Patulin production by Byssochlamys spp. in fruit juices

Ten strains of Byssochlamys fulva and three strains of B. nivea were cultured in a laboratory medium and tested for their ability to produce patulin. Two strains of B. fulva and all three strains of B. nivea produced the mycotoxin. One strain of B. fulva produced patulin in 11 of 13 processed fruit juices, with greatest amounts being produced in blueberry, red raspberry, and boysenberry juices, whereas no patulin was detected in prune or tomato juices. Grown in Concord grape juice at 18, 25, 30, and 38 degrees C, this strain produced the highest patulin concentration at 18 degrees C after 25 days, whereas biomass production was greatest at 25 and 30 degrees C after 20 and 25 days.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices

The objective of this study was to compare high pressure resistance of Listeria monocytogenes strains at 25 degrees C and 50 degrees C at 350 MPa and to use high pressure (250 MPa and 350 MPa) at 30 degrees C and 40 degrees C for the inactivation of the relatively most pressure resistant strain inoculated in pasteurized apple, apricot, cherry and orange juices. L. monocytogenes CA was found to be the relatively most pressure resistant strain and increasing pressurization from 250 MPa to 350 MPa at 30 degrees C had an additional three to four log cycle reduction in viability, still leaving viable cells after 5 min. When 350 MPa at 40 degrees C for 5 min was applied more than eight log cycle reduction in cell population of all fruit juices was achieved. This study demonstrated that low temperature (40 degrees C) high pressure (350 MPa) treatment has the potential to inactivate relatively pressure resistant L. monocytogenes strains inoculated in different fruit juices within 5 min.     

Existence commune,sur les fraises,d'ascospores de Byssochlamys nivea pouvant produire de la patuline

Tha auteurs have obtained a strain of Byssochlamys nivea from heat processed strawberries. The mold grown on synthetic media shows antibiotic properties. The metabolite responsible is patulin which has been purified and characterized. Several strains of B. nivea produced this mycotoxin. Patulin seems to be stable in strawberries juice.     

Influence of temperature and water activity on growth and patulin production by Byssochlamys nivea in apple juice.

The influence of temperature and water activity (aw) on growth and patulin production by Byssochlamys nivea in apple syrups was determined over a 44-day incubation period. The minimum aw at which the mold was capable of growing was 0.915 and 0.886 at 21 and 30 degrees C, respectively. Growth at 37 degrees C was observed at 0.871 aw. Minimum aw values for patulin production were 0.978, 0.968, and 0.959 at 21, 30 and 37 degrees C, respectively.     

Influence of temperature and water activity on growth and patulin production by Byssochlamys nivea in apple juice

The influence of temperature and water activity (aw) on growth and patulin production by Byssochlamys nivea in apple syrups was determined over a 44-day incubation period. The minimum aw at which the mold was capable of growing was 0.915 and 0.886 at 21 and 30 degrees C, respectively. Growth at 37 degrees C was observed at 0.871 aw. Minimum aw values for patulin production were 0.978, 0.968, and 0.959 at 21, 30 and 37 degrees C, respectively.     

Effect of Penicillium expansum culture conditions on patulin production.

Penicillium expansum strains grown on Capek-Dox liquid medium excreted patulin to the medium. Its amount increased until day 12, then smaller amounts of the toxin were observed. Maximum patulin excretion took place at 25 degrees C, pH 6, although at 5 degrees C, pH 3 the presence of this mycotoxin was also observed. The highest amount of patulin produced was observed in medium containing fructose.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain.

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.     

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Production of eicosapentaenoic acid by marine bacteria

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.     

Influence of temperature on the development, reproduction and longevity of Ceratothripoides claratris (Thysanoptera: Thripidae) on tomatoes

Ceratothripoides claratris (Shumsher) is a serious pest attacking tomatoes in Thailand. Temperature-dependent development of C. claratris was studied at seven constant temperatures, i.e. 22, 25, 27, 30, 34, 35 and 40 degrees C. Pre-adult survivorship was greatest (95%) at 25 and 30 degrees C and shortest at 22 degrees C. Egg-to-adult time decreased within the range of 20 to 30 degrees C and at 34 degrees C it started to increase. The lower thermal threshold for egg-to-adult development was estimated at 16 and 18 degrees C by linear regression and the modified Logan model, respectively. The optimum temperature for egg-to-adult development was estimated at 32-33 degrees C by the modified Logan model. The influence of temperature on reproduction and longevity of C. claratris was determined at 25, 30 and 35 and 40 degrees C. Both inseminated and virgin females failed to reproduce at 40 degrees C. Virgin females produced only male offspring, confirming arrhenotoky. The sex ratio of the offspring of fertilized females was strongly female-biased, except at 25 degrees C. Mean total fecundity per female and mean daily total fecundity per female were highest for both virgin and inseminated females at 30 degrees C. Female longevity was longest at 25 degrees C and shortest at 40 degrees C. Male longevity was longest at 30 degrees C and shortest at 40 degrees C. The net reproductive rate (R0) and intrinsic rate of natural increase (rm) was greatest at 30 degrees C while, mean generation time (G) and the doubling time (t) were highest at 25 degrees C. The finite rate of increase (lambda) was fairly constant (1.1-1.5 days) over the three temperatures tested. The pest potential of C. claratris for tropical Asia is discussed.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense.

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060-5010 mg/kg dry wt of culture) at ambient temperature (16-29 degrees C) in daylight than at ambient temperature (18-23 degrees C) in darkness or at controlled temperatures of 11 degrees C, 20 degrees C or 25 degrees C in darkness. Yields at 25 degrees C were low. Incubation at 11 degrees C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 degrees C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 degrees C (2460-21 360 mg/kg), 1 strain at 11 degrees C (6570 mg/kg). Yields at a temperature oscillating daily from 10-20 degrees C were less than at 15 degrees C. One of the 5 strains produced appreciable amounts of a-zearlaenol (1645 mg/kg at 20 degrees C) and 2 of nivalenol (340 and 499 mg/kg at 20 degrees C).     

Study of conditions of production of roquefortine and other metabolites of Penicillin roqueforti.

Experiments to determine optimum yields of roquefortine, isofumigaclavine A, and PR toxin, metabolites from Penicillum roqueforti Thom, were performed. Four strains, isolated from blue cheese, and five liquid media were evaluated, although not all permutations were studied. Sucrose (15%)-yeast extract (2%) was the medium chosen for time-course studies at 25 and 15 degrees C using one favorable strain. At 25 degrees C, maximum estimated yields of roquefortine were about 100 mg/liter in the mycelium by 16 days, and no subsequent degradation of this alkaloid was observed. On the other hand, production of PR toxin in the medium peaked at 770 mg/liter at 21 days. At 15 degrees C, yields of roquefortine and PR toxin after 49 days were 60 to 70% of the maximum yields obtained at 25 degrees C. However, about three times more isofumigaclavine A (up to 11 mg/liter) was formed in the mycelium at 15 degrees C than at 25 degrees C. All four strains of P. roqueforti procedure both roquefortine and PR toxin on the sucrose-yeast extract medium at 25 degrees C; isofumigaclavine A was detected in all but one strain grown on this medium.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.     

Prototheca zopfii Krüger Strain UMK-13 Growth on Acetate or n-Alkanes

A new strain of Prototheca zopfii Krüger was grown on acetate or on pure n-alkanes. A maximum acetate-supported exponential growth of 12 divisions day occurred at pH 5 and 30 degrees C. At 25 degrees C, growth on n-alkanes was almost as fast, but no growth occurred at 30 degrees C. After 4 days at 25 degrees C, 34 to 45% of the n-alkanes had been removed, whereas at 21 degrees C and slower growth, utilization was twofold greater after 15 days. Rates of growth and utilization increased markedly after a point of sudden emulsification.     

Comparison of growth and lipid content in three <Emphasis Type="Italic">Botryococcus braunii</Emphasis> strains

The growth and lipid content of three Botryococcus braunii strains from China (CHN), United Kingdom (UK) and Japan (JAP) were compared at three temperatures (20, 25 and 30 ^∘C), three irradiances (60, 100 and 300 W m⁻²) and four salinities (0, 0.15, 0.25, and 0.5 M NaCl) for 30 days, respectively. In the temperature trial, the UK strain and JAP strain grew faster at 25 ^∘C than at other temperatures, while the CHN strain performed equally well at 20 and 25 ^∘C. The JAP strain grew slowest among the three strains at all temperatures, whereas the growth rate of the CHN and UK strains was similar at all temperatures except at 20 ^∘C. The UK strain contained the highest lipid content, but the CHN strain had the lowest lipid content at most temperatures. In the light trial, the highest growth rate was found in the UK strain and the lowest growth rate was observed in the JAP strain at most irradiances. The UK and JAP strains contained more lipids than the CHN strain at 60 and 100 W m⁻², but the lipid content was not significantly different among the three strains at 300 W m⁻². In the salinity trial, both the CHN and UK strains grew faster than the JAP strain at all salinities, but the growth rate between the CHN and UK strains was not different. However, the CHN strain had the lowest lipid content whereas the UK strain produced the highest lipids at most salinities. Our results indicate that the CHN strain and the UK strain grow faster than the JAP strain, but the UK and JAP strains produce more lipids than the CHN strain. The UK strain should be considered as a potential B. braunii strain for the exploitation of renewable energy.     

Byssochlamys nivea as a source of mycophenolic acid

Byssochlamys species are responsible for spoilage and degradation of fruits and silages and can also produce the mycotoxin patulin. We analyzed secondary metabolite production by Byssochlamys nivea. Mycophenolic acid and its precursors, 5-methylorsellinic acid and 5,7-dihydroxy-4-methylphthalide, were identified in all of the B. nivea strains that we examined.