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Bacteria, yeast, fungi, microalgae and insects sources of biocolours

Thursday, 17 May, 2018, 08 : 00 AM [IST]

Sajad A Rather, Syed Muzaffar and F A Masoodi

Biocolours
Colour is the main feature of any food item as it provides its primary identification. It assists in judging the nature, quality and safety of a food product in question. Addition of colours to food products means to achieve some of the aims - 1) Replacement of colour lost during processing, 2) Enhancement of the colour already present, 3) Minimisation of batch variations among foods and 4) To colour otherwise uncoloured food.

For the said purpose, colours, both naturally obtained and chemically synthesised, have been put to tremendous use. However, concerns centering on the potential toxicity and unsuitability of synthetic colours for human consumption have propelled research efforts towards more natural sources. Running parallel is the increasing global trend towards ecofriendly and biodegradable commodities. Together these factors are leading to increasing demands for natural colours and consequently putting thrust on the food and allied research fields to find alternatives to synthetic colours. Biocolours are those colouring agents, which are obtained from the biological sources. Biocolours are sourced from insects, plants and microbes. These sources form encouraging alternatives to synthetic colours, provided certain technological and economical issues are dealt with.

History of Biocolours
From time immemorial, colour has been an important criterion for acceptability of products like textiles, cosmetics and food. In Europe, it was practiced during the Bronze Age. The earliest written record of the use of natural dyes was found in China dated 2600 BC. According to Aberoumand, in Indian subcontinent, dyeing was known even in the Indus Valley period (2500 BC) and has been substantiated by findings of coloured garments of cloth and traces of madder dye in the ruins of Mohenjodaro and Harappa civilisation (3500 BC). In Egypt, mummies have been found wrapped in coloured cloth. Chemical tests of red fabrics found in the tomb of King Tutankhamen in Egypt showed the presence of alizarin, a pigment extracted from madder.

The cochineal dye was used by the people of Aztec and Maya culture period of Central and North America. By the 4th century AD, dyes such as woad, madder, weld, Brazilwood, indigo and a dark reddish-purple were known. Brazil was named after the woad found there. Henna was used even before 2500 BC, while saffron is mentioned in the Bible. Use of natural biocolourants in food is known from Japan in the Shosoin text of the Nara period (8th century), which contains references regarding colouring soybean and adzuki-bean cakes. Thus, it appears that coloured processed foods had been taken by the people of some sections during that period. According to Aberoumand and Mortensen, the study of colour was intensified in the late 19th century. Today, dyeing is a complex and specialised science.

Sources of Biocolours
Bacterial Pigments: Bacteria have immense potential to produce diverse bioproducts, among which one such product is pigment. Bacterial pigment production is an emerging field of research to demonstrate its potential for industrial applications. Most of the bacterial pigment production is in the R&D stage and hence requires intensive work in especially finding cheap and suitable growth medium, to reduce the cost and increase industrial applicability. Carotenoids are very well known and highly popular as food colourants. The carotene production by a pigmented strain of bacteria Bacillus (alkaliphilic yellow) has been documented.

Yeast Pigments: The Carotenoids astaxanthin, produced by yeast Phaffia Xhodoxyma, is considered as an important source of the natural pigment for colouring foods. The growth of Phaffia rhodozyma on 7-10% B or C grade molasses gives 2-3 times more astaxanthin than with glucose or sugar blend. In addition to Phaffia rhodoxyma even food spoiling xerophytic yeast produces highly pigmented colonies, which may also likely to contribute in the natural colouring for food in near future. Yeasts in the genus Rhodotorula synthesise carotenoid pigments. Most of the research is focussed on the species Rhodotorula glutinis. The main compounds produced by these red yeasts are torulene and torularhodin, with minute quantity of ß-carotene.

Fungal Pigments: Among fungi, pigments of basidiomycetes have been used for dyeing wool and silk, but such fungi are difficult to be cultivated under lab and industrial scale conditions. Therefore, the attention is drawn towards ascomycetes or filamentous fungi. Several characteristic non-carotenoid pigments produced by filamentous fungi include quinines such as anthraquinones and naphthaquinones, dihydroxy naphthalene melanin (a complex aggregate of polyketide) and flavin compounds like riboflavin. Monascus spp. has been known to produce well known pigments like monascorubrin, rubropunctatin and recently monascusones from yellow Monascus mutant have been identified. Monascorubrin and rubropunctatin have a unique structure responsible for their high-affinity for compounds with primary amino groups (so-called aminophiles). Reactions with amino acids yield the water-soluble red pigments, monascorubramine and rubropunctamine.

In fact, various pigment derivatives with improved functional properties in the colour range of orange-red to violet-red can be produced by Monascus fermentations in the presence of different amino acids. In the case of pigment production from fungi knowledge could be used to engineer improved biosynthetic pathways by selectively controlling the production of pigments and uncoupling their formation from the synthesis of toxins, as has been done in the case of Monascus ruber. Concerns with stability and toxicity of Monascus pigments have led to the identification of non-toxic and non-pathogenic fungi such as Penicillium aculeatum, Penicillium pinophilum, P.purpurogenum and P.funiculosum.

Pigments from Microalgae: As algal cultures are ecofriendly and renewable, there is increasing trend towards using them as a source of natural colours e.g., phycocyanin (blue pigment from Spirulina), ß-carotene from Dunaliella and astaxanthin from Haematococcus. Astaxanthin produced from Haematococcus pluvialis is more stable, has high antioxidant potential and other nutraceuticals properties. Some of the pigments produced by microorganisms are shown in Table 1:

Table 1: Pigments Produced by Microorganisms

Microorganisms	Pigments/Molecule	Colour/Appearance
Bacteria
Agrobacterium aurantiacum	Astaxanthin	Pink-red
Bradyrhizobium sp.	Canthaxanthin	Dark-red
Flavobacterium sp. Paracoccus zeaxanthifaciens	Zeaxanthin	Yellow
Rugamonas rubra	Prodigiosin	Red
Chromobacterium violaceum	Violacein	Purple
Algae
Dunaliella salina	ß-carotene	Red
Chlorococcum	Lutein
Fungi
Blakeslea trispora	ß-carotene, Lycopene	Cream, Red
Fusarium sporotrichioides	Lycopene	Red
Monascus sp.	Monascorubramin Rubropunctatin	Red Orange
Ashbya gossypi	Riboflavin	Yellow
Yeast
Rhodotorula glutinis	Torularhodin	Orange-red

Plant and Insect Sources: The colourants that occur naturally in food plants have been the source of the traditional colourants of raw as well as the processed foods since ancient times. Plant biocolourants are extracts from fruits, vegetables, seeds and roots. Plant pigments, by virtue of their natural occurrence in edible plant are generally considered to be harmless. Nature produces a variety of brilliant coloured pigments viz., water-soluble anthocynanins, betanins and fat-soluble pigments carotenoids and chlorophylls used for colouring foods. Some of the natural colours from plant and insect sources are shown in Table 2: The proportion of different plant parts used for source of biocolourants is shown in Figure 1.

Table 2: Biocolourants from Plant and Insect Sources

Source	Biocolourants	Colour/Appearance
Red beet (Beta vulgaris)	Betalains	Red
Marigold, (Tagetes erecta)	Lutein	Yellow
Bixa orellana	Annatto (bixin and norbixin)	Yellow to orange colour
Reseda luteola	Luteolin	Yellow dye
Dunaliella salina, Euglena, Blakeslea trispora	ß-carotene	Yellow to orange depending upon colour formulations
Taxus baccata	Rhodoxanthin	Orange-yellow
Haematococcus pluvialis	Astaxanthins	Orange pink to red
Tomato	Lycopene	Red
Cape jasmine (Gardenia jasminoides) and red stigmas of saffron (Crocus sativus L.)	Crocin	Yellow
Turmeric plant (Curcuma longa)	Curcumin	Yellow
Cochineal insect (female) Peru	Carminic acid	Orange to red
Cochineal insect (female) Peru	Carmine	Pink to red

Application of Biocolours in Food Industry
Carotenoids have extensive applications as antioxidants in dietary supplements and as colours in foods and beverages as well as pigments in poultry and fish. Colourants containing anthocyanins have been suggested for beverages - in particular fruit drinks - which represent the biggest market of commercial applications in the United States, fruit preparations, jellies, jams, preserves, ice cream, yoghurt, gelatin desserts, canned fruits and so on. Annatto is considered essentially non-toxic. In the United States, it is a natural colourant exempt from certification (regulated by FDA in 21CFR 73.30).

Annatto is especially suited for colouring in dairy products such as cheese, ice cream and yoghurt besides popcorn, confectionery products, snack foods, dry mixes and soft drinks. Betanin is used in the colouration of some sugar confectionery products such as fondants, sugar strands, sugar coatings and fruit or cream fillings, dry mixes, sauces, salad dressings, seasoning bases, soup mixes, instant desserts and snack foods.

Beet juice can be used successfully in hot processed candies if added at the end of the process. In almost every country of the world, cochineal extract and carmine are listed as exempt from certification (US: regulated by FDA in 21CFR 73.100) and permitted as safe colour additive mixtures for colouring foods. Turmeric and saffron find use by developers as natural yellow colourants. ß-carotene has been used in various food products including margarines, beverages, cakes, soups, milk-based products, desserts, candies, salad dressings, and pastas. The potential for a wider application of naturally-derived colourants would seem to be unlimited.

Microbial colours find use not only as colours but also as potential nutraceuticals owing to the health benefits associated with them in being antioxidants, antibiotics, anticancerous, immunosuppressants and so on. Canthaxanthin is used for egg yolk and broiler pigmentation, colourant in food, beverage, pharma and in fish feed and fish. Astaxanthin is used in drinks for lowering cholesterol and medication for autoimmune diseases. Prodigiosin is used as antimalarial, antibacterial activity, anticancerous activity and encapsulated particles used in yoghurt, milk and carbonated drinks. Violacein used in medicine, cosmetics, food and encapsulated pigment applied in yogurt and jelly.

(Rather is research fellow and Masoodi is dean, School of Applied Sciences & Technology, University of Kashmir, and Muzaffar is associate professor, Amar Singh College, Srinagar. They can be contacted at rsajad.mt@gmail.com)