Higly efective extraction of oil from soybean by pretreatment of solidstate fermentation with fungi

Abstract
The solid-state prefermentation by aspergillus niger (CICC 2377) and aspergillus
flavus (CICC 40536) was employed to increase the oil extraction yield from
soybean. The influence of incubation time on oil yield was investigated. The
maximum oil yield extracted from the substrate prefermented by aspergillus
niger for 96 h was 23%, which increase by 47,4% compared with control (15,6%).
In the same fermentation conditions, the maximum oil yield extracted from
substrate prefermented by aspergillus flavus was 21,6%, wich increased by
38,5% compared with control (15,6%). The quality of soybean oil was not
changed obviously by pretreatment of fermentation with fungi.
Keyword : soybean, aspergillus niger, aspergillus flavus, solid-state
fermentation, extraction of oil.
Introduction
Biodiesel is an excellent subtitute for fossil fuels, because of its feature of
environmental protection and regeneration. Many researchers engaged in
exploiting biodiesel, such as using food residues, planting energy crops, applying
oleaginous microorganism to produce microbial oil, etc. However, these have not
been applied ever to produce biodiesel in industry. Oil crops, such as rapeseed,
soybean, and Jatropha curcas seeds are the main raw materials for the
productions of biodiesel. Improving the extraction of oil from oil crops has
important and actual significance. Moreover, the demand for oil crops could offer
a significant opportunity for agriculture in developing economies.
It was definite that oil bodies in oil seeds are trapped in the organelles calle lipid
and protein bodies. Oil from oil seeds is conventionally extracted by mechanical
pressing. However, mechanical pressing cannot break down thw cwll wall and the

protein efficiently, leading to low release oil. Some scholars have reported that
enzyme-assisted extraction has been successfully employed to extract oil from
soybean by using proteases, from rice bran by using proteases, amylase, and
cellulose. And from Jatropha curcas seed kernels by combination of
ultrasonication and aqueous enzymatic. Although the above methods can
improve the oil yield extracted from plant seeds, they need energy and enzymes,
which will increase the extra cost. Soybean contains 20% of crude fat, 20-30% of
carbohydrate, and 30% of protein; this composition is appropriate to support
good microbial growth and enzyme production. Solid-state fermentation has
been employed to produce extracelluler enzymes like roteases, xylanase, and
cellulose. It is woth to explore the use of soybean as substrate for solid-state
fermentation. The present work demonstrate the effect of microorganism (asp.
Niger and asp. Flavus) fermentation pretreatment in extraction of oil from
soybean.
Material and methods
1. Microorganisms
Aspergillus niger (CICC 2377) and aspergillus flavus (CICC 40536), two
protease producers, were purchased from the Chna Centre of Industry Culture
Collection (CICC).
2. Inoculum
a. A mother culture was repared by inoculating 2 loopful of stock culture of
aspergillus niger in the medium containing glucose (20 g/L), KH2PO4 (3
g/L). And MgSO4 (1,5 g/L), followed by incubation overnigth grown culture
was used as inoculum in further studies.
b. A mother culture was prepared by inoculation 2 loopful of stock culture of
aspergillus flavus in the medium containing sucrose (30 g/L), NaNo3 (2
g/L), MgSO4 (0,5 g/L), KCL (0,5 g/L), K2HPO4 (1 g/L) and FeSO4 (0,01 g/L),
followed by incubation at 280 C and 180 rpm. This overnight grown culture
was used as inoculum in further studies
3. Substrate reparation and solid-state fermentation

Soybean was purchased from the lokal market of Guiyang, Guizhou province ,
China. Ten grams of well-ground dry substrate was taken into a 250 ml conical
flask and 15 ml of tap water was added, shaken, and autoclaved at 121 0C for
20min. After coolig, sterile media was inoulated with 2 ml inoculum and then
incubated for 6 d in an incubator at 28 0C.
4. Analytical method
To anlyze the change of composition in the substrate, five gram of fermented
substrate was taken from the flask every 24 h, then 50 ml distilled water was
added, and the mixture was stirred in a magnetic blender at 200 rpm for 30
min. The suspension was then centrifuged at 4000 rpm for 20 min, and the
supernatant used for assaying reducing sugar content, ammonia nitrogent
content, and protease activity. Reducing sugar content was determining using
the method described by reference. Formol titration method was use for thr
detrmination of ammonia nitrogen content, while proteate activity was
determined by folin-phenol method. One unit of protease activity is defined as
the amount of enzyme required to roduce one microgram of tyrosine per
minute under the conditions described. Soybean oil was extracted by soxhlet
extraction. Acid value and peroxide value were determined as described,
respectively. All chemicals and solvent used were of analytical grade.