The application of staphylococci with flavour-generating potential is affected by acidification in fermented dry sausages

Differences in the production of bacterial metabolites with potential impact on fermented sausage flavour were found in meat simulation medium when comparing different strains of Staphylococcus xylosus and Staphylococcus carnosus as starter cultures. Overall, higher levels of 3-methyl-1-butanol and acetoin were found for S. xylosus, with some intraspecies variability. In addition, sausage fermentation parameters affected staphylococcal growth and metabolism. Strong acidification, as in Northern-European types of fermented dry sausage, inhibited S. xylosus 3PA6 but not S. carnosus 833. During a milder, Southern-European type of acidification, both strains displayed survival over time. During in situ sausage trials, variations in the degree of acidification and the choice of starter microorganisms were of importance, whereas modifications in fat and salt contents had no effects. Staphylococcus sciuri αSg2, Staphylococcus succinus 4PB1, and S. xylosus 3PA6 were unable to survive the fermentation of a Northern-European type of fermented dry sausage, characterized by low or no 3-methyl-1-butanol and acetoin production. Inoculation with S. sciuri αSg2, S. succinus 4PB1, or S. xylosus 3PA6 led to 3-methyl-1-butanol and acetoin production in Southern-European type of fermented dry sausages, which was not observed with S. carnosus 833.     

Effect of selected dairy starter cultures on microbiological, chemical and sensory characteristics of swine and venison (Dama dama) nitrite-free dry-cured sausages

The aim of this study was the evaluation of selected lactic acid bacteria (LAB) starter culture of dairy origin in the production of nitrite-free low-acid fermented venison (Dama dama) sausage (salame di daino) produced in a small-scale plant in Umbria (Italy), and their effect on microbiological, physico-chemical and sensorial properties of the products. Salame di daino was obtained with two different processes: with and without the addition of selected LAB starter cultures. Microbial counts of Enterobacteriaceae, coliform organisms and Pseudomonas spp. were lower in salami made with the addition of starter cultures. Staphylococcus aureus, Salmonella spp, and Listeria monocytogenes after the first week of ripening were only detected from control salami. Control salami were paler and harder, whereas those made with the addition of starter cultures were slightly saltier, juicier and in general more acceptable. Selected dairy-origin starter (SDS) cultures did prevent the growth of both indicators of food safety and of process hygiene and increased the acceptability of full-ripened salami.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Effect of selected autochthonous starter cultures on processing and quality characteristics of Greek fermented sausages

Selected autochthonous starter (SAS) cultures (i.e. Lactobacillus sakei 8416, Lactobacillus sakei 4413, and L. sakei 8426, L. plantarum 7423 and L. curvatus 8427) were used as starter cultures in addition to a control treatment in the production of fermented sausages. The SAS cultures had a rapid growth and dominated the fortuitous population of LAB during the whole fermentation and ripening process improving the sensory attributes in comparison to control. Apart from the treatment produced with L. sakei 8416, all other SAS cultures prevented the lipid oxidation to values lower than 1 mg malonaldehyde/kg. The Micrococcaceae count and the redness of the sausages was not affected by the smoking and the acidification during the fermentation in the treatments produced with L. sakei 8416 and L. sakei 4413. The treatment of L. sakei 4413 had the lowest (*P < 0.05) content of all biogenic amines. In comparison to the control, the reduction of tyramine was 13%, tryptamine 55%, cadaverine 60% and putrescine 72%. Sausages produced with SAS cultures L. sakei 4413 and L. sakei 8416 had the highest scores for all sensory attributes. The results indicated that the SAS culture of L. sakei 4413 is the best autochthonous starter culture for fermented sausages.     

发酵剂对发酵香肠挥发性风味物质形成的作用及影响机制研究进展

发酵香肠是指将搅碎的猪肉（或牛、羊肉等）、动物脂肪、盐、糖、发酵剂和香辛料等混合后灌进肠衣,经过微生物发酵而成的香肠制品。因其具有独特的风味和较高的营养价值,深受国内外消费者的青睐。风味作为发酵香肠的重要品质属性之一,其形成与发展与发酵过程中微生物群落的代谢活动密切相关。本文对发酵香肠发酵、成熟过程中风味物质的形成和发展以及与微生物代谢相关的反应进行了概述,总结了几种主要微生物对发酵香肠风味品质形成的作用及影响机制,并对其未来研究进行展望,以期为发酵香肠风味品质的研究提供参考。     

Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 in liquid broth medium and during processing of fermented sausage using autochthonous starter cultures

The antimicrobial effect of two autochthonous starter cultures of Lactobacillus sakei was evaluated in vitro (in liquid broth medium) and in situ assays. The inactivation of foodborne pathogens Listeria monocytogenes (serotype 4ab No 10) and Escherichia coli O157:H7 ATCC 43888 was investigated during the production of fermented sausage according to a typical Greek recipe using L. sakei strains as starter cultures. The inactivation kinetics were modeled using GInaFiT, a freeware tool to assess microbial survival curves. By the end of the ripening period, the inhibition of L. monocytogenes was significant in treatments with L. sakei 8416 and L. sakei 4413 compared to the control treatment. A 2.2-log reduction of the population of E. coli O157:H7 resulted from the autochthonous starter culture L. sakei 4413 during sausage processing. The use of the autochthonous starter cultures constitutes an additional improvement to the microbial safety by reducing foodborne pathogens.     

Tyrosine- and histidine-decarboxylase positive lactic acid bacteria and enterococci in dry fermented sausages

Lactic acid bacteria (LAB) and enterococci were isolated immediately after stuffing (day 0), at the end of ripening (28th day) and at the end of storage (112th day) from dry fermented sausages produced by two different producers (K; R) in two diameters (4.5 and 7 cm) using either of two spice mixtures (P; H) and either of two starter cultures (Pediococcus pentosaceus, C; Lactobacillus curvatus + Staphylococcus carnosus, F), resulting in a total of 16 different combinations. Tyrosine-decarboxylase DNA sequence (tyrdc) was identified on average in 88% and 44% of enterococci and LAB isolates, respectively at the end of ripening, the corresponding figures regarding histidine-decarboxylase gene sequence (hisdc) was 71% and 16%, respectively. Lactobacillus plantarum, L. brevis and L. casei/paracasei, and Enterococcus faecium and Enterococcus faecalis were identified as tyramine/histamine producers in the sausages.     

Microbial and Physicochemical Characterization of the Horse Meat in Fermented Sausage

In this study, 150 lactic acid bacterial strains, originating from horse meat, were subjected to a step-by-step screening and characterization to search for potential protective cultures to be used in the meat industry. Strains were first tested for their homofermentation and salt tolerance. Second, the antibacterial capacities towards Listeria monocytogenes were determined in an agar spot test. In total, 50% of the tested strains were inhibitory towards Listeria monocytogenes. However, only 12 strains produced a bacteriocin. Finally, three strains with strong bacteriocin activity were competitively evaluated by comparing their growth rate, acidifying character and lactic acid production at 15°C under anaerobic conditions in a liquid broth. All three strains had a fast growth rate with rapid acidification caused by the production of high levels of lactic acid. Based on this Lactobacillus sakei was used as starter culture for producing sausage horse meat. In this study, fermentations were followed analyzing the microbiological and physicochemical aspects of this product. The sausages were characterized based on microbial activity of lactic acid bacteria that resulted in a product with a final pH of about 4.56. No Listeria monocytogenes, Salmonella spp. nor Sulfite reducing clostridia were ever isolated from the raw materials or the fermented sausages during the maturation, reflecting the safety of this product. The final water activity of the product was 0.85. Lactobacillus sakei was efficient in reducing amine production since this strain caused a quick pH drop during sausage fermentation.     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

Use of molecular methods to characterize the bacterial community and to monitor different native starter cultures throughout the ripening of Galician chorizo

The development of Lactobacillus and Staphylococcus strains used as starter cultures throughout the ripening of Galician chorizo, a traditional dry fermented sausage from the north-west of Spain, was monitored combining different molecular-based techniques. The bacterial diversity occurring in the inoculated sausages at the beginning and the end of the ripening was also studied and compared to the indigenous population in an uninoculated control batch.     

The effect of kimchi on the microbiological stability of fermented sausage

The effects of kimchi and freeze-dried kimchi-powder added to raw meat mixtures on the microbiological quality of fermented sausage were studied. The results clearly demonstrated that the lactic acid bacteria (LAB) integrated via the addition of kimchi as well as kimchi-powder were well adapted to the new habitat of fermenting sausage, reaching maximum numbers of 8.65-8.80log(10)cfu/g after 1-2days of fermentation. In all kimchi and kimchi-powder sausages, the growth of Enterobacteriaceae was completely inhibited throughout the processing period (<2log(10)cfu/g). The sausage batches containing more than 10% kimchi and 2% kimchi-powder showed no growth of S. aureus, whereas the control and another kimchi sausage batch reflected the growth of S. aureus (3.68-4.72log(10)cfu/g). As a result, the addition of kimchi (≥10%) and kimchi-powder to the sausage mixture prior to fermentation produced the microbiological stability required for fermented sausages.     

Characterization of Proteolytic Effect of Lactic Acid Bacteria Starter Cultures on Thai Fermented Sausages

The objective of this study was to investigate the effect of starter culture addition on proteolysis of Thai fermented sausages. Sausages inoculated with six different external starter cultures—Pediococcus pentosaceous, Pediococcus acidilactici, Weissella cibaria, Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus sakei—were compared with naturally fermented sausages. The results of microbiological analysis indicated that the dominance of lactic acid bacteria (LAB) could inhibit the growth of pathogens and spoilage. Proteolysis was observed during fermentation by the reduction of myofibrillar and sarcoplasmic proteins and the increase in nonprotein nitrogen (NPN) and total free amino acids. The highest increase in concentration of NPN and free amino acids was obtained from sausages inoculated with LAB. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed a similar pattern of proteolysis of sarcoplasmic proteins in all sausages, while that of the inoculated sausages with L. plantarum, L. pentsus, and L. sakei exhibited increased degradation of myofibrillar protein bands at 200 and 45 kDa.     

腌腊肉制品中乳酸菌的筛选鉴定及其在腊肠中的应用

为筛选适合传统腌腊肉制品的优良乳酸菌菌株,从多种农家自制传统腌腊肉制品中分离纯化出9株优势乳酸菌。通过发酵特性筛选,得到一株性状优良菌株10M-7,并制备该菌株的干粉发酵剂,以未接种发酵剂腊肠为对照,分析此发酵剂对腊肠感官品质和微生物变化的影响。结果表明,10M-7菌株具有良好的产酸特性和抑菌性能。根据形态学、生理生化特征和16S rRNA序列分析,鉴定其为植物乳杆菌,采用冷冻干燥法制备纯种发酵剂,并制作人工发酵腊肠。发酵剂组pH值在初期便迅速下降,且始终低于对照组;发酵剂组乳酸菌迅速生长繁殖,且葡萄球菌和大肠杆菌数量与对照组相比明显降低。感官评价表明,当添加量为10~4CFU/g原料肉时,能够很好地保持和改善产品风味,使产品整体感觉更好。     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

Molecular Characterization of Lactic Acid Bacteria in Levačka Sausage

Levačka sausage is traditionally fermented dry sausage which is produced in central Serbia. It is made of beef and pork with the addition of solid fat and natural spices. The whole manufacturing process lasted for 21 days. The goal of this study was to create a collection of lactic acid bacteria isolated during the fermentation and identify them using molecular methods. A total of 50 isolates from different stages of fermentation were identified by molecular methods. Pediococcus pentosaceus, Ln. mesenteroides were predominant microorganisms in levačka sausage. Lb. curvatus, Lb. sakei and Lb. carnosum were also identified.     

Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H⁺-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 °C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.     

Novel starter cultures to inhibit biogenic amines accumulation during fish sauce fermentation

Bacteria with amine oxidase activity have become a particular interest to reduce biogenic amines concentration in food products such as meat and fish sausages. However, little information is available regarding the application of these bacteria in fish sauce. Hence, our study was aimed to investigate the effect of such starter cultures in reducing biogenic amines accumulation during fish sauce fermentation. Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were used as starter cultures in this study. Fermentation was held for 120 days at 35 °C. The pH value increased in all samples, while salt concentration remained constant throughout fermentation. Aerobic bacteria count was significantly lower (p < 0.05) in the control than in inoculated samples as a result of starter cultures addition. However, it decreased during fermentation due to the growth inhibition by high salt concentration. Proteolytic bacterial count decreased during fermentation with no significant difference (p > 0.05) among samples. These bacteria hydrolyzed protein in anchovy to produce free amino acid precursors for amines formation by decarboxylase bacteria. The presence of biogenic amines producing bacteria in this study was considered to be indigenous from raw material or contamination during fermentation, since our cultures were negative histamine producers. Amino acid histidine, arginine, lysine and tyrosine concentration decreased at different rates during fermentation as they were converted into their respective amines. In general, biogenic amines concentration namely histamine, putrescine, cadaverine and tyramine increased throughout fermentation. However, their concentrations were markedly higher (p < 0.05) in the control (without starter cultures) as compared to the samples treated with starter cultures. Histamine concentration was reduced by 27.7% and 15.4% by Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively. Both cultures could also reduce other amines during fermentation. After 120 days of fermentation, the overall biogenic amines concentration was 15.9% and 12.5% less in samples inoculated with Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively, as compared to control samples. These findings emphasized that application of starter cultures with amines oxidase activity in fish sauce fermentation was found to be effective in reducing biogenic amines accumulation.     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.     

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.     

发酵剂对发酵香肠微生物及理化特性的影响

采用3种复合型发酵剂(VBL-97、VBM-60、SHI-59)和1种单一型发酵剂(BOM-13)生产发酵香肠,研究不同发酵剂对发酵香肠微生物及理化性质的影响。结果表明4组发酵香肠在发酵和成熟过程中,乳酸菌始终保持为优势菌,当达到成熟时,乳酸菌数在7.21~8.13 lgcfu/g范围内,葡萄球菌(除BOM-13外)高于5.50 lgcfu/g,肠杆菌科菌数低于3.00 lgcfu/g。BOM-13型发酵剂由单一清酒乳杆菌组成,发酵和成熟时间长于其他三组,所以成品的硫代巴比妥酸值(TBARS值,0.84 mg/100 g)和非蛋白氮值(NPN,0.46%)高于其它3组产品。由此可知由于发酵剂组成不同,相应地发酵条件(温度和时间)和成熟时间亦不同,但最终四种发酵剂都可生产出高品质产品(p H值5.3;AW0.82;NPN值高;TBARS值1.0 mg/kg;乳酸菌为优势菌且7.0 lgcfu/g;肠杆菌科菌3.0lgcfu/g)。VBL-97型发酵剂产酸能力和抗氧化能力强,而BOM-13型发酵香肠蛋白分解程度最高。