Study on Natural Microbial Pigment

Pigments are important additives in food, medicine, and feed, and mainly include synthetic and natural pigments. Synthetic pigments are mostly made from chemical products such as benzene, toluene, and naphthalene, and are formed through a series of organic reactions such as sulfonation, nitration, halogenation, and nitration [1. They have the advantages of good stability, high color strength, being economical, and easy to use. However, with the continuous development of toxicological research and analytical techniques, it has been found that synthetic pigments are toxic and carcinogenic[2] and can also lead to decreased fertility and teratogenicity[3]. Therefore, the safety of synthetic pigments is becoming a growing concern, and their use is gradually being strictly restricted.

Compared with synthetic pigments, natural pigments have the following advantages [34]: (1) Most natural pigments are highly safe and have no toxic side effects; (2) many natural pigments retain many active substances (such as small molecule active peptides, amino acids, vitamins, aromatic substances and certain essential elements) or are themselves a kind of nutrient. For example, riboflavin is a kind of vitamin itself, and β-carotene has the activity of vitamins and and has certain nutritional value and health functions. (3) Some natural pigments also have certain pharmacological functions and can prevent and treat certain diseases. For example, carotene has therapeutic effects on cardiovascular disease and tumors; anthocyanins have been made into an oral liquid abroad that can resist radiation and treat eye strain; and tea pigments have been used in clinical settings. [5; (4) Natural pigments provide a more natural coloring, closer to the colors of natural substances. It can be seen that natural pigments have become a research hotspot, and the development and utilization of natural pigments has become the general trend of development in the pigment industry.

Natural pigments are widely found in various organisms, including plant pigments, animal pigments, and microbial pigments. There are two constraints to extracting pigments from plants and animals: (1) the growth and reproduction of plants and animals are affected by various factors such as season, climate, and place of origin, and their resources are limited; (2) the content of the target product in the raw material is low, the production process is complex, the extraction rate is low, and the cost is high. Many microorganisms in nature can produce pigments of various colors during normal metabolic processes. Microorganisms are diverse and widespread; they have many metabolic types and strong metabolic abilities; they grow and reproduce quickly and are easy to cultivate. Microorganisms can easily produce pigments through large-scale cultivation and fermentation, and are not limited by resources, environment or space. They are an effective way to obtain target pigments efficiently and at low cost. Therefore, the development of microbial pigments has incomparable advantages over plant pigments and animal pigments, and is therefore attracting more and more attention.

1 Research progress of major microbial pigments

Microbial pigments include red, orange, yellow, green, blue, purple, brown, black, and various colors in between.

The main microbial pigments are red yeast rice pigment, β-carotene, melanin, and indigo.

1.1 Red yeast rice pigment

Red yeast rice pigment is a general term for a series of pigments produced by the filamentous fungus Monascus during its metabolic process. It is a polyketide compound that It can be divided into three categories according to chemical structure and properties: red pigments Monascorubramine and Rubropunctamine, orange pigments Monascorubin and Rubropunctatin, and yellow pigments Ankaflavin and Monascin. Monascus pigments are stable to alkalis, heat-resistant, unaffected by metal ions, oxidants and reducing agents, safe and have good coloring properties. At the same time, red yeast rice pigment has the advantages of many natural pigments and is mainly used for coloring meat products, flour products, as well as beverages and seasonings. It is currently the only food coloring in the world produced by microbial fermentation.

Red yeast rice pigment has been used in the food industry for more than a thousand years. However, its coloring effect is limited because it is unstable to light. Some scholars have added antioxidants to red yeast rice pigment, but its light stability has not been improved. Other scholars have proposed adding bioactive substances such as amino acids and polypeptides during the fermentation process, so that when the red yeast rice secretes polyketide enzymes, the color-protecting substances are bound to the pigment structure, thereby improving the light stability of red yeast rice pigment. At the same time, some scholars believe that the organic solvent extraction method for red yeast rice pigment extracts a small amount of pigment with low purity. Therefore, a large amount of research has been carried out on improving the yield of red yeast rice pigment. Minghongmei 6 and others used a microwave-assisted method to increase the yield of red yeast rice pigment by 72.2%. Yang Chenglong [7 and others used an ultrasonic-assisted extraction method to extract red yeast rice pigment from the liquid fermentation of red yeast rice, which causes less structural damage to the active ingredients and is easy to operate. Rosa M8 and others successfully extracted red yeast rice pigment through deep fermentation of wheat substrates; Donghua Jiang [9 and others used high-γ-aminobutyric acid to produce red yeast rice.

With further research into the water solubility and light resistance of red yeast rice pigment, the range of applications for red yeast rice pigment will become even wider.

1.2 β-Carotene

β-Carotene is one of many carotenoids and is a precursor of vitamin A, also known as a provitamin. It is a type of xanthophyll pigment, a highly unsaturated compound that is insoluble in water but soluble in organic solvents. It contains a series of conjugated double bonds and methyl branches. Its dilute solution is orange-yellow to yellow in color, and the color of the pigment varies with the number of conjugated double bonds. Carotenoids have health benefits and are the most promising microbial food coloring additives. Beta-carotene is widely found in plants, algae and fungi.

Currently, there are two main methods for extracting β-carotene: one is to extract β-carotene by culturing salt algae, but this method requires high salt content in the sea and has great limitations in production; the other is to use microbial fermentation to produce β-carotene. Currently, the main microorganisms that can ferment to produce β-carotene are fungi, bacteria, and yeasts [10]. Tang Tang [1 believes that the red yeast strain has low nutritional requirements, fast metabolism, a short fermentation cycle, and the bacteria themselves are nutrient-rich, etc., making it suitable for high-density cultivation in fermenters and easy to industrialize. Eon Seon Jin [121, etc. used the green algae Dunaliella salina to produce carotenoids. However, this method ultimately did not achieve industrial production due to expensive fermentation facilities and long toxicological research times.

With the development of microbial fermentation technology, the demand for carotenoid products produced by microbial methods is increasing year by year, which indicates that the use of microbial methods to produce β-carotene will have a good future.

1.3 Black Coloring

Black coloring is a biopigment formed by a series of chemical reactions of tyrosine, which ranges from brown to black and usually exists in a polymerized form. It is insoluble in acidic solutions and common organic solvents, soluble in alkaline solutions, and slightly soluble in water [13]. Melanin is widely found in animals, plants and microorganisms. Natural black pigment is extracted from animal and plant species, mainly based on its property of dissolving in alkaline solutions and precipitating in acidic solutions. The extraction rate is low and the color of black is not good, which is costly and not easy to produce industrially. However, the production of black pigment using microbial methods is not restricted by geographical or seasonal conditions, and is more conducive to industrial production. The black color-producing microorganisms mainly include bacteria such as bacilli, pseudomonads, and nitrogen-fixing bacteria, as well as some molds and actinomycetes. The black pigment produced by microorganisms is mainly divided into wall (membrane)-bound melanin and extracellular melanin. For example, Aspergillus niger secretes melanin while forming spores. RCR Gon alves[14 and others successfully extracted melanin using Aspergillus niger. Dong-Seok Kim[15 increased the yield of melanin by regulating the temperature.

Black coloring is currently mainly used in cosmetics or hair dyes for decorative purposes, protection against ultraviolet radiation, scavenging of free radicals, and as a photoprotectant in biopesticides. In addition, it has been found in recent years that some soluble melanins have a significant inhibitory effect on the AIDS virus in vitro. Therefore, with the rapid development of biotechnology, the microbial extraction of melanin has broad application prospects.

1.4 Indigo

Indigo is a bright, vivid and durable blue dye that is insoluble in water, alcohol and ether, but soluble in chloroform, nitrobenzene and aniline. It is one of the earliest discovered natural pigments and is widely used in the dyeing, pharmaceutical and food industries. Its traditional production method is to extract it from plants in the genus Indigo, and after the chemical structure was elucidated, it was chemically synthesized using aniline and sodium amide as raw materials. Studies have shown that aniline is a potential carcinogen. The microbial synthesis of indigo not only facilitates the modification of the production process for indigo, reduces costs, and reduces energy consumption, but also promotes research on the microbial degradation of aromatic compounds and also finds new ways to develop microbial synthetic dyes.

Many microbial species and strains capable of synthesizing indigo have been isolated and identified, and many of these strains are bacteria that can degrade aromatic hydrocarbons. For example, the P. putida strain PpG7, which uses naphthalene as a carbon source [161], S. monacensis, which uses 1,2,3,4-tetrahydronaphthalene as a carbon source [17], P. putida mt-2, which uses toluene-xylene or other derivatives of toluene as a carbon source [18], the toluene-degrading strain of Pseudomonas mandelssohnii KRI[19] and the styrene-degrading strains of Pseudomonas putida S12 and CA-3[20].

The synthesis of indigo pigments is the result of tryptophan metabolism catalyzed by tryptophan hydrolase and a class of dioxygenases that can catalyze aromatic compounds, mainly monooxygenases and dioxygenases, which can add a single or double oxygen atom to an indole or indole derivative molecule, respectively. The naphthalene dioxygenase is one of the most studied enzymes in indigo biosynthesis. The cloning of the naphthalene dioxygenase gene in a bacterial strain enabled the rapid and efficient synthesis of indigo from indole. However, the by-product indirubin hindered the industrial production of indigo from indole. This problem has been solved by cloning the styrene monooxygenase gene from Pseudomonas fluorescens, which only catalyzes the synthesis of 3-hydroxyindole.

The microbial indigo synthesis system still needs to be further studied and developed, such as how to construct and select a reasonable enzyme system and efficient engineered strains, optimize fermentation parameters, indigo extraction or extraction procedures, etc., so as to greatly reduce production costs and improve production efficiency.

2 Main problems with microbial pigments

At present, the number of microbial pigments discovered far exceeds the number of known plant pigments, but the red pigment produced by red yeast rice is the only pigment in the world that is used in the food industry.

2.1 Stability of the properties of natural pigments

Pigments are susceptible to a variety of physical and chemical factors during the extraction, processing and application, such as light, temperature, oxidants and reductants, pH, polar media, metal ions and various additives, which can cause changes in hue, absorbance and physiological function.

2.2 Pigments are accompanied by the production of toxins

Pigments are a kind of secondary metabolite produced by microorganisms. They are generally synthesized at the later stage of bacterial growth. However, the production of pigments by microorganisms is often accompanied by the production of toxins, which are secreted into the extracellular space or remain within the cell. This often makes the fermentation broth complex in composition, and the process of purifying pigments is highly demanding. It is difficult to obtain a relatively pure pigment product using conventional methods.

2.3 Lack of good strains

The pigment yield of wild strains is generally low. As production bacteria, they not only increase the difficulty of purification, but also increase product investment and reduce profits. Therefore, it is necessary to screen for strains with excellent properties such as strong reproductive ability, easy cultivation, production of large amounts of fermentation products in a short period of time, no or low production of by-products, and stable genetic characteristics.

2.4 Safety testing

As a newly developed microbial pigment, it not only has high purity requirements, but also high safety requirements. Before it is approved, there must be sufficient evidence to prove that the pigment does not contain toxins. It has been proven to be non-toxic through acute toxicity tests, safety toxicity tests and chronic toxicity tests, and it has no mutagenic effect. In addition, it should also have multiple physiological functions such as lowering blood lipids and blood pressure, anti-mutagenic, antiseptic and preservation.

2.5 Optimization of culture conditions

Microorganisms have different cell biomass and pigment yields in different media and culture conditions. Nutrients such as carbon and nitrogen sources in the culture medium provide the material basis for the growth of microorganisms and the accumulation of pigments, and determine the direction of pigment synthesis. BuzziniPl21], Chen Jianjun[22], Zeng Qiangsong[23], Hu Aihong24], Salguero A[25], Fbregas J[26 The results show that the optimal culture medium and culture conditions can increase the yield of microbial pigments.

2.6 Optimization of purification methods

The main extraction methods for natural pigments are aqueous extraction, organic solvent extraction, alkaline extraction, acid extraction, supercritical CO2 extraction, and microwave extraction. The water extraction method is the simplest method, requiring no special equipment and being easy to operate. However, the extraction efficiency is low and the process is time-consuming. The organic solvent extraction method uses cheap extraction agents, simple equipment and easy-to-follow operating procedures, and has a high extraction rate. However, the quality of some products extracted using this method is poor, with low purity and an unpleasant smell or solvent residue, which affects the scope of application of the product.

The main extraction efficiency of acid and alkali extraction methods is not as high as that of organic solvent extraction, and the processing process consumes a large amount of acid and alkali, and the waste liquid is difficult to recycle. Supercritical carbon dioxide fluid extraction has the advantages of a simple extraction process, low energy consumption, cheap extraction agents, high purity of the extracted product, low solvent residue, and no toxic side effects, and is therefore attracting more and more attention. However, due to problems such as imperfect technology, complex and expensive equipment, and high operating costs, the development and application of this extraction method in this field has been limited. Microwave extraction has the advantages of being able to extract multiple sample components in a short time, using a small amount of solvent, and having good reproducibility. It has broad prospects for application research. However, it is currently limited to laboratory research, and its scope of application has been somewhat affected. Therefore, in order to improve the extraction efficiency of natural microbial pigments and maintain the effective pigment activity, the extraction method still needs to be further studied.

3 Development trends of microbial pigments

3.1 Selection and breeding of excellent strains

Microorganisms that produce pigments and are isolated and selected from nature have low pigment yields and unstable properties. On the other hand, the production of pigments is often accompanied by the production of other metabolites such as toxins. Therefore, the use of genetic engineering techniques to modify wild strains is an effective way to select and breed excellent strains. For example, the genome of a microbial pigment with high application value can be cloned and transferred to an E. coli that does not produce pigments, so that it can efficiently express pigments and obtain a large amount of pigment products.

3.2 Improvement of fermentation technology

The traditional fermentation method for microbial pigments is low in production efficiency, labor-intensive, and quality is controlled. The immobilized cell technology has the advantages of high cell density, fast reaction rate, good stability, long service life, reusability, and ease of product separation. Therefore, the use of cell immobilization technology, which combines the advantages of solid-state fermentation and liquid deep fermentation, can effectively increase the yield and color value of the pigment. The fermentation method after immobilization can be optimized, and the cells can be immobilized in liquid fermentation under optimal conditions to increase the yield of the pigment.

3.3 Research on the metabolic mechanism of pigments

The relevant gene composition and regulatory elements of the metabolic synthesis process of microbial pigments in living organisms are sought, and the unknown links of pigment metabolism are studied in depth unknown links, and explore potential regulatory methods to provide a theoretical basis for revealing its pigment synthesis mechanism.

4 Conclusion

With the improvement of people's living standards and the deepening of research, it has gradually been discovered that natural pigments have multiple biological effects such as improving human immunity, antibiosis, and antitumor. Their development and utilization have attracted more and more widespread attention, so research on natural pigments has made rapid progress in recent years. However, natural pigments derived from animal and plant sources are difficult to produce in large quantities due to limitations in conditions and yields. The microbial fermentation method for producing natural pigments has advantages in terms of quality, technology, resources and cost. Red yeast rice (Blakeslea trispora) is currently the only high-yield filamentous fungus that can achieve industrial production of natural pigments from microbial sources. Further research is needed to open up the use of other microbial natural pigments.

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