Production of microbial colors or pigments

Production of pigments by microorganisms.

1. Production of microbial colors or pigments PRESENTED BY : RITU L V

2. Introduction Color is the most vital attribute of any article particularly food .  Pigments from natural sources have been obtained since long time ago, and their interest has increased due to the toxicity problems caused by the synthetic pigments. In this way the pigments from microbial sources are a good alternative .

3.   The various microorganisms such as  Micrococcus, Bacillus, Rhodotorula , Monascus , Phaffia , Sarcina  and  Achromobacter   have the capability to produce different pigments. These colors have number of beneficial properties like anti-cancerous, immunosuppressive, antibiotic, anti-proliferative, bio-degradability etc.

4. Microbial sources of natural color Microorganisms are the most versatile tools in biotechnology to produce variety of molecules including enzymes, antibiotics, organic acids and pigments . The presence of pigments has been reported among the entire microbial world including Bacteria, Fungi, Yeast, algae & protozoa These microorganisms can be isolated/cultured/purified from various environmental sources such as water bodies, soil, plants, insects and animals.

5. Production 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 submerged fermentation.

6. Fermentation strategy 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. I n submerged fermentation, microorganisms are cultivated in liquid medium aerobically with proper agitation to get homogenous growth of cells and media component. T he influence of various process parameters such as carbon source, temperature, pH, aeration rate on pigment production as well.

7. However, due to the high cos t 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 .

8. Microorganism Pigments Achromobacter Creamy Bacillus sp Brown Brevibacterium sp. Orange, yellow Corynebacterium michigannise Greyish to creamish Pseudomonas sp Yellow Rhodococcus maris Bluish red Streptomyces sp. Yellow, red, blue Microbial pigments and their colour shades 1. Bacteria

9. Microorganism Pigments Aspergillus sp. Orange, red A. glaucus Dark red Blakeslea trispora Cream Helminthosporium catenarium Red colour H. gramineum Red H. cynodontis Bronze colour H. avenae Bronze colour H. catenarin Dark maroon Monascus purpureus Yellow, orange, red P. cyclopium Orange P. nalgeovensis Yellow Microbial pigments and their colour shades 2. Molds

10. Microorganism Pigments Cryptococus sp. Red Phaffia rhodozyma Red Rhodotorula sp . Red Yarrowia lipolytica Brown Microorganism Pigments Dunaliella salina Red Microbial Pigments and their colour shades 3. Yeast Microbial Pigments and their colour shades 4. Algae

11. Monascus purpureus (fungi) The common names of this fungal product are Red Yeast Rice (RYR), Monascus spp. produce many pigments of industrial importance and these pigments are mainly of three types i.e. red colorants, orange colorants and yellowish colorants Monascus pigments are used in traditional oriental foods as a natural colouring agent. These are extracted from M. purpureus grown on steamed rice by solid state fermentation.

12. Monascus fungi, can convert starchy substrates into several metabolites such as alcohols, antibiotic agents, antihypertensives, enzymes, fatty acids, flavor compounds, flocculants, ketones, organic acids, pigments and vitamins . Red yeast rice

13. . Medium (broth) (maltose, yeast extract, glucose, KH2P04, MgS04, CaCh ) Sterilization Medium (yeast extract, sucrose, agar) Inoculation Plates (5 +mm disc) Incubation (28°C for 14 days in darkness) Harvesting of mycelium Mycelium coloured Drying Grinding Extraction with solvent (methanol) Shaking (1 min.) Filtration Drying ( for 24 hrs.)   Pigment P ackaging Flowsheet for the production of Monascus pigment

14. R ed Monascus pigment production  by  Monascus   purpureus   was carried out under submerged fermentation using soybean meals as nitrogen sources to replace yeast extracts.

15. Torularhodin Orange-red Produced by Rhodotorula  spp . (yeast) Antioxidant, Anti-microbial Apple pomace , a waste from apple juice processing industry, which is cheap, rich in sugar, acid and minerals, has been employed in pigment production. Rhodotorula   spp

16. The production of Rhodotorula glutinis by batch fermentation in rotatory shaker has resulted in good yield of carotenoids . The Rhodotorula cells are grown at 30°C for 5 days and, after centrifugation, washed with distilled water . The optimum conditions in the fermenter for the carotenoid production are: fermenter capacity, 1 litre ; temperature, 30°C; air flow rate, 1 minI ; magnetic stirrer, 220 rev/min; and, antifoaming agent, 0.3% w/ v.

17. Phaffia rhodozyma ( yeast) Phaffia rhodozyma ferments glucose and other sugars to produce pigment, astaxanthin . The pigment is produced in shake flask at and an increase in glucose concentration in the medium gave higher yield of pigment rather than growth only.  

18. IN FOOD INDUSTRY β – Carotene production known as pro-vitamin A acts as antioxidant and has potential positive properties against certain diseases Following microbes are mainly using for β –Carotene production : Blakeslea trispora Mucor circinelloides Phycomyces blakesleeanus

19. β-carotene from Blakeslea trispora Inocula of spores of mating types + and − are used in the ratio in a suitable culture medium, such as (in g/L) d -glucose 50, olive oil 5.4, yeast extract 1.0, l -asparagine 2.0, KH2PO4 1.5, MgSO4 ·7H2O 0.5, BHT 0.02, and thiamine-HCl 0.005. After 8 days in aerated culture with moderate agitation at 26 °C, the cells are separated from the broth by filtration, partially dehydrated and slowly extracted with a suitable solvent such as chloroform . The solvent is separated from the biomass and evaporated; the dry raw extract is further processed. From 62 kg of corn steep liquor and 38 kg of glucose, there should be produced 20 kg of biomass which, extracted with 50 L chloroform, would give approximately and 8 kg extract containing 90 g of β-carotene .

20. β – Carotene Blakeslea trispora Mucor circinelloides

21. Riboflavin (Vitamin B2) Production yellow food colourant used mainly for cereal-based products . Applications - limited due to its bitter taste and naturally slight odour . many microorganisms have potential to produce riboflavin by fermentation . Fermentation with Ashbyagossypi . Corn steep liquor, peptone, soybean oil as culture medium Ashbya gossypi

22. S.No . Producer microorganism Amount produced Examples 1 Weak overproducer 100 mg/L or less Clostridium acetobutylicum 2 Moderate overproducer Upto 600mg/L Candidaguilliermundiior Debaryomycessubglobosus 3 Strong overproducer Over 1g/L Eremotheciumashbyii and Ashbyagossypi Different level of Riboflavin production from microbes

23. Lycopene Production red open-chain unsaturated carotenoid also known as psi-carotene very sensitive to heat and oxidation and is insoluble in water Genetically modified fungus Fusarium sporotrichioides was used by Jones et al to manufacture the colourant and antioxidant lycopene. They used the cheap corn fiber material as the substrate. Cultures in lab flasks produced 0.5 mg (lycopene)/g ofdry mass within 6 days and such a production will be increased within the next years

24. In parmaceutical industry Prodigiosin : potential pigment having many pharmacological properties . broad range of cytotoxic activity is produced by Vibrio psychroerythrus , Serratia marcescens, Pseudomonas magnesiorubra , and other eubacteria as Maltose and peanut oil based , Powdered peanut medium , Sucrose and peptone based culture medium.

25. Violacein violet pigment violacein is an indole derivative isolated mainly from bacteria Chromobacterium violaceum , which exhibits important antitumoural , antiparasitary , antiprotozoan , anticancer, antiviral, antibacterial and antioxidant activities.

26. Factor affecting Microbial pigment production Temperature Depends on the type of microorganism The growth of Monascus spp. requires 25-28˚C , Pseudomonas requires 35-36˚C for its growth and pigment production .

27. pH pH of the medium affects the growth of the microbe and type of pigment production. Optimum pH for Monascus sp. – 5.5-6.5 Rhodotorula is 4.0- 4.5 . Neutral to slight alkaline pH favours lycopene formation acidic pH favours β-Carotene synthesis

28. Carbon source The mycelial growth of pigment producing microorganism is affected by the type of carbon sources like glucose, fructose, maltose, lactose, galactose, etc.

29. Type of Fermentation The solid state fermentation yields 3 fold more pigment than submerged fermentation In M. purpureus , yields are superior in solid cultures than submerged , though media composition, pH and agitation also affect pigment production

30. 5. Minerals 6. Nitrogen source Ammonium chloride is the best for production of Monascus pigment followed by ammonium nitrate and then glutamate. Potassium nitrate is the poorest nitrogen source, while glutamate proved outstanding for the pigment production. 7 .Moisture content In solid state fermentation, the higher level of pigment production by Monascusruber occurs at the 70% initial moisture level in substrate i.e. rice 8. Aeration rate

31. APPLICATIONS

32. Application Pharmaceutical industry:  T reating various diseases. These have many properties like antibiotic, anticancer and immune-suppressive properties.   Adonirubin  and astaxanthin act as nutraceuticals , prevent carcinogenesis, prevent problems like heart attacks and strokes. Monascus purpureus   produce pigments which help in the inhibition of hepatitis virus replication by interfering with viral RNA polymerase activity. P rodigiosins are known for their immune suppressive anticancer properties. Dairy industry:   Monascus species  are known to produce nontoxic pigments, which can be used as food colorants, flavour enhancers and as a food preservative .  Monascus ruber  is used to prepare flavoured milk by utilizing rice carbohydrate for its metabolism and produces pigment as a secondary metabolite.

33. Textile industry: These synthetic dyes are used in industries, due to their easy and cheap synthesis, stability towards light, temperature and advanced colors covering whole color spectrum. However, these synthetic dyes have many drawbacks like toxicity, mutagenicity and carcinogenicity properties leading to various health problems like skin cancer and allergies. Hence, consumers demand for dyes of natural origin as colorants.

34. Nutritional supplements - β- carotene , Riboflavin , Yellow β- xanthins (  essential dietary amino acids) Food colorants :  An important goal of food industry is to produce food with an attractive appearance. Food producers are opting for natural food colors, as artificial ones shows many negative impacts on health when consumed. Penicillium oxalicum   is a fungus which produces red color used in cosmetic and food industry. These colors are useful in different products like baby foods, breakfast cereals, pastas, sauces, processed cheese, fruit drinks, vitamin-enriched milk products, and some energy drinks, as probiotcs and so on .

35. S.No Name of dye Source Application 1 Prodigiosin Vibrio spp. Wool, nylon, acrylics and silk 2 Red Prodigiosin Serratia marcescens Acrylics, polyester, polyester microfiber, silk and cotton 3 Pigments from Janthinobacterium lividum Silk, cotton and wool (bluish-purple, all natural fibers) and nylon and vinylon (both synthetic fibers) 4 Violacein (violet pigment) Chromobacterium violaceum Pure cotton, pure silk, pure rayon, jacquard rayon, acrylic, cotton, silk stain and polyster 5 Pink pigment Roseomonas fauriae Cotton fabrics 6 Pigments from Fusarium oxosporum , Trichoderma viride and Alternaria spp Cellulosic fibers 7 Anthraquinone Dermocybe sanguine Wool fibers 8 Pigment from Trichoderma spp Wool and silk fibers Sources and uses of dyes in textile industry

36. CONCLUSION Microbial pigments are not only used as food colorant, flavoring agent and dying agents they are widely applied in medicinal aspects . lower blood cholesterol concentration, Anti-Diabetic Activity, Anti-Inflammation etc.

37. References http://www.ijpcbs.com/files/volume5-1-2015/24.pdf https://ijpsr.com/bft-article/microbial-pigments-as-a-natural-color-a-review/?view=fulltext https://www.frontiersin.org/articles/10.3389/fnut.2019.00007/full https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519520/ https://www.researchgate.net/profile/Shashi_Bhushan6/publication/228730339_Microbial_Pigments/links/0fcfd5090a422deb9b000000/Microbial-Pigments.pdf?origin=publication_detail