Question

• This is a report about Production of color pigments by biological systems, aiming to decrease emissions of synthetic paint from

  the chemical industry

• Production of pigments. How do we produce color pigment?. Discuss the different methods to produce color pigment. Some methods are Strain development, fermentation, Metabolic engineering of microbes for natural product biosynthesis. Current technology and challenges for example some studies have shown that microorganisms are a promising source for natural colors.

Answer 1

PRODUCTION OF COLOUR PIGMENT BY BIOLOGICAL SOURCES-

Synthetic colors have been widely used in various industries including food, textile, cosmetic and pharmaceuticals. However toxicity problems caused by synthetic pigments have triggered intense research in natural colors and dyes.Organizations like the World Health Organization (WHO), the U.S. Food and Drug Administration (FDA), and the European Food Standards Authority (EFSA) have recommended the safe dosage of artificial colors in food, drug and cosmetic items . However, many synthetic colorants have been banned or being banned due to their hyper-allergenicity, carcinogenicity and other toxicological problems. These adverse effects of synthetic colors have made the scientific community skewed towards natural colors Among the natural sources, pigment producing microorganisms hold a promising potential to meet present day challenges. Furthermore natural colors not only improve the marketability of the product but also add extra features like anti oxidant, anti cancer properties etc.

METHODS-

1.Strain development & fermentation-

Using fermentation tanks for large scale production of pigments & use of strain improving techniques and strain development through random mutagenesis and multiple selection rounds has helped to develop a cost effective and industrially viable production process for pigments and other natural compounds. Strain development is important because the pigments produced by wild type strains are often too low in quantity and take longer fermentation times, making the process uneconomical. Strain improvement is done by common mutagens like 1-methyl-3-nitro-1-nitrosoguanidine (NTG), Ethyl methyl sulfonate (EMS) and Ultraviolet (UV), which can lead to a several-fold increase in pigment production

Fermentation strategy

Advancements in fermentation techniques have lead to the easy production and isolation of color pigments. Microbial pigments can be produced either by solid substrate fermentation or by submerged fermentation. In solid substrate fermentation (SSF), the cultivation of microbial biomass occurs on the surface of a solid substrate. This SSF technique has many potential advantages including savings in wastewater and yield of the metabolites. On the other hand, in submerged fermentation, microorganisms are cultivated in liquid medium aerobically with proper agitation to get homogenous growth of cells and media components. Furthermore researchers investigated the influence of various process parameters such as carbon source, temperature, pH, aeration rate on pigment production as well. However, due to the high cost of using synthetic medium, there is a need to develop new low cost process and extraction procedure for the production of pigments. Efforts are on to utilize the waste organic material for large scale production of microbial pigments. Some studies have focused on production of carotenoids from waste material such as whey, apple pomade and crushed pasta. Such kind of waste utilization procedures not only lower down the production cost but also act as potent waste management tool as well.

2.Cost-Effective Downstreaming-

Developing more cost-effective recovery and separation techniques for microbial pigments are also needed. Large-scale separation and recovery of pigments using conventional methods is costly. Extraction using organic solvents is a complicated and time-consuming process, in which substantial amounts of organic solvents are exhausted while the yield of the high purity product can be extremely low. In addition, using solvents other than water and ethanol can defeat the purpose of obtaining a natural pigment for regulatory purposes, since most organic solvents are not natural. The technique of using non-ionic adsorption resins for an efficient separation and purification has been applied to many nucleic acids, organic acids, peptides, and others. These resins have a high loading ability, thereby helping in recovering of compounds in large quantities. In addition, these resins can directly be used to adsorb compounds from the culture broth. It helps in lowering the cost of separation, by lessening the consumption of extraction solvents and increasing its reusability.

3.Metabolic Enginerring of microbes for natural product biosynthesis-

Cloning of genes responsible for pigment biosynthesis and enabled overproduction of these pigments by gene manipulations. Pigment biosynthetic pathways have been extensively studied and engineered to overproduce a pigment and to change the pigments' molecular structure and color. Blue pigment Actinorhodin, produced by Streptomyces coelicolor, has been genetically manipulated to produce a related bright yellow polyketide known as kalafungin, that is used to produce an antraquinone, which is a reddish-yellow color. Heterologous expression has been used to develop cell factories to efficiently produce pigments by expressing biosynthetic pathways from novel or known pigment producers .Cloning the genes responsible for pigment biosynthesis into microbial vectors, like bacterial or yeast cells, has become a cost-effective and more economical industrial production process. Industrially reliable micro-organisms such as E.coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum, and Pichia pastoris, can be used to developof tailor-made recombinants, genetically engineering the production of pigments .

CHALLANGES

Even though there are many types of natural pigments from various microbial sources, the commercial development of natural pigments as food colorants is challenging. Regulatory hurdles are high for the development of any new compounds for food use, including as a colorant.

The cost of using natural colors is five times more than using synthetic colors, especially when used in confectionary items, where it can be 20 times more expensive.

Substantial quantities of raw materials are required to produce equal quantities of natural colors than synthetic colors. Higher dosages of a natural color are normally needed for the desired hue, thereby increasing the cost.

Microbial pigments have a weaker tinctorial strength and may react on different food matrices, causing undesirable flavors and odors.

Replacement of synthetic colors with natural colors in the food industry is challenging particularly with regard to the relatively low range of natural colors approved for food use.

Deodorization is another issue that arises in natural pigment products as many of the available natural pigments have an odor that is undesired in the food products.

Natural colors are generally more sensitive to light, pH, UV, temperature, oxygen, and heat, leading to color loss caused by fading and a decreased shelf life. Some natural pigments are sensitive to other ambient conditions like metal ions, proteins or organic compounds.

ADDRESSING INSTABILITY OF NATURAL PIGMENTS

To be industrially useful, microbial pigments need to be stable against environmental factors like light, pH, temperature, UV, and food matrices. Many microbial pigments are rendered useless because of their instability against ambient conditions and have short shelf life. There are various techniques available that can produce a more stable natural pigment, which has a higher shelf life and market value in terms of the cost-effective stability measures taken.

Microencapsulation, Nanoemulsions, and the Formation of Nanoformulations

Micro-encapsulation and nano-formulations can be applied to stabilize, improve solubility and deliver natural pigments to food matrices. Natural colors like anthocyanins and carotenoids, have stability issues in various environmental conditions and also present solubility problems in some matrices.

Micro-encapsulation can be defined as packing any solid, gas or liquid in sealed capsules of sizes ranging from millimeters to nanometers. The core or the active compound becomes the packaging material, in this case the microbial pigment and the packaging material, is called the wall or shell material. The wall material used should have emulsifying properties, low viscosity, be biodegradable, should have film forming properties, should resist GIT, be low cost and should show low hygroscopicity. There are various wall materials that are currently used to encapsulate microbial pigments for use as food color such as maltodextrins, modified starch, inulin, furcellaran and others.

Encapsulated colors are easier to handle, have better solubility, and show improved stability to ambient conditions, leading to an increased shelf life. The wall material protects the active core material from light, temperature, oxygen, humidity, and matrix interactions.

Encapsulated microbial pigments, such as anthocyanin, in which maltodextrin has been micro-encapsulated as the wall material, using spray-drying. B-Carotene has been reported to be encapsulated in modified starch as the wall material using freeze drying.

Nano-encapsulation or nano-emulsions are droplet size, 100 nm or less, and can also be prepared to encapsulate microbial pigments. Nano-emulsions contain three constituents, water, oil, and emulsifier. The addition of an emulsifier is the most critical step in forming a nano-emulsion, as it helps to decrease the tension between the water and oil phases of the emulsion. It also stabilizes the nano-emulsion by negating the steric hindrance and repulsive electrostatic interactions. The emulsifiers used are mostly surfactants, but proteins and lipids are also used.

Compared to micro- and macro-emulsions, nano-emulsions have improved applications because of their large surface area per unit, stronger kinetic stability and resistance to any chemical or physical change. Importantly, nano-emulsions, and nano-capsules are small enough to be invisible in solutions and are therefore useful vehicles for the dispersion of poorly water-soluble pigments in aqueous solutions.