Aspergillus flavus dose-response curves to selected natural and synthetic antimicrobials

The effects of selected concentrations of antimicrobials from natural (vanillin, thymol, eugenol, carvacrol or citral) or synthetic (potassium sorbate or sodium benzoate) origin on Aspergillus flavus lag time inoculated in laboratory media formulated at water activity (a(w)) 0.99 and pH 4.5 or 3.5, were evaluated. Time to detect a colony with a diameter > 0.5 mm was determined. Mold response was modeled using the Fermi function. Antimicrobial minimal inhibitory concentration (MIC) was defined as the minimal required inhibiting mold growth for 2 months. Fermi function successfully captured A. flavus dose-response curves to the tested antimicrobials with a highly satisfactory fit. Fermi equation coefficients, Pc and k, were used to compare antimicrobials and assess the effect of pH. Important differences in Pc and k were observed among antimicrobials, being natural antimicrobials less pH dependent than synthetic antimicrobials. A large Pc value represents a small antimicrobial effect on A. flavus lag time; thus, high concentrations are needed to delay growth. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate (at pH 3.5), and sodium benzoate (at pH 3.5) than to vanillin or citral. MICs varied from 200 ppm of sodium bcnzoate at pH 3.5 to 1800 ppm of citral at both evaluated pHs.     

Synergistic inhibitory effect of citral with selected phenolics against Zygosaccharomyces bailii

Antifungal susceptibilities of Zygosaccharomyces bailii to individual and binary mixtures of citral with selected phenolics were evaluated to identify synergistic combinations. Individual effects of citral, vanillin, thymol, carvacrol, and eugenol concentrations and combined effects of citral with the other phenolic compounds on the growth of Z. bailii were evaluated in potato dextrose agar, adjusted with sucrose to a water activity of 0.99 or 0.95, and hydrochloric acid to pH 4.5 or 3.5. MICs for individual and binary antimicrobial mixtures were identified and then transformed to fractional inhibitory concentrations. Inhibitory concentrations of citral and vanillin were higher than 650 ppm, whereas for thymol, eugenol, and carvacrol, concentrations were lower than 250 ppm for several of the studied water activity-pH conditions. Combining citral with the other phenolic compounds, fractional inhibitory concentration (FIC) and FIC(Index) varied from 0.216 to 0.582. FIC(Index) demonstrated synergistic effects on Z. bailii inhibition when citral was used in combination with vanillin, thymol, carvacrol, or eugenol. Therefore, the relative amount of antimicrobials could be greatly reduced.     

柠檬醛对花生酱的防腐效果

采用平板法比较柠檬醛与合成食用防腐剂苯甲酸钠、山梨酸钾对8种霉菌的抗菌效力。结果表明，在花生酱pH5.5时，柠檬醛对多数霉菌的最低抑菌浓度（MIC）为0．10％，山梨酸钾为0．10％，而苯甲酸钠的MIC值在0．35％或0．35％以上。同时，从柠檬醛与黄曲霉产毒关系的试验中发现，柠檬醛对黄曲霉产生黄曲霉毒素有较强的抑制作用。     

Vanillin and pH Synergistic Effects on Mold Growth

The combined effects of pH (3.0–4.0) and vanillin concentration (500–1,000 ppm) on the growth of Aspergillus flavus, A. niger, A. ochraceus and A. parasiticus were evaluated in potato-dextrose agar adjusted to a water activity 0.98. Mold germination time and radial growth rates (RGR) were significantly affected by the variables (p<0.05). For equal concentration of the antimicrobial the reduction in pH had important effects lowering the RGR. The lag time increased as vanillin concentration increased and pH decreased. The inverse lag times of each mold could be described by second order polynomials.     

Inhibitory effects of selected Turkish spices and oregano components on some foodborne fungi

The inhibitory effects of 10 selected Turkish spices, oregano essential oil, thymol and carvacrol towards growth of 9 foodborne fungi were investigated in culture media with pH 3.5 and 5.5. The antifungal effects of sodium chloride, sorbic acid and sodium benzoate and the combined use of oregano with sodium chloride were also tested under the same conditions for comparison. Of the spices tested, only sodium chloride were also tested under the same conditions for comparison. Of the spices tested, only oregano at 1.0, 1.5, 2.0% (w/v) levels showed effect on all fungi. 8% (w/v) sodium chloride was less effective than oregano. Oregano essential oil, thymol or carvacrol at concentrations of 0.025% and 0.05% completely inhibited the growth of all fungi, showing greater inhibition than sorbic acid at the same concentrations. The combined use of oregano and sodium chloride exhibited a synergistic antifungal effect.     

In vitro antifungal activity of several antimicrobial compounds against Penicillium expansum

Fungicides used in the prevention and control of mold rots in stored apples are subjected to legal, social, and biological limitations. The aim of this study was to find an alternative to postharvest fungicides currently used in the prevention and control of blue mold rot caused by Penicillium expansum in apples. For this purpose, the antimicrobial activity and MIC of several substances against P. expansum were evaluated in vitro using different end point methods: agar diffusion assay, volatility method, and agar dilution and broth dilution MIC assays. Most of the substances tested are common food ingredients and have a recognized antimicrobial activity. Essential oils, such as thymol, eugenol, citral and cineole, vanillin, sodium hypochlorite, acetic acid, potassium sorbate, and hydrogen peroxide, were the substances evaluated. Thymol and citral were the essential oil components that showed the greatest inhibitory effects. The effectiveness of 5 and 10% hydrogen peroxide in growth inhibition of P. expansum in the agar diffusion assay was total, and its MIC as determined by the agar and broth dilution assays was less than 0.025%. These results indicate that the application of small quantities of hydrogen peroxide to the apple skin might be an alternative to fungicides in the elimination of P. expansum.     

SURVIVAL OF LISTERIA INNOCUA IN APPLE JUICE AS AFFECTED BY VANILLIN OR POTASSIUM SORBATE

Survival of stationary phase Listeria innocua ( as surrogate microorganism for L. monocytogenes) inoculated in apple juice (pH 3.3 or 3.8) supplemented with vanillin (1,500 ppm or 3,000 ppm) or potassium sorbate (500 ppm or 1,000 ppm) and stored at room temperature was studied. L. innocua survived in apple juice without the preservatives at pH 3.3 or 3.8, with minimal population reductions. In the juices with the incorporation of potassium sorbate or vanillin , L. innocua behavior depended on the pH value, the type of antimicrobial and its concentration. At pH 3.3, the presence of vanillin (3,000 ppm) or potassium sorbate (1,000 ppm or 500 ppm) decreased L. innocua counts, with population reductions ranging from 4 to 5 log cycles after a 4 h – 8 h exposure at 30C. However, at pH 3.8 , L. innocua showed sensitivity only to 3,000 ppm vanillin. Survival curves were successfully fitted using a Weibull type distribution of resistances.
The results suggest that the use of potassium sorbate or vanillin could prevent the survival of L. innocua in contaminated unpasteurized and pasteurized apple juice. Vanillin, a natural antimicrobial, would be particularly suitable as an antilisterial additive for less acidic apple juice.     

Study of benzoate, propionate, and sorbate salts as mould spoilage inhibitors on intermediate moisture bakery products of low pH (4.5-5.5)

A hurdle technology approach has been applied to control common mold species causing spoilage of intermediate moisture bakery products (Eurotium spp., Aspergillus spp., and Penicillium corylophilum), growing on a fermented bakery product analogue (FBPA). The factors studied included a combination of different levels of weak acid preservatives (potassium sorbate, calcium propionate, and sodium benzoate; 0-0.3%), pH (4.5-5.5), and water activity (a(w); 0.80-0.90). Potassium sorbate was found to be the most effective in preventing fungal spoilage of this kind of products at the maximum concentration tested (0.3%) regardless of a(w). The same concentration of calcium propionate and sodium benzoate was effective only at low a(w) levels. On the other hand, potassium sorbate activity was slightly reduced at pH 5.5, the 0.3% being only effective at 0.80 a(w). These findings indicate that potassium sorbate may be a suitable preserving agent to inhibit deterioration of a FBPA of slightly acidic pH (near 4.5) by xerophilic fungi. Further studies have to be done in order to adjust the minimal inhibitory concentration necessary to obtain a product with the required shelf life.     

Individual and combined effects of vanillin and potassium sorbate on Penicillium digitatum, Penicillium glabrum, and Penicillium italicum growth.

The individual and combined effects of potassium sorbate and vanillin concentrations on the growth of Penicillium digitatum, P. glabrum, and P. italicum in potato dextrose agar adjusted to water activity 0.98 and pH 3.5 were evaluated. Inhibitory concentrations of potassium sorbate varied from 150 ppm for P. digitatum to 700 ppm for P. glabrum, and for vanillin from 1,100 ppm for P. digitatum and P. italicum and 1,300 ppm for P. glabrum. Fractional inhibitory concentration (FIC) isobolograms show curves deviated to the left of the additive line. Calculated FIC index varied from 0.60 to 0.84. FIC index as well as FIC isobolograms show synergistic effects on mold inhibition when vanillin and potassium sorbate are applied in combination.     

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.     

Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium: a synergistic effect?

The use of many antimicrobial compounds in combination can achieve a good microbiological safety in food while decreasing the doses of each compound in the product. This is interesting in terms of consumer demand. In that context, combinations of five aromatic compounds (thymol, carvacrol, citral, eugenol, geraniol) and four acidic compounds (acetic acid, citric acid, lactic acid, pyropolyphosphoric acid) were investigated for their ability to inhibit the growth of Salmonella sv. Typhimurium. Many combinations of either acidic or aromatic compounds appear to be promising. Some antimicrobials, like citric acid or thymol, lead to a better amplification of inhibition than others, but no real synergistic effect has been demonstrated between compounds. Moreover, our results underline that it is important to take into account the cumulative effect of each compound when compounds are mixed.     

Bacterial microflora of carp (Cyprinus carpio) and its shelf-life extension by essential oil compounds

The microflora of common carp (Cyprinus carpio) skin, gill and intestine were analysed and the antimicrobial activities of garlic oil and nine constituents of essential oils (allyl isothiocyanate, carvacrol, cinnamaldehyde, citral, cuminnaldehyde, eugenol, isoeugenol, linalool and thymol) against the carp isolates were studied to identify compounds that might extend the shelf-life of carp fillet. A total of 90 isolated strains were identified to belong to seven genera: Acinetobacter (6), Alcaligenes (2), Bacillus (2), Flavobacterium (20), Micrococcus (2), Moraxella (6) and Pseudomonas (4), and two families Enterobacteriaceae (14) and Vibrionaceae (34). The dominant micro-organisms of carp were found to be Flavobacterium (37%) and Vibrionaceae (33%) in skin, Flavobacterium (33%) in gill and Vibrionaceae (63%) and Flavobacterium (37%) in intestine. Against these isolates, thymol, carvcarol and cinnamaldehyde had the strongest antimicrobial activities, followed by isoeugenol, eugenol, garlic oil, and then citral. The antimicrobial properties of the other constituents tested (cuminnaldehyde, linalool and allyl isothiocyanate) were low. In tests of mixed compounds, a combination of carvacrol and thymol had the highest antimicrobial activity. Moraxella, Flavobacterium and Vibrionaceae were more sensitive to the compounds, whereas Alcaligenes strains were resistant. Dipping carp fillets in a solution of 0.5% carvacrol and 0.5% thymol before storage at 5°C and 10°C reduced both the total microbial load by about 100-fold and the Volatile Bases Nitrogen (VB-N), as compared with controls. In addition, dipping treatment delayed bacterial growth and extended the shelf-life of the fillets from 4 to 12 days at low temperature (5°C). However, the treated and control fillets showed little difference during storage at 10°C. Data from sensory evaluation showed that dipped fillets in 1% (carvacrol+thymol) extended the shelf-life of carp fillets by 8 and 4 days at 5° and 10°C, respectively. Thus, carvacrol and thymol dipping can improve the microbial stability of fish fillets by removing bacteria and by inhibiting bacterial growth.     

Inhibition by gamma-irradiation and antimicrobial food additives of aflatoxin B1 production by Aspergillus flavus in poultry diet

The effect of gamma-irradiation and the antimicrobial food additives on growth and aflatoxin B₁ production by A. flavus NRRL 5520 in poultry diet was investigated. By increasing the irradiation doses the viable population as well as aflatoxin B₁ production decreased greatly. No growth and no detection of aflatoxin production occurred in poultry diet after treatment with 6 kGy. The addition of the antimicrobial food additives resulted in a significant decrease in aflatoxin B₁. Growth and toxin production was completely inhibited by 0.64% sodium propionate, 0.134% potassium sorbate and 0.1% sodium bisulfite. The combination treatments between gamma-irradiation (3 kGy) and sodium propionate (0.48%), potassium sorbate (0.022% and 0.044%) and sodium bisulfite (0.0334% and 0.0667%) inhibited completely the fungal growth and aflatoxin B₁ production. At 5 kGy, no growth was recorded and aflatoxin B₁ production was completely inhibited at all tested levels of priopionate, sorbate and bisulfite. The combined effects between gamma-irradiation and the antimicrobial agents had a synergistic effect on the mould growth and aflatoxin production in poultry diet.     

丁香和肉桂挥发油的提取、主要成分测定及其抗菌活性研究

本文采用水蒸汽蒸馏法提取丁香和肉桂的挥发油,并对所得挥发油主要成分进行气相测定,通过体外抗菌试验,比较了这两种挥发油及其主要成分和目前常用化学防腐剂苯甲酸钠和山梨酸钾对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、沙门氏菌和志贺氏菌等5株食品中常见污染菌的抗菌作用。结果表明丁香油中丁香酚含量为78.1%,肉桂油中肉桂醛含量为86.8%;这两种挥发油及其主要成分丁香酚和肉桂醛的抗菌作用均强于两种化学防腐剂,其中肉桂油与其主要成分肉桂醛表现出的抗菌活性最强,最小抑菌浓度(MIC)为200～1600mg/L;仅为苯甲酸钠和山梨酸钾MIC(6400～25600mg/L)的1/64～1/16;丁香油及其主要成分丁香酚的抗菌活性次之,MIC为800～1600mg/L,相当于苯甲酸钠和山梨酸钾MIC的1/16～1/8。     

Effects of Combined Antimicrobial Agents on Fermentation Initiation by Saccharomyces cerevisiae in a Model Broth System

Effects of sodium benzoate, potassium sorbate, sodium metabisulfite (SO₂), butyl p-hydtoxybenzoic acid and their dual combinations were observed against the ability of the wine yeast, Saccharomyces cerevisiae, Montrachet 522, to begin a fermentation in yeast nitrogen base-glucose broth at pH 3.30. Antimicrobial activities of equal molar concentrations of compounds and combinations of compounds were compared. Butylparaben and combinations involving butylparaben most effectively inhibited fermentation initiation. Combinations involving SO₂ had lower antimicrobial activities than either SO₂ or the other individual compound alone indicating antagonistic effects between SO₂ and the other antimicrobial agents tested. Apparent additive antimicrobial effects were observed for butylparaben/sorbate, butylparaben/ benzoate and benzoate/sorbate.     

Effects of antimicrobial components of essential oils on growth of Bacillus cereus INRA L2104 in and the sensory qualities of carrot broth

The possible use of antimicrobials from seven plant essential oils as food preservatives was studied by examining their effects on the growth kinetics of activated Bacillus cereus INRA L2104 spores inoculated into tyndallized carrot broth. The effects of various concentrations of borneol, carvacrol, cinnamaldehyde, eugenol, menthol, thymol, and vanillin were determined. Five microliters of cinnamaldehyde, 15 microl of carvacrol, or 30 mg of thymol per 100 ml of inoculated carrot broth completely inhibited bacterial growth for more than 60 days at 16 degrees C. Lower concentrations of the three antimicrobials prolonged the lag phase and reduced both the exponential growth rate and the final population densities of cultures. The study of the sensory characteristics of the supplemented broths suggested that low concentration of cinnamaldehyde enhanced the taste of carrot broth, and that it did not have any adverse effect on the taste and smell of carrot broth at concentrations less than 6 microl 100 ml(-1).     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

Aspergillus Flavus, Aspergillus Niger, and Penicillium Corylophilum Spoilage Prevention of Bakery Products by Means of Weak-Acid Preservatives

ABSTRACT: Apart from Eurotium species, Aspergillus and Penicillium isolates are common contaminants of bakery products. This paper deals with the use of weak-acid preservatives (potassium sorbate, calcium propionate, and sodium benzoate) to prevent spoilage by Aspergillus niger, A. flavus , and Penicillium corylophilum in analogs of a bakery product. A hurdle technology approach has been considered in which factors other than preservatives are pH and water activity. Potassium sorbate has been found to be the most effective in preventing fungal spoilage of this kind of products at the maximum concentration tested (0.3%). Suboptimal doses (0.03%) of all preservatives tested led to an enhancement of growth of Aspergillus and Penicillium isolates. The characteristics of the products involved must be carefully considered before making the decision of adding weak-acid preservatives; moreover, they must be added at the right concentrations. Furthermore, more research is needed on the use of alternative natural preservatives, such as essential oils.     

Effect of potassium sorbate and sodium benzoate on microbial population and fermentation of black olives

Black olive fermentation characteristics and diffusion of preservatives into olives were evaluated in brines containing 500 ppm potassium sorbate, 1000 ppm sodium benzoate, 500 ppm sodium benzoate + 250 ppm potassium sorbate and no preservative (control). Changes in brine pH, acidity and microbial population (lactic acid bacteria, yeasts, moulds) were followed during fermentation and storage. Results indicated that K‐sorbate when used at 500 ppm concentration in black olive fermentation had a slight stimulatory effect on the growth of lactic acid bacteria. The yeast counts of brines containing 500 ppm K‐sorbate and 1000 ppm Na‐benzoate were lower than for the brine containing 250 ppm K‐sorbate + 500 ppm Na‐benzoate and for the control with no preservative, while mould growth was completely inhibited in all treatments during fermentation. Mould counts stayed below the detection limit (<10 cfu g⁻¹) during 214 days of vacuum‐packaged storage. Yeast counts showed a progressive decline within 28 days of storage and then stayed relatively constant in all treatments thereafter. The level of yeast population was higher in the control sample than in the sample containing both preservatives during storage. The diffusion of Na‐benzoate and K‐sorbate into the olives after equilibrium was 64% and 80% during fermentation respectively. © 1999 Society of Chemical Industry     

Preservation of acidified cucumbers with a combination of fumaric acid and cinnamaldehyde that target lactic acid bacteria and yeasts

The naturally occurring compound, fumaric acid, was evaluated as a potential preservative for the long-term storage of cucumbers. Fumaric acid inhibited growth of lactic acid bacteria (LAB) in an acidified cucumber juice medium model system resembling conditions that could allow preservation of cucumbers in the presence of sodium benzoate. Forty millimolars of fumaric acid were required to inhibit growth of an extremely aciduric Lactobacillus plantarum LA0445 strain at pH 3.8. Half of this concentration was required to achieve inhibition of L. plantarum LA0445 at pH 3.5. As expected growth of the spoilage yeasts Zygosaccharomyces globiformis and Z. bailii was not inhibited by fumaric acid at near saturation concentrations in the same cucumber juice medium. To usefully apply fumaric acid as a preservative in acidified foods it will be necessary to combine it with a food grade yeast inhibitor. The antimicrobial agent, cinnamaldehyde (3.8 mM) prevented growth of Z. globiformis as well as the yeasts that were present on fresh cucumbers. Acidified cucumbers were successfully preserved, as indicated by lack of yeasts or LAB growth and microbial lactic acid or ethanol production by a combination of fumaric acid and cinnamaldehyde during storage at 30 °C for 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate, potassium sorbate, or sodium metabisulfite for preservation of acidified vegetables without a thermal process. PRACTICAL APPLICATION: This study evaluates the potential application of alternative preservatives for the long-term storage of cucumbers in a reduced NaCl cover brine solution and without a thermal process.