Characterization of the Nutritional Composition of a Biotechnologically Produced Oyster Mushroom and its Physiological Effects in Obese Zucker Rats.

SCOPE: Sustainable protein sources are needed to meet the increasing protein demands of a continuously growing world population. This study is focused on the biotechnological production of a protein rich oyster mushroom (Pleurotus sajor-caju; PSC) by valorization of an agricultural side stream and the evaluation of the physiological effects of PSC in a rat model of metabolic syndrome. METHODS AND RESULTS: PSC is produced via submerged cultivation in a 150 L bioreactor that utilizes isomaltulose molasses as its sole carbon source, and is further analyzed for its nutritional composition. A feeding trial is performed using Zucker rats which are fed a 5% PSC supplemented diet, for 4 weeks. Biochemical analyses reveal a significant reduction of the liver lipid concentrations and liver inflammation in the PSC fed obese rats in comparison to the obese rats from the control group. Hepatic qPCR analyses, differential transcript profiling, and enzyme activity measurements reveal a number of altered pathways that may be responsible for these anti-steatotic and anti-inflammatory effects of the mushroom. CONCLUSION: Bioconversion of a low quality agricultural side stream to an improved protein source is performed by submerged cultured PSC, and the obtained mycelium shows strong anti-steatotic and anti-inflammatory effects.

Biotechnological production, characterization and in vitro antitumor activity of polysaccharides from a native strain of Lentinus crinitus.

This work deals with the submerged cultivation, extraction and antitumor activity of polysaccharides from Lentinus crinitus. The fungus was isolated from a tropical forest (Antioquia, Colombia), cultivated in laboratory conditions, and classified by classical and molecular taxonomy. Then, it was cultivated in a bioreactor of 5 L using a ligninolytic residue as substrate. The fermentation conditions were 30 ±â€¯1 °C, pH 4.5, 300 rpm and 1.5 vvm for 4 days. The yields of fermentation were 20 g/L of biomass. After extraction, 0.65 g/L of water-soluble exopolysaccharide (LEPS) and 3.3 mg/100 g of water-soluble intrapolysaccharide (LIPS) were obtained. In each extract total carbohydrate, glucans and protein contents were determined. Also, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), X-ray diffractometry (XRD), high performance liquid chromatography with refraction index detection (HPLC-RI) and high performance gel permeation chromatography (HPGPC) analysis for characterization were performed. The antitumor activity was evaluated and polysaccharides not only showed anti-proliferative activity in breast cancer cells but also they activate J774 macrophages as evidenced by the increase of nitric oxide and tumor necrosis factor-α (inducers of tumor cell apoptosis). Our findings suggest that polysaccharides can activate macrophages to release nitric oxide (NO) and tumor necrosis factor alpha (TNF-α), which directly blocks cancer cell growth. These findings enhance our knowledge about new sources of fungal metabolites that serve as coadjuvant, cheap and less harmful alternatives to cancer treatment.

Genomics and Development of Lentinus tigrinus: A White-Rot Wood-Decaying Mushroom with Dimorphic Fruiting Bodies.

Lentinus tigrinus is a species of wood-decaying fungi (Polyporales) that has an agaricoid form (a gilled mushroom) and a secotioid form (puffball-like, with enclosed spore-bearing structures). Previous studies suggested that the secotioid form is conferred by a recessive allele of a single locus. We sequenced the genomes of one agaricoid (Aga) strain and one secotioid (Sec) strain (39.53-39.88 Mb, with 15,581-15,380 genes, respectively). We mated the Sec and Aga monokaryons, genotyped the progeny, and performed bulked segregant analysis (BSA). We also fruited three Sec/Sec and three Aga/Aga dikaryons, and sampled transcriptomes at four developmental stages. Using BSA, we identified 105 top candidate genes with nonsynonymous SNPs that cosegregate with fruiting body phenotype. Transcriptome analyses of Sec/Sec versus Aga/Aga dikaryons identified 907 differentially expressed genes (DEGs) along four developmental stages. On the basis of BSA and DEGs, the top 25 candidate genes related to fruiting body development span 1.5 Mb (4% of the genome), possibly on a single chromosome, although the precise locus that controls the secotioid phenotype is unresolved. The top candidates include genes encoding a cytochrome P450 and an ATP-dependent RNA helicase, which may play a role in development, based on studies in other fungi.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr = 9.84 mm day-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22 U g-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

Effect of Tree Species on Enzyme Secretion by the Shiitake Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

We compared cold and hot wood extracts of 3 endemic Siberian trees-namely, Prunus padus (bird cherry), Populus tremula (aspen), and Betula sp. (birch)-on biomass production and laccase and peroxidase secretion in submerged cultures by the medicinal mushroom Lentinus edodes. Of the conditions tested, only hot Prunus extracts stimulated biomass production, whereas all extracts stimulated laccase and peroxidase secretion, albeit to different extents. A large, differential stimulation of manganese peroxidase was observed by hot Prunus extracts. The results highlight important differences between tree species in the stimulation of biomass and enzyme production by L. edodes and point to potentially interesting stimulatory factors present in hot Prunus extracts. These findings are of relevance in the use of L. edodes for medicinal or biotechnological applications.

[Antioxidant Effect on the Growth And Formation of Lipids in the Fungus Lentinus tigrinus Growing on Medium with Lignosulfonate].

The addition of an antioxidant (2-ethyl-6-methyl-3-hydroxypyridine hydrochloride) to a culture of the fungus Lentinus tigrinus growing on a medium with lignosulfonate inhibited growth and changed the composition of cell phospholipids. The ratio of lipid messengers also changed, the phosphatidic acid level decreased, and the content of phosphatidylinositol dramatically increased. The substitution of lignosulfonate with glucose and the addition of an antioxidant increased the biomass yield of L. tigrinus, as well as that of another fungus, Cunninghamella japonica, which was incapable of biodegrading the biopolymer. The obtained results indicate the specificity of growth processes in the presence of lignosulfonate and confirm the role of free radical oxidation reactions in the biodegradation of this biopolymer by L. tigrinus.

Comparative Study of Worldwide Species of Genus Lentinus (=Lentinula, Higher Basidiomycetes) Based on Linear Mycelium Growth.

Species of mushroom genus Lentinus (=Lentinula) are best known for the commercially important and extensively studied culinary-medicinal shiitake, L. edodes. A few mycelium growth studies have focused on Lentinus boryana, but information is lacking for L. raphanica and L. aciculospora, endemic to the Americas. In this study, 14 dikaryon strains representing 5 Lentinus species were grown on 5 nutritive agar media at increments of 5°C. Growth for each species was significantly slower on corn meal agar, but no differences were found among malt extract, potato dextrose, malt peptone, and yeast malt extract agars. Lentinus aciculospora and L. boryana consistently exhibited the slowest mycelium growth rates among all species and across all temperatures tested, with optima at 15°C and 20°C. The fastest mycelium growth rates for L. edodes, L. novaezelandiae, and L. raphanica occurred at 25°C. Strains of the latter continued to grow well at 30°C, whereas growth of the other 2 species declined significantly. Differences in mycelium growth rates for American strains could be partially explained by their geographic locations, indicating that understanding this physiological parameter has important ramifications for the edible mushroom industry.

Phylogenetic relationships and morphological evolution in Lentinus, Polyporellus and Neofavolus, emphasizing southeastern Asian taxa.

The genus Lentinus (Polyporaceae, Basidiomycota) is widely documented from tropical and temperate forests and is taxonomically controversial. Here we studied the relationships between Lentinus subg. Lentinus sensu Pegler (i.e. sections Lentinus, Tigrini, Dicholamellatae, Rigidi, Lentodiellum and Pleuroti and polypores that share similar morphological characters). We generated sequences of internal transcribed spacers (ITS) and partial 28S regions of nuc rDNA and genes encoding the largest subunit of RNA polymerase II (RPB1), focusing on Lentinus subg. Lentinus sensu Pegler and the Neofavolus group, combined these data with sequences from GenBank (including RPB2 gene sequences) and performed phylogenetic analyses with maximum likelihood and Bayesian methods. We also evaluated the transition in hymenophore morphology between Lentinus, Neofavolus and related polypores with ancestral state reconstruction. Single-gene phylogenies and phylogenies combining ITS and 28S with RPB1 and RPB2 genes all support existence of a Lentinus/Polyporellus clade and a separate Neofavolus clade. Polyporellus (represented by P. arcularius, P. ciliatus, P. brumalis) forms a clade with species representing Lentinus subg. Lentinus sensu Pegler (1983), excluding L. suavissimus. Lentinus tigrinus appears as the sister group of Polyporellus in the four-gene phylogeny, but this placement was weakly supported. All three multigene analyses and the single-gene analysis using ITS strongly supported Polyporus tricholoma as the sister group of the Lentinus/Polyporellus clade; only the 28S rRNA phylogeny failed to support this placement. Under parsimony the ancestral hymenophoral configuration for the Lentinus/Polyporellus clade is estimated to be circular pores, with independent transitions to angular pores and lamellae. The ancestral state for the Neofavolus clade is estimated to be angular pores, with a single transition to lamellae in L. suavissimus. We propose that Lentinus suavissimus (section Pleuroti) should be reclassified as Neofavolus suavissimus comb. nov.

Optimum conditions for inducing laccase production in Lentinus crinitus.

Laccases are environmentally friendly alternatives in many important applications such as in bioremediation, biopulping, textile, and the food industry. They have wide substrate specificity, can oxidize a broad range of compounds, and show potential for use in various industrial processes. Therefore, developing methods to increase laccase production is important. In the current study, we aimed to identify optimum conditions for inducing laccase production in the basidiomycete Lentinus crinitus cultivated under varying nitrogen concentrations and in the presence of potential inducers of laccase production, including copper and phenolic compounds. Peak enzymatic activity (11,977 U/L) occurred at higher nitrogen concentrations (2.8 g/L nitrogen). Regardless of the nitrogen concentration, addition of copper increased the laccase activity and decreased mycelial growth, with maximum laccase activity (14,320 U/L) observed at the highest nitrogen concentration combined with 150 mM CuSO4. In addition, ethanol (0.5 or 1.0 mM) and guaiacol (1.5 mM) increased laccase production to 15,000, 14,800, and 14,850 U/L, respectively. Our findings highlighted the optimum conditions for producing L. crinitusderived laccase as potential alternatives to the conventional production and application of the enzyme.

HS/GC-MS analyzed chemical composition of the aroma of fruiting bodies of two species of genus Lentinus (Higher Basidiomycetes).

The chemical composition of the aroma of fresh fruiting bodies of the cultivated mushroom Lentinus boryanus is described here and compared with medicinal shiitake mushroom L. edodes. Volatile compounds were analyzed through headspace sampling coupled with gas chromatography-mass spectrometry. The mushrooms under study were grown on different substrates based on barley straw, sugarcane bagasse, oak wood sawdust, and beech leaf litter. It was determined that L. boryanus as well as L. edodes contain an abundant amount of a volatile compound identified as 3-octanone with a sweet fruity aroma. On the other hand, only L. boryanus produced 3-octanol a characteristic aroma of cod liver oil. In total, 10 aromatic compounds were identified, some of which were obtained exclusively in one species or substrate.

[Influence of lignin and oxygen on the growth and the lipid formation of the fungus Lentinus tigrinus].

During cultivation of the filamentous fungus Lentinus tigrinus on a medium containing lignin, a high oxygen content stimulated the growth of the fungus and contributed to the yield of lipids. A high content of phosphatidic acid and a reduction in the level of phosphatidylethanolamine and phosphatidylserine were first detected in the composition of phospholipids. Changes in the composition of neutral lipids, such as variation in the ratio of esterified and free sterols, have occurred; thus, the amount of sterol esters reduced simultaneously with a decrease in the content of free fatty acids. Based on the obtained results, the possible role of phosphatidic acid as a second messenger in the process of the consumption of lignin by the fungus Lentinus tigrinus is discussed.

Mycelial biomass and biochemical properties of proteases produced by Lentinus citrinus DPUA 1535 (Higher Basidiomycetes) in submerged cultivation.

The cultivation of Lentinus citrinus for mycelial biomass and protease production under different carbon and nitrogen sources was studied in submerged cultivation. The nutritional source concentration for protease production was evaluated using a full factorial design. For mycelial biomass maltose (4.94 mg/mL) and beef extract (5.45 mg/mL), carbon and nitrogen sources presented the best results, respectively. The maximum protease activity was 73.33 U/mL with fructose (30.0 g/L) and beef extract (10.0 g/L). Proteases showed maximum activity at 40°C and pH 7.0, which exhibited high stability at experimental conditions. The final part of this work was devoted to estimating the main thermodynamic parameters of the irreversible enzyme inactivation (ΔH* = 17.86 kJ/mol, ΔG* =102.09 kJ/mol, ΔS* = -260.76 J/mol×K) through residual activity tests carried out at 25-70°C, by making use of Arrhenius and Eyring plots.

Biodegradation potential and ligninolytic enzyme activity of two locally isolated Panus tigrinus strains on selected agro-industrial wastes.

The degradation potential and ligninolytic enzyme production of two isolated Panus tigrinus strains (M609RQY and M109RQY) were evaluated in this study. These strains were grown on three selected abundant agro-industrial wastes (rice straw; rice husk and cassava peel) under solid-state fermentation conditions. Degradation potential was determined by analyzing the chemical composition of the selected substrates before and after fermentation along with ligninolytic enzyme production. The strain M609RQY led to the highest lignin degradation of 40.81% on cassava peel, 11.25% on rice husk and 67.96% on rice straw. Both strains significantly increased the protein content of cassava peel. Rice husk stimulated maximum laccase (2556 U/L) and lignin peroxidase (24 U/L) production by the strains M109RQY and M609RQY, respectively. Furthermore, cassava peel stimulated maximum manganese-dependent peroxidase (141 U/L) production by the strain M109RQY. The de-lignified rice straw and the nutritionally-improved cassava peel could serve as potential animal feed supplements.

The tropical white rot fungus, Lentinus squarrosulus Mont.: lignocellulolytic enzymes activities and sugar release from cornstalks under solid state fermentation.

Lentinus squarrosulus Mont., a high temperature tolerant white rot fungus that is found across sub-Saharan Africa and many parts of Asia, is attracting attention due to its rapid mycelia growth and potential for use in food and biodegradation. A solid state fermentation (SSF) experiment with L. squarrosulus (strain MBFBL 201) on cornstalks was conducted. The study evaluated lignocellulolytic enzymes activity, loss of organic matter (LOM), exopolysaccharide content, and the release of water soluble sugars from degraded substrate. The results showed that L. squarrosulus was able to degrade cornstalks significantly, with 58.8% LOM after 30 days of SSF. Maximum lignocellulolytic enzyme activities were obtained on day 6 of cultivation: laccase = 154.5 U/L, MnP = 13 U/L, peroxidase = 27.4 U/L, CMCase = 6.0 U/mL and xylanase = 14.5 U/mL. L. squarrosulus is a good producer of exopolysaccharides (3.0-5.13 mg/mL). Glucose and galactose were the most abundant sugars detected in the substrate during SSF, while fructose, xylose and trehalose, although detected on day zero of the experiment, were absent in treated substrates. The preference for hemicellulose over cellulose, combined with the high temperature tolerance and the very fast growth rate characteristics of L. squarrosulus could make it an ideal candidate for application in industrial pretreatment and biodelignification of lignocellulosic biomass.

The influence of different submerged cultivation conditions on mycelial biomass and protease production by Lentinus citrinus Walleyn et Rammeloo DPUA 1535 (Agaricomycetideae).

The influence of different carbon and nitrogen sources, pH of the culture medium, and temperature and period of cultivation on mycelial biomass production and protease activity by Lentinus citrinus DPUA 1535 were investigated in submerged culture. A 2(5) full factorial design with three central points was employed, and the results showed that at a significance level of 95% only nitrogen source and temperature were statistically significant for mycelial biomass production. On the other hand, for protease activity all factors and some interactions were significant, and the temperature and nitrogen source had the most significant effect. The best condition for mycelial biomass production (5.76 mg mL(-1)) and protease activity (32.3 U mL(-1)) was obtained in medium formulated with 0.5% soluble starch, 0.2% gelatin, pH 7.0, 25 degrees C, in 5 days.

Mineralization of mono-nitrophenols by Bjerkandera adusta and Lentinus squarrosulus and their extracellular ligninolytic enzymes.

Nitroaromatic compounds constitute a major class of widely distributed environmental contaminants. Fifty fungal strains were screened for their potential to tolerance with 2-nitrophenol, 3-nitrophenol and 4-nitrophenol on solid medium supplemented with 2% malt extract (MEA). Growth rate (mm/day) was determined at three concentrations (0.25, 0.5 and 1 mM) of all the three nitrophenols. From the fifty fungal strains only Bjerkandera adusta and Lentinus squarrosulus were able to tolerate all the three nitrophenols (NPs). These white-rot fungi (WRF) were chosen for liquid medium studies for the mineralization of mono-nitrophenols and ligninolytic enzyme activity at 0.25 mM concentration. Both varieties completely removed 2-NP and 3-NP while 4-NP was hard to mineralize. AAO (Aryl Alcohol Oxidase) is the main oxidase enzyme in B. adusta while laccase plays important role in L. squarrosulus. MnP (Manganese peroxidase) is the main peroxidase enzyme in both varieties. These fungal strains were capable to degrade nitrophenols and could be used for bioremediation applications on large scale.

[The role of laccase and peroxidase of Lentinus (Panus) tigrinus fungus in biodegradation of high phenol concentrations in liquid medium].

The possibility of the usage of Lentinus tigrinus fungus strain VKM F-3616D for biodegradation of high (up to 5%) phenol concentrations in liquid medium and the involvement of laccase and peroxidase in this process have been studied. L. tigrinus fungus was demonstrated to effectively digrade phenol with easy biomass separation from the liquid. Decrease in phenol concentration was accompanied by increased secretion level and laccase activity at the preliminary stages of biodegradation, while that of peroxidase was at the latest stages of biodegradation. These enzyme secretions in distinct ratios and consequences are necessary for effective phenol biodegradation. An effective approach for phenol concentration decrease in the waste water of smoking shops in meat-processing factories using L. tigrinus fungus was described.

Lentinus (Panus) tigrinus augmentation of a historically contaminated soil: matrix decontamination and structure and function of the resident bacterial community.

The ability of Lentinus tigrinus to grow and to degrade persistent aromatic hydrocarbons in aged contaminated soil was assessed in this study. L. tigrinus extensively colonized the soil; its degradation activity after 60 d incubation at 28°C, however, was mostly limited to dichloroaniline isomers, polychlorinated benzenes and diphenyl ether while the fungus was unable to deplete 9,10-anthracenedione and 7-H-benz[DE]anthracene-7-one which were the major soil contaminants. Although clean-up levels were limited, both density of cultivable heterotrophic bacteria and richness of the resident bacterial community in L. tigrinus microcosms (LtM) increased over time to a significantly larger extent than the respective amended incubation controls (1.9×10(9) CFU g(-1) vs. 1.0×10(9) CFU g(-1) and 37 vs. 16, respectively). Naphthalene- and catechol 2,3-dioxygenase gene copy numbers, however, decreased over time at a higher rate in LtM than in incubation controls likely due to a higher stimulation on heterotrophs than xenobiotics-degrading community members.

Temperatura e meio de cultura mais favoráveis ao crescimento micelial de uma linhagem de Lentinus strigosus de ocorrência na Amazônia / Most favorable temperature and culture medium for the mycelial growth of a strain of Lentinus strigosus from the Amazon region

O objetivo do trabalho foi avaliar o crescimento micelial de uma linhagem de Lentinus strigosus, de ocorrência na Amazônia, em cinco meios de cultura à base de malte, serragem de marupá (Simarouba amara), serragem de pau de balsa (Ochroma piramidale), estipe de pupunheira (Bactris gasipaes Kunth) e bagaço de cana-de-açúcar (Saccharum officinarum), submetidos às temperaturas de 20, 25, 30, 35, 40, 45 e 50º C. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 5 x 7. Cada tratamento constou de quatro repetições, correspondente a uma placa de Petri, totalizando 140 unidades experimentais. Verificou-se que a temperatura de 35° C foi a mais favorável para o crescimento micelial de L. strigosus e que o meio à base de estipe de pupunheira foi o mais promissor para o cultivo deste fungo.

The objective of this work was to evaluate the mycelial growth of a strain of Lentinus strigosus occurring in the Amazon region in five culture mediums made on the basis of malt, "marupá" sawdust (Simarouba amara), "pau de balsa" sawdust (Ochroma piramidale), peach palm stipe (Bactris gasipaes Kunth) and crushed sugarcane (Saccharum officinarum), when submitted to different temperatures (20, 25, 30, 35, 40, 45 and 50º C). The experimental design was totally randomized, in a 5 x 7 factorial scheme. Each treatment corresponded to a Petri dish with four repetitions, totaling 140 experimental units. The temperature of 35º C was found to be the most favorable for the mycelial growth of L. strigosus, and the medium with peach palm stipe was the most promising for the cultivation of this fungus.