Heat resistance of five spoilage microorganisms in a carbonated broth.

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO2 on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO2 and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment. Five microorganisms were selected for this study: Alicyclobacillus acidoterrestris (spores), Aspergillus niger (spores), Byssochlamys fulva (spores), Saccharomyces cerevisiae (vegetative cells), and Zygosaccharomyces parabailii (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance. The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO2 concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for As. niger, for which the presence of dissolved CO2 reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO2.

Evaluating changes in growth and pigmentation of Cladosporium cladosporioides and Paecilomyces variotii in response to gamma and ultraviolet irradiation.

Melanin-containing fungi (black molds) have the capacity to thrive under extreme environmental conditions such as the elevated radiation levels inside the former Chernobyl reactors. These fungi have been hypothesized to grow toward and use gamma radiation as an energy source, but the literature does not clearly address which energies of the electromagnetic spectrum, if any, positively affect fungal growth. The goal of this work was to characterize the response of non-melanized and melanized fungi to two distinct electromagnetic wavelengths, i.e., ultraviolet (UV) and gamma ray, keeping absorption and other potentially confounding variables constant. Exposure to UV or gamma radiation induced significant changes in fungi pigmentation, but not growth rate of Cladosporium cladosporioides and Paecilomyces variotii. Specifically, increased pigmentation of both fungi was observed in samples exposed to UV, while decreased pigmentation was observed for gamma-irradiated samples. These results provide new insights into the role of electromagnetic energies on growth of fungi and provide an impetus to examine additional energies and types of radiation to develop a fundamental understanding of this phenomenon.

Transformation of lignocellulose to starch-like carbohydrates by organic acid-catalyzed pretreatment and biological detoxification.

Corn dry milling provides a mature model for lignocellulose biorefinery process. To copy this technical success, a crucial step is to transform lignocellulose into starch-like carbohydrates (SLC), similar to milled corn grain and in a similar fashion to corn dry milling. The transformation process should be zero wastewater generation and sufficient fermentable sugar conservation; the product should be in solid particle form, free of toxic residues, and high enzymatic hydrolysis yield and fermentability. Here we designed and verified a SLC transformation process by (i) biodegradable oxalic acid-catalyzed pretreatment, and (ii) simultaneous biodegradation of inhibitors and oxalic acid catalyst. The oxalic acid catalyst was effective on disrupting the lignocellulose structure and also biodegradable at low pH value. The biodetoxification fungus Paecilomyces variotii FN89 was capable of degrading the furan/phenolic aldehydes and oxalic acid simultaneously and ultimately, while the fermentable sugars were well preserved. The obtained SLC from wheat straw and corn stover were similar to dry milled corn meal in terms of morphological properties, fermentable sugar contents, enzymatic hydrolysis yield, elemental contents, and free of inhibitors and acid catalyst. The bioconversion of starch-like wheat straw and corn stover produced 78.5 and 75.3 g/L of ethanol (9.9% and 9.5%, v/v) with the yield of 0.47 and 0.45 g ethanol/g cellulose/xylose, respectively, compared with 78.7 g/L (10.0%, v/v) from corn meal and the yield of 0.48 g ethanol/g starch. Mass balances suggest that the ethanol yield, wastewater generation, and elemental recycling of the SLC from lignocellulose were essentially the same as those of corn meal.

Catalytic and thermodynamic properties of ß-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis.

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.

Inter- and intra-species variability in heat resistance and the effect of heat treatment intensity on subsequent growth of Byssochlamys fulva and Byssochlamys nivea.

The major aims of this study were to assess inter- and intra-species variability of heat resistant moulds (HRMs), Byssochlamys fulva and Byssochlamys nivea, with regards to (i) heat resistance and (ii) effect of heat treatment intensity on subsequent outgrowth. Four-week-old ascospores were suspended in buffered glucose solution (13° Brix, pH 3.5) and heat treated in a thermal cycler adjusted at 85 °C, 90 °C and 93 °C. Two variants of the Weibull model were fitted to the survival data and the following inactivation parameters estimated: b (inactivation rate, min-1), n (curve shape) and δ (the time taken for first decimal reduction, min). In addition to the assessment of heat resistance, outgrowth of Byssochlamys sp. from ascospores heated at 70 °C, 75 °C, 80 °C, 85 °C and 90 °C for 10 min and at 93 °C for 30 and 70 s was determined at 22 °C for up to 30 days. The Baranyi and Roberts model was fitted to the growth data to estimate the radial growth rates (µmax, mm.day-1) and lag times (λ, days). Inter-species variability and significant differences (p < 0.05) were observed for both inactivation and growth estimated parameters among B. fulva and B. nivea strains. The effect of heat treatment intensity on outgrowth of B. fulva strains was more apparent at the most intense heat treatment evaluated (90 °C/10 min), which was also the condition in which greater dispersion of the estimated kinetic parameters was observed. On the other hand, B. nivea strains were more affected by heating, resulting in greater variability of growth parameters estimated at different heating intensities and in very long lag phases (up to 25 days). The results show that inter- and intra-species variability in the kinetic parameters of Byssochlamys sp. needs to be taken into account for more accurate spoilage prediction. Furthermore, the effect of thermal treatments on subsequent outgrowth from ascospores should be explored in combination with other relevant factors such as °Brix and oxygen to develop thermal processes and storage conditions which can prevent the growth of HRMs and spoilage of heat treated food products.

Inactivation of Byssochlamys nivea ascospores in strawberry puree by high pressure, power ultrasound and thermal processing.

Byssochlamys nivea is a mold that can spoil processed fruit products and produce mycotoxins. In this work, high pressure processing (HPP, 600 MPa) and power ultrasound (24 kHz, 0.33 W/mL; TS) in combination with 75°C for the inactivation of four week old B. nivea ascospores in strawberry puree for up to 30 min was investigated and compared with 75°C thermal processing alone. TS and thermal processing can activate the mold ascospores, but HPP-75°C resulted in 2.0 log reductions after a 20 min process. For a 10 min process, HPP-75°C was better than 85°C alone in reducing B. nivea spores (1.4 vs. 0.2 log reduction), demonstrating that a lower temperature in combination with HPP is more effective for spore inactivation than heat alone at a higher temperature. The ascospore inactivation by HPP-thermal, TS and thermal processing was studied at different temperatures and modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature in spore inactivation, alone or combined with other physical processes. The Weibull model described the spore inactivation by 600 MPa HPP-thermal (38, 50, 60, 75°C) and thermal (85, 90°C) processing, whereas the Lorentzian model was more appropriate for TS treatment (65, 70, 75°C). The models obtained provide a useful tool to design and predict pasteurization processes targeting B. nivea ascospores.

Modeling the growth of Byssochlamys fulva and Neosartorya fischeri on solidified apple juice by measuring colony diameter and ergosterol content.

Byssochlamys fulva and Neosartorya fischeri are heat-resistant fungi which are a concern to food industries (e.g. apple juice industry) since their growth represents significant economic liabilities. Although the most common method used to assess fungal growth in solid substrates is by measuring the colony's diameter, it is difficult to apply this method to food substrates. Alternatively, ergosterol contents have been used to quantify fungal contamination in some types of food. The current study aimed at modeling the growth of the heat-resistant fungi B. fulva and N. fischeri by measuring the colony diameter and ergosterol content, fitting the Baranyi and Roberts model to the results, and finally establishing a correlation between the parameters of the two analytical methods. Whereas the colony diameter was measured daily, the quantification of ergosterol was performed when the colonies reached diameters of 30, 60, 90, 120 and 150 mm. Results showed that B. fulva and N. fischeri were able to grow successfully on solidified apple juice at 10, 15, 20, 25 and 30 °C, and the Baranyi and Roberts model showed good ability to describe growth data. The correlation curves between the parameters of colony diameter and ergosterol content were obtained with satisfactory statistical indexes.

Nutrient-Dependent Efficacy of the Antifungal Protein YvgO Correlates to Cellular Proliferation Rate in Candida albicans 3153A and Byssochlamys fulva H25.

YvgO is a recently characterized antifungal protein isolated from Bacillus thuringiensis SF361 that exhibits a broad spectrum of activity and pH stability. Customized colorimetric metabolic assays based on standard broth microdilution techniques were used to determine the variable tolerance of Byssochlamys fulva H25 and Candida albicans 3153A to YvgO exposure under select matrix conditions impacting cellular proliferation. Normalization of the solution pH after antifungal challenge expanded the available pH range under consideration allowing for a comprehensive in vitro assessment of YvgO efficacy. Indicator susceptibility was examined across an array of elementary growth-modifying conditions, including media pH, incubation temperature, ionic strength, and carbohydrate supplementation. Under suboptimal temperature and pH conditions, the indicator growth rate reduced, and YvgO-mediated susceptibility was attenuated. While YvgO association but not efficacy was somewhat influenced by solution ionic strength, carbohydrate supplementation was shown to be the most influential susceptibility factor, particularly for C. albicans. Although the specific choice of carbohydrate/nutrient supplement dictated the extent of enhanced YvgO efficacy, D-glucose additionally improved the association between antifungal and target. Indeed, when exposed to YvgO under conditions that lead to increased cellular proliferation, both indicators displayed a stronger association and susceptibility to YvgO when compared to carbohydrate-deprived media or suboptimal incubation environments. With further study, YvgO may have the capacity to function as a prophylaxis for food safety and preservation, as well as a pharmaceutical agent against opportunistic fungal pathogens either independently or in combination with other established treatments applied to both livestock and human health concerns.

Purification and identification of a novel antifungal protein secreted by Penicillium citrinum from the Southwest Indian Ocean.

A novel antifungal protein produced by the fungal strain Penicillium citrinum W1, which was isolated from a Southwest Indian Ocean sediment sample, was purified and characterized. The culture supernatant of P. citrinum W1 inhibited the mycelial growth of some plant pathogenic fungi. After saturation of P. citrinum W1 culture supernatants with ammonium sulfate and ion-exchange chromatography, an antifungal protein (PcPAF) was purified. The N-terminal amino acid sequence analysis showed that PcPAF might be an unknown antifungal protein. PcPAF displayed antifungal activity against Trichoderma viride, Fusarium oxysporum, Paecilomyces variotii, and Alternaria longipes at minimum inhibitory concentrations of 1.52, 6.08, 3.04, and 6.08 µg/disc, respectively. PcPAF possessed high thermostability and had a certain extent of protease and metal ion resistance. The results suggested that PcPAF may represent a novel antifungal protein with potential application in controlling plant pathogenic fungal infection.

Isolation of novel benzo[a]anthracene-degrading microorganisms and continuous bioremediation in an expanded-bed bioreactor.

In the present work, several samples from lab waste containers polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals were investigated as potential sources of PAH-degrading microorganisms. After isolating, two fungal strains were selected as the best degrading microorganisms. Genetic identification by sequencing was carried out and they were identified as Trichoderma longibrachiatum and Byssochlamys spectabilis. Their degradation ability was determined in liquid cultures with 100 µM of benzo[a]anthracene. T. longibrachiatum cultures showed highest degradation values (around 97%) after 9 days, furthermore in a second batch the time was reduced to 6 days. To analyse the viability of industrial application, a continuous treatment in an expanded-bed bioreactor was developed operating at different residence times with T. longibrachiatum immobilised on cubes of nylon sponge. It is noticeable that the bioreactor working in continuous mode was able to operate without operational problems and attaining high degradation levels depending on the residence time.

Optimization of production and reaction conditions of polygalacturonase from Byssochlamys fulva.

In the present study, the optimization of production and reaction conditions of polygalacturonase produced by a fungus Byssochlamys fulva MTCC 505 was achieved. The production of polygalacturonase with a considerable activity of 1.28 IU/ml was found when the culture was shaken at 30°C for 5 days in 100 ml of medium containing (w/v) 10 g/l pectin, 2 g/l NaNO3, 1 g/l KH2PO4, 0.5 g/l KCl, 0.5 g/l MgSO4. 7H2O, 0.001 g/l FeSO4. 7H2O, 0.001 g/l CaCl2. The best carbon and nitrogen source for this enzyme were pectin (1%) and Ca(NO3)2 (0.1%), respectively. The enzyme gave maximum activity at incubation time of 72 h, temperature of 30°C and pH 4.5. During the optimization of reaction conditions, the enzyme showed maximum activity in sodium citrate buffer (50 mM) of pH 5.5 at 50°C reaction temperature for 15 minutes of incubation. The enzyme showed greater affinity for polygalacturonic acid as substrate (0.5%). Km and Vmax values were 0.15 mg/ml and 4.58 µmol/ml/min. The effect of various phenolics, thiols, protein inhibitors and metal ions on the enzyme activity was investigated. The enzyme was quite stable at 4°C and 30°C. At 40°C the half life of the enzyme was 6 h and at 60°C it was 2 h.

Modelling the effect of temperature and water activity on the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea.

The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 degrees C) and a(w) levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) (Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 degrees C, 46.4 degrees C and 32.1 degrees C for B. fulva and 10.5 degrees C, 43.2 degrees C and 32.1 degrees C for B. nivea. The respective values for a(w) were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 a(w) regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 degrees C irrespective of a(w). Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4-4.2% false positive and 2.1-3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.

Use of a logistic model to assess spoilage by Byssochlamys fulva in clarified apple juice.

The percentage P (%) of spoiled bottles (n=40) of clarified apple juice due to Byssochlamys fulva, was modeled by using a logistic model: P = P(max)/1 + exp (k(tau-t)) where P(max) (%) the maximum percentage of spoiled bottles, k (h(-1)) a slope parameter and tau (h) the time for P=P(max)/2. Bottles of pasteurized apple juice were inoculated with B. fulva IOC 4518 ascospores for low and high initial loads, 4.8+/-2.3 ascospores/100mL and 19.3+/-4.6 ascospores/100mL respectively and incubated at 21 degrees C and 30 degrees C. P(max) was not significantly different from 100% except for a low initial load at 21 degrees C. Model parameters were estimated with a good accuracy, RMSE in the range 3.89-7.50. Then the model was used to determine the time for 10% bottles spoiled, t(10%). This time was greater at low initial loads, 57.4 and 104 h at 30 and 21 degrees C respectively, than at high initial loads 23.9 and 75.1h at 30 and 21 degrees C respectively. This study demonstrated that even at a very low initial contamination, clarified apple juice can be easily spoiled by B. fulva highlighting the importance of controlling critical control steps of fruit juice processing (i.e., fruit washing, juice filtration and pasteurization).

Heat resistance and the effects of continuous pasteurization on the inactivation of Byssochlamys fulva ascospores in clarified apple juice.

AIMS: To determine thermal resistance, the effect of pasteurization temperature variations (c. 2 degrees C) in a continuous system in the number of decimal reductions (n) of a Byssochlamys strain in clarified apple juice (CAJ). METHODS AND RESULTS: Thermal destruction kinetics of Byssochlamys fulva IOC 4518 in thermal death tubes were determined at 85 degrees , 90 degrees , 92 degrees and 95 degrees C by using Weibull distribution frequency model. Three processes with different heating and holding temperatures (A: 94 degrees , 92 degrees C; B: 95 degrees , 93 degrees C; C: 96 degrees , 94 degrees C, respectively) were performed in a continuous system. Process time was 30 s. delta (time of first decimal reduction) values were: 42.98, 8.10, 3.62 and 1.81 min. Variable n ranged from 0.16 to >4.78 for process B (equivalent to industrial). Variable n (0.95-2.66 log CFU ml(-1)) were obtained in CAJ bottles processed under condition B, while process A resulted in total heat-resistant mould (HRM) survival and process C in total HRM destruction. CONCLUSIONS: This study demonstrates that small variations in temperature during the CAJ pasteurization could result in elimination or survival of HRM due to its nonlogarithmic behaviour. SIGNIFICANCE AND IMPACT OF THE STUDY: This was the first study to use Weibull frequency method to model inactivation of HRM in fruit juices. Temperature variations could culminate in the presence of HRM in pasteurized juices even when low counts (<10 spores per 100 ml) were present in the raw materials.