Isolation Of Pigments Research Articles

Identification, isolation and extraction of pigments producing bacteria from egg and use for dyeing

Identification, isolation and extraction of pigments producing bacteria from egg and use for dyeing

Selective Removal of Chlorophyll and Isolation of Lutein from Plant Extracts Using Magnetic Solid Phase Extraction with Iron Oxide Nanoparticles.

In recent years, there has been a growing interest in plant pigments as readily available nutraceuticals. Photosynthetic pigments, specifically chlorophylls and carotenoids, renowned for their non-toxic antioxidant properties, are increasingly finding applications beyond their health-promoting attributes. Consequently, there is an ongoing need for cost-effective methods of isolation. This study employs a co-precipitation method to synthesize magnetic iron oxide nanoparticles. Scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS) confirms that an aqueous environment and oxidizing conditions yield nanosized iron oxide with particle sizes ranging from 80 to 140 nm. X-ray photoelectron spectroscopy (XPS) spectra indicate the presence of hydrous iron oxide FeO(OH) on the surface of the nanosized iron oxide. The Brunauer-Emmett-Teller (BET) surface area of obtained nanomaterial was 151.4 m2 g-1, with total pore volumes of pores 0.25 cm3 g-1 STP. The material, designated as iron oxide nanoparticles (IONPs), serves as an adsorbent for magnetic solid phase extraction (MSPE) and isolation of photosynthetic pigments (chlorophyll a, lutein) from extracts of higher green plants (Mentha piperita L., Urtica dioica L.). Sorption of chlorophyll a onto the nanoparticles is confirmed using UV-vis spectroscopy, Fourier transform infrared photoacoustic spectroscopy (FT-IR/PAS), and high-performance liquid chromatography (HPLC). Selective sorption of chlorophyll a requires a minimum of 3 g of IONPs per 12 mg of chlorophyll a, with acetone as the solvent, and is dependent on a storage time of 48 h. Extended contact time of IONPs with the acetone extract, i.e., 72 h, ensures the elimination of remaining components except lutein, with a spectral purity of 98%, recovered with over 90% efficiency. The mechanism of chlorophyll removal using IONPs relies on the interaction of the pigment's carbonyl (C=O) groups with the adsorbent surface hydroxyl (-OH) groups. Based on molecular dynamics (MD) simulations, it has been proven that the selective adsorption of pigments is also influenced by more favorable dispersion interactions between acetone and chlorophyll in comparison with other solutes. An aqueous environment significantly promotes the removal of pigments; however, it results in a complete loss of selectivity.

Compressed fluid-based technology for downstream isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus.

A dynamic compressed fluid-based separation process combining carbon dioxide and ethanol was explored to isolate portisins previously hemi-synthesized from blueberry surplus anthocyanins. The influence of process parameters such as pressure (100-500 bar), temperature (40-60 °C), and ethanol content in the compressed fluid mixture (20-50 wt%) on extraction yield, portisins yield, and portisins content in the extract was investigated. The two-step isolation process includes (1) a first step at 100 bar, 60 °C, and 20 wt% ethanol content in the compressed fluid mixture to remove the low polarity compounds; and (2) a second step at 500 bar, 40 °C, and 100 wt% ethanol to recover portisins, resulting in a 1.5-fold increase in portisins content. The performance of the two-step separation process was compared to centrifugal partitional chromatography and conventional reverse phase liquid chromatography already reported in terms of portisins content in the extract, process throughput, process efficiency, and total solvent used. The two-step separation process decreased the total solvent used, although with a decrease in the throughput and efficiency. Nevertheless, the choice of the best separation technology depends on the application, as these techniques result in different portisins purities. Overall, this study contributed to a scalable and more sustainable process for natural colorant production, specifically focusing on blue pigments, with several industrial applications.

Isolation and Identification of Pigments from Marine Actinomycetes, Along with Their Potential Applications

Natural dyes produced by marine microorganisms particularly actinomycetes—have gained significance as a source of active chemicals and pharmaceuticals with potential for medicinal purposes in recent years. This significant finding served as the foundation for the investigation, which aimed to determine the cytotoxic, antibacterial, and antioxidant properties of a methanolic pigment extract obtained from the marine actinomycetes VES 01 and VES 04. Based on the Brine Shrimp Lethality Test (BSLT), both extracts showed substantial toxicity activity. The actinomycetes VES 01 and VES 04 pigment crude extracts had lethal concentration 50% (LC50) values of 92.64µg/mL and 134.21 mg/mL, respectively, according to the data. When those extracts were evaluated for antibacterial efficacy against a variety of microorganisms, Bacillus subtilis and Escherichia coli showed the best inhibition rates. Additionally, we used 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals to measure the antioxidant activity. The findings demonstrated that the actinomycetes VES 01 and VES 04 pigment crude extracts had inhibitory concentration 50% (IC50) values of 228.08 µg/mL and 346.3 µg/mL, respectively. We identified the chemical components of actinomycete pigment crude extracts using GC-MS analysis. The results showed that the most prevalent chemicals were 5-Methoxypyrrolidin-2-one (30.23%) for VES 04, 1H-Purine-2,6-dione, and 3,7-dihydro-1,3,7-trimethyl- (CAS) (44.65%) for VES 01 and VES 04. This work showed the antibacterial, antioxidant, and toxicological properties of two pigment crude extracts obtained from actinimycetes actinomycetes VES 01 and VES 04. In terms of antibacterial activity against E. coli ATCC 8739, actnomycete VES 04 pigment crude extract was the most effective, whereas actnomycete VES 01 pigment crude extract revealed the highest level of antioxidant activity.

Sustainable approach towards isolation of photosynthetic pigments from Spirulina and the assessment of their prooxidant and antioxidant properties

Carotenoids, chlorophyll and phycocyanin are three types of photosynthetic pigments found in Spirulina that differ in colour, composition, stability, solubility, and commercial importance. Such diversity of structures creates a challenge to extract these pigments simultaneously from the same batch of raw material in an efficient and sustainable manner. This study demonstrates that water can be successfully used as a single solvent together with combined (non)mechanical cell membrane disruption techniques (ultrasonication, centrifugation, freezing/thawing cycle) to extract these photosynthetic pigments from the same batch. This water-based approach delivers a significant improvement in isolating green pigments, which are often overlooked during extraction due to a preference for blue and yellow pigments. Chlorophyll was quantitatively converted to its stable derivatives to carry out a comparative analysis of antioxidant properties (DPPH, TEAC, FRAP), singlet oxygen production and intracellular activities (MTT, ROS assays) using Caco-2 cells.

Fungal Anthraquinone Photoantimicrobials Challenge the Dogma of Cationic Photosensitizers.

The photoantimicrobial potential of four mushroom species (i.e., Cortinarius cinnabarinus, C. holoxanthus, C. malicorius, and C. sanguineus) was explored by studying the minimal inhibitory concentrations (MIC) via a light-modified broth microdilution assay based on the recommended protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The extracts were tested against Candida albicans, Escherichia coli, and Staphylococcus aureus under blue (λ = 428 and 478 nm, H = 30 J/cm2) and green light (λ = 528 nm, H = 30 J/cm2) irradiation. Three extracts showed significant photoantimicrobial effects at concentrations below 25 μg/mL. Targeted isolation of the major pigments from C. sanguineus led to the identification of two new potent photoantimicrobials, one of them (i.e., dermocybin) being active against S. aureus and C. albicans under green light irradiation [PhotoMIC530 = 39.5 μM (12.5 μg/mL) and 2.4 μM (0.75 μg/mL), respectively] and the other one (i.e., emodin) being in addition active against E. coli in a low micromolar range [PhotoMIC428 = 11.1 μM (3 μg/mL)]. Intriguingly, dermocybin was not (photo)cytotoxic against the three tested cell lines, adding an additional level of selectivity. Since both photoantimicrobials are not charged, this discovery shifts the paradigm of cationic photosensitizers.

Isolation and chemistry of plant pigments

Purpose This paper aims to critically review the isolation and chemistry of plant pigments. Design/methodology/approach A literature survey from 1974 to 2022 was carried out and studied thoroughly. The authors reviewed literature in various areas such as isolation methods and catalytic properties of pigments. Findings With vast growing research in the field of catalytic activities of various pigments like chlorophyll, anthocyanin and flavonoids, there is still scope for further research for the pigments such as Lycopene, carotenoids and xanthophyll as there has not been any significant work in this area. Research limitations/implications Plant pigments may be used as an ecofriendly catalyst for chemical reactions. Practical implications One can get the direction of pigment research. Social implications Plant pigments are natural and ecofriendly catalyst which can reduce the pollution. Originality/value This is an original work. This paper precisely depicts the advantages as well as disadvantages of the isolation techniques of pigments. This study also presents the chemistry of plant pigments. Graphical abstract

Isolation and Characterisation of Pigments from Pigment-producing Microorganisms Isolated from Environment and Their Antibacterial Activity

Abstract In the food industry, there is always a demand for food products which are colourful, have an attractive appearance, and also have nutritional and health-enhancing properties to attract the attention of consumers. Synthetic pigments are widely used in the global market, however, they can cause many side effects such as hyperallergenicity, carcinogenicity and other toxicological problems. Recent studies have revealed that microorganisms are an abundant source of natural colours that allow the industrial production of safe, environmentally friendly biodegradable pigments. The aim of the work was to isolate pigmented microorganisms from environmental samples, select fermentation conditions, isolate pigments from microorganisms and check their antimicrobial activity. Pigments have been isolated from various sources such as soil, food waste, flour, etc. Growth parameters of pigment-producing microorganisms such as growth temperature, pH, tryptone and NaCl concentration in the medium were optimised to evaluate pigment production. After fermentation, five types of pigments were isolated by cell lysis with an ultrasonic bath and solvent extraction. The antimicrobial activity of the extracted pigments was investigated. During the study, the optimal conditions for the growth of microorganisms were determined: temperature of 30 °C, pH of 7, concentration of 3% tryptone and 6% NaCl in the culture medium. Glycerol was found as an additional carbon source, which had a positive effect on pigments production. The results of the antibacterial effect of the extracted pigments showed that P. aeruginosa was the most sensitive to the effect of the pigments. The pink-red pigment showed the highest antimicrobial activity against the tested pathogenic bacteria.

Isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus using centrifugal partition chromatography

Replacement of synthetic colorants with natural ones is a current marketing trend. Nevertheless, the naturally occurring blue color is rare compared to other colours. In this work, centrifugal partition chromatography (CPC) process was developed as a more efficient and sustainable alternative to reversed phase column chromatography (RP-CC) for the preparative-scale purification of portisins. The strategy began with the extraction of anthocyanins from blueberry surplus and hemi-synthesis of respective portisins. Then, the CPC method development started with the biphasic solvent system selection followed by the optimization of the operating parameters and ended up with a comparison with RP-CC. Aiming at maximizing the portisin content, process throughput, efficiency, and minimizing the environmental risk factor, the effect of sample load (100–500 mg/100 mL of column volume), mobile phase flow rate (10–20 mL/min), and rotation speed (1000–1600 rpm) was evaluated. The two-phase solvent system consisted of tert‑butyl‑methyl ether, n-butanol, acetonitrile, and water (volume ratio 2:2:1:5) acidified with 0.1 vol.% of HCl was selected. The best conditions were 464 mg of sample/100 mL of column volume, 20 mL/min of mobile phase flow rate, and 1600 rpm of rotation speed at reversed phase mode, allowing the purification of portisins by 5-fold. Compared to the RP-CC, the CPC process efficiency was 2.4 times higher, while the CPC process environmental risk factor was 5.5 times lower. Overall, this study suggests that CPC can be considered an effective, and sustainable alternative process for the preparative isolation of portisins. With this purification approach, the blueberry surplus has been valorized and a naturally derived product has been prepared, allowing its subsequent use as a natural blue colorant.

The Influence of Cryogrinding on Essential Oil, Phenolic Compounds and Pigments Extraction from Myrtle (Myrtus communis L.) Leaves

The aim of this study was to investigate the influence of cryogrinding pretreatment on the recovery of essential oil, phenolics and pigments from myrtle leaves. The duration of cryogrinding (3, 6 and 9 min) in combination with the duration of hydrodistillation (30, 60 and 90 min) for the isolation of essential oils and the duration of hydroethanolic extraction (5, 10 and 15 min) for the isolation of phenols and pigments were studied as independent factors in a full factorial design. The major volatile components detected in myrtle leaf essential oil were myrtenyl acetate, 1,8-cineole, α-pinene and linalool. The most abundant phenols detected were myricetin derivatives (myricetin 3-O-galactoside and myricetin 3-O-rhamnoside), galloylquinic acid, myricetin and digalloylquinic acid, while the major pigments were chlorophyll b, pheophytin a and lutein. A 3 min cryogrinding pretreatment significantly increased the yield and concentrations of essential oil volatile compounds and reduced the distillation time to 30 min. A 9 min cryogrinding pretreatment and 15 min extraction resulted in at least 40% higher concentrations of phenolic compounds and pigments in the extracts when compared to the untreated control. According to the results obtained, cryogrinding can significantly increase the yield of myrtle EO and extracts and also modulate their composition.

Extraction of natural pigments from red sorghum (Sorghum bicolor) husk by ultrasound and microwave assisted extraction: A comparative study through response surface analysis

AbstractSorghum (Sorghum bicolor) is an important source of bioactive compounds, such as the phenolic compounds. The phenolic compounds in sorghum grain are concentrated in its hull; however, its hull is usually being discarded as a waste. In this study, ultrasonication power intensity (916, 1018, and 1120 W/cm2), sonication time (20, 25, and 30 min), and particle size (100, 300 and 500 μm) for ultrasound assisted extraction and, microwave power (50, 60 and 70 W), microwave time (90, 105, and 120 s), and particle size (100, 300, and 500 μm) for microwave assisted extraction were investigated and optimized using Box–Behnken design. For ultrasound assisted extraction, optimized condition was found as 1078 W/cm2 power intensity, 23.6 min sonication time and 100 μm particle size, and corresponding values of responses were 8.55 mg LE/g dw total anthocyanin content, 779.0 mg GAE/g dw total phenolic content, 172.0 mg CE/g dw total flavonoid content, 70.0 and 73.4% antioxidant activity by DPPH and ABTS methods, respectively and 11.1 redness (a*). For microwave assisted extraction, optimized condition was found as 63 W power, 101 s time and 100 μm particle size, and the responses were 10.5 mg LE/g dw total anthocyanin content, 606.0 mg GAE/g dw total phenolic content, 195.0 mg CE/g dw total flavonoid content, 79.5 and 82.2% antioxidant activity by DPPH and ABTS methods, respectively and 13.6 redness (a*). For validation of model at optimum conditions, predicted values were insignificantly (p > .05) differ than actual values for all the responses for both ultrasound and microwave assisted extraction.Practical ApplicationsSorghum consists of a rare class of natural pigments (3‐deoxyanthocyanidins) in its husk. These natural pigments have antioxidant, anti‐proliferative activity, and thus, can be utilized as natural food colorant in food industry. Extraction and isolation of these natural pigments from sorghum husk are prerequisites for their application in food industry. In order to obtain an efficient extraction of sorghum anthocyanins, resulting in minimal degradation and high antioxidant capacity, the choice of the extraction method is important. This research provides information about standardization of novel extraction techniques (ultrasound and microwave assisted extraction) for effective utilization of sorghum husk for extraction of natural pigments, which can be utilized for application in various food products.

Antibacterial Activity and Potential Of Natural Textile Dyes From Sea Water Bacteria

This research aims to determine the potential of bacterial isolates from Marina beach seawater to be used as natural textile dyes and to test their antibacterial ability. The media used for culturing bacteria is a zobel synthesis medium with a mixture of seawater. The results of the isolation and extraction of color pigments obtained a red color with a characteristic wavelength of 535 nm and has a variety of chemical content results. Pigment immersion trials using 3 types of fabric, namely cotton, primisima and dobby fabric. The results of immersion with ethanolic solvent obtained optimum results for 12 hours and with the addition of mordant (Fe.H2O4S.7H2O), the color of the fabric is strong enough to withstand washing with detergent and sunlight. From the results of the study, data showed that the dyed primisima fabric gave a reduction or inhibition of the growth of E. coli bacteria by 9% while the inhibition of S. aureus bacteria was 116%. The pigment has the potential to be used as a dye for batik cloth with the provisions of optimizing to find the right reinforcement or mordant. This study has implications for the discovery of red pigment-producing bacteria from the isolation of seawater at Marina Semarang beach.

Isolation, Characterization, and Optimization Studies of Bacterial Pigments

Recent concern for human safety and environmental protection has rekindled interest in natural pigment sources. In comparison to synthetic pigments, microbial pigments show better biodegradability and environmental compatibility and are used in a variety of applications ranging from food to cosmetics. The areas of attention for economical pigment synthesis include the identification of novel microbiological sources and improvement of process parameters. The purpose of this research was to screen and identify microbial isolates capable of generating pigments with antimicrobial activity from a variety of soil samples. A total of six pigment-producing bacterial sps were able to isolate from various soil samples such as bore well digging sites, river shores, river beds, forest areas, dumping yards using the enrichment culture technique. All the isolates were morphologically and biochemically identified as Micrococcus sp producing two-color pigments i.e., yellow and orange, Serratia sp producing red and pink color pigments, Salinococcus sp producing orange color pigment, and Exiguobacterium sp producing yellow color pigment respectively. During optimization studies maximum pigment production was observed at pH 7, agitation at 90 rpm (rotations per minute) and 120 rpm, the temperature of 30°C and 37°C, inoculum size up to 2% with NaCl concentration of 2%, 4%, and 6% respectively. Optimization of nutritional parameters such as carbon source and nitrogen source it was found that glucose (1%) and yeast extract (0.1%) work the best. Extraction of the pigment from the fermented broth was done by solvent-solvent extraction method. UV-Visible spectrophotometry and Silica gel Thin-layer chromatography was used to detect the presence of carotene and prodigiosin in the extracted bacterial pigment. The crude bacterial pigments were tested for antimicrobial activity against clinical pathogens including E. coli, Klebsiella sp, Bacillus sp, Staphylococcus sp, and pseudomonas sp respectively. Among all the isolates, pigments of Micrococcus sp and Salinococcus sp showed comparatively good results. Further purification of the pigment will lead to discovering a promising drug in the pharmaceutical industry.

Isolation and purification of phycocyanin pigments from Spirulina sp. biomass and evaluation of its anticancer and antioxidant potential

Isolation and purification of phycocyanin pigments from Spirulina sp. biomass and evaluation of its anticancer and antioxidant potential

Front Cover: A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography

DOI: 10.1002/jssc.202100869 The cover picture shows a visual representation of the vivid colors found in basidiomycetes. Starting from fruiting bodies of Cortinarius sanguineus, a separation strategy for anthraquinones was developed based on centrifugal partition chromatography (scheme in the middle). The methanolic crude extract (top right) was fractionated (bottom right) using a five-component solvent system, allowing the isolation of fungal anthraquinone pigments (bottom left).

Screening, Characterization, and Isolation of Pigments from Bacteria in Mesophotic Depths of the Benham Bank Seamount, Philippine Rise Region

The exploration for pigment-producing bacteria and structurally novel pigment continues to increase, and the marine environment has recently become an attractive research site for these investigations because of its rich yet untapped biodiversity. In this study, 16S rRNA gene sequencing and HPLC (high-performance liquid chromatography) profiling of the microbial pigments produced by marine heterotrophic bacteria were described. Seven phenotypically distinct isolates of marine pigmented heterotrophic bacteria (MPHB) were isolated from near-bottom waters and coral reef sediments at mesophotic depths of the Benham Bank Seamount – namely, isolates BR14, BR61, BR63, BR100, BR101, BR144, and BR146. Four promising isolates were subjected to 16S rRNA gene sequencing and revealed that strains BR61 and BR100 were related to members of the genus Cytobacillus, whereas isolates BR144 and BR146 clustered with Meridianimaribacter flavus and Pseudoalteromonas rubra, respectively. Growth of the seven isolates on three media – natural sea water (NSW) Reasoner’s 2A (R2A), marine agar (MA), and tryptic soy agar – revealed variable pigment production and growth yield. MA appeared to be a superior substrate, resulting in darker pigmentation and higher biomass yield. Through a liquid-liquid partitioning approach, isolates BR14 and BR100 produced pigments that were acetone-soluble, whereas isolates BR101, BR144, and BR146 are hexane soluble. Moreover, the BR146 red pigment was proposed to be a mixture of putative prodiginine analogs. The putative prodiginines produced by isolate BR146 can stain fabrics, supporting a proof-of-concept that marine bacteria can be utilized as fabric colorants. The data presented here provided new insights into the utilization of local Philippine marine microbial resources for natural marine pigments with industrial applications.

Targeted isolation of photoactive pigments from mushrooms yielded a highly potent new photosensitizer: 7,7\u2032-biphyscion

Pigments of fungi are a fertile ground of inspiration: they spread across various chemical backbones, absorption ranges, and bioactivities. However, basidiomycetes with strikingly colored fruiting bodies have never been explored as agents for photodynamic therapy (PDT), even though known photoactive compound classes (e.g., anthraquinones or alkaloids) are used as chemotaxonomic markers. In this study, we tested the hypothesis that the dyes of skin-heads (dermocyboid Cortinarii) can produce singlet oxygen under irradiation and thus are natural photosensitizers. Three photosensitizers based on anthraquinone structures were isolated and photopharmaceutical tests were conducted. For one of the three, i.e., (–)-7,7′-biphyscion (1), a promising photoyield and photocytotoxicity of EC50 = 0.064 µM against cancer cells (A549) was found under blue light irradiation (λexc = 468 nm, 9.3 J/cm2). The results of molecular biological methods, e.g., a viability assay and a cell cycle analysis, demonstrated the harmlessness of 1 in the dark and highlighted the apoptosis-inducing PDT potential under blue light irradiation. These results demonstrate for the first time that pigments of dermocyboid Cortinarii possess a so far undescribed activity, i.e., photoactivity, with significant potential for the field of PDT. The dimeric anthraquinone (–)-7,7′-biphyscion (1) was identified as a promising natural photosensitizer.

Isolation and identification of natural green and yellow pigments from pineapple pulp and peel

The isolation of pineapple pulp and peels can produce natural pigments which contain bioactive compounds that have an antimicrobial effect and benefit human health. Therefore, the aim of this study is to isolate the green and yellow pigments from the pineapple pulp and peels. The isolated pigments were used for phytochemical analysis and antimicrobial activity. These pigments were identified by Fourier transform infrared (FTIR) spectroscopy and Ultraviolet–Visible (UV–Vis). These pigments present phytochemicals have the potential to act as antimicrobial agents. This study would be one of the alternatives in developing food colouring that is beneficial in preserving the quality of food against foodborne pathogens using both isolated pigments.

Isolation, Characterization, and Antibiofilm Activity of Pigments Synthesized by Rhodococcus sp. SC1.

Infections from multi-drug resistant bacteria and biofilms constitute a serious problem worldwide. There is a need for new antibacterial and antibiofilm compounds in the fight against infectious diseases. In recent years, pigment-producing microorganisms have drawn a great deal of attention as a promising source for antibacterial and antibiofilm compounds. Here, we report the antibacterial and antibiofilm activity of pigments synthesized by bacteria isolated from soil. This study aimed to perform an evaluation of the antibacterial, antibiofilm, and characteristic of crude pigments from Rhodococcus sp. SC1 isolates. The total pigment extract exhibited antibacterial activity against Gram-positive and Gram-negative reference bacteria with required minimum inhibitory concentration (MIC) values ranging from 64 to 256µg/ml. Moreover, it reduced biofilm formation of Gram-negative reference bacteria at sub-MIC concentration. For characterization of the pigments, UV-absorbance, thin layer chromatography, fourier transform infrared spectroscopy, and QTOF-LC/MS analyses were performed. The results of this study showed that pigments of Rhodococcus sp. SC1 isolates can be a candidate for medical applications.

Isolation and identification of pigments from oyster mushrooms with black, yellow and pink caps

The genus Pleurotus, namely oyster mushroom, is widely cultivated and consumed worldwide. Cap color is an important commercial trait for oyster mushroom. Diverse color is determined by various pigment constituents. However, the pigments of oyster mushrooms are still ambiguous. In this study, we extracted and identified pigments of oyster mushroom species with black, yellow and pink cap color. The extracted pigments appearing the three color types correspondingly to the cap color, which were all identified as melanin using a panel of spectroscopic and physical/imaging techniques. Nevertheless, HPLC and elemental analysis indicated that the melanin in oyster mushrooms was actually a mixture of eumelanin and phaeomelanin. Differences in the quantities and relative proportions of eumelanin and phaeomelanin resulted in the color variation in oyster mushroom caps. Electron microscopy studies showed that the melanin units are likely located in the cell wall, as reported in other fungi. The pigments in oyster mushrooms with three different cap color were extracted and identified for the first time in this study, which provided fundamental knowledge for future studies on the mechanism of color formation in mushrooms.