Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

The amine content of fresh and vacuum-packaged beef of normal pH stored at 1 degree C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, spermine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities less than log10 6/cm2 indicated its potential as an objective measure of acceptability/spoilage.     

Temperature-dependent changes of amine levels during early seedling development of the cold-adapted subantarctic crucifer Pringlea antiscorbutica

Pringlea antiscorbutica R. Br., an endemic crucifer from the Kerguelen Archipelago in the subantarctic, has been previously shown to be unable to acclimatize to 25°C when transferred after several months cultivation under cold conditions. Furthermore, the polyamine composition was greatly modified in such high-temperature-treated plants. The development of seedlings of this species was investigated under a regime mimicking the subantarctic summer thermoperiod (5/10°C night/day) and a regime with high temperatures (22/25°C night/day). In parallel, the associated changes in polyamine composition that occurred during the first 6 days of seedling life were determined. Marked acceleration of seedling growth and intense cotyledon greening were observed at day 4 in 5/10°C-grown seedlings but not in 22/25°C-grown seedlings. Seedlings grown at high temperature accumulated agmatine and putrescine, whereas cold-cultivated seedlings maintained high levels of spermidine. Cold-cultivated seedlings accumulated the uncommon long-chain polyamines norspermidine and homospermidine. These seedlings also accumulated free 1,3-diaminopropane, cadaverine, N¹-acetylspermidine, N¹-acetylspermine and bound polyamines, whereas seedlings under high temperature accumulated N¹-acetylputrescine. Aromatic amine metabolism also appeared to be very responsive to temperature: seedlings under a cold regime accumulated free dopamine and bound phenylethylamine and tyramine, whereas seedlings grown at high temperature accumulated free tyramine. The possible relationships between the observed amine patterns and seedling growth under low and high temperature are discussed.     

Polyamines in Rhizobium, Bradyrhizobium, Azorhizobium and Argobacterium

Abstract Eighteen strains of Rhizobium including four species, R. leguminosarum, R. meliloti, R. loti and R. fredii , nine strains of Bradyrhizobium japonicum and three strains of Azorhizobium caulinodans contained putrescine and honospermidine as major polyamines. All these nodulating N₂-fixing rhizobia lack spermidine. Spermidine and cadaverine were present only in a limited number of R. meliloti and B. japonicum . Polymanine-synthetic activity was not affected by the differences in ability to produce phytoxine (rhizobitoxine and dihydrorhizobitoxine) H₂-uptake-hydrogenation in the organisms. Putrescine and homospermidine were major polyamined in a strain of Agrobacterium rhizogenes . All the eight strains of Agrobacterium tumefaciens as well as A. rubi, A. radiobacter and two other strains of A. rhizogenes contained putrescine and spermidine as major polyamines and homospermidine and spermine (and thermospermine) as minor polyamines.     

Polyamine biosynthesis in the ectomycorrhizal fungus Paxillus involutus exposed to zinc

Following exposure of the ectomycorrhizal fungus Paxillus involutus to 3·75 μg ml^-1 or 15 μg ml^-1 zinc for 2 weeks, growth was unaffected, putrescine and spermidine concentrations remained unaltered, while spermine concentration increased substantially. This was accompanied by small changes in the activities of ornithine decarboxylase and S -adenosylmethionine decarboxylase. There was, however, reduced synthesis of aminopropylcadaverine and N,N' bis (3-aminopropyl)cadaverine at the higher zinc concentration. It is possible that the increased spermine concentration may have resulted from displacement of the amine from the cell wall by the metal ion.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

两种常见海水鱼高温贮存过程中挥发性盐基氮和生物胺含量变化

棘头梅童鱼(Collichthys lucidus)和龙头鱼(Harpodon nehereus)是我国沿海常见的两种小型海水鱼, 常被作为水产动物饵料, 也可被人类食用。研究检测了这两种鱼在30℃下贮存48h 每隔6h 的挥发性盐基氮(T-VBN)和9 种生物胺(尸胺、腐胺、组胺、酪胺、5-羟色胺、亚精胺、精胺、多巴胺、章鱼胺)的含量变化, 并对这两种鱼的T-VBN 和生物胺含量与时间的相关性进行分析, 为水产品类饵料安全投喂和人类食品安全提供基础资料。结果表明: 两种鱼在相同贮存条件中T-VBN 和生物胺含量均存在一定差异。T-VBN 含量随着贮存时间的延长而逐渐增加, 棘头梅童鱼T-VBN 含量从0h 的8.19 mg/100 g 增加到48h 的568.05 mg/100 g,龙头鱼从0h 的13.16 mg/100 g 增加到48h 的361.34 mg/100 g, 棘头梅童鱼增长值显著高于龙头鱼(PPPP>0.05);多巴胺在两种鱼体内均未检测到。这两种鱼体内T-VBN、腐胺、尸胺、组胺、酪胺含量与时间的相关性均极其显著(P<0.01)。       

Putrescine and cadaverine formation in vacuum packed beef

Bacterial numbers, putrescine and cadaverine concentrations and pH were measured at regular intervals during the chill storage of vacuum packed beef. Odours on opening the packs were also assessed. Cadaverine concentration increased more rapidly than that of putrescine and measurable increases were evident before maximum bacterial numbers were attained and before any permanent off-odours were detected. Diamine concentrations correlated better with total viable count (TVC) than with counts of Gram negative organisms.     

Putrescine and cadaverine formation in vacuum packed beef

Bacterial numbers, putrescine and cadaverine concentrations and pH were measured at regular intervals during the chill storage of vacuum packed beef. Odours on opening the packs were also assessed. Cadaverine concentration increased more rapidly than that of putrescine and measurable increases were evident before maximum bacterial numbers were attained and before any permanent off-odours were detected. Diamine concentrations correlated better with total viable count (TVC) than with counts of Gram negative organisms.     

Correlation between microbial flora, sensory changes and biogenic amines formation in fresh chicken meat stored aerobically or under modified atmosphere packaging at 4 °C: possible role of biogenic amines as spoilage indicators

This study evaluated the formation of biogenic amines (BAs) in breast chicken meat during storage under aerobic and modified atmospheric packaging (MAP) conditions at 4 °C, the correlation of microbial and sensory changes in chicken meat with formation of BAs and the possible role of BAs as indicators of poultry meat spoilage. Poultry breast fillets were stored aerobically or under MAP (30%, CO₂, 70% N₂) at 4 °C for up to 17 days. Quality evaluation was carried out using microbiological, chemical and sensory analyses. Total viable counts, Pseudomonads and Enterobacteriaceae, were in general higher for chicken samples packaged in air whereas lactic acid bacteria (LAB) and Enterobacteriaceae were among the dominant species for samples under MAP. Levels of putrescine and cadaverine increased linearly with storage time and were higher in aerobically stored chicken samples. Spermine and spermidine levels were also detected in both aerobically and MAP stored chicken meat. Levels of tyramine in both chicken samples stored aerobically and or under MAP were low (< 10 mg kg⁻¹) whereas the formation of histamine was only observed after day 11 of storage when Enterobacteriaceae had reached a population of ca. 10⁷ CFU g⁻¹. Based on sensory and microbiological analyses and also taking into account a biogenic amines index (BAI, sum of putrescine, cadaverine and tyramine), BAI values between 96 and 101 mg kg⁻¹ may be proposed as a quality index of MAP and aerobically-packaged fresh chicken meat. Spermine and spermidine decreased steadily throughout the entire storage period of chicken meat under aerobic and MAP packaging, and thus these two amines cannot be used as indicators of fresh chicken meat quality.     

Bacterial sources of putrescine and cadaverine in chill stored vacuum-packaged beef

Of the meat strains of streptobacteria, leuconostocs, Enterobacteriaceae and Brochothrix thermosphacta tested, only Hafnia alvei and Serratia liquefaciens showed diamine-producing potential during growth in pure culture on beef stored in vacuum packs at 1°C. Both organisms produced cadaverine at concentrations similar to those reported previously in naturally contaminated beef stored under the same conditions. Putrescine concentrations produced by the two organisms, however, were an order of magnitude lower. During the growth on beef of either H. alvei or S. liquefaciens in mixed culture with arginine-utilizing strains of streptobacteria, putrescine as well as cadaverine concentrations were similar to those detected in naturally contaminated samples.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL V. BREAKDOWN OF GRISEOFULVIN IN SOIL

When griseofulvin (I; R = Cl, R '= OCH₃), a chlorine-containing antibiotic produced by Penicillium nigricans , was added to fresh garden loam, after an initial lag it disappeared rapidly. When further griseofulvin was added it was inactivated from the start and at rates which increased with each successive addition, suggesting that it was degraded biologically. The numbers of one organism, a Pseudomonas sp., increased in the soil steadily after adding griseofulvin.
When a little soil was added to a solution (pH 7·0) containing inorganic salts and griseofulvin as the sole carbon source, bioassays showed that the griseofulvin disappeared within 5 days. An organism isolated from the broth was identified as the Pseudomonas sp. thought to break down griseofulvin in soil. Griseofulvin also disappeared from a broth at pH 5·0 inoculated with soil, but at this lower pH value a dematiaceous fungus was responsible for its breakdown.
The Pseudomonas sp. also degraded two derivatives of griseofulvin, dechlorogriseofulvin (I; R = H, R'= OCH₃) and the amine (I; R = Cl, R '= NH₂). Cl^– was detected in the solutions after breakdown of griseofulvin by the Pseudomonas ; the amount present agreed well with that calculated on the assumption that all the chlorine in the griseofulvin supplied was liberated as Cl^–. Spectrophotometric examination of the solutions showed no metabolites with the aromatic ring intact, and confirmed the complete breakdown of griseofulvin suggested by the liberation of Cl-.     

Bacterial sources of putrescine and cadaverine in chill stored vacuum-packaged beef

Of the meat strains of streptobacteria, leuconostocs, Enterobacteriaceae and Brochothrix thermosphacta tested, only Hafnia alvei and Serratia liquefaciens showed diamine-producing potential during growth in pure culture on beef stored in vacuum packs at 1 degree C. Both organisms produced cadaverine at concentrations similar to those reported previously in naturally contaminated beef stored under the same conditions. Putrescine concentrations produced by the two organisms, however, were an order of magnitude lower. During the growth on beef of either H. alvei or S. liquefaciens in mixed culture with arginine-utilizing strains of streptobacteria, putrescine as well as cadaverine concentrations were similar to those detected in naturally contaminated samples.     

Tyramine functions as a toxin in honey bee larvae during Varroa-transmitted infection by Melissococcus pluton

From wounds of honey bee pupae, caused by the mite Varroa destructor, coccoid bacteria were isolated and identified as Melissococcus pluton. The bacterial isolate was grown anaerobically in sorbitol medium to produce a toxic compound that was purified on XAD columns, gelfiltration and preparative HPLC. The toxic agent was identified by GC-MS and FTICR-MS as tyramine. The toxicity of the isolated tyramine was tested by a novel mobility test using the protozoon Stylonychia lemnae. A concentration of 0.2 mg/ml led to immediate inhibition of mobility. In addition the toxicity was studied on honey bee larvae by feeding tyramine/water mixtures added to the larval jelly. The lethal dosis of tyramine on 4-5 days old bee larvae was determined as 0.3 mg/larvae when added as a volume of 20 microl to the larval food in brood cells. Several other biogenic amines, such as phenylethylamine, histamine, spermine, cadaverine, putrescine and trimethylamine, were tested as their hydrochloric salts for comparison and were found to be inhibitory in the Stylonychia mobility test at similar concentrations. A quantitative hemolysis test with human red blood cells revealed that tyramine and histamine showed the highest membranolytic activity, followed by the phenylethylamine, trimethylamine and spermine, while the linear diamines, cadaverine and putrescine, showed a significantly lower hemolysis when calculated on a molar amine basis. The results indicate that tyramine which is a characteristic amine produced by M. pluton in culture, is the causative agent of the observed toxic symptoms in bee larvae. Thus this disease, known as European foulbrood, is possibly an infection transmitted by the Varroa destructor mite.     

Incidence and Spoilage Potential of Isolates from Vacuum-packaged Meat of High pH Value

Meat of high pH value (6·6) showing dark-cutting characteristics was vacuum-packaged and stored for up to 8 weeks at 0–2°C. 'Off'-odours were detected on opening the packages after 6 weeks of storage. Total counts at this stage were ca. 10⁷/cm² of which lactobacilli were the major component, with ca. 10⁶/cm² Gram negative organisms. Psychrotrophic Enterobacteriaceae represented a major proportion of the microflora only after the full 8 weeks of storage and were not detected previously. Aerobic storage of steaks cut from the vacuum packaged meat stored for 8 weeks resulted in a predominantly Gram negative spoilage flora.
Inoculation studies on meat of normal pH value (5·4) and appearance using representative isolates from the vacuum-packaged meat microflora indicated that most of the test organisms were capable of causing spoilage under aerobic conditions but few under vacuum-packaging when incubated at 4°C. On meat of higher pH value (6·15) many of the Gram negative isolates did not grow as well, whereas the Gram positive isolates grew better than on meat of normal pH value when held under aerobic conditions. Under vacuum-packaging all but one isolate grew as well or better on meat of high pH value than on normal meat at 4°C and objectionable odours were more marked.     

Biogenic amine production by wild lactococcal and leuconostoc strains

Two qualitative and one quantitative HPLC methods were evaluated for the detection of biogenic amine producers among wild dairy lactococcal and leuconostoc strains. High tyramine producers ranging from 370 to 807 mg l⁻¹ were detected by qualitative methods and confirmed by HPLC analysis. Tyramine levels detected throughout the incubation time depended on the concentration of the amino acid precursor available and no tyramine production was observed when strains were grown in milk. However, increasing amounts of tyramine were detected in cultures grown in milk supplemented with different concentrations of tyrosine. Qualitative methods failed to detect weak producers so that tryptamine production (<7 mg l⁻¹) could only be determined by HPLC. None of the tested strains was able to produce histamine. Simultaneous production of different amines was observed by HPLC although no colour change was observed in the specific decarboxylase media. Thus, it was concluded that the amine forming ability should be taken into account when selecting starters for milk fermentations. Qualitative methods could be used as a first screening step to eliminate the highest amine producers while the quantitative methods would detect any producing strain.     

Biogenic amine formation by poultry-associated spoilage and pathogenic bacteria

The production of biogenic amines by 50 poultry-associated bacterial strains (25 Pseudomonas , 13 Salmonella and 12 Listeria ) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy-four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine-producing bacterial strains associated with poultry.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

产生物胺乳酸菌的筛查与检测

目的对上海市场上食品和药品中分离出的20株乳杆菌,13株链球菌,3株乳球菌和3株肠球菌的产生物胺能力进行检测,以揭示其潜在的安全性问题。方法检测的生物胺共包括6种：分别为酪胺、精胺、尸胺、组胺、腐胺和色胺。利用添加了前体氨基酸的氨基酸脱羧酶筛选培养基对各菌株的产胺能力进行初筛,通过培养基中指示剂的颜色变化判定产胺能力。结果在检测的39株菌中,8株菌具有产酪胺的能力,7株菌具有产精胺的能力,1株菌具有产组胺的能力,1株菌具有产腐胺的能力。尤其是精胺和酪胺的产量较为引人关注。结论生物胺的危害水平取决于个体解毒的能力,但在筛选食品药品用菌株时应运用规范的方法来检测其产生物胺的能力,以保障相应食品药品的安全问题。     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.