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.     

The effect of different starter cultures and ripening temperatures on formation of biogenic amine in Turkish fermented sausages

Different starter culture added groups (Group I: Lactobacillus sake, Staphylococcus xylosus; Group II: Lactobacillus plantarum, Staphylococcus carnosus; Group III: Lactobacillus curvatus, Staphylococcus carnosus, and Staphylococcus xylosus) and control group sausage samples were produced experimentally at two different temperatures (22 and 26 °C). In raw materials and all groups, by the 0th, 2nd, 5th and 7th days of the ripening period, the analysis of HPLC and biogenic amine (tyramine, histamine, cadaverine, putrescine, phenylethylamine, tryptamine, spermine, and spermidine) amounts, microbiological (Lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, Enterococcus spp., and moulds–yeasts) and chemical (pH, dry matter, and salt) have been done. During the ripening period, there is no statistical discrepancy (P > 0.05) in terms of biogenic amine amounts, microbiological, and chemical values detected from the sausage samples produced at 26 and 22 °C. However, in both levels of temperature, there is statistical discrepancy (P < 0.001) detected in terms of tyramine, putrescine values and the count of Enterococcus spp. between the starter culture added samples and control group samples. By this study, it has been stated that the ripening temperature does not make any statistical discrepancy (P > 0.05) for all values but the use of starter culture prevents the formation of biogenic amine in Turkish fermented sausages.     

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.     

Effect of starter culture, spice mix and storage time and temperature on biogenic amine content of dry fermented sausages

Two types of dry fermented sausage differing in spicing mixture and the diameter (low content of red pepper + diameter 80 mm, H-sausage; high content of red pepper + diameter 55 mm, P-sausage, respectively) were produced in parallel with two different starter cultures (Pediococcus pentosaceus + Staphylococcus carnosus, B-samples and S. carnosus + Staphylococcus xylosus + Lactobacillus farciminis, F-samples, respectively). The sausages were ripened 21 days and subsequently stored 91 days at the room temperature. Concentration of both most abundant amines, putrescine and tyramine (y; mg/kg DM) increased significantly (P<0.01) in HB-sausage during ripening (x; days): y=2.5+18.13x−0.3144x² (R²=0.99) and y=0.7+8.17x−0.1130x² (R²=0.99), and also during storage: y=127.3+5.123x (R²=0.79) and y=26.0+3.211x (R²=0.74), respectively. At the end of ripening, putrescine (247 mg/kg DM) and tyramine (123 mg/kg DM) content in the HB-sausage was higher (P<0.05) than in the PB-sausage (12 and 9 mg/kg DM, respectively), concentration of either of these amines was negligible (1 mg/kg DM) in either type of F-inoculated sausage. Both starter culture and sausage type influenced significantly (P<0.001) both putrescine and tyramine content in the sausage; starter accounted for 57% and 55% of total variability in putrescine and tyramine content, respectively. Due to the significant (P<0.05) increase of total aerobic counts in the HB-sausage between the end of ripening and the 7th day of storage, followed by the significant (P<0.01) increase of the sum of total biogenic amines between the end of ripening (425 mg/kg DM) and the end of storage (1029 mg/kg DM), the storage of the dry fermented sausages at the room temperature should not be recommended.     

发酵剂对熏马肠成熟过程中蛋白质降解的影响

对熏马肠发酵成熟过程中蛋白质的降解变化进行了研究。结果表明,非蛋白氮含量在加工过程中持续增加,并且发酵剂组的非蛋白氮值上升较快;通过SDS-PAGE图谱分析,肌浆蛋白和肌原纤维蛋白均发生了降解,发酵剂组的蛋白降解程度明显高于空白组,尤其是肌浆蛋白,分子质量在14.4 ku、27～35 ku、66～90 ku附近的蛋白条带均降解消失;通过对组织蛋白酶B和L活力测定,得出空白组的酶活力高于发酵剂组。     

发酵剂对熏马肠成熟过程中生物胺含量变化的影响

利用从熏马肠中分离出的具有抑菌作用并可以产生物胺氧化酶的菌株,1株表皮葡萄球菌(Staphylococcus epidermidis),3株模仿葡萄球菌(Staphylococcus simulans)作为发酵剂加入到熏马肠,在熏马肠成熟过程中采用高效液相色谱检测不同发酵剂对生物胺含量变化的影响,进而阐明发酵剂对熏马肠中生物胺累积的控制作用。从研究结果可以看出,在马肠成熟过程中,B组对色胺、苯乙胺、腐胺、组胺、酪胺都有较显著的减少作用;C组对色胺、腐胺、尸胺、组胺、酪胺减少作用显著;D组对色胺、苯乙胺、腐胺、组胺、酪胺的减少作用显著;E组对组胺、苯乙胺、色胺的减少作用显著。添加发酵剂组对精胺、亚精胺的减少作用均不显著。结果表明,上述菌种对熏马肠成熟过程中生物胺产生有较好的抑制作用,具有肉品发酵剂的良好特性,为熏马肠以及其他发酵肉制品的工业化生产和安全性奠定了基础。     

一种混合发酵剂对干发酵香肠中生物胺含量的影响

研究了复合发酵剂(戊糖片球菌Pediococcuspentocaceus和木糖葡萄球菌Staphylococcusxylosus)对发酵香肠中生物胺含量的影响。通过与单一发酵剂戊糖片球菌和空白组的产胺、微生物生长情况的比较,分析了木糖葡萄球菌对戊糖片球菌在发酵过程中降胺作用的影响。结果表明,木糖葡萄球菌有助于戊糖片球菌进一步降低发酵香肠中的酪胺和色胺的量,分别比单一发酵菌株降低了6.7%、22.83%;三种胺的量比空白组分别降低了94.04%,29.53%,46.59%,从而证实了戊糖片球菌具有降酪胺和色胺的能力。结合微生物菌数可知,木糖葡萄球菌是通过抑制乳酸杂菌的生长促使戊糖片球菌菌数的增加而有利于胺量的降低。     

Effect of starter culture on proteolytic changes during processing of fermented beef sausages

Fermented beef sausages inoculated with four different starter cultures (Pediococcus acidilactici,Lactobacillus curvatus, Lactobacillus sake, or Streptomyces griseus) were evaluated for proteolysis during process stages (prefermentation, fermentation, drying and heating). Increases (p ? 0.05) in the nonprotein nitrogen (NPN) fraction were found at sequential stages of processing, while starter cultures had no major effects on NPN content. Concentrations of most free amino acids increased (p ? 0.05) during fermentation and drying, and culture effects were found for differences among concentrations of some individual free amino acids. From SDS–PAGE analysis of sarcoplasmic and myofibrillar protein fractions after fermentation and drying, myosin heavy and light chains, actin and troponin were degraded during processing. However, starter culture effects were absent from SDS–PAGE protein patterns.     

Relationship between biogenic amine contents and the size of dry fermented sausages

Three trials were carried out to study the influence of the diameter on biogenic amine contents and related parameters (pH, humidity and proteolysis) in fermented sausages. The first trial was done on three groups of Spanish dry fermented sausages with different diameter. In the second, two sections (centre and edge) of salchichón sausages were examined. The last trial consisted in the study of the ripening of two batches of sausages fermented under the same conditions but with two different diameters. Biogenic amine contents varied among the different type of products as well as among the same type of samples. Generally, amine levels in the biggest diameter sausages were higher than in the thinnest sausages. Amine contents were higher in the central part of the sausages than in the edge. During the ripening, larger tyramine amounts were formed in sausages with the biggest diameter. Statistical correlations were found among the diameter, the pH, the proteolysis and some amines. The results of the three trials agree with the hypothesis that the diameter is a factor that may affect the formation of biogenic amines during sausage fermentation.     

Accelerated ripening of dry fermented sausages

Research on accelerated ripening of dry fermented sausages started in the early 1990s. Fermented sausages manufacture is a very important part of meat industry in many countries and the acceleration of ripening would result in a reduction of the storage time and would increase the profit margin and the competitiveness of the end product. The different strategies that have been assayed with this purpose include ripening at elevated temperature, use of genetically modified starter bacteria, addition of enzymes and addition of slurry systems. Over the last decade, numerous studies have been carried out, especially on the addition of enzymes. The aim of this paper is to review and update the knowledge on this topic. The more recent approaches in this field, such as the use of microbial extracts, are also presented.     

Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening

Two commercial starters were investigated for their potential ability to decarboxylate amino acids during goat cheese ripening. Two batches of goat cheese were produced with identical pasteurized milk but different starter cultures. One of them contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and the other Lactococcus lactis subsp. lactis. The amine contents, microbial counts, proteolysis-related parameters, pH, total solids and salt content were studied in raw materials and cheeses. In raw materials, polyamines were the prevailing amines, whereas the main amines in cheeses were putrescine, tryptamine and, in particular, tyramine (94.59 mg/kg). Aerobic mesophilic microorganisms and Lactococcus counts increased throughout ripening, while Enterobacteriaceae were no longer detectable in cheese after 30 days of ripening. Amine concentration rose during cheese ripening in both batches. Moreover, the decarboxylase activity of microorganisms isolated from samples during cheese ripening was assayed and discussed.     

Flavour profiles of dry sausages fermented by selected novel meat starter cultures

Probiotic or bioprotective Lactobacillus rhamnosus strains GG, LC-705 and E-97800 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 were studied for their ability to act as main fermenting organisms in the manufacturing process of dry sausages. In the preliminary tests, their abilities to produce lactic acid and biogenic amines, histamine or tyramine, were studied in MRS broth and analysed by high-performance liquid chromatography. The strains produced higher or equal amounts of lactic acid compared to control and were amine negative. During the actual fermentation process of dry sausages the numbers of inoculated bacteria increased from the level 6.5-7.0 log cfu/g to 8.0-9.0 log cfu/g. The most fast growing strains were P. pentosaceus E-90390 and the control while the growth of L. plantarum E-98098 and L. rhamnosus LC-705 were the slowest. The pH value of the sausages decreased from 5.6 to 4.9-5.0. The presence of these experimental strains as major organisms in the sausages after fermentation and ripening was confirmed on the bases of their genetic fingerprints. The flavour profiles of the experimental sausages produced by these probiotic or protective strains were similar with that produced by the commercial meat starter culture and commercial North European dry sausage recipe.     

The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages

The main objective of this work was to examine the effects of using five types of commercial starter cultures in fermented sausages. During the fermentation stage, changes in proteolytic characteristics were observed in fermented sausages. Proteolytic activity was high in Ls_b + Sc:(Lactobacillus sakei + Staphylococcus carnosus) and Pp + Sx:(Pediococcus pentosaceus + Staphylococcus xylosus) starter-inoculated sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both these sausages. Sarcoplasmic and myofibrillar proteins were also affected by this starter culture addition, during the fermentation, ripening and intense proteolysis were observed in both the fermented sausages. The content of free amino acids was similar at the beginning of the fermentation stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the starter culture activity.     

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.     

Influence of starter cultures on the free fatty acids during ripening in Tea sausages

The influence of starter cultures on the free fatty acids content during ripening of Tea sausages, typical dry sausages produced in the south of Croatia, was studied. Three batches of Tea sausages were produced using different starter mixtures (Staphylococcus xylosus S81 and Lactobacillus sakei G20; Staphylococcus xylosus S142 and Lactobacillus sakei G20; Staphylococcus xylosus S206 and Lactobacillus sakei G20), while the control batch was produced without a starter. The amounts of free fatty acids present in the samples at the end of the ripening period were not significantly different, suggesting that the lipolytic enzymes naturally occurring in meat could play a predominant role in the free fatty acids release. Oleic and linoleic acids were present in the highest concentrations, while only small quantities of short-chain fatty acids were detected, with acetic acid being the most representative one.     

Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits

In this study, three starter formulations including Lactobacillus curvatus and Staphylococcus xylosus strains selected in vitro on the basis of their lipolytic and proteolytic activities were employed for the manufacture of traditional fermented sausages of southern Italy. Microbial population, proteolysis, lipolysis, changes in free amino acids (FAA) and free fatty acids (FFA) and development of characteristic taste and flavor of the final product were investigated. Proteolysis and lipolysis were observed in sausages inoculated with proteolytic and lipolytic S. xylosus coupled with L. curvatus, while the sausage started with only S. xylosus without lactobacilli was identical to the non-inoculated control, indicating that the proteolysis could be due to both microbial activity and endogenous proteases activated by the decrease in pH. The statistical analysis applied to the instrumental and sensory data showed that there was an effect of the starter used on the characteristics of the sausage obtained. In particular, the control samples showed very close features different from the sausages obtained by adding starter cultures. Finally, analyzing the sensory parameters the sausages ripened without starter addition and those started without the L. curvatus AVL3 showed similar features indicating an influence of the presence of the lactobacilli on the final organoleptic quality of the sausages. An appropriate choice of a combination of strains in a starter formulation is fundamental to obtain products of the expected quality.     

复合发酵剂和黑胡椒对羊肉发酵干香肠挥发性风味成分的影响

以内蒙古羊肉和羊尾脂肪为原料,接种复合发酵剂和黑胡椒生产羊肉发酵干香肠,利用动态顶空制样（DHS）/吹扫捕集（P＆T）技术结合气-质联机（GC-MS）,分析鉴定了羊肉发酵干香肠在成熟后挥发性风味成分的变化情况,结果检出了52种挥发性风味成分,主要由醛类、酮类、酸类、醇类、萜类、烷烃类、酯类、芳烃类、呋喃类和含氮化合物等十类化合物组成,种类数最多的是烷烃类（10种）,占总种类数的19.23%,其次是萜类（9种）。同时检出了来源于脂肪水解氧化和氨基酸的Strecker降解以及碳水化合物代谢产生的戊醛、3-甲基丁醛、3-羟基-2-丁酮等关键芳香化合物以及来源于黑胡椒的3-蒈烯和D-柠檬精油等,这些成分对羊肉发酵干香肠的风味有重要贡献。     

Use of Staphylococcus xylosus as a starter culture in dried sausages: effect on the biogenic amine content

The main objective of this work was to investigate if the use of Staphylococcus xylosus S81 as a starter culture in sausage production can influence the amount of histamine during ripening, and the concentration of the other most important biogenic amines, by influencing the activity of the microbial amino oxidases as well as the aminoacid decarboxylases. The results confirm that the biogenic amines presence in foods is the consequence of a complex equilibrium between the composition of the medium and the enzymatic activities of the microbial population. In addition, the results suggest that the presence and relative activity of amino oxidases should be considered as an important characteristic in the selection of starter cultures used in the production of fermented foods.     

Microorganisms and lipolysis in the ripening of dry fermented sausages

The actual roles of two strains of microorganisms ( Micrococcus -12 and Lactobacillus -56) isolated from Spanish dry sausages have been studied by using an aseptic method for making experimental sausages. The fermentation of sugars by lactobacilli is clearly demonstrated by the pH changes and the nitrate and nitrite reduction by micrococci is inferred by the changes in the sausage colour. The lipolysis occurring during ripening of experimental sausages seems to be mainly due to the lipases from meat rather than those elaborated by the organisms used.     

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。