Preservative tolerance and resistance

Preservative tolerance is defined as a situation in which a formerly effective preservative system no longer controls microbial growth. Tolerance can have several potential causes: destabilization of the preservative, establishment of biofilms in the manufacturing system, and the development of resistance. As part of an effort to understand and control tolerance, a collection of preservative-resistant bacteria was established. Pseudomonas and related genera constitute 92% of the isolates in the collection. The collection includes bacteria isolated from products preserved with dimethoxy dimethyl hydantoin (DMDMH), dibromodicyanobutane (DBCB), glutaraldehyde (GLUT), benzisothiazolone (BIT), and methylchloroisothiazolone/methylisothiazolone (MCI/MI). Several isolates were also obtained from products preserved with combinations of DMDMH/iodopropargyl butylcarbamate (IPBC) and DMDMH/IBPC/MCI/MI. Bacteria were deemed resistant if they were isolated from a matrix in which the preservative was stable, and their minimal inhibitory concentrations (MIC) were significantly elevated as compared to a group of conspecific strains from the American Type Culture Collection. Isolates resistant to DMDMH, DBCB, or GLUT had normal outer membrane protein (OMP) profiles, and were cross-resistant to formaldehyde (FA) and several other FA-releasing preservatives, a pattern typical of strains producing elevated levels of FA dehydrogenase. The BIT-resistant isolates were cross-resistant to a number of structurally dissimilar preservatives but retained susceptibility to MCI. MCI-resistant isolates had altered OMP profiles, displayed reduced uptake of MCI, and were cross-resistant to some other preservatives. Restoration of sensitivity to MCI by treatment with EDTA confirmed the role of the outer membrane in MCI resistance.     

Control of Some Filamentous Fungi and Yeasts by Dehydrated Allium Extracts

The antifungal activities of dehydrated garlic (Allium sativum L.), onion (Allium cepa L.) and leek (Allium porrum L.) against Aspergillus niger, Fusarium oxysporum, Candida albicans ATCC 10231 and Metschnikowia fructicola were investigated. Inhibition activities of the ethyl alcohol or acetone extracts of dehydrated Allium species were studied by disc-diffusion and broth dilution methods. The Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (MFC) were found in the range of 75 and 100 mg/mL (w/v). Ethyl alcohol extracts of dehydrated onion (Allium cepa L.) in the range of 75 and 175 mg/mL were determined as the most inhibitory MIC and MFC for A. niger, F. oxysporum and C. albicans respectively. The extracts possess antifungal activity against some of the tested filamentous fungi and yeasts at various concentrations.     

Control of Some Filamentous Fungi and Yeasts by Dehydrated Allium Extracts

The antifungal activities of dehydrated garlic (Allium sativum L.), onion (Allium cepa L.) and leek (Allium porrum L.) against Aspergillus niger, Fusarium oxysporum, Candida albicans ATCC 10231 and Metschnikowia fructicola were investigated. Inhibition activities of the ethyl alcohol or acetone extracts of dehydrated Allium species were studied by disc-diffusion and broth dilution methods. The Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (MFC) were found in the range of 75 and 100 mg/mL (w/v). Ethyl alcohol extracts of dehydrated onion (Allium cepa L.) in the range of 75 and 175 mg/mL were determined as the most inhibitory MIC and MFC for A. niger, F. oxysporum and C. albicans respectively. The extracts possess antifungal activity against some of the tested filamentous fungi and yeasts at various concentrations. Received: July 8, 2008; accepted: July 30, 2008     

Determination of seven preservatives in cosmetic products by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method using cetyltrimethylammonium bromide (CTAB) as a cationic surfactant, coupled with UV–Vis detection, was developed for the simultaneous determination of seven preservatives, including methyl‐, ethyl‐, propyl‐ and butyl‐paraben and phenol, phenoxyethanol and resorcinol. The method involved optimizing the pH of the phosphate buffer and concentrations of CTAB, ethanol and 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD). The preservatives were well separated using optimum conditions and separated within 10 min at a separation voltage of ?12.5 kV with the 1.0 mM phosphate buffer (pH 7.0) containing 90 mM CTAB, 25 mM HP‐β‐CD and 10% (v/v) ethanol. Satisfactory recoveries (84.1–103.0%), migration time (RSD < 3.1%) and peak area (RSD < 4.5%) repeatabilities were achieved. Detection limits of the preservatives were between 0.31 and 1.52 μg mL^?1 (S/N = 3, n = 5). The optimized method was successfully applied to the simultaneous determination of these preservatives in 10 commercial cosmetic products.     

不同防腐剂对3 种模式腐败菌抑菌效果的比较

目的：研究食品防腐剂对腐败菌的抑菌效果。方法：采用微量稀释法测定不同防腐剂对藤黄微球菌、枯草芽孢杆菌和大肠杆菌的最小抑菌质量浓度(MIC)。结果：对于藤黄微球菌(G+),抑菌效果最好的是乳酸链球菌素(Nisin)与ε- 聚赖氨酸,MIC 均为12.5μg/mL;对于枯草芽孢杆菌(G+),抑菌效果最好的是丙酸钙、丁二酮、柠檬酸、柠檬醛和D- 异抗坏血酸钠,MIC 均为6.25μg/mL;对于大肠杆菌(G －),抑菌效果最好的是过氧化氢和丁二酮,MIC 均为25μg/mL。结论：不同防腐剂对3 种模式腐败菌的抑制效果有所差异,应复配使用。     

酶标比浊法评价月桂酸单甘油酯对肉葡萄球菌的抑菌活性

以分离自鱿鱼丝的肉葡萄球菌S1为试验菌株,采用酶标比浊法测定了月桂酸单甘油酯(GML)、乳酸链球菌素(Nisin)、双乙酸钠、柠檬酸和山梨酸钾对试验菌的最低抑菌浓度(MIC),GML的抑菌生长曲线,以及GML与Nisin、双乙酸钠和柠檬酸复配物对试验菌的抑菌增效性;结合涂布计数法确定选用抑菌剂的最低杀菌浓度(MBC)及GML对试验菌的动态杀菌曲线。试验结果表明:GML对试验菌的MIC和MBC分别为25μg/mL和50μg/mL,均低于其它抑菌剂;GML对试验菌抑制生长曲线及动态杀菌曲线表明,较低浓度的GML的存在即可有效延长试验菌的延滞期,2×MBC浓度的GML可在4.5h内使试验菌全部失活;同时,GML与Nisin、柠檬酸和双乙酸钠均显示出较好的复配增效性。GML对试验菌具有极强的抑制作用,且与多种抑菌剂显示出较好的增效性。     

Evaluation of Natural Antimicrobials on Typical Meat Spoilage Bacteria In Vitro and in Vacuum-Packed Pork Meat

ABSTRACT: The aim of this study was to investigate the inhibitory effect of natural antimicrobials on the growth of typical spoilage bacteria from marinated pork. Minimum inhibitory concentrations (MIC) of thymol, cinnamaldehyde, allyl isothiocyanate, citric acid, ascorbic acid, a rosemary extract, and a grapefruit seed extract against Lactobacillus algidus, Leuconostoc mesenteroides, Leuconostoc carnosum, Carnobacterium maltaromaticum, Carnobacterium divergens, Brochothrix thermosphacta, and Serratia proteamaculans were determined in a microplate assay. Combinations of antimicrobials were tested and several combinations showed synergistic effects in inhibiting bacterial growth. Single and combined antimicrobials were added to vacuum-packed pork meat to evaluate preserving effects. Antimicrobial concentrations of up to 10 times the MIC values showed no effect on total bacterial growth in vacuum packed pork meaning that although most antimicrobials inhibited the growth of spoilage bacteria in vitro, results from the microplate assay could not be transferred to the meat system. Most natural antimicrobials possess strong odor and flavor that limit their use as a food preservative. In conclusion, this study showed that the use of natural antimicrobials in meat products is limited and that bacterial quality and shelf life was not enhanced under the chosen conditions.     

Effects of methanolic extract of sour cherry (Prunus cerasus L.) on microbial growth

The antimicrobial activity of a methanolic extract of the common edible fruit Prunus cerasus L. was evaluated. HPLC analysis of the extract revealed the abundant presence of anthocyanins. Minimum Inhibitory Concentrations (MIC) for Gram‐positive and Gram‐negative pathogenic bacteria was in the range 2–6.6 mg mL^?1, whereas the time‐kill assay revealed that the bactericidal effect was exerted only at concentrations higher than 2× MIC. Interestingly, at concentrations lower than MIC, P. cerasus L. extract exerted a stimulating effect on bacterial proliferation and on the ability to form biofilms. In particular, a multidrug‐resistant Acinetobacter baumannii strain showed a 30% increase in growth at low concentrations. In the light of this finding, we speculate that the use of berry extracts (rich in anthocyanins) may present some risks to human health when used as preservatives in food and cosmetic products.     

Czech Ethanol‐Free Propolis Extract Displays Inhibitory Activity against a Broad Spectrum of Bacterial and Fungal Pathogens

Propolis acts primarily as a biocide against invasive bacteria and fungi in the hive, suggesting its potential for industrial applications. In food application, propolis is considered as a chemical preservative in meat products, extending shelf life of frozen meat and other food. The mechanism of action is still unclear due to the synergy of multiple compounds contained in propolis and due to parallel targeting of multiple pathways within each affected organism. Here, we examined the antimicrobial properties of dimethylsulfoxide (DMSO) Czech propolis extract. Until recently, DMSO was only rarely used in the propolis studies, although the other solvents tested (mostly ethanol) may significantly affect the observed inhibitory effects, notwithstanding the antimicrobial effects of ethanol itself. Here, we provide results of zone inhibition tests against Aspergillus fumigatus, Microsporum gypseum, Microsporum canis, Candida albicans, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. Although we determined inhibitory effects against all the microorganisms tested, the dose‐dependent response curves were not similar to each other. While inhibitory effects against C. albicans or S. aureus were strictly dose‐dependent, responses of M. gypseum and E. faecalis displayed plateau across the broad range of concentrations tested. Interestingly, response of E. coli revealed the double‐peak dose‐dependent curve, and responses of M. canis and L. monocytogenes decreased at the highest concentrations tested. Suggested is evaluation of DMSO propolis extracts in experimental treatment of human and veterinary infections, preferably in multitherapy with antibiotics.     

防腐剂对地衣芽孢杆菌的抑制效果

通过药敏纸片法、最小抑菌质量浓度(MIC)法测定地衣芽孢杆菌对不同防腐剂的敏感性,利用正交试验设计筛选出最佳的防腐剂组合,并对获得的最佳防腐剂组合的抑菌效果进行验证。结果表明:地衣芽孢杆菌对山梨酸钾、乳酸钠不敏感;对Nisin、ξ-聚赖氨酸和尼泊金乙酯较为敏感,在20mg/mL时其抑菌圈直径分别为9.25、10mm和7.5mm,MIC分别为0.078、0.078mg/mL和2.5mg/mL。正交试验结果表明:当Nisin、ξ-聚赖氨酸和尼泊金乙酯质量浓度分别为0.313、0.313mg/mL和0.125mg/mL时,对地衣芽孢杆菌的抑制效果最佳,可使基质中的地衣芽孢杆菌数(空白样品中为5.48(lg(CFU/g))降低2(lg(CFU/mL))。选择这一最佳组合分别对其在肉制品、乳制品、果汁中的应用效果进行测定,结果显示在0～4℃条件下,复配防腐剂的效果较好。     

Isolation of antibacterial components from infusion of Caesalpinia paraguariensis bark. A bio-guided phytochemical study

The antimicrobial activities and toxicity of infusion, decoction and tincture of Caesalpinia paraguariensis Burk. bark (CPBEs) were investigated to validate its traditional use as drink additive and to identify microbicidal component(s). The minimum inhibitory concentrations (MIC) of CPBEs against aerobic bacteria (Gram-negative and Gram-positive species) were determined using standardised dilution methods. The LC₅₀ were determined by Brine Shrimp Test. CPBEs showed bacteriostatic and bactericidal activity against tested strains. The highest activity was observed for infusion (MIC:200 μg/mL) against Morganella morganii, Erwinia carotovora, Bacillus spp., Staphylococcus spp. and Enterococcus spp. The bacterial species were susceptible to CPBEs (MIC:200–1993 μg/mL) at lower concentration than sodium benzoate, a known food preservative. Two bioactive components were isolated from liophylised infusion by bio-guided chromatographic procedures; these were identified by spectrometric techniques as ellagic and 3-O-methylellagic acids. This study demonstrated that C. paraguariensis bark infusion it is safe for human consumption and a possible source of food natural preservatives.     

Inhibition of Listeria monocytogenes by food antimicrobials applied singly and in combination

Abstract: Combining food antimicrobials can enhance inhibition of Listeria monocytogenes in ready-to-eat (RTE) meats. A broth dilution assay was used to compare the inhibition of L. monocytogenes resulting from exposure to nisin, acidic calcium sulfate, ɛ-poly-L-lysine, and lauric arginate ester applied singly and in combination. Minimum inhibitory concentrations (MICs) were the lowest concentrations of single antimicrobials producing inhibition following 24 h incubation at 35 °C. Minimum bactericidal concentrations (MBCs) were the lowest concentrations that decreased populations by ≥3.0 log₁₀ CFU/mL. Combinations of nisin with acidic calcium sulfate, nisin with lauric arginate ester, and ɛ-poly-L-lysine with acidic calcium sulfate were prepared using a checkerboard assay to determine optimal inhibitory combinations (OICs). Fractional inhibitory concentrations (FICs) were calculated from OICs and were used to create FIC indices (FIC_Is) and isobolograms to classify combinations as synergistic (FIC_I < 1.00), additive/indifferent (FIC_I= 1.00), or antagonistic (FIC_I > 1.00). MIC values for nisin ranged from 3.13 to 6.25 μg/g with MBC values at 6.25 μg/g for all strains except for Natl. Animal Disease Center (NADC) 2045. MIC values for ɛ-poly-L-lysine ranged from 6.25 to 12.50 μg/g with MBCs from 12.50 to 25.00 μg/g. Lauric arginate ester at 12.50 μg/g was the MIC and MBC for all strains; 12.50 mL/L was the MIC and MBC for acidic calcium sulfate. Combining nisin with acidic calcium sulfate synergistically inhibited L. monocytogenes; nisin with lauric arginate ester produced additive-type inhibition, while ɛ-poly-L-lysine with acidic calcium sulfate produced antagonistic-type inhibition. Applying nisin along with acidic calcium sulfate should be further investigated for efficacy on RTE meat surfaces. Practical Application: This study demonstrates the potential for combinations of antimicrobials to result in greater pathogen inhibition as compared to the application of a single antimicrobial. The data presented in this study can aid the food industry in developing more efficient and effective application of antimicrobials. These findings should also prompt further studies validating the inhibitory effect of combinations of antimicrobials on ready-to-eat surfaces.     

冷却肉天然复合保鲜剂配比的优化

根据中心复合设计原理,在琼脂扩散法得出3 种天然保鲜剂最小抑菌浓度基础上,运用Minitab14 数据统计分析软件,采用四因素二水平中心复合响应曲面分析法,确定保鲜剂的最佳配比为Nisin 0.2773g/100mL、溶菌酶0.46g/100mL、乳铁蛋白0.604g/100mL、pH5.16。pH 值与乳铁蛋白、pH 值与溶菌酶、pH 值与Nisin、乳铁蛋白与溶菌酶以及溶菌酶与Nisin 之间均存在显著的交互作用(P＜ 0.01),而乳铁蛋白与Nisin 交互作用不显著(P＞0.05)。单因素对细菌总数影响的大小次序为Nisin ＞溶菌酶＞ pH 值＞乳铁蛋白。     

Antimicrobial activity and cytotoxicity towards cancer cells of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> (tea tree) oil

Antimicrobial and in vitro anti-tumor activities of Melaleuca alternifolia (tea tree) oil (TTO) were analyzed. The results indicated that TTO possessed significant antimicrobial activity towards all tested microorganisms except Aspergillus niger. The minimum inhibitory concentration (MIC) of TTO towards Staphylococcus epidermidi, Bacillus subtilis, Escherichia coli and Candida albicans was 0.25% (v/v). Time kinetic experiments showed that Staphylococcus epidermidi, Escherichia coli and Candida albicans were completely killed after exposure to TTO at 2× MIC for 1, 4 and 8 h, repectively. TTO also exhibited strong cytotoxicity towards human lung cancer cell line (A549), human breast cancer cell line (MCF-7) and human prostate cancer cell line (PC-3). The IC₅₀ values obtained were 0.012% (v/v), 0.031% (v/v) and 0.037% (v/v), respectively. Flow cytometric analysis demonstrated that TTO restrained the cell cycle progression of PC-3 and MCF-7 cell lines at the G0/G1 phase and A549 cell at the S phase. X. Liu and Y. Zu contributed equally to this paper.     

桦菌芝多糖抗氧化性及抑菌活性研究

以桦菌芝为原料,采用水提取乙醇沉淀法提取桦菌芝多糖,分析其清除DPPH自由基和ABTS自由基能力,并用滤纸片法测定不同浓度桦菌芝多糖对大肠杆菌、金黄色葡萄球菌、枯草杆菌和四联球菌的抑菌活性及最低抑菌浓度(MIC)。结果表明,桦菌芝多糖对DPPH自由基和ABTS自由基均具有消除作用,当浓度为1.0mg/mL时,桦菌芝多糖对DPPH自由基和ABTS自由基的清除能力均超过BHT对照品,但较弱于VC对照品;不同浓度桦菌芝多糖的抗氧化活性具有显著性差异。桦菌芝多糖对4种受试菌均具有抑制作用且存在显著性差异,其中对大肠杆菌的抑制活性最强。大肠杆菌、金黄葡萄球菌、枯草杆菌和四联球菌的最小抑菌浓度分别为1.25,1.25,2.50,5.00mg/mL。综上,桦菌芝多糖具有较好的抗氧化及抑菌活性,且抗氧化及抑菌活性随多糖浓度的增大而逐渐加强。     

Minimum Inhibitory Concentration Studies of Antimicrobic Combinations Against Saccharomyces cerevisiae in a Model Broth System

Minimum inhibitory concentration (MIC) studies were conducted to observe synergistiC., antagonistic or additive effects produced by combinations of butylparaben, ethanol, potassium sorbate and sodium metabisulfite against growth of two Saccharomyces cerevisiae strains (Montrachet 522 and NCSU 76) in yeast nitrogen base-glucose broth at pH 3.30. Results suggested antagonistic effects between SO₂/sorbate, SO₃/butylparaben and butylparaben-ethanol. Additive effects were observed between ethanol/sorbate, ethanol/SO₂, and butylparaben/sorbate. No synergistic effects were detected. NCSU 76 had higher MICs than Montrachet 522 for all individual antimicrobial agents indicating greater resistance to these compounds. On an individual molar basis, MIC results indicated butylparaben was more inhibitory than the other antimicrobial agents alone.     

In vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms

The aim of this study was to evaluate the antimicrobial efficacy of selected plant essential oil (EO) combinations against four food-related microorganisms. Ten EOs were initially screened against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae using agar disk diffusion and broth dilution methods. The highest efficacy against all the tested strains was shown when testing the oregano EO. EOs of basil and bergamot were active against the Gram-positive bacteria (S. aureus and B. subtilis), while perilla EO strongly inhibited the growth of yeast (S. cerevisiae). The chemical components of selected EOs were also analyzed by GC/MS. Phenols and terpenes were the major antimicrobial compounds in oregano and basil EOs. The dominant active components of bergamot EO were alcohols, esters and terpenes. For perilla EO, the major active constituents were mainly ketones. The checkerboard method was then used to investigate the antimicrobial efficacy of EO combinations by means of the fractional inhibitory concentration index (FICI). Based on an overall consideration of antimicrobial activity, organoleptic impact and cost, four EO combinations were selected and their MIC values were listed as follows: oregano–basil (0.313–0.313 μl/ml) for E. coli, basil–bergamot (0.313–0.156 μl/ml) for S. aureus, oregano–bergamot (0.313–0.313 μl/ml) for B. subtilis and oregano–perilla (0.313–0.156 μl/ml) for S. cerevisiae. Furthermore, the mechanisms of the antimicrobial action of EO combinations to the tested organisms were studied by the electronic microscopy observations of the cells and the measurement of the release of cell constituents. The electron micrographs of damaged cells and the significant increase of the cell constituents' release demonstrated that all EO combinations affected the cell membrane integrity.     

Inhibition of individual and combination of cell free supernatants of phenyllactic acid,pediocin- and nisin-producing lactic acid bacteria against food pathogens and bread spoilage molds

Consumer demand has led to an increase in the use of natural antimicrobials for baked good preservation. In this study, the cell-free culture supernatants of three antagonistic bacterial strains were tested, and their individual and combined antimicrobial effects on Escherichia coli, Bacillus cereus, Aspergillus neoniger, and Penicillium roqueforti were examined. The mixture of phenyllactic acid and pediocin with ethylenediaminetetraacetic acid (EDTA) exhibited a strong inhibitory effect on E. coli growth (RIA 85%). Nisin mixed with phenyllactic acid or pediocin with EDTA exhibited a strong inhibitory effect on B. cereus growth with RIA 85% and 80%, respectively. Phenyllactic acid had a stronger inhibitory effect on P. roqueforti than on A. neoniger at 50 mg/ml MIC. The antipathogenic effects of phenyllactic acid, nisin, and pediocin (alone or in combination, with or without adjuncts) were stronger than their mold-controlling effects. A combination of such biopreservatives with EDTA is a suitable alternative to the preservatives used to improve the shelf life of perishable food products.     

Antimicrobial activity of binary combinations of natural and synthetic phenolic antioxidants against Enterococcus faecalis

This study investigated the antimicrobial activity of 3 natural (thymol, carvacrol, and gallic acid) and 2 synthetic [butylated hydroxyanisole (BHA) and octyl gallate] phenolic compounds, individually and in binary combinations, on 4 dairy isolates of Enterococcus faecalis with different virulence factors (β-hemolytic, gelatinase, or trypsin activities; acquired resistance to erythromycin or tetracycline; and natural resistance to gentamicin). A checkerboard technique and a microdilution standardized method were used. All compounds individually tested exhibited antimicrobial activity against E. faecalis, with minimal inhibitory concentrations (MIC) ranging from 30 μg/mL (octyl gallate) to 3,150 μg/mL (gallic acid), although no significant differences were detected among strains to each phenolic compound. Carvacrol in combination with thymol or gallic acid, and gallic acid combined with octyl gallate showed partial synergistic inhibition of all E. faecalis strains. The most effective combinations were thymol + carvacrol and gallic acid + octyl gallate, as the MIC for each of these compounds was reduced by 67 to 75% compared with their respective individual MIC. These results highlight the possibility of using combinations of these phenolic compounds to inhibit the growth of potential virulent or spoilage E. faecalis strains by reducing the total amount of additives used in dairy foods.     

Characterisation and cooperative antimicrobial properties of chitosan/nano-ZnO composite nanofibrous membranes

Chitosan was combined with nano-ZnO to increase its antimicrobial activity, using polyvinyl alcohol as a support, and then were electronspun to form composite nanofibres. Through SEM, EDX and XRD observations, chitosan was seen to be able to incorporate nano-ZnO in the composite nanofibres. Escherichia coli, expressing recombinant enhanced green fluorescent protein, and Candida albicans were used to test the antimicrobial efficacy of the newly synthesised chitosan/nano-ZnO antimicrobial composite. The CdTe quantum dots were used to rapidly detect the residual changes of C. albicans and determine the end point of using antimicrobial agents. Minimal minimum inhibitory concentration (MIC), post-antibiotic effect and continuous agent effect of the composite were determined. The MIC of chitosan/nano-ZnO against C. albicans was 160 μg/ml, close to the concentration of the treated composite with the lowest fluorescence intensity. The cell damage was observed by SEM, which indicated that nano-ZnO in the nanofibrous membranes played a cooperative role in the antimicrobial process of chitosan.