RU2322489C1 - Arthrospira platensis (nordst.) geitl. 1/02-t/03-5 strain as producer of protein biomass - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes the strain Arthrospira platensis (Nordst.) Geitl. 1/02-T/03-5 isolated form the clone culture Arthrospira platensis 1/02. The strain is stored in collection of NIL VIE geographic faculty of Moscow State University. The strain is characterized by the enhanced yield of protein biomass wherein the mass part of protein is 65.6%.

EFFECT: increased content and yield of protein.

3 tbl, 3 dwg, 5 ex

Description

The invention relates to microalgal biotechnology and is a new strain of blue-green alga / cyanobacterium Arthrospira platensis (Nordst.) Geitl, which has not been described previously and is stable in morphological characteristics. 1/02-T / 03-5 (collection of the Research Laboratory of Renewable Energy Sources (RES) of the Geographical Department of Moscow State University), which can be used to produce biomass for food, feed, medical, pharmaceutical purposes.

и т.д. Представителен список публикаций, касающихся масштабов использования спирулины (коммерческое название биомассы A.platensis) по различному назначению [1].The biomass value of various A.platensis strains for these purposes is determined by the high content of easily digestible protein, including all essential amino acids, carbohydrates, lipids, polyunsaturated fatty acids (especially a large number of valuable linoleic and γ-linolenic acids), a wide range of B vitamins, and β- carotene, phycocyanin, chlorophyll

etc. A representative list of publications on the extent of use of spirulina (the commercial name for A.platensis biomass) for various purposes is representative [1].

A distinctive feature of the strains used to produce spirulina biomass on an industrial scale, including the A. platensis 1/02 strain (collection of the NIL RES), is their high polymorphism, which manifests itself both under stationary cultivation conditions and is a consequence of altered physicochemical growing conditions [2, 3, 4]. The geometry of the spiral undergoes a change, up to the appearance of direct trichomes, which can comprise from 10 to 100% biomass [5, 6]. The appearance in cultivators of direct variants of trichomes creates certain difficulties in the technological process of obtaining biomass associated with the establishment of algological purity of the culture [7], the deterioration of the filterability of the culture fluid [5]. Maintaining crops in the collection requires frequent reseeding, with the complete replacement of spiral forms with straight ones.

The subject of the invention is A.platensis strain 1/02-T / 03-5 of a spiral shape different from that of the original A.platensis 1/02 strain, during which long trichomes, as well as any other morphological variants, were not observed during long-term cultivation and long-term storage. . At the same time, all the beneficial properties of biomass inherent in A.platensis pcs. 1/02 are preserved (table 1).

The origin of the strain.

From the production facility of the Scientific Research Laboratory of RES, where Spirulina A.platensis (Nordst.) Geitl was grown. strain IPPAS B-287 (IGF RAS collection) with a high content of direct trichomes (up to 90%), two clonal cultures were isolated on Zarruk solid agar medium [8]: helical strain 1/02 and direct strain 1/02-P / 03 forms. Genetic analysis of isolated cultures and collection strain A.platensis (Nordst.) Geitl. strain IPPAS B-287 showed that all three cultures belong to the same species (table 2).

Table 2.
The genomic characteristics of strains of A.platensis (Nordst.) Geitl. Strain Genome Size, Daltons HZ, mol% DNA hybridization level,% pcs. 287 pcs. 1/02-P / 03 A.platensis pc. B-287 3.510 ⁹ 43.8 one hundred A.platensis pcs. 1/02 3.8 × 10 ⁹ 44.5 96 85 A.platensis pcs. 1/02-P / 03 3.7 · 10 ⁹ 44.0 92 one hundred

In the process of studying the natural variability of the clone culture of A.platensis pcs. 1/02 according to morphological characteristics, the strain A.platensis 1/02-T / 03-5 was obtained, which differs in spiral geometry from the original strain and is stable during long-term cultivation and storage in the collection according to morphology of trichome (three years of observation). Genetic analysis of both strains according to the results of ARDRA with two restriction enzymes showed their identity (figure 1).

Morphological features of the strain.

When growing strain 1/02-T / 03-5 in flasks on Zarruk liquid medium at a temperature of 24-30 ° С, illumination of 2.0 kl and periodic shaking, the culture is represented by weakly spiralized dark green trichomes, granular, without constrictions or with weakly expressed constrictions at transverse partitions. The width of the cell (trichome) is 8.5-10.5 microns; cell length - 2.5-4.0 microns; the inner diameter of the spiral is 7.0-8.5 microns; external - 15.0-19.0 microns; the distance between the turns of the spiral (the height of the spiral) is 36.0-57.0 microns. The length of the trichome is up to 500 microns, the number of turns of the spiral is 2.0-20.0, mainly 5-8 turns.

On the Zarruk agar medium (1.2% agar-agar), the culture is presented both as individual trichomes and in the form of bunches. Comparative data on the morphology of trichomes of the original strain 1/02 and strain 1/02-T / 03-5 are given in table 3 and figure 2.

When stored in liquid Zarruk medium in a refrigerator bar at a temperature of 12-14 ° C and illuminated with a 0.5 kl daylight lamp, the culture for 5 months without reseeding is represented by dark green trichomes granulated at a speed of 2 to 20.

A characteristic feature of strain 1/02-T / 03-5 is that the geometry of the trichome helix does not change during multiple transfers during prolonged cultivation in a liquid nutrient medium and long-term storage on liquid nutrient media for 3 years (observation time).

Reproduction.

Vegetative, cell division and the subsequent fragmentation of trichomes by rupture or in the area of non-credential cells. Under optimal conditions, the relative growth rate is 0.20 h ^-1 , which corresponds to a minimum doubling time of 5.0 hours.

Physiological properties of the strain.

The arthrospira strain 1/02-T / 03-5 is an autotroph, carbon source is sodium and potassium carbonates and bicarbonates, assimilates nitrogen in nitrate (sodium nitrate and potassium nitrate) and ammonia (urea, ammonium nitrate) forms. Mesophile: optimal growing temperature 26-32 ° C. The optimum pH is 8.5-10.5. Alkalization above 11.0 inhibits the growth and accumulation of biomass.

Zarruk medium [8] and its modifications are used for cultivation. The composition of the medium, ensuring the productivity of strain 1/02-T / 03-5 at the level of 0.9-1.5 g / l of dry biomass, includes (in g / l):

- NaHCO ₃ - 8.0-32.0,

- KNO ₃ - 1.0-6.0,

- K ₂ NRA · 3H ₂ O - 0.5-2.0,

- K ₂ SO ₄ - 0.5-2.0,

- MgSO ₄ · 7H ₂ O - 0.1-0.4,

- NaCl - 0.5-2.0,

- CaCl ₂ - 0.02-0.04

- FeSO ₄ · 7H ₂ O - 0.01-0.04,

- Trilon B - 0.04-0.16,

- trace elements (solutions A and B in 1 ml / l of medium).

Solution A (g / l):

- H ₃ VO ₃ - 2.86,

- MnCI ₂ · 4H ₂ O - 1.81,

- ZnSO ₄ · 7H ₂ O - 0.22,

- CuSO ₄ · 5H ₂ O - 0.08,

- MoO ₃ - 0.015.

Solution B (g / l):

- K ₂ Cr ₂ (SO ₄ ) ₄ · 24H ₂ O - 0.096,

- NH ₄ VO ₃ - 0.023,

- NiSO ₄ · 7H ₂ O - 0.048,

- Na ₂ WO ₄ · 2H ₂ O - 0.018,

- Ti ₂ (SO ₄ ) ₃ - 0.04,

- Co (NO ₃ ) ₂ · 6H ₂ O - 0.044.

The invention can be illustrated by the following examples.

Example 1

The clonal culture of A.platensis strain 1/02 (initial strain) was grown for 5 months in ten test tubes of size (20 × 2) cm with a volume of Zarruk medium of 15 ml under illumination of 2.0 kl, temperature 26-28 ° С and periodic shaking with reseeding on a fresh nutrient medium every two weeks. Microscopic examination of the state of culture in the Goryaev chamber after 2-week cultivation showed direct morphological variants in the amount of 0.1-0.3% after the 3rd passage; 1.0-3.0% after the 4th passage; up to 6.0% after the 5th passage; 12.0-20.0% after the 6th passage; 18.0-37.0% after the 7th passage; over 50.0% after the 10th passage (5-month cultivation). Microscopic analysis was performed on an Axioplan 2 Imaging microscope with an AxioCam MBm high-resolution camera and an AxioVision 3.1. Modular image processing and analysis system. The culture density at the end of the 2-week cultivation was 0.9-1.6 g / l dry for each passage biomass.

Example 2

A 2-week clonal culture of A. platensis pcs. 1/2, grown in ten test tubes of size (20 × 2) cm with a volume of Zarruk's medium of 15 ml under illumination of 2.0 cells, at a temperature of 26-28 ° C, was stored in a refrigerator a bar with a glass door at a temperature of 12-14 ° C and illuminated by a fluorescent lamp 0.5 clk. The density of the culture was 1.2-1.5 g / l of dry biomass. After three months of storage, direct variants were found in the amount of 1.5–2.0%, which were revealed by reseeding on fresh nutrient medium of Zarruk (10 vol.% Of seed) and growing in test tubes with periodic shaking at T 26–28 ° C and illumination of 2.0 cl for 2 weeks. After 5 months of culture storage, direct variants were detected in the initial culture by microscopy, and when reseeding on fresh Zarruk nutrient medium (10 vol.% Inoculum), direct variants were detected in an amount of up to 20% after two weeks of growing.

Example 3

The clonal culture of A.platensis strain 1/02-T / 03-5 was grown for 5 months in ten test tubes of size (20 × 2) cm with a volume of Zarruk's medium of 15 ml under illumination of 2.0 kl, temperature 26-28 ° С and periodic shaking with reseeding on a fresh nutrient medium every two weeks. The density of the culture at the end of the 2-week cultivation at each passage was 0.9-1.3 g / l of dry biomass. Microscopic examination did not reveal direct and other morphological variants in any of the passages. The geometry (character) of the spiral was maintained throughout the entire experiment.

Example 4

A 2-week clonal culture of A.platensis pcs. 1/02-T / 03-5, grown in ten test tubes measuring 20 × 2 cm in size with a volume of Zarruk medium of 15 ml under illumination of 2.0 cells, at a temperature of 26-28 ° С, was laid in storage in a fridge-bar with a glass door at a temperature of 12-14 ° C and illuminated by a fluorescent lamp 0.5 cl. The density of the culture was 0.9-1.3 g / l of dry biomass. During 5 months of storage, the culture was analyzed monthly microscopically directly, as well as after plating on fresh nutrient medium of Zarruk and growing under conditions similar to those described in example 2. During the entire shelf life of the culture, no direct or other morphological variants were found in it. The culture was morphologically stable.

Example 5

2-week-old clonal culture of A. platensis pcs. 1/02-T / 03-5, grown in ten test tubes of size (20 × 2) cm with a volume of Zarruk's medium of 15 ml under illumination of 2.0 kl, temperature 26-28 ° С, was stored in a refrigerator-bar with a glass door at a temperature of 12-14 ° C and lighting with a fluorescent lamp 0.5 klx. The density of the culture was 0.9-1.3 g / l of dry biomass. For 3 years of storage, every five months, the culture was analyzed microscopically and reseeded. Throughout the entire shelf life of the culture, no direct or other morphological variants were found in it. The culture was morphologically stable.

Information sources

1. R. Henrikson. Earth Food Spirulina. Kenwood, California, 1994.180 p.

2. Levin R.A. Uncoiled variants of Spirulina platensis (Cyanophyceae: Oscillatoriaceae), Arch. HydrobioL, Suppl. 60. Algol. Stud., 1980.26, P. 48-52.

3. Vrio Ciferri. Spirulina, the Edible Microorganism. Microbiological Reviews. Dec., 1983. Vol. 47, No.4. P.551-578.

4. N.Jeeji Bai. Competitive exclusion or morphological transformation? A case study with Spirulina fusiformis, Arch. Hydrobiol, Suppl. 71, Algol. Stud, 1985. 38/39, P.191-199.

5. Jourdan J-P. Grow your own spirulina. http://perso.orange.fr/petites-nouvelles/manuel/grow.htm

6. A.c. USSR No. 1347204. A01H 13/00, A23K 1/00, C12N 1/12 // C12N 1/12, C12R 1:89. 1985.

7.Sirenko L.A., Shevchenko T.F. Algological pollution of industrial crops Spirulina Turp. and difficulties in identifying them. Algology, 1996. V.6, No. 1. S.91-96.

8. Pinevich V.V., Verzilin N.N., Mikhailov A.A. The study of Spirulina platensis - a new facility for high-intensity cultivation. Plant physiology. 1970.V.17, Issue 5. S.1007.

Claims (1)

Strain Arthrospira platensis (Nordst.) Geitl. 1/02-T / 03-5 (collection of research laboratories RES of the geographical faculty of Moscow State University) - producer of protein biomass.

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