The ปริมาณs of พาราไมลัม and ไลปิด (แวกซ์เอสเตอร์) produced by the
culture in Example 20 and Comparative Example 7 were measured every day.
The พาราไมลัม ปริมาณ after the culture was measured by the following procedure. That is, a mixture (40 mL) of the บรอธ and the ไมโครแอลจี after the culture was put into a centrifugal tube, followed by
การปั่นเหวี่ยง. Pure water was added to the precipitate after the
5 การปั่นเหวี่ยง to yield a suspension, and the operation of centrifugal
re-separation was repeated twice. Then, a small ปริมาณ of pure water was added to the precipitate after the การปั่นเหวี่ยง to yield a suspension, and the suspended solids were freeze-dried. Thus, the components of the บรอธ were removed.
10 Next, about 400 mg of the cells of the freeze-dried ไมโครแอลจี was
accurately weighed out in a centrifugal tube (which had been weighed as a blank value). Acetone was added thereto to yield a suspension, and the supernatant fluid after การปั่นเหวี่ยง was removed. Until the color of the
supernatant fluid disappears, the washing operation with acetone was repeated 15 about 5 times. Thus, the รงควัตถุ components produced by the ไมโครแอลจี
were removed.
Subsequently, using a dodecyl sodium sulfate solution, an operation to
remove components other than พาราไมลัม was performed. That is, 20 mL of
a 1% dodecyl sodium sulfate (SDS) solution was added to the residue after the 20 รงควัตถุ components were removed so as to yield a suspension, which was
thereafter heated at 100°C for 10 minutes. Then, the supernatant fluid after
การปั่นเหวี่ยง was removed. Such an operation was repeated twice, and
thereafter the same operation was repeated three times using pure water
instead of the SDS solution. Thus, the SDS was washed away.
25 Finally, the whole centrifugal tube was put in a dryer at 105°C so that
the moisture was removed, and the weight of the centrifugal tube containing พาราไมลัม was measured. Then, the ปริมาณ of พาราไมลัม was determined from the difference from the aforementioned blank value.
Meanwhile, the ปริมาณ of แวกซ์เอสเตอร์ after the culture was measured 30 by the BLIGHT-DYER method.
[0099]
29
รูป 8A and รูป 8B show the measurement results of the พาราไมลัม ปริมาณ over time in the culture of Example 20 and Comparative Example 7. Further, รูป 9A and รูป 9B show the measurement results of the ไลปิด (wax ester) ปริมาณ over time.
5 As seen from รูป 8A and รูป 8B, the ไมโครแอลจี of the จีนัส ยูกลีนา
(ยูกลีนา gracilis) strain EOD-1 have a higher พาราไมลัม production speed
than the conventional ไมโครแอลจี of the จีนัส ยูกลีนา (ยูกลีนา gracilis)
strain NIES-48. Further, the strain EOD-1 has a พาราไมลัม content per cell of about 55% or more, which is higher than in the strain NIES-48.
10 Further, as seen from รูป 9A and รูป 9B, the ไมโครแอลจี of the จีนัส
ยูกลีนา (ยูกลีนา gracilis) strain EOD-1 have a higher ไลปิด (wax ester) production speed, and thus the content of แวกซ์เอสเตอร์ per cell is increased within a shorter period.
[0100]
15 Further, the culture step was performed using the อาหารเพาะเลี้ยง
having the composition shown in Table 4 and Table 5 below under the following culture conditions.
[0101]
Example 21
20 The aforementioned ไมโครแอลจี, strain EOD-1, were cultured for two
days by photoเฮเทอโรโทรฟic culture. [0102]
Comparative Example 8
The aforementioned ไมโครแอลจี, strain NIES-48, were cultured for two 25 days by photoเฮเทอโรโทรฟic culture.
[0103]
As the อาหารเพาะเลี้ยง, a อาหารเพาะเลี้ยง obtained by adding 30 g/L of
glucose to a modified Hutner อาหารเพาะเลี้ยง (hereinafter, referred to as
"Modified Hutner อาหารเพาะเลี้ยง") was employed. Specifically, the
30 composition was as shown in Table 4, and the component in the liquid other
than nutrients was water. As the trace metal solution in Table 4, a trace metal solution having the composition in Table 5 below was used. The component other than trace metal was water.
The detailed culture method was as follows.
บรอธ: with a pH adjusted to 4.0 using hydrochloric acid
5 Culture container: 500-mL Sakaguchi flask
Introduction before the culture: 200 mL of the บรอธ and the ไมโครแอลจี before the culture were put into the Sakaguchi flask (in order to adjust the
initial ปริมาณ of biomass, 220 mL in total was used for the strain EOD-1 and 236 mL in total was used for the strain NIES-48).
10 Temperature during culture: 28°C
Light-dark conditions during culture: cultured under light-shielded dark conditions
Aerobic conditions during culture: The Sakaguchi flask was placed on a shaker, and air was supplied into the บรอธ by operating the shaker with
15 reciprocal shaking at 130 rpm.
[0104]
Table 4
Modified Hutner อาหารเพาะเลี้ยง: Glucose Added
Glucose 30 g/L
Asparagine 2 g/L
Glutamic acid 5 g/L
Malic acid 5 g/L
Glycine 2.5 g/L
Urea 0.4 g/L
Succinic acid 0.1 g/L
KH2PO4 0.4 g/L
MgSO4.7H20 0.14 g/L
MgCO3 0.4 g/L
CaCO3 0.1 g/L
(NH4)2Fe(SO4)2.6H20 0.021 g/L
Vitamin B1 0.6 mg/L
Vitamin B12 0.05 mg/L
Trace metal solution 10 mL/L
20
31
[0105] Table 5
ZnSO4.7H20 1.1 g/L
MgSO4.H20 0.58 g/L
(NH4)6Mo7024'4H20 0.18 g/L
CoSO4.7H20 0.024 g/L
CuSO4.5H20 0.077 g/L
H3B03 0.029 g/L
NaNO3' 41120 0.074 g/L
[0106]
5 The dry weight (biomass production) of the algae after two days from
the culture and the glucose conversion were calculated in the same manner as above. Table 6 shows the results.
[0107]
Table 6
After two days Ex. 21 C. Ex. 8
from the culture (Strain EOD-1) (Strain NIES-48)
Dry weight (g/L) 24.50 11.50
Glucose conversion (%) 76 33
10
[0108]
As seen from Table 6, the ไมโครแอลจี of the จีนัส ยูกลีนา (ยูกลีนา
gracilis) strain EOD-1 have a better biomass production performance and a
better glucose conversion than the conventional ไมโครแอลจี of the จีนัส 15 ยูกลีนา (ยูกลีนา gracilis) strain NIES-48.
INDUSTRIAL APPLICABILITY
[0109]
The ไมโครแอลจี of the การประดิษฐ์นี้, the วิธีเพื่อผลิต
20 โพลีแซคคาไรด์ of the การประดิษฐ์นี้, and the วิธีเพื่อผลิต an
สารประกอบอินทรีย์ of the การประดิษฐ์นี้ are suitably used for obtaining
สารประกอบอินทรีย์s such as โพลีแซคคาไรด์ such as พาราไมลัม, ไลปิด such as แวกซ์เอสเตอร์, and proteins by culture.
32
The obtained สารประกอบอินทรีย์s can be used in applications such as health food, pharmaceutical, feed, chemical products, or fuel, while remaining stored in the cells of the ไมโครแอลจี, or after being extracted by an extraction
process or the like. Specifically, the ไลปิด stored in the cells of the ไมโครแอลจี 5 as สารประกอบอินทรีย์s by culture are suitably used, for example, as a raw
material of fuel by being extracted from the cells.