Membrane toxicity of antimicrobial compounds from essential oils

Natural antimicrobial compounds perform their action mainly against cell membranes. The aim of this work was to evaluate the interaction, meant as a mechanism of action, of essential oil antimicrobial compounds with the microbial cell envelope. The lipid profiles of Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas fluorescens, and Brochothrix thermosphacta cells treated with thymol, carvacrol, limonene, eugenol, and cinnamaldehyde have been analyzed by gas chromatography. In line with the fatty acids analysis, the treated cells were also observed by scanning electron microscopy (SEM) to evaluate structural alterations. The overall results showed a strong decrease of the unsaturated fatty acids (UFAs) for the treated cells; in particular, the C18:2trans and C18:3cis underwent a notable reduction contributing to the total UFA decreases, while the saturated fatty acid C17:0 raised the highest concentration in cinnamaldehyde-treated cells. SEM images showed that the used antimicrobial compounds quickly exerted their antimicrobial activities, determining structural alterations of the cell envelope.     

Vapor-phase activities of cinnamon, thyme, and oregano essential oils and key constituents against foodborne microorganisms

The aim of the study presented here was to gain knowledge about the vapor-phase antimicrobial activity of selected essential oils and their major putatively active constituents against a range of foodborne bacterial and fungal strains. In a first step, the vapor-phase antimicrobial activities of three commercially available essential oils (EOs)-cinnamon (Cinnamomum zeylanicum), thyme (Thymus vulgaris), and oregano (Origanum vulgare)-were evaluated against a wide range of microorganisms, including Gram-negative bacteria (Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa, and Salmonella choleraesuis), Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis), molds (Penicillium islandicum and Aspergillus flavus), and a yeast (Candida albicans). The minimum inhibitory concentrations (MICs) were generally lower for oregano EO than for the thyme and cinnamon EOs, especially against the relatively resistant Gram-negative. The persistence of the EOs' antimicrobial activities over time was assessed, and changes in the composition of the atmosphere they generated over time were determined using single-drop microextraction (SDME) in combination with gas chromatography-mass spectrometry (GC-MS) and subsequent analysis of the data by principal component analysis (PCA). More relevant chemicals were selected. In addition, the vapor-phase activities of putatively key constituents of the oils were screened against representative Gram-positive (L. monocytogenes) and Gram-negative (S. choleraesuis) bacteria, a mold (A. flavus), and a yeast (C. albicans). Of the tested compounds, cinnamaldehyde, thymol, and carvacrol showed the strongest antimicrobial effectiveness, so their MICs, defined as the minimum vapor concentrations that completely inhibited detectable growth of the microorganisms, were calculated. To check for possible interactions between components present in the EOs, cinnamon EO was fortified with cinnamaldehyde and thyme EO with thymol, and then the antimicrobial activities of the fortified oils were compared to those of the respective unfortified EOs using fractional inhibitory concentration (FIC) indices and by plotting inhibition curves as functions of the vapor-phase concentrations. Synergistic effects were detected for cinnamaldehyde on A. flavus and for thymol on L. monocytogenes, S. choleraesuis, and A. flavus. In all other cases the fortification had additive effects, except for cinnamaldehyde's activity against S. choleraesuis, for which the effect was antagonistic. Finally, various microorganisms were found to cause slight changes over time to the atmospheres generated by all of the EOs (fortified and unfortified) except the fortified cinnamon EO.     

Development of flexible antimicrobial films using essential oils as active agents

The antimicrobial activity in the vapor-phase of laboratory-made flexible films of polypropylene (PP) and polyethylene/ethylene vinyl alcohol copolymer (PE/EVOH) incorporating essential oil of cinnamon ( Cinnamomum zeylanicum), oregano ( Origanum vulgare), clove ( Syzygium aromaticum), or cinnamon fortified with cinnamaldehyde was evaluated against a wide range of microorganisms: the Gram-negative bacteria Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa, and Salmonella choleraesuis; the Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis; the molds Penicillium islandicum, Penicillium roqueforti, Penicillium nalgiovense, Eurotium repens, and A spergillus flavus and the yeasts Candida albicans, Debaryomyces hansenii, and Zigosaccharomyces rouxii. Films with a nominal concentration of 4% (w/w) of fortified cinnamon or oregano essential oil completely inhibited the growth of the fungi; higher concentrations were required to inhibit the Gram-positive bacteria (8 and 10%, respectively), and higher concentrations still were necessary to inhibit the Gram-negative bacteria. PP films were more effective than PE/EVOH films. The atmospheres generated by the antimicrobial films inside Petri dishes were quantitatively analyzed using headspace-single drop microextraction (HS-SDME) in combination with gas chromatography-mass spectrometry (GC-MS). The analyses showed that the oregano-fortified PP films released higher levels of carvacrol and thymol, and the cinnamon-fortified PP films released higher levels of cinnamaldehyde, during the first 3-6 h of incubation, than the corresponding PE/EVOH films. Shelf-life tests were also performed, demonstrating that the antifungal activities of the films persisted for more than two months after their manufacture. In addition, migration tests (overall and specific) were performed, using both aqueous and fatty simulants, to ensure that the films meet EU regulations regarding food contact materials. Following contact with the tested films, the substances that had migrated into the aqueous simulants were recovered by direct immersion-single drop extraction (DI-SDME) and then analyzed by GC-MS. The fatty stimulant (isooctane) was directly injected into the chromatographic system.     

Antimicrobial paper based on a soy protein isolate or modified starch coating including carvacrol and cinnamaldehyde

Soy protein isolates (SPI) and octenyl-succinate (OSA) modified starch were used as paper coating and inclusion matrices of two antimicrobial compounds: cinnamaldehyde and carvacrol. Antimicrobial compound losses from the coated papers were evaluated after the coating and drying process, and the two matrices demonstrated retention ability that depended on the compound nature and concentration. Whereas carvacrol losses ranged between 12 and 45%, cinnamaldehyde losses varied from 43 to 76%. The losses were always higher from OSA-starch-coated papers than from SPI-coated papers. During storage in accelerated conditions, at 30 degrees C and 60% relative humidity, carvacrol retention from coated papers was found to be similar whatever the coating matrices and the carvacrol rate. In contrast, the retention from SPI-coated papers was particularly high for the cinnamaldehyde concentration of 30% (w/w) compared to the lowest (10% w/w) or highest concentration (60% w/w). Compared to carvacrol, faster release was observed, particurlarly when OSA-starch was used. The antimicrobial properties of the coated papers were shown against Escherichia coli and Botrytis cinerea and explained by favorable conditions of total release of the antimicrobial agents.     

Kombucha fermentation and its antimicrobial activity

Kombucha was prepared in a tea broth (0.5% w/v) supplemented with sucrose (10% w/v) by using a commercially available starter culture. The pH decreased steadily from 5 to 2.5 during the fermentation while the weight of the "tea fungus" and the OD of the tea broth increased through 4 days of the fermentation and remained fairly constant thereafter. The counts of acetic acid-producing bacteria and yeasts in the broth increased up to 4 days of fermentation and decreased afterward. The antimicrobial activity of Kombucha was investigated against a number of pathogenic microorganisms. Staphylococcus aureus, Shigella sonnei, Escherichia coli, Aeromonas hydrophila, Yersinia enterolitica, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus epidermis, Campylobacter jejuni, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Helicobacterpylori, and Listeria monocytogenes were found to be sensitive to Kombucha. According to the literature on Kombucha, acetic acid is considered to be responsible for the inhibitory effect toward a number of microbes tested, and this is also valid in the present study. However, in this study, Kombucha proved to exert antimicrobial activities against E. coli, Sh. sonnei, Sal. typhimurium, Sal. enteritidis, and Cm. jejuni, even at neutral pH and after thermal denaturation. This finding suggests the presence of antimicrobial compounds other than acetic acid and large proteins in Kombucha.     

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.     

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.     

Cinnamaldehyde content in foods determined by gas chromatography-mass spectrometry

trans-Cinnamaldehyde, the principal component of cinnamon flavor, is a potent antimicrobial compound present in essential oils such as cinnamon. In the course of studies designed to discover its maximum microbial lethality under food-processing conditions, a gas chromatographic-mass spectrophotometric procedure was developed for the extraction and analysis of essential oil components such as cinnamaldehyde from commercial cinnamon-containing foods (several brands of cinnamon breads, cereals, cookies, puddings, applesauces, and fruit juices). The cinnamaldehyde content ranged from trace amounts in orange juice to 12.2 mg/100 g (122 ppm) in apple cinnamon cereals and 31.1 mg/100 g (311 ppm) for cinnamon swirl bread (highest value). To ascertain the heat stability of cinnamaldehyde, pure cinnamaldehyde, pure eugenol, cinnamon oil, and mixtures consisting of cinnamaldehyde plus eugenol or cinnamon oil were heated at graded temperatures up to 210 degrees C and 60 min, and then possible compositional changes were examined. Eugenol was stable to heat, as were the components of cinnamon oil: carvone, eugenol, and linalool. In contrast, starting at approximately 60 degrees C, pure cinnamaldehyde undergoes a temperature-dependent transformation to benzaldehyde under the influence of heat. Eugenol, both pure and in cinnamon oil, when added to pure cinnamaldehyde protected the aldehyde against heat destruction. The protection may due to an antioxidative action of eugenol. The possible mechanism of this effect and the significance of these findings for food chemistry and microbiology are discussed.     

Eggshell matrix proteins as defense mechanism of avian eggs

This study focused on the role of eggshell matrix proteins as a function of potential natural antimicrobial defenses of avian eggs. The electrophoretic profile of SDS-PAGE showed that the soluble eggshell matrix proteins had three major bands of 15 000, 36 000, and 66 000 and several minor bands comprising 17 000, 25 000, 30 000, and 75 000, while insoluble matrix proteins were consisting of various bands comprising at least 16 distinct migration bands between 10 000 and 200 000. Three bacteria species, Pseudomonas aureginosa, Bacillus cereus, and Staphylococcus aureus, were found to be inhibited in the presence of soluble eggshell matrix proteins (100 microg/mL). On the other hand, Escherichia coli and Salmonella enteritidis were weakly inhibited at only an early stage of incubation time (up to 4 h). Scanning electron microscopy revealed that eggshell matrix proteins might interact and disrupt the membrane integrity of bacteria. The present study clearly indicated that avian eggshell matrix proteins possess a potential of novel antimicrobial defensin mechanism.     

Enzyme-assisted processing increases antimicrobial and antioxidant activity of bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.     

Structural requirements for the antimicrobial activity of carvacrol

Carvacrol is a component of several essential oils and has been shown to exert antimicrobial activity. The structural requirements for the activity of carvacrol were determined by comparison to structurally related (nonessential oil) compounds. Removal of the aliphatic ring substituents of carvacrol slightly decreased the antimicrobial activity. The effect of the hydroxyl group of carvacrol on activity could not be determined by simply comparing it to p-cymene, because this compound is immiscible with water; therefore, 2-amino-p-cymene, the amino analogue of carvacrol, which has a similar hydrophobicity and structural characteristics, was used. 2-Amino-p-cymene had similar membrane disruption and bacterial killing characteristics as carvacrol showing that, contrary to previous reports, the hydroxyl group of carvacrol itself is not essential for the antimicrobial activity. However, the observed 3-fold lower activity for 2-amino-p-cymene as compared to carvacrol indicates special features in the antimicrobial mode of action of carvacrol due to the hydroxyl group.     

Efficient screening of a novel antimicrobial peptide from Jatropha curcas by cell membrane affinity chromatography

A novel method named cell membrane affinity chromatography was used to screen antimicrobial peptides from Jatropha curcas . A cationic antimicrobial peptide (KVFLGLK, JCpep7) was successfully isolated and identified. Antimicrobial assays indicated that JCpep7 was active against the tested microorganisms ( Salmonella typhimurium ATCC 50013, Shigella dysenteriae ATCC 51302, Pseudomonas aeruginosa ATCC 27553, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 23631, and Streptococcus pneumoniae ATCC 49619) with minimal inhibitory concentration (MIC) values ranging from 24 to 64 μg/mL. The antimicrobial mechanisms based on Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) techniques showed that JCpep7 killed microbes principally via breaking of their cell walls and membranes, followed by cell lysis. The results indicated that cell membrane affinity chromatography could be a promising approach for high-throughput screening of antimicrobial peptides from J. curcas .     

Comparative study on the antioxidant and biological activities of carvacrol, thymol, and eugenol derivatives

Four derivatives of thymol, carvacrol, and eugenol were synthesized: 4-(hydroxymethyl)-5-isopropyl-2-methylphenol, 4,4'-methylenebis(5-isopropyl-2-methyl)phenol, 4-allyl-6-(hydroxymethyl)-2-methoxyphenol, and 4-(hydroxymethyl)-2-isopropyl-5-methylphenol. The obtained derivatives showed remarkably better antioxidative properties according to 1,1-diphenyl-2-picrylhydrazyl assay (50% inhibitory concentrations = 4-156 microg/mL) and Rancimat assay (protection factors = 1.55-5.84) when compared with parent compounds and values similar to or better than those of butylated hydroxytoluene and vitamin C. At concentrations of 10 mM carvacrol derivatives had no toxic effect on viability of Escherichia coli K-12 (determined by minimum inhibitory concentrations). Other phenol derivatives showed reduced cytotoxic effect on E. coli K-12 at concentrations of 2-5 mM on the basis of 50% lethal dose measurements. In comparison with the parent compounds, phenol derivatives showed reduced cytotoxic effect for Saccharomyces cerevisiae cells (determined by yeast colony reduction). On the other hand, the majority of synthesized compounds had dose-dependent antiproliferative effects on human uterine carcinoma cells (HeLa), which makes them potentially interesting for the adjuvant experimental cancer treatments. The 4,4'-methylenebis(5-isopropyl-2-methyl)phenol derivative of carvacrol showed lower inhibiting capacity also for the HeLa cells, which makes this particular derivative attractive as an efficient antioxidant with negligible cytotoxic effects.     

Chemical composition and antifungal activity of Arnica longifolia, Aster hesperius, and Chrysothamnus nauseosus essential oils

Essential oils from three different Asteraceae obtained by hydrodistillation of aerial parts were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). Main compounds obtained from each taxon were found as follows: Arnica longifolia carvacrol 37.3%, alpha-bisabolol 8.2%; Aster hesperius hexadecanoic acid 29.6%, carvacrol 15.2%; and Chrysothamnus nauseosus var. nauseosus beta-phellandrene 22.8% and beta-pinene 19.8%. Essential oils were also evaluated for their antimalarial and antimicrobial activity against human pathogens, and antifungal activities against plant pathogens. No antimalarial and antimicrobial activities against human pathogens were observed. Direct bioautography demonstrated antifungal activity of the essential oils obtained from three Asteraceae taxa and two pure compounds, carvacrol and beta-bisabolol, to the plant pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides. Subsequent evaluation of antifungal compounds using a 96-well micro-dilution broth assay indicated that alpha-bisabolol showed weak growth inhibition of the plant pathogen Botrytis cinerea after 72 h.     

Suppression effect of Cinnamomum cassia bark-derived component on nitric oxide synthase

The inhibitory effects of Cinnamomum cassia bark-derived material on nitric oxide (NO) production in RAW 264.7 cells was determined through the evaluation of NO production and expression of inducible nitric oxide and compared to the effects of three commercially available compounds, cinnamyl alcohol, cinnamic acid, and eugenol. The biologically active constituents of C. cassia extract were characterized as trans-cinnamaldehyde by spectral analysis. The inhibitory effects varied with both chemical and concentration used. Potent inhibitory effects of cinnamaldehyde against NO production were 81.5 and 71.7% at 1.0 and 0.5 microg/microL, respectively, and a 41.2% inhibitory effect was revealed at 0.1 microg/microL. However, little or no activity was observed for cinnamic acid and eugenol. Suppression effects of cinnamaldehyde on inducible nitric oxide synthase expression were revealed by Western blot analysis. As a naturally occurring therapeutic agent, trans-cinnamaldehyde could be useful for developing new types of NO inhibitors.     

Weakening of salmonella with selected microbial metabolites of berry-derived phenolic compounds and organic acids

Gram-negative bacteria are important food spoilage and pathogenic bacteria. Their unique outer membrane (OM) provides them with a hydrophilic surface structure, which makes them inherently resistant to many antimicrobial agents, thus hindering their control. However, with permeabilizers, compounds that disintegrate and weaken the OM, Gram-negative cells can be sensitized to several external agents. Although antimicrobial activity of plant-derived phenolic compounds has been widely reported, their mechanisms of action have not yet been well demonstrated. The aim of our study was to elucidate the role of selected colonic microbial metabolites of berry-derived phenolic compounds in the weakening of the Gram-negative OM. The effect of the agents on the OM permeability of Salmonella was studied utilizing a fluorescence probe uptake assay, sensitization to hydrophobic antibiotics, and lipopolysaccharide (LPS) release. Our results show that 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid (3,4-diHPP), 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and 3-(3-hydroxyphenyl)propionic acid efficiently destabilized the OM of Salmonella enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Infantis as indicated by an increase in the uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN). The OM-destabilizing activity of the compounds was partially abolished by MgCl2 addition, indicating that part of their activity is based on removal of OM-stabilizing divalent cations. Furthermore, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-diHPP increased the susceptibility of S. enterica subsp. enterica serovar Typhimurium strains for novobiocin. In addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid, were shown to be efficient permeabilizers of Salmonella as shown by an increase in the NPN uptake assay and by LPS release.     

鸵鸟异嗜白细胞抗菌肽的分离纯化及部分性质研究

目的：分离纯化出鸵鸟异嗜白细胞抗菌肽,并对其部分特性进行研究,为开发新一代高效肽类抗菌药提供依据。方法：使用氯化铵裂解、超声波破碎、醋酸提取、CM-Sepharose F F弱酸性阳离子交换层析和反相高效液相色谱（RP-HPLC）等方法,从鸵鸟异嗜白细胞中分离纯化出了抗菌肽,使用微量琼脂糖弥散法进行了抗菌活性与最小抑菌浓度（MIC）的测定,应用二级质谱（MS/MS）对抗菌肽的分子量进行测定。结果：在鸵鸟异嗜白细胞中存在抗菌肽,其对金黄色葡萄球菌S.aureus 1056MRSA、鸡大肠杆菌E.coli O78和白色念珠菌C.albicans ATCC10231具有较强的抑制作用;抗菌肽具有阳离子性质;热稳定性良好;经RP-HPLC纯化得到峰6、峰16和峰24,峰6对白色念珠菌的MIC为0.065μg/mL,峰16的分子量为4012.472Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为3.971μg/mL和5.245μg/mL,峰24的分子量为3542.479Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为26.472μg/mL和21.561μg/mL。结论：本研究得到的抗菌肽与已报道的鸵鸟抗菌肽ostricacins相比显示出更强的抑菌活性,是否为同一物质需要进一步的验证。     

Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria

The in vitro antimicrobial activity of commercial coffee extracts and chemical compounds was investigated on nine strains of enterobacteria. The antimicrobial activity investigated by the disc diffusion method was observed in both the extracts and tested chemical compounds. Even though pH, color, and the contents of trigonelline, caffeine, and chlorogenic acids differed significantly among the coffee extracts, no significant differences were observed in their antimicrobial activity. Caffeic acid and trigonelline showed similar inhibitory effect against the growth of the microorganisms. Caffeine, chlorogenic acid, and protocatechuic acid showed particularly strong effect against Serratia marcescens and Enterobacter cloacae. The IC(50) and IC(90) for the compounds determined by the microtiter plate method indicated that trigonelline, caffeine, and protocatechuic acids are potential natural antimicrobial agents against Salmonella enterica. The concentrations of caffeine found in coffee extracts are enough to warrant 50% of the antimicrobial effect against S. enterica, which is relevant to human safety.     

Chemical composition of the essential oils of Juniperus from ripe and unripe berries and leaves and their antimicrobial activity

The composition of the essential oil from ripe and unripe berries and leaves of Juniperus oxycedrus L. ssp. oxycedrus, Juniperus phoenicea ssp. turbinata and Juniperus communis ssp. communis was analyzed by GC-MS, and microbiological assays were carried out. Samples were collected in different localities (Sardinia, Italy) and hydro distilled. The yields ranged between 2.54% +/- 0.21 (v\w dried weight) and 0.04% +/- 0.00. A total of 36 components were identified. The major compounds in the essential oils were alpha-pinene, beta-pinene, delta-3-carene, sabinene, myrcene, beta-phellandrene, limonene, and D-germacrene. Both qualitative and quantitative differences between species and between different parts of the plant were observed. The essential oils and their major compounds were tested against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the minimum inhibitory concentration and minimum bactericidal concentration were determined. The results obtained led to a nonsignificant inhibitory effect, although all the essential oils from Juniperus phoenicea ssp. turbinata and the essential oil from leaves of Juniperus oxycedrus ssp. oxycedrus exhibited rather good or weak activity against Candida albicans and Staphylococcus aureus.     

Solid- and vapor-phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains

The antimicrobial activity of essential oils (EOs) of cinnamon (Cinnamon zeylanicum), clove (Syzygium aromaticum), basil (Ocimum basillicum), rosemary (Rosmarinus officinalis), dill (Anethum graveolens), and ginger (Zingiber officinalis) was evaluated over a range of concentrations in two types of contact tests (solid and vapor diffusion). The EOs were tested against an array of four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and Listeria monocytogenes), four Gram-negative bacteria (Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis, and Pseudomonas aeruginosa), and three fungi (a yeast, Candida albicans, and two molds, Penicillium islandicum and Aspergillus flavus). The rationale for this work was to test the possibility of creating a protective atmosphere by using natural compounds that could extend the shelf life of packaged foodstuffs while minimizing organoleptic alterations. In the solid diffusion tests, cinnamon and clove gave the strongest (and very similar) inhibition, followed by basil and rosemary, with dill and ginger giving the weakest inhibition. The fungi were the most sensitive microorganisms, followed by the Gram-positive bacterial strains. The Gram-negative strain P. aeruginosa was the least inhibited. The composition of the atmosphere generated by the EOs, and their minimum inhibitory concentrations (MICs), were determined using a disk volatilization method, in which no inhibition from rosemary or basil was observed. Cinnamon and clove, once again, gave similar results for every microorganism. As a general rule, MIC (fungi) < MIC (bacteria) with no clear differences between Gram-positive or -negative strains except for P. aeruginosa, which was not inhibited by any of the EOs in the vapor phase. The atmosphere generated from the EOs was analyzed by means of solid-phase microextraction combined with gas chromatography-ion trap mass spectrometry. Differences among the volatiles in the EOs, which may be responsible for the differences in their antimicrobial performances, were found.