Survival of Penicillium expansum and Patulin Production on Stored Apples after Wash Treatments

ABSTRACT: Penicillium expansum is a widespread fungus found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, patulin. This study was undertaken to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing wash treatments that would inhibit P. expansum growth and subsequent patulin production on Empire apples destined for cider. Wash treatments included 200 ppm NaOCl, 1% StorOx®, 0.5% potassium sorbate, 300 ppm SO₂, and 0% to 5% acetic acid. Spores of P. expansum or inoculated apple slices were dipped in sanitizing wash solution for 5 min, and mold growth and patulin production was monitored on subsequent storage. It was found that 0.5% potassium sorbate and 300 ppm SO₂ did not affect mold survival or patulin production; 1% StorOx® was effective against mold spores in solution (4 log Most Probable Number destruction of spores), but there was no significant reduction in spore count when the same solution was used to sanitize mold‐inoculated apple discs. Washing with 200 ppm NaOCl delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solution (2% to 5%) was the most efficient chemical against P. expansum. A wash treatment with ≥2% acetic acid for more than 1 min is recommended to completely inhibit growth of P. expansum and subsequent patulin production on apples destined for cider.     

Thermal Inactivation of Yeasts in Fruit Juices Supplemented with Food Preservatives and Sucrose

Thermal inactivation of five yeasts (Candida krusei, Hansenula anomala, Saccharomyces bailii, S. cerevisiae and Torulopsis magnoliae) suspended in five fruit juices (apple, apricot, grape, orange and pineapple) as affected by potassium sorbate, sodium benzoate and sucrose was investigated. Yeasts were most sensitive to heat when suspended in orange juice. Both preservatives, at a concentration as low as 100 ppm, enhanced the rate of inactivation in juices containing no added sucrose. Supplementation of juices with sucrose (30 and 50%) resulted in protection against death of yeasts. The detrimental effects of potassium sorbate and sodium benzoate were reduced in the presence of high concentrations of sucrose. While neither preservative exhibited a consistent superior lethal effect compared with the other, overall, sodium benzoate was generally more effective than was potassium sorbate.     

The microbiology of apples and apple products

The apple industry has reached an annual production level of 8.5 billion pounds. CA storage of 25% of this crop has enabled a fresh market on a year‐round basis. To achieve high quality in raw fruit and processed apple products, careful attention must be paid to maintaining a microbiologically stable environment. The ecology of the microflora associated with the apple is a reflection of the orchard, handling, harvesting, and storage practices. Yeasts#lbpredominate on orchard fruit, molds may become a storage problem, and bacteria cause spoilage, off flavors, and loss of quality in juice products. Despite the microbial problems inherent in producing of quality product, the apple industry is faced with the occurrence of patulin. Patulin, a mycotoxin produced by Penicillium and Aspergillus species, has been associated with damaged fruit. Decreased temperatures, coupled with CA storage; can deter mold growth and patulin production. Laboratory detection methods for derivations of patulin are able to detect microgram quantities. Means to eliminate patulin formed in apple products include addition of ascorbate and SO₂, extending fermentation, or charcoal filtering. However, degradation products of patulin have not been evaluated toxicologically.     

A Fast and Reliable UHPLC-PDA Method for Determination of Patulin in Apple Food Products Using QuEChERS Extraction

A fast and reliable method for the quantification of patulin using ultra high performance liquid chromatography coupled to a photodiode array detector was developed and validated for the analysis of several apple-based foodstuffs. Sample preparation was based on the QuEChERS procedure. Samples were extracted with acetonitrile and partitioned with a mixture of sodium citrate, NaCl, and Mg₂SO₄. The cleanup step was performed using dispersive SPE mixed with Mg₂SO₄ and PSA. This step was carried out twice in order to improve chromatographic results. The method was validated in spiked cloudy apple juice, apple puree, apple yogurt, beer with apple juice, and cider and applied to 24 commercial samples. The limits of detection and limits of quantification were ≥0.4 and ≥2 ng/g, respectively. Both were below the maximum legal limit established by the European Union. Recoveries for all the matrices were between 78.4 and 94.7 % while relative standard deviations were between 3.8 and 10.4 %. Patulin was detected on four apple juices from concentrate, one cloudy apple juice from an eco-store, and one beer. Nevertheless, the highest concentration found was 25.4 ng/g.     

Modulation of thermal resistance of ascospores of Neosartorya fischeri by acidulants and preservatives in mango and grape juice

Minimal thermal processing is desirable for near natural organoleptic and nutritional qualities of fruit based products. In the present investigation, the effect of heat (85°C) in combination with acidulants or common preservatives on inactivation of ascospores of Neosartorya fischeri, a heat resistant mould isolated from grapes, has been studied in mango and grape juice. The ascospores were found to survive for >300 min of heating at 70, 75 and 80°C in these fruit juices and complete inactivation required 120 min of heating at 85°C. The synergistic effect of heat and organic acids or preservatives in fruit juices was noticed. The thermal death rate (1/k_85°C) values did not vary much in the presence of lactic (20), malic (20) and citric (19) acids, but tartaric acid showed least inactivation effect (1/k_85°C=54 min) in mango juice. The 1/k_85°Cvalues for ascospores of N. fischeri in mango juice containing 0·1% of potassium sorbate or sodium benzoate or combination of both at 0·05% were found to be 44, 35 and 29 min respectively. These values were respectively, 32, 13 and 14 min in grape juice. Nearly 50 and 67% of the heating time was reduced by the use of potassium sorbate and sodium benzoate (0·05% each) in mango and grape juice to inactivate 3 log number of ascospores of N. fischeri. These results may be useful in thermal processing of fruit juices.     

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.     

Inhibition of Salmonella by Sodium Nitrite and Potassium Sorbate in Frankfurters

All beef frankfurters and beef-pork frankfurters containing various levels of sodium nitrite (0, 50, or 156 ppm) or potassium sorbate (0, 0.26, or 0.39%) alone or in combination (50 ppm nitrite + 0.26% sorbate) were prepared. Frankfurters were inoculated with nalidixic acid resistant Salmonella and incubated at 15°C and 27°C for up to 21 days. Frankfurters formulated with 50 or 156 ppm nitrite and incubated at 27°C, or 50 ppm nitrite and incubated at 15°C failed to inhibit the growth of Salmonella. Sorbate alone and sorbate in combination with 50 ppm nitrite were equally effective in inhibiting Salmonella at either temperature and were equivalent to 156 ppm nitrite in inhibiting Salmonella at 15°C.     

Effects of apple and pear varieties and pH on patulin accumulation by Penicillium expansum

BACKGROUND: Accumulation of patulin in apples and pears and subsequently in their by‐products is caused mostly by Penicillium expansum which causes blue mould. Fruit pH and other parameters are sometimes characteristic of a certain variety and thus the use of particular varieties might affect patulin content in the final products. The aim of this study was to evaluate the influence of fruit variety and pH on patulin accumulation. Patulin accumulation in both apple and pear juices at different pH and also in different apple varieties (Golden, Gala and Fuji) and pear varieties (Blanquilla and Conference) was assessed. RESULTS: The pH of juices significantly influenced patulin accumulation, especially in apple juice in which the highest amounts of patulin were detected at pH 3.5. In fruits, the pH values of the substrate were determinant only under cold storage. Thus, Golden apples, which presented a lower pH, accumulated more patulin at 1 °C. However, this trend was not observed at other temperatures in which varieties with higher amounts of organic acids (Golden and Fuji apples) accumulated most patulin. In the pear varieties, significant differences in pH did not lead to significant differences in patulin accumulation. CONCLUSION: Although pH influences patulin accumulation, other factors such as organic acid content may play an important role. Concerning the apple and pear varieties we studied, it seems that rather than variety, other parameters like pH or acidic content may be more important. These parameters vary significantly depending on the degree of ripeness of the fruit. Gala apple, which is used in Spanish juice production, was the only variety that accumulated dramatically higher amounts of patulin. Copyright © 2008 Society of Chemical Industry     

Citric Acid and Antimicrobials Affect Microbiological Stability and Quality of Tomato Juice

Acidification, addition of dimethyl dicarbonate and a mixture of potassium sorbate and sodium benzoate and storage temperature were related to microbiological stability and quality of tomato juice. Whether iuices were acidified CDH 4.0 to 3.7) or nonacidified. dimethyl dicarbonate and sorbate/benzoate were highly effective in diminishcng mold and yeast count at 5°C and 20°C. Dimethyl dicarbonate in juices acidified to pH 3.7 and stored at 5°C was most effective in controlling plate counts, followed by sorbate/benzoate in juices acidified to pH 3.7 and stored at 5°C and 20°C. Tomato juice treated with dimethyl dicarbonate had lower amounts of ascorbic acid, total amino acids, fructose, glucose, lycopene and β-carotene.     

Control of Native Spoilage Yeast on Dealcoholized Red Wine by Preservatives Alone and in Binary Mixtures

In order to preserve a commercial dealcoholized red wine (DRW), a study with 4 preservatives and binary mixtures of them were performed against 2 native spoilage yeasts: Rhodotorula mucilaginosa and Saccharomyces cerevisiae. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for potassium sorbate, sodium benzoate, sodium metabisulfite and dimethyl dicarbonate (DMDC) were evaluated in DRW stored at 25 °C. MICs of potassium sorbate and sodium metabisulfite were 250 and 60 mg/kg, respectively for both target strains. However for sodium benzoate, differences between yeasts were found; R. mucilaginosa was inhibited at 125 mg/kg, while S. cerevisiae at 250 mg/kg. Regarding MFC, differences between strains were only found for sodium metabisulfite obtaining a MFC of 500 mg/kg for R. mucilaginosa and a MFC of 250 mg/kg for S. cerevisiae. Potassium sorbate and sodium benzoate showed the MFC at 1000 mg/kg and DMDC at 200 mg/kg. Regarding the effect of binary mixtures the Fractional Fungicidal Concentration Index (FFC_i) methodology showed that binary mixtures of 100 mg/kg DMDC/200 mg/kg potassium sorbate (FFC_i = 0.7) and 50 mg/kg DMDC / 400 mg/kg sodium benzoate (FFC_i = 0.65) have both synergistic effect against the 2 target strains. These binary mixtures can control the growth of spoilage yeasts in DRW without metabisulfite addition. The results of this work may be important in preserving the health of DRW consumers by eliminating the use of metabisulfite and reducing the risk of growth of R. mucilagosa, recently recognized as an emerging pathogen.     

Effect of heat treatment and evaporation on patulin and some other properties of apple juice

Patulin, hydroxymethylfurfural (HMF), colour and clarity values of 20 apple juice samples with different Brix contents were determined after heat treatment (90 and 100 °C for 5, 10, 15 and 20 min under atmospheric pressure) and evaporation (70 and 80 °C for 5, 10, 15 and 20 min) processes. As the heating and evaporation times increased, the concentration of patulin in the apple juice samples decreased. The 90 and 100 °C heat treatments resulted in reductions in patulin concentration of 18.81 and 25.99% respectively after 20 min; the corresponding values were 9.40 and 14.06% for 70 and 80 °C evaporation respectively. The increase in HMF was higher with heat treatment than with evaporation. Evaporation decreased the colour values of the samples, whereas heat treatment increased them. © 2003 Society of Chemical Industry     

Kultur vonAspergillus parasiticus im Apfelsaft

Zusammenfassung Kulturen vonAspergillus parasiticus NRRL 2999 in Apfelsaft (aus Sirup) wurden Natriumbenzoat bzw. Kaliumsorbat (100, 200, 300 und 400 mg/l zugesetzt, danach wurden diese bei 25 °C, 3, 6, 9, 12, oder 15 Tage bebrütet und Myceltrokkengewicht, pH und Konzentration der Aflatoxine B₁ und G₁ bestimmt. Der pH-Anfangswert von 2,5 blieb in allen Fällen während der ganzen Inkubationsdauer unverändert. Natriumbenzoat unterdrückte bei allen getesteten Konzentrationen das Wachstum und förderte die Biosynthese von Aflatoxin G₁, während es die Anhäufung von B₁ wenig beeinflußte. Kaliumsorbat förderte das Wachstum bei 100 mg/kg, doch alle getesteten Konzentrationen inhibierten die Produktion der Toxine erheblich (keine nachweisbaren Mengen von B₁ und 3- bis 5 mal weniger G₁ als in der Kontrollreihe). Ausnahmslos wurde Aflatoxin G₁ stärker als B₁ angehäuft.

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The cultivation ofAspergillus parasiticus on apple juiceI. Influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Summary Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures ofAspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 °C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B₁ and G₁. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, supressed fungal growth and stimulated the biosynthesis of G₁, whereas little influence was exerted upon the accumulation of B₁. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B₁ and 3 to 5 times less G₁ than in controls). The concentration of G₁ surpassed that of B₁ without exception.

     

Storage stability of pineapple slices preserved by combined methods

Storage stability during 3 months was evaluated for pineapple slices preserved by combined methods (blanching for 1 min with saturated vapour, a_w reduction by osmotic concentration in sucrose syrup, pH reduction by addition of citric acid, and the addition of 1000 parts per million (ppm) of potassium sorbate and 150 ppm of sodium bisulphite). Pineapple slices were microbiologically sound and their moisture content, °Brix, pH and a_w remained almost constant during storage with mean values of 71.8%, 26.1, 3.65 and 0.978, respectively. Total sulphite and potassium sorbate concentrations of the pineapple slices were reduced during storage at 5 and 25 °C, being final concentrations 51% and 38% and 63% and 48% of the initial concentrations, respectively. Colour, texture and sensory evaluation revealed that there were no significant (P < 0.05) changes between samples stored at 5 or 25 °C. Good overall acceptability (initial mean score 7.21) of preserved pineapple slices even after 3 months storage at 25 °C (final mean score 7.02) was observed.     

Effect of Sodium Acid Pyrophosphate in Combination with Sodium Nitrite or Sodium Nitrite/Potassium Sorbate on Clostridium botulinum Growth and Toxin Production in Beef/Pork Frankfurter Emulsions

Combinations of sodium acid pyrophosphate (SAPP) with added sodium nitrite and/or potassium sorbate were tested at various pH levels to determine effectiveness in delaying Clostridium botulinum growth and toxin production in frankfurter emulsions. Formulations containing sodium nitrite (40 ppm), potassium sorbate (0.26%) and SAPP (0.4%) resulted in a greater delay of toxin production (12–18 days) than other combinations (6–12 days) having similar pH values. Treatments containing 0.4% SAPP appeared to be more inhibitory than their counterparts without SAPP, displaying less numbers of toxic samples during the 53-day storage period at 27°C. Aerobic mesophilic colony counts and residual nitrite data showed little difference among treatments.     

Sensory evaluation of high-moisture dried prunes preserved with sodium benzoate

Both sodium benzoate and potassium sorbate were significantly detected (P < 0.05) in prune juice at levels of 500 to 600 parts/million. Detection levels for both preservatives in high-moisture dried prunes were higher (about 800 parts/million). No significant flavour differences were found between prunes treated with similar amounts of sodium benzoate and potassium sorbate. Since it was necessary to use more sodium benzoate than potassium sorbate to preserve prunes, comparisons were made between prunes containing equally effective levels of the two preservatives. No significant flavour differences were found between prunes effectively preserved with sodium benzoate and potassium sorbate. This suggests that sodium benzoate could be used as a preservative for high-moisture dried prunes with essentially no flavour problem.     

Mathematical Modeling of Microbial Growth in Packaged Refrigerated Orange Juice Treated with Chemical Preservatives

ABSTRACT Microbial flora of refrigerated orange juice was analyzed during storage at 10 °C and the effects of the following factors were discussed: 1) the previous washing process of the orange peel, 2) the different levels of the added preservatives (citric acid, ascorbic acid, potassium sorbate, sodium benzoate), 3) the gaseous permeabilities of the packaging film. Gompertz equation was applied to model molds and yeasts growth for the different treatments and packaging conditions. The washing procedure with sodium hypochlorite extend 2–3 d the storage life of the juice (time to reach microbial counts of 10⁶ CFU/ml) in both packaging films. The use of organic acids and potassium sorbate or sodium benzoate (1.66–6.94 mM) led to storage life values > 11 d in polyethylene and > 20 d in the low gaseous permeability film, maintaining good sanitary conditions.     

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [¹⁴C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a λ_max = 271nm, identical to that of ascladiol. Thenmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.     

利用Saccharomyces cerevisiae KD控制苹果汁中棒曲霉素的污染

研究一株食品生产用酿酒酵母Saccharomyces cerevisiae KD在培养基及市售100%苹果汁中对棒曲霉素污染的控制作用。通过高效液相色谱法对棒曲霉素进行定量,分析起始棒曲霉素浓度、菌体接种量和培养基pH对S. cerevisiae KD去除棒曲霉素活力的影响;利用酶标仪监测S. cerevisiae KD的生长状况,且通过检测可溶性固形物、酸度、总酚、黄酮含量对菌体发酵后苹果汁的品质进行了评估。结果表明:有氧条件下S. cerevisiae KD能够在28 h内完全去除培养基中的棒曲霉素,其去除机理包括物理吸附和酶解;在较低的起始棒曲霉素浓度和较高的菌体接种量条件下,S. cerevisiae KD对棒曲霉素的去除率较高,但在培养后期,不同菌体接种量下棒曲霉素的去除率接近一致;实验还发现酸性条件有利于S. cerevisiae KD去除棒曲霉素。此外,S. cerevisiae KD对棒曲霉素的耐受性较强,甚至在棒曲霉素浓度高达100 mg/L的环境中依然能较好生长。在市售100%苹果汁中,S. cerevisiae KD也能高效控制棒曲霉素的污染,且与Lactococcus lactis MG1363联合发酵2 d后,果汁中已无棒曲霉素检出,总酚含量显著高于发酵前苹果汁(p<0.05),发酵果汁的品质较好。结论:S. cerevisiae KD可有效控制食品中棒曲霉素的污染,具有潜在的应用前景。     

REDUCTION OF PATULIN IN APPLE JUICE CONCENTRATES DURING STORAGE

In this study, the reduction of patulin content in apple juice concentrates during 6 months of storage at 22 and 30C was investigated. Results demonstrated that reduction in patulin content was dependent on the storage temperature and time. Patulin reductions after 1 month of storage at 22 and 30C were in the ranges of 45–64% and 66–86%, respectively. Levels of patulin were below detectable limits after 4 months of storage at 22 and 30C.