Factors influencing leucine catabolism by a strain of Staphylococcus carnosus.

The metabolism of leucine by resting cells of Staphylococcus carnosus 833 was studied according to three physicochemical factors: preculture condition (defined medium; complex medium), nitrate concentration (0% and 0.03%) and stirring condition (static or shaking). A factorial design was set up to test the effects of these factors, each at two levels. The results showed that resting cells of S. carnosus 833 produced 3-methyl butanal, 3-methyl butanol and 3-methyl butanoic acid from leucine. Whatever the incubation conditions, there was greater quantity of 3-methyl butanoic acid than 3-methyl butanal and 3-methyl butanol. The preculture and incubation conditions influenced the level of production of the 3 metabolites. The highest overall production of the 3 metabolites was observed when cells were incubated without nitrate in the reaction mixture. 3-methylbutanoic acid production was enhanced when S. carnosus 833 was precultivated in complex medium. 3-methylbutanal was only detected when cells were precultivated in defined medium. Stirring condition had no effect on leucine catabolism of S. carnosus 833.     

Relationship between fat depots in carcasses of beef bulls and effect on meat colour and tenderness

The growth and the effects of four species of staphylococci and six lactic acid bacteria (LAB) of the genus Carnobacterium, Lactobacillus and Pediococcus on unsaturated free fatty acids were studied. The strains were grown in complex medium supplemented either with oleic, linoleic or linolenic acids. Growth was followed and oxidation of the substrates measured by TBARS. The strains of Staphylococcus xylosus 873, 16, Staphylococcus warneri 863 and Staphylococcus saprophyticus grew well on all the substrates. Whereas, the growth of the two strains of Staphylococcus carnosus and Staphylococcus xylosus 831 was inhibited in the media with linolenic acid. The addition of manganese to this media allowed good growth of these strains. All the LAB did not grow well in the media with linoleic acid, but their growth was favoured by addition of manganese to the media. Under our conditions, only linoleic and linolenic acids were oxidised. All the strains had no prooxidant activity. All the staphylococci limited oxidation of linoleic acid and had a small effect on linolenic acid. LAB did not limit oxidation of linoleic acid. With manganese in the media: the oxidation of the sterile controls was delayed for 2 days and then increased; strains of S. carnosus and S. xylosus inhibited oxidation of linolenic acid; and Lactobacillus plantarum and Pediococcus pentosaceus limited oxidation of linoleic acid. The two Carnobacterium, whatever the conditions, had no antioxidant properties.     

Effects of starter cultures on the biochemical characteristics of French dry sausages

Bacterial starter cultures, consisting of lactic acid bacteria (Lactobacillus, Pediococcus) and staphylococci (Staphlylococcus carnosus. S. saprophyticus, S. warneri), have an important effect on the pH, lactate, acetate and free fatty acid contents of sausages. Sausages made with L. sake had the lowest pH whereas no change of pH was noticed in the controls and in the sausages inoculated with P. acidilactici. Inoculation of S. saprophyticus led to sausages with a high acetate content. Lipolysis occurred not only in inoculated samples but also in the controls, but it was the highest in the sausages inoculated with S. warneri.     

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。     

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.     

Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

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.     

RELEASE OF ENTEROTOXIN A AND THERMONUCLEASE FROM GROWING AND NON-GROWING CELLS OF STAPHYLOCOCCUS AUREUS

Enterotoxin A and thermonuclease were produced by Staphylococcus aureus cell populations in all growth phases. Synthesis of entero-toxin was more rapid than that of thermonuclease early in the exponential phase, whereas later in the exponential phase the thermonuclease synthesis was more rapid than that of enterotoxin. The optimum temperature for enterotoxin production was 42°C, while that for thermonuclease production was in the range 27–33°C. Small amounts of enterotoxin and thermonuclease were released at temperatures up to 45°C.
During heat treatment of staphylococcal cells, enterotoxin production was not observed at temperatures in excess of those permitting growth, although small amounts of thermonuclease were produced. Intracellular enterotoxin was not detected after disintegration of cells under pressure at –30°C. Enterotoxin at detectable concentration, was not bound to the cell membranes. All the results obtained indicate that the synthesis of enterotoxin by S. aureus is intimately associated with cell growth.     

DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci

Aim of the work was to design a polynucleotide based DNA microarray as screening tool to detect genes in food associated coagulase-negative staphylococci (CNS). A focus was laid on genes with potential health concern and technological relevance. The microarray contained 220 probes for genes encoding antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (e.g. biogenic amine formation), binding proteins to extracellular matrix (ECM), lipases, proteases, stress response factors, or nitrate dissimilation. Hybridization of genomic DNA isolated from 32 phenotypically characterized CNS permitted to detect numerous genes, corresponding with the phenotype. However, numerous hybridization signals were obtained for genes without any detectable phenotype. The antibiotic resistance genes blaZ, lnuA, and tetK involved in ?-lactam, lincomycin and tetracycline resistance, respectively, were rarely identified in CNS, however, all species contained some strains with such resistance genes. Decarboxylase genes involved in biogenic amine formation were detected regularly in Staphylococcus carnosus, S. condimenti, S. piscifermentans and S. equorum, but was rarely correlated with the phenotype. The same applied for the fibrinogen (clf) and fibronectin (fbp) binding protein genes, whose phenotype (binding assay) was only correlated in S. equorum and Staphylococcus succinus. Although some CNS showed hemolytic activity and enterotoxin production (Immunoblot) the corresponding genes could not be verified. Technological relevant genes such as proteases or lipases revealed good hybridization signals. In addition, genes involved in nitrate dissimilation (nre, nar, nir), catalase (kat), or superoxide dismutase (sod) were well detected. Interestingly, genes involved in dissimilatory nitrate reduction were more prevalent in strains of S. carnosus, S. condimenti and S. piscifermentans than of S. equorum, S. succinus and S. xylosus.     

Molecular, technological and safety characterization of Gram-positive catalase-positive cocci from slightly fermented sausages

The population of Gram-positive catalase-positive cocci from slightly fermented sausages was characterized at species and strain level by molecular techniques and some technological and hygienic aspects were also considered. Staphylococcus xylosus was the predominant species (80.8%) followed by Staphylococcus warneri (8.3%), Staphylococcus epidermidis (5.8%) Staphylococcus carnosus (4.6%), and Kocuria varians (0.4%). Proteolytic activity was observed in 23% of the isolates. The species with the highest percentage of proteolytic strains was S. warneri. Lipolytic activity was found in 45.8% of the isolates and S. xylosus was the species with the highest percentage of lipolytic isolates. Biogenic amine production was not widely distributed (only 14.6% of the isolates). Tyramine was the most intense amine produced, although by only 4.6% of the isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. Some strains also produced putrescine (3.3%), cadaverine (2.9%), histamine (1.3%) and tryptamine (0.4%). All isolates were susceptible to linezolid and vancomicin and over 70% were resistant to penicillin G, ampicillin and sulphonamides. Most of the mecA+ strains (only 4.6% of isolates) also displayed resistance to multiple antibiotics. A reduced enterotoxigenic potential was found. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene. The combination of RAPD-PCR and plasmid profiling allowed the discrimination of 208 different profiles among the 240 Gram-positive catalase-positive cocci characterized, indicating a great genetic variability.     

溶氧对巨大芽孢杆菌发酵亚硝酸还原酶的影响

通过振荡间歇静置培养,研究不同静置时间对巨大芽孢杆菌发酵生产亚硝酸还原酶(nitrite reductase,NiR)的影响,并在适宜间歇静置条件下,分析细胞生长、基质消耗与NiR酶量的关系.结果表明,在发酵前12h每2h振荡培养间歇60min静置培养的条件下,20h发酵时所得亚硝酸盐还原酶的酶活最高:在每2h振荡培养间歇60min静置培养的条件下,周期为20h的该菌的发酵特性研究表明,菌体的细胞生长量及发酵液中的亚硝酸盐消耗量和葡萄糖消耗量均同步低于一直振荡培养的对照组,但发酵20h后所得亚硝酸还原酶的酶活为98.7U/mL,比一直振荡培养的对照组的酶活高出103.4%.     

传统湖南腊肉中产香葡萄球菌的筛选及鉴定

从传统湖南腊肉中共分离到289株葡萄球菌,按照肉用发酵剂筛选的基本原则进行初筛,再以产3-甲基丁醛的能力为依据,测定初筛获得的菌株产香能力,然后以产硝酸还原酶、过氧化氢酶和抑制脂肪酸氧化的能力为指标进行复筛,得到1株安全的,产香性能优良,适用于肉制品发酵的葡萄球菌S52。生化鉴定和16S rRNA鉴定的结果表明,S52为模仿葡萄球菌。     

Characterization and technological properties of Staphylococcus xylosus strains isolated from a Tunisian traditional salted meat

The technological properties of strains of Staphylococcus xylosus were studied to select the most suitable for use as starter cultures for the production of dried fermented meat products. Strains of S. xylosus were isolated from traditional salted Tunisian meat and were identified by biochemical and molecular methods. Thirty strains of S. xylosus were studied to evaluate their catalase, nitrate reductase, lipolytic, proteolytic and antibacterial activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All strains of S. xylosus had catalase activity and were able to reduce nitrates to nitrites. The nitrate reductase activity increased when the strains were kept under anaerobic conditions. Proteolytic activity on milk and on gelatin agar was demonstrated for 100% and 83.3% of the S. xylosus isolates, respectively. However extracellular proteolytic activity as assessed by the azocasein method was poor in all the strains. Lipolytic activity as assessed by the agar method showed that 76.6% of strains of S. xylosus could hydrolyze Tween 20 against 33.3% that could hydrolyze tributyrin. Tween 80 was hydrolyzed by only 10% of strains. Strains of S. xylosus hydrolyzed pork fat better than beef and lamb fat. The majority of strains had antibacterial activity against Salmonella arizonae, Staphylococcus aureus, Pseudomonas aeuroginosa, Escherichia coli and Enterococcus faecalis.     

Growth and aroma production by Staphylococcus xylosus, S. carnosus and S. equorum--a comparative study in model systems

A laboratory medium inoculated with 20 different Staphylococcus strains was prepared in accordance with a full factorial experimental design investigating the effect of temperature, pH, NaCl and glucose on growth. The 12 strains most suited to growth in a fermented meat environment were inoculated in sausage minces together with Pediococcus pentosaceus, incubated at 25 degrees C for 1 week and the produced aroma compounds collected. The data were analysed by multiple linear regression and partial least squares regression analysis. The results showed that increasing pH and temperature from 4.6 to 6.0 and 10 to 26 degrees C, respectively, increased growth of all strains with strong synergy between temperature and pH. Increasing salt concentration from 5% to 15% w/v decreased growth of most strains, but the effect of pH and temperature was much stronger than the effect of salt. Strains of S. carnosus were more salt tolerant than strains of S. equorum and S. xylosus, especially at high pH and temperature. Addition of glucose up to 0.5% w/v had no significant influence on growth of any of the strains. With regard to aroma production, species characteristics were detected. S. carnosus and S. xylosus were quite different regarding the overall aroma profiles, whereas the profiles of S. equorum lied somewhere in-between. Contrary to S. carnosus, S. xylosus and S. equorum did not produce 2-methyl-1-butanol. On the other hand, in particular, S. xylosus produced more 3-methyl-1-butanol. Except for one of the strains of S. equorum, S. xylosus and S. equorum formed more diacetyl, 2-butanone and acetoin and also more of the methyl-branched ketones arising from degradation of leucine, isoleucine and valine. S. carnosus produced more methyl-branched aldehydes, acids and corresponding esters from leucine, isoleucine and valine compounds that have been correlated with fermented sausage maturity in former studies. S. equorum produced the least of the methyl-branched aldehydes.     

The nitrate reductase activity of human saliva]

Investigations of human saliva reductase activity report the enzyme inactivation in 5-min heating at 50 degrees C, its maximal activity in neutral medium, reduced specific activity in the saliva dilution. Routine centrifugation inactivated nitrate reductase in supernatant, its activity in the precipitate hardly reached 10%. Combination of supernatant with precipitate recovered the baseline activity completely. Nitrates reduction into nitrites requires donor electrons. The latter are suggested to be present in the supernatant. Dehydrogenases substrates (lactate, pyruvate, malate, etc.) may act as donor electrons for nitrate reductase reaction. The addition of lithium lactate instead of supernatant to the precipitate partially recovered nitrate reductase activity. Biological role of salivary nitrate reductase is considered.     

不同发酵肉中优良葡萄球菌的筛选鉴定

为获得适用于发酵香肠的优势葡萄球菌,本研究从国内外6种具有代表性的不同来源的发酵肉样品中分离到120株葡萄球菌.按照肉用发酵剂的基本原则进行初筛后共得到10株性能优良菌株.对这些菌株的蛋白酶、脂肪酶、硝酸还原酶和过氧化氢酶活性以及其在高盐和亚硝酸盐环境中的生长情况进行测定.发现菌株RG18和SL4有较高的蛋白酶、脂肪酶...     

Identification and characterization of potential autochthonous starter cultures from a Croatian "brand" product "Slavonski kulen"

The microbial population of a traditional Croatian fermented sausage "Slavonski kulen" was isolated, identified and subjected to technological and functional characterization in order to select potential autochthonous functional starter cultures. Dominant microflora were lactic acid bacteria (LAB), followed by staphylococci. Identification of isolated lactobacilli showed domination of Leuconostoc mesenteroides and Lactobacillus acidophilus while Staphylococcus xylosus and Staphylococcus warneri outnumbered the staphylococcal microbiota. Most of the isolated LAB and Staphylococcus species displayed good growth in the presence of 5% of NaCl and at 12, 18 and 22°C. All LAB and most of the staphylococci possess proteolytic activity and only Staphylococcus xylosus had lipolytic activity. All lactobacilli and staphylococci isolates produced significant concentrations of lactic acid (as determined by HPLC) and showed antimicrobial activity against pathogenic test microorganisms. Dominant LAB and Staphylococcus species displayed growth in the presence of 1% bile. Most of the staphylococci and all of lactobacilli showed sensitivity to all antibiotics tested.     

肉品发酵剂葡萄球菌和微球菌的筛选

应用一种快速半定量平板方法初步筛选可作为肉品发酵剂的葡萄球菌和微球菌。从中式传统肉制品中分离、纯化了 1 0 0多株葡萄球菌和微球菌 ,将菌株点种在加有KNO3的琼脂平板 ,据菌落周围显示的红圈大小挑选出硝酸盐还原酶活性较高的菌株 ,同时具有一定蛋白酶和脂肪酶活性的菌株进行发酵适应性试验。筛选出 1株过氧化氢酶阳性球菌 ,经初步鉴定为肉糖葡萄球菌S1 5。通过在模拟肉汤培养基中和乳酸菌混合培养确定了一种复配方案P2 0 +S1 5。将混合发酵剂用于发酵香肠的制作 ,测定微生物和理化指标 ,并用仪器测定标准化部分感官指标     

Effects of age and variety on nitrate reductase and nitrogen fractions in potato plants

The effects of plant age and variety on the level of nitrate reductase activity in three varieties of Solanum tuberosum (Kennebec, Norchip and Early Ohio) have been studied by assaying nitrate reductase at different intervals during growth and development. Nitrate reductase appears to be localised in leaves and young stems. Negligible nitrate reductase activity was recorded in roots and tubers. The varieties differed significantly in nitrate reductase activity and the tuber protein nitrogen content paralleled the level of activity in leaves. The levels of nitrate reductase activities in leaves were also highly positively correlated with quantities of leaf inorganic nitrogen, protein and free amino acids. The activity began to decline at about 50 days after emergence, and this may be a useful indicator of when to apply nitrogen fertiliser so as to increase tuber protein content.     

Staphylococcal community of a small unit manufacturing traditional dry fermented sausages

The level and the diversity of the staphylococcal community occurring in the environment and meat products of a small unit manufacturing traditional dry fermented sausages were investigated at two seasons: winter and spring. Gram-positive cocci were enumerated and a collection of 412 Staphylococcus isolates was made. Multiplex PCR, pulse-field gel electrophoresis (PFGE) and sequencing of the sodA gene were used to identify and characterize the isolates. High counts of Staphylococcus were found in final traditional sausages, reaching about 6 log CFU/g in winter and about 8 log CFU/g in spring. In the environment, the counts varied from 2 log to 7 log/100 cm(2), the higher colonisation being observed on the surface of the drying and cold rooms, cutting tables and the butcher's block. The combination of the three methods allowed the identification of seven species of Staphylococcus in spring and five in winter. S. equorum and S. succinus dominated both in environment and in meat products, 49% and 33% of the isolates, respectively. The other identified species were in decreasing order S. saprophyticus (6%), S. xylosus (5%), S. carnosus (5%), S. simulans (1%) and S. warneri (1%). The two species S. xylosus and S. carnosus were sporadically isolated during the spring. PFGE allowed the assignment of S. equorum to eight pulsotypes showing a wide diversity among this species. But the entire environment and the meat products were dominated by one pulsotype. For S. succinus, three pulsotypes were found with one dominant mainly isolated during the spring sampling. This study highlighted the diversity of staphylococci isolated in the environment and the meat products of a small processing unit manufacturing traditional dry fermented sausages. The S. equorum and S. succinus species rarely described in meat products and never in the environment had great capacity to colonise the entire small processing unit and the meat products.