Inte rnatio nal Jo urnal o f Sc ie ntific & Eng inee ring Re se arc h, Vo lume 3, Iss ue 2, February -2012 1

ISS N 2229-5518

Study of Inoculum size, Incubation temperature and Nucleic Acid Concentration on the Single Cell Protein produced by using Soymilk residue (Okara)

Mahmood Khan Yousufi

Abs tract-Okara, a soymilk residue is produced in huge quantities in soymilk industries. In the present investigation three combinations of Okara and w heat grits w here prepared in the ratio of 3:1 (60% moisture content), 1:1 (40% moisture content), 1:3 ( 25 % moisture cont ent) respectively. The combinations w ere inoculated w ith tw o fungal species viz. Rhizopus oligosporus and Aspergillus oryzae, w ith an inoculum size of 1.0 × 10 3 , 1.0 × 10 4 and 1.0 × 10 5 cf u per g substrate. The combinations w ere incubated at diff erent temperatures i.e. 200 c, 250 c and 300 c. The combinations w ere f inally analyzed f or the eff ective inoculum size, incubation temperature and nucleic acid concentration. The results obtained depict that the best inoculum size

f or maximu m SCP yield w as 1.0 × 10 3 cf u per g substrate. The best incubation temperature w as f ound to be 250 c. The maximu m nucleic acid concentration w as f ound to be 249 mg f or R. oligosporus and 260 mg f or A. oryzae.

Ke ywords : Inoculum size, Incubation temperature, Nucleic acid, SCP, Okara. Rhizopus oligosporus and Aspergillus oryzae.

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INTRODUCTION

The term “ Single Cell Pr otein” (SCP) was coined at Massachusetts Institute of Technology(MIT) to r epr esent the cells of algae, bacteria, yeast and fungi gr own for the pr otein contents (Schr imshaw 19 ,1975). Due to the incr easing population and shor tage of pr oteins, the wor ld`s attention has been dr awn to micr obial sources of pr oteins. Hedenskog17 et al., (1973) described some methods of pr ocessing the single cell pr otein. Huang8 (1974) utilized acid br ine for the pr oduction of food yeast. Bellamy 1 (1975) has studied the conversion of insoluble agricultural wastes to SCP by thermophilic micr oor ganisms. Cooney 4 et al., (1975) pr oduced SCP fr om methanol by using yeast. Bodw ell2 (1977) evaluated the pr oteins for humans. Chen and Peppler 3 (1977) highlighted the application of single cell pr otein in food. Ethanol was used as a substr ate for the pr oduction of single cell pr otein by Laskin13 (1977B). Dimmling5 (1978) examined the r aw mater ials for the pr oduction of SCP. Formation of single cell pr otein filament was detected by Huang8 et al., (1978). Khar atyan (1978) explor ed the micr obes used as foods for humans.
Resear ch on SCP has been stimulated by a concer n over an eventual food cr isis or food shortage that will occur if the wor ld`s population is not controlled (Fr azier6 ,1995). W ills22 (1999) highlighted some advantages of SCP over plant and animal sources of pr otein, which ar e as follows :
1. Micr oor ganisms can pr ovide rapid mass incr ease.
2. Micr oor ganisms can easily be genetically modified
to pr oduce cell that br ing about desirable r esults.
3. Micr obial pr otein content is high.
4. For the production of SCP, raw mater ial available
in lar ge quantities can be utilized.
5. SCP pr oduction can be carr ied out in a continuous
cultur e, and ther efor e it is independent of climatic changes.
The nutritional status of SCP obtained fr om certain
microor ganisms was described by Singh 21 (2002). His r eport is depicted in table- 1

Table 1 – The nutritional s tatus of SCP(%) obtaine d from some microorganisms.

Nutrient Microorganisms

Suitability of single cell protein as a feed for human beings

Paec ilomyc es varioti Candida utilis Methylophilus

methylotrophus

Spirulina maxima

include industrial and agricultural pr oducts/byproducts.
Schueger l and Rosen18 , (1997) have investigated the use of agr icultural bypr oducts for fungal pr otein pr oduction
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Aut hor- Professor & Head, Depart ment of Microbiolog y,

Jawaharlal Nehru Colleg e, Bhopal(M.P.) India. E-mail: mk ha nyo usuf i@g ma il.co m

Ash 6 8 3 2

In the pr esent investigation, okara, a bypr oduct of soymilk industry was used as the substr ate. okara is pr oduced in lar ge quantities in soymilk industr ies, and poses a big disposal pr oblem. Each kg of soybean pr ocessed

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Inte rnatio nal Jo urnal o f Sc ie ntific & Eng inee ring Re se arc h, Vo lume 3, Iss ue 2, February -2012 2

ISS N 2229-5518

for soymilk pr oduction yields 1.1 kg of okara. The pr oduction of soymilk is descr ibed in Fig. 1
Washed soybean Tap water



Soaked for 16 hrs at r oom temper atur e

Pulver ised in water

Heated to the boiling point for 20 minutes or steamed


Str ained thr ough cheese cloth
Insoluble r esidue(okara) soybean milk

Fig.1 Showing protocol for soymilk production.

The high moistur e content of okara (Shurtleff and Aoyagi20 , 1979) acts as a drawback in its utilizat ion. Nutr itionally okara is r ich and it contains 79.6 % moistur e,
19.91% pr otein, 8.37 % fat, 2.87% star ch and 9.53 %
carbohydrate. In addition to this it is also contains maj or
miner als like calcium, ir on, copper and zinc. Taking into consideration the nutr itional quality of okara, Righelato and Elsw orth17 (1970) successfully utilized it for the pr oduction of some fer mentation products. Kinoshita 12 et al ., (1985) have also used okara for the pr oduction of r iboflavin and lipase. Matsuo15 , (1997) has r eported the in vivo antioxidant activity of okara by Aspergillus oryzae. Matsuo (loc. Cit) has further r eported the consumption of okara
fermented w ith Actinomuco r elegans (meitauza). Ma16 et. al., (1997) has studied the isolation and character ization of pr otein fr om soymilk r esidue (okara). The fibr inolytic activity of natto produced from okara fer mented by Bacillus subtilis has been investigated by Miyamura 16 et al., (1998). Yousufi23 et al., 2003 have investigated the pr oduction of SCP using okara-wheat gr it combinations contr olling its moistur e content.
Keeping into consider ation the above evidences on the utility of okara, the pr esent investigation was undertaken.

M ATERIAL AND M ETHODS:

The mater ial used in the pr esent investigation include okara, wheat gr its w er e mixed in ratios of 3:1 (150 g okara / 50 g
wheat gr its ), 1:1 (100 g okara / 100 g wheat grits) and 1:3 ( 50 g okara / 150 g wheat gr its ), so as to pr epar e thr ee combinations with moistur e content, 60 %, 40 % and 25 % r espectively. The w heat grits not only r educed the moistur e content but also pr ovided fermentable carbohydrates. The combinations w er e filled in Petr i plates, which w er e autoclaved at standar d temperatur e, pr essur e and time. After autoclaving, the combinations w er e aseptically inoculated with tw o fungal cultur es of R. oligosporus and A. oryzae separately. The inoculum size of the inoculum was
1.0 × 10 3 , 1.0 × 10 4 and 1.0 × 10 5 cfu per g substrate. The
combinations w er e then incubated at differ ent
temperatur es i.e. 200 c, 250 c and 300 c. After incubation,
small amount of samples w er e r emoved fr om each combination. A part of the sample was used for fr esh w eight analysis, wher eas another part was dried and used for dry weight analysis. The samples w er e subj ected to determination of the nucleic acid concentration using diphenylamine r eagent.

RESULT AND DI SCUSSION:

The best inoculum size for maximum SCP yield was found to be 1.0 × 103 (Table-2).

Table-2 Effe ct of inoculum s izes on SCP yie ld.

Note: In itia l mo is ture contents are given in parenthes es
The maximum SCP yield was obtained with the inoculum size of 1.0 × 10 3 In case of R. oligosporus the maximum SCP yield w as found to be 19.88 % (150 / 50 combination) wher eas in case of A. oryzae the maximum SCP yield was found to be 22.50 % (150/50 combination).
The best incubation temperatur e for maximum SCP
yield was found to be 25 0 C (Table-3).

Table-3 Effe ct of incubation temperature in SCP yie ld.

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Note: In itia l mo is ture contents are given in parenthes es
The maximum SCP yield was obtained at 250 C
temperatur e, in case of R. oligosporus it was found to be
15.23 % (150/50 combination) and in case of A. oryzae it was found to be 18.30 % (100/100 combination).
The nucleic acid concentrations obtained in differ ent combinations ar e depicted in Table -4.

Table-4 Nucle ic acid conce ntration in SCP yie ld.

SCP yield (g per 100 g substrate)

3. Chen,S.L. and Peppler , H.J. 1977. Single cell pr oteins in food applications. Dev. Ind. Microbiol. 19:79-94
4. Cooney, C.L. and Levine, D.W . 1975. SCP pr oduction fr om Methanol by yeast. In single cell pr otein II. S.R. Tannenbaum and D.I.C. Wang (Editors), MIT pr ess, Cambridge, Mass.
5. Dimmling, W . and Seipenbusch, R. 1978. Raw mater ial for the pr oduction of SCP. Precess Biochem. 13 (3) 9-15.34
6. Frazier ,W .C. and W esthoff,D.C. 1995. Food Micr obiology. Tata Mc Gr awhill Publishing Co. Ltd., New Delhi.
7. Hedenskog, G. and Mogr en, H. 1973. Some methods of pr ocessing for single cell pr otein. Biotechnol. Bio eng. 15:129-
142.

Organism Okara / w heat

grits (g)

R.

oligosporus 150 / 50(59.9)

100/100(40.1)

50 / 150(25.1)

A. oryzae

150 / 50(60.5)

100/100(40.1)

50 / 150(25.0)

Fresh w eight basis(in mg)

218

225

240

226

247

245

Dry w eight

Basis (in mg)

245

249

246

250

228

260

8. Huang, G. and Rha, C. 1978. For mation of single cell pr otein filament. J. Food Sci. 43:780-782.
9. Huang, Y.D. 1974. Pr uduction of food yeast fr om acid br ine. Pr oc. 4 th Inter n. Symp. Yeasts Vienna, 1974, Part I,
157-158, Inter n. Comm. Yeasts (ICY).
10. Kacmpfel, U.; Ber ghausen, K.H.; Lieflander ,M. (1995). Ar e cyanobacteria Suitable for feeding human beings ? A contr ibution on essential amino acids of.cyanobacter ia. Deutsche Lebensmittel- Rundschau, 91:(2) 50-52.
11. Kharatyan, S.G. 1978. Micr obes as foods for humans.
Ann. Rev. Micr obiol..32:301-327.
Note: In itia l mo is ture contents are given in parenthes es
The maximum nucleic acid concentration achieved for R. oligosporus was 249 mg (100/100 combination) on dry w eight basis and for A. oryzae it was found to be 260 mg (50/150 combination) on dry w eight basis. Accor ding to Food and Agr icultural Or ganisation (FAO) 2 gram nucleic acid per day from SCP for an adult has been given as safe practical limit. Since the r esults in Table-4 shows that nucleic acid concentration is far below the toler ance limit, ther efor e the SCP pr oduced can be consider ed safe for dietary intake. The over all r esult is that Okara w as successfully utilized to produce SCP.

ACKNOWL EDGEM ENT :

The author is thankful to the Dir ector , Mr . Aasif Zaki and Principal, Dr . Y.P. Singh of Jawahar lal Nehru College, Bhopal for pr oviding libr ary and labor atory facilities dur ing the tenur e of this investigation .

REFERENC E :

1. Bellamy, W .D. 1975. Conver sion of insoluble agricultural wastes to SCP by thermophilic micr oor ganisms. In single cell pr otein II. S.R. Tannenbaum and D.I.C. Wang (Editor s). MIT Pr ess, Cambridge, Mass.

2. Bodw ell, C.E. 1977. Evaluations of pr oteins for Humans.
AVI Publishing Co. W estport. Conn.
12. Kinoshita, S., H-Kittikun, A and Pithong, R. (1985). Pr oduction of r iboflavin fr om waste of tofu (soy cur d). Annual Report IC Biotechnology, 8: 322:324.
13. Laskin, A.I. 1977B. Ethanol as substrate for single cell pr otein pr oduction. In Single cell pr otein fr om Renewable and Non r enewable Resources. E.L. Gaden. Jr . (Editor ), Biotechnol. Bioeng. Symp. 7. John wiley and sons, New Yor k.
14. Ma,C.Y., Liu, W .S., Kw ok, K.C. and Kw ok, F. 1997. Isolation and Character ization of pr otein fr om soymilk r esidue (okara). Food r esearch International, 29:799-805.
15. Matsuo, M. 1997. In vivo antioxidant activity of okara
Kogi, a fermented okara, By Aspergillus oryzae. Bioscience, Biotechnology and Biochemistry, 61:1968-1972.
16. Miyamura, H., Takenaka, Y. and Takenaka, T. 1988. Fibrinolytic activity of okara fer mented by Bacillus subtilis II. The utility of okara, bypr oduct of the soybean pr ocessing industry. Journal of Japenese society of food science and technology (Nippon Shokuhin Kagaku Kogaku Kaishi), 45:100-107.
17. Righelato, R.H. and Elsworth, R. 1970. Adv. In Appl. Micr obiology, 13:399-417.
18. Schueger l, K. and Rosen, W . 1997. Investigation of the use of agr icultur al Bypr oducts for fungal pr otein
pr oduction. Process Biochemistry. 32:705-714.
19. Scr imshaw , N.S. 1975. Single cell pr otein for human
consumption – an overview . In single cell pr otein II. S.R.

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Tannenbaum and D.I.C. W ang (Editors) MIT Pr ess, Cambridge, Mass.
20. Shurtleff, W . and Aoyagi, A. 1979. Tofu and soymilk pr oduction. New Age Food Study Centr e. Lafayette, CA, USA.
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New Delhi.
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Sar up and Son Publishers, New Delhi.
23. Yousufi, Mahmood Khan: Khan, Shaukat Saeed and
Jha, Kr ishna, 2003. The Effect of moistur e content on the pr oduction of single cell pr otein using Rhizopus oligospor us and Aspergillus oryzae gr own on okara – wheat
gr it Combinations. Indian J. Applied and Pure Bio. 18(1) 81-84.

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