Evaluation of Lignin-Modifying Enzyme Activity of Trametes spp. (Agaricomycetes) Isolated from Georgian Forests with an Emphasis on T. multicolor Biosynthetic Potential.

In this study, a wide diversity in lignin-modifying enzyme (LME) secretion by 11 Trametes spp. strains isolated from the forests of Georgia was revealed in their submerged cultivation in both synthetic and lignocellulose-based media. Among them, T. multicolor BCC 511 was distinguished by simultaneous production of laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP) in the presence of high carbon and nitrogen concentrations. Mannitol at the concentration of 15 g/L provided an accumulation of 23.7 U/mL laccase and 0.56 U/mL MnP. Significant modulation of LME activity by lignocellulosic substrates, metals, aromatic compounds, and their concentrations was established. Mandarin peels manifold increased the fungus laccase and LiP activities, while the ethanol production residue and banana peels activated manganese-oxidizing and Phenol Red-oxidizing manganese peroxidases, respectively. The addition of 2 mM of copper sulfate to the control medium induced the laccase production 28-fold and did not significantly affect the MnP and LiP activities. Fe2+ at a concentration of 0.1 mM enhanced the fungus volumetric and specific laccase activities almost 8-fold; at a concentration of 0.25-0.5 mM, there was a 2-fold increase in the MnP activity. Mn2+ appeared to be an effective inducer of the Mn-oxidizing MnP, increasing specific activity of the enzyme 14-fold. Supplementation of the copper-containing medium with 1 mM veratryl alcohol or guaiacol favored laccase and MnP production. The high yields of laccase (110 U/mL), MnP (0.62 U/mL), and LiP (0.71 U/mL) obtained in a laboratory fermenter make T. multicolor 511 useful for industrial and environmental applications.

Physiological Peculiarities of Lignin-Modifying Enzyme Production by the White-Rot Basidiomycete Coriolopsis gallica Strain BCC 142.

Sixteen white-rot Basidiomycota isolates were screened for production of lignin-modifying enzymes (LME) in glycerol- and mandarin peel-containing media. In the synthetic medium, Cerrena unicolor strains were the only high laccase (Lac) (3.2-9.4 U/mL) and manganese peroxidase (MnP) (0.56-1.64 U/mL) producers while one isolate Coriolopsis gallica was the only lignin peroxidase (LiP) (0.07 U/mL) producer. Addition of mandarin peels to the synthetic medium promoted Lac production either due to an increase in fungal biomass (Funalia trogii, Trametes hirsuta, and T. versicolor) or enhancement of enzyme production (C. unicolor, Merulius tremellosus, Phlebia radiata, Trametes ochracea). Mandarin peels favored enhanced MnP and LiP secretion by the majority of the tested fungi. The ability of LiP activity production by C. gallica, C. unicolor, F. trogii, T. ochracea, and T. zonatus in the medium containing mandarin-peels was reported for the first time. Several factors, such as supplementation of the nutrient medium with a variety of lignocellulosic materials, nitrogen source or surfactant (Tween 80, Triton X-100) significantly influenced production of LME by a novel strain of C. gallica. Moreover, C. gallica was found to be a promising LME producer with a potential for an easy scale up cultivation in a bioreactor and high enzyme yields (Lac-9.4 U/mL, MnP-0.31 U/mL, LiP-0.45 U/mL).

Anti-Inflammation Properties of Fruiting Bodies and Submerged Cultured Mycelia of Culinary-Medicinal Higher Basidiomycetes Mushrooms.

This research shows the phenolic composition and anti-inflammation properties of fruiting bodies and mycelia of 15 strains of 12 species of higher Basidiomycetes medicinal mushrooms. In this research, 15 extracts were prepared and their effects on inflammation-related mediators in RAW 264.7 cells were evaluated. In the extracts, amounts of total phenols ranged from 8.47 to 70.32 gallic acid equivalents mg/g and amounts of flavonoids ranged from 0.13 to 15.21 rutin equivalents mg/g. The production of nitric oxide, tumor necrosis factor-α, and interleukin-6 was decreased at different levels by these extracts, whereas the production of interleukin-10 was increased by 6 of the extracts. Overall, Cordyceps militaris fruiting bodies, Grifola frondosa fruiting bodies, and Ophiocordyceps sinensis mycelia might be used to ameliorate inflammatory responses.

Chemical Composition and Medicinal Value of Fruiting Bodies and Submerged Cultured Mycelia of Caterpillar Medicinal Fungus Cordyceps militaris CBS-132098 (Ascomycetes).

In this paper, we report the results of a proximate analysis (i.e., moisture, ash, protein, fat, carbohydrates, and energy); a bioactive compounds analysis (i.e., cordycepin and ergothioneine); fatty and amino acid analysis; and analyses of vitamin content, macro- and microelement composition of fruiting body (FB), and mycelial biomass (MB) of medicinal caterpillar fungus Cordyceps militaris strain CBS-132098. These results demonstrate that the FB and MB of C. militaris are good sources of proteins: 59.8% protein content in the FB and 39.5% in the MB. The MB was distinguished by its carbohydrate content (39.6%), which was higher than that of the FB (29.1% carbohydrate). In the FB of C. militaris, the total amino acid content was 57.39 mg/g and in the MB it was 24.98 mg/g. The quantification of the identified fatty acids indicated that palmitic acid, oleic acid, linoleic acid, and linolenic acid were the major fatty acids. The micro- and macroelement compositions were studied. The highest results were calcium (797 mg/kg FB; 11 mg/kg MB); potassium (15,938 mg/kg FB 12,183 mg/kg MB); magnesium (4,227 mg/kg FB; 3,414 mg/kg MB); sodium (171 mg/kg FB; 1,567 mg/kg MB); phosphorus (7,196 mg/kg FB; 14,293 mg/kg MB); and sulfur (5,088 mg/kg FB; 2,558 mg/kg MB). The vitamin composition was studied, and the most abundant vitamins were vitamin A, vitamin B3, and vitamin E. The bioactive components were cordycepin, cordycepic acid (D-mannitol), and ergothioneine. There were differences in cordycepin and ergothioneine contents between the FB and the MB. The cordycepin concentration was 0.11% in the FB and 0.182% in the MB, the cordycepic acid was 4.7 mg/100g in the FB and 5.2 mg/100 g in the MB, and the ergothioneine content was 782.37 mg/kg in the FB and 130.65 mg/kg in the MB. The nutritional values of the FB and the MB of C. militaris detected indicate its potential use in well-balanced diets and sources of bioactive compounds.

Chemical Composition and Medicinal Value of the New Ganoderma tsugae var. jannieae CBS-120304 Medicinal Higher Basidiomycete Mushroom.

In this research, the chemical composition and anticancer and antioxidant activity of the new medicinal mushroom Ganoderma tsugae var. jannieae CBS-120304 were evaluated. The chemical composition assay includes amounts of total carbohydrates and proteins, amino acids, fatty acids, micro- and macroelements, and vitamins. The investigated medicinal mushroom seemed to be a rich source of nutritional components. Mycelium accumulated more than 2-fold more total protein compared with the fruiting body and reached 37% and 16% of dry weight, respectively. Carbohydrate content in the fruiting body seemed to be conspicuously higher than in the mycelium (50% of dry weight) and reached 80% of dry weight. Quantification of the identified fatty acids indicated that, in general, palmitic acid, oleic acid, and linoleic acid were the major fatty acids. Toxic elements, such as silver, arsenic, cadmium, and mercury, were found only in trace amounts in mycelium and were not detected in the fruiting body. Furthermore, the 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay was used to evaluate antioxidant activity. The highest radical scavenging activity was 9.0 mg/mL (65.9%) by ethanol extract. In addition, mycelial extracts were tested to inhibit MCF7 breast cancer cells. Ganoderma tsugae var. jannieae ethyl acetate extract (GTEAE) extract showed high potential by inhibiting reporter activity by more than 70%. Results demonstrated that GTEAE had a strong effect on inhibitory protein κΒα level in the higher concentration used (200 gg/mL), which could be compared with the effect of parthenolide. Furthermore, GTEAE demonstrated strong inhibition of IκΒα phosphorylation.

Shiitake Medicinal Mushroom, Lentinus edodes (Higher Basidiomycetes) Productivity and Lignocellulolytic Enzyme Profiles during Wheat Straw and Tree Leaf Bioconversion.

Two commercial strains of Lentinus edodes have been comparatively evaluated for their productivity and lignocellulolytic enzyme profiles in mushroom cultivation using wheat straw or tree leaves as the growth substrates. Both substrates are profitable for recycling into shiitake fruit bodies. L. edodes 3715 gave the lowest yield of mushroom during tree leaves bioconversion with the biological efficiency (BE) 74.8% while the L. edodes 3721 BE achieved 83.4%. Cultivation of shiitake on wheat straw, especially in the presence of additional nitrogen source, increased the L. edodes 3721 BE to 92-95.3% owing to the high hydrolases activity and favorable conditions. Despite the quantitative variations, each strain of L. edodes had a similar pattern for secreting enzymes into the wheat straw and tree leaves. The mushrooms laccase and MnP activities were high during substrate colonization and declined rapidly during primordia appearance and fruit body development. While oxidase activity decreased, during the same period cellulases and xylanase activity raised sharply. Both cellulase and xylanase activity peaked at the mature fruit body stage. When mushrooms again shifted to the vegetative growth, oxidase activity gradually increased, whereas the hydrolases activity dropped rapidly. The MnP, CMCase, and FP activities of L. edodes 3721 during cultivation on wheat straw were higher than those during mushroom growth on tree leaves whereas the laccase activity was rather higher in fermentation of tree leaves. Enrichment of wheat straw with an additional nitrogen source rather favored to laccase, MnP, and FPA secretion during the vegetative stage of the L. edodes 3721 growth.

Chemical composition and nutritional and medicinal value of fruit bodies and submerged cultured mycelia of culinary-medicinal higher Basidiomycetes mushrooms.

This research gives the results of a proximate analysis (moisture, ash, crude protein, fat, total carbohydrates, and total energy); a bioactive compounds analysis (γ-aminobutyric acid [GABA], ergothioneine, lovastatin, and cordycepin); fatty acid and amino acid analysis; and an analysis of macro- and microelement content of fruit bodies and mycelia of 15 higher Basidiomycetes medicinal mushroom strains belonging to 12 species. The results obtained demonstrate that almost all investigated mushrooms were found to be good sources of proteins and carbohydrates, with content varying in the ranges of 8.6-42.5% and 42.9-83.6%, respectively. Different species exhibited distinct free amino acid profiles. The total amino acid content was highest in Ophiocordyceps sinensis (MB) (23.84 mg/g) and Cordyceps militaris (FB) (23.69 mg/g). The quantification of the identified fatty acids indicated that, in general, palmitic acid, oleic acid, stearic acid, and linoleic acid were the major fatty acids. The micro- and macroelement compositions were studied, and the highest results were (as milligrams per kilogram) 224-7307 for calcium, 1668-38564 for potassium, 1091-11676 for phosphorus, and 5-97 for zinc. Bioactive components were lovastatin, GABA, and ergothioneine, which are commonly found in most mushrooms. C. militaris (FB), Pleurotus ostreatus (FB), and Coprinus comatus (FB) were most abundant and contained a high amount of GABA (756.30 µg/g, 1304.99 µg/g, 1092.45 µg/g, respectively) and ergothioneine (409.88 µg/g, 2443.53 µg/g, 764.35 µg/g, respectively). The highest lovastatin content was observed in Hericium erinaceus (FB) (14.38 µg/g) and Ganoderma lucidum (FB) (11.54 µg/g). In contrast to C. militaris (FB), cordycepin was not detected in O. sinensis (MB). The fruit body biomass of C. militaris cordycepin content reached 1.743 mg/g dry weight. The nutritional values of the mushroom species studied here could potentially be used in well-balanced diets and as sources of bioactive compounds.

Comparative study of contents of several bioactive components in fruiting bodies and mycelia of culinary-medicinal mushrooms.

Mushrooms (including fruiting bodies and mycelia) contain several bioactive components such as lovastatin, γ-aminobutyric acid (GABA), and ergothioneine. This article reports the results of 49 samples, including 9 fruiting bodies, 39 mycelia, and 1 vegetative cell, of 35 species of culinary-medicinal mushrooms from 18 genera: Agaricus, Agrocybe, Coprinus, Cordyceps, Cyathus, Daedalia, Flammulina, Fomes, Ganoderma, Grifola, Laetiporus, Lentinus, Morchella, Ophiocordyceps, Pleurotus, Trametes, Tremella, and Verpa. The results show that Cyathus striatus strain 978 contained the highest amount of lovastatin (995.66 mg/kg) in mycelia. Among fruiting bodies, 6 samples contained a high amount of GABA (274.86-822.45 mg/kg), whereas among mycelia, contents of GABA in 27 samples ranged from 215.36 to 2811.85 mg/kg. Among mycelia, Pleurotus cornucopiae strain 1101 contained the highest amount of ergothioneine (3482.09 mg/kg). Overall, these 3 bioactive components were commonly found in most mushrooms, and the results obtained might be related to their beneficial effects.

Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle.

Digital dermatitis (DD) is an infectious claw disease of cattle that causes painful lesions, principally along the coronary band of the claws. In the US alone, the estimated economic impact of DD is estimated to be $190 million. The etiology of DD remains unclear and there is no reliable laboratory test, so DD is most often diagnosed clinically. Spirochetal bacteria of the genera Treponema have been implicated in DD infections following their isolation using culture techniques, serological detection of bovine antibodies against treponemes, and amplification of treponemal 16s DNA sequences by PCR. During in vitro growth of spirochetes and treponemes isolated from DD, morphological changes have been observed indicating the presence of a spiral form and an encysted form. It is not known why encysted forms appear or what role they have in the progression of DD. The current study established growth curves for three subtypes of treponemes, Treponema denticola-like, Treponema phagedenis-like, and Treponema medium-like, while photographically monitoring changes in morphology. In addition to observing spiral and encysted forms, two intermediate forms were also observed. These appeared as either spiral forms with spherical bodies or as enveloped clusters of granules. The observation of encysted forms adds further support to the theory that treponemes causing recurrent infections deep in bovine skin have mechanisms to facilitate persistence and the chronic character of DD.

The Shaggy Inc Cap medicinal mushroom, Coprinus comatus (O.F.Mull.: Fr.) Pers. (Agaricomycetideae) substances interfere with H2O2 induction of the NF-kappaB pathway through inhibition of Ikappaalpha phosphorylation in MCF7 breast cancer cells.

Breast cancer is the most commonly diagnosed cancer among women. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. In our study, we used hydrogen peroxide, a well-known strong oxidative reagent capable of activating the nuclear factor kappa B (NF-kappaB) transcription factor. The IC50 value of the culinary-medicinal Shaggy Inc Cap mushroom Coprinus comatus culture liquid crude extract on MCF7 cell viability was found to be as low as 76 microg/mL, and the IC50 value of C. comatus ethyl acetate extract was only 32 microg/ mL. Our results also showed that both extracts significantly affected IkappaBalpha phosphorylation in a dose-dependent manner. The effect of ethyl acetate extract was comparable to the effect of curcumin, a known NF-kappaB pathway inhibitor, and seemed to be the most active inhibitor of H2O2-dependent IkappaBalpha phosphorylation. In addition, the data obtained showed that only ethyl acetate extract inhibited the activity of IKK complex, at close to 90% as compared to the control of the untreated sample. These results suggest that C. comatus contains potent compounds capable of inhibiting NF-kappaB function and also possibly acts as an antitumor agent.

Effects of signal transducer and activator of transcription (STAT) genes STAT1 and STAT3 genotypic combinations on fertilization and embryonic survival rates in Holstein cattle.

Infertility is a major cause of dairy cow culling and economic loss. Signal transducer and activator of transcription (STAT) proteins are transcription factors that play an important role in fertility and early embryonic development, among many other functions. Previous studies have reported the association of several genes from the JAK/STAT signaling pathway with fertility traits in cattle. The STAT1 and STAT3 genes are members of this pathway and are known to interact with each other by forming a heterodimer complex that enters the nucleus and controls expression of specific genes. Thus, the objective of this study was to investigate the effects of the interactions between polymorphisms in these genes on fertilization and early embryonic survival rates using an in vitro fertilization system. A total of 7,519 oocytes, collected from 445 ovaries, were exposed to sperm and a total of 5,075 embryos were produced. Fertilization rate was calculated as the number of cleaved embryos at 48 h post-fertilization out of the total number of oocytes exposed to sperm. Early embryonic survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the total number of embryos cultured. Effects of ovary genotypes on fertilization and early embryonic survival rates were evaluated. Single-SNP analysis revealed a statistically significant association between SNP25402 in STAT3 and fertilization rate. Oocytes produced from ovaries with AA genotype showed a 0.701 fertilization rate versus 0.666 and 0.663 for oocytes produced from AC and CC ovaries, respectively. The interaction between STAT3 SNP (SNP19069/SNP25402) was highly significant for survival rate but not for fertilization rate. Also, the interaction between STAT1 SNP and SNP19069 was highly significant for survival rate. Genotype combinations found to promote fertilization and embryonic survival could be incorporated into breeding programs aimed at improving fertility performance in dairy cattle.

Free-radical scavenging activity of submerged mycelium extracts from higher basidiomycetes mushrooms.

Twenty-four Basidiomycetes strains were evaluated to determine their free-radical scavenging capacity in submerged cultivation. The scavenging capacity of the extracts varied from 1 to 85% depending on the mushroom species, solvent used, and concentration. A calculation of EC(50) of extracts from several wood-rotting Basidiomycetes showed high scavenging abilities at low effective concentration.