Use of autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter cultures in the production of "chorizo" in 2 different traditional industries

The present study determined how the different ripening conditions affected the growth and development of 3 autochthonous starter cultures, and the physico-chemical and sensory characteristics of chorizo. Each of 3 strains of Pediococcus acidilactici (MC184, MS198, and MS200) and one of Staphylococcus vitulus (RS34) were associated to prepare the starter cultures, P184S34, P198S34, and P200S34. Then, chorizo was prepared following 2 manufacturing procedures. The autochthonous starter cultures were able to compete and colonize the sausages in both ripening procedures. The use of the starter cultures showed evident differences by the texture analysis, with the control batches being generally tougher than the starter culture batches. Also, the highest biogenic amine (BA) levels were found in control batches and the lowest in P200S34 batches. While the use of these starter cultures does not change the sensory characteristics of these traditional fermented sausages, it improves their homogeneity and safety, except for P184S34 batch in which more BAs are detected in industry 2. PRACTICAL APPLICATION: The 3 autochthonous starter cultures selected could be used in traditional industries because they are able to compete well and colonize the dry fermented sausages "chorizo." The use of these starter cultures improves the texture and homogeneity of traditional fermented sausages. Biogenic amines decreased in the starter cultures batches improving the safety.     

Comparison of the effects of a commercial and an autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter culture on the sensory and safety properties of a traditional Iberian dry‐fermented sausage “salchichón”

The effect of the addition of an autochthonous starter culture and commercial starter culture on the physico‐chemical and sensory characteristics of the dry‐fermented sausage ‘salchichon’ was investigated. Sausages were prepared with a mixture of Pediococcus acidilactici MC184 and Staphylococcus vitulus RS34 as the autochthonous starter culture (P184S34), ripened for 86 days, and compared with a commercial starter batch (CS1). Strains of acid lactic bacteria and Staphylococcus were identified in the samples along the ripening process which demonstrated that the inoculation of sausages assures their implantation during the ripening process. However, P184S34 starter culture showed a higher inhibitory effect on enterobacteria and coliform flora than the CS1 batches, guaranteeing a better quality hygienic sausages. Dry‐fermented sausages ripened with P184S34 showed higher amounts of NPN and volatile compounds derived from amino acid catabolism than the CS1 batches. Especially important was the result found in biogenic amines, since the P184S34 reduced their accumulation compared to the commercial starter batch. The inoculation of a decarboxylase‐negative autochthonous starter culture P184S34 reduced the biogenic amine accumulation guaranteeing safety and homogeneity of the products without producing a negative effect on the sensory characteristics of the traditional fermented sausages.     

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.     

Effects of muscle shortening and proteolysis on Warner-Bratzler shear force in beef longissimus and semitendinosus

The effect of the addition of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage "salchichón" ripened with commercial starter cultures was investigated. Sausages were prepared with purified EPg222 and Staphylococcus carnosus, Staphylococcus xylosus, and Lactobacillus sakei as starter cultures, ripened for 145 days and compared with a control batch only inoculated with the starter cultures. Dry fermented sausages ripened with EPg222 and starter cultures showed higher amount of NPN and volatile compounds derived from amino acid catabolism, than control ripened only with starter cultures. Several branched aldehydes, acids and alcohols such as 2- and 3-methylbutanoic acid and 2-methylpropanol were detected only in enzyme treated samples. Sensory analysis reflected higher values for aroma intensity of sausages treated with EPg222 and lower values of hardness than control. The effect of EPg222 may be of great interest to improve sensory characteristics of dry fermented sausages ripened with starter cultures.     

Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.     

Starter cultures and high-pressure processing to improve the hygiene and safety of slightly fermented sausages

The effectiveness of selected starter cultures and high hydrostatic pressure after ripening was evaluated to improve the safety and quality of slightly fermented sausages. Inhibition of common foodborne pathogens, spoilage bacteria, and biogenic amine content was studied. Random amplification of polymorphic DNA and plasmid profiles were used to monitor the competitiveness of the starter cultures during fermentation and ripening. Lactobacillus sakei CTC6626 and Staphylococcus xylosus CTC6013 dominated L. sakei CTC6469 and S. xylosus CTC6169 independently of the product assayed. Starter cultures were able to control the growth of Listeria monocytogenes, Enterobacteriaceae, Enterococcus, and the biogenic amine content. A pH decrease below 5.3 at the seventh day of fermentation was crucial. Salmonella spp. counts decreased significantly during ripening independently of the use of starter culture and product. High hydrostatic pressure treatment was necessary to ensure absence of Salmonella spp. in final products.     

Functional meat starter cultures for improved sausage fermentation

Starter cultures that initiate rapid acidification of the raw meat batter and that lead to a desirable sensory quality of the end-product are used for the production of fermented sausages. Recently, the use of new, functional starter cultures with an industrially or nutritionally important functionality is being explored. Functional starter cultures offer an additional functionality compared to classical starter cultures and represent a way of improving and optimising the sausage fermentation process and achieving tastier, safer, and healthier products. Examples include microorganisms that generate aroma compounds, health-promoting molecules, bacteriocins or other antimicrobials, contribute to cured meat colour, possess probiotic qualities, or lack negative properties such as the production of biogenic amines and toxic compounds. The vast quantity of artisan fermented sausages from different origins represents a treasure chest of biodiversity that can be exploited to create such functional starter cultures.     

Dry sausage fermented by Lactobacillus rhamnosus strains

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.     

合成菌群的构建及其对发酵香肠风味的改良效果

以自然发酵香肠为研究对象,通过分析其细菌群落优势菌属（相对丰度＞1%）、共现菌属（Spearman相关性系数|ρ|＞0.5,P＜0.05）,以及与挥发性风味物质显著相关（P＜0.05）的菌属,确定出调控自然发酵香肠典型风味的核心菌群,据此构建合成菌群,与进口商业发酵剂发酵香肠进行对比,分析合成菌群对发酵香肠感官和风味的改良效果。结果表明：自然发酵香肠的核心菌属为葡萄球菌属（Staphylococcus）、乳杆菌属（Lactobacillus）、假单胞菌属（Pseudomonas）和链球菌属（Streptococcus）,以上述菌属对应的代表性菌种清酒乳杆菌（Lactobacillus sakei）、嗜热链球菌（Streptococcus thermophilus）、马胃葡萄球菌（Staphylococcus equorum）和木糖葡萄球菌（Staphylococcus xylosus）构建的合成菌群制备改良发酵香肠,其感官特性及风味明显优于商业发酵剂制备发酵香肠,具体表现为：前者的清新味和甜香味较为明显,酸味、咸味和金属异味较淡,特别是具有潜在清新味和果香味的醛类和芳香烃类物质含量显著高于后者（P＜0.05）。     

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。     

Effects of starter cultures and nitrite levels on formation of biogenic amines in sucuk

The effects of starter cultures, starter A (Lactobacillus sakei+Staphylococcus carnosus) and starter B (Pediococcus acidilactici+Staphylococcus xylosus+Lactobacillus curvatus), nitrite levels (0, 75, and 150ppm) and ripening period on biogenic amine production were investigated in sucuk (Turkish dry-fermented sausage) production. Changes in biogenic amines, pH, water activity, moisture, non-protein nitrogen, nitrite and counts of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, and moulds-yeasts were determined. Ripening period had a very significant effect (P<0.01) on the all biogenic amines. Sausages produced by fermentation with starter cultures, as compared to natural fermentation (control), had lower amounts of putrescine, cadaverine and tyramine (P<0.05). Starter cultures A and B were found to have almost the same effect on the formation of biogenic amines. Use of nitrite in sucuk production affected the formation of biogenic amines except for spermidine and spermine (P<0.01). Lactic acid bacteria counts in the control group were lower than that of starter culture groups. Lactic acid bacteria, Micrococcus/Staphylococcus, and moulds-yeasts counts increased during ripening period while levels of species of Enterobacteriaceae decreased, which was also found to be under the detectable level (<100cfug(-1)), by day 3 of ripening.     

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.     

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。     

Binding to extracellular matrix proteins and formation of biogenic amines by food-associated coagulase-negative staphylococci

In connection with a study on the DNA microarray based detection of genes involved in safety and technologically relevant properties (Seitter (née Resch) et al., 2011), food-associated coagulase-negative staphylococci (CNS) were investigated phenotypically with regard to their ability to bind to the extracellular matrix proteins (ECM) and to produce biogenic amines. The properties have been shown to be involved in the colonization of injured tissue and invasion into host cells as well as in pharmacologic effects on humans, respectively. The CNS exhibited a low, but nevertheless clearly measurable ECM binding capacity, except for strains of Staphylococcus equorum and Staphylococcus succinus, which show a comparable or even higher binding to fibrinogen and fibronectin than that of the control strain Staphylococcus aureus Cowan. Formation of biogenic amines could be often detected in S. carnosus, S. condimenti and S. strains, but rarely in S. equorum and not in S. succinus and S. xylosus strains. Mostly, 2-phenylethylamine, tyramine and tryptamine were formed by resting cells in amounts < 25 mg/l, whereas growing cells formed high amounts (> 100 mg/l) of 2-phenylethylamine and putrescine. This study confirmed the need of consideration of ECM binding and biogenic amine formation in the safety assessment of CNS used in the production of fermented foods.     

熏马肠成熟过程中产生物胺氧化酶菌对生物胺作用的研究

该研究以从新疆熏马肠中筛选出来的6株产生物胺氧化酶菌株(鼠李糖乳杆菌、枯草芽孢杆、腐生葡萄球菌、木糖葡萄球菌、戊糖片球菌、植物乳杆菌)作为发酵剂加入熏马肠中,采用PCR-DGGE技术监测菌落动态变化,高效液相色谱法检测熏马肠中生物胺(色胺、腐胺、苯乙胺、组胺、酪胺、尸胺、精胺、亚精胺)含量变化。结果表明,在熏马肠成熟过程中发酵剂对生物胺有一定的抑制作用。其中以腐生葡萄球菌对色胺、腐生葡萄球菌对苯乙胺、木糖葡萄球菌对尸胺、鼠李糖乳杆菌对腐胺、腐生葡萄球菌对组胺和枯草芽孢杆菌对酪胺的抑制作用最为显著,减少程度分别为45.49%、57.54%、40.75%、51.03%、49.28%、64.33%。本研究为熏马肠以及其他发酵肉制品的提高安全性奠定了基础。     

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.     

传统中式香肠中产生物胺氧化酶菌的分离鉴定

结合培养基显色法和氧化酶试验,对发酵香肠中产生物胺氧化酶优势菌进行了初步分离。进而利用肉品发酵剂标准剔除不符合要求的菌株。利用高效液相色谱检测符合要求的产生物胺氧化酶菌株对生物胺的氧化减少能力。最后采用16S rDNA分子鉴定方法,通过同源性比对,得到4株符合发酵剂标准的产生物胺氧化酶菌株,即1株表皮葡萄球菌,3株模仿葡萄球菌。     

Application of Autochthonous Mixed Starter for Controlled Kedong Sufu Fermentation in Pilot Plant Tests

Traditional sufu is fermented by back‐slopping and back‐slopping has many defects. The objective of this study was to apply autochthonous mixed starter to control Kedong sufu fermentation. Sufu was manufactured using back‐slopping (batch A) and autochthonous mixed starter (batch B) with Kocuria kristinae F7, Micrococcus luteus KDF1, and Staphylococcus carnosus KDFR1676. Considering physicochemical properties of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B met the standard requirements, respectively. Considering sensory characteristics of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B showed no significant differences (P > 0.05). The maturation period of sufu was shortened by 60 d. Profiles of free amino acids and peptides partly revealed the mechanism of typical sensory quality and shorter ripening time of sufu manufactured by autochthonous mixed starter. In final products, content of total biogenic amines was reduced by 48%. Autochthonous mixed starter performed better than back‐slopping. Fermentation had a positive influence on the quality, safety, and sensory properties of sufu. The application of autochthonous mixed starter does not change the sensory characteristics of traditional fermented sufu. In addition, it reduces maturation period and improves their homogeneity and safety. It is possible to substitute autochthonous mixed starter for back‐slopping in the manufacture of sufu.     

Role of an autochthonous starter culture and the protease EPg222 on the sensory and safety properties of a traditional Iberian dry-fermented sausage "salchichón"

The effect of the addition of an autochthonous starter culture and the protease EPg222 on the physico-chemical and sensory characteristics of dry-fermented sausage ‘‘salchichon” was investigated. Sausages were prepared with purified EPg222 and Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34 as starter culture (P200S34), separately and together, ripened for 90 days, and compared with a control batch. Dry-fermented sausages ripened with EPg222 and starter culture showed higher amounts of AN and volatile compounds derived from amino acid catabolism than the control, especially in samples in which was added the association of enzyme and starter culture (P200S34 + EPg222). There were clear differences shown by the texture analysis, with the P200S34 + EPg222 batch being less hard. Especially important was the result found in biogenic amines, since the association P200S34 + EPg222 reduced their accumulation compared to the EPg222 batch. The use of EPg222 may be of great interest to improve the sensory characteristics of dry-fermented sausages, but its association with the selected starter culture with low decarboxylase activity is necessary to guarantee healthiness and homogeneity.     

Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages

The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.