Extraction and identification of fungal pigment from Penicillium europium using different spectral studies

1 Introduction

There is growing scrutiny over the detrimental impact of synthetic colourants on both the environment and consumers' health, which has shifted the focus towards natural colouring alternatives. Hence, international demand for natural pigments is quickly growing in the cosmetic, pharmaceutical, food, and textile industries. Natural colourants can be used for diverse industrial applications, such as dyes for fabric and non-fabric substrates such as paper, leather, coatings and paints, and as additives in food and cosmetics (Kalra et al., 2020).

Colour plays a vital role in the food production and processing sector, contributing to food's sensory attribute. It signifies the nutritional value, freshness, safety, and aesthetic value of food, directly influencing the coloured food product's market value. Natural colours are considered safe if they are non-toxic, non-allergic, biodegradable, and non-carcinogenic, thereby rendering no risk to the environment. Due to the lower risk advantage of natural colours and consumers' changing attitudes toward consuming natural products, there is an expanding interest in discovering new natural colours. The consumer demand for natural colours and their growth as a category is expected to increase by 7% annually. Natural food colours have varied food industry applications, with almost all major natural pigment classes being used in at least one food industry sector (Aberoumand, 2011). Micro-organisms derived pigments commonly used as food colorants include violacein, riboflavin, astaxanthin, canthaxanthin, phycocyanin, prodigiosin, beta-carotene, lycopene, and melanin (Sen et al., 2019).

All life on earth ultimately depends on light energy from the sun, converted to other forms of energy or biomass by plants and photosynthetic microorganisms. The electromagnetic spectrum includes the wavelength range humans recognize as visible light (380–750 nm) (Orna, 1984). When pigments are illuminated by visible light, part of the energy may be absorbed, and the emitted light will appear as colour. Pigments contain a chromophore, a colour-bearing group, which directly produces colour by light absorption or transfer energy to auxochromes which are colour increasers, giving characteristic colours. Various chemical classes of pigments occur in nature, of which the highest is produced by microorganisms (Gill and Steglich, 1987).

Microbial pigments can be classified into relatively few major biosynthetic and structural classes; the pyrroles and tetrapyrroles, tetraterpenoids or carotenoids, N- or O-heterocyclic pigments and metalloproteins. Some microbial pigments have important biological functions for the producing organisms, such as the production of energy and fixation of matter by bacteriochlorophylls and phycobilins in microbial photosynthesis. Whereas, in some organisms, the only known attribute of pigments is to give colour. Some microbial pigments such as the phenazines are secondary metabolites, and their natural functions are not well understood (Turner and Messenger, 1986). Most fungal pigments have economic importance, such as food, feed and drug colourants, or essential nutrients (Hendry and Haugton, 1996). Thus, the microbes producing pigments could contribute to biotechnology and add value to the food, feed, and pharmaceutical industries. Different types of fungi show the presence of different kinds of pigments like carotenoids (Britton, 1991), ubiquinones (Takizawa et al., 1992), anthraquinones (William et al., 1994; Brauers et al., 2000; Gill and Saubern Simon, 2000), xanthorin (Gill and Qureshi, 1992; Gill and Morgan, 1999), indigo, monascins, quinines, phenazines, violacein, flavins and melanins (Dufosse et al., 2014). Fungi and bacteria offer a tremendous resource in that they produce hundreds to thousands of various pigments, and it has been estimated that only 5–7% of fungi and 10–12% of bacteria have been isolated from nature (Demain, 1981). Many more natural pigments will likely be isolated from microorganisms. Microorganisms offer advantages over plants and animals for pigment production. Microorganisms have an exceedingly small size compared to animal and plant cells with high growth rates. Microbial cells provide excellent host environment for the expression of foreign genes. Certain mammalian and plant pigments, such as haemoglobin and nonmicrobial carotenoids, have been expressed in microorganisms (Kushwaha et al., 2014). Microbes will undoubtedly be more extensively utilized in the future for basic and applied studies of pigments.

This research objective deals with the study of pigment produced by the fungal strain that has been used for longifolene fermentation, characterization of purified pigment and toxicity studies.

2 Materials and methods

3 Results and discussion

The fungus Penicillium europium producing yellowish pink pigment in Byrde medium was isolated in our laboratory. The TLC analysis of acetone extract of Penicillium europium cultural broth revealed the presence of two pigments. Further, the light pink pigment was separated into a pure form by subjecting acetone fraction to silica gel chromatography eluted with methanol. The purity was checked by TLC (R_f = 0.70) with a single spot.

The UV–visible spectrum shows absorption at 343 nm, indicating a (3-unsaturated ketone (Benzoquinone moiety) (Cooksey et al., 1998) (Fig. 1). The IR spectrum shows bands at 3406 cm⁻¹ corresponding to the hydroxyl group (O–H), bands at 1641 cm⁻¹ correspond to quinone structure (C = 0), bands at 1387 cm⁻¹, 1337 cm⁻¹ correspond to p-unsaturation and bands at 2935 cm⁻¹ correspond to C–H stretching (Fig. 2).

Close

Fig.1

UV–Vis Spectra of pigment.

Export to PPT

Close

Fig. 2

IR spectra of pigment.

Export to PPT

Further ¹H NMR (CDCl₃) (Figure −3) shows peaks (9 ppm) at 0.89 (d, 6H, J = 8 Hz, 2CH₃), 1.34 (d, 6H, J = 7 Hz, (CH₂)₃), 1.31 (d, 3H, J = 7 Hz, l'–CH₃), 1.65 (s, 3H, 5-CH₃), 1.75 (s, 3H, 3-CH₃), 1.87 (s, 3H, –CH₃), 2.10 (m, 1H, CH(CH₃)₂), 2.40 (m,1H, l'–CH), 3.5 (s, 1H, OH) exchangeable with D₂O (Fig. 3).

Close

Fig. 3

¹H NMR spectra of pigment.

Export to PPT

Table 1 shows the molecular ion peak at 264 M/Z (25%).

Thus based on UV, IR, PMR and mass spectra, the pigment was tentatively identified as 2- (1,5, dimethyl hexyl) −3, 5-dimethyl-6- hydro −1, 4 - benzoquinone having the molecular formula C₁₆H₂₄O₃. (Fig. 4)

Close

Fig. 4

Structure of 2- (1,5, dimethyl hexyl) −3,5-dimethyl-6- hydro −1,4 – benzoquinone.

Export to PPT

The proposed compound structure was further confirmed by comparison of its NMR spectral data with those published for synthetic O-methyl perezone (Perri et al., 1989; Escobedo-González et al., 2019) Such compound is believed to be isolated for the first time from the fungus.

Using the pigment (quinone), the toxicity was studied using LD₅₀. The pigment was diluted in water and a single dose (300 mg/Kg) was administered orally to 3 Albino rats. Animals were observed individually after dosing at least once during the first 30 min, periodically during 24 h, with special attention given during the initial 4 h.

Even after 48 h of observation, there was no toxic effect of pigment on rats. Then three other rats were subjected for a limit test at one dose level of 5000 mg/Kg and kept under observation. After 48 h, there was no toxic effect of pigment on the rats.

Majority of the natural pigments obtained from fungi are reported to have a broad spectrum of pharmacological activities. These pigments render fungi diverse biological roles such as enzyme cofactors (flavins) (Rao et al., 2017); protection against the damaging effects of photo-oxidation (carotenoids) (Gmoser et al., 2017), and the protection against environmental stress (melanins) (Dufosse et al., 2014). Though these fungal pigments are associated with distinct biological activities, the factors regulating their production and their physiological role remain largely unstudied (Sen et al., 2019). Current advances in biotechnological and analytical tools employing computational and molecular techniques help decipher the components responsible for the pigment colour, de-novo pathways and genome accountable for its production.

To reap maximum benefit, alternative routes for these metabolites' mass production may be achieved using heterologous expression, manipulating culture conditions and co-culturing. Fungi proficient of producing pigments can be collected from various environmental ecosystems and habitats and explored as a source of commercial pigments.

4 Conclusions

Based on this study, we conclude that this pigment is non-toxic, and it can be used as food colourant and additive. The fungus Penicillium europium which transforms longifolene was also found to produce yellowish pink pigment in Byrde medium. This pigment was isolated and characterized tentatively as 2- (1,5, dimethyl hexyl) -3, 5-dimethyl 1-6-hydro-l, 4-benzoquinone having the molecular formula C₁₆H₂₄O₃based on UV, IR, ¹HNMR and Mass spectral studies. This compound was isolated for the first time from the fungus. The pigment is nontoxic, which was confirmed by the LD₅₀ test. Thus our research has shown that this pigment can be used as food, feed, colourant, and as essential nutrient. Microbes producing pigments could contribute to biotechnology and add value to the food, feed, and pharmaceutical industries.