A fermented meat model system for studies of microbial aroma formation

A fermented meat model system was developed, by which microbial formation of volatiles could be examined. The model was evaluated against dry, fermented sausages with respect to microbial growth, pH and volatile profiles. Fast and slowly acidified sausages and models were produced using the starter cultures Pediococcus pentosaceus and Staphylococcus xylosus. Volatiles were collected and analysed by dynamic headspace sampling and GC-MS. The analysis was primarily focused on volatiles arising from amino acid degradation and a total of 24 compounds, of which 19 were quantified, were used for multivariate data analysis. Growth of lactic acid bacteria was comparable for model and sausages, whereas survival of S. xylosus was better in the model. Multivariate analysis of volatiles showed that differences between fast and slowly acidified samples were identical for model and sausage. For both sausage and model, fast-acidified samples had a high content of ketones, sulphides and methyl-branched acids, whereas slowly acidified samples had the highest content of methyl-branched alcohols, aldehydes, their ethyl esters, phenylacetaldehyde and methional. Furthermore, model repeatability with respect to pH, microbial growth and volatile profiles was similar to sausage production. Based on these findings, the model system was considered valid for studies of aroma formation of meat cultures for fermented sausage.     

Growth and production of volatiles by Staphylococcus carnosus in dry sausages: Influence of inoculation level and ripening time

Three sausage batches inoculated with normal inoculation level of Pediococcus pentosaceus (5×10(6) CFU/g) and with low, intermediate, and high inoculation levels of Staphylococcus carnosus (10(5), 5×10(6), 5×10(7) CFU/g, respectively) were produced. Cell counts and formation of volatiles were followed throughout a ripening period of three weeks. The staphylococci exhibited the fastest growth in sausages with a low inoculation level, whereas growth was only moderate in sausages with a high initial level. Analysis of volatiles showed that methyl-branched aldehydes and acids, phenylacetaldehyde, 2-methyl-1-butanol, dimethyldisulphide and dimethyltrisulphide were produced in higher amounts in sausages with a high inoculation level of S. carnosus, whereas a low inoculation level correlated with high amounts of diacetyl, ethanol and ethyl esters. The levels of most compounds increased over time, but the amount of diacetyl was negatively correlated to ripening time. A negative interaction effect between inoculation level and ripening time was observed for the amounts of methyl-branched aldehydes.     

Effect of ascorbate, nitrate and nitrite on the amount of flavour compounds produced from leucine by Staphylococcus xylosus and Staphylococcus carnosus

Resting cells of Staphylococcus xylosus and S. carnosus were incubated with ascorbate, nitrate and nitrite in defined reaction medium and their degradation of (3)H-labelled leucine into methyl-branched catabolites were studied using HPLC/radiometric detection. The experiments were carried out with and without addition of α-ketoglutarate. The main catabolic product of leucine degradation was 3-methylbutanoic acid but also small amounts of α-hydroxy isocaproic acid were produced. Nitrite addition lowered the concentration of 3-methylbutanoic acid for both Staphylococcus species and this effect was strongly amplified by ascorbate for S. xylosus but not for S. carnosus. For both species ascorbate alone had little if any effect. Also nitrate lowered the concentration of 3-methylbutanoic acid for S. xylosus. The concentration of α-hydroxy isocaproic acid was, however, increased by addition of nitrite and nitrate for S. xylosus. Addition of α-ketoglutarate generally increased the concentration of 3-methylbutanoic acid for both S. xylosus and S. carnosus.     

Antibiotic resistance of coagulase-negative staphylococci associated with food and used in starter cultures

The resistance of 330 coagulase-negative staphylococci (CNS) associated with food or used in starter cultures and belonging to the species Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus piscifermentans, Staphylococcus equorum, Staphylococcus succinus and Staphylococcus xylosus, against 21 antibiotics was determined using the disk diffusion method. The incidence and number of resistances was found to be species and source of isolation dependent. Most strains of S. equorum (63%), S. succinus (90%) and S. xylosus (95%) exhibited resistances against up to seven antibiotics, whereas only few strains of S. carnosus (12%) and S. piscifermentans (27%) were antibiotic resistant. Resistances to lincomycin, penicillin, fusidic acid, oxacillin, ampicillin and tetracycline were predominant. Among strains of S. xylosus, the incidence of resistance ranged from 22% for tetracycline up to 69% for penicillin. Concerning the source of isolation, resistances were often determined in strains of S. equorum, S. succinus and S. xylosus isolated from cheese (87%) and sausage (83%), and strains of S. xylosus obtained from meat starter cultures (93%). Remarkably, all CNS were sensitive to the clinically important antibiotics chloramphenicol, clindamycin, cotrimoxazol, gentamicin, kanamycin, linezolid, neomycin, streptomycin, synercid and vancomycin. The phenotypic resistances to ss-lactam antibiotics, lincomycin and tetracycline were verified by PCR amplification and could be traced back to the genes blaZ, lnuA and tetK, respectively. This study permitted a comprehensive insight into the incidence of antibiotic resistances in food-associated CNS.     

应用多重PCR快速鉴定自然发酵肉制品中6 种葡萄球菌

肉葡萄球菌（Staphylococcus carnosus）、木糖葡萄球菌（S. xylosus）、腐生葡萄球菌（S. saprophyticus）、表皮葡萄球菌（S. epidermidis）、马胃葡萄球菌（S. equorum）和松鼠葡萄球菌（S. sciuri）是发酵肉制品中常见的葡萄球菌,为准确、快速地检测和鉴定这些菌种,建立6?种葡萄球菌的多重聚合酶链式反应（polymerase chain reaction,PCR）方法并进行验证。以上述菌种的gap、rpoB、SodA和SNc为靶基因,设计和筛选菌种特异性引物6?对,优化PCR体系,建立6?种葡萄球菌的多重PCR鉴定方法。实验结果显示多重PCR的基因组DNA检测灵敏度可达到4?pg,活菌检测灵敏度可达到2×102?CFU;采用建立的多重PCR方法对传统肉制品中分离的8?株葡萄球菌进行鉴定,结果与16S rDNA序列分析、生理生化鉴定结果一致。建立的多重PCR方法具有较高的种间特异性,可快速、准确地用于发酵肉制品中肉葡萄球菌、木糖葡萄球菌、表皮葡萄球菌、马胃葡萄球菌、松鼠葡萄球菌和腐生葡萄球菌的检测和鉴定,具有较好的应用前景。     

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）。     

Maturity Acceleration of Italian Dried Sausage by Staphylococcus carnosus—Relationship Between Maturity and Flavor Compounds

ABSTRACT: The mature flavor of Salame Milano, an Italian dried sausage, was increased in two ways: by increasing maturation time or with a strain of Staphylococcus carnosus. The sensory and volatile profiles of the sausages were determined and the data analyzed by analysis of variance and chemometrics. Sausages with S. carnosus 833 matured more than 2 wk faster than control sausages. Maturity correlated significantly with higher amounts of branched-chain aldehydes and alcohols and both branched- and straight-chain methyl ketones—compounds arising from the breakdown of the amino acids leucine, isoleucine, and valine, or from microbial β-oxidation of fatty acids. Also, sulfur compounds arising from added garlic correlated positively with mature flavor.     

Addition of α-ketoglutarate enhances formation of volatiles by Staphylococcus carnosus during sausage fermentation

The effect of leucine and α-ketoglutarate addition on transamination of branched-chain amino acids was studied in model minces inoculated with Pediococcus pentosaceus and Staphylococcus carnosus. Leucine addition changed the ratio of volatile breakdown products of leucine, isoleucine and valine but not the total amount of the volatiles and it was concluded that the amount of free amino acids does not limit transamination of amino acids. The addition of α-ketoglutarate resulted in increased levels of methyl-branched aldehydes and insignificant positive changes in methyl-branched acid production. The results were verified in real fermented sausages with no, low (0.09% w/w) and high (0.36% w/w) addition of added α-ketoglutarate since the levels of the flavour intensive methyl-branched aldehydes and acids were drastically increased in sausages added α-ketoglutarate. The catabolism of phenylalanine was also induced by α-ketoglutarate and there were further indications of increased transamination of aspartate. A triangular test showed that the flavour of sausages with no and low addition of α-ketoglutarate could be clearly distinguished from one another. Altogether the results presented in this paper point to glutamate dehydrogenase, the enzyme catalyzing regeneration of α-ketoglutarate, as a key enzyme in catabolism of amino acids and thereby also in aroma formation during sausage processing.     

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。     

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.     

木糖葡萄球菌和肉葡萄球菌混合发酵剂对腊肉品质的影响

以不添加发酵剂的腊肉为空白对照,从理化特性、微生物分析、感官评价、香气活性化合物组成及电子鼻分析角度,研究木糖葡萄球菌和肉葡萄球菌混合发酵剂对腊肉品质的影响。结果表明,添加葡萄球菌混合发酵剂对产品的品质影响较大。与空白组比,葡萄球菌混合发酵剂可显著提高产品的酸价（2.12→3.28?mg/g）、蛋白质降解指数（16.32%→19.24%）、红度值a*（9.25→11.08）、香气和接受度得分及氨基酸代谢源香气活性化合物的种类和含量（3-甲基丁醛、3-甲基丁酸）,同时显著降低产品的过氧化值（0.15→0.07?g/100?g）、硫代巴比妥酸反应物含量（0.25→0.17?mg/kg）和脂肪氧化源香气活性化合物的含量（己醛和壬醛）,而对成品的基本理化指标（水分含量、水分活度、pH值）没有显著影响。电子鼻传感器能够将2?组产品区分开,说明加入发酵剂后,腊肉的风味有所改变。因此,通过添加木糖葡萄球菌和肉葡萄球菌混合发酵剂可以显著促进腊肉中蛋白质和脂肪的水解、改善产品的色泽,促进发酵风味快速形成,同时延缓脂肪氧化。     

木糖葡萄球菌和肉糖葡萄球菌的生理特性及其转化硝酸盐影响因素的研究

对普遍存在于发酵肉制品中具有硝酸还原酶活性的2种外源性木糖葡萄球菌和肉糖葡萄球菌的生理生化特性进行了鉴定,并研究了不同温度、pH、盐含量对两菌株及不同混合配比菌株生成亚硝酸盐量的影响。结果表明:两菌株均具有较强的耐盐性、耐亚硝性、耐硝酸盐性,且在30、40℃,pH6.5,2%NaCl时生成亚硝酸盐速率最快。生理生化等鉴定实验表明,木糖葡萄球菌和肉糖葡萄球菌可为发酵肉制品生产中的优良菌株选择和应用提供理论参考。     

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.     

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.     

Protease and esterase activity of staphylococci

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing L-methionine, L-leucine and L-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were rho-nitrophenyl (rho-NP) butyrate and rho-NP caprylate and, secondly, rho-NP palmitate. The extracellular extracts of most of the strains were only active against rho-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed rho-NP caprylate and rho-NP palmitate. The cell-free extracts and whole cells were mainly active against rho-NP butyrate and rho-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.     

Molecular and technological characterization of Staphylococcus xylosus isolated from naturally fermented Italian sausages by RAPD, Rep-PCR and Sau-PCR analysis

Coagulase-negative cocci (CNC) are important microorganisms in fermented sausages because they release lipases and proteases that are able to free short-chain fatty acids and peptides and aminoacids, respectively, that are responsible for the aroma of fermented sausage. The purpose of this study was to characterize Staphylococcus xylosus strains isolated from naturally fermented sausages, produced in three different processing plants in the Friuli Venezia Giulia region in the Northeast of Italy. Two hundred and forty-nine strains of S. xylosus were identified by species-specific PCR and subjected to molecular and technological characterization. RAPD-PCR with primer M13, Rep-PCR using primer (GTG)(5) and Sau-PCR with primer SAG(1) were used for the molecular analysis, while the capability of the strains to grow at different temperatures and in the presence of NaCl and their lipolytic and proteolytic activity were tested in order to define the technological characteristics. The results obtained allowed us to differentiate strains coming from different plants, thereby admitting the presence of strains that are plant-specific.     

The influence of Debaryomyces hansenii and Candida utilis on the aroma formation in garlic spiced fermented sausages and model minces

The influence of the yeast starter cultures Debaryomyces hansenii and Candida utilis on fermented meat aroma was studied in model minces and in commercial-type fermented sausages. Volatile compounds from model minces and sausages were collected using diffusive and dynamic headspace sampling respectively and were identified by gas chromatography/mass spectrometry (GC/MS). A triangle test was carried out on the sausages to detect whether the yeast influenced the sausage odour. C. utilis demonstrated high metabolic activity in the model minces, producing several volatile compounds, in particularly esters. C. utilis also seemed to ferment the amino acids valine, isoleucine and leucine into compounds important for the aroma of sausages. D. hansenii on the contrary, had very little effect on the production of volatile compounds in the model minces. In the sausage experiment both yeast cultures died out before the ripening process ended and the sensory analysis showed only a slight difference between the sausages. A fungistatic test of the garlic powder added to the sausages indicated that garlic inhibits the growth of the yeast starter cultures.     

Aroma components from dried sausages fermented with Staphylococcus xylosus

Sausages with and without Staphylococcus xylosus were manufactured with four replicates. Antibiotics and a fungicide to inhibit growth of naturally occuring microorganisms were added to the control sausages. The volatile compounds from the sausages were collected and identified by gas chromatography/mass spectrometry, the effluent evaluated by sniffing and the sausages assessed by a trained panel. The study showed that sausages fermented with Staphylococcus xylosus contained several fragrant esters that were not found in control sausages without microbial growth. Control sausages had an unpleasant, rancid odour compared to sausages with Staphylococcus xylosus, indicating that the esterase activity of Staphylococcus xylosus or other microorganisms is very important in order to obtain the proper fermented sausage aroma. Although sausages with Staphylococcus xylosus contained the highest amounts of free fatty acids, it seemed to be of no importance to the aroma development. It is therefore questionable whether lipolytic activity of starter cultures has any influence on sausage flavour.     

Dried sausages fermented with staphylococcus xylosus at different temperatures and with different ingredient levels. Part IV. Amino acid profile

Sausages with added Staphylococcus xylosus were fermented at different temperatures and with different added levels of salt, glucose, nitrite, nitrate and Pediococcus pentosaceus in accordance with a six-factor fractional design. The amounts of individual amino acids were measured and the effects of temperature and different ingredients on the amino acid profile were tested using multiple linear regression and analysis of variance. Also, the amino acid profile was correlated to the level of volatile compounds by partial least squares analysis. The study showed that the level of free amino acids was significantly affected by the different factors. High fermentation temperature and nitrite content increased the amount, while high contents of salt, nitrate, glucose and P. pentosaceus lowered the amount of free amino acids. In general, temperature and nitrate had the greatest influence. It was shown that the amounts of the volatile compounds, 2-methyl propanal, 2- and 3-methyl butanal, were inversely correlated with the amounts of valine, isoleucine and leucine, respectively, indicating that those volatiles were degradation products of the latter.     

Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci

The objective of this work was to study the production of catalase and nitrate reductase by staphylococci in order to understand their role in lipid oxidation during sausage manufacturing. Catalase and nitrate reductase were measured in resting cells and supernatants of staphylococci grown in different conditions. All staphylococci (except S. warneri) synthetized nitrate reductase. In static condition, the synthesis was maximal during exponential growth phase, whereas in shaking condition, the synthesis was maximal at the beginning of stationary phase. The production of nitrate reductase was increased in presence of nitrate, this effect was particularly important for the two S. carnosus strains which exhibited the highest activity. For all staphylococci, the production of catalase was maximal at the end of the exponential growth phase. The lowest amount of catalase was produced by S. warneri and the highest by S. carnosus. Only S. xylosus 873 and S. saprophyticus 852 released high amounts of catalase in the supernatant growth. Staphylococci produced higher amounts of catalase in shaking conditions. Addition of nitrate in the growth media favoured the synthesis of catalase, with a pronounced effect for S. carnosus. Nitrate also favoured the release of catalase.