CZ2017331A3 - A production strain of the Bracteacoccus bullatus alga for the production of oils containing essential unsaturated fatty acids, a method of producing these oils and the use of this strain for the industrial production of these oils - Google Patents

Abstract

The production strain deposited in the Collection of Autotrophic Organisms of the Institute of Botany AS CR, Třeboň, Dukelská 135, under the accession number CCALA 1120 :, strain Cepák et Lukavský 2011/13 is usable as a source for industrial production of oils with high content of polyunsaturated fatty acids, especially linoleic acid and α-linolenic acid, oleic acid and cis-vaccenic acid, for human, animal, or other food supplements. The invention also discloses a method of producing high unsaturated fatty acid oils in a pilot plant.

Description

Technical field

The present invention relates to a production strain of Bracteacoccus bullatus algae producing oils with a high content of polyunsaturated fatty acids.

BACKGROUND OF THE INVENTION

Essential fatty acids are indispensable for the proper functioning of the body, but humans and most mammals cannot synthesize these substances. Strictly speaking, only two fatty acids are essential, ie α-linolenic acid - polyunsaturated omega-3 (ω-3) and linoleic acid - polyunsaturated omega-6 (ω6). Conditionally essential, however, is the majority of polyunsaturated fatty acids, or PUFAs, ie Unsaturated Fatty Acids, which contain more than one double bond in their carbon chain. These acids not only serve as a source of energy, but are precursors of biologically active substances, such as thromboxanes or prostacyclins, blood coagulation control agents.

According to the chemical composition, respectively. There are several groups in the structure, ω-3 Fatty acids contain a double bond on the third carbon atom from the end of the hydrocarbon chain. These include α-linolenic acid with the abbreviation ALA, ie 18: 3ω-3, which contains eighteen carbon atoms and three double bonds, and hexadecatetraenoic acid, ie 16: 4ω-3, which contains sixteen carbon atoms and four double bonds bonds, ω-6 Polyunsaturated fatty acids have the last double bond at position ω-6, which means the sixth carbon from the methyl end. These include mainly linoleic acid with the abbreviation LA, ie 18: 2ω-6, which contains eighteen carbon atoms and two double bonds, and γ-linolenic acid with the abbreviation GLA, ie 18: 3ω-6, which contains eighteen carbon atoms and three double bonds.

The polyunsaturated α-linolenic acid is biosynthesized by a cascade of oleic acid reactions using the delta 12- and delta 15-desaturases enzymes. It is part of some so-called drying vegetable oils. The polyunsaturated hexadecatetraenoic acid is biosynthesized from palmitoleic acid with the aid of desaturase enzymes. The only source of this acid is only lower photosynthetic microorganisms, especially sea or freshwater algae, where the acid content reaches tens of percent. These essential substances are necessary as a supplement to healthy human nutrition as well as in addition to feeding fish in aquaculture or zooplankton, which then serves as fish food.

Oleic acid is a higher monounsaturated acid that contains one double bond in its molecule and is present in various animal fats and vegetable oils. This acid constitutes, for example, 55 to 80% of olive oil. Cocoa beans contain 35%. It is a precursor of the synthesis of essential α-linolenic acid, another important potential component of nutritional products. Another important monounsaturated fatty acid is cis-vaccinic acid, which is commonly found in ruminant fat, mammalian milk, butter and yoghurt. Polyunsaturated fatty acids are the main factor in the beneficial effects of the so-called Mediterranean diet, which includes eg sea fish. However, the fish themselves do not form them, but receive them through zooplankton from marine phytoplankton. However, in this handover, there are regular losses in the various stages of the food chain, and therefore it is advantageous to skip these intermediate steps. For vegans, vegetarians and those who do not like to eat fish, there is also the possibility of substitution in the form of quality food supplements.

Fatty acids are valuable products, but usually individual organisms mainly produce only one group of fatty acids depending on their biosynthetic capabilities. However, the content of the desired substances is only one of the conditions for the success of biotechnological production. The outdoor cultivation organism must furthermore easily grow in the plant, it must have some resistance to technological treatment, such as shearing forces (mechanical influences) generated during pumping, it must tolerate temperature and light intensity oscillations, etc. Costly provisioning is not necessary for outdoor cultivations. thermal and light energy, because the sun delivers energy in a natural way. In temperate conditions, however, outdoor algae cultivation can only be operated for a limited period of the year, when temperatures and sunlight are high enough for the growth of these microorganisms. Therefore, new organisms with advantageous properties are constantly sought and tested.

Algae have great potential for use in biotechnology due to their high growth rate and the possibility of industrial cultivation in suspension cultures that are easy to operate and automate. In addition, they can be cultivated in locations unsuitable for conventional crops because they do not require fertile soil, eg in deserts, roofs of industrial buildings, etc. In cultivators with fully artificial light, they can then be grown anywhere. There are known solutions using strains of algae for the production of oils containing unsaturated fatty acids, where, for example, algae of the genus Monoraphidium are used to produce

Similarly, CN 105296553 describes a method of cultivating an algae of the genus Monoraphidium to produce another acid, namely fulvonic acid used subsequently for the preparation of biodiesel. The disadvantages of these solutions are, in particular, that these algae produce only one or two types of fatty acids and the cultivation of these algae is therefore used to produce oils containing one or two specific fatty acids.

With the change in diet in recent centuries, the type of fatty acids consumed has also changed. The rapid development of society, especially Western civilization, after World War II also led to a change in the consumption rates of ω-6 and ω-3 PUFA, mainly due to the large consumption of vegetable oils. The use of vegetable oils has shifted the ratio from the ideal ω-6 / ω-3 ratio of 1: 1 to 1: 0.25 as recommended by the World Health Organization (WHO) to almost unbelievable 1: 0.1 to 1: 0.03, typical for European and US residents. This change in ratio brings with it a number of complications, mainly due to an increased incidence of cardiovascular disease, an increased risk of cancer and autoimmune diseases, mental disorders, memory impairment in the form of Alzheimer's disease, developmental retardation, visual disturbances, muscular weakness and tremor, dyslipidemia , insulin resistance, high blood pressure, thrombosis, diabetes, asthma, overweight, etc.

To confirm the above, we present examples of ratios of ω-6 and ω-3 PUFA in some biotechnologically cultivated algae based on Lang et al. 2011 (DOI: 10.1186 / 1471-2229-11-124): For commonly large-scale cultured algae such as Chlorine, the ratio of ω-6 / ω-3 is 1: 0.40, Parachlorella kessleri (formerly Chloreila kessleri) 1: 0.19, Scenedesmus 1: 0.45. In cyanobacteria, the ratio of these fatty acids ranges from 1: 0.30 -Spirulina (Arthrospira) to cases where the cyanobacteria produces no ω-3 PUFA. Thus, they have a much worse ratio and almost similar to conventional vegetable oils such as rapeseed 1: 0.5, soybean 1: 0.14, olive 1: 0.07 or sunflower oil that does not contain ω-3 PUFA.

SUMMARY OF THE INVENTION It is an object of the present invention to find a suitable organism which is capable of producing oils with a high content of unsaturated fatty acids when cultivated in open systems in central European climatic conditions throughout the year, which is advantageously able to grow even at lower temperatures and lower radiation. it was able to produce oils with a high content of polyunsaturated fatty acids even during the cold period when common cyanobacteria - 4 - and algae practically did not grow, and which would grow autotrophically and be sufficiently resistant to contamination by other algae, cyanobacteria and bacteria.

SUMMARY OF THE INVENTION

This object is solved by the strain of algae Bracteacoccus bullatus, which in the sense of the present invention is a production microorganism for obtaining oils with a high content of unsaturated fatty acids. The production strain of algae Bracteacoccus bullatus is deposited in the collection of the Institute of Botany AS CR, Třeboň, Dukelská 135, under the accession number CCALA 1120: Bracteacoccus bullatus strain Cepák et Lukavský 2011/13. The invention further relates to the industrial use of this strain and to a process for producing oils and to the content of unsaturated fatty acids in an industrial strain of this algae.

This strain preferably produces oils in which, of the total fatty acid content, unsaturated fatty acids are 53% w / w. These are considered essential ie necessary or at least favorable for the health of vertebrates including humans. In a preferred embodiment, the strain produces an oil with a content of greater than 18.3% by weight. % linoleic acid, greater than 17.4 wt. % α-linolenic acid, greater than 22.6 wt. % of oleic acid and greater than 2.4 wt. of acetic acid. Monounsaturated vaccine acid reduces both total and LDL cholesterol and triglycerides. Monounsaturated oleic acid is a precursor of α-linolenic acid. Linoleic acid and α-linoleic acid are polyunsaturated essential fatty acids. The strain Bracteacoccus bullatus CCALA 1120 contains the PUFA ω-6 / ω-3 at a ratio of 1: 1.16, ie it is relatively close to the ideal 1: 1 ratio recommended by WHO.

Cells of the Bracteacoccus bullatus CCALA 1120 strain are shown in Figure 1 as seen by light microscopy. They are spherical, sparsely pear-shaped, as shown in Fig. 1d, with an adult cell diameter of 15 to 17 µm, as shown in Figs. 1a to 1e and 1i, where Fig. 1b shows young cells, and 1a and 1e are aplanospores. Chloroplasts are about ten in an adult cell, they are disc-like, wall-like, without a pyrenoid ₍ as shown in Figure 1c. Reproduction is the formation of a larger number of zoospores, which can be up to 128 as shown in Figures 1d, 1f, 1j, spherical, oval to tear-shaped, with two identical apex-based flagella, the papilla is bland, a tiny spherical, red eye patch is deposited in the back of the cell on a wall-mounted chloroplast having a number of 1-2 patterns, as shown in Figs. 1g and 1h. There are two pulsating vacuoles in the apex, which are located in the apex, and the zoospores begin to move inside the mother cell and sometimes relax and swim actively.

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A pairing tendency was observed, so they can also function as gametes as shown in Figures 11 and 1m. After some time of movement within the parent cell, the zoospores cease to move as represented in Fig. 1j, lose the flagella, turn round and gradually turn into aplanospores, that is, reproductive cells that have zoospore appearance still have red eye spots and pulsating vacuoles for some time, but they are immobile because they have lost the flagella, as shown in Figures 1a and 1e. They then lose both the eye patch and the pulsating vacuoles and resemble their parent cell and can therefore be considered as autospores, i.e. asexual reproductive cells morphologically similar to the parent cell with a diameter of 3 to 5 µm in Fig. 1i. Zoospores, aplanospores, autospores, adult cells merge smoothly. They then grow to a size of 15 to 17 µm. Dark red dyes are gradually formed in the old cells.

Based on analysis of the nuclear molecular marker ITS2 rDNA, the CCALA 1120 strain was identified as Bracteacoccus bullatus. ITS2 - 'internal transcribed spacer' - is a non-coding region between two coding regions 5.8S and 26S rDNA and is used as a suitable marker for the study of closely related species according to Coleman 2003 (DOI: 10.1016 / S0168-9525 (03) 00118-5). Figure 2 shows a comparison of the secondary structure of the ITS2 rDNA transcript of CCALA 1120 strain with other closely related 14 strains of Bracteacoccus bullatus included in the study by Fucik et al. (2012) (DOI: 10.1127 / 0029-5035 / 2012/0067). Nucleotide changes of these strains to CCALA 1120 strain are shown by features along the secondary structure. If the change occurred only in certain strains, the affiliation is indicated by a small italic letter (a - KF10, b - SAG 2032, c - KF65, d - KF22, e - KF72, f - KF34, g - KF3, h - CCALA 69). The ITS2 sequence was identical between KF65 strains,

SAG 2317, UTEX 345, BCP-CNP2-VF8, BCP-UT8-26, and Broady 420, all thus designated c. Furthermore, this sequence was identical between the KF3 and BCP-CNP1-VF10 strains, collectively referred to herein as g. Secondary structure The ITS2 of the compared strains showed typical features for eukaryotic organisms, namely the presence of four helices (I to IV) and the pyrimidine base-ipyrimidine base mismatch on the second helix, indicated by arrows. - The secondary structure of ITS2 is associated with the so-called CBC generic concept (Coleman 2000, DOI: 10.1078 / 1434-4610-00002). CBC, or compensatory base change, is a change on both sides of a structure that provides nucleotide pairing. No CBCs were found for the strains of Bracteacoccus bullatus, suggesting that all these strains are one species.

Most of the strains of the genus Bracteacoccus bullatus were isolated from the soil, the strain Bracteacoccus bullatus CCALA 1120 was isolated from the snow in the Sierra Nevada mountains in Spain in 2011. Bracteacoccus bullatus belongs to the class Chlorophyceae, ie the class of green algae where oil or starch. The total oils content in the biomass of intensively growing Bracteacoccus bullatus culture is over 30% in dry matter, unsaturated fatty acids form

45.8% of the total fatty acid. The course of the temperature of the culture medium and the intensity of photosynthetically active radiation or FAR in the wavelength range 400 to 700 nm are shown in Fig. 3. Bracteacoccus bullatus grows in a wide range of temperatures and radiation, short-term survival of -1 ° C and +35 ° C.

The present invention also relates to a process for producing oils containing unsaturated fatty acids, in particular linoleic acid, α-linolenic acid, oleic acid and vaccinic acid in a production strain of Bracteacoccus bullatus CCALA 1120. The present invention is characterized in that the production strain of algae is cultivated in temperature. in the range of 5 to 30 ° C, preferably in the temperature range of 10 to 16 ° C. Since the given strain of Bracteococcus bullatus CCALA 1120 was obtained from snow, its cultivation is possible even at low temperatures. The strain is psychrotolerant, which means that short-term cooling even below 0 ° C does not bother him. A typical example is the spring frosts that algae will survive without difficulty, unlike Chlorella algae and similar cultures that freeze at temperatures below 0 ° C. The reason for this resistance is apparently a high content of unsaturated fatty acids in the cell.

The advantages of the Bracteacoccus bullatus CCALA 1120 production strain according to the present invention lie mainly in the ability to produce oils with a high content of polyunsaturated and monounsaturated fatty acids, when cultivated in open systems in Central European climatic conditions throughout the year, ie the ability to grow at lower temperatures and lower radiation . The production strain Bracteacoccus bullatus CCALA 1120 is able to produce unsaturated fatty acids even during the cold period when common cyanobacteria and algae practically do not grow, grows autotrophically and are sufficiently resistant to contamination by other algae, cyanobacteria and bacteria. Its use can extend the cultivation season to the relevant industrial plant. At the same time, this strain is resistant to higher temperatures and therefore there is no danger of dying during temperature oscillation on a sunny winter day.

Literature

Coleman A. W (2000): The significance of coincidence between evolutionary landmarks found in mating affinity and DNA sequence. - Protist 151: 1-9. DOI: 10.1078 / 14344610-00002

Coleman, A.W. (2003): ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends Genet 19: 370-375. DQI: 10.1016 / S0168-9525 (03) 00118-5

J

Fučíková, K., Flechtner, V.R. & Lewis, L.A. (2012): Revision of the genus Bracteacoccus

Tereg (Chlorophyceae, Chlorophyta) based on phylogenetic approach. - Nova Hedwigia

96: 15-59. DOI: 10.1127 / 0029-5035 / 2012/0067

Lang, I., Hodac, L., Friedl, T. & Feussner, I. (2011): Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. - BMC Plans Biology 11: 124. DOI: 10.1186 / 1471-2229-11-124

Clarification of drawings

The present invention will be explained in more detail in the following drawings, where:

Figure 1 shows the different life cycle stages of the Bracteacoccus bullatus strain CCALA 1120 J2.

observed under a light microscope; and pulsating vacuole aplanospora; b young cell; c - adult cell; d - maternal adult pear-shaped cell dividing into zoospores; e-aplanospores with a light spot; f - a cell with zoospores; g, h - zoospores; i - autospores; j - adult cell with zoospores; k zoospora; I, m - coupling of gametes; the 10 µm scale applies to all images except g, h, where the dimensions are 3x6 µm; Fig. 2 shows a comparison of the secondary structure of the ITS2 rDNA of CCALA 1120 transcript with other strains within Bracteacoccus bullatus; media during outdoor cultivation, March 22 to May 1.5. Fig. 5 shows the table of fatty acids and lipids content in the pilot plant cultivation of Bracteacoccus bullatus CCALA 1120, Fig. 6 shows the table of the composition of the stock solutions for outdoor cultivation. preparation of Z8 medium, Fe-EDTA solution and Gaffron's solution of microelements, Fig. 7 shows a table of composition of stock solutions for preparation of SS1 / 2 medium and microelement solution.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the specific embodiments of the invention described and illustrated below are presented by way of illustration and not by way of limitation of the invention to the examples given. Those skilled in the art will find or will be able to provide, by routine experimentation, more or less equivalents to the specific embodiments of the invention described herein. These equivalents will also be included within the scope of the following claims.

Preparation of inoculum

The strain Bracteacoccus bullatus CCALA 1120 is kept in the Collection of Autotrophic Organisms of the Institute of Botany of the ASCR in Třeboň as a growing culture in a tube on sloping agar with medium Z8, at about 15 ° C, under fluorescent lighting 46 pmol.m ~ ² .sec ^-1 . From this culture, the strain is pre-grown by batch, stationary culture on Z8 liquid medium, the composition of which is shown in the table in Fig. 6, in 2L glass bottles. To 700 mL of distilled water is added 10 mL of the individual microelements of FIG. 6 of the stock solutions, 0.2 mL of Fe-EDTA solution and 0.08 mL of Gaffron's microelement solution, the composition of which is also shown in Fig. 6. The entire mixture is then made up to 1000 mL with distilled water. Two-liter bottles initially contain 250 mL of medium, and gradually, after thickening of the suspension, the volume is replenished to a total volume of 2000 mL. Mixing of the suspension and carbonation is ensured by bubbling excess air with 2% CO2 by means of a tube introduced into the bottom of the bottle, the aeration mixture is filtered through a Milipore membrane bacterial filter (Midisart 2000, PT FE IN) with pores of 0.2 µm. The bottles are placed in a water bath where the temperature is kept below 10 ° C by a compressor cooling circuit. The glass wall is continuously illuminated from the sides by LED panels with FAR intensity with energy of 180 pmol.m ^-2 .sec · ¹ , which represents about one fifth of solar intensity. At the beginning of cultivation and after every increase of light intensity for the first 5 days reduced to 90 pmol.m ^_2 .s ^-1 until culture visibly thickened.

Pilot cultivation - -

The pilot plant Bracteacoccus bullatus CCALA 1120 was grown on a platform of BCS Engineering Brno, located in the greenhouse of the Institute of Botany in Trebon, ie in the climatic conditions of Central Europe. The greenhouse was only heated if the internal temperature dropped below 8 ° C. It is a platform cultivator with an inclined slope of 12 m length and 1 m width, gradient 15 cm, volume 150 L. This type is designed for very thick suspensions in a thin layer up to 10 mm. The cultivation from the collection culture is carried out analogously to that described above.

The grown inoculum was transferred completely into 150 L SS1 / 2 medium, which is optimal for higher nutrient content for industrial cultivation, its composition is shown in the table in Fig. 7. For large-scale cultivation on the platform, weighed chemicals can be poured directly onto the platform individually dissolved in tap water with open CO2 flow. CO2 is fed into the medium by introducing it in front of a centrifugal pump for rapid absorption of algae suspensions and its concentration is regulated to 5 L.hr ^-1 . Throughout the cultivation, suspension temperature and FAR intensity are recorded at 10 minute intervals using Minikin dataloggers (EMS, Brno).

The algae was grown on a pilot scale on 22 March to 1 May 2017. Fig. 3 shows the temperatures of the culture medium and the intensity of photosynthetically active FAR radiation. The temperature of the suspension varied between 5 and 30 ° C and 35 ° C, respectively, and was usually around 15 ° C, the arithmetic mean being 15.8 ° C. FAR values oscillated in the range of 0 to 1000 pmol.m- ² .sec · ¹ or 1700 pmol.m- ² .sec · ^{1, respectively} . Giant. 4 shows the course of a growth curve in outdoor culture. The growth curve was evaluated daily by measuring the optical density at 750 nm with a Shimadzu UV-1650 PC in blooms with a suspension thickness of 1 cm, possibly after dilution. The grown biomass was harvested by centrifugation in an EVODOS 10 centrifuge at 7000 rpm, frozen to -20 ° C and later lyophilized or vacuum defrosted at 0.05 hPa. Harvesting, total 1271 g dry weight after 40 days, i.e., 1.76 gm ^_2 .day ^-1.

For the volume of the suspension is 0.14 g. L ^-1. day ' ¹ . The content of unsaturated fatty acids determined in the dry matter of the strain Bracteacoccus bullatus CCALA 1120 is shown in the table in Fig. 5. At the content of polyunsaturated fatty acids, linoleic (18.3%) and α-linolenic (17.4%) of the total amount of fatty acids ( 30% of dry matter), their production was ALA = 0.15 and LA = 16 gm @ ^{2 / day} . Microscopic analysis has shown that algae well tolerate mechanical stress by a centrifugal pump, so it can be grown in conventional types of cultivators that are already commonly used for commercial production of algae and cyanobacterial biomass such as Spirulina or Chlorella. - ·. ·;

Biomass dry matter

Biomass dry weight is determined by gravity centrifugation of the sample for 20 min at 3000 µg in 2 mL plastic, Eppendorf preweighed tubes. The sediment is dried at 105 ° C to constant weight and weighed. _z . \ (h jC. 7

10Identification of fatty acids

100 mg of lyophilized biomass is saponified with 10% KOH in methanol at room temperature overnight. Neutral and babic compounds are shaken from diethyl ether from the pH 9 solution and the aqueous fatty acid solution is acidified to pH 2 and the acids are subsequently extracted into hexane. Fatty acids are methylated with BF3-methanol and identified by GC-MS, ie gas chromatography with mass spectrometry, with an ion trap detector with electron impact ionization. The sample is injected into a capillary column with a polar stationary phase, 25 m * 0.25 mm χ 0.1 µm and a temperature gradient of 5 min at 50 ° C is used for elution, heating the column at a rate of 10 ° C min ^-1 to 240 ° C and isothermally at 240 ° C for 15 min. The carrier gas is helium with a flow rate of 0.52 mL.min ^-1 . All spectra are scanned in the 50 to 600 Da range. The structure of methyl esters is determined based on retention times, fragmentation, and comparison of mass spectra with mass spectra of commercially obtained standards.

Industrial applicability

The breeding strain of Bracteacoccus bullatus CCALA 1120 according to the present invention can be used to produce oils with a high content of polyunsaturated fatty acids, in particular linoleic acid and α-linoleic acid and monounsaturated fatty acids, in particular oleic acid and vaccinic acid, at normal and low culture temperatures, addition of human and animal diet, fish and zooplankton feed in aquaculture or cosmetics.

Claims (6)

PATENT CLAIMS 1. A production strain of algae Bracteacoccus bullatus for the production of oils containing unsaturated fatty acids deposited in the Collection of Autotrophic Organisms of the Institute of Botany AS CR, Třeboň, Dukelská 135, under the accession number CCALA 1120 Bracteacoccus bullatus, strain Cepák et Lukavský 2011/13. The seaweed production strain of claim 1, wherein the total sum of unsaturated fatty acid fractions in the oil produced is greater than 53% by weight. The seaweed production strain according to claim 1, wherein the produced oils contain more than 18.3 wt. % linoleic acid, more than 17.4 wt. % α-linolenic acid, more than 22.6 wt. % oleic acid and more than 2.4 wt. of acetic acid. Use of a production strain of Bracteacoccus bullatus algae according to one of claims 1 to 3 for the production of oils containing polyunsaturated fatty acids, in particular linoleic acid and α-linolenic acid, oleic acid and vaccine acid. A process for producing oils containing unsaturated fatty acids, in particular linoleic acid, α-linolenic acid, oleic acid and vaccinic acid in an algae production strain according to any one of claims 1 to 3, characterized in that: The method according to claim 1, wherein the algae production strain is cultured in a temperature range of 5 to 30 ° C. The method according to claim 5, characterized in that the algae production strain is preferably cultivated in a temperature range of 10 to 16 ° C.