Enhancing Eritadenine Production in Submerged Cultures of Shiitake (Lentinula edodes Berk. Pegler) Using Blue LED Light and Activated Charcoal. Revealing Eritadenine's Novel In Vitro Bioherbicidal Activity Against Chrysanthemum morifolium.

Eritadenine from shiitake mushroom is a secondary metabolite with hypocholesterolemic, hypotensive and antiparasitic properties, thus promising for pharmaceutical and agricultural applications. Eritadenine is obtained from submerged mycelial cultures of shiitake, but the actual yields remain unsatisfactory to explore potential applications or industrial-scale production. In this study, green and blue LED lights were tested to increase yields of eritadenine in submerged cultures of shiitake. Notably, blue LEDs increased yields by 13-14 times, reaching 165.7 mg/L, compared to darkness (11.2 mg/L) and green light (12.1 mg/L) (p < 0.05, Tukey test). Nitrogen sources yeast extract (YE) and peptone (at 2 g/L) increased eritadenine production. YE promoted 22.6 mg/L, while peptone 18.3 mg/L. The recovery of eritadenine was evaluated using amberlite and activated charcoal (AC) adsorption isotherms. AC demonstrated the highest adsorption rate, with 75 mg of eritadenine per gram of AC, according to the Freundlich isotherm. The desorption rate reached 93.95% at pH 10. The extract obtained from submerged cultures had eritadenine content of 63.31%, corresponding to 87.86% of recovery, according to HPLC analysis. Furthermore, the novel bioherbicidal potential of eritadenine was tested on in vitro Chrysanthemum morifolium plants. The cultures extract containing eritadenine had a detrimental impact on plant development, generating mortality of 100% at 3%, 0.5%, and 0.25%. Moreover, pure eritadenine exhibited a phytotoxic effect similar than glyphosate on leaves, stems and roots. These findings highlight the significant bioherbicidal properties of eritadenine. Further studies are needed to understand the biosynthetic pathway of eritadenine and its bioherbicidal properties on weeds and illicit crops.

Preparation and characterization of water vapor-responsive methylcellulose-polyethylene glycol-400 composite membranes and an indication of freshness of shiitake mushrooms.

Intelligent packaging with freshness indication capability can help consumers purchase fresh food. However, current research primarily focuses on carbon dioxide-sensitive intelligent packaging, with limited research on water vapor-sensitive indication packaging. In this study, the water vapor-sensitive indicator membrane was prepared and used to determine the freshness of mushrooms. The results of this study showed that the water permeability of the indicator membrane decreased from 33.17 % to 21.59 % with the increase of Polyethylene glycol-400(PEG-400) content in methylcellulose(MC) membrane, and the contact angle of the indicator membrane increased from 87 % to 98 % with the addition of PEG-400. The addition of plasticizer PEG-400 increased the hydrophobicity of the indicator film, which could be attributed to the improvement of the molecular arrangement and crystallinity of the indicator film by the addition of PEG-400. After encountering water, the transparency of the indicator membrane changes from completely opaque (white) to transparent. Addition of PEG-400 reduces the rate of change in the transparency of the indicator membrane. The indicator membrane was successfully used to indicate the freshness of mushrooms and effectively reflected the freshness of mushrooms during storage. This technology could be applied to measure the freshness of other foods.

Impact of Incorporating Shiitake Mushrooms (Lentinula edodes) on Microbial Community and Flavor Volatiles in Traditional Jiuqu.

Jiuqu is one of the important raw materials for brewing Chinese rice wine (Huangjiu), often known as the "bone of wine". In this study, the microbial community and flavor substances of Jiuqu made with different amounts of shiitake mushroom (Lentinula edodes) were investigated through high-throughput sequencing technology and headspace gas chromatography-ion migration spectroscopy (HS-GC-IMS), using traditional wheat yeast as a control. The results showed that 1593 genera and 5507 species were identified among the four types of yeast, with Aspergillus and Paecilomyces being the most dominant microorganisms at the genus level. Carbohydrate, coenzyme, and amino acid metabolism may be the main metabolic processes of the dominant microorganisms in Jiuqu. In terms of flavor, a total of 79 volatile substance monomers and some dimers were detected from four types of Jiuqu raw materials, with the main substances being 12 aldehydes, 19 ketones, 13 alcohols, 19 esters, 4 olefins, 1 acid, 3 ethers, 4 furans, 1 pyrazine, 1 pyridine, 1 triethylamine, and 1 thiazole. The correlation results indicate that Aspergillus, Lactobacillus, and Vibrio correlate significantly with the volatile flavor compounds unique to shiitake mushrooms and also have a positive effect on alcohol, esters, and furans. These results could shed light on the selection of Lentinula edodes as a fermentation starter for Huangjiu in the Qinba Mountain area.

Near-gapless genome and transcriptome analyses provide insights into fruiting body development in Lentinula edodes.

Fruiting body development in macrofungi is an intensive research subject. In this study, high-quality genomes were assembled for two sexually compatible monokaryons from a heterokaryotic Lentinula edodes strain WX1, and variations in L. edodes genomes were analyzed. Specifically, differential gene expression and allele-specific expression (ASE) were analyzed using the two monokaryotic genomes and transcriptome data from four different stages of fruiting body development in WX1. Results revealed that after aeration, mycelia sensed cell wall stress, pheromones, and a decrease in CO2 concentration, leading to up-regulated expression in genes related to cell adhesion, cell wall remodeling, proteolysis, and lipid metabolism, which may promote primordium differentiation. Aquaporin genes and those related to proteolysis, mitosis, lipid, and carbohydrate metabolism may play important roles in primordium development, while genes related to tissue differentiation and sexual reproduction were active in fruiting body. Several essential genes for fruiting body development were allele-specifically expressed and the two nuclear types could synergistically regulate fruiting body development by dominantly expressing genes with different functions. ASE was probably induced by long terminal repeat-retrotransposons. Findings here contribute to the further understanding of the mechanism of fruiting body development in macrofungi.

Dried shiitake mushroom grade recognition using D-VGG network and machine vision.

Grading dried shiitake mushrooms is an indispensable production step, as there are large quality differences between different grades, which affect the product's price and marketability. Dried shiitake mushroom samples have irregular shapes, small morphological differences between different grades of the same species, and they may occur in mixed grades, which causes challenges to the automatic grade recognition using machine vision. In this study, a comprehensive method to solve this problem is provided, including image acquisition, preprocessing, dataset creation, and grade recognition. The osprey optimization algorithm (OOA) is used to improve the computational efficiency of Otsu's threshold binarization and obtain complete mushroom contours samples efficiently. Then, a method for dried shiitake mushroom grade recognition based on the improved VGG network (D-VGG) is proposed. The method uses the VGG16 network as the base framework, optimizes the convolutional layer of the network, and uses a global average pooling layer instead of a fully connected layer to reduce the risk of model overfitting. In addition, a residual module and batch normalization are introduced to enhance the learning effect of texture details, accelerate the convergence of the model, and improve the stability of the training process. An improved channel attention network is proposed to enhance the feature weights of different channels and improve the grading performance of the model. The experimental results show that the improved network model (D-VGG) can recognize different dried shiitake mushroom grades with high accuracy and recognition efficiency, achieving a final grading accuracy of 96.21%, with only 46.77 ms required to process a single image. The dried shiitake mushroom grade recognition method proposed in this study provides a new implementation approach for the dried shiitake mushroom quality grading process, as well as a reference for real-time grade recognition of other agricultural products.

Development of bio-based PLA/cellulose antibacterial packaging and its application for the storage of shiitake mushroom.

This study presents the extraction of cellulose from water bamboo byproducts to prepare polylactic acid (PLA)/cellulose antibacterial packaging material. The cellulose was modified using a silane coupling agent, which improved the interfacial compatibility between cellulose and PLA. Upon coating the PLA onto the modified cellulose sheet, the water contact angle of the composite material increased from 11.42° to 132.12° and the water absorption rate decreased from 182.52% to 55.71%, which improved the water resistance performance of the material. The addition of cinnamaldehyde in the PLA layer imparted antibacterial activity to the PLA/cellulose packaging material. This packaging material effectively inhibited the mycelial growth and spore germination of Aspergillus niger and Trichoderma harzianum isolated from shiitake mushroom. Additionally, the study investigated the effects of the composite on the postharvest quality of shiitake mushroom. Overall, the packaging material contributed to shiitake mushroom storage and can be applied to other perishable food products.

Changes in Physicochemical Characteristics and Antioxidant Activities of Dried Shiitake Mushroom in Dry-Moist-Heat Aging Process.

Shiitake mushrooms are prized for their unique flavor and bioactive properties. While there has been extensive research on drying methods, a comprehensive investigation of the effects of drying parameters in the dry-moist-heat system on shiitake quality is still needed. This study aimed to investigate the effects of dry-moist-heat aging on dried shiitake mushrooms comprehensively. Four aging temperatures, specifically 50, 60, 70, and 80 °C, were applied to the mushrooms, maintaining a constant humidity level of 75% RH and aging duration of 20 days. Color analysis revealed a progressive decrease in measured values as aging temperature increased, indicating noticeable changes in visual characteristics. Regarding amino acid composition, glutamic acid was found to be the predominant amino acid in shiitake mushrooms in the range of 90.29-467.42 mg/100 g. However, aging led to a reduction in overall amino acid content, with higher aging temperatures resulting in greater decline. Similarly, the equivalent umami content (EUC) also decreased (from 123.99 to 7.12 g MSG/100 g) with the increase in aging temperatures up to 80 °C, suggesting a decline in the overall umami taste sensation. Interestingly, despite the reduction in amino acid levels and umami content, the aging process positively impacted the phenolic compounds and the antioxidant activity of dried shiitake mushrooms. The antioxidative abilities of all aged mushroom extracts for DPPH, ABTS, and FRAP ranged from 65.01 to 81.39 µg TE/mL, 87.04 to 258.33 µg GAE/mL, and 184.50 to 287.68 µg FeSO4/mL, respectively. The utilization of aged temperature at 60 °C for 20 days with controlled relative humidity (~75%) should be a suitable aging condition of this edible mushroom with both antioxidant and umami qualities. Nevertheless, the control sample demonstrated higher levels of amino acid content and EUC compared to the aged samples. Conversely, the aged samples exhibited higher polyphenol content and greater antioxidant activity. Depending on specific requirements, these powders can be used in food formulation as flavor enhancers for control samples or as enriching agents for polyphenols and antioxidant activity in matured samples. Therefore, all of the powders obtained have potential applications in the field of nutrition.

Preparation and Identification of Peptides with α-Glucosidase Inhibitory Activity from Shiitake Mushroom (Lentinus edodes) Protein.

The shiitake mushroom is the most commonly cultivated edible mushroom in the world, and is rich in protein. This study aims to obtain the peptides with α-glucosidase inhibition activity from shiitake mushroom protein hydrolysate. The conditions of enzymatic hydrolysis of shiitake mushroom protein were optimized by response surface test. The results showed that the optimal conditions were as follows: the E/S was 3390 U/g, the solid-liquid ratio was 1:20, the hydrolysis temperature and time were 46 °C and 3.4 h, respectively, and the pH was 7. The active peptides were separated by gel filtration and identified by LC-MS/MS analysis and virtual screening. The results indicated that fourteen peptides were identified by LC-MS/MS. Among them, four new peptides (EGEPKLP, KDDLRSP, TPELKL, and LDYGKL) with the higher docking score were selected and chemically synthesized to verify their inhibition activity. The IC50 values of EGEPKLP, KDDLRSP, TPELKL, and LDYGKL for α-glucosidase inhibition activity ranged from 452 ± 36 µmol/L to 696 ± 39 µmol/L. The molecular docking results showed that the hydrogen bond and arene-cation bond were the two major interactions between four peptides and 2QMJ. The hydrogen bonds were crucial to the inhibition activity of α-glucosidase. The results indicate the potential of using the peptides from shiitake mushroom protein as functional food with α-glucosidase inhibition activity.

Effect of Different Cooking Methods on the Bioactive Components, Color, Texture, Microstructure, and Volatiles of Shiitake Mushrooms.

The effects of different cooking methods (steaming, boiling, air frying, and oven baking) and cooking times (0, 5, 10, 15, and 20 min) on the bioactive components (total phenol, total flavonoid, crude polysaccharides, and eritadenine), color, texture, microstructure, and volatiles in shiitake mushrooms were investigated in this study. Steaming, boiling, and air frying for 5-20 min could decrease the contents of all the four bioactive components in the shiitake mushroom. However, oven baking for 5 min and 10 min showed the highest contents of total phenolics and total flavonoids, respectively. Moreover, the lowest losses of crude polysaccharides and eritadenine were observed for oven baking for 5 min and 15 min, respectively. The lightness of shiitake mushrooms was decreased by all treatments; however, steaming could keep a higher brightness compared with other methods. The microstructure was damaged by all cooking methods, especially air frying for 20 min. Meanwhile, steaming for 20 min decreased the hardness mostly, and there was no significant difference with air frying for 20 min. All cooking treatments decreased the complexity of the flavors and the relative contents of volatile compounds; the lowest contents were found when boiling for 5 min. From these results it can be seen that the physical, histological, and chemical features in shiitake mushroom were influenced by cooking methods and times. In addition, our results provide valuable information for the cooking and processing of shiitake mushrooms and other fungi.

Effect of oxyresveratrol on the quality and membrane lipid metabolism of shiitake mushroom (Lentinus edodes) during storage.

The effect of oxyresveratrol on postharvest quality and membrane lipid metabolism of shiitake mushroom was investigated. The result exhibited that oxyresveratrol retarded browning, maintained firmness and alleviated occurrence of decay of shiitake mushroom. The oxidation and hydrolysis of membrane phospholipids were suppressed by oxyresveratrol treatment, which was associated with reduced LOX and PLD activities and increased SOD and CAT activities. The membrane lipidomics of shiitake mushroom was determined by LC-MS. 385 lipid species and 13 fatty acids in membrane lipids were identified by multiple reaction monitoring method. Compared with control group, the phospholipic acid and lysophospholipid reduced by 29.24% and 21.29% in oxyresveratrol-treated group, respectively, which alleviated hydrolysis of phospholipid. Meanwhile, oxyresveratrol maintained the unsaturation of fatty acids and alleviated oxidation of phospholipid. These results demonstrated that oxyresveratrol could play a dual role of inhibiting the oxidation and hydrolysis of phospholipids to mitigate cellular damage of shiitake mushroom.

Insights into the Global Transcriptome Response of Lentinula edodes Mycelia during Aging.

The spawn of Lentinula edodes and other basidiomycete fungi tend to age with long-term culture. This causes heavy yield losses if aging spawn is used for propagation. In this study, we cultivated dikaryotic L. edodes mycelia in plates for 60 days to produce intrinsic aging phenotypes. We found that intracellular reactive oxygen species levels increased in contrast to mitochondrial depolarization and also observed greater DNA fragmentation with longer culture time. Transcriptome analysis of mycelia at different growth stages revealed pronounced expression differences between short- and long-term cultures. In particular, "phenylalanine, tyrosine, and tryptophan biosynthesis", "mitophagy and autophagy", "MAPK signaling pathway", and "ABC transporter" were among the enriched terms in the mycelial aging process. Weighted correlation network analysis identified LeAtg8, LeHog1, LePbs2, and LemTOR as key genes during aging. Western blotting confirmed that LeATG8 and phosphorylated LeHOG1 protein levels were significantly upregulated in aging mycelia. Our combined analytical approach provides insights into the mechanisms that regulate mycelial aging, indicating that autophagy/mitophagy plays a major role in counteracting the effects of age on mycelial growth development.

Characterization of an active film prepared with Lentinus edodes (shiitake) polysaccharide and its effect on post-harvest quality and storage of shiitake.

The aim of this study was to prepare a film based on shiitake (Lentinus edodes) stalk polysaccharides (LEP) for mushroom preservation. The effects of different LEP concentrations on physical, mechanical, antioxidant, and antimicrobial properties of the prepared film were evaluated. Using scanning electron microscopy, it was revealed that the addition of 1.5 % LEP resulted in homogeneous distribution in the prepared film, as well as greatly improved its antimicrobial properties. Moreover, LEP film resulted in superior mushroom preservation by regulating enzyme activities related to mushroom browning and softening, thereby decaying these processes. In addition, the prepared film maintained mushroom quality by reducing the accumulation of H2O2 and activating the regulatory system against oxidative stress. Collectively, the findings of the present study highlight the potential benefits of LEP films as a strategy to improve mushroom quality and prevent post-harvest spoilage, hence constituting a novel prospect for the development of shiitake by-products.

Proteomic characterization of Shiitake (Lentinula edodes) post-harvest fruit bodies grown on hardwood logs and isolation of an antibacterial serine protease inhibitor.

Lentinula edodes (Shiitake) is one of the most heavily cultivated mushrooms in the world with proven antioxidant and antibacterial properties, among others. Evidence indicates that the choice of mushroom cultivation technique strongly influences the production of bioactive compounds, but to date the nature of many of these compounds has not been fully established. This work focuses on the proteomic characterization of L. edodes to highlight the main active processes two days after harvest and elucidates the proteins involved in the known antioxidant and antibacterial proprieties of Shiitake fruit bodies cultivated on oak logs. A label-free approach allowed us to identify a total of 2702 proteins which were mainly involved in carbohydrate and protein metabolism, cell growth and replication, indicating that several developmental processes remain active in fruit bodies post-harvest. Proteins with antioxidant activities were identified, indicating the contribution of proteins to the antioxidant properties of L. edodes extracts. Antibacterial assays also reveal the activity of a serine protease inhibitor that strongly accumulates in the post-harvest fruit body grown on oak logs. Overall, this study contributes to the understanding of the impact of the log cultivation method on the production of Shiitake mushrooms richest in high-value bioactive compounds.

Lignin Promotes Mycelial Growth and Accumulation of Polyphenols and Ergosterol in Lentinula edodes.

It has been demonstrated that lignin was efficiently degraded by Lentinula edodes (L. edodes). However, the process of lignin degradation and utilization by L. edodes has not been discussed in detail. Therefore, the effects of lignin on L. edodes mycelium growth, chemical compositions, and phenolic profiles were investigated herein. It has been revealed that 0.10% lignin acted as the most effective concentration to accelerate mycelia growth, which yielded the highest biomass of 5.32 ± 0.07 g/L. Furthermore, a 0.10% concentration of lignin promoted the accumulation of phenolic compounds, especially protocatechuic acid, with peak value of 48.5 ± 1.2 µg/g. In contrast, the higher concentration of lignin (0.20%) exerted an inhibitory effect on the growth of L. edodes. Overall, the application of lignin at the optimal concentration of 0.10% could not only enhance the mycelial growth but also accumulate the phenolic acids and raise the nutritional and medical values of L. edodes.

Evaluation of Shiitake Mushroom (Lentinula edodes) Supplementation on the Blood Parameters of Young Thoroughbred Racehorses.

The aim of this study was to evaluate the effect of shiitake mushroom (Lentinula edodes) supplementation on the hematology and biochemical blood parameters of young Thoroughbred racehorses. The study was conducted with 20 horses divided into two groups: the supplemented and the control group. The supplemented group was given 30 g of L. edodes daily for four months. One blood sample was collected four times from each horse at four-week intervals. The hematology analysis in the supplemented group showed a higher level of monocytes at day 56 when compared to the control group (p = 0.000986). Biochemical analysis showed that alkaline phosphatase is most sensitive to shiitake mushroom supplementation, with statistically significant lower levels in supplemented group compared to the control group on all individual days of blood sampling. It was also found that supplementation had an effect on the decrease of glucose levels on days 28 (p = 0.009109) and 56 y (p = 0.025749), on reduction aspartate aminotransferase level on day 56 (p = 0.017258) and a decrease of lactic acid on day 28 of sampling (p = 0.037636). Cholesterol levels decreased consistently in all individual days of blood sampling. Further studies are needed to show the influence of supplementation with shiitake mushroom in larger groups of horses over a longer period.

A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes.

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

A new method for the preparation of a purified glucosylceramide and ceramide from shiitake mushroom.

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinula edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2'-hydroxypalmitoyl-1-O-ß-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], we developed a new method to purify this fungal glucosylceramide using ethanol precipitation and high-performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Effect of shiitake mushrooms polysaccharide and chitosan coating on softening and browning of shiitake mushrooms (Lentinus edodes) during postharvest storage.

We investigated the browning and softening of fresh Lentinula edodes (LE) coated with polysaccharides (LEP) isolated from LE stalks and stored at 4 °C for 15 days. The results showed that compared to the chitosan-coated and uncoated LE, the LEP-treated mushrooms showed significant improvements in several qualities during storage, such as reduced weight loss, retention of hardness and springiness, improved soluble protein content, and reduced browning, malondialdehyde content, and electrolyte leakage rate. The best results were obtained with 1.5 % LEP. LEP improved the activities of peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, and phenylalanine ammonialyase and significantly reduced the accumulation of hydrogen peroxide during storage compared to the control samples. In addition, the LEP treatment maintained the high antioxidant activity of LE during storage. Notably, LEP inhibited browning-related enzymes (polyphenol oxidase and tyrosinase) to reduce browning. It also maintained high levels of cellulase, chitinase, and ß-1,3 glucanase to improve softening during storage. These findings suggest the potential of LEP to improve the post-harvest quality of mushrooms, allowing a storage period of up to 15 days (extending the shelf life by six days) and indirectly suggesting that the polysaccharide component of LEP can act as a self-defense additive to protect against spoilage during storage.

Astragalus Shiitake-A Novel Functional Food with High Polysaccharide Content and Anti-Proliferative Activity in a Colorectal Carcinoma Cell Line.

The chemical and nutritional constituents of mushrooms can alter significantly when grown on different substrates. Based on this fact, an approach was made to cultivate a new type of mushroom, Hengshan Astragalus Shiitake, by growing Shiitake mushrooms on beds supplemented with the roots of an edible herbal plant, Astragalus membranaceus. In this study, three green extraction techniques, including microwave-enzyme assisted (MEA), ultrasound-enzyme assisted (UEA) and microwave-ultrasound-enzyme assisted (MUEA) extractions, were used to compare both the yield and antiproliferative activity of the polysaccharide-rich extracts (PREs) from HAS in human colorectal carcinoma cells (HCT 116). Both HAS-A and HAS-B extracts contain significantly higher amounts of polysaccharides when compared to the control (Shiitake extract), regardless of the extraction methods. The PREs from HAS-B have significantly higher anti-proliferative activity in HCT 116 compared to the control when using the UEA extraction method. Our findings demonstrate that HAS-B can become a novel functional food with anti-proliferative activities and the optimization of UEA extraction would help to develop new active extract-based health products.

Effect of Lentinula edodes on Morphological and Biochemical Blood Parameters of Horses.

Shiitake mushrooms have been highly regarded as possessing enormous nutritive and medicinal values. No clinical studies have yet investigated the effect of shitake supplementation on the health of horses. The aim of this study was to evaluate the effect of shiitake mushroom supplementation on the morphological and biochemical blood properties in horses. A total of 17 adult horses were divided into two groups: supplemented and control. The supplemented group was fed 60 g of shiitake mushrooms per day for 5 months. Blood samples were collected in five sessions. Blood morphological analysis showed higher levels of lymphocytes in session 3 and monocytes in session 4 in the supplemented group. In addition, basophils, hemoglobin, and hematocrit were elevated compared to the control group. Biochemical analysis showed that the shiitake mushrooms affected a large number of parameters. In particular, alkaline phosphatase was found to be the most sensitive to shitake mushroom supplementation, for which the statistical differences were significant for sessions 2, 4, and 5. Furthermore, calcium was found to be affected by supplementation only in session 4, and gamma-glutamyl transferase in session 2. In addition, the bilirubin and glucose levels were lower in the supplemented group, and the albumin/globulin ratio was higher compared to the control group. The differences between the supplement and the control group in various sessions suggest that shiitake mushrooms are a beneficial nutritional supplement for horses.