Table of Content: Content 1. Abstract 2. Introduction 3. Short statement of problem and objectives 4. Review of research literature 5.

Material and method 6. Prediction of results 7. Discussion 8. Conclusion 9. Reference Page 1 2 3 4 6 9 9 12 12

Abstract: Food demand increase as population increases, however supply may not be plentitude to support the increasing population. A decrease in protein intake in diet can lead to protein malnutrition problems thus unconventional way of protein production has been discovered to substitute or replace the conventional protein source at the same time to overcome malnutrition problems and food crisis. Fermentation of carbon rich component by microorganisms is used to produce unconventional protein which is called bio-protein where it is a high quality nutritive protein. Rice husk is the chosen carbon source to produce bioprotein due to its high availability in our country. It is first dried at 550C and milled to smaller particle size and this is then followed by a series of pretreatment of rice husk before the addition of inoculums, Aspergillus oryzae. The fermentation occurs at 430C for 3 days and sample will be filtered for biomass separation and drying. The dried mixture is then centrifuged at 8000rpm for 15 minutes, and the supernatant were kept for further analysis of protein concentration using Bradford method, while on the other hand total nitrogen content is determined by microKjeldahl method. Expected amino acid obtain are Methionine, Leucine, Isoleucine, Cysteine, Threonine, lysine, valine and phenylalanine.

1.0 Introduction: Worlds population is increasing year by year and this leads to an increase in food demand. However, food supplies from natural resources are limited in quantity thus required sustainable development in order to provide enough food sources for future needs. Protein is one of the important dietary nutrients needed to sustain life yet, there is a race between demand and supply of protein in real life. Protein sources from both animal and plant based might not be plentitude enough to sustain worlds population in one day and shortage of protein in diet can lead to the development of protein malnutrition related problems. World Health Organization (WHO) cites malnutrition as the greatest single threat to the world's public health. Time is needed to harvest and yield from both protein production are affected by the environment such as weather, diseases and natural disaster. Thus it provides an idea of producing alternative protein source from non-conventional way to supplement the conventional protein sources. There are a lot of research has been done in these few years by other countries to prove that solid state fermentation was available for reprocessing and reuse of different agriculture and food waste for the conversion of valuable and nutritive products. By the application and help of biotechnology, bio-proteins, a cheaper source of protein is able to synthesize from carbon source by the aids of selected microorganisms in the process of fermentation. This will help to deal with protein shortage problem in future and lowered down the dependence on animal protein. Rice husk has been chosen due to it is high availability in our country and also due to its high content of cellulose, hemi-cellulose, lignocellulosic (Badal C.S. [Link] 2008) which can provide carbon source to produce bioprotein by fermentation. Recycling it into production of bio-protein can increase the value of it, reducing the wastage of valuable food source and preventing environmental decay. Bio-protein, which is also called microbial protein or single cell protein, is the protein extracted from cultivated microbial mass and can be produced using a number of different microorganisms such as bacteria, yeast, fungi and algae (S. Hafiza [Link] 2011). Bio-protein is a new healthy source of proteins for the country facing nutrition crisis mainly due to high quality protein content with good nutritive value that can be digested maximally and absorbed optimally by our bodies. Besides high quality of protein, carbohydrates, fatty acid, vitamins and minerals are also found in it and therefore it is effective in solving malnutrition and food crisis problem in the world (D. Dhanasekaran [Link] 2010). The production of bioprotein by microorganisms is also much higher and rapid as compared to conventional ways. Thus bio-protein is a good source of protein supplement to supplement those that face lack of
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enough protein in their diet and replacing costly conventional protein source. Bio-protein is also useful to be as food addictive, fat binding, addictive in certain chemical and pharmaceutical products. Thus the benefits and usage of bio-protein is been highly highlighted in previous study due to its large market value in food industry.

2.0 Objective: (1) To produce a cheaper source of protein by incorporate selection strain and undergo careful formulation of fermentation. (2) To solve malnutrition and food crisis problem in the world and achieve the high demand of nutrition among the population. (3) To increase the availability of affordable quality proteins and reduce dependence on animal proteins. (4) Prevent environmental decay and increase food supplies. (5) To dispose rice husk in a better way.

2.1 Short Statement of Problem: A huge increase of population over the past two thousand years especially in developing countries has lead to increasing in demand of foodstuff. Due to the high cost of protein, scientists have been work out to search for non-conventional protein sources to supplement the conventional protein sources. The high quality of protein, carbohydrates, fatty acid, vitamins and minerals in bioprotein supplement is effective in solving malnutrition and food crisis problem. According to an Overview of Solid Waste Management in Malaysia, the estimation of 9th Malaysia Plan stated that there is about 45% of the waste is made up of food waste. The proper management and recycling of huge volumes of food waste became a challenges faced by Malaysia. In Malaysia, rice husk is usually used for heat energy production by burning. Due to its high ash content, only a small proportion of rice husk is utilized for non-energy related and low value applications, for instance, production of chicken litter, animal roughage, mulching and bedding materials. Some country dispose rice husk by burning. This practice is strongly opposed and prohibited in some countries under protection legislation. It is crucial to find a better and profitable way to dispose rice husk to prevent pollution issue. Bioprotein production can be a best idea as it converts its nutritive value into valuable product. Moreover, rice husk is extremely prevalent and easily available in Malaysia because of the rice production in Kedah.
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3.0 Review of Research Literature The increased demand for food production and the world shortage of protein have stimulated the interest of scientists for the single cell protein (SCP) production. Cultivation of microorganisms on hydrocarbon substrate was realized to have a great potential as a food protein source. The production of SCP by microorganisms has been investigated by many researchers and the utilization of some agricultural waste as carbon and nitrogen source for microorganisms growth have been described in the published papers. For example, coconut dregs (S. Hafiza et al., 2011), pineapple skin waste (D. Dhabasekaran et al., 2011), parboiled rice effluent (Zepka et al., 2010), waste paper (Ivarson and Morita, 1982), pineapple cannery effluent (Nigam, 1998), vegetable and fruit processing wastes (O. Stabnikova, 2005), ram horn (Kurbanoglu and Algur, 2002), rice bran (Anupama and Ravindra, 2001) and rice husk (M. Y. Khan and M. U. Dahot, 2010). The requirements for the usage of agricultural wastes as the substrate for SCP production includes non-toxic, presence in nature on large scale, cheap cost and ability to support microorganisms growth. There are various potential strains and substrates that could be utilized for SCP production. A variety of microorganisms are used to produce SCP at commercial scale which include algae, fungi and bacteria. Their rapid growth and high protein content have made them suitable for use as sources of SCP production. Among the algae, the two most extensively used species are the unicellular green alga, Chlorella and the filamentous bluegreen alga, Spirulina. Biomass from Chlorella and Spirulina have been harvested and utilized as food in many parts of the world (Anupama and Ravindra, 2000). Some advantages of algae species are their high nutrition sources, high quality protein content and very low nucleic acid content. However, there are some limitations of algae for human consumption. One of the main limitations is the presence of their cell wall. Algae have cellulosic cell walls which is indigestible by human beings and other non-ruminants. This poses a problem in their biomass utilization as humans lack the cellulose enzyme to digest the cellulose component of the cell wall. Hence, in order to be used for human consumption, effective treatments are necessary to disrupt the cell wall to make the protein and other constituents digestible. Other than that, algae production is generally done outdoors and is dependent on the climatic conditions. They have limitations such as the need for warm temperatures, plenty of sun light and carbon dioxide. Many fungal species are also used as SCP production and the most popular among them are the yeast species. Many other filamentous fungi also serve as sources of SCP. In
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general, fungi are capable to degrade a wider range of complex plant materials. They can also tolerate low pH. The main limitation in their use is the presence of mycotoxins in certain fungal species as these toxins will produce allergic reactions and diseases in humans and animals. Fungi generally have lower nucleic acid content than bacteria. However, to make the fungal species to be used as SCP sources on commercial basis, removal of mycotoxins and reduction of nucleic acid content to levels comparable of algae are important (A.T. Nasseri et al., 2011). Yeasts are probably the most widely used microorganisms for SCP. These include strains of Candida utilis and Saccharomyces cerevisiae. Yeast is suitable for SCP production because of its low production costs, high cell density and relatively high protein content, ability to grow at acidic pH and their wide acceptability due to its long history in traditional use. Its limitation for animal and human consumption includes their nucleic acid content and low cell wall digestibility due to the complex and thick cell envelope of yeast. In general, bacteria are capable of growth on a wide variety of substrates and have a rapid growth rate than fungi. They also produce higher protein content than yeast or fungi. Among the bacterial species, Cellulomonas and Alcaligenes are the most frequently used as SCP source. The main limitation of use of bacterial SCP is due to its high cost. Their small cell size and low density makes the harvesting difficult and costly. To harvest the protein from bacteria, it requires the use of sedimentation techniques and centrifugation. Other than that, bacterial cells have a relatively high nucleic acids content compare to yeast and fungi. Their high nucleic acid content makes them less desirable as food for human and animal. Additional processing step has to be introduced to decrease the nucleic acid level and this increases the cost. In addition, some bacteria will produce endotoxins which can promote allergenic reactions in humans. Lastly, there is also a psychological barrier to the use of bacterial protein as food source due to poor public acceptance of bacteria as food (Anupama and Ravindra, 2000).

4.0 Materials: Mutant strains of Aspergillus oryzae, ethyl methyl sulphonate, pretreated rice husks with dilute acid of dilute H2SO4 (15%, w/v), Carboxymethyl cellulose (CMCase), 1% oat spelt xylan, acetate buffer, Na2CO3 , 10M NaOH Element Carbon Hydrogen Nitrogen Sulfur % 38.68 5.4 0.67 0.21
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Carbon:nitrogen ratio of the rice husks is 58:1. Thus, the ammonium sulfate is added as nitrogen sources to achieve C: N ratio become 5:1. 4.1 Methods 4.1.1 Milling of rice hull: The rice hulls were dried in forced-air oven at 550C for 24 hours and milled in a hammer mill to pass through a 1.27 rom screen. The milled rice hulls were stored at room temperature. 4.1.2 Dilute acid pretreatment of rice hulls: 3.0g of milled rice hulls were slurried in dilute H2SO4 (15%, w/v) and pretreated in an autoclave at 1210C or sand bath at a desired temperature (140 0C, 1600C, 1800C). The pretreated rice hull slurry was adjusted to pH 5.0 with 10M NaOH before enzymatic saccharification. For two- stage pretreatment, rice hulls (15%, w/v) slurried in 1.0% (v/v) H2SO4 were replaced in the sand bath at 1400C for 15 minutes and cooled. The liquid was collected by centrifugation (15,000 x g for 10 minutes). The solid residue after washing with water was slurried with 1.0% (v/v) H2SO4 and then treated at 1900C for 10 minutes. 4.1.3 Enzyme assays: Carboxymethyl cellulose (CMCase) and xylanase activities were assayed in reaction mixture (0.5,mL) containing 1% (w/v) carboxymethyl cellulose and 1% (w/v) oat spelt xylan , respectively 50mM acetate buffer, pH 5.0, and appropriately diluted enzyme solutions. After incubation at 500C for 30 minutes, the reducing sugar liberated in the reaction mixture was measured by the dinitrosalicylic acid (DNS) method. One unit (U) of each enzyme activity is defined as the amount of enzyme, which produces 1 mol of reducing sugar as glucose (xylose in the case of xylanase) in the reaction mixture per minute under the above-specified conditions. -Glucosidase, -xylosidase and -L-arabinofuranosidase activities were assayed in the reaction mixture (1 ml) containing 4 mM p-nitrophenyl -D-glucoside, 2 mM p-nitrophenyl -D-xyloside or 1 mM p-nitrophenyl--L-arabinofuranoside, respectively, 50 mMacetate buffer, pH 5.0 ; and appropriately diluted enzyme solutions. After incubation at 50 0C for 30 minuntes, the reaction was stopped by adding 1 mL of ice-cold 0.5 M Na2CO3, and the color that developed as a result of p-nitrophenol liberation was measured at 405 nm. One unit (U) of each enzyme activity is defined as the amount of enzyme, which releases 1 mol pnitrophenol per minute in the reaction mixture under these assay conditions.
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4.1.4 Enzymatic saccharification/ enzymatic hydrolysis: The enzymatic saccharification of the diluted acid pretreated rice hulls with enzymes was performed by shaking slowly at 100 rpm for 72 hours at 45 0C after adjusting the pH to 5.0 with NaOH. After that all of the enzymes were used at a dose of 2 mL/100 g of the rice hull. 0.5 mL of the samples were withdrawn and kept at-20 0C before processing for analysis. 4.1.5 Over liming of hydrolyzate with calcium hydroxide: Calcium hydroxide solution was added to the dilute acid pretreated and enzyme saccharified rice hull hydrolyzate to increase the pH to 10.5. The whole mixture was stirred for 30 min at 90 0C, allowed to cool slowly to room temperature and then adjusted back to pH 6.5 with HCl. It was then centrifuged (15,000 x g for 30 minutes) to remove any precipitate formed before using as a substrate for fermentation. 4.1.6 Preparation of inoculums: Aspergillus oryzae were maintained on potato dextrose agar (PDA) subcultures for 30 days and stored at 4 0C. Spores were harvested from 7 days-old PDA slants with distilled water. 4.1.7 Mutation of Aspergillus oryzae with ethyl methyl sulphonate(EMS): 109 spores per mL of Aspergillus oryzae were collected from 7 days-old slant and 300 L ethyl methyl sulphonate (EMS) was added. This was incubated at equal time intervals. The spores were diluted, spread on PDA and incubated for 60 hours at 300C. The survival percentage was calculated from the number of colonies obtained at each time interval. Spores incubated for an hour were used for screening. 4.1.8 Screening of microorganisms: Screening of microorganisms was carried out to determine the best and potential microorganism that could produce highest bioprotein by glucose from the rice husks as a substrate and applying the solid state fermentation method. 4.1.9 Fermentation experiments: An amount of 10 gram of pretreated rice husk was taken in 500mL flask and the moisture was maintained by adding inoculums medium containing per 1 L of distilled water: FeSO4. 7H2O 0.1 g, MgSO4.7H2O 5g, (NH4)2SO4 3g and K2HPO4 1 g. After that, it was autoclaved at 1210C for 15 minutes. 100mL of inoculums medium in separate flask was adjusted to pH 5.0 and inoculated with spores 3x 10 9 /mL of Aspergillus oryzae and then incubated at 430C for 3 days. The sample was removed for biomass separation by filtration at regular intervals
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of 10 hours. The contents of the flask were transferred into carbonate bicarbonate buffer. Furthermore, the biomass of Aspergillus oryzae grown was used for determination of amino acid composition. 4.1.10 Protein recovery: The fermentation samples in the flasks were dried for 24 hours at 650C. Dried ssample was added with 50mL phosphate buffer and macerated in pestle mortar. The mixture was centrifuged at 8000rpm for 15 minutes. The supernatant obtained were kept in the refrigerator for further analysis of protein concentration using Bradford method. 4.1.11 Analytical methods: Protein was measure by method of Lowry and the total nitrogen was determined by microKjeldahl method. The amino acids composition was analyzed with 6 HCI at 1100C with Biocal Automatic amino acid analyzer.

5.0 Prediction of result: Amino acid Met Leu Ile Cts Tyr Lys Val Phe Table 2: Mutant strains of Soy bean meal [Link] (mg/g) (mg/g) 15 8 62 68 48 64 13 0 30 32 62 64 53 50 55 48 FAO (mg/g) 22 48 42 20 28 42 42 28

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composition of amino acid (mg/g)

70 60 50 40 30 20 10 0 Met Leu Ile Cts Tyr Lys Val Phe Mutant of [Link] Soy bean meal FAO

Graph 1: The comparison of the composition of amino acid (mg/g) of mutant strain of [Link] and soy bean meal.

6.0 Discussion: 6.1 Technique difficulties: The most important and difficult problem in overall process is it requires microorganisms that can utilize natural lignocelluloses in the rice husk at rates of commercial interest. Unfortunately there are no known microorganisms which can utilize lignocelluloses up to now. Hence, it is necessary to pre-treat the rice husk before fermentation process start. There are two ways of pre-treatment which are exposing the cellulose by removal or modification of the lignin, and to reduce the crystalline fraction of the cellulose. Among several methods of pre-treatment, the most effective pre-treatment method to release most of the cellulose are hot alkali/ acid and both ball milling to micron size particle. Hot alkali/acid can direct convert maize, cereal, rice husk, etc, into a more digestible feed. However, this method does not increase protein content due to no microbial growth. It requires relatively large amounts of alkali/acid and its economic value is still under investigation. Wet and dry milling methods are usually most used because there are more economical. After wet and drying method, 90 percent of cellulose is made available for microbial digestion.

On the other hand, development of low labour processes, high technology and capitalintensive of an industry for the production of single cell protein from lignocelluloses is rare in last few years. This is because economic consideration and technical problems. These all problems can be solved when the economic climate improves. Another problem in the overall process is the carbon: nitrogen of the rice husks is 58:1. It is too least of nitrogen component in the rice husk to synthesis single cell protein. Hence, the ammonium sulfate is added as nitrogen sources to achieve C: N ratio become 5:1. 6.2 Advantages: Malnutrition is a major problem of most of the developing countries in the world. On the other hand, the environment is getting worst due to the tremendous increase amount of waste of the world from time to time. Therefore waste recycling is proposed as a new method that prevents environment decay and increases food supplies. Single cell protein produced from lignocelluloses waste is one of the waste recycling methods that can use one stone to kill two birds; it can at once solve malnutrition problem and environment problem. Single cell protein (SCP) refers as used of dried microbial cells or total protein extracted from pure microbial cell culture (algae, yeasts, fungi and bacteria) that can be used as food supplement or animal feed. Fermentation methods are one of the methods to produce high quality protein; it is the method for large scale protein production which does not require a concomitant increase in energy consumption. In addition, microorganisms have a high rate of multiplication hence growths of microorganism are higher than plants. In this technique, Aspergillus oryzae filamentous fungi are selected as the microorganism which is used in fermentation process. This is because Aspergillus oryzae is a strain with high yield and protein can produce relatively easy. It can produce very high protein content around 43-85 % in the dry mass; hence, the overall cost of protein production from the Aspergillus oryzae is more economical as compared to that of single celled organism. On the other hand, microbial biomass production occurs in continuous cultures and quality of protein does not change due to the growth is independent of climatic variations. Protein is easily produced by Aspergillus oryzae rather than plants or animals whose growth is dependent of seasonal or climatic variations. Another advantage of using Aspergillus oryzae is it has greater penetrating power into insoluble substrates. Therefore, it is suitable for solid state fermentation of lignocelluloses materials. In addition, compared to yeast in terms of flavour with Aspergillus oryzae, faint mushroom like odor and taste of Aspergillus oryzae are more readily acceptable as a new source of food. It also can help in removal of pollutant
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due to its ability to utilize various waste products as carbon sources. It saves cost to manage waste and is friendlier to the earth. 6.3 Commercial viability: Bio-protein is a profitable business that worth to develop in our country. Rice husk is usually sold as animal feed which is selling at a very low price. Value of rice husk will be greatly increase if it is used in the production of bio-protein because bio-protein gives a higher return rate and generates higher profit as compare to animal feed. Bio-protein is common in other country but not for Malaysia. Due to the new concept of Bio-protein technology, large scale production of bio-protein is not seen in our country yet and thus it does not become a popular supplement product. Eventually leading to a low market competition for bio-protein products in Malaysia and thus hardly found any bio-protein supplement in our country. Bio-protein has great market value if it is sells as supplement because it provides lots of benefits to consumers health and promoting greener environment by recycling waste into consumable product. From a medical view, bio-protein will provide wholesome nutrients that increase body health. By processing rice husk and turning them into a health-giving product will contribute to conservation of environmental resources directly and provide an alternative way for people to decrease the dependence on animal protein source indirectly. Peoples become more concern about health issue as we entered the 20th century. Peoples are also more environmentally conscious hence they will be more incline to support this product. As bio-protein is a low cost and environmentally friendly product, it also has a huge potential to attract Green Corporation to invest in this product. Children and adolescence requires higher protein needs for their rapid growing stage and bio-protein can provides all of the essential minerals they need. While for adult, bioprotein can be consumed as a supplement daily to supplement the essential nutrient missed in their diet. The target market for Bio-protein is huge because it is suitable for customers of all ages and vegetarians. Bio-protein supplement will be a new alternative source of protein for vegetarians besides legumes.

6.4 Novelty: Production of bio-protein by fermentation using agriculture waste is not a new idea in biotechnology field. It is well known in other countries such as India to overcome malnutrition crisis and replacing costly conventional protein. However, the production of bio11

protein is still considering as a new market in Malaysia. We usually import bio-protein from other country instead of producing it locally. Research has been done on using agriculture waste such as coconut dregs (S. Hafiza et al., 2011), pineapple skin waste (D. Dhabasekaran et al., 2011), parboiled rice effluent (Zepka et al., 2010), waste paper (Ivarson and Morita, 1982), pineapple cannery effluent (Nigam, 1998), vegetable and fruit processing wastes (O. Stabnikova, 2005), ram horn (Kurbanoglu and Algur, 2002), rice bran (Anupama and Ravindra, 2001) and rice husk (M. Y. Khan and M. U. Dahot, 2010). However the idea of producing bio-protein from rice husks is new and has not tried in our country.

7.0 Conclusion: As a conclusion, bio-protein is able to synthesize via fermentation using rice husk as a carbon source by the aids of microorganism. Aspergillus oryzae is the microorganism selected which belongs to the fungus family. It has rapid growth rate and contributes to high amount of protein content. The expected essential amino acids found from the product are Methionine, Leucine, Isoleucine, Lysine, Valine and Phenylalanine and Cysteine and Tyrosine as the nonessential amino acids.

Reference: Badal C. Saha, Loren B. Iten, Michael A. Cotta, Y. Victor Wu, 2008. Dilute Acid Pretreatment, Enzymatic Saccharification, and Fermentation of Rice Hulls to Ethanol. Journal of Biotechnology, 21(3), pp.816-822. Israelidis, C. (n.d.). NUTRITION - SINGLE CELL PROTEIN, TWENTY YEARS LATER. Retrieved March 7, 2011. Available from: [Link] Bhalla, T.C., Sharma, N.N., and Sharma, M., 2007. Production of Metabolites, Industrial enzymes, Amino acid, Organic acids, Antibiotics, Vitamins and Single Cell Proteins. National Science Digital Library, India. Kavanagh, K., 2005. Fungi: Biology and Applications. England: John Wiley and Sons Ltd, pg 102-104. Anupama and Ravindra P., 2001. Studies on production of single cell protein by Aspergillus niger in solid state fermentation of rice bran. Braz. Arc. Biol. Tech. 44: 79-88. Anupama and Ravindra P., 2000. Value-added food: single cell protein. Biotechnology Advances 18, 459479.
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A.T. Nasseri, S. Rasoul-Amini, M.H. Morowvat and Y. Ghasemi, 2011. Single Cell Protein: Production and Process. American Journal of Food Technology, 6: 103-116. D. Dhanasekaran, S. Lawanya, S. Saha, N. Thajuddin and A. Panneerselvam, 2011. Production of Single Cell Protein from Pineapple Waste Using Yeast. Innovative Romanian Food Biotechnology. 8, 26-32. Ivarson, K.C. and H. Morita, 1982. Single-Cell Protein Production by the Acid-Tolerant Fungus Scytalidium acidophilum from Acid Hydrolysates of Waste Paper. Appl. Environ. Microbiol., 43(3): 643-647. Kurbanoglu, E.B. and Algur, O.F., 2002. Single-cell protein production from ram horn hydrolysate by bacteria. Biores. Technol. 85, 125129. M. Yakoub Khan and M. Umar Dahot, 2010. Effect of Various Agriculture Wastes and Pure Sugars on the Production of Single Cell Protein by Penicillium Expansum, World Appl. Sci. J., 8 (Special Issue of Biotech. & Genet. Engineer.): 80-84. Nigam, J.N., 1998. Single cell protein from pineapple cannery effluent. World J. Microbiol. Biotechnol. 14, 693696. S. Hafiza, N. G. Ahmad Anas and B. Nor Hidayah, 2011. Screening of Potential Strain for Bioprotein Production from Coconut Dregs. International Conference on Food Engineering and Biotechnology. Vol.9. Stabnikova, J.Y. Wang, H.B. Ding, J.H. Tay, 2005. Biotransformation of vegetable and fruit processing wastes into yeast biomass enriched with selenium, Bioresour. Technol. 96:747 751. Zepka, L.Q., Jacob-Lopes, E., Goldbeck, R., Souza-Soares, L.A., Queiroz, M.I., 2010. Nutritional evaluation of single-cell protein produced by Aphanothece microscpica Ngeli. Bioresour. Technol. 101, 71077111.

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