Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat extract-based substrate

Penicillium commune, a mold frequently found on dry-cured meat products, is able to synthesize the mycotoxin cyclopiazonic acid (CPA). To evaluate the hazard due to CPA on such foods, the ability of P. commune to grow and produce CPA at water activities (a(w)) in the range of 0.99 to 0.90 with a meat extract-based medium from 12 to 30 degrees C was determined. CPA was quantified by high-pressure liquid chromatography and mass spectrometry. P. commune was able to grow at every a(w) and temperature tested. The optimal environmental conditions for growth were 20 to 25 degrees C, at 0.97 to 0.96 a(w), but the highest amount of CPA was produced at 30 degrees C, 0.96 a(w). No direct correlation between growth rate and CPA production was assessed. Temperature seems to be the most important factor influencing CPA production. However, there was an interaction between temperature and a(w) that significantly (P < 0.001) affected growth and CPA production. An a(w) of 0.90 had a marked effect, depressing growth and CPA production. Meat extract-based medium proved to be an appropriate substrate for CPA biosynthesis by P. commune under a wide range of conditions.     

Preliminary Characterization of Novel Extra-cellular Lipase from Penicillium crustosum Under Solid-State Fermentation and its Potential Application for Triglycerides Hydrolysis

Current studies about lipase production involve the use of agro-industrial residues and newly isolated microorganisms aimed at increasing economic attractiveness of the process. Based on these aspects, the main objective of this work is to perform the partial characterization of enzymatic extracts produced by a newly isolated Penicillium crustosum in solid-state fermentation. Lipase extract presented optimal temperature and pH of 37?°C and 9?C10, respectively. The concentrated enzymatic extract showed more stability at 25?°C and pH?7. The enzymes kept 100% of their enzymatic activity until 60?days of storage at 4 and ?10?°C. The stability under calcium salts indicated that the hydrolytic activity presented decay with the increase of calcium concentration. The specificity under several substrates indicated good enzyme activities in triglycerides from C4 to C18.     

Glucose Production from Treated Sago Starch Granules by Raw Starch Digesting Amylase from Penicillium brunneum

Several microorganisms have been found to produce raw starch digesting amylase. We have isolated Penicillium brunneum from sago palm tree at a sago processing site, which was used as a source of starch digesting amylase. All the raw starch digesting enzymes were effective for cereal starches, but root starches and sago starch were resistant to the enzyme reaction. Treatment of sago starch by heating to temperature below gelatinization temperature at lower pHs resulted in an increase in the ability of enzyme to digest sago starch granules. Heating to 60°C at pH 2.0 resulted in a conversion rate of sago starch granules to glucose near to the conversion rate of raw corn starch to glucose. At higher concentration, the degree of hydrolysis of treated sago starch granules was about 275% as compared to that of untreated sago starch granules. Addition of the enzyme in large amount or small portion at various time intervals was found effective in the hydrolysis of treated sago starch granules.     

Production of mycotoxins by Penicillium expansum inoculated into apples

We investigated the production of mycotoxins in apple fruits inoculated with spores of 40 strains of apple blue mold, Penicillium expansum. Patulin and citrinin contents in the extracts from apples stored at 25 degrees C for 12 days after inoculation were determined by high-performance liquid chromatography (HPLC) analysis with UV and fluorescence detection. Patulin and citrinin were produced by 90% (36) and 80% (32) of the 40 strains, indicating that P. expansum is a consistent producer of these mycotoxins. The patulin content in the extracts was substantially higher than the citrinin content. Other mycotoxins whose production in pure culture has been reported were simultaneously detected with high-resolution liquid chromatography-mass spectrometry (LC-MS) analysis with the positive ion mode of electrospray ionization. Along with patulin and citrinin, expansolides A and B were identified based on the HPLC and LC-MS spectral data and detected in 88% (35) of the extracts. The results indicate that P. expansum is a consistent producer of expansolides A and B in rotten areas of apple fruits. The findings raise the possibility that products from decayed apples might contain expansolides A and B in addition to patulin and citrinin.     

Production of antilisterial bacteriocins by staphylococci isolated from bovine milk

A collection of 111 staphylococcal isolates recovered from healthy cows in 41 dairy herds in Brazil was surveyed for the production of bacteriocins. The group included 94 coagulase-positive and 17 coagulase-negative strains of staphylococci. All cultures were grown in tryptic soy broth for 18 h at 37°C, and cell-free supernatants were tested for antimicrobial activity against several target organisms by using the agar diffusion method. Filtrates of 57 staphylococci showed strong activity against Listeria monocytogenes Scott A, and 52 isolates also inhibited the growth of Stapylococcus aureus Newbould 305, a major causative agent of bovine mastitis in the United States. The plasmid profiles of staphylococci invariably included an 8-kb plasmid. Staphylococcal isolates were tested for the production of aureocins A70 and A53, 2 bacteriocins of coagulase-positive staphylococci known to be associated with 8-kb and 10.2-kb plasmids, respectively. The presence of the A70 or A53 bacteriocin gene was checked by PCR techniques using primers based on nucleotide sequences flanking the structural gene of each bacteriocin. Agarose gel analysis of amplified PCR products of plasmid templates from all 58 isolates showed only a 525-bp fragment corresponding to the structural gene of the bacteriocin aureocin A70. The results indicated that the apparently widespread association of A70-producing staphylococci with healthy cows in Brazil may be beneficial in controlling undesirable bacteria in dairy herds.     

棘孢青霉菌发酵产右旋糖酐酶的条件优化

为优化棘孢青霉菌F1001产右旋糖酐酶的发酵条件,以培养基装载量、蛋白胨和右旋糖酐T70添加量为主要影响因素,结合其他发酵条件,在单因素试验的基础上,运用Box-Behnken试验设计原理,探讨培养基装载量、蛋白胨和右旋糖酐T70添加量的最佳组合。结果表明,蛋白胨添加量3 g/L、右旋糖酐T70添加量14 g/L、培养基装载量85 mL/250 mL,右旋糖酐酶活力最高可达到453 U/mL,比优化前提高88%。通过测定发酵过程中酶活力、pH值、蛋白质含量的变化,进一步验证发酵84 h酶活力达到最大,此时蛋白质含量达到最高,pH值达到3.9。     

Genotoxicity assessment of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verruculogen, and verrucosidin) produced by molds isolated from fermented meats

A number of toxinogenic fungal species, particularly producers of tremorgenic mycotoxins, have been isolated from traditional fermented meats. Tremorgenic mycotoxins are a group of fungal metabolites known to act on the central nervous system, causing sustained tremors, convulsions, and death in animals. However, the mode of action of these mycotoxins has not been elucidated in detail, and their genotoxic capacity has hardly been investigated. Because genotoxicity is one of the most prominent toxicological end points in food safety testing, we assessed the genotoxicity of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verrucosidin, and verruculogen) associated with molds found in fermented meats. The mycotoxins were tested in two short-term in vitro assays with the use of different genotoxic end points in different phylogenetic systems (the Ames Salmonella/mammalian-microsome assay and the single-cell gel electrophoresis assay of human lymphocytes). According to the results obtained in this study, all of the investigated mycotoxins except penitrem A exhibited a certain degree of genotoxicity. Verrucosidin appeared to have the highest toxic potential, testing positive in both assays. Verruculogen tested positive in the Salmonella/mammalian-microsome assay, and paxilline and fumitremorgen B caused DNA damage in human lymphocytes. The use of fungal starter cultures to avoid tremorgen contamination in fermented meats is recommended.     

Production of trichothecenes and zearalenone by Fusarium species isolated from wheat

Fusaria isolates from wheat were tested for ability to produce trichothecenes and zearalenone. Four isolates of F. culmorum out of 13 produced vomitoxin (DON) and 3 Ac-DON, one produced diacetoxysirpenol and 12 zearalenone. Particularly high yield of zearalenone was observed in cultures of sever pathogenic isolates. Higher temperature (20 °C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.     

Production of equol from daidzein by gram-positive rod-shaped bacterium isolated from rat intestine

Isoflavones (mainly daidzein and genistin) belong to the flavonoid group of compounds and are classified as phytoestrogens. In the intestine, daidzin is converted to daidzein by beta-glucosidase, and then daidzein is converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by enzymes of intestinal bacteria. We isolated, for the first time, an anaerobic gram-positive rod-shaped strain capable of producing equol from daidzein. Its 16S rDNA gene sequence (1428 bp) showed 99% similarity with that of the human intestinal bacterium SNU-Julong 732 (AY310748) and 93% similarity with that of Eggerthella lenta ATCC 25559(T) (AF292375). This strain converted daidzein to equol via dihydrodaidzein in an equol-assay medium anaerobically. The addition of butyric acid and arginine increased the conversion ratio of daidzein to equol 4.7- and 4.5-fold, respectively.     

Production of alternaria mycotoxins by Alternaria alternata isolated from weather-damaged wheat

The production of Alternaria mycotoxins by Alternaria alternata isolated from Chinese weathered wheat kernels were first investigated on polished rice and durum wheat grains. These mycotoxins included alternariol (AOH) and its monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), and tenuazonic acid (TA). Of 25 isolates tested, all were AOH and AME producers, 21 (84%) coproduced ALT and ATX-I, and 8 (32%) produced TA in rice culture. TA was the most abundant toxin produced at a level ranging from 1,369 to 3,563 mg/kg. Much smaller amounts of AOH, AME, ALT, and ATX-I were present with average concentrations of 54, 40, 44, and 8 mg/kg, respectively. There were linear correlations between the level of AOH and AME (r = 0.846), alternariols (AOH plus AME) and ALT (r = 0.785), and ATX-I and TA (r = 0.553). Polished rice medium seems to support a bit more production of Alternaria metabolites than wheat but with an insignificant difference in concentrations (P > 0.05). A study of the time-course of toxin production by A. alternata isolates indicated that AOH production began faster than any other toxins monitored, and ALT production exhibited a progressive increase throughout the experiment. TA producers might reveal their considerably higher ability to produce toxin in the field despite their low frequency.     

Production of cyclopiazonic acid by molds isolated from Taleggio cheese.

Twenty-seven strains of Penicillium were isolated from the rind of Taleggio, a typical Italian cheese, so that we could test their capacity to produce cyclopiazonic acid (CPA); all strains produced CPA. The production was strongly influenced by the strain variety and growth conditions. Strains incubated at 25 degrees C for 7 days always produced CPA in mannitol broth, with concentrations ranging from 0.02 to 1 microg/ml, whereas only 33% of strains grown in yeast-extract broth produced CPA, with a maximum value of 0.1 microg/ml. In milk, maximum production (1.6 microg/ml) was observed after 14 days of incubation at 25 degrees C. In order to evaluate the presence of the toxin and its capacity for migrating into the cheeses, the rind, the cheese near the rind, and the cores from six Taleggio cheeses were analyzed. CPA was present in five cheeses, with a maximum concentration of 0.25 mg/kg in one rind, and in one cheese, the toxin migrated to the core. A positive correlation between CPA production and surface mold was found.     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Production of D-iditol from D-sorbose by Rhodotolura rubra RY10 isolated from miso paste

The yeast strain RY10 that can convert D-sorbose to D-iditol was isolated from miso paste and identified as Rhodotolura rubra. The cells grown on D-fructose were found to have relatively high conversion potential. Addition of ethanol to the reaction mixture significantly accelerated the conversion rate of D-sorbose to D-iditol. During the conversion reaction, ethanol was added to the reaction mixture at 48 h intervals to maintain the concentration of ethanol at 1.0%. The final conversion ratios were 82.7%, 95.0%, 93.7%, and 78.0% using washed cells when the concentration D-sorbose were 1.0%, 2.0%, 3.0% and 5.0%, respectively. The product produced from D-sorbose was identified as D-iditol by high performance liquid chromatography analysis, infrared spectrum, optical rotation and melting point measurements.     

Production of a yogurt-like product from plant foodstuffs and whey. Substrate preparation and fermentation

A mixed substrate composed of soya milk, oat flour and dried cheese whey (82, 11 and 7% respectively) had a content of lactose and protein similar to that of milk used for yogurt manufacture. Heat treatment for 20 min at 80°C resulted in a viscosity similar to that of yogurt whilst removing coliform and mesophilic aerobic bacteria, moulds and yeasts. Fermentation with traditional yogurt bacteria did not increase viscosity further, and the final product had similar acidity and texture to yogurt. Acid development, carbohydrate consumption, proteolysis and starters counts were followed during fermentation. The fermentation profile of the mixed substrate was very similar to that of milk.     

Production of bacteriocin-like metabolites by lactic acid cultures isolated from sucuk samples

A total of 51 sucuk samples, obtained from different regions of Turkey, were examined for lactic acid bacteria (LAB) strains producing putative bacteriocin-like metabolites. For detection of antagonistic activity, agar spot and well diffusion assay tests were used. Lactobacillus sake Lb790, Listeria monocytogenes Li6, Staphylococcus aureus St44 and Escherichia coli NRRL B-3704 were used as indicator organisms. Strains having antimicrobial activity were also tested against Clostridium perfringens 4TTK, Clostridium botulinum type A. 7948, Bacillus cereus NRRL B-3711, Micrococcus luteus NRRL B-4376 and Yersinia enterocolitica type 103. 57 of 424 isolates from sucuk samples were putative producers of bacteriocin-like metabolistes and were identified as the following: 19 Lactobacillus plantarum, 4 L. curvatus, 4 Pediococcus pentosaceus, 3 P. acidilactici, 2 L. pentosus, 2 L. sake, 1 L. delbrueckii, 1 L. rhamnosus and 21 unidentified Lactobacillus spp. P. pentosaceus 416, P. acidilactici 413, 419 and 446, L. curvatus 348, L. plantarum 452 and 495, and Lactobacillus spp. 411 strains have the best potential for use as sucuk starter cultures.     

Penicillium sp.1407产纤维素酶的发酵条件优化

利用廉价的麸皮作为培养基的碳源,采用单因素试验分析了发酵影响因素摇床转速、发酵温度、接种量、初始pH、发酵时间对Penicillium sp.1407产纤维素酶的影响;利用Box-Behnken试验设计及响应面分析法进行了优化,确定最佳发酵条件为:发酵温度32℃、发酵时间142 h、初始pH 5.2、转速230 r/min,纤维素酶活力为(136±0.74)IU/mL,验证试验结果与模型预测相符。     

Production of L-talitol from L-psicose by Metschnikowia koreensis LA1 isolated from soy sauce mash

A strain LA1 that can convert L-psicose to L-talitol was isolated from soy sauce mash and identified as Metschnikowia koreensis. The cells grown on L-arabitol were found to have relatively high conversion potential. Addition of D-sorbitol to the reaction mixture considerably accelerated the conversion rate of L-psicose to L-talitol. During the conversion reaction, D-sorbitol was added to the reaction mixture at 12-h intervals to maintain the concentration of D-sorbitol at 1.0%. The final conversion ratios were 81.4%, 75.2%, 73.0%, 60.4% and 43.5% using washed cells when the concentrations of L-psicose were 0.5%, 1.0%, 2.0%, 3.0% and 5.0%, respectively. The product from L-psicose was identified as L-talitol by HPLC analysis, and infrared spectroscopy, optical rotation and melting point measurements.     

Mycological examination of domestic unpolished rice and mycotoxin production by isolated Penicillium islandicum

Fungi growing on domestic rice were examined from April to June, 2003. One hundred samples of rice, which had been harvested in the autumn of 2002, were collected from the local market, and 15 samples of stored rice, which had been harvested in 2001 and stored in warehouses under government control, were used as samples. From each sample, 50 grains (100 grains in total) were plated on potato-dextrose agar (PDA) and malt yeast 40% sucrose agar (M40YA) containing chloramphenicol after being washed with sterile distilled water to remove any microorganisms on the surface, and incubated at 25 degrees C for a week. For most of the rice samples harvested in the preceding year, the proportion of grains infected with fungi was less than 20% of the total grains tested. In about half the samples of rice stored for one and half years, more than 80% of the grains were infected with fungi that grew on M40YA. The major genera of fungi isolated from the rice harvested in the preceding year were Penicillium and Alternaria, and those from the rice stored for one and a half years were Aspergillus, Penicillium and Eurotium. P. islandicum, A. versicolor, A. ochraceus and others were isolated as possible mycotoxin-producers in the mycoflora of domestic rice. P. islandicum was isolated from 3 samples, and 82% of the grains were infected with this fungus in one sample. All three isolates from these samples appeared to produce luteoskyrin on Czapek yeast extract agar, based on TLC and HPLC analysis.     

Production of L-sorbitol from L-fructose by Aureobasidium pullulans LP23 isolated from soy sauce mash

A strain LP23 that can convert L-fructose to L-sorbitol was isolated from soy sauce mash and identified as Aureobasidium pullulans. The cells grown on L-arabinose were found to have relatively high L-fructose to L-sorbitol conversion potential. Addition of erythritol to the reaction mixture considerably accelerated the conversion rate of L-fructose to L-sorbitol. During the conversion reaction, erythritol was added to the reaction mixture at 8-h intervals to maintain the concentration of erythritol at 1.0%. The final conversion ratios were 82.8%, 95.3%, 92.4%, and 42.6% using washed cells when the concentrations of L-fructose were 1.0%, 2.0%, 5.0% and 10.0%, respectively. The product from L-fructose was identified as L-sorbitol by HPLC analysis, infrared spectroscopy, optical rotation and melting point measurements.