Biogenic amines produced by Enterobacteriaceae isolated from meat products

Biogenic amines in ground meat and processed meat products are one of the indicators to determine the poor quality raw material. Major histamine forming bacterium was Escherichia coli (strain EC04 with 65.88 mg/100 ml in Brain Heart Infusion medium), followed by the microorganisms Morganella morganii (strain MM4 with 8.40 mg/100 ml and strain MM7 with 8.28 mg/100 ml) and Proteus mirabilis (strain PM02 with 8.76 mg/100 ml), respectively. The highest putrescine production level was found in Citrobacter freundii and Enterobacter spp. strains, followed by Serratia grimesii, Proteus alcalifaciens, E. coli, Escherichia fergusonii, Morganella morganii, Proteus mirabilis, Proteus penneri and Hafnia alvei, respectively. The most important cadaverine producer was E. coli EC03 with a production level of 45.48 mg/100 ml, and of the strains Escherichia vulnaris EV01, Escherichia fergusonii EF06 produced 37.92 mg/100ml and 35.40 mg/100 ml, respectively. On the basis of our results, it can be concluded that the major biogenic amines produced by Enterobacteriaceae are putrescine, cadaverine, tyramine and histamine, both in culture medium and meat products.     

2-羟丙基-β-环糊精/阿魏酸包合物对熏马肠中阴沟肠杆菌产酪胺的影响机制

制备2-羟丙基-β-环糊精（2-hydroxypropyl-β-cyclodextrin,HP-β-CD）/阿魏酸（ferulic acid,FA）包合物（HP-β-CD/FA）,探究其对阴沟肠杆菌（Enterobacter cloaca）L63的pH值、生长量、酪氨酸脱羧酶相关基因表达和酪胺积累的影响,以及对熏马肠中酪胺积累的影响。采用实时荧光定量聚合酶链式反应检测酪氨酸脱羧酶（tyrosine decarboxylase,tdcA）、酪氨酸/酪胺逆向转运体（tyrosine/tyramine antiporter,tyrP）、酪氨酸t-RNA合成酶和Na＋/H＋逆向转运体基因在酪胺产生途径中的表达水平。此外,采用高效液相色谱法检测酪胺在48 h内的累积水平。结果表明：FA和HP-β-CD/FA包合物在纯菌体系下均能够降低tdcA和tyrP基因的表达（P＜0.05）,对阴沟肠杆菌L63有较强的抑菌活性,在纯菌体系和熏马肠中均能够抑制pH值升高,同时有效减少酪胺积累,FA包埋后抑制酪胺积累效果更显著（P＜0.05）。     

Comparison of newly isolated strains of Lactobacillus delbrueckii subsp. lactis for hydrogen peroxide production at 5 degrees C

Isolates of Lactobacillus delbrueckii subsp. lactis obtained from raw milk samples were compared for the ability to produce hydrogen peroxide (H2O2) at 5 degrees C. Nineteen out of 101 lactobacilli isolated were identified as L. delbrueckii subsp. lactis. The isolates of L. delbrueckii subsp. lactis from most raw milk samples produced more H2O2 than did isolates of other species of lactobacilli from the same samples. Seven isolates of L. delbrueckii subsp. lactis, which produced the highest levels of H2O2 at 5 degrees C were selected for comparison with a laboratory strain, L. delbrueckii subsp. lactis I. In 24 h, isolate RM2-5 produced 7.0 microg/10(9) cfu in buffer containing 5 mM sodium lactate and 4.4 microg/10(9) cfu in buffer containing 5 mM glucose. Three other isolates also produced more H2O2 on sodium lactate than on glucose. However, three remaining new isolates produced more H2O2 on glucose than on sodium lactate. All seven of the most active new isolates of L. delbrueckii subsp. lactis produced significantly higher concentrations of H2O2 than did L. delbrueckii subsp. lactis I in both solutions. Strain RM2-5 produced more H2O2 than did the other six most active newly isolated strains of L. delbrueckii subsp. lactis in this comparison.     

Growth of Carnobacterium divergens V41 and production of biogenic amines and divercin V41 in sterile cold-smoked salmon extract at varying temperatures, NaCl levels, and glucose concentrations

A complete factorial design 2(3) was used to study some aspects of Carnobacterium divergens V41 metabolism (growth, biogenic amine production, and divercin V41 production) in sterile cold-smoked salmon extract (SSE) at varying temperatures (3 to 9 degrees C), NaCl levels (2.5 to 6.5%), and glucose concentrations (2 to 6 g liter(-1)). The results showed that temperature and NaCl content were the most influential factors on growth parameters in SSE. Predictive models are suggested for the assessment of C. divergens lag time (t(lag)) and maximum specific growth rate (micro(max)) Among the biogenic amines studied, only tyramine was found to be produced by C. divergens in SSE. Furthermore, we showed that temperature, NaCl, and glucose variations did not greatly affect tyramine and divercin V41 production by the bacteria under the experimental conditions used. Indeed, divercin V41, a bacteriocin from C. divergens V41 that is highly active against some Listeria strains, was produced in SSE even under harsh culture conditions. Similarly, tyramine production in SSE was delayed at 3 degrees C but reached 35 microg ml(-1) in all experiments after 27 days of storage. However, this final tyramine concentration in SSE is low compared with the threshold values of 100 to 800 microg g(-1) reported as the potentially toxic dose in foods. Thus, we have found that C. divergens V41 is a promising strain for the biopreservation of refrigerated cold-smoked salmon.     

Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli.

Minimum inhibitory contentrations (MICs) of selected inhibitors of cell wall synthesis (benzylpenicillin, ampicillin, and vancomycin), protein synthesis (gentamicin, streptomycin, tetracycline, chloramphenicol, and erythromycin), and nucleic acid synthesis (co-trimoxazole, rifampicin, and metronidazole) were determined by gradient diffusion (E test; AB Biodisk, Solna, Sweden) on deMan, Rogosa, Sharpe (MRS) agar for Lactobacillus strain GG and 11 closely related, rapidly growing, facultatively anaerobic, potentially probiotic Lactobacillus rhamnosus strains. All strains were resistant to vancomycin (MIC90 > or = 256 microg/ml), co-trimoxazole (MIC90 > or = 32 microg/ml), metronidazole (MIC90 > or = 32 microg/ml), gentamicin (MIC90 > or = 128 microg/ml), and streptomycin (MIC90 > or = 256 microg/ml), and sensitive to pencillin G (MIC90 > 0.375 microg/ml), ampicillin (MIC90 > 0.750 microg/ml), rifampicin (MIC90 > 0.375 microg/ml), tetracycline (MIC90 > 1.5 microg/ml), chloramphenicol (MIC90 > 8 microg/ml), and erythromycin (MIC90 > 2 microg/ml). E test MICs were also determined for L. acidophilus National Collection of Food Bacteria (NCFB) 1748 and L. reuteri Deutsche Sammlung von Mikroorganismen 20016T by the inoculum application method recommended by the manufacturer (swabbing), with and without antibiotic prediffusion for 1 h at room temperature, and by an alternative inoculum application (agar overlay) method, without antibiotic prediffusion. Antibiotic prediffusion increased the MICs for penicillin G, ampicillin, tetracycline, and chloramphenicol by up to 2 log2 MIC dilutions without changing antibiotic susceptibility category. Agar overlay application also increased the MICs for these antibiotics as well as for gentamicin by up to 3 log2 MIC dilutions without changing antibiotic susceptibility category. Exact agreement between MICs determined by swab and agar overlay application without antibiotic prediffusion was strain dependent: 54.5% for strain DSM 20016T and 72.7% for strain NCFB 1748. The swab and agar overlay gradient diffusion methods provide a reliable basis for antibiotic susceptibility testing of rapidly growing, facultatively anaerobic lactobacilli, using MRS agar as test medium and are readily applicable for testing individual isolates as needed.     

Efficacy of enterocin AS-48 against bacilli in ready-to-eat vegetable soups and purees

The broad-spectrum bacteriocin enterocin AS-48 was tested for biopreservation of ready-to-eat vegetable foods (soups and purees) against aerobic mesophilic endospore-forming bacteria. By adding AS-48 (10 microg/ml), Bacillus cereus LWL1 was completely inhibited in all six vegetable products tested (natural vegetable cream, asparagus cream, traditional soup, homemade-style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22 degrees C. A collection of strains isolated from spoiled purees showed slightly higher resistance to AS-48 in the order Paenibacillus sp. > Bacillus macroides > B. cereus, although they were also completely inhibited in natural vegetable cream by AS-48 at 10 microg/ml. However, cocktails of five or eight strains composed of B. cereus (three strains), B. macroides (two strains), and Paenibacillus sp., Paenibacillus polymyxa, and Paenibacillus amylolyticus showed higher bacteriocin resistance with AS-48 of up to 50 microg/ml required for complete inactivation in natural vegetable cream stored at 22 degrees C. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) analysis showed that paenibacilli (along with some B. cereus) was the predominant survivor in the cocktails after bacteriocin treatment. To increase the effectiveness of enterocin AS-48, the bacteriocin was tested (at 20 microg/ml) against the eight-strain cocktail in natural vegetable cream in combination with other antimicrobials. The combination of AS-48 and nisin had a slight but significant additive effect. Bactericidal activity was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a rapid and complete inactivation of bacilli in the tested puree at 22 degrees C.     

蔬菜发酵剂乳酸菌产生物胺的检测与评价

通过联合采用聚合酶链式反应(PCR)技术和高效液相色谱分析(HPLC)技术对本实验室筛选保藏的60 余株拟准备用于蔬菜发酵的乳酸菌形成生物胺的能力和水平进行检测和评价。氨基酸脱羧酶基因的PCR 检测结果表明,受检菌株中有3 株组氨酸脱羧酶阳性菌和22 株酪氨酸脱羧酶阳性菌;同时,利用HPLC 法对受检乳酸菌在MRS 培养基体系和模式蔬菜发酵体系中发酵形成生物胺的水平进行分析。受试乳酸菌在MRS 培养基中组胺产生量在4.32～32.15mg/L 之间,酪胺产生量在9.22～114.02mg/L 之间。在发酵蔬菜体系中,组胺和酪胺的产生量均小于40mg/L。PCR 检测结果与HPLC 分析结果具有较好的一致性。     

Production of cyclopiazonic acid by molds isolated from Taleggio cheese.

Twenty-seven strains of Penicillium were isolated from the rind of Taleggio, a typical Italian cheese, so that we could test their capacity to produce cyclopiazonic acid (CPA); all strains produced CPA. The production was strongly influenced by the strain variety and growth conditions. Strains incubated at 25 degrees C for 7 days always produced CPA in mannitol broth, with concentrations ranging from 0.02 to 1 microg/ml, whereas only 33% of strains grown in yeast-extract broth produced CPA, with a maximum value of 0.1 microg/ml. In milk, maximum production (1.6 microg/ml) was observed after 14 days of incubation at 25 degrees C. In order to evaluate the presence of the toxin and its capacity for migrating into the cheeses, the rind, the cheese near the rind, and the cores from six Taleggio cheeses were analyzed. CPA was present in five cheeses, with a maximum concentration of 0.25 mg/kg in one rind, and in one cheese, the toxin migrated to the core. A positive correlation between CPA production and surface mold was found.     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 microg in 2002 to 500-5000 microg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5alpha harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 microg/ml and 6.3 microg/mg dry cell mass respectively) and the lowest was for LB (2.8 microg/ml and 3.3 microg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 microg/ml and 11.2 microg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.     

Combinations of antimycotics to inhibit the growth of molds capable of producing 1,3-pentadiene

Some species of molds are capable of degrading sorbic acid to produce 1,3-pentadiene, a volatile compound with an unpleasant hydrocarbon-like odor. The effectiveness of reduced concentrations of sorbate, in combination with other antimycotics, to control the growth of these molds has not been described. We did a study to evaluate potassium sorbate, sodium benzoate, calcium propionate, disodium ethylenediaminetetraacetic acid (EDTA), and natamycin, alone and in combination, for their effectiveness in preventing the growth of five molds isolated from Parmesan cheese and a lemon-flavored drink subjectively judged to contain 1,3-pentadiene. Growth of Penicillium brevicompactum, Penicillium roqueforti, Paecilomyces variotii, Aspergillus niger, and Cephaloascus fragrans on model agar media containing Parmesan cheese (PRM agar) (pH 5.5) and lemon-flavored drink (LD agar) (pH 2.6) supplemented with antimycotics was studied. All molds grew well at 21 degrees C on PRM agar containing potassium sorbate (3500microg/ml), calcium propionate (3000microg/ml), or natamycin (20microg/ml). Combinations of potassium sorbate (250-1000microg/ml), calcium propionate (250-1000microg/ml), and/or natamycin (10-18microg/ml) greatly inhibited or prevented growth of molds on PRM agar, indicating their potential as preservative systems at pH values resulting in large percentages of the acids in dissociated forms. Three of the five molds grew on LD agar containing potassium sorbate or sodium benzoate at a concentration of 200microg/ml. Growth did not occur within 70 days on LD agar containing EDTA (30microg/ml) in combination with potassium sorbate and sodium benzoate at 50 and 175microg/ml, respectively, or 175 and 50microg/ml, respectively. Results of this study show that preservative systems containing a reduced concentration of potassium sorbate, in combination with other antimycotics, particularly natamycin, have potential for controlling the growth of molds thought to be capable of producing 1,3-pentadiene.     

Antimicrobial activity of foodborne Paenibacillus and Bacillus spp. against Clostridium botulinum

The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.     

江西产腐乳生物胺水平调查及产胺菌的分离与鉴定

采用高效液相色谱法对江西产腐乳生物胺含量水平进行调查分析,通过微生物学分离结合高效液相色谱确认,筛选出生物胺产生菌,并对其进行鉴定。结果表明,12种腐乳样品中总生物胺含量范围为68.5～1084.0 mg/kg,色胺、腐胺、组胺和酪胺是主要生物胺,其中腐胺和酪胺的含量较高。筛选出的9株产胺菌产生物胺的含量范围为93.8～369.2 mg/L。对产胺量较高的6株菌进行表型鉴定和基因型16S rDNA序列测定,得到耐热芽孢杆菌（Bacillus sporothermodurans）1株,鲁梅利杆菌（Rummeliibacillus stabekisii）1株,耐硼赖氨酸芽孢杆菌（Lysinacillus boronitolerans）3株和皮脂葡萄球菌（Staphylococcus piscifermentans）1株。研究结果对腐乳食用安全性评价提供了理论依据,为进一步探清如何抑制腐乳中产胺菌的活性提供了理论研究基础。     

Tyramine and phenylethylamine biosynthesis by food bacteria

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.     

Tyramine reduction by tyrosine decarboxylase inhibitor in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> for tyramine controlled <Emphasis Type="Italic">cheonggukjang</Emphasis>

This study was carried out to find a method to control tyrosine decarboxylase activity (TDC) of a strain of Enterococcus faecium capable of producing high levels of tyramine. To select a TDC inhibitor, enzyme assay was first performed using purified TDC enzyme and 0.1% of TDC inhibiting chemicals. When 0.23% of nicotinic acid was added, tyramine content (363 ug/mL) was lower than that of the control group (873 ug/mL). At the same time, bacterial growth was decreased 1 log cycle from 8.62 to 7.56 log CFU/mL. TDC expression level in E. faecium was measured by using RT-qPCR. Lower expression level (below 0.7) was observed after the addition of 0.23% nicotinic acid (in vitro). When cheonggukjang was manufactured with addition of nicotinic acid, tyramine contents were decreased from 698.67 to 117.27 mg/kg when the concentration of nicotinic acid added was increased from 0.10 to 0.30%. These results suggest that nicotinic acid could be used as an agent (TDC inhibitor) to reduce tyramine content in cheonggukjang.     

Amino acid decarboxylase activities and biogenic amine formation abilities of lactic acid bacteria isolated from shalgam

56 lactic acid bacteria were isolated during shalgam fermentation and identified as Lactobacillus spp. (51 isolates), Lactococcus spp. (3 isolates), Streptococcus sp. (one isolate), and Leuconostoc sp. (one isolate). 53 of all isolates decarboxylated both arginine and tyrosine, while others decarboxylated one of arginine or tyrosine. None of the isolates could decarboxylate histidine, ornithine, lysine, phenylalanine, or tryptophan. All isolates produced both agmatine (105.8–867.5 mg L^–1) and tyramine (24.5–649.7 mg L^–1). Although none of the isolates displayed ornithine decarboxylase activity, putrescine was produced (2.1–33.3 mg L^–1) by all isolates, except one Lactobacillus strain. Therefore, lactic acid bacteria seem to be responsible mainly for tyramine and agmatine formation during shalgam fermentation, as well as a small amount of putrescine.     

阿魏酸对粪肠球菌和屎肠球菌产酪胺机制的影响

摘 要：研究阿魏酸对高产酪胺的粪肠球菌XL-M66和屎肠球菌XL-M76生长、基因表达以及产酪胺的影响。利用反转录实时荧光定量聚合酶链式反应技术分析2 株菌在阿魏酸作用下的酪氨酸脱羧途径相关基因表达情况,并使用高效液相色谱法检测2 株肠球菌培养48 h期间酪胺积累量。结果表明：未添加酪氨酸底物时,阿魏酸对酪氨酸脱羧酶（tyrosine decarboxylase,tyrDC）和酪氨酸/酪胺透性酶（tyrosine/tyramine permease,tyrP）基因的转录影响不大（P＞0.05）,但能促进酪氨酰-tRNA合成酶（tyrosyl-tRNA synthetase,tyrS）基因的转录（P＜0.05）。反之存在酪氨酸时,阿魏酸对tyrS基因表达的影响不大（P＞0.05）,却能显著抑制tyrDC和tyrP基因的表达（P＜0.05）。同时,阿魏酸能显著抑制粪肠球菌XL-M66和屎肠球菌XL-M76的生长（P＜0.05）,最终使得酪胺产量分别降低27.0%和19.9%。     

Synthesis of enzymatically-gellable carboxymethylcellulose for biomedical applications

We synthesized a carboxymethylcellulose with phenol moieties by covalently incorporating tyramine into carboxymethylcellulose using aqueous-phase carbodiimide activation chemistry. The resulting hydrogel was obtained from an aqueous solution of the conjugate via the horseradish peroxidase-catalyzed oxidation reaction of phenols by consuming H(2)O(2), where the gelation speed depended on the concentrations of enzyme and H(2)O(2). The viability of the mammalian cells enclosed within the hydrogel prepared from 1.5% (w/v) conjugate solution containing 5 units/ml horseradish peroxidase and 1 mM H(2)O(2), was 80% after 24 h. These results demonstrate that this carboxymethylcellulose with phenol moieties has potential for biomedical applications including tissue-engineering.     

琼脂糖酶产生菌的筛选和培养基优化

从太岁中筛选得到一株初始产酶较高的琼脂糖酶产生菌,经形态和分子生物学分析方法鉴定之后,认定其属于类芽孢杆菌属(Paenibacillus),命名为Paenibacillus sp.P1(简称为P1).P1为革兰氏阴性菌,短杆状细胞,对明胶和纤维素都没有水解活性.研究该菌的生长及产酶过程发现,该菌株最适发酵产酶时长是40...     

Mutagenicity of meat flavorings with natural and/or nature-identical flavorings

The mutagenic potential of twenty commercially available meat flavours from three different producers was determined using the Ames test. The flavours contained natural and/or nature-identical components as recorded on their label. Twelve flavours showed mutagenic activity with at least one of the four employed test strains (TA1535, TA100, TA97, and TA98). Flavours containing natural components yielded positive results in this mutation test in all cases but one. On the other hand flavours produced from nature-identical substances with one exception did not demonstrate mutagenic effects.