JPH06277075A - Yield increase of phycocyanin - Google Patents

Abstract

PURPOSE:To increase the yield of phycocyanin by remarkably increasing the content of phycocyanin per unit algal cells. CONSTITUTION:In relation to a method for producing phycocyanin by culturing and growing a diatom in an aqueous medium and extracting phycocyanin from the resultant algal cells, this method for increasing the yield of phycocyanin involves one process among (1) a process for increasing the concentrations of the whole medium components, (2) a process for increasing the concentration of only a specified medium component, (3) a process for adding a culture medium increased in the concentrations of the whole components during culture and (4) a process for adding only a specified medium component during culture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention cultivates and proliferates microalgae in an aqueous medium.
The present invention relates to a method for increasing the yield of a target substance by increasing the content of the target substance in the alga in a method for extracting and producing phycocyanin, which is one of the methods.

[0002]

2. Description of the Related Art Phycocyanin is one of pigment proteins constituting phycobilisomes of cyanobacteria or cryptoalga and plays a role of supplementing light energy in photosynthesis of cyanobacteria or cryptoalga. . Usually, since it has an absorption maximum wavelength at 615 nm and exhibits a bright blue color, it is used as a natural dye (see Japanese Patent Publication Nos. 55-47866 and 56-5143). Further, since a physiologically active action is also drawing attention, technological development for producing phycocyanin by growing cyanobacteria is under way, but further increase in yield is desired.

Conventionally, in the study of phycocyanin production by cyanobacteria, only the culture for a fixed period such as 1 to 2 weeks has been studied, and the change in the phycocyanin content per alga body during the culture period can be detailed over time. There was no study, and it was thought that the phycocyanin content per alga was constant throughout the culture period.

The present inventors investigated the phycocyanin content in detail over time through the culture period of cyanobacteria, and the phycocyanin content reached a maximum on the 2nd to 4th days in the early stage of the culture, and thereafter, as the cyanobacteria grew, the algae increased. It was found that the phycocyanin content per body decreased (see Fig. 1). That is, conventionally, the culture was stopped when the phycocyanin content decreased within the culture period of about 1 to 2 weeks, and the cyanobacteria were collected to extract the phycocyanin. However, in order to maintain a high content of phycocyanin while growing cyanobacteria, the present inventors have
It has been found that a nutrient substance containing a nitrogen source or a medium component may be added during the culture, and the present invention has been completed.

[0005]

Means for Solving the Problems The present invention comprises culturing and proliferating microalgae in an aqueous medium and extracting phycocyanin from the obtained algal cells to produce phycocyanin.
Any of the following steps, (1) increasing the concentration of all the medium components, (2) increasing the concentration of only specific components in the medium, (3) adding a medium in which the concentration of all the components is increased during the culture Or (4) a step of adding only a specific component in the medium during the culture, to a method for increasing the yield of phycocyanin.

[0006]

EXAMPLES The present invention provides a method for increasing the yield of phycocyanin in which the productivity of phycocyanin is markedly increased by growing microalgae and maintaining a high phycocyanin content per alga body.

The microalgae that can be used in the present invention may be any algae that produce phycocyanin, but cyanobacteria are preferable, and the American type culture collection (American) which is an international depository institution.
The well-known cyanobacteria deposited in the Type Culture Collection (hereinafter referred to as ATCC) or its variants or mutants are not limited, and marine and freshwater cyanobacteria isolated from nature can also be used. Moreover, you may use these algal bodies together by 1 type (s) or 2 or more types. For example, Synechococ
cus) genus, Synechocystis (Synechocystis) genus Spirulina (Spirulina) genus, Nostoc (Nostoc) genus,
The genus Fischerell , Phordium
midium) genus, Gureokapusa (Gloeocapsa) genus, Karosurikku (Calothrix) genus, Purekutonema (Plectonema)
Examples include cyanobacteria belonging to the genus Oscillatoria . Specific examples of such algae include ATCC 2 belonging to the genus Synechococcus sp .
7144, ATCC 27192, ATCC 27180, ATCC 294
04 or ATCC 29534, Synec
ATCC 27150 or ATCC belonging to hocystis sp .
27266, AT belonging to the genus Spirulina sp .
CC 29541 or ATCC 29542, Nostoc
oc sp .) ATCC 27904, genus Fischerella ( Fi
schirella sp .) belonging to ATCC 27929 or ATC
C 29161, ATCC 29409 belonging to the genus Phormidium sp ., And genus Gloeocapsa sp .
Belongs to ATCC 27269, Calothrix
AT CC 27901 belonging to sp .), Plecto nema ( Plecto
nema sp.) belonging to ATCC 18200, Osushiratoria the genus
ATCC 27906 and the like belonging to ( Oscillatoria sp .).

The medium component for maintaining and growing cyanobacteria used in the present invention may be any known medium for cyanobacteria that is commonly used for culturing cyanobacteria, for example, Table 1
BG-11 medium shown in 2 or C medium of National Institute for Environmental Studies can be used. Further, in the case of seawater or halophilic cyanobacteria, 0.1 to 5% of NaCl may be appropriately added, and in the case of algae requiring vitamin B ₁₂ ,
Vitamin B ₁₂ of 1 to 50 μg / l may be added.

[0009]

[Table 1]

[0010]

[Table 2]

The present invention relates to N in a medium for culturing cyanobacteria.
The phycocyanin content is maintained high by increasing the concentration of the source material or the medium containing them, or by adding the N source material in the medium or the medium containing them several times during the culture to increase the phycocyanin yield. Is to measure.

The N-source substance may be any substance as long as it supplies nitrogen into cyanobacteria in the assimilation action of cyanobacteria. Specific examples of such an N-source substance include NaNO ₃ and
Examples thereof include NH ₄ NO _{3 and} KNO ₃ .

When the concentration of the N source substance in the medium or the medium containing them is increased and the culture is carried out, the concentration of the N source substance is 0.5 to 12.5 g / l, preferably 0.5
Culture may be performed using a medium prepared to have a concentration of 2.5 g / l. For example, when BG-11 medium is used, it is 2 to 10 times, preferably 2 to 10 times the standard medium concentration.
Culture may be performed at a 5-fold concentration.

When the N-source substance or the medium containing them is added several times during the culture, the final total amount of the N-source substance added is nitrogen every 2 to 7 days after the start of the culture. As an element, it may be added several times, preferably 2 to 5 times, so as to have an amount of 0.5 to 12.5 g / l.

However, the salt concentration in the seawater medium is 1 to
It is preferably doubled.

Cyanobacteria may be cultured by a usual method. Although light irradiation is required during culture, sunlight, halogen lamps, fluorescent lamps, incandescent lamps, etc. are used. Light during cyanobacterium culture may have an intensity of 4 to 200 μEinstein / m ² / sec, preferably 5 to 60 μEinstein / m ² / sec, but it should be weakened at the beginning of the culture and adjusted according to the growth of cyanobacteria. You may strengthen it. The growth rate can also be increased by appropriately blowing carbon dioxide into the culture solution to increase the amount of carbonate ions in the solution.

For high-density culture, a semipermeable semipermeable membrane or dialysis membrane or the like is used, or a centrifuge is used to collect algal cells, and a part of the medium is removed, followed by N source. It is preferred to add a solution or medium.

The cultured algal cells may be filtered or by using a centrifuge in a conventional manner, for example, 3000 rpm, 15 rpm.
Centrifuge for about a minute.

Phycocyanin is produced in the cultured alga body, and the extraction and content of phycocyanin are measured as follows.

30 algae suspensions sampled over time
Centrifuge at 00 rpm and 4 ° C for 15 minutes to discard the supernatant and obtain an algal suspension. The algal suspension is washed with the same amount of water as the obtained algal suspension, and then freeze-dried. Lyophilized algal cells (10 mg) are suspended in 1 mg / ml lysozyme solution (0.1 M phosphate buffer, pH 6.5) and shaken at 30 ° C. for 3 to 6 hours in the dark to lyse the cells. This was centrifuged at 3000 rpm for 15 minutes at 4 ° C, and the resulting supernatant extract was adjusted to 615 nm.
The phycocyanin content per dry alga is determined by measuring the absorbance at 652 nm.

Next, the method of the present invention will be specifically described by way of examples, but the invention is not limited to the following examples.

Comparative Example 1 AT belonging to the genus Synechococcus sp .
CC27144 in BG-11 medium at 25 ° C under fluorescent light 40μ
The cells were cultured at Einstein / m ² / sec. After the start of the culture, a part of the culture solution was withdrawn with time, centrifuged at 3000 rpm, 4 ° C. for 15 minutes to collect algal cells, washed with water and freeze-dried. The algal growth was evaluated by this dry weight.
Furthermore, 10 mg of this freeze-dried algal cells was added to 10% of 0.1% lysozyme solution.
ml (0.1 M phosphate buffer containing 0.1 M Na-EDTA, p
The cells were suspended in H6.5), shaken at 30 ° C. for 4 hours in the dark, and lysed. The phycocyanin content was determined by centrifuging this in the same manner as above, separating it into a precipitate and an extract, and measuring the absorbance of the obtained extract at 615 nm and 652 nm. The results are shown in Fig. 1. The phycocyanin content per unit dry algal weight reached a maximum on the third day after the start of the culture, and thereafter, the phycocyanin content per unit dry algal weight decreased with the growth of algae.

After 3 weeks of culturing, the concentration of dried algal cells was 2.0 g / l,
The phycocyanin content was 7.1% and the phycocyanin produced was 142 mg / l.

Example 1 AT belonging to the genus Synechococcus sp .
After culturing was started in the same manner as in Comparative Example 1 using CC 27144, the culture solution was diluted to 1/4, 1 and 4 days 4, 7, 11 and 16, respectively.
/ 3, 1/2, 1/2 each, collected alga bodies by centrifugation, returned the alga bodies to the incubator again, and collected the medium containing BG-11 medium at double concentration And added the same amount. twenty one
The culture was stopped on the day, and the culture solution was treated in the same manner as in Comparative Example 1,
The amount of dried algal cells and the content of phycocyanin were measured to determine the amount of phycocyanin produced. The concentration of dry algal cells was 2.1 g / l, the phycocyanin content was 18.8%, and the phycocyanin produced was 395 mg / l. Compared with the results of Comparative Example 1, the production amount of the method of the present invention was remarkably improved by 2.8 times.

After culturing was started in the same manner as in Comparative Example 1, a part of the culture solution was withdrawn over time to determine the phycocyanin content (%) per dried alga. The results are shown in Fig. 1.

Examples 2-9 In Example 1, the genus Synechococcus
sp .) was replaced with ATCC 27144 belonging to Table 3 and the same procedure as in Example 1 was carried out except that the cyanobacteria in Table 3 was used, and the results in Table 3 were obtained.

[0027]

[Table 3]

Example 10 As in Comparative Example 1, B was measured 4, 7, 11, 16 days after the start of culture.
A medium containing G-11 medium at a double concentration was added by 1/4 volume of the culture solution, the culture was stopped on the 21st day, the culture solution was treated in the same manner as in Comparative Example 1, and the amount of dried algal cells and phycocyanin content were changed. I asked. The culture solution has increased about 2.4 times, and the concentration of dried algal cells is
The phycocyanin content was 0.91 g / l, the phycocyanin content was 15.5%, and the phycocyanin production was 141 mg / l.

Example 11 Example 1 was repeated except that the medium of BG-11 medium containing only NaNO _{3 in} double concentration was added instead of the medium containing BG-11 medium of double concentration. I went the same way. The culture was stopped on the 21st day and the concentration of dried algal cells was 2.0 g /
1, phycocyanin content 12.8%, phycocyanin production
256 mg / l was obtained.

Example 12 and Comparative Example 2 Synechocystis sp . ATCC 2
7266 was used to make the BG-11 medium a 3-fold concentration, and 2% (w / v) NaCl and 10 μg / l vitamin B ₁₂ were added to the medium.
Culture was performed in the same manner as in Comparative Example 1 except that was added. As Comparative Example 2, Synechocystis s
p .) ATCC 27266 in BG-11 medium at 2% (w
/ V) NaCl and 10 μg / l vitamin B ₁₂ were added, and the same culture as in Comparative Example 1 was performed. The results are shown in Table 4.

[0031]

[Table 4]

Example 13 Synechocystis sp . ATCC 2
Using 7266, culturing was performed in the same manner as in Example 12 except that NaNO ₃ in the BG-11 medium was used at a 3-fold concentration. Cultivation was stopped on the 21st day, and a dry algal cell concentration of 1.8 g / l and a phycocyanin content of 11.5% phycocyanin production amount of 207 mg / l were obtained.

[0033]

INDUSTRIAL APPLICABILITY According to the method of the present invention, the phycocyanin content per alga body can be remarkably increased, and the phycocyanin yield can be increased, so that the phycocyanin production cost can be remarkably reduced.

[Brief description of drawings]

FIG. 1 is a diagram showing changes in phycocyanin content per weight of dry algal cells in culture of Synechococcus sp . ATCC 27144 described in Comparative Example 1 and Example 1 over time in culture.

Claims (3)

[Claims] 1. A microalgae is cultured and grown in an aqueous medium,
A method for increasing the yield of phycocyanin, which comprises the step of any one of the following in producing phycocyanin by extracting phycocyanin from the obtained algal cells. (1) A step of increasing the concentration of all medium components, (2) A step of increasing the concentration of only specific components in the medium, (3) A step of adding a medium in which the concentration of all the components is increased during the culture, or (4) Culture A step of adding only specific components in the medium in the middle of. 2. The method for increasing the yield of phycocyanin according to claim 1, wherein the microalgae are cyanobacteria. 3. The method for increasing the yield of phycocyanin according to claim 1, wherein the specific component is a substance containing N element or a medium.