Translate this page into:
Bio-characterization of food grade pyocyanin bio-pigment extracted from chromogenic Pseudomonas species found in Pakistani native flora
⁎Corresponding author. saniamazharr@gmail.com (Sania Mazhar),
⁎⁎Corresponding author. ahmadadnan@gcu.edu.pk (Ahmad Adnan)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
The consumer sensitivity toward application of synthetic colors led to exploitation of food grade bio-colors from bacteria. Pseudomonas aeruginosa is an opportunistic, gram-negative bacteria that secrete a variety of redox-active phenazine pigmented compounds, which are significant for a variety of biological activities. Pyocyanin, a water soluble blue green phenazine pigment producing P. aeruginosa was screened from aquatic habitats of isolation of Pakistan, identified and compared by 16S rRNA gene sequence for genetic variability. The similarity of selected strain was found 99% with P. aeruginoas DSM 50071 with accession no CP012001.1 in gene bank. Pyocyanin from the identified strain was extracted after 72 h of incubation by chloroform and purified with 0.1 N HCl and 1 N NaOH. Protective effects of the extracted pyocyanin as food colorant were evaluated against multiple biological activities. Pyocyanin showed anti-oxidant potential with 58.0% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical compare to Trolox (68.5%) and BHT (88.1%) and 52.5% free radical scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6- sulfonic acid) in comparison to Trolox (67.4%) and BHA (86.0%) at 50 μg/ml concentration. The anti-microbial efficacy of pyocyanin was assessed against food borne pathogenic bacteria and fungi by agar well diffusion method. Pyocyanin exhibited anti-bacterial activity with distinct zone of inhibition against B. spizizenii (16 mm), S. aureus (19 mm), E. aerogenes (14 mm), S. enterica (13 mm), P. aeruginosa (13 mm) and E. coli (12 mm) at 50 μg/ml concentration. Pyocyanin was more susceptible at the same concentration against fungal strains with zone of inhibition measuring 21 mm, 18 mm and 17 mm for F. oxysporum, A. niger and A. fumigatus respectively. Anti-biofilm profile of pyocyanin exhibited significant inhibition of the biofilm formation against biofilm forming bacteria B. cereus (81%), S. aureus (80%), P. aeruginonsa (78%) and K. pneumonia (76%) when assessed by crystal violet assay at 50 μg/ml concentration. Similar effects at the same concentration were observed in disruption of pre-formed biofilm against B. cereus (77%), S. aureus (76%), P. aeruginonsa (74%) and K. pneumonia (73%). The presented remarkable biological activities of pyocyanin against food borne pathogens augment the utilization of chromogenic microbes existing in Pakistani aquatic resource as an alternative potent source for efficient production of natural pigment and their application as natural color and bio-preservative in food industries.
Keywords
Microbial pigments
Food color
Natural colorants
Pigment production
Pyocyanin
Pseudomonas aeruginosa
Evolutionary ecology
Color extraction
Biological activities
Water resources
1 Introduction
Color is the most appealing characterization of any object for its aesthetic purposes and to attract consumers that directly affect the market value of the finished product (Downham and Collins, 2000; Sigurdson et al., 2017). Coloring was originated in 1500 BE (Burrows, 2009) and most of the colors were presumed to be derived from natural sources likes berries and flowers at that time (Aberoumand, 2011; Gulrajani, 2001). In 1856 the development of synthetic colors (Burrows, 2009) resulted in their broad uses due to their stability, low cost of production and less usage to give the final color to the product. However, with passage of time the unfavorable environmental and human health effects along with toxicity led to the banning of many synthetic colors (McCann et al., 2007; Potera, 2010; Amchova et al., 2015) resulting in re-emergence and increase attraction for the usage of natural pigments (Zhang et al., 2020). Thereby, the advocated dose of color compounds usage in drugs, cosmetics and food is highly regulated by the United States Food and Drug Administration (FDA), The European Food Standards Authority (EFSA) and the World Health organization (WHO), whether the colors are produced synthetically or derived naturally (Stachowiak-Oplatowska and Elliot, 2017; Wrolstad and Culver, 2012; Lehmkuhler et al., 2020).
Natural colors are gradually making its way in the global market as a subsequent alternative of synthetic colors through green chemistry approaches (Yusuf et. al., 2017). The worldwide utilization of natural colors in food, pharmaceutical, cosmetics, textile and printing dye is assumed safe because they are non-toxic, non-carcinogenic and bio-degradable (Unagul et al., 2005). The current global marketing trend of manufacturers as well as consumers to replace synthetic color with natural colorants for eco-preservation, eco-safety and health concerns speculates that by 2020 the market for natural colorant in food industry alone will reach 1.7 billion USD (Hasmida et al., 2018).
Natural colors are also called as bio-color or bio-pigments because of their biological origin like ores, insects, fruits, vegetables, seeds, roots and microorganisms (Sen et al., 2019) Classification of bio-colors is often based on their source of origin, color and chemical structure of chromophore (Madadi et al., 2020). Plant/vegetable and microorganism are widely considered the suitable source for the biotechnological production of natural pigments because of understanding of cultural techniques and processing. Although there are a variety of plant based natural pigments such as red, blue, yellow, indigo, green, white and brown (Hasmida et al., 2018), only few are accessible in sufficient quantities throughout the year to be useful for industries because they are usually extracted from seasonal fruits and vegetables (DeMejia et al., 2020). Hereby, microbial colorants are more preferable source because of their easier extraction without seasonal variation problems, low production cost and effective yield of natural colors (Galaffu et al., 2015; Panesar et al., 2015).
Algae, fungi, yeast and various types of pigmented bacteria isolated from different environmental sources are in use for commercial production of bio-pigments for food additive, colorants and bio-medical applications (Du et al., 2011; Chidambaram et al., 2013; Hardeep et al., 2014; Narsing et al., 2017; Novoveská et al., 2019; Morales-Oyervides et al., 2020; Kalra et al., 2020). These microorganisms can produce a variety of pigments likes astaxanthin, canthaxanthin, carotenoids, flavins, lycopene, melanins, monascins, prodigiosin and violacein (Nigam and Luke, 2016; Sen et al., 2019, Maoka, 2020). These pigments not only work as coloring agents but also imparts additional health benefits with diverse range of their activities including anti-oxidant, anti-cancer, anti-fungal, anti-bacterial, anti-inflammatory, anti-cholesterol, anti-biofilm and immuno-regulatory effects (Vendruscolo et al., 2016; Narsing et al., 2017; Zhang et al., 2020). In spite of the availability of many types of natural pigments from various microbial sources, the current range of natural food grade colors is relatively small compared to the large range of synthetic colors. The discovery of new and novel natural colors from microbes is therefore important to improve the cost effectiveness of food products.
P. aeruginosa is gram negative and aerobic rod shaped opportunistic pathogenic bacteria measuring 0.5–0.8 μm wide and 1.5–3.0 μm long (Moore et al., 2006; Recio et al., 2020), which thrives in normal as well in hypoxic atmospheric conditions. Its habitat is widespread and it is found in soil, water, humans, animals, plants, sewage and hospitals (Green et al., 1974; Crone et al., 2020). P. aeruginosa can cause infections in respiratory and urinary tract of immune-deficient and immune-compromised host (Faure et al., 2018; Mittal et al., 2009). However, despite being pathogenic bacteria, P. aeruginosa is one of the most commercially and biotechnologically valuable microorganisms (Jayaseelan et al., 2014). P. aeruginosa attracts attention due to their characteristic to produce extracellular pigments of different shades like pyocyanin (blue-green), pyomelanin (light brown), pyoverdin (yellow, green and fluorescent) and pyorubrin (red brown) (Meyer, 2000) and their subsequent wide range of industrial applications in food, pharmaceuticals, textiles, leather and other industries (Anayo et al., 2019).
Nearly 90–95% isolates of P. aeruginosa produce biologically active water soluble pyocyanin which is not only a virulence factor but also is significant by terms of biotechnology (Jayaseelan et al., 2014; Alatraktchi et al., 2020; Sood et al., 2020). Pyocyanin has the ability to act as a bio-control agent because of its pharmacologic effect on phytopathogens and prokaryotic as well as eukaryotic cells (Sudhakar et al., 2013; Zhao et al., 2014). The other use of pyocyanin is it applications as a major anti-fungal molecule (Sass et al., 2020) and biosensors (Ohfuji et al., 2004; Wang et al., 2020). The versatile uses of pyocyanin (Marrez and Mohamad, 2020; Hamad et al., 2020) prophesy potential for new insight into application of pyocyanin bio-pigment from P. aeruginosa. However, P. aeruginosa isolates that experience different ecologies may produce differential adaptive traits including motility, Na+ pump changes, pigment production as well as phenotypic diversity (Argenio et al., 2007; Kidd et al., 2012; Friman et al., 2013; DeBritto et al., 2020). This scenario assumes that P. aeruginosa bacteria tend to suffer growth trade-offs across their environment. Bio-diversity of P. aeruginosa hereby determines the continuous wide research interest in P. aeruginosa, colonize in variable terrestrial and aquatic habitats for its commercial and industrial applications (Anayo et al., 2019; Soliev, 2012).
Evenly, recent advances such as identification of microbial pigments, their extraction, purification and stability techniques has been performed for the development and socio-economic viability of natural colorants at commercial scale (Yusuf et al., 2017) In Pakistan there is scarcity of knowledge regarding sustainable utilization of bio-resources for the production of natural color from chromogenic microbes and then to use them in different products for healthy life style and eco-safety. The bio-colors, which are being used in Pakistani industries, are being imported at high cost that ultimately have impact on the price of the finished product. Research is therefore required to produce color through pigmented microbes while using indigenous bio-resources to establish new understanding in the area of biotechnology and to provide import substitute.
Pakistan is endowed with profound blend of diversified natural bio-resources spanning from plains, mountains, desserts and forests to several lakes and rivers that join the largest lndus River. Indus river flows through the entire length of Pakistan and merges with the Arabian Sea in the south of Pakistan. This wide spread water resources encompassing 2.86% of the total area of Pakistan is known to be rich in nutrient as a result of prevailing moon soon dynamics. The nutrient rich water resources result in surface productivity and rich plant and animal life along with provision of favorable environment for the growth of microorganisms (Ali and Dinshaw, 2016). Though the existing water resources constitute an integral part of Pakistan’s economy, however Pakistan is facing yearly about 29 billion USD economic losses on account of un-utilized flow of river water into the Arabian Sea (Kiani, 2020). Hereby, effective utilization of aquatic resource of Pakistan for the identification of prevailing chromogenic microbes and thereafter extraction of pigments from them as natural colors will be a cost effective approach. Therefore, regarding the cumulative effects of ecology on genetic diversity, total growth and subsequent productivity of bio-pigments (Buchanan et al., 1997; Ciofu et al., 2010; Fouly et al., 2015) and utilization of water resources for the production of microbial natural pigments, the present study was aimed on elucidation of the possible genetic diversity of chromogenic P. aeruginosa found in aquatic environments of Pakistan along with characterization of bioactivities of extracted Pyocyanin pigment to explore the potential use of pigmented microbes existing in aquatic resource of Pakistan as an alternative potent source for the effective production of natural food grade pigments.
2 Materials and method
2.1 Detail and specifications of media, chemicals and reagents
Microbiological and analytical grade media, chemical and reagents were used in the present study. Butterfield phosphate buffer, asparagine broth, cetrimide agar, and nutrient agar used for serial dilutions, isolation and purification of P. aeruginosa were procured from Merck, Germany. Solutions used for Gram staining were hucker’s crystal violet, gram’s iodine and hucker’s counter stains. Hucker’s crystal violet solution was prepared by dissolving crystal violet (BDH, UAE) in 95% ethanol (Merck, Germany) and thereafter mixing it in aqueous solution of ammonium oxalate (Scharlau, Spain). Gram’s iodine solution was made from iodine (Merck, Germany) and potassium iodide (AlfaAesar, Germany) in dH2O. Hucker’s counter stain was prepared with safranin O (BDH, UAE) in 95% ethanol. Biochemical and physiological identification was performed by using kovac’s reagent (Merck, Germany) for indole reaction, simmons citrate agar (Oxoid, UK) for citrate utilization, 30% hydrogen peroxide (Scharlau, Spain) for catalase and N,N,N′,N′-tetramethyl-p-phenylenediamine.2HCl (Merck, Germany) for oxidase test. MR-VP media (Oxoid, UK) was used for methyl red and vogus proskuaer reactive compound tests. 0.02% methyl red (BDH, UAE) indicator was prepared in 95% ethanol and dH2O for methyl red test. 5% α-Naphtol (Merck, Germany) mixed in 95% alcohol was used as color intensifier while 40% aqueous solution of potassium hydroxide (Merck, Germany) was used as oxidizing agent with few crystal of creatine (BDH, UAE) to intensify and speed the reaction of vogus proskuaer test. Nutrient broths (Oxoid, UK), chloroform (Sigma-Aldrich, USA), 0.1 N HCl (Sigma-Aldrich, USA), 1 N NaOH (bioWORLD, USA) were used for the production, purification and extraction of pyocyanin. Diphenyl-1-picrylhydrazyl (DPPH) stable free radical, potassium per sulfate and reference standard butylated hydroxytoluene (BHT) were purchased from (Sigma-Aldrich, USA). While 2,2ile ldrich,-(3-ethylbenzothiazoline-6-sulfonic acid and reference standards butylated hydroxyanisole (BHA) & trolox were procured from Merck, Germany. Methanol used to prepare the required solutions of scavenging assay was purchased from Fisher Scientific, UK. Potatoes dextrose agar, ketoconazole and chloramphenicol used for anti-fungal and anti-bacterial activities were purchased from Merck, Germany, Shaigan pharma, Pakistan and Oxoid, UK respectively. Anti-biofilm assay media and reagents were tryptic soy broth, PBS, glacial acetic acid and DMSO. Tryptic soy broth was purchased from Oxoid, UK, glacial acetic acid and DMSO from Merck, Germany while analytical grade chemicals for the preparation of PSB were NaCl procured from AlfaAesar, Germany, KH2PO4 from PanReac AppliChem, Germany and KCl and Na2HPO4 from Merck, Germany.
2.2 Isolation and molecular identification of pyocyanin producing Pseudomonas aeruginosa strain
River, Swimming pool and drainage water samples representative of Pakistani aquatic flora were collected from different location of district Punjab, Pakistan in sterilized bottles for isolation of blue green pyocyanin pigment producing P. aeruginosa bacterial strain. Collected samples were processed according to the methods provided in the manual of standard methods for the examination of water and wastewater (Rodger et al., 2017). Water sample producing water soluble blue green pyocyanin in asparagine broth was selected for isolation of characteristic blue green purified colony of P. aeruginosa. The selected sample was serially diluted upto 1–106 in Butterfield phosphate buffer, plated on cetrimide agar and incubated at 35 °C for 24–48 h. The characteristic appeared blue green colonies of P. aeruginosa were isolated and re-streaked for purification and isolation on nutrient agar. The purified isolated colony of P. aeruginosa bacterial strain was further processed for morphological characterizations including colony shape, pigment color, gram staining and cell shape followed by biochemical and physiological identification encompassing indole production, citrate utilization, catalase, oxidase, methyl red and vogus proskuaer reactive compounds tests (Cappuccino and Sherman, 2014; Schaad et al., 2001). The molecular identification of the selected strain was performed while using AGA GTT TGA TCM TGG CTC AG and TAC GGY TAC CTT GTT ACG ACT T forward and reverse primer for PCR and GGA TTA GAT ACC CTG GTA and CCG TCA ATT CMT TTR AGT TT forward and reverse primers for 16S rRNA sequencing followed by blast function.
2.3 Pyocyanin production, purification and extraction
P. aeruginosa was cultured in nutrient broth, incubated at 35 °C for 72 h and observed for color production in the broth. After incubation broth was centrifuged at 55,000 rpm for 15 min to obtain cell free supernatant. Pyocyanin containing supernatant was mixed well with equal volume of chloroform for blue green pigment extraction in chloroform. The chloroform extracted pyocyanin was purified with 0.1 N HCl followed by 1 N NaOH until the solution color was changed from deep pink to blue green. Afterward, the solution was filtered to obtain clear blue green solution of pyocyanin, which was extracted again in chloroform. After extraction chloroform was evaporated and pyocyanin powder was collected (Feghali and Nawas, 2018a).
2.4 Free radical scavenging activities of pyocyanin
Free radical scavenging activity of pyocyanin was screened through DPPH and ABTS radical scavenging assays (Tirzitis and Bartosz, 2010). The reductions in absorbance indicating higher free radical scavenging activity were calculated and analyzed after triplicate experiments as follows: Scavenging (%) = (Ablank - Asample/Ablank) × 100. Where Ablank is the absorbance of the control reaction (containing all reagents except tested sample) and Asample is the absorbance of the tested concentration of the pyocyanin or reference standard.
2.4.1 DPPH radical scavenging assay
The anti-oxidant activity of pyocyanin was measured by its capacity to scavenge 2,2׳-diphenyl-1-picrylhydrazyl (DPPH) stable free radical. The assay was carried out spectrophotometrically at 517 nm by the method described by Liyana and Shahidi (2005) with some modification. Briefly, tested concentration (50 μg/ml) of extracted pyocyanin and reference standards butylated hydroxytoluene (BHT) and trolox were prepared in methanol. 0.1 ml of test concentration of extracted pyocyanin and standards were mixed separately with 3 ml of the methanolic DPPH solution (0.5 mM) and left at room temperature for 30 min in the dark. Thereafter, the absorbance at 517 nm was measured and the %age scavenging of DPPH free radial was calculated.
2.4.2 ABTS radical scavenging assay
Free radical scavenging activity of pyocyanin was determined by 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical cation decolorization assay as well while using the method of Rice-Evans et al. (1997) with slight modifications. ABTS.+ cation radical produced by the reaction between equal volume of 7 mM ABTS and 2.4 mM potassium per sulfate was stored in the dark for 12–16 h at room temperature before use. ABTS.+ solution was then diluted with methanol until an absorbance of 0.700 ± 0.02 was obtained at 734 nm. 0.2 ml of tested concentration (50 μg/ml) of extracted pyocyanin solution was added to 2.8 ml of diluted ABTS+ solution, mixed for 30 min and the absorbance was measured at 734 nm. Methanolic solutions of butylated hydroxyanisole (BHA) and trolox were used as reference standards. Percent inhibition of absorbance indicating %age scavenging of ABTS nm was calculated at 734.
2.5 Anti-bacterial activity of pyocyanin
The anti-bacterial effect of extracted pyocyanin was performed against ATTC culture of gram positive Staph. aureus (ATCC 25923) and Bacillus spizizenii (ATCC 6633) and gram negative E. coli (ATCC 8739), Enterobacter aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 27853) and Salmonella enterica (ATCC 14028) food borne pathogenic bacterial strains by agar well diffusion method (Zaika, 1988). The test strains were incubated in nutrient broth at 35 °C for 24 h. After incubation 1x106 cells were loaded with nutrient agar in petri plates and agar was let to solidify. Wells of 8 mm diameter were made and filled with extracted pyocyanin at concentration of 50 μg/ml to measure anti-bacterial activity. The wells of control plates were loaded with distilled water for negative control and chloramphenicol for positive controls. The plates were incubated at 35 °C for 24 h and zones of bacterial growth inhibition around wells were measured in mm.
2.6 Anti-fungal activity of pyocyanin
The degree of anti-fungal activity of extracted pyocyanin was tested by agar well diffusion method (Zaika, 1988) against Aspergilus niger (Accession No. 1109), Aspergillus fumigatous (Accession No. 0079) and Fusarium oxysporum (Accession No. 1175) fungal strains purchased from the fungal bank, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. Sterilized potatoes dextrose agar was inoculated with 1x 106 spore suspension of the fungal isolate, allowed to solidify and punctured to make wells of 8 mm diameter. The wells were filled with extracted pyocyanin at concentration of 50 μg/ml compare to well of control plates, which were loaded with distilled water and ketoconazole for negative and positive controls. The zones of fungal growth inhibition were measured in mm after 48 h of incubation at 25 °C.
2.7 Anti-biofilm activity of pyocyanin
The potential activity of extracted pyocyanin to disrupt the pre-formed biofilm or inhibit biofilm formation by strongest biofilm producers P. aeruginonsa, Staphylococcus aureus, Bacillus cereus and Klebsiella pneumoniae was checked by Crystal violet (CV) assay in a 96-well microtitre plate (Coraca-Huber et al., 2012; Masadeh et al., 2013; Ghosh et al., 2015). Overnight cultures having 1x106 cells of P. aeruginonsa (ATCC 27853), S. aureus (ATCC 25923), B. cereus (ATCC 14579) and K. pneumoniae (ATCC 13882) were diluted into 100 μl tryptic soy broth (TSB), dispensed into wells and incubated at static condition for 24 h at 37 °C for bacterial growth and biofilm formation. After incubation plate was washed thrice with PBS (pH 7.2) to remove non-adherent cells, loaded with 50 μl of pyocyanin (50 μg/ml) and incubated again at 37 °C for 24 h. The following day plate was stained with 1% CV for 15 min at room temp. The stained wells were washed thrice with PBS to remove unbound dye and air dried for 30 min. The bound dye was re-suspended in 33% acetic acid solution and absorbance was recorded at 450 nm by multimode reader (Enspire, Perkin Elmer). The inhibition of biofilm formation was assessed using the same method with the exception that 1x106 cells of said bacterial strains diluted into tryptic soy broth (TSB) were mixed with 50 μl of pyocyanin (50 μg/ml), loaded into wells and let to incubate at static condition for 24 h at 37 °C. After incubation the wells were washed with PBS, stained by 1% CV and quantified similarly at 450 nm. Mean absorbance values of each triplicate assay were calculated and compared with the average value of the DMSO negative control and pyocyanin standard positive control.
3 Results and discussion
3.1 Isolation, identification and comparison of Pseudomonas aeruginosa isolated from different ecology
P. aeruginosa bacteria thrive in a wide variety of environments, however there is accumulating evidence that the extent of phenotypic variability among P. aeruginosa is associated with type of their ecology (Khan et al., 2007; Remold et al., 2011; Rodriguez-Rojas et al., 2012; Selezska et al., 2012; Poulsen et al., 2019). Here to address that the phenotypic variability of P. aeruginosa bacteria is correlated with origin of their geographical habitat, we isolated blue green pyocyanin producing P. aeruginosa colonies from three aquatic habitats of isolation (river, swimming pool and drainage water) of Pakistan. A very similar growth performance, phenotype and biochemical profile was observed across P. aeruginosa isolates taken from three different environments. The observed pigmented blue green morphological distinct circular colonies with smooth margin of strains isolated from three aquatic habitats exhibited specific rod cell shape of gram negative P. aeruginosa. Similarly characteristics biochemical identification of selected stains was found positive for citrate utilization, catalase and oxidase while negative for indole production, methyl red and vogus proskuaer reactive compound tests. Hereby, these results underscore the hypothesis that variability of P. aeruginosa is associated with the variable habitat of origin in the same geographical area. To elucidate further that the geographical area of Pakistan represents genetic diversity of P. aeruginosa from that observed in other continents, one of the selected strains that showed comparatively intense pigment production was chosen for the molecular identification and comparison of nucleotide sequence with sequence databases through BLAST, NCBI. The comparison of the 16S rRNA gene sequences was found to show 99% high sequence similarity with P. aeruginoas DSM 50071 with accession no CP012001.1 (Nakano et al., 2015) in gene bank that has been previously reported for beneficial applications such as a potent source of L- asparaginase for in vitro and in vivo anti-cancer consideration (Bessoumy et al., 2004, Dalfard et al., 2016). The phylogenetic tree (Fig. 1) of the identified P. aeruginoas strain (query name A_contig_1) matched with other strains available in public database as well and no variability was observed. Hereby, these results suggest that P. aeruginosa bacteria appear to be broad generalists irrespective to their habitat of growth. Crone et al. (2020) similarly concluded from 16S rRNA data analysis that P. aeruginosa sequences are present in all habitats and their performance traits across environments are not correlated with their habitat of isolation (Diaz et al., 2018; Kidd et al., 2012; Ruimy et al., 2001).
Phylogenetic tree of 16S rRNA gene showing resemblance of isolated P. aeruginosa to various isolates of pyocyanin producing bacterium P. aeruginosa. A_contig_1 = query name of the identified P. aeruginoas strain; gi = Gene info identifier number assigned to each sequence record of P. aeruginosa processed by NCBI.
3.2 Pyocyanin production, purification and extraction
The amount of pyocyanin produced by P. aeruginosa is time dependent (Feghali and Nawas, 2018a). P. aeruginosa was cultured in nutrient broth and incubated at 35 °C for color production in the broth. The production of Pyocyanin pigment in this study revealed that the appearance of the pigment from inoculated P. aeruginosa strain started in nutrient broth culture media within 10 h of incubation. However, a gradual increase in pyocyanin concentration was observed from 24 to 72 h with increase in cell proliferation and thereafter bacterial density. Longer incubation of P. aeruginosa was avoided with consideration that long holding time of incubation would start producing other pigments like pyomelanin (light brown), pyorubin (red- brown) and pyoverdin (green, yellow and uorescent) which could have affected the production rate of pyocyanin and interfered with its extraction (Meyer 2000). The maximum production with a steady change in color from light to dark green color was observed at 72 h (Fig. 2) which is in accordance with observation of Gahlout et al. (2017) who studied the effect of incubation period on pyocyanin production and observed that maximum pyocyanin production occurs at 72 h which decreases with increase in incubation time. Onbasli and Belma (2008) and Saha et al. (2008) similarly reported that highest pyocyanin production occurred at 72 h of incubation. DeBritto et al., in 2020 compared nutrient supplements in both King’s A medium as well as nutrient broth and provided the evidence that improved growth and cell proliferation of P. aeruginosa result in increased bacterial density and maximal production of pyocyanin.
Incubation of selected P. aeruginosa strain in nutrient broth from 10 h to 72 h. A steady increase in pyocyanin bio-pigment production from light to dark blue green color was observed with increase in incubation period.
The extraction procedure used in this study was based on the redox properties of pyocyanin and on the fact that only pure pyocyanin changes its color when it passes from basic to acidic pH. These properties gave pyocyanin the special characteristic to have different solubility based on its state. When in the red state, pyocyanin is more soluble in aqueous solvents compare to organic solvents, whereas in its blue state, it is more soluble in organic solvents than aqueous solvents. This duality in solubility makes the purification of pyocyanin possible and highly effective through changing the pH and water/chloroform extraction (Feghali and Nawas, 2018a). Thereby, following the extraction procedure (Fig. 3A), the blue green pigment produced by the inner part of the cells was extracted by chloroform followed by the acid/base extraction steps (Fig. 3B & 3C) to optimize the final yield of pure pyocyanin. The observed change in solubility with changing pH and change in chloroform extracted pigment color from blue to deep pink upon addition of 0.1 N HCl confirmed the extraction of pure pyocyanin as reported by Raoof and Latif (2010), Fouly et al. (2015) and Raouia et al. (2020) as well.
Extraction and purification of Pyocyanin. (A) Schematic diagram showing the overall extraction process of pyocyanin pigment (B) Chloroform extraction of bluish green pyocyanin from 72 h old culture of P. aeruginosa (C) Purified pyocyanin after acid/base extraction method.
3.3 Free radical scavenging activities of pyocyanin
Free radical plays an important role in the origin of numerous lifestyle diseases and physiological diseases like cancer, cardiovascular defects, high blood pressure, diabetes and neurodegeneration etc. Therefore it is important to search for bioactive anti-oxidant compounds with reduced or no side effects. Microbial pigments possess a great potential for application because of their safety, low production cost and anti-oxidant activity due to their free radical scavenging capacity (Tirzitis and Bartosz, 2010; Gupta et al., 2013). The extracted microbial pigment pyocyanin was evaluated for its free radical scavenging activity against DPPH and ABTS. Pyocyanin at 50 μg/ml concentration exhibited a moderate inhibition potential (58.0%) compare to Trolox (68.5%) and BHT (88.1%) in DPPH test (Fig. 4A). Results obtained by the ABTS showed that Pyocyanin is more effective agent in DPPH test (Fig. 4B) compare to ABTS test with observed 52.5% free radical scavenging activity in comparison to Trolox (67.4%) and BHA (86.0%). However, the observed difference in DPPH and ABTS scavenging activities might be due to the reaction media. Sengupta and Bhowal (2021) similarly carried out DPPH and ABTS anti-oxidant assay to evaluate the free radical scavenging potential of pyocyanin extracted from P. aeruginosa. Likewise, Laxmi and Bhat (2016) recorded the results that pyocyanin isolated from P. aeruginosa strain BTRY1 has higher radical scavenging activities at concentration very much lower compare to ascorbic acid standard. Rani and Azmi (2019) reported the maximum activity to be 74.9% at 30 μg/ml concentration of pyocyanin while Chandran et al. (2014) evaluated the anti-oxidant activity of pyocyanin pigment and found it to be 55% at 500 μg/mL concentration. Hereby, the obtained high anti-oxidant activity at 50 μg/ml concentration of pyocyanin is a positive indication for the effective use of compound (Liyana and Shahidi, 2005).
Scavenging of free radicals by pyocyanin. (A) Scavenging of DPPH radical representing %age inhibition potential of pyocyanin versus trolox and BHT (B) Scavenging of ABTS radical representing %age inhibition potential of pyocyanin versus trolox and BHA. BHA = Butylated hydroxyanisole; BHT = butylated hydroxytoluene; Error bar=±Standard deviation.
3.4 Anti-microbial activities of pyocyanin
Pyocyanin, the growth pigment produced by P. aeruginosa is one of the important chemicals that allow it to survive in environments with competitive organisms (Feghali and Nawas, 2018a). Pyocyanin has the capacity to arrest the electron transport chain of the different microorganisms (Kerr, 1994; Kerr et al., 1999). The extracted pyocyanin was subjected to agar well diffusion method for anti-bacterial activity against four gram negative and two gram positive food spoilage bacteria. The data presented in Fig. 5A & B revealed that pyocyanin exhibited distinct activity against gram positive bacteria B. spizizenii and S. aureus with 16 mm and 19 mm zone of inhibition at 50 μg/ml concentration. Pyocyanin induced moderate zone of inhibition at the same concentration against gram negative bacteria E. aerogenes (14 mm) followed by S. enterica (13 mm), P. aeruginosa (13 mm) and E. coli (12 mm) which is in accordance to the observation of Devnath et al. (2017), Darwesh et al. (2019) and Hamad et al. (2020) that gram negative bacteria are less sensitive to pyocyanin than gram positive bacteria. Pyocyanin exhibits intracellular oxidant stress and initiates a redox cycle that results in production of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide. These ROS compound inhibit the growth of pyocyanin sensitive microbes (Hassan and Fridovich, 1980; Norman et al., 2004; Das and Manefield, 2012). Hereby, the possible mechanism of differentiating resistance of bacteria to pyocaynin would be the levels of catalase and superoxide dismutase production by the gram positive and gram negative organism and on the presence of oxygen (Baron and Rowe, 1981). The difference in lipid content of cell wall of gram negative and gram positive bacteria might be associated with different level of sensitivity of bacteria to pyocyanin as well (Das and Manefield, 2012).
Anti-bacterial activity of pyocyanin against food borne pathogenic bacteria. (A) Zone of inhibition measured in mm (including 8 mm well diameter) through agar well diffusion method exhibiting distinct anti-bacterial activity of pyocyanin against gram positive and gram negative bacteria (B) Anti-bacterial susceptibility profile of pyocyanin against gram positive and gram negative bacteria. B = B. spizizenii; EB = E. aerogenes; EC = E. coli; Pseudo = P. aeruginosa; Sal = S. enterica; Staph = S. aureus; Phy = Pyocyanin; Error bar=±Standard deviation.
Compare to bacteria, tested fungal strains were found more susceptible to anti-microbial action of pyocyanin with formation of 16 mm to 21 mm zone of inhibition. F. oxysporum displayed 21 mm zone of inhibition whereas A. niger and A. fumigatus exhibited 18 mm and 17 mm zone of inhibition respectively at 50 μg/ml concentration of pyocyanin (Fig. 6A & B). Sass et al. (2020) reported that mutant strain of P. aeroginosa PAO1 that lacked the production of pyocyanin under nonlimiting iron conditions, was found less inhibitory to A. fumigatus. However, when blood as a natural source of iron was supplied during P. aeruginosa supernatant production, pyocyanin appeared and resulted in an anti-fungal effect on A. fumigatus. Similarly, Ozyurek et al. (2016) analyzed the sensitivity of A. niger strain to pyocaynin at 0.005 μg to 50 μg concentration by performing tube dilution and agar well diffusion methods. Mahmoud et al., reported in 2016 that pyocyanin is a broad spectrum pigment which acted as a bio-control agent on pathogenic microbes, importantly on wilt disease which is caused by F. oxysporum. The possible reason of suppression of fungal growth by pyocyanin is a reduction in camp in hyphae formation (Kerr et al., 1999). Another reason of fungal sensitivity to pyocyanin is inhibition of pleiotropic drug resistance subfamily FgABC3 transporter gene that is known to mediate fungal resistance to anti-fungal drugs (Houshaymi et al., 2019).
Anti-fungal activity of pyocyanin against food borne pathogenic fungal strains. (A) Zone of inhibition measured in mm (including 8 mm well diameter) through agar well diffusion method representing distinct anti-fungal activity of pyocyanin (B) Anti-fungal susceptibility profile of pyocyanin against fungal strains. AN = A. niger; AFG = A. fumigatus; FO = F. Oxysporum; Phy = Pyocyanin; Error bar=±Standard deviation.
3.5 Anti-biofilm activity of pyocyanin
Cell to cell bacterial adhesion and physico-chemical interactions results in biofilm formation in a wide range of bacterial species (Tsuneda et al., 2003; Das et al., 2013). Bacterial adhesion and biofilm formation represent lethal bacterial infections due to increased tolerance to antibiotics while targeting bacteria that are enclosed within formed biofilm (Stoodley et al., 2004; Abou et al., 2018; Kalia et al., 2019). Therefore, it is essential to explore the potential compounds having anti-biofilm activity for a better treatment option in the future (Haney et al., 2018). The extracted pyocyanin at 50 μg/ml, concentration was tested for considerable effect on disruption of pre-formed biofilm as well as inhibition of biofilm formation against biofilm forming food borne pathogens. The results of crystal violet (CV) assay for the pyocyanin exhibited significant inhibition of the biofilm formation against biofilm forming bacteria when assessed spectrophotometrically. Highest inhibition (Fig. 7A) was observed against gram positive bacteria B. cereus (81%) and S. aureus (80%) followed by gram negative bacteria P. aeruginonsa (78%) and K. pneumonia (76%). The pyocyanin was found similarly highly effective in disruption of pre-formed biofilm as well (Fig. 7B) against gram positive bacteria B. cereus (77%) and S. aureus (76%) as well as gram negative bacteria P. aeruginonsa (74%) and K. pneumonia (73%). Likewise, Sood et al. (2020) also checked the ability of pyocyanin to inhibit the formation of biofilm by Bacillus species which are well known for their ability to produce biofilm (Kalia et al., 2017). Corroborating to this study Abou et al. (2018) reported the similar use of pyocyanin for the inhibition of biofilm formed by S. saprophyticus and K. pneumonia. Cell lysis appears to have a role in the biofilm lifecycle (Guilhen et al., 2017). Issa et al. (2019) similarly reported that the loss of viable bacterial cells is associated biofilm reduction. Das and Manefield (2012) reported that P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H2O2) generation that leads to cell lysis. Similarly Zegans et al. (2009) exhibited that P. aeruginosa PA14 phage mediated cell lysis inhibits bacterial adhesion, cellular aggregation and biofilm formation. The possible modes of this action could be that bactericidal activity of pyocyanin modify the biotic or abiotic surface properties of biofilm included cells that mediates cell death or inhibition of cell growth in mature biofilm or during biofilm maturation, leading to dispersal of surviving cells and reducing the subsequent cell-cell interactions as well as adhesion (Bayles, 2007; Bhattacharyya et al., 2013; Feghali and Nawas, 2018b; Doghri et al., 2020).
Anti-biofilm activity of extracted pyocyanin determined by crystal violet (CV) assay against gram positive and gram negative bacteria. (A) Inhibition (in %age) of biofilm formation (B) Disruption (in %age) of pre-formed biofilm. Error bar=±Standard deviation.
4 Conclusion
The consumer sensitivity toward application of synthetic food additives has led to great demand of natural foods with natural ingredients and additives. Majority of the natural food additives are derived from bacteria, yeast and fungi etc. because microbial sources can be scaled up and more readily manipulated for commercial production of pigments/colorants compare to fruits and vegetables. Exploitation of novel food grade bio-colors from bacteria is the most focus point in the rapidly growing food category that requires natural ingredients and additives. Higher water soluble blue green pyocyanin pigment producing P. aeruginosa was selected from aquatic habitats of isolation of Pakistan and proceeded for extraction of pyocyanin using chloroform followed by purification with 0.1 N HCl and 1 N NaOH. Protective effects of pyocyanin as food colorant in food industry were checked against multiple food borne bacterial and fungal pathogens. The remarkable anti-bacterial and anti-fungal activities of pyocyanin augment their potency for application in food industries as natural food grade color or food preservative to control several potent food pathogens. In addition to this, pyocyanin exhibited effective anti-oxidant and anti-biofilm activities even at small concentration, which is a positive sign for its use in future as an alternative to synthetic colors. However, in all case, there is an imperative need for toxicological evaluation of the extracted pyocyanin through organoleptic and oral toxicity tests to pave the way for their regulatory approval.
Acknowledgement
We are grateful to Dr. Quratulain Syed (Chief Scientific Officer), Director General PCSIR Laboratories Complex Lahore, Pakistan for providing the opportunity to work on her novel R & D project entitled “Bio-process development of color pigments from eco-friendly microbes for industrial use” in PCSIR Laboratories Complex Lahore, Pakistan.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- A review article on edible pigments properties and sources as natural biocolorants in foodstuff and food industry. World J. Dairy Food Sci.. 2011;6:71-78.
- [Google Scholar]
- Pyocyanin: a powerful inhibitor of bacterial growth and biofilm formation. Int. J. Case Reports. Stud.. 2018;3:101-107.
- [CrossRef] [Google Scholar]
- Electrochemical detection of pyocyanin as a biomarker for pseudomonas aeruginosa: a focused review. Sensors (Basel).. 2020;20:5218.
- [CrossRef] [Google Scholar]
- A handbook on Pakistan’s coastal and marine resources. Daccan Printers (Pvt) Ltd.; 2016.
- Health safety issues of synthetic food colorants. Regul. Toxicol. Pharmacol.. 2015;73:914-922.
- [CrossRef] [Google Scholar]
- Anayo, F.O., Scholastica, C.E., Peter, C.O., Nneji G.U., Obinna, A., Mistura, O.L., 2019. The beneficial roles of pseudomonas in medicine, industries, and environment: A. review. Pseudomonas aeruginosa. In: Sriramulu, D., ebook. https://doi.org/10.5772/intechopen.85996.
- Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients. Mol. Microbiol.. 2007;64:512-533.
- [CrossRef] [Google Scholar]
- Antibiotic action of pyocyanin. Antimicrob. Agents Chemother.. 1981;20:814-820.
- [CrossRef] [Google Scholar]
- The biological role of death and lysis in biofilm development. Nat. Rev. Microbiol.. 2007;5:721-726.
- [CrossRef] [Google Scholar]
- Production, isolation, and purification of L- asparaginase from Pseudomonas aeruginosa 50071 using solid state fermentation. J. Biochem. Mol. Bio.. 2004;37:387-393.
- [CrossRef] [Google Scholar]
- Inhibition of candida biofilms by pyocyanin: an invitro study. Int. J. Cur. Res. Rev.. 2013;5:31-36.
- [Google Scholar]
- When is simple good enough: a comparison of the Gompertz, Baranyi, and three-phase linear models for fitting bacterial growth curves. Food Microbiol.. 1997;14:313-326.
- [CrossRef] [Google Scholar]
- Palette of our palates: A brief history of food coloring and its regulation. Comprehensive Reviews in Food Science and Food Safety.. 2009;8:394-408.
- [CrossRef] [Google Scholar]
- Microbiology: A Laboratory Manual (tenth ed.). UK: Pearson; 2014.
- Production and extraction of bacterial pigments from novel strains and their applications. Res. J. Pharmaceut. Biolog Chem. Sci.. 2014;5:584-593.
- [Google Scholar]
- Bacterial pigments and their applications. Process Biochem.. 2013;48:1065-1079.
- [CrossRef] [Google Scholar]
- Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology. 2010;156:1108-1119.
- [CrossRef] [Google Scholar]
- Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces. J. Appl. Microbiol.. 2012;112:1235-1243.
- [CrossRef] [Google Scholar]
- The environmental occurrence of pseudomonas aeruginosa. APMIS. 2020;128:220-231.
- [CrossRef] [Google Scholar]
- Gene sequencing, cloning, and expression of the recombinant L- Asparaginase of Pseudomonas aeruginosa SN4 strain in Escherichia coli. Biol. J. Microorganism. 2016;4:11-20.
- [Google Scholar]
- Production of antimicrobial blue green pigment pyocyanin by marine pseudomonas aeruginosa. Biointer. Res. In. App. Chem.. 2019;9:4334-4339.
- [CrossRef] [Google Scholar]
- Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation. PLoS One. 2013;8:e58299
- [CrossRef] [Google Scholar]
- Pyocyanin promotes extracellular DNA release in Pseudomonas aeruginosa. PLoS One. 2012;7:1-9.
- [CrossRef] [Google Scholar]
- Isolation and characterization of nutrient dependent pyocyanin from Pseudomonas aeruginosa and its dye and agrochemical properties. Sci. Rep.. 2020;10:1542.
- [CrossRef] [Google Scholar]
- The colors of health: chemistry, bioactivity, and market demand for colorful foods and natural food sources of colorants. Annu. Rev. Food. Sci. Technol.. 2020;11:145-182.
- [CrossRef] [Google Scholar]
- Extraction, purification and characterization of pyocyanin produced by Pseudomonas aeruginosa and evaluation for its antimicrobial activity. Int. Res. J. Biological. Sci.. 2017;6:1-9.
- [Google Scholar]
- Generalized growth of estuarine, household and clinical isolates of Pseudomonas aeruginosa. Front. Microbiol.. 2018;9:1-8.
- [CrossRef] [Google Scholar]
- Anti-biofilm activity in the culture supernatant of a marine Pseudomonas sp. Bacterium. Microbiol.. 2020;166:239-252.
- [CrossRef] [Google Scholar]
- Colouring our foods in the last and next millennium. Int. J. Food Sci. Tech.. 2000;35:5-22.
- [CrossRef] [Google Scholar]
- Engineering microbial factories for synthesis of value-added products. J. Ind. Microbiol. Biotechnol.. 2011;38:873-890.
- [CrossRef] [Google Scholar]
- Pseudomonas aeruginosa in chronic lung infections: how to adapt within the host? Front. Immunol.. 2018;9:2416.
- [CrossRef] [Google Scholar]
- Extraction and purification of pyocyanin: a simpler and more reliable method. MOJ. Toxicol.. 2018;4:417-422.
- [CrossRef] [Google Scholar]
- Pyocyanin: A Powerful inhibitor of bacterial growth and biofilm formation. Madridge J. Case Rep. Stud.. 2018;3:101-107.
- [CrossRef] [Google Scholar]
- Biosynthesis of pyocyanin pigment by Pseudomonas aeruginosa. J. Radiation Res. Applied Sci.. 2015;8:36-48.
- [CrossRef] [Google Scholar]
- Pseudomonas aeruginosa adaptation to lungs of cystic fibrosis patients leads to lowered resistance to phage and protist enemies. PLoS One. 2013;8:e75380
- [CrossRef] [Google Scholar]
- Isolation and screening of pyocyanin producing Pseudomonas spp. from soil. Int. J. Adv. Res. Biol. Sci.. 2017;4:147-152.
- [CrossRef] [Google Scholar]
- Galaffu, N., Bortlik, K., Michel, M., 2015. An industry perspective on natural food colour stability. Colour Additives for Foods and Beverages. Woodhead Publishing. https://doi.org/10.1016/b978-1-78242-011-8.00005-2.
- Dioscorea bulbifera mediated synthesis of novel AucoreAgshell nanoparticles with potent antibiofilm and antileishmanial activity. J. Nanomater. 2015
- [CrossRef] [Google Scholar]
- Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa. Appl. Microbiol.. 1974;28:987-991.
- [Google Scholar]
- Biofilm dispersal: Multiple elaborate strategies for dissemination of bacteria with unique properties. Mol. Microbiol.. 2017;105:188-210.
- [CrossRef] [Google Scholar]
- Functional foods enhanced with microbial antioxidants. Acad. J. Nutr.. 2013;2:10-18.
- [CrossRef] [Google Scholar]
- Toxicity evaluation and antimicrobial activity of purified pyocyanin from pseudomonas aeruginosa. Bioint. Res. App. Chem.. 2020;10:6974-6990.
- [CrossRef] [Google Scholar]
- Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides. Biomolecules. 2018;8:29.
- [CrossRef] [Google Scholar]
- Microbial pigments as natural color sources: Current trends and future perspectives. J. Food Sci. Technol.. 2014;52:4669-4678.
- [CrossRef] [Google Scholar]
- Natural colorants from plants for wellness industry. Int. J. Pharm. Sci. Res.. 2018;9:836-843.
- [CrossRef] [Google Scholar]
- Pyocyanin, a metabolite of Pseudomonas aeruginosa, exhibits antifungal drug activity through inhibition of a pleiotropic drug resistance subfamily FgABC3. Drug Res.. 2019;69:658-664.
- [CrossRef] [Google Scholar]
- Antibiofilm potential of purified environmental bacteriophage preparations against early stage Pseudomonas aeruginosa biofilms. J. Appl. Microbiol.. 2019;126:1657-1667.
- [CrossRef] [Google Scholar]
- Pyocyanin: production, applications, challenges and new insights. World J. Microbiol. Biotechnol.. 2014;30:1159-1168.
- [Google Scholar]
- Quorum sensing inhibitors as antipathogens: biotechnological applications. Biotechnol. Adv.. 2019;37:68-90.
- [CrossRef] [Google Scholar]
- Simple and rapid method for detecting biofilm forming bacteria. Indian J. Microbiol.. 2017;57:109-111.
- [CrossRef] [Google Scholar]
- Fungi as a potential source of pigments: harnessing filamentous fungi. Front Chem.. 2020;8:369.
- [CrossRef] [Google Scholar]
- Suppression of fungal growth exhibited by Pseudomonas aeruginosa. J. Clin. Microbiol.. 1994;32:525-527.
- [Google Scholar]
- Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. J. Clin. Pathol.. 1999;52:385-387.
- [CrossRef] [Google Scholar]
- Isolation of Pseudomonas aeruginosa from open ocean and comparison with freshwater, clinical and animal isolates. Microb. Ecol.. 2007;53:173-186.
- [CrossRef] [Google Scholar]
- Kiani, K., 2020. Unutilized water flow into sea causes $29bn yearly loss to economy: Irsa. www.dawn.com, (assessed 4th November 2020).
- Pseudomonas aeruginosa exhibits frequent recombination, but only a limited association between genotype and ecological setting. PLoS One. 2012;7:e44199
- [CrossRef] [Google Scholar]
- Characterization of pyocyanin with radical scavenging and antibiofilm properties isolated from P. aeruginosa strain BTRY1. 3 Biotech.. 2016;6:27.
- [CrossRef] [Google Scholar]
- Certified food dyes in over the counter medicines and supplements marketed for children and pregnant women. Food. Chem. Toxicol.. 2020;143:111499
- [CrossRef] [Google Scholar]
- Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L.) as affected by gastric pH conditions. J. Agric. Food Chem.. 2005;53:429-443.
- [CrossRef] [Google Scholar]
- Therapeutic application of betalains: a review. Plants (Basel).. 2020;9:1219.
- [CrossRef] [Google Scholar]
- Antifungal activity of pyocyanin produced by pseudomonas aeruginosa against Fusarium oxysporum Schlech a root-rot phytopathogenic fungi. Int. J. Pharm. Tech. Res.. 2016;9:43-50.
- [Google Scholar]
- Biological activity and applications of pyocyanin produced by Pseudomonas aeruginosa. J. Bio. Sci.. 2020;1:140-144.
- [CrossRef] [Google Scholar]
- In vitro determination of the antibiotic susceptibility of biofilm forming Pseudomonas aeruginosa and Staphylococcus aureus: Possible role of proteolytic activity and membrane lipopolysaccharide. Infect. Drug Resist.. 2013;6:27-32.
- [CrossRef] [Google Scholar]
- Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo- controlled trial. Lancet. 2007;370:1560-1567.
- [CrossRef] [Google Scholar]
- Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species. Arch. Microbiol.. 2000;174:135-142.
- [CrossRef] [Google Scholar]
- Contribution of macrophage secretory products to urovirulence of pseudomonas aeruginosa. FEMS. Immunol. Med. Microbiol.. 2009;57:156-164.
- [CrossRef] [Google Scholar]
- Biotechnological approaches for the production of natural colorants by Talaromyces/Penicillium: a review. Biotechnol. Adv.. 2020;43:107601
- [CrossRef] [Google Scholar]
- First complete genome sequence of Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071 T), determined using PacBio single-molecule real-time technology. Genome. Announc.. 2015;3:e00932-e1015.
- [CrossRef] [Google Scholar]
- Fungal and bacterial pigments: secondary metabolites with wide applications. Front. Microbiol.. 2017;8:1113.
- [CrossRef] [Google Scholar]
- Food additives: production of microbial pigments and their antioxidant properties. Curr. Opin. Food Sci.. 2016;7:93-100.
- [CrossRef] [Google Scholar]
- Effect of pyocyanin on a crude-oil degrading microbial community. Appl. Environ. Microbiol.. 2004;70:4004-4011.
- [CrossRef] [Google Scholar]
- Microalgal carotenoids: a review of production, current markets, regulations and future direction. Mar. Drugs. 2019;17:640.
- [CrossRef] [Google Scholar]
- Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa. Biosens. Bioelectron.. 2004;19:1237-1244.
- [CrossRef] [Google Scholar]
- Determination of antimicrobial activity and production of some metabolites by Pseudomonas aeruginosa B1 B2 in sugar beet molasses. Afr. J. Biotechnol.. 2008;7:4614-4619.
- [Google Scholar]
- Investigation of antimicrobial activity of pyocyanin produced by pseudomonas aeruginosa strains isolated from different clinical specimens. Hacettepe. J. Biol. Chem.. 2016;1:1-6.
- [CrossRef] [Google Scholar]
- Production of microbial pigments utilizing agro-industrial waste: a review. Curr. Opinion Food Sci.. 2015;1:70-76.
- [CrossRef] [Google Scholar]
- Defining the core essential genome of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA. 2019;116:10072-10080.
- [CrossRef] [Google Scholar]
- Isolation of Pseudomonas aeruginosa for production of a remarkable antioxidant molecule pyocyanin. Int. J. Adv. Res. Biolog. Sci.. 2019;6:46-51.
- [Google Scholar]
- In vitro study of the swarming phenomena and antimicrobial activity of pyocyanin produced by Pseudomonas aeruginosa isolated from different human infections. Eur. J. Sci. Res.. 2010;47:405-442.
- [Google Scholar]
- Effect of static magnetic field (200 mT) on biofilm formation in Pseudomonas aeruginosa. Arch. Microbiol.. 2020;202:77-83.
- [CrossRef] [Google Scholar]
- Pathogenic characteristics of Pseudomonas aeruginosa bacteraemia isolates in a high-endemicity setting for ST175 and ST235 high-risk clones. Eur. J. Clin. Microbiol. Infect. Dis.. 2020;39:671-678.
- [CrossRef] [Google Scholar]
- Differential habitat use and partitioning by Pseudomonas species in human homes. Microb. Eco.. 2011;62:505-517.
- [CrossRef] [Google Scholar]
- Rodger, B.B., Andrew, D.E., Eugene, W.R., 2017. Standard methods for the examination of water and wastewater, Twenty third ed. American Public Health Association.
- Intrinsic and environmental mutagenesis drive diversification and persistence of Pseudomonas aeruginosa in chronic lung infections. J. Infect. Dis.. 2012;205:121-127.
- [CrossRef] [Google Scholar]
- Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infect. Immun.. 2001;69:584-588.
- [CrossRef] [Google Scholar]
- Phenazine pigments from Pseudomonas aeruginosa and their application as antibacterial agent and food colorants. Res. J. Microbiol.. 2008;3:122-128.
- [Google Scholar]
- Under nonlimiting iron conditions pyocyanin is a major antifungal molecule, and differences between prototypic Pseudomonas aeruginosa strains. Med. Mycol.. 2020;myaa066
- [CrossRef] [Google Scholar]
- Laboratory guide for identification of plant pathogenic bacteria (third ed.). USA: St. Paul American Phytopathological Society Press; 2001.
- Pseudomonas aeruginosa population structure revisited under environmental focus: Impact of water quality and phage pressure. Environ. Microbiol.. 2012;14:1952-1967.
- [CrossRef] [Google Scholar]
- Microbial Pigments in the food industry-challenges and the way forward. Front. Nutri.. 2019;6
- [CrossRef] [Google Scholar]
- Study on the antioxidant and cytotoxic properties of Pyocyanin extracted from pseudomonas aeruginosa. In: Ramkrishna D., Sengupta S., Dey B.S., Ghosh A., eds. Advances in Bioprocess Engineering and Technology. Singapore: Springer; 2021. p. :133-141.
- [CrossRef] [Google Scholar]
- Natural colorants: food colorants from natural sources. Annu. Rev. Food. Sci. Technol.. 2017;8:261-280.
- [CrossRef] [Google Scholar]
- Soliev, A.B., 2012. Pigmented bioactive compounds from marine bacteria and their mechanisms of action involved in cytotoxicity. A dissertation, Kochi, Japan, pp. 120.
- Rapid and solitary production of mono-rhamnolipid biosurfactant and biofilm inhibiting pyocyanin by a taxonomic outlier Pseudomonas aeruginosa strain CR1. J. Biotechnol.. 2020;307:98-106.
- [CrossRef] [Google Scholar]
- Food colours: existing and emerging food safety concerns. Crit. Rev. Food Sci. Nutr.. 2017;57:524-548.
- [CrossRef] [Google Scholar]
- Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol.. 2004;2:95-108.
- [CrossRef] [Google Scholar]
- Analysis of pyocyanin compound and its antagonistic activity against phytopathogens. Int. J. Chem. Tech. Res.. 2013;5:1101-1106.
- [Google Scholar]
- Determination of antiradical and antioxidant activity: basic principles and new insights. Acta Biochem. Polon.. 2010;57:139-142.
- [CrossRef] [Google Scholar]
- Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. FEMS Microbiol. Lett.. 2003;223:287-292.
- [CrossRef] [Google Scholar]
- Production of red pigments by the insect pathogenic fungus Cordyceps unilateralis BCC 1869. J. Ind. Microbiol. Biotechnol.. 2005;32:135-140.
- [CrossRef] [Google Scholar]
- Monascus: a reality on the production and application of microbial pigments. Appl. Biochem. Biotechnol.. 2016;178:211-223.
- [CrossRef] [Google Scholar]
- Visual detection of Hg2+ by manipulation of pyocyanin biosynthesis through the Hg2+ dependent transcriptional activator MerR in microbial cells. J. Biosci. Bioeng.. 2020;129:223-228.
- [CrossRef] [Google Scholar]
- Alternatives to those artificial FD & C food colorants. Ann. Rev. Food. Sci. Technol.. 2012;3:59-77.
- [CrossRef] [Google Scholar]
- Natural colorants: historical, processing and sustainable prospects. Nat. Prod. Bioprospect.. 2017;7:123-145.
- [CrossRef] [Google Scholar]
- Spices and herbs: their antimicrobial activity and its determination. J. Food Saf.. 1988;9:97-118.
- [Google Scholar]
- Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol.. 2009;191:210-219.
- [CrossRef] [Google Scholar]
- Microbial astaxanthin biosynthesis: recent achievements, challenges and commercialization outlook. Appl. Microbiol. Biotechnol.. 2020;104:5725-5737.
- [CrossRef] [Google Scholar]
- Anticancer effects of pyocyanin on HepG2 human hepatoma cells. Lett. Appl. Microbiol.. 2014;58:541-548.
- [CrossRef] [Google Scholar]