The effect of three starter culture Lactobacillus plantarum plus Bifidobacterium lactis, L. plantarum plus Bifidobacterium bifidum or L. plantarum plus B. lactis and B. bifidum on amines production were investigated. Tyramine (TY), Cadaverine (CA), Putrescine (PU), Histamine (HIS) and Tryptamine (TR) contents increased during the fermentation and storage stage, and TY was the prevailing amine in the final product. Sausages produced by fermentation with starters, as compared to natural fermentation (control), had a lower amount of biogenic amine. A great reduction in biogenic amine content was achieved when Bifidobacterium sp. were inoculated, also levels of species of Enterobacteriaceae and Pseudomonas decreased, while lactic acid bacteria counts increased during fermentation and storage.     

Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria

The effect of controlled fermentation processes on the microbial association and biochemical profile of cv. Conservolea naturally black olives processed by the traditional anaerobic method was studied. The different treatments included (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a strain of Lactobacillus plantarum isolated from a fermented cassava product and (c) uninoculated spontaneous process. Microbial growth, pH, titratable acidity, organic acids and volatile compounds were monitored throughout the fermentation. The initial microbiota consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all processes. Both starter cultures were effective in establishing an accelerated fermentation process and reduced the survival period of Gram-negative bacteria by 5 days compared with the spontaneous process, minimizing thus the likelihood of spoilage. Higher acidification of the brines was observed in inoculated processes without any significant difference between the two selected starter cultures (113.5 and 117.6mM for L. plantarum and L. pentosus, respectively). L. pentosus was also determined as the major species present during the whole process of spontaneous olive fermentation. It is characteristic that lactic acid fermentation was also initiated rapidly in the spontaneous process, as the conditions of fermentation, mainly the low salt level (6%, w/v) favored the dominance of lactic acid bacteria over yeasts. Lactic, acetic and propionic were the organic acids detected by HPLC in considerable amounts, whereas citric and malic acids were also present at low levels and degraded completely during the processes. Ethanol, methanol, acetaldehyde, ethyl acetate were the major volatile compounds identified by GC. Their concentrations varied among the different treatments, reflecting varying degrees of microbial activity in the brines. The results obtained from this study could help the Greek table olive industry to improve the existing processing schemes in order to increase product consistency and quality expanding the international market for naturally black olives.     

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMβ1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.