Food coloration is an age-old process to make a food more lucrative to consumers without altering its palatability and nutritional value. The colour, odor of food stimulate our salivary gland through the resulting impulse from the optic and oculomotor nerve. Most bakery, beverages, and confectionery industries use food colour to make their product presentable to consumers. The coloration of food and drinks was initially done using various phytochemicals until the invention of Perkin’s Mauve Pigment, the first ever synthetic colour in 1856.¹ As per Food Safety and Standard Act, 2006, only eight synthetic dyes can be used for food coloration within their prescribed limit – Brilliant Blue FCF (Blue 1), Indigo Carmine (Blue 2), Fast Green FCF (Green 3), Tartrazine (Yellow 5), Sunset Yellow FCF (Yellow 6), Erythrosine (Red 3), Carmoisine (Red 10) and Ponceau 4R (Red 18).² World Health Organization (WHO), U.S. Food and Drug Administration (FDA), European Food Standards Authority (EFSA), and Food Safety and Standard Authority of India (FSSAI) have conclusive and specific guidelines for using synthetic artificial dyes in the food industry. As per FSSAI guidelines, the permissible limit of these synthetic dyes in food products is 100 ppm only in the final product. But unregulated use of these dyes has been proven to be detrimental to human health,³ mostly due to the presence of unidentified impurities which could not be removed even after purification procedure.⁴ Excessive consumption of synthetic dyes may lead to abdominal pain, asthma, and renal failure.⁵ It was reported that consumption of such synthetic pigments above 50 mg per day may elevate allergic and hypersensitive reactions and also lead to several neurological disorders.⁶ Other risks associated with synthetic food dyes involve disruption of the natural immune system, increased inflammation, and oncogenicity.⁷ Synthetic food colours are also toxic to liver, kidney, and testes in human.⁸ They lower the level of high-density lipoprotein cholesterol, secretion of glutathione and alternatively increase the level of low-density lipoprotein cholesterol, plasma urea, cretin, creatinine and blood glucose. Simultaneously these synthetic food dyes significantly increase the activities of alkaline phosphatase, acid phosphatase, and lactate dehydrogenase enzymes.⁹ Tartrazine and Carmoisine-derived food colours are toxic against normal microflora of human beings, like Escherichia coli, and Staphylococcus albus also.¹⁰ As a consequence, most of the European countries have banned the use of synthetic dyes as food colouring agents.

In this background, natural dyes have got their importance to be used as food colorant again. Initially, different plants derivatives like lycopene, β-carotene, anthocyanin, chlorophyll were used as colouring agents for various packaged food particles. All these chemicals are extracted and processed from different plants parts for which a regular supply of sufficient amountof raw materials are necessary. Moreover, the plant-derived pigments are sensitive to various weather conditions like light, temperature, and pH. As a result, the focus of the research regarding the bio-colouration of foods has shifted towards microorganisms. Microorganisms are considered as the most industrially beneficial organic colour producers due to their neutral effects on seasonal variation, ease of handling, high growth rate, genetically and metabolically modifiable nature, greater stability than their synthetic counterparts and last but not the least highly cost-effective production rate.¹¹ By monitoring various cultural conditions like carbon/nitrogen source, rate of aeration, temperature, pH, one can regulate pigment production according to his/her need. By using the genetic modification technique, pigment production capacity per microbial cell can be increased many folds. Not only by minimizing the health hazards, but microbial pigments have also proved their efficiency as anti-cancerous, antioxidant, antimicrobial, antitumor and antifouling agents.^12,13 Different microbial pigments like carotenoids, melanin, flavins, quinines, monascin, violacein are widely being used in the food industry for colouring different food products, nowadays.¹⁴ A list of commercially available food-grade dyes of microbial origin is mentioned in Table 1.

Table (1):
List of synthetic colours and their biological counterpart

Microbes that can produce food grade pigments
Pigment production by microorganisms is quite common in nature. A diverse range of microorganisms like bacteria, fungi, and algae produce pigments as primary or secondary metabolitesby-products which play a crucial role in the survival of producing organisms in many aspects.

Bacteria
In many bacteria, pigment production occurs to activate photoreactive mechanisms to overcome DNA damages caused by UV rays,¹⁵ to give protection against oxidative damage¹⁶ and many others. Pigments like melanin are produced by a few strains of Pseudomonas stutzeri,¹⁷ Azotobacter chroococcum,¹⁸ Burkholderia cenocepacia,¹⁹ and many species of Actinobacteria,²⁰ Aeromonas, Alteromonas, Bacillus, and Vibrio.¹⁷ Other than melanin, many pigments like chinonenes, phenazines, flavins, carotenoids, and heterocyclic compounds like prodigiosins from Serratia marcescens²¹ and many other bacteria are produced as secondary metabolites, which help the bacteria to overcome different environmental and physiological stresses.¹³ Photosynthetic pigments of bacteria like Phycocyanin, Allophycocyanin, Phycoerythrin, bacteriochlorophyll²² are also being used in the food industry. Beside photosynthesis, these bacterial pigments also have antiviral, antibacterial and antifungal activities.²³ Zeaxanthene and lutein, two carotenoids produced by Flavobacterium sp. are widely used in the food industry for the yellow or orange colouration of many common foods like pasta.²⁴ Many carotenoid pigments are known for their antioxidant, antitumorigenic activities. The azaphilone pigment produced by Monascus sp. is commercially sold in dry powder form which is widely used in the food industry.²⁵ Pyocyanin produced by Pseudomonas aeruginosa is currently used as colouring agent in confectionaries, cakes, puddings, and beverages.²⁶

Fungi
Fungi including yeasts and molds are more industrially economical than other microorganisms for pigment production. Pigmentous fungi have been isolated from a diverse range of habitats including marine to mangrove sediments, endophytic to invertebrate inhabitant, and geothermal to halophilic soil. Monascus purpureus, commonly known as ‘Red Yeast’ produces three types of pigments- yellow-coloured ankaflavin and monascine, orange-coloured rubropunctatine and monascorubrine and purple coloured rubropunctamine²⁷ and monascorubramine.²⁸ This yeast is widely used as a colour intensifier and food additive in the food industry. Commercially used ‘Natural Red’ (chemically anthraquinone) is the product of the fungi Penicillium oxalicum.²⁹ The use of fungi for production of different edible pigments is an age-old practice. Pigments like azaphilone, anthraquinones, chromene, hydroxyanthraquinone, napthoquinone, carotenoids, and many others are produced as secondary metabolites by different classes of fungi (Table 2). Though the initial research regarding the industrial use of fungi was restricted within the Basidiomycetes group, however, the Ascomycetes group of fungi exhibit better potency of pigment production than the former.³⁰ While selecting a fungus for industrial production of edible biopigment, it is necessary to assay the toxicity of the fungus, if any. For example, commercial production of food-grade pigment is avoided from Penicillium and Fusarium as they have been detected with some toxicity.³¹

Table (2):
List of common fungi and pigments produced by them

Algae
Among all the pigments of microbial origin, microalgae are the most reliable, easily extractable and industry-friendly source. The practice of consumption of whole microalgae as a potential source of all essential amino acids⁴¹ and highly digestible mono and polysaccharides⁴² is quite primitive. But the use of microalgae as a food colorant flourished when the use of biocolorant gained importance in the food sector. Algae are reliable and primary sources of phycobilin and phycobiliproteins. Though commercially extracted from plants, algae can also act as an alternative for commercial chlorophyll production. In chlorophyta group of algae, the biomass-to-pigment ratio is equivalent to that of the seed plant.⁴³ Spirulina platensis, Chlorella vulgaris, Dunaliella salina, and Haematococcus pluvialis are widely used in the food industry as a potential sources of different biocolorants namely phycocyanin, carotenoids, astaxanthin, lutein, lycopene etc.⁴⁴ Chlorella vulgaris is used to provide a green tone in butter cookies.⁴⁵ Combination of these phycobilin varies in different members of Glaucophyta, Cryptophyta and Rhodophyta.⁴⁶ Besides phycobiliproteins, microalgal pigments like carotenoids, xanthophyll, and chlorophyll have high commercial utilization and market value. Algal pigments have been widely used as supplements of synthetic food colours and are additionally preferred for their anti-inflammatory, antidiabetic, and anticarcinogenic activity.⁴⁷

Types of microbial pigments used in food industry
Whatever be the source, most commercially produced microbial pigments can be broadly categorized as melanin, phycobiliproteins, xanthenes and its derivatives, quinone and its derivatives, and carotenoids.

Melanin
Melanin is a universally occurring dark polymeric pigment (oxidized phenolic/indolic compound). In our system produced in the melanosomes of the melanocytes. The ratio of two of its naturally occurring forms- eumelanin and pheomelanin regulates the type of pigmentation of hair and skin.⁴⁸ The pigment is produced from its precursor L-tyrosine⁴⁹ by enzymatic modification where the enzyme tyrosinase is a product of the tyr gene present in chromosome number 11 of humans. The tyrosinase enzyme converts L-tyrosine to L-DOPA which is then oxidized to produce dopachrome. Melanin is a polymerized product of this dopachrome.⁵⁰ Not only in pigmentation, but melanin is also well known for its free radical scavenging activity,⁵¹ anti-inflammatory activity by inhibiting cox and lox genes, protective activity against anti-microbial drug,⁵² anti-cancer activity⁵³ and many others. Recent studies have shown that different melanin derivatives can interact with SARS-CoV-2 spike protein and can act as antiviral compounds.⁵⁴

Though direct melanin-containing food grains are unavailable, but, Vitamin C⁵⁵ and Vitamin D⁵⁶ rich foods and other antioxidants can increase melanin production in our system. Industrial production of melanin is principally done using Aeromonas media and Pseudomonas maltophila.⁵³ Genetically modified bacteria are now being considered to increase the yield of melanin in industries. Recombinant E. coli has been prepared by cloning two melanin-producing genes mel and ORF438 from Streptomyces antibioticus.⁵⁷

Chlorophyll
Chlorophyll, the major photosynthetic pigment of plants and algae largely prevails in its different forms like Chlorophyll a, Chlorophyll b, Chlorophyll c, and Chlorophyll d. A modified form of chlorophyll, known as Chlorophyllides is produced by different Cyanobacteria.⁵⁸ The pigment is highly soluble in organic solvents like ether and alcohol due to its long hydrocarbon chain attached to the central porphyrin ring. With the overgrowing urge to use biopigments in food stuffs, industrial production of chlorophyll gets hyped to give green colouration to the foods. Chlorophyll is not only being used to imply green colour to foods like cakes and beverages but it is also being added to retain the colour of many packaged foods.⁵⁹ Initially, ethanolic extraction of chlorophyll from plants was used for this purpose but with due course of time it was found that the chlorophyta group of algae can produce an almost equivalent amount of chlorophyll compared to seed plants but in less time. As maintenance of algal culture is easier than seed plants, it is preferred over the second for commercial chlorophyll production.

Use of chlorophyll as a food colour is beneficial for human health also. Reports in favour of chlorophyll as an antitoxin, anticancer and anti-inflammatory compound have already published. Chlorophyll protect the liver and intestine by reducing and/or neutralizing the toxicity of certain toxic compounds. Besides that, chlorophyll can regulate the blood pressure, the pancreas, and abnormal release of thyroid hormone. The healing effect of chlorophyll against chronic ulcers has also been established.⁶⁰

Commercially chlorophyll is available in the market as Semi-synthetic sodium copper chlorophyllins (SCC). In this compound the magnesium ion of the chlorophyll’s tetrapyrrole ring is replaced by copper and the hydrocarbon chain is deleted to increase the stability and water solubility.⁶¹ However, the bioavailability of this modified form may not be similar to that of the natural one we get from our diet.⁶² For chlorophyll production large scale cultivation of filtered microalgae like Chlorella, Dunaliella, and Spirulina is done either in an ‘Open Air System’ or in a ‘Photobioreactor’ depending on the availability of nutrients, sunlight, carbon dioxide, and water. However, construction, maintenance and handling of the first one is easier than the second one.⁶³ After cultivation, the cells are lysed by homogenization, sonication, or grinding, and chlorophyll is extracted using organic solvents like acetone or ethanol. However, dimethyl formamide (DMF) shows best extraction ability than other solvents. However, the use of DMF is restricted due to its toxic nature. Usually, extraction is performed in multiple steps and the degree of extraction depends on several physical parameters.⁶⁴ Nowadays, Supercritical Fluid Extraction (SFE) has gained importance to overcome the negative impact of organic solvent extraction in nature. In this method, liquid carbon dioxide is used for extraction purposes at around 31 °C temperature and 7.38 MPa pressure in a specially designed apparatus. After the extraction, the fluid is removed from the extract by keeping the mixture at atmospheric pressure.⁶⁵

Phycobiliproteins and phycobilin
Phycobiliproteins are water-soluble protein-bound forms of phycobilin, a group of light-harvesting pigments, located within the phycobilisome of Cyanobacteria and red algae and help the host organism to capture the light of specific wavelength (usually higher than that of the chlorophyll) and transfer the same into photosynthetic reaction center with high efficiency. Depending upon the wavelength of light they absorb, phycobiliproteins are classified into four major groups – phycocyanin, phycoerythrin, phycoerythrocyanin, and allophycocyanin.⁶⁶ Among them, phycocyanin has extensive nutraceutical and pharmaceutical value due to its anticarcinogenic, antioxidative, and anti-inflammatory activities.⁶⁷ Phycocyanin is commonly produced from chlorophyll A containing dried cyanobacteria Arthrospira platensis. The first report of extraction of phycobilin was done from Oscillatoria sp. for industrial production of Brilliant blue.⁶⁸ Later on, phycobilin extraction was started from the cell culture of different cyanobacteria and red algae (Table 3) to fulfil the needs of the food and cosmetic industry.⁴⁶

Table (3):
Different phycobiliproteins and their producer algae

Metabollically phycobilin is produced from heme which is obtained from its precursor protoheme IX by the action of the enzyme ferrochelatase. The heme is then converted into biliverdin IXa by heme oxygenase. Phycobilin is the reduced product of biliverdin IXa which is catalyzed by the enzyme ferrodoxin-dependent bilin reductase. Covalent attachment of phycobilin with apoprotein produces phycobiliproteins by phycobiliprotein reductase.⁶⁹

Phycoerythrin is used in the food industry as a natural red colorant and is preferred over anthocyanin due to its stability in a wide range of pH. This pigment is extensively used in the preparation of candy, lollipops, and decoration of cakes at lower pH.^70,71 Another phycobiliprotein, phycocyanin is used as a natural non-toxic blue colorant of chewing gum, various soft and alcoholic beverages, milkshakes and many other milk products.⁷⁰ In the USA, increasing demand for natural blue colorants for preparing candy and chewing gum, Spirulina extract, another source of phycobiliprotein is currently being used.⁷² Antioxidant and anti-cancerous effects of phycocyanin have been successfully tested in vitro on liver cells, renal cells, cardiac cells, blood cells, macrophages, neuronal cells, and other.⁷³

For industrial production of phycobilin and phycobiliproteins, the cells are disrupted by maceration, sonication and freeze-thaw technique⁷⁴ and sometimes by enzymatic hydrolysis. To increase the yield of the product in fewer cases both the mechanical technique and enzymatic hydrolysis are used in combination.⁷⁵ Extraction of the pigments are commonly done in water and 100 mM acetate buffer of pH 6.0.⁷⁶ However, whatever the means of extraction, the purity obtained after extraction is poor, and purified phycobilin and phycobiliproteins which should be used in the food industry are highly expensive. The stability of phycobilin and phycobiliproteins are major concern as these pigments are readily degradable under adverse environmental and physical conditions. To increase the stability of phycocyanin, the extract is mixed with 20% glucose, 20% sucrose or 2.5% sodium chloride.⁷⁷ Mixing of high sugar concentrations increases the stability of phycocyanin for about 2 months. The addition of benzoic acid, citric acid, and sucrose increases the thermal stability of the pigments.⁷⁸

Carotenoids
Carotenoids are another group of important accessory light-harvesting pigments used by photosynthetic microorganisms. It implies yellow, orange, or red colouration on the food staffs like cakes, cheese, ice cream, and many non-alcoholic beverages. Carotenoids act as the precursor of Vitamin A. In addition, the role of carotenoids in minimizing oxidative damage by neutralizing the superoxide radicals is also well established. Metabolically carotenoids are derivatives of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In archaebacteria and fungi, IPP is produced enzymatically in cytosol from Acetyl CoA via mevalonate (MVA pathway). IPP is then isomerized to form DMAPP and both these molecules act as a conjugant to produce lycopene and β-carotene.^79,80 On the other hand, in eubacteria, malarial parasites, and microalgae, carotenoid precursor IPP is produced from pyruvate and glyceraldehyde 3 phosphate via the intermediate methylerythritol 4 phosphate (MEP pathway).⁸¹

Microbial carotenoids include β-carotene, astaxanthin, zeaxanthin, etc. β-carotene, commonly sold as CI Natural yellow 26, should contain all trans β-carotene and no cis β-carotene in the mixture. However, no such regulation has still been implied for other carotenoids. Increased production of commercial β-carotene has been achieved by the implementation of Recombinant DNA Technology. The highest recombinant β-carotene production was reported from a fungus Yarrowia lipolytica under defined environmental and culture condition.⁸² Other microbes reported for commercial production of β-carotene include Blakeslea trispora, Dunaliella salina, and Dunaliella bardawil.

Lycopene is another lipophilic carotenoid which structurally intermediate between β-carotene and Xanthophyll. Industrially, lycopene is produced by modifying β-carotene biosynthetic pathway in filamentous fungi like Blakeslea trispora. In this fungus, lycopene accumulation is directed by the addition of imidazole as an inhibitor of the fermentation of glucose via MEP pathway.⁸³ In a different study, the efficacy of different inhibitors of the enzyme lycopene cyclase has been evaluated for accumulation of lycopene in the system where it has been established that around 270 mg/L of lycopene accumulation can be obtained at 100 to 1000 ppm of inhibitors concentration.⁸⁴

Astaxanthin is a red coloured carotenoid that belongs to the Xanthophyll sub class and is well known for its antioxidant activity. Commercial production of astaxanthin is done from the yeast Xanthophyllomyces dendrorhous,⁸⁵ Phaffia rhodozyma,⁸⁶ and also from a few microalgae like Haematococcus pluvialis, Chromochloris zofingiensis, etc.⁸⁷ For commercial production of astaxanthin, yeast cells are lysed by the enzyme Accellerase or Glucanex under optimum conditions. The fermentation was best reported at 20 °C, pH 6 at a constant aeration rate of 1 L/min. Biomass and product formation both can be increased by increasing the agitation speed upto 600 rpm at controlled environment. In addition to Accellerase or Glucanex, the extraction of astaxanthin can be more intensified using supercritical CO₂.⁸⁸

Zeaxanthin is an orange-coloured modified β-carotene commercially being produced by genetically engineered Escherichia coli. Besides that, other bacteria of the family Flavobacteriaceae and Sphingomonadaceae⁸⁹ family are also used for commercial Zeaxanthin production. These cells are not only engineered for overexpression of related genes but also metabolically engineered to modify the existing biochemical pathway that leads towards increased production of Zeaxanthin.⁹⁰

Fucoxanthin is another important Xanthophyll type of carotenoid that is being used in the food industry to give brownish colour to the food stuffs. Fucoxanthin is produced by different types of Brown algae like Sargassum, Laminaria, Undaria, and some other microalgae like diatoms.⁹¹ Around 420 µg/g Fucoxanthin can be commercially extracted from the brown algae Eisenia bicyclis, in 90% ethanol at 110 °C under 1500 psi pressure.⁹²

In most cases, carotenoids are extracted by non-polar or organic solvents from different plant sources like Kabocha pumpkin, Cassava leaves, Marigold flowers and many others.⁹³ Extraction can be done using either the Soxhlet, Supercritical fluid extraction or Green method.⁹⁴ However, industrial production of carotenoids from microbes like cyanobacteria, microalgae, and bacteria are preferred over the plants due to lower overall production cost. These microbes, not only use low-cost agricultural wastes as substrate for fermentation, but also the extraction of pigments from the microbes are relatively cheaper than that of the plant sources.⁹⁵ For large-scale production of carotenoids, microbes like Dunaliella salina, Xanthophyllomyces dendrorhous, Haematococcus pluvialis, Blakeslea trispora, Bradyrhizobium sp., Paracoccus zeaxanthinifaciens, Chlorella zofingiensis and Brevibacterium aurantiacum are currently being used extensively.⁹⁶

Riboflavin
Riboflavin, commonly known as Vitamin B12, is a dietary supplement used in baby foods, dairy products, and packaged fruit juices.⁷ In addition to being used as a nutritional supplement, it also imparts a yellow colour to the food. Metabolically, riboflavin is used as a precursor of flavin, an important co-enzyme required in different dehydrogenation reactions. Industrially riboflavin is produced mostly by submerged fermentation of simple sugar by Bacillus subtilis, Candida famata, Ashbya gossypii,⁹⁷ Eremothecium ashbyii, Debaryomyces subglobosus, Clostridium acetobutylicum.⁹⁸ However, genetically modified Escherichia coli, Corynebacterium ammoniagenes are also used for riboflavin production nowadays.⁹⁹ Though mostly purified (97%), as per FSSAI guidelines, a marketable purified riboflavin may contain 5 ppm of arsenic and 20 ppm of lead as metallic impurities.

Other smaller groups
Apart from these major classes, bacteria produce a few other pigments also, which are known to have antimicrobial properties and hence used as food pigments, as well as, protect related food from being contaminated by other microbes. Prodigiosins produced by Serratia marcescens and Hahella chejuensis are widely used in food industry as immunosuppressants, anti-cancer, and antibiotic agents.^100,101

Factors that regulate pigment production in microbes
All pigments are metabolic products/by-product of certain biosynthetic pathways.So, pigment production mostly depends on the smooth running of the concerned metabolic pathway. To regulate the quantitative extent of pigment, the concerned metabolic pathway should properly be directed. For industrial production of microbial pigments to be used in the food industry, the nature of fermentation, media composition, temperature, pH, moisture content, aeration and the incubation period play crucial roles.

Carbon and Nitrogen source
For fungal growth and pigmentation, the selection of mono/poly/oligosaccharides is essential. In most cases Glucose is preferred over other carbon sources like maltose, fructose, etc.¹⁰² The carbon requirement also varies from species to species. For Rhodotorulla glutinis, it has been reported that sucrose is preferred over other carbon sources.¹⁰³ Sometimes, it may happen that the carbon requirement for biomass production does not match the carbon requirement of pigment production per cell. In those cases, a suboptimum condition may be maintained concerning both biomass and pigment production. For β-carotene production from the fungi Blakeslea trispora Glucose and corn strip liquor served as best carbon and nitrogen source,²⁷ however, if strains are acclimatized with lactose, then whey can also be used.¹⁰⁴

Nitrogen, one of the key components for microbial growth and biomass production, can also influence the extent of pigments produced by certain organisms. It has been reported that glutamic acid and 6-furfurylaminopurine convincingly increase monascin in a specific strain of Monascus.¹⁰⁵ The effect of different nitrogen sources on the growth and pigment production by Serratia nematodiphila was studied where yeast extract was reported as the best nitrogen source from all the view point.¹⁰⁶ Though reports are very few in support of the utilization of nutrients by every individual microbe in association with their pigment production, the optimization of carbon and nitrogen sources during fermentation still needs standardization.

Temperature
All the biochemical processes are best performed at their optimum temperature where their concerned enzymes show maximum activity. Most of the pigment production so far reported is done at mesothermal temperature. During fermentation, the temperature is controlled by the exposure to light where the time and intensity of exposure are directly proportional to the temperature. Similarly in a few bacteria and fungi like Myxococcus xanthus, Mycobacterium marinum, Rhodotorulla glutinis positive carotenoid production was evident in the light.¹⁰⁷ A psychrotolerant Paenibacillus was reported to grow maximum at 25 °C and its pigment was successfully extracted with a lab scale bioreactor.¹⁰⁸ Few cold-adopted bacteria and fungi are found to produce FDA-approved colour at psychrophilic temperature, however, the absence of proper policies regarding the utilization of resources from psychrophilic areas, appears as a challenge.

pH
Maintenance of proper pH of the medium is not only required for the growth of the concerned microorganism, but it also influences the stability of the pigment depending upon its acidic or basic nature. Lycopene production gets promoted at neutral to slightly alkaline pH, while acidic pH facilitates β-carotene production.¹⁰⁹ An orange pigment produced by halophile Salinococcus roseus changes to yellow when grown at acidic pH.¹¹⁰ Similarly, the green pigment produced by Pseudomonas aeruginosa at neutral pH will be turned into dark red and yellow if grown at pH 2 and pH 13, respectively.¹¹¹ Not only the stability, but pH also influences the overall pigment production concerning the control. Micrococcus luteus produces a carotenoid pigment at pH 7, however, drifting the pH from pH 7 will decrease the pigment production by lowering the overall biomass.¹¹²

Besides these major physical regulators of pigment and biomass production, many other minor parameters like dissolved oxygen, humidity, and availability of different micronutrients also regulate microbial pigment production.

Improving the production of microbial pigment
Every industrial production aims to increase the yield of the product cost-effectively. Basic improvements in the strategy of industrial production of any commercial microbial product involve the modification of the producer organism so that it can produce increased amount of target product than the original. So screening and selection of the desired microbial strain followed by subsequent genetic modifications are important for improving its production threshold.

Screening of pigment producing microorganisms
Numerous screening strategies have been employed to select the desired producer organisms. Microbes that can produce edible pigments can be isolated using bioprospecting programs. Source microorganisms are allowed to grow in an optimum environment for pigment production in a selective media. Bacteria like Micrococcus sp., Serratia sp., Salinococcus sp., etc. are optimized for maximum pigment production in the presence of 1% glucose, 0.1% yeast extract, 2-6% NaCl at 30 °C-37 °C at pH 7.¹¹³ Phycobilin-producing cyanobacteria like Anabaena, Nostoc, Oscillatoria, Chrococcus, etc. can be screened out using BG11 and CFTRI media at 30 °C in the presence of light intensity of 25 L mol photons/m² /s following 12:12 hour light/dark regime for 27 days.¹¹⁴ The screening strategies also involve prior knowledge about any toxic by-product, and should be selected accordingly. For example, the producer organism of commercial bio colour Quoron TM, Fusarium venenatum produces a cytotoxin 4, 15-diacetoxyscirpenol.¹¹⁵ Modern biophysical and spectrophotometric techniques like LCMS, NMR, and UV VIS spectroscopies are employed for the detection and screening of microbial pigments.¹¹⁶

Strain improvement strategies
Traditional techniques related to the genetic modification of microbes are the key strategy for increasing the production of pigments. Plenty of chemical mutagens like 1-methyl-3-nitro-1-nitrosoguanidine (NTG), ethyl methane sulfonate (EMS), and physical mutagens like UV rays of specific wavelength have widely been employed as mutagens.¹¹⁷ Recent developments in molecular biological techniques and biological databank and data management systems play a crucial role in the identifying and revealing genetic information of the producer organism, which in turn helps in modifying the strain for better production. The entire PKS-FAS gene cluster of Monascus purpureus was identified for azaphilone pigment production including the pivotal enzymes MppR1 and MpPKS5. This information helps to modify the organism at the genetic level which lead to better azaphilone production.¹¹⁸ Mining and reconstructing metabolic pathways by insertions and deletions of certain genes also improve the overall pigment productivity of certain organisms. Modifications of the promoter, RBS region of the pivotal enzyme of pigment producing pathways may guide the efficiency and amount of the enzyme produced and thereby control the pigment production.¹¹⁹

Modification of cultural conditions
Not only with modification of producer organism but enhanced production of commercial grade microbial pigment also requires the modification of culture conditions aiming towards the increased production of desired pigment. Pigments are produced both by solid-state fermentation and submerged fermentation technique. The media compositions and associated physical, chemical, and environmental factors can be modified to get the product at optimum concentration. Pigment production by different molds can be optimized by implying suitable immobilization techniques that minimize the cell loss and increase cost-effective production of desired pigment. Controlling other physical and environmental factors like moisture content, temperature, pH of the medium, etc. can also affect pigment production. Choosing a suitable substrate for the fermentation technique also plays an important role in pigment production. Apple pomace is used in solid-state fermentation for the production of pink shed from Rhodotorula, red shed from Chromobacteria, yellow pigment from Micrococcus.¹²⁰ Penicillium purpurogenum normally produce a brick red pigment, however by modifying the culture condition, one can channel the production towards violet and orange pigment.¹²¹ Designing new metabolic pathways and modification of existing pathways are also being employed to bypass the blockage in production due to feedback inhibition of traditional biosynthetic pathways. For example, β-carotene production in Escherichia coli can be increased by introducing a novel mevalonic acid pathway where the first part of the pathway resembles that of Streptococcus pneumoniae and the end part resembles that of Enterococcus faecalis.¹²² In recent years, multiple pigment-producing genes have been identified by advanced biotechnological techniques and biosynthetic pathways for pigment production have been discovered using isotope labelling.¹²³ Most of the pigments produced by the microbes are insoluble. However, chemical modifications have been done to make them soluble in various solvents like aminoacetic acid, and aminobenzoic acid, so that, they can be used as food colorants.

In very recent studies, it has been stated that for large scale production of microbial pigments, agro-industrial waste materials as a substrate for fermentation has been preferred. For producing food-grade pigments from Monascus ruber, Penicillium resticulosum¹²⁴ corn steep liquor obtained from different cereal processing industries can act as a good nitrogen and vitamin source. During carotenoid production from Rhodotorula glutinis mung bean waste is being used.¹²⁵ Whey protein is used for submerged fermentation by Rhodotorula rubra that produces a characteristic yellow pigment.¹²⁶ Similarly, waste from the fruit and vegetable industry like jackfruit seed, Kinnow peel, apple pomace, grape waste, etc. are currently being used as substrates for fermentation by various microbes for the commercial production of pigment.¹²

Controlling other physical parameters of fermentation
Besides the screening and strain improvement strategies, the functionality of the fermenter like aeration rate, agitation speed, etc. also play an important role in regulating yield. At an agitation rate of 1000 rpm, more red pigment can be recovered from Monascus purpureus than that with a lower agitation rate.¹²⁷ In a mathematical model presented in 2002, it has been stated that the aeration and agitation rate play a crucial role in the production of β-carotene by Blakeslea trispora. Maximum β-carotene production was reported at lower agitation speed with higher aeration rate.¹²⁸ In addition to aeration and agitation, pigment production from edible fungus Neurospora intermedia can be enhanced by post-treatment with 95% humidity.¹²⁹ However, solid-state fermentation for commercial bio-pigment production is favoured over submerged fermentation due to the ease in isolation of pigment from the reactor.

Advantages and challenges of using microbial pigments
Advantages
Microbial pigments are preferred over their synthetic counterpart mostly due to their availability, non-toxic nature. Microbial pigments are evident for having lower downstream processing costs with higher yield than the synthetic pigments.¹³⁰ Microbial pigments also exhibit antioxidant, anticancer, immunosuppressive and inflammatory properties in different cases.

The antioxidant activity of a compound is related to the presence of conjugated systems that are susceptible to electrophilic attack. Staphyloxanthin produced by Staphylococcus aureus reduces oxidative stress in Swiss albino mice caused by carbon tetrachloride.¹³¹ Another group of microbial pigments like β-carotene, astaxanthin and zeaxanthin also show some extent of antioxidant properties due to their reactive hydroxyl group.¹³² A recent study with carotenoid pigments of Staphylococcus sp. and Pseudomonas sp. has demonstrated their free radical scavenging activity using DPPH assay. Different red pigments like monascorubramine, lycopene, undecylprodigiosin, orange pigments like orevactaene, neurosporaxanthin, and many others produced by certain groups of fungi, actinomycetes are well evident for their antioxidant nature.¹³³ The addition of natural microbial pigments produced from whey in the food chelates the free radicals from the body and thus acts as a potential antioxidant.¹³⁴

Anti-cancerous activity of microbially produced pigments is another important advantage of using them as food colorants, whereas synthetic colours are sometimes carcinogenic. Extracted pigment from Monascus purpureus mixed with coix seed was tested for its anti-cancerous activity over HEp-2 cell line of human laryngeal carcinoma and it was observed that the IC₅₀ value of the pigmented seed was decreased about 42%.¹³⁵ Pigments like anthocyanin, and violacein produced by different microbes, can be used as food colorant and have anti-cancerous, anti-inflammatory properties. Prodigiosin produced by Serratia marcescens is currently used as a dietary supplement for treating diabetes mellitus. This particular pigment has also been successfully tested as an inducer of the apoptotic pathway and an inhibitor of cell cycle.¹³⁶ Cytotoxic activity of different bacterial pigments has been successfully tested against different mammalian and human cell lines. Melanin produced by Bacillus sp. and Pseudomonas sp. is being used for treating metastatic melanoma. Pigments like prodigiosin and its analogs, violacein and many other carotenoids are reported for their immunosuppressive activities through inhibition of proliferation of T cells. The anti-inflammatory and antiproliferative effect of scytonemin, produced from cyanobacteria, is well established for reducing cytokine production which upregulated during the onset of carcinoma.¹⁴

Microbial pigments like prodigiosin compounds produced by Serratia and Vibrio, tambjamines produced by many marine bacteria like Pseudoalteromonas, and violacein produced by Janthinobacterium and Chromobacterium have antibacterial activities against wide range of gram-positive and gram-negative bacteria. They are effective against multiple human pathogens also. Not the antibacterial properties, but all these compounds are also well known for their antifungal activities against a broad range of pathogenic fungi like Candida, Penicillium, Aspergillus, Cryptococcus, Histoplasma, and many others.¹³ Flexirobin, ant342, a yellow pigment produced by Flavobacterium sp. is currently being used as a chemotherapeutic agent for the treatment of tuberculosis. Carotenoid pigments from Staphylococcus sp. and Pseudomonas sp. are evident to exhibit antimicrobial properties against Bacillus subtilis, Streptococcus pyogenes, Shigella sp., Klebsiella sp. and Escherichia coli.¹³² Staphyloxanthin isolated from Staphylococcus gallinarum has been reported to have growth inhibition against Escherichia coli, Staphylococcus aureus, and Candida albicans.¹³⁷ Similarly, the antimicrobial activity of violacein was successfully tested against a broad range of bacteria namely Bacillus lichenniformis, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Pseudomonas aeruginosa, and viruses like polio virus, herpes simplex virus and simian rotavirus SAII.^138,139 Pyocyanin, a blue pigment produced by Pseudomonas aeruginosa is widely used as natural food colorant. This compound shows distinct antibacterial efficacy against different food pathogen like Bacillus spizizennii, Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes, and even against other strains of Pseudomonas aeruginosa also.¹⁴⁰ Pigments produced from endophytic fungi have shown better antimicrobial potential than commercially available streptomycin against many human pathogens.

Few microorganisms that produce edible pigments also possess some nutritional value. Spirulina, a widely used algae as green/blue colorant can act as a potential source of essential amino acids (single cell protein), calcium, potassium, magnesium, iron, and vitamins.¹⁴¹ Other than Spirulina, microalgae like Chlorella, Dunaliella, and Aphanizomenon can also act as rich sources of protein. Microalgae can act as potential source of other macronutrients like polyunsaturated fatty acids, and vitamins also.¹⁴²

Challenges
Although edible pigments of microbial origin are associated with numerous health benefits for consumers, their use in the food industry is not widely accepted by industrialists due to high production and maintenance costs. The use of microbial pigments in the food industry increases the overall production cost by at least five times, while in confectionery items, it can increase up to twenty times.¹³¹ The production and maintenance of raw materials (bacterial culture) and the low yield per unit for the producing organism make these pigments more expensive than synthetic dyes.¹⁴³ Additionally, natural pigments often have inherent odors that can affect the quality and palatability of food. Most foods that contain natural pigments as coloring agents require deodorization, a technique used to mask unpleasant odors. Another significant drawback of using microbial pigments in the food industry is their natural instability. These pigments do not maintain the same consistency as synthetic pigments. The quality and stability of microbial pigments are greatly influenced by heat, light, air, water activity, and pH levels, making the use of these pigments in the food industry more challenging. Various modifications have been implemented to enhance the stability of these natural pigments. In many cases, slight changes in media composition can alter the associated structure of the pigments while keeping the skeleton intact, which, in turn, increases their stability under harsh environmental conditions.¹⁴⁴

Presently most of the fermentation are performed using different synthetic and chemically defined media which needs extreme expertise to formulate and prepare. Additionally, they are costly enough to increase the price of the product. To make these biocolours available to all for use as a food colour, new synthetic or semisynthetic media must be formulated to reduce the production cost.

Isolation and purification of pigments from microbes is another technical challenge. Conventional separation techniques that involve different types of solvents like acetone or ethyl acetate have significant negative effects on human health. Thus, some alternative strategies like spray drying, solid phase extraction may be tested to overcome the challenge. Another disadvantage of using microbial pigment for food coloration is that many microorganisms show their pathogenicity in the human body under some specified condition. Melanin producing organisms like Vibrio cholerae, Cryptococcus neoformans, Aspergillus fumigatus are known for their pathogenicity in human.¹³ Organisms like Serratia marcesens that produce prodigiosin causes urinary tract infection, Staphylococcus aureus that produces staphyloxanthin, Chromobacterium violaceum that produce violacein are all known human pathogen.¹⁴⁵ It was reported in a recent publication, that Stenotrophomonas maltophilia produces an orange pigment, but the organism causes serious fatal infections in human beings.¹⁴⁶ So, while dealing with these microbes for their pigment production, trials are going on to reduce the pathogenicity of these organisms while being used for industrial purposes. To reduce the microbial toxicity generated from the source of isolation, genetically modified non-toxic non-pathogenic microbes like Escherichia coli, Saccharomyces cerevisiae, Candida albicans and Zymomonas mobilis are preferred over other microbial sources.¹⁴⁷