Production of Single Cell Protein from Orange Peels Using Aspergillus niger and Saccharomyces cerevisiae

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1 Global Journal of Biotechnology & Biochemistry 9 (1): 14-18, 2014 ISSN X IDOSI Publications, 2014 DOI: /idosi.gjbb Production of Single Cell Protein from Orange Peels Using Aspergillus niger and Saccharomyces cerevisiae Sadiq Azam, Zeeshan Khan, Bashir Ahmad, Ibrar Khan and Javid Ali 1 Center of Biotechnology and Microbiology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan 2 Pakistan Council of Scientific and Industrial Research Laboratories Complex, Peshawar, Khyber Pakhtunkhwa, Pakistan Abstract: The bioconversion of fruit wastes into certain valuable products like single cell protein (SCP) has the ability to solve the worldwide food protein deficiency by obtaining an economical product for food and feed. However using wastes as substrate for the production of high nutritious product may also alleviate environmental pollutant up to some extent. In the light of this, an attempt was made in this study by selecting Aspergillus niger and Saccharomyces cerevisiae for the production of SCP. The orange peels were used as sole carbon source for preparation of fermentation media on which the two selected fungal strains were grown. A. niger biomass was produced by solid state fermentation while the S. cerevisiae biomass was obtained by submerged fermentation. The present research work can helps in SCP production from inexpensive and cheap agro waste material. Key words: Aspergillus niger Saccharomyces cerevisiae Citrus aurantium Citrus sinensis Citrus paradisi Single cell protein INTRODUCTION bacteria, developed on substrates containing carbon. This technique has been in vogue in the contemporary Proteins are mainly called as building blocks of all world at the commercial level [4]. living organisms. Proteins are necessary for the growth Pakistan is one of the major producers of different and development of living organisms and carry out a fruits and vegetables. The country is now an important number of different biochemical reactions in the form of global citrus producer and contributing fulfilling its enzyme. As compared to other macromolecules, our body demand by exporting it as well. Apart from that these requires abundant quantity of proteins and its destruction fruits are also utilized by different industries for producing is sometime lethal [1]. different industrial products and discard orange peels Though post-modern world is replaced from the [5].These discarded peels can cause pollution which can modern but still poverty is rampant in the world. The data affect the environment and also results various infectious collected by the Food and agriculture organization, UN diseases due to growth of microorganisms. These fruit (FAO), 25% of the world population has protein wastes contain carbohydrates that are natural substrates deficiency, which is a glaring example of protein gap. for microorganisms. So by utilizing these cheap available To rectify this deficiency, microbial proteins can be substrates for the production of SCP will help in reducing utilized to provide an alternative of protein to the pollutant up to certain extent [6, 7]. economically marginalized segments of the societies [2]. For the production of SCP a wide variety of For this purpose SCP was developed to produce microorganisms are used. Using algae for the SCP microbial proteins [3]. These are the dead, dry cells of production it was revealed that it has high protein content microorganisms like yeast, fungi, algae and as compared to other microorganisms and can be easily Corresponding Author: Sadiq Azam, Center of Biotechnology and Microbiology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan. 14

2 harvested, but apart from these merits such proteins are chances of toxin production by A. niger. As a result some indigestible due to cellulosic wall and high concentration of proteins were also digested into small peptides and of metals [8, 9]. make it fit for digestion. Mixture of each fungal biomass A number of different types of agro-industrial was then filtered through filter paper (Whattsman filter wastes, fruits and wooden pulps are used for growing paper). For drying these cells adhered with filter paper, yeast. Such cells are larger in size but similar to algae cells were transferred to aluminum foil and were kept at these cells are also poorly digestible. Temperature 50 C to remove all moisture contents. These cells were required for growing yeast cells ranges C and ph under regular check to avoid destruction of protein [19] Similar to yeast cells, bacteria can also be grown on wide range of different substrates [10, 11]. Analysis of Fungal Biomass: The fungal biomass of each Throughout the world a large number of different batch was analyzed for its ash, crude fiber, crude fat researches are going on in which low value products such and protein contents using the procedure mentioned in as agro-industrial wastes are utilized and are successfully AOAC methods [17]. converted into products of high nutritious value [12-15]. RESULTS AND DISCUSSION MATERIALS AND METHODS Ash Contents: Ash content of C. aurantium, C. sinensis Collection of Orange Peels and Microbial Culture: and C. paradisi peels were 4.4, 4.8 and 4.0%, respectively, Orange peels (Citrus aurantium, Citrus sinensis and as presented in figure 1. Ash content reflects the presence Citrus paradisi) were collected from local market of of organic matter in orange peels. As organic compounds District Peshawar in sterile bottles. By gently washing are natural substrate for microorganisms so it will lower these peels with distilled water, removed dust particles. its content up to certain level. The peels were shade dried and crushed into fine powder The ash contents of the fungal biomass obtained in in an electric grinder [16]. three different batches were also determined. The results A. niger and S. cerevisiae were obtained from revealed that the ash contents were decreased as shown Microbial Type Culture Collections (MTCC). in Figure 1. The cultures were maintained on slant of Potato In batch 1 the ash contents of A. niger was 3.62, 4.00 Dextrose Agar medium (Potato infusion 200g, Dextrose and 4.20% for C. paradisi, C. aurantium and C. sinensis, 20g, Agar 20g, Distilled water 1000ml) and stored at 4 C. respectively. Similarly the ash content of S. cerevisiae was 3.60, 3.80 and 3.80% for C. sinensis, C. paradisi and Sample preparation for S. cerevisiae: S. cerevisiae lacks C. aurantium, respectively. In batch 2, ash content of the enzyme system which hydrolyzes the polysaccharides into monosaccharide. The peels were treated with 30% HCl and were kept in water bath at 75 C for one hour to obtain monosaccharides [16]. Proximate Analysis of the Substrate: The ash, crude fiber, crude fat and protein contents of the substrate was determined by AOAC methods [17]. Production and harvesting of SCP: Orange peels were inoculated with A. niger and S. cerevisiae and were incubated at various temperature and duration; 32 C for 8 days (Batch I), 30 C for 6 days (Batch II) and 28 C for 4 days other required conditions for the growth of these microorganisms were also provided. Each batch was observed on regular basis to avoid contamination [18].After production the A. niger and S. cerevisiae biomass was autoclaved at 121 C for 20 minutes [18]. Autoclavation of fungal biomass were done to reduce A. niger biomass was 3.38, 3.6 and 3.6% for C. sinensis, C. paradisi and C. aurantium, respectively, while ash content of S. cerevisiae biomass was 2.80, 3.0 and 3.2% for C. sinensis, C. paradisi and C. aurantium respectively (Figure 1). Ash content of batch 3 was also determined the results showed that, for A. niger biomass it was 3.80, 4.00 and 4.20% for C. paradisi, C. sinensis and C. aurantium respectively, while for S. cerevisiae biomass it was 3.00, 3.20 and 3.40% for C. paradisi, C. aurantium and C. sinensis, respectively (Figure 1). Crude Fat Contents: Crude fat content of citrus peels was determined and shown in Figure 2. The results revealed that all the samples showed a very low level of fat contents; 1.0% (C. paradisi), 1.5% (C. aurantium) and 1.5% (C. sinensis). As SCP must have low content of fats therefore by comparing crude fat contents of each batch it was found that A. niger biomass obtained in batch 2 15

3 Fig. 1: Percent ash contents of citrus peels, A. niger and S. cerevisiae biomass of batch 1-3. Fig. 2: Percent crude fate contents of citrus peels, A. niger and S. cerevisiae biomass of batch 1-3. have lowest fat contents; 0.50, 0.50 and 0.75% for biomass produced in batch 2 showed lowest crude fiber C. sinensis, C. aurantium and C. paradisi, respectively contents; 1.85% (C. paradisi), 1.85% (C. aurantium) and while in case of S. cerevisiae biomass, the fat contents 2.0% (C. sinensis) while S. cerevisiae biomass also were recorded as; 0.65, 0.75 and 0.80% for C. paradisi, showed low fat contents; 2.0, 2.1 and 2.2% for C. sinensis, C. aurantium and C. sinensis respectively. For batch 1, C. paradisi and C. aurantium, respectively. The other the fat content of A. niger biomass was 0.60, 0.75 and two batches showed relatively high crude fat contents as 1.00% for C. paradisi, C. aurantium and C. sinensis, shown in Figure 3. respectively. Similarly low fat contents were shown by the S. cerevisiae biomass; 0.75% (C. paradisi) 1.00% Protein Contents: Single Cell Proteins are recommended (C. aurantium) and 1.00% (C. sinensis). In batch 3 the as protein source, but like other organisms it also contains A. niger biomass the percent fate contents were; other biological macromolecules, like carbohydrate, lipids, 0.75% (C. paradisi), 0.85% (C. sinensis) and 0.95% nucleic acids and vitamins. However these (C. aurantium) while in S. cerevisiae biomass the macromolecules are given little importance by the percent fat contents recorded were; 0.65, 0.70 and 0.80% nutritionists. Results obtained for protein contents of for C. paradisi, C. sinensis and C. aurantium, each citrus peel and fungal biomass are presented in respectively. figures 4. The C. aurantium showed highest protein content (13.37%) while in rest of the citrus peels the Crude Fiber Contents: As the fiber contents of fruits are protein contents were recorded as 11.9 and 11.53% for high, the results obtained (figure 3) showed that high C. paradisi and C. sinensis, respectively. The protein crude fiber content was recorded for C. paradisi (2.95%). contents of each fungal batch were also determined. The remaining citrus peels also showed high level of fiber In case of A. niger it is revealed that highest protein contents; 2.85% (C. aurantium)and 2.65% (C. sinensis). contents were obtained in batch 2 that is 29.75% for The fungal biomass showed a low level of crude fiber C. aurantium, 27.15% for C. sinensis and 29.00% for contents as compared to the citrus peels. A. niger C. paradisi. Similarly the S. cerevisiae biomass of 16

4 Fig. 3: Percent crude fiber contents of citrus peels, A. niger and S. cerevisiae biomass of batch 1-3. Fig. 4: Percent protein contents of citrus peels, A. niger and S. cerevisiae biomass of batch 1-3. the same batch, also showed highest protein contents; aurantium, C. sinensis and C. paradisi which was 13.37, 23.50% (C. paradisi).22.50% (C. sinensis) and 22.06% and 11.99% for its original peels. (C. aurantium).the A. niger biomass of batch 1 also For S. cerevisiae protein contents were increased showed high protein contents as compared to citrus to 22.06, and 23.50% from 13.37, and 11.99% for peels; (C. aurantium), 21.56% (C. paradisi) C. aurantium, C. sinensis and C. paradisi, respectively. and 20.63% (C. sinensis). The protein contents of the Such increase in protein contents is due to the availability S. cerevisiae biomass were recorded as 20.37% of high monosaccharide for A. niger as compared to S. (C. paradisi), 18.68% (C. sinensis) and 18.0% (C. cerevisiae. Apart from it SCP had shown a remarkable aurantium). In batch 3 for A. niger biomass, the percent decrease in crude fat, crude fiber and ash content. protein contents were; 26.81% (C. paradisi), 26.37% (C. sinensis) and 25.37% (C. aurantium) while in S. cerevisiae biomass the percent protein contents REFERENCES recorded were; 21.06, and 17.75% for C. paradisi, C. sinensis and C. aurantium, respectively. 1. Solomons, G.L., Single Cell Protein. CRC Critical Reviews in Biotechnology; CRC Press: Boca Raton, CONCLUSION USA. 2. Najafpur, G.D., Single Cell Protein. Regarding SCP nutritionist are more concerned with Biotechnology advances. Biochemical its protein contents as compared to other characteristics. Engineeringand Biotechnology Advances, By comparing different batches of each inoculated sample pp: of both microorganisms, it was found that in case of A. 3. Riviere, J., In Industrial Applications of niger biomass it had shown a tremendous increase in its Microbiology. Microbial Proteins. Surrey University protein contents that is 29.75, and 29.0% for C. Press, Chap., 4:

5 4. Jamel, P., M.Z. Alam and N. Umi, Media 12. Khan, M.Y., To determine protein content of optimization for bio proteins production from single cell protein produced by using various cheaper carbon source, Journal of Engineering, combinations of fruit wastes in the production of Science and Technology, 3(2): SCP by using two standard food fungi A. oryzae and 5. Barton, A.F.M., Industrial and Agricultural R. oligospora. International Journal of Advanced Recycling Processing In: Resources Recovery Biotechnology and Research, 3(1): andrecycling, John Wiley and Sons. New York. 13. Jaganmohan, P., B.P. Daas and S.V. Prasad, Callihan, C.D. and J.E. Clemmer, In Microbial Production of Single Cell Protein with Aspergillus Biomass. Economic Microbiology, Biomass from terreus Using Solid State Fermentation. European Cellulosic Materials; Academic Press: London, Journal of Biological Sciences, 5(2): pp: Ravi, N.S., V.K. Kumar, R. Shailaja, K. Saritha and 7. Bacha, U., M. Nasir, A. Khalique, A. Anjum and N.V. Naidu, Single cell proteins production by M.A. Jabbar, Comparative assessment of Trichoderma harzianum using waste Banana peel. various agro-industrial wastes for Saccharomyces International Journal of Microbiological Research, cerevisiae biomass production and its quality 2(1): evaluation as single cell protein. The Journal of 15. Irfan, M., M. Nadeem, Q.A. Syed and B. Shahjahan, Animal & Plant Sciences, 21(4): Submerged cultivation of Aspergillus niger on 8. Gad, A.S., E.A. Hasan and A. Abd El Aziz, pretreated Sugarcane Bagass. World Journal of Utilization of Oputinaficus indica waste for Agricultural Sciences, 6(4): production of Phanerochaete chrysosporium 16. Lenihan, P., A. Orozco, E.O. Neill, M.N.M. Ahmed, bioprotein. Journal of American Sciences, 6(8): D.W. Rooney and G.M. Walker, Dilute acid 9. Moebus, O. and M. Teuber, General aspects of hydrolysis of lignocellulose biomass. Chemical production of biomass by yeast fermentation from Engineering Journal, 156(2): whey and permeate. In Production and Feeding of 17. AOAC; The Association of Official Analytical Single Cell Protein, Applied Science London, pp: 153. Chemists, The Official Methods of Analysis of 10. Harris, E.E., Food Yeast Production from Wood th AOAC International, 18 edn. Arlington, U.S.A. Processing By-Products United States Department 18. Pearson., D., Pearson s chemical analysis of of Agriculture Forest Service. th Foods 8 (Edn). Essan, H. E. Kiriltris, Sawyer, R. 11. Soumalainen, H. and E. Oura, Yeast nutrition Churchhill Livingstone London, pp: st and solute uptake. In the Yeasts 1 Edn, Yeast 19. Anupama, R.P., Value added food: Single cell nutrition and solute uptake, Academic Press: protein. Biotechnology Advances, 18(6): London, pp: 3. 18

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