FUNGAL PROTEIN PRODUCTION BY SMALL SCALE FERMENTATION TECHNOLOGY USING ASPERGILLUS NIGER

Abstract

Protein is a nitrogenous compound and made up of 20 different amino acids. It is required to all living organisms for their multiplication. On the industry level protein produced by fermentation technology using many microorganisms including fungi and bacteria. In this study isolates Aspergillus niger from soil and protein produced by small scale fermentation process using two different culture medium during various incubation times. Crude protein was harvested by filtration method and estimated via Spectrophotometer using Lowry reagents. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Protein is a nitrogenous compound and made up of 20 different amino acids. It is required to all living organisms for their multiplication. On the industry level protein produced by fermentation technology using many microorganisms including fungi and bacteria. In this study isolates Aspergillus niger from soil and protein produced by small scale fermentation process using two different culture medium during various incubation times. Crude protein was harvested by filtration method and estimated via Spectrophotometer using Lowry reagents.

KEYWORDS: Aspergillus niger, Protein production, Lowry method, Incubation time, Fermentation technology.

Cite this article:

Chouhan Pawan Kumar, Das Prakash (2013), FUNGAL PROTEIN PRODUCTION BY SMALL SCALE FERMENTATION TECHNOLOGY USING ASPERGILLUS NIGER, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 642-647

INTRODUCTION:

Generally fungi are filamentous multicellular achlorophyllus organism grown by tacking nutrients through absorption from dead and living cells. They multiply on surface or submerge condition during fermentation process and produce their metabolites intracellularly as well as extracellularly which are harvested, purified and used as supplements. Similar proteins are produced by A. niger extracellularly and quantitatively estimated by Lowry method (Lowry et al., 1951).

Metz and Kossen (1977) reported that many parameters influence fungal pellet formation including inoculum level, initial pH of medium, agitation, medium composition and use of polymer additives or surface-active agents. Nielsen and Carlsen (1996), Jimenez-Tobon et al. (1997) studied fungal growth and reported that filamentous growth of fungi observed at high initial spore levels whereas increased pellets size were usually formed with reduced inoculum level. Fungi have been used in a variety of industries e.g. food, chemical, detergent, textiles and paper industries for production of protein, enzyme and other products (Moreira et al., 1999 & 2001; Kathiresan and Manivannan, 2006).

Filamentous fungi have been widely used in the fermentation industry as it becomes a principal source of protein, enzymes and other metabolites. Therefore, fungi have been widely investigated by various researchers due to low cost and high productivity which attracted many other researchers to improve fungal strains by molecular techniques and also bioprocess (Finkelstein and Ball, 1992; Banerjee et al., 2003).

Whitaker & Long (1973) and Wainwright et al. (1993) reported that the initial pH of medium plays an important role in fungal morphology as the higher pH values (5.0 to 6.0) produce pellets while low pH values (2.0 to 3.0) leads to filamentous mycelium growth, meant the surface properties of the spores are influenced by pH.

Proteins are nitrogen or amino acid supplements and most of the enzymes are made of proteins. Besides energy source, protein required for fungi, bacteria, actinomycetes and other unicellular and multicellular cells for their membrane and enzyme synthesis. Protein present in cells as lipoprotein, glycoprotein and other forms. Therefore, proteins are produced by fermentation technology and used in a lot of industries such chemical, detergent and food as nutrient supplements for human diet (Moreira et al., 2001). The objectives of our study to isolate fungal strain from soil for protein production in laboratory via small scale fermentation technology using two different types culture. Fungal isolate was inoculated in both production media and incubated for various incubation times at 30°C. Total protein was harvested by filtration method and quantitatively estimated by spectrophotometer using Lowry reagents.

MATERIAL AND METHODS:

Isolation, Purification & Identification of fungi:

Soil samples were collected from Botanical garden of Govt. RGPG College, Mandsaur, Madhya Pradesh, India. All soil samples were mixed together then formed one composite sample. Isolation of fungi was done by serial dilution technique. 0.1 ml sample was transferred from 10-3 dilution to sterilized Petri plates and poured melted Potato dextrose agar (PDA) medium then solidified at room temperature. All plates were incubated at 30°C for 5 days and purified by streak plate method. After purification, fungal cultures were observed on the basis of morphological structures by microscopic method (Fig.-1) and identified using laboratory manual of filamentous soil fungi (Gilman, 1944 and Smith et al., 1983). Identified cultures were transferred to PDA slant and incubated at 30°C for 5 days then stored in refrigerator for further experiments.

Media preparation:

An amount of 34.501 g and 102.2 g of Czapeks Dox medium and Starch peptone medium was weighted into Erlenmeyer conical flasks respectively. One thousand milliliter of glass distilled water was added to conical flasks. The mixtures were poured into conical flasks, 100 ml each and plugged with cotton wool. The cotton wools were covered with brown paper and the conical flasks were autoclaved at 15 lb steam presser for 15 min. The Czapeks Dox medium and Starch peptone medium were cooled at room temperature before use.

Protein production by fermentation technology:

Aspergillus niger spores were obtained from PDA slants using sterilized distilled water and inoculation loop. Spores were inoculated to Czapeks Dox medium and Starch peptone medium respectively (Fig.- 2). All inoculated conical flasks were incubated at 30°C for 3, 6, 9 and 12 days.

Protein estimation by Lowry method:

After incubation time, the Lowry method (Lowry et al., 1951) was used for total protein estimation. Culture filtrates were obtained by filtration method using Whatman filter paper No 1 and then 1 ml culture filtrate of Czapeks Dox medium and Starch peptone medium was mixed with Lowry reagents. The color density was recorded at 750 nm by Spectrophotometer and compared to standard curve of protein (BSA 1mg /ml).

RESULT AND DISCUSSION:

1. Total protein estimation from culture filtrate of Czapex Dox production medium:

After inoculation and incubation, culture filtrates were obtained by filtration method using Whatman No.1 filter paper and 0.1 ml culture filtrate was used for total protein estimation by Lowry method at 750 nm. It is evident from table-1 and Fig.- 3 that 15.4 mg/ml protein was produced by Aspergillus niger within 3 days incubation period and also 2.15 mg/ml protein was recorded in 9 day incubation. Further table- 1 indicates that synthesized protein was used by fungi for their building blocks. Hence, the amount of protein was decreased on 6 and 12 days.

2. Total protein estimation from culture filtrate of Starch peptone production medium:

It is evident from table- 2 and Fig.-4 that 1.23 mg/ml protein was produced by Aspergillus niger within 3 day incubation and also 2.73 mg/ml protein was recorded in 9 day incubation. The amount of protein was recorded maximum on 9 (2.82 mg/ml) day incubation period. Starch peptone medium have starch, a polysaccharide, which was initially not metabolized. Therefore, the growth of Aspergillus niger was slow which affect protein production initially on three day incubation time.

These data are supported by Finkelstein and Ball (1992); Moreira et al. (1999 and 2001); Banerjee et al. (2003) and Karthiresan and Manivannan (2006) which were reported that fungi have been used for their metabolite production. A. niger produced maximum amount of protein (15.4 mg/ml) in Czapex Dox medium while minimum (1.23 mg/ml) in Starch peptone medium within three incubation time. In Czapex Dox medium the amount of protein was decreased with increased incubation time and slightly increased on 12th day while amount of protein was increased till 9th incubation time in starch peptone medium and slightly decreased on 12th day.

The growth of Aspergillus niger was fast in Czapex Dox medium as compared to starch peptone medium due to availability of oligosaccharide instead of polysaccharide as carbon source. Starch is polysaccharide and initially not metabolized, it metabolized after protein (amylase enzyme) synthesis. Therefore, the amount of protein was more in Czapex Dox medium as compared to starch peptone medium.

CONCLUSION:

It is suggested that microbial proteins are easily produced by fermentation technology on small and industrial scale. It is available on low cost for humans, animals as well as other living cells where it completes nutrient requirement of cells. Further it is suggested that Czapex Dox medium was better for protein production by fungi where it produced maximum amount of protein within 3 days incubation time.

References

REFERENCES:

Banerjee AC, Kundu A, Ghosh SK (2003). Genetic manipulation of filamentous fungi. In: Roussos S, editor. New horizons in biotechnology. Dordrecht (Neth)Kluwer Academic Publishers. 193-8.

Finkelstein DB, Ball C (1992). Biotechnology of filamentous fungi. Boston Butterworth-Heinemann. 221 -416.

Gilman JC, (1944). A manual of soil fungi, Revised 2nd edition, Oxford and IBH publishing Co.

Jimenez-Tobon GA, Penninckx MJ, Lejeune R (1997). The relationship between pellet size and production of Mn(II) peroxidase by Phanarochaete chrysosporium in submerged culture. Enzyme Microb. Technol. 21: 537-42.

Kathiresan K, Manivannan S (2006). a-Amylases production by Penicillium fellutanum isolated from mangrove rhizoshere soil. Afr. J. Biotechnol. 5 (10): 829-832.

Lowry OH, Rosbrough NJ, Farr AL, Randall RJ (1951). J. Biol. Chem. 193: 265.

Moreira FG, De Lima FA, Pedrinho SRF, Lenartovicz V, De Souza CGM, RM Peralta CGM (1999). Production of amylases by Aspergillus tamari. Rev. Microbiol. 30: 157-162.

Moreira FG, Lenartovicz V, De Souza CGM, Ramos EP, Peralta RM (2001). The use of a-methyl-D-glucoside, a synthetic analogue of maltose, as inducer of amylase by Aspergillus sp. In solid-state and submerged fermentation. Braz. J. Microbiol. 32: 15-19.

Metz B, Kossen NWF (1977). Biotechnology review: the growth of molds in the form of pellets- A literature review. Biotechnol Bioeng. 19: 781-99.

Nielsen J, Carlsen M (1996). Fungal pellets In: Willaert RG, Baron GV, De Backer L editors. Immobilised living cell systems: modeling and experimental methods. Chichester (NY) John Wiley & Sons. 273-93.

Smith JE, Berry DR, Kristiansen B (1983). Filamentous fungi Vol. IV, Fungal Technology. Edward Arnold. London.

Whitaker A, Long PA (1973). Fungal pelleting. Proc Biochem, 8: 27-31.

Wainwright MP, Trinci APJ, Moore D (1993). Aggregation of spores and biomass of Phanerochaete chrysosporium in liquid culture and the effect of anionic polymers on this process. Mycol Res. 97: 801-6.