Mushrooms

蘑菇
  • 文章类型: Journal Article
    目的:探讨3种羊肚菌提取物(羊肚菌,使用凋亡生物标志物在乳腺癌和结肠癌细胞系上的羊肚菌和羊肚菌)。
    方法:用浓度范围为0.0625至2mg/mL的3种羊肚菌的甲醇和乙醇提取物处理人乳腺细胞系(MCF-7)和结肠癌细胞系(SW-480)。之后,它们对凋亡相关标志物(促凋亡标志物包括Bax,使用逆转录聚合酶链反应确定caspase-3,caspase-7和caspase-9以及抗凋亡标志物,包括Bcl-2)。
    结果:所有羊肚菌提取物在0.02±0.01至0.68±0.30mg/mL的半抑制浓度(IC50)下都能降低乳腺癌和结肠癌细胞的增殖。不出所料,所有羊肚菌提取物均显著增加Bax的基因表达,caspase-3,caspase-7和caspase-9下调了MCF-7和SW-480细胞系中Bcl-2的基因表达(P<0.05)。
    结论:羊肚菌提取物通过凋亡诱导机制对乳腺癌和结肠癌细胞系具有显著的抗增殖活性。羊肚菌提取物的抗癌活性以及乳腺癌和结肠癌细胞凋亡的激活表明,它将来可能用于开发抗癌的化学治疗剂。
    OBJECTIVE: To explore the potential apoptotic mechanisms of 3 Morchella extracts (Morchella conica, Morchella esculenta and Morchella delicosa) on breast and colon cancer cell lines using apoptotic biomarkers.
    METHODS: Human breast cell line (MCF-7) and colon cancer cell line (SW-480) were treated with methanol and ethanol extracts of 3 Morchella species with concentration ranging from 0.0625 to 2 mg/mL. After that their effects on gene expression of apoptosis related markers (pro-apoptotic markers including Bax, caspase-3, caspase-7, and caspase-9, and the antiapoptotic marker including Bcl-2) were determined using reverse transcription polymerase chain reaction.
    RESULTS: All Morchella extracts reduced breast and colon cancer cells proliferation at half inhibitory concentration (IC50) of 0.02 ±0.01 to 0.68 ±0.30 mg/mL. As expected, all Morchella extracts significantly increased gene expressions of Bax, caspase-3, caspase-7, and caspase-9 and downregulated the gene expression of Bcl-2 in MCF-7 and SW-480 cell lines (P<0.05).
    CONCLUSIONS: Morchella extracts demonstrated significant anti-proliferative activity against breast and colon cancer cell lines via an apoptosis induction mechanism. Anticancer activity of Morchella extracts and activation of apoptosis in breast and colon cancer cells suggest that it may be used to develop chemotherapeutic agents against cancer in future.
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  • 文章类型: Journal Article
    几个世纪以来,蘑菇已被用作护肤配方的成分。环境压力和现代生活方式使皮肤加速老化。为了减缓这个过程,天然抗衰老护肤成分正在寻找。在这次审查中,选择了52种有关从大型真菌子实体中提取的化合物对皮肤细胞的影响的科学出版物。已经描述了来自9个物种的提取物的抗衰老效果测试的效果。根据现有文献数据,大型真菌含有许多多糖,酚类化合物,多糖肽,游离氨基酸,固醇,蛋白质,糖苷,三萜,生物碱,它可以通过作为抗氧化剂对皮肤产生抗衰老作用,光保护,皮肤美白,保湿,抗炎和稳定胶原蛋白,皮肤中的弹性蛋白和透明质酸水平。
    For centuries, mushrooms have been used as a component of skincare formulations. Environmental stresses and a modern lifestyle expose the skin to accelerated aging. To slow down this process, natural anti-aging skincare ingredients are being sought. In this review, 52 scientific publications about the effects of chemical compounds extracted from the fruiting bodies of macrofungi on skin cells were selected. The effects of extracts from nine species that are tested for anti-aging effects have been described. According to available literature data, macrofungi contain many polysaccharides, phenolic compounds, polysaccharide peptides, free amino acids, sterols, proteins, glycosides, triterpenes, alkaloids, which can have an anti-aging effect on the skin by acting as antioxidants, photoprotective, skin whitening, moisturizing, anti-inflammatory and stabilizing collagen, elastin and hyaluronic acid levels in the skin.
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  • 文章类型: Journal Article
    蘑菇由于其巨大的营养和治疗特性而引起了研究人员的极大兴趣。生物活性的初级和次级代谢物的存在,其中包括几种微量营养素,包括维生素,必需矿物质,和其他膳食纤维,使它们成为极好的功能性食物。此外,据报道,在饮食中加入蘑菇可以减少与衰老和生活方式相关的疾病的发病率,比如癌症,肥胖,和中风,以及通过促进免疫调节来提供整体健康益处,抗氧化活性,和增强肠道微生物菌群。已经通过使用细胞系以及无脊椎动物和脊椎动物模型系统的体外和体内研究来评估几种蘑菇提取物的多功能活性,以在功能和分子水平上解决人类疾病和病症。尽管每种模型都有自己的优势和不足,各种研究已经产生了大量的数据,关于调节球员,以提供各种保护活动;因此,这篇综述旨在汇编和概述蘑菇衍生的生物活性物质的作用机理,这将有助于未来的医学探索。
    Mushrooms have garnered considerable interest among researchers due to their immense nutritional and therapeutic properties. The presence of biologically active primary and secondary metabolites, which includes several micronutrients, including vitamins, essential minerals, and other dietary fibers, makes them an excellent functional food. Moreover, the dietary inclusion of mushrooms has been reported to reduce the incidence of aging- and lifestyle-related diseases, such as cancer, obesity, and stroke, as well as to provide overall health benefits by promoting immunomodulation, antioxidant activity, and enhancement of gut microbial flora. The multifunctional activities of several mushroom extracts have been evaluated by both in vitro and in vivo studies using cell lines along with invertebrate and vertebrate model systems to address human diseases and disorders at functional and molecular levels. Although each model has its own strengths as well as lacunas, various studies have generated a plethora of data regarding the regulating players that are modulated in order to provide various protective activities; hence, this review intends to compile and provide an overview of the plausible mechanism of action of mushroom-derived bioactives, which will be helpful in future medicinal explorations.
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  • 文章类型: Journal Article
    在这里,我们强调了在体外和体内研究之前分子建模方法的重要性;特别是,在没有公认的治疗方法的疾病,如神经系统异常。阿尔茨海默病是一种神经退行性疾病,可导致不可逆转的认知功能下降。毒性和ADMET研究分别使用Maestro软件和DiscoveryStudio2.0中的Qikprop平台进行,从过去十年中从蘑菇中分离出的超过45种经过审查的化合物中选择有前途的骨骼。使用刚性和柔性分子对接方法,例如在β-分泌酶(BACE1)和乙酰胆碱酯酶(ACHE)的结合位点中诱导对接(IFD),使用Maestro和CressetFlare平台,与标准药物多奈哌齐和(2E)-2-亚氨基-3-甲基-5,5-二苯基咪唑烷-4-酮(OKK)相比,通过高精度分子对接筛选了有希望的结构。分子相互作用,绑定距离,测量和RMSD值以揭示两种神经变性酶的结合位点处的关键相互作用。对IFD结果的分析显示,在4ey7的口袋中,双基喹唑A和GensetinI的结合一致,而对单酚A,成霉素,和fornicin在4dju中非常吻合,表明在ACHE和BACE1上的结合与天然配体非常接近。根据其吉布斯自由结合能,相应的关键氨基酸接触表现出最小的空间问题,GlideXP分数,RMSD值,和关键氨基酸的分子取向。分子动力学模拟进一步证实了我们的发现,并展望了这些化合物在未来的药理学研究中显示出显著的体外结果。
    Herein, we highlight the significance of molecular modeling approaches prior to in vitro and in vivo studies; particularly, in diseases with no recognized treatments such as neurological abnormalities. Alzheimer disease is a neurodegenerative disorder that causes irreversible cognitive decline. Toxicity and ADMET studies were conducted using the Qikprop platform in Maestro software and Discovery Studio 2.0, respectively, to select the promising skeletons from more than 45 reviewed compounds isolated from mushrooms in the last decade. Using rigid and flexible molecular docking approaches such as induced fit docking (IFD) in the binding sites of β-secretase (BACE1) and acetylcholine esterase (ACHE), promising structures were screened through high precision molecular docking compared with standard drugs donepezil and (2E)-2-imino-3-methyl-5,5-diphenylimidazolidin-4-one (OKK) using Maestro and Cresset Flare platforms. Molecular interactions, binding distances, and RMSD values were measured to reveal key interactions at the binding sites of the two neurodegenerative enzymes. Analysis of IFD results revealed consistent bindings of dictyoquinazol A and gensetin I in the pocket of 4ey7 while inonophenol A, ganomycin, and fornicin fit quite well in 4dju demonstrating binding poses very close to native ligands at ACHE and BACE1. Respective key amino acid contacts manifested the least steric problems according to their Gibbs free binding energies, Glide XP scores, RMSD values, and molecular orientation respect to the key amino acids. Molecular dynamics simulations further confirmed our findings and prospected these compounds to show significant in vitro results in their future pharmacological studies.
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  • 文章类型: Journal Article
    出于可持续性和环境原因,需要降低动物源性食品在人类饮食中的比例。然而,同样重要的是,从动物源性食品的过渡不会导致任何不利的营养影响。在这项研究中,研究了将乳清蛋白分离物(WPI)与香菇(SM)或牡蛎蘑菇(OM)混合以创建具有增强营养和理化特性的混合食品的潜力。OM或SM添加对地层的影响,微观结构,因此检查了热定形乳清蛋白凝胶的物理化学属性。使用蘑菇粉是因为它们含有相对较高的维生素,矿物,植物化学物质,和膳食纤维,可以提供营养益处,而WPI用于提供蛋白质和良好的热凝胶化性质。采用多种分析方法对WPI-蘑菇杂种的结构和理化性质进行了表征,包括共聚焦显微镜,粒子电泳,光散射,近似分析,差示扫描量热法,热重分析,动态剪切流变学,纹理轮廓分析,和比色法。当pH从3升高到9时,乳清蛋白和蘑菇颗粒上的电荷从正变为负,但是乳清蛋白具有更高的等电点和电荷大小。OM略微增加了WPI的热稳定性,但SM效果不大。两种蘑菇都降低了乳清蛋白凝胶的亮度并增加了棕色。蘑菇粉的加入也降低了乳清蛋白凝胶的硬度和杨氏模量,这可能是因为蘑菇颗粒充当了柔软的填充物。这项研究为具有理想的生理化学和营养属性的混合乳清蛋白-蘑菇产品的形成提供了有价值的见解。
    There is a need to reduce the proportion of animal-derived food products in the human diet for sustainability and environmental reasons. However, it is also important that a transition away from animal-derived foods does not lead to any adverse nutritional effects. In this study, the potential of blending whey protein isolate (WPI) with either shiitake mushroom (SM) or oyster mushroom (OM) to create hybrid foods with enhanced nutritional and physicochemical properties was investigated. The impact of OM or SM addition on the formation, microstructure, and physicochemical attributes of heat-set whey protein gels was therefore examined. The mushroom powders were used because they have relatively high levels of vitamins, minerals, phytochemicals, and dietary fibers, which may provide nutritional benefits, whereas the WPI was used to provide protein and good thermal gelation properties. A variety of analytical methods were used to characterize the structural and physicochemical properties of the WPI-mushroom hybrids, including confocal microscopy, particle electrophoresis, light scattering, proximate analysis, differential scanning calorimetry, thermogravimetric analysis, dynamic shear rheology, textural profile analysis, and colorimetry. The charge on whey proteins and mushroom particles went from positive to negative when the pH was raised from 3 to 9, but whey protein had a higher isoelectric point and charge magnitude. OM slightly increased the thermal stability of WPI, but SM had little effect. Both mushroom types decreased the lightness and increased the brownness of the whey protein gels. The addition of the mushroom powders also decreased the hardness and Young\'s modulus of the whey protein gels, which may be because the mushroom particles acted as soft fillers. This study provides valuable insights into the formation of hybrid whey protein-mushroom products that have desirable physiochemical and nutritional attributes.
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  • 文章类型: Journal Article
    已经开发了可食用的比色标签来确定蘑菇的新鲜度水平,即白牡蛎蘑菇(平菇)。可食用指示标签是基于固定在壳聚糖和玉米淀粉制成的可食用薄膜上的紫甘薯(IpomoeabatatasL.)花青素(PSPA)制成的,并添加了PVA。蘑菇的新鲜度参数是pH值,减肥,纹理,和感官评价。结果表明,当蘑菇新鲜时,比色标签为深紫色,当蘑菇仍然新鲜时,变成浅紫色,当蘑菇不再新鲜时,最后是绿色的。使用ImageJ程序测量标签的颜色值(平均红色),其颜色值(平均红色)随着蘑菇新鲜度水平的降低而增加。食用标签可以区分新鲜蘑菇和腐败,使其适合用于包装的蘑菇作为新鲜度指标。
    An edible colorimetric label has been developed to determine the freshness level of mushrooms, i.e. white oyster mushrooms (Pleurotus ostreatus). The edible indicator label has been fabricated based on purple sweet potato (Ipomoea batatas L.) anthocyanins (PSPA) immobilized onto an edible film made of chitosan and cornstarch with added PVA. The freshness parameters of the mushrooms were pH, weight loss, texture, and sensory evaluation. The results showed that the colorimetric label was dark purple when the mushroom was fresh, and turn to light purple when the mushroom was still fresh, and finally green when the mushroom was no longer fresh. The color value (mean Red) of the label was measured using the ImageJ program, where its color value (mean Red) increased with decreasing freshness level of the mushrooms. The edible label can distinguish fresh mushrooms from spoilage, making it suitable to be used in a packaged mushroom as a freshness indicator.
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  • 文章类型: Journal Article
    蘑菇的遗传研究,在CRISPR-Cas9基因组编辑和RNA干扰等创新的推动下,改变我们对这些神秘真菌及其在农业中的多方面作用的理解,医学,和保护。这篇综合综述探讨了蘑菇遗传研究的基本原理和意义,深入研究道德,监管,和这个领域的生态维度。CRISPR-Cas9作为一种改变游戏规则的技术出现,实现精确的基因组编辑,靶向基因敲除,和路径操纵。RNA干扰通过下调基因以提高作物产量和增强病虫害抗性来补充这些努力。遗传研究还有助于保护稀有物种和开发更强大的蘑菇菌株,促进可持续种植实践。此外,它们释放了发现新的药用化合物的潜力,为制药和营养品提供新的视野。随着新兴技术和伦理考虑塑造了蘑菇研究的未来,这些研究有望彻底改变我们与这些真菌的关系,为更可持续和创新的世界铺平道路。
    Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world.
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  • 文章类型: Journal Article
    Schizphyllumcommune是一种蘑菇形成的真菌,以其独特的子实体和裂开的g而著称。它被用作研究蘑菇发育的模式生物,木质纤维素降解和交配型基因座。它是一种超变物种,菌株之间具有相当大的遗传和表型多样性。在这项研究中,在蘑菇发育方面,我们系统地对16个原核菌株进行了表型分析,对18个单核菌株进行了木质纤维素降解。关于这些表型,菌株之间存在相当大的异质性。大多数菌株培养出形态不同的蘑菇,尽管某些菌株仅在测试条件下营养生长。在各种碳源上的生长显示出菌株特异性曲线。对七个单核菌株的基因组进行了测序,并与六个先前发表的基因组序列一起进行了分析。此外,对相关物种筋膜裂竹进行了测序。尽管基因组组装之间存在相当大的遗传变异,与蘑菇形成和木质纤维素降解相关的基因保存良好。这些测序的基因组,结合高表型多样性,将为S.commune菌株的功能基因组学分析提供坚实的基础。
    Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes, in combination with the high phenotypic diversity, will provide a solid basis for functional genomics analyses of the strains of S. commune.
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  • 文章类型: Journal Article
    高级真菌木素营养担子菌已知是生物活性代谢物的储库。目前,菌丝真菌产品作为作物保护的创新替代品的开发受到了广泛的关注。没有关于木养蘑菇糖聚物质与植物之间相互作用背后机制的数据。在这项研究中,研究了担子菌代谢产物对小麦植株形态生理和生化变量的影响。小麦(小麦。Saratovskaya29)的幼苗用从20个担子菌菌株的浸没培养物中分离的细胞外多糖(EPS)处理,这些菌株分为13种和8属。将最终浓度为15、40和80mg/L的EPS溶液施用于小麦幼苗,然后使其生长10天。在植物样本中,生物量,胚芽鞘的长度,芽和根,根号,丙二醛浓度的脂质过氧化率,过氧化氢的含量,和总酚进行测量。定义了过氧化物酶和超氧化物歧化酶活性。大多数EPS制剂提高了生物量产量,以及检查的形态参数。EPS的应用加强了抗氧化酶的活性,下降了对脂质的氧化毁伤。从其对小麦植株生长指数和氧化还原系统的总体影响来看,与其他浓度相比,EPS浓度为40mg/L已被证明是最有益的。这项研究证明,可以开发基于蘑菇EPS的新型生物制剂,并且对小麦生长和抗氧化反应有效。针对EPS发现的植物刺激特性使人们有理由考虑在木营养担子菌培养物中产生的细胞外代谢物作为能够诱导植物对胁迫的反应的活性成分。
    Higher-fungi xylotrophic basidiomycetes are known to be the reservoirs of bioactive metabolites. Currently, a great deal of attention has been paid to the exploitation of mycelial fungi products as an innovative alternative in crop protection. No data exist on the mechanisms behind the interaction between xylotrophic mushrooms\' glycopolymeric substances and plants. In this study, the effects of basidiomycete metabolites on the morphophysiological and biochemical variables of wheat plants have been explored. Wheat (Triticum aestivum L. cv. Saratovskaya 29) seedlings were treated with extracellular polysaccharides (EPSs) isolated from the submerged cultures of twenty basidiomycete strains assigned to 13 species and 8 genera. The EPS solutions at final concentrations of 15, 40, and 80 mg/L were applied to wheat seedlings followed by their growth for 10 days. In the plant samples, the biomass, length of coleoptile, shoot and root, root number, rate of lipid peroxidation by malondialdehyde concentration, content of hydrogen peroxide, and total phenols were measured. The peroxidase and superoxide dismutase activity were defined. Most of the EPS preparations improved biomass yields, as well as the morphological parameters examined. EPS application enhanced the activities of antioxidant enzymes and decreased oxidative damage to lipids. Judging by its overall effect on the growth indices and redox system of wheat plants, an EPS concentration of 40 mg/L has been shown to be the most beneficial compared to other concentrations. This study proves that novel bioformulations based on mushroom EPSs can be developed and are effective for wheat growth and antioxidative response. Phytostimulating properties found for EPSs give grounds to consider extracellular metabolites produced in the xylotrophic basidiomycete cultures as an active component capable of inducing plant responses to stress.
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  • 文章类型: Journal Article
    实验研究一直专注于开发用于种植香菇的新基质,作为标准橡木锯末基质的替代品。合适的木质纤维素材料的选择基于它们在生产区域中的可用性以及它们与所栽培的蘑菇物种的要求的相容性。与橡木锯末基板(OS)作为对照相比,这项研究评估了橡树橡子(OA)的潜力,橄榄修剪(OLPR),和葡萄园修剪(VIP),和各种组合:OA-OLPR:1-1、OA-VIP:1-1、OS-OLPR:1-1和OS-VIP:1-1,基于干重制备。与操作系统相比,在OA中加速了完整的菌丝体发育,OA-VIP:1-1和OS-VIP:1-1由9.5、7.9和4.2天延迟,而在OLPR和OS-OLPR:1-1由11.3和7.0天延迟,分别。此外,在OA中收获较早,OA-VIP:1-1,OS-VIP:1-1到9.3、6.7和3.3天,分别,虽然它在OLPR中被显著延迟,VIP,和OS-OLPR:1-1由12.3、3.7和8.0天,分别。虽然OLPR中的总生物产量显著降低,OS-OLPR:1-1,VIP,和OS-VIP:1-1,与OA中的OS相当,OA-OLPR:1-1,OA-VIP:1-1(597.0、552.0、532.2和556.2g/kg,分别)。在操作系统连续两次刷新中,产量一直很高,OA,和OA-VIP:1-1。OA-OLPR:1-1比OS-OLPR:1-1高,OA-VIP:1-1比OS-VIP:1-1高。OA增加了蘑菇数量和硬度,VIP和OLPR增加了蘑菇的重量,和OA-VIP:1-1增加了绒毛厚度。在所有底物中,蘑菇蛋白质和纤维含量均高于OS,在OA-OLPR中最高:1-1(8.7%)和OLPR(2.8%),分别。最后,基材OA,OA-VIP:1-1和OA-OLPR:1-1可能会交替使用橡木木屑;但是,由于较早的收获,前两种底物比第三种具有优势。此外,与OA结合使用VIP和OLPR比单独使用它们更有利。
    Experimental research has been focusing on developing new substrates for growing shiitake mushrooms as alternatives to the standard oak sawdust substrate. The selection of appropriate lignocellulosic materials is based on their availability in the production area and their compatibility with the requirements of the mushroom species being cultivated. In comparison to oak sawdust substrate (OS) as the control, this study evaluated the potential of oak acorns (OA), olive pruning (OLPR), and vineyard pruning (VIP), and various combinations: OA-OLPR:1-1, OA-VIP:1-1, OS-OLPR:1-1, and OS-VIP:1-1, prepared on a dry weight basis. In comparison to OS, complete mycelial development was hastened in OA, OA-VIP: 1-1, and OS-VIP:1-1 by 9.5, 7.9, and 4.2 days and delayed in OLPR and OS-OLPR:1-1 by 11.3 and 7.0 days, respectively. Also, harvest was earlier in OA, OA-VIP:1-1, and OS-VIP:1-1 by 9.3, 6.7, and 3.3 days, respectively, while it was significantly delayed in OLPR, VIP, and OS-OLPR:1-1 by 12.3, 3.7, and 8.0 days, respectively. While the total biological yield was significantly reduced in OLPR, OS-OLPR:1-1, VIP, and OS-VIP:1-1, it was comparable to OS in OA, OA-OLPR:1-1, and OA-VIP:1-1 (597.0, 552.0, 532.2, and 556.2 g/kg, respectively). Production was consistently high over two consecutive flushes in OS, OA, and OA-VIP: 1-1. Total biological yields were higher in OA-OLPR: 1-1 than OS-OLPR:1-1 and in OA-VIP:1-1 than OS-VIP:1-1. OA increased mushroom number and firmness, VIP and OLPR increased mushroom weight, and OA-VIP:1-1 increased pileus thickness. Mushrooms\' protein and fiber contents were higher than OS in all substrates and the highest in OA-OLPR:1-1 (8.7 %) and OLPR (2.8 %), respectively. Conclusively, the substrates OA, OA-VIP: 1-1, and OA-OLPR:1-1 may alternate oak sawdust; however, the first two substrates have an advantage over the third due to earlier harvests. Also, it is more favorable to use VIP and OLPR in combination with OA than to use them alone.
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