TWI589692B - Raphidocelis sp. and use thereof - Google Patents

Description

Microalgae and its use

The present invention relates to a novel microalgae isolate which can produce high amounts of 1,3-bisguanidinoglycerine (1,3-DAG) and C16~C18 fatty acids, so that the cultured product can be used as a health product. Raw materials for oils and raw fuels.

The microscopic microscopic size needs to be seen by the microscope, and it can exist in almost any environment. At present, it is estimated that there are between 200,000 and 800,000 microalgae species, but only about 35,000 species have been discovered. The idea of using microalgae to generate energy originated from the 1970 American algae biodiesel program, which has regained attention in recent years under energy shortages and the greenhouse effect. The biofuels produced by microalgae are now called third-generation energy to distinguish between the first generation of energy (materials for food crops) and the second generation of energy (materials are non-food crops). Microalgae has the advantages of rapid growth rate, high carbon dioxide utilization rate, high-density culture, small required land area, marine aquaculture, utilization of flue gas and wastewater, and a small chance of contamination by pathogens. It has simple cell structure and lack of cell differentiation, is simpler in genetic engineering operation than plant cells, and has a similar glycation post-translational modification mechanism to facilitate human manipulation of eukaryotic gene expression (Yen, HW, et al .2013.Microalgae-based biorefinery-From biofuels to natural products.Bioresource Technology 135:166-174), the current microalgae are mainly locked as algae polysaccharides, carotenoids, phycobilins and multiple One of the natural sources of saturated fatty acids (DHA and EPA) (Spolaore et al., 2006. Commercial applications of microalgae. J. Biosci. Bioeng..101:87-96), and the successful microalgae industry needs to have the most suitable algae species, optimized culture conditions and optimized active high-priced substances. Under the blessing of bio-genetic transformation, rapid screening of algae and artificial culture technology, the application of microalgae materials with potential for development into the industries of health care, food processing, aquaculture, animal feed and beauty has become a future development. The new blue ocean category accelerates the exploration of microalgae special active substances and enhances its purification technology, which will open up a broader market application prospect. At present, the focus of the development of algae species is to reduce the pollution of wastewater and waste gas through the large-scale cultivation process of microalgae, to reduce the emission of carbon dioxide, and to refine the energy of birth or high-priced active substances from algae to achieve the goal of environmental protection and industrial win-win (Farrelly) , DJet.al., 2013. Carbon sequestration and the role of biological carbon mitigation: A review. Renewable and Sustainable Energy Reviews 21: 712-727).

The microalgae life cycle depends on the photosynthesis, so carbon dioxide, sunlight and water are the three essential elements for cultivating microalgae. In general, the amount of algae is doubled every 6 to 72 hours in microalgae. The faster the algae grows, the higher the frequency of harvesting. The higher the oil content of algae means more algae oil. It is converted into biofuels, and it is necessary to use microalgae to produce high-quality algae, which is fast-growing and high in oil content. However, algae species with high oil content usually grow slower than algae species with low oil content. At the same time, considering the two factors of growth rate and oil content, the appropriate algae species are selected. In addition, the process of algae recovery is a part of the current energy consumption. Therefore, the microalgae species suitable for biofuel production must have the characteristics of high biomass yield, high algal oil content, and easy recovery of algae. The saturation of fatty acids in the algae oil and the ratio of triglyceride (TAG) must also be taken into account. The total amount of oil in the algae is composed of various chemical compounds such as triglycerides to sterols, but not all chemical compounds. Both are suitable for the production of biofuels, in which lipids containing fatty acids are preferred compounds, since they can be converted into biodiesel by transesterification, so the fatty acid profile in algal oil can be used as one of the indicators for algae screening (Ramos , MJ, et al., 2009. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour. Technol. 100: 261-268), in 2013, the research literature pointed out that Monoraphidium contortum (SAG 47.8) With 300mg/L/day of biomass energy production, oil content accounts for 22.2% of dry weight of algae and main fatty acid composition is C16:0 to C18:1 fatty acid, which can be used as biomass fuel potential algae strain (Bogen, C., et al., 2013. Identification of Monoraphidium contortum as a promising species for liquid biofuel production. Bioresource Technology 133: 622-626), another study indicates that even a single algae has a large total amount of oil. Oligomeric and fatty acid constituents are affected by the composition of the medium and the culture process (Dhup S. and Dhawan V., 2014. Effect of nitrogen concentration on lipid productivity and fatty acid composition of Monoraphidium sp. Bioresource Technology 152: 572-575).

The traditional classification methods of microalgae are mainly through the morphology of algae cells and filaments, the length and width of vegetative cells, the morphology of terminal cells, cell size, mitotic pattern, cell shape and arrangement, spacing of shaped cells, irregularities and The closest distance between thick-walled spores, the presence of atypical cells, thick-walled spores, pigments, bubbles, and thick sheaths, and their morphological characteristics such as colony can be classified. The common algae in the Selenastraceae family are the capricornutum shape or the crescent shape of the genus such as Ankistrodesmus , Selenastrum , Monoraphidium and hoof. Algae such as Kirchneriella , although algae can be distinguished by algae as a single or aggregated community, the presence or absence of a mucilage pad, and algal cell morphology, but due to algal morphology Some may change due to the composition of the medium and the length of the culture, resulting in the polymorphism of the form of the algae. However, due to the vigorous development of molecular biology in recent years, the high retention of repeats in the biological genome makes it It can be used as a molecular marker for DNA fingerprinting. Currently, the most commonly used molecular markers in the field of microalgae are 18S rRNA sequences and ITS region sequences, which are used to assist the identification of algae species (Krienitz, L., et al., 2011; Yu , X. et al., 2012. SSU rRNA Gene Phylogeny of Morphospecies Affiliated to The Bioessay Alga " Selenastrum capricornutum " Recovered the Polyphyletic Origin of Crescent-Shaped Chlorophyt a.J.Phycol. 47:880-893).

Obesity is a concern of many people today. Body fat accumulation can cause metabolic abnormalities such as diabetes, hyperlipidemia, cardiovascular disease, and hypertension, and circulatory diseases. Less activity and excessive intake of energy are the main causes of obesity. Excessive intake of fat is one of the main reasons for excessive energy intake. However, fat is also one of the important nutrients, in addition to providing energy, it is also related to the absorption of fat-soluble vitamins. At the same time, fat has a unique flavor, which provides a rich taste and texture of food, and is also an indispensable heat medium for cooking food. Natural fats contain many different triglycerides. In order to solve the problem of excessive fat intake, many research and manufacturers have successively developed fat substitutes, hoping to reduce the absorption of oil. For example, the sucrose polyester of U.S. Patent No. 3,600,186, which has the property of being not digested and absorbed and excreted into the feces, has the function of "low calorie". However, sucrose fatty acids may cause problems of abdominal cramps or soft stools, and have disadvantages such as absorption of fat-soluble vitamins. 1,3-Dicylglycerol (1,3-DAG) is a natural oil, which is not high in general oils and fats. However, due to its structural characteristics, it is metabolized into energy after being digested and absorbed by the human body. The triglyceride will be re-synthesized, so the intake of 1,3-bismercaptoglyceride is considered to be a preferred edible oil type. There have been many studies on how to replace triglycerides with 1,3-bismercaptoglycerides as the main component of healthy fats and oils. As published by Peng Xuanfu et al. ( Burkholderia sp. lipase and its accompanying progeny performance and application. Taiwan Chemistry and Food Science 49 (6): 316-328, 2011) and Taiwan Patent No. I423983 is an isolated lipase gene and a conserved gene of the lipase, which encodes a polypeptide having lipase activity and can be used for preparing 1,3-bismercaptoglyceride.

There is currently no literature on the accumulation of 1,3-bisguanidinoglycerides in the fat composition of algae. Therefore, whether algae culture can be used as a raw material for healthy fats and raw fuels remains to be further developed and discussed.

One object of the present invention is to provide a microalgae isolate which can be used as a raw material for producing healthy fats and biodiesel, and which can fix carbon dioxide as a carbon reduction tool.

Another object of the present invention is to provide a method of culturing the microalgae isolate to obtain a culture product containing microalgae.

Another object of the present invention is to provide a microalgae culture product obtained by the above method.

Another object of the present invention is to provide a process for obtaining 1,3-bismercaptoglyceride from the above cultured product of microalgae.

Another object of the present invention is to provide a method for obtaining a fatty acid from the above cultured product of microalgae.

The invention is described in detail in the following sections. Other features, objects, and advantages of the invention are apparent from the embodiments of the invention and the appended claims.

Fig. 1 shows a microscopic examination of the FP-7MA strain, wherein A is a bright field observation, the cell length is about 10-15 μm, the width is about 5-8 μm, the microscopic magnification is 1,000X; and B is stained with Nile Red to Under the fluorescence microscope, there is a yellow oil droplet distribution inside the algae, and the microscopic magnification is 1,000X.

Figure 2 shows the growth of FP-7MA strains in C medium at different culture temperatures.

The present invention can be understood from the various aspects of the invention, the embodiments and the description of the embodiments disclosed herein. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with the present invention shall have the meaning as understood by those of ordinary skill in the art. And, as will be appreciated, unless the definitions provided herein are otherwise specified, in the case of any potential ambiguity, the definition of terms should be consistent with such commonly used terms (as defined in the dictionary). For further understanding, the terminology used in this case is only It is used to describe a particular embodiment and is not intended to be limiting.

It must be noted that the singular forms "a" and "the" Therefore, unless the context requires otherwise, the singular terms shall include the plural and the plural terms also include the singular.

The scope of the present invention is expressed by "a particular value" and / or another "about" specific value. When the range is expressed in the above manner, it includes a range from a particular value and/or to another particular value. Similarly, when a value can be approximated by the term "about", it will be understood that it is another aspect of a particular value. It can be further appreciated that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful. According to the present invention, "about" can mean ± 20%, preferably ± 10%, more preferably ± 5%.

In the present invention, the term "isolated" or "isolated" means that the substance is removed from its original environment (for example, the natural environment if it is naturally present). The term "isolated" or "isolated" does not necessarily mean that the material is purified.

A first object of the present invention is to provide a microalgae isolate comprising an 18S rRNA sequence having at least 95% similarity to the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide represented by SEQ ID NO: The sequence has an ITS region sequence with at least 95% similarity. In other words, the 18S rRNA sequence in the microalgae isolate has at least 95%, 96%, 97%, 98%, 99% or 100% similarity to the nucleotide sequence shown in SEQ ID NO: 1, and the ITS region The sequence has a similarity to the nucleotide sequence set forth in SEQ ID NO: 2 of at least 95%, 96%, 97%, 98%, 99% or 100%.

The difference between the two nucleic acid sequences may occur at the 5' or 3' end position of the reference nucleotide sequence, or may be interspersed among the nucleotides in the reference sequence or interspersed with one or more adjacent groups within the reference sequence. Anywhere between the end positions of the group. Whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98%, 99%, or 100% similar to a reference nucleotide sequence refers to the use of a standard algorithm well known in the art between two molecules. Compare and routinely use publicly available computer programs (such as BLASTN calculations) Method) to judge.

In a preferred embodiment of the present invention, the microalgae isolate is a strain deposited in the Food Industry Development Research Institute of the Corporation and registered as BCRC 980034, or deposited with the Food Industry Development Institute of the Corporation. The strain numbered BCRC 980034 has a variant of substantially identical characteristics.

The above "mutant strain" means any microalgae strain in which all cytogenetic compositions have been altered by chemical mutation induction, spontaneous mutation, genetic engineering, transformation or transfection to affect their physical or biochemical properties. However, the variant strain should have all the identification features of the strain deposited under the accession number BCRC 980034 in the Food Industry Development Institute.

Another object of the present invention is to provide a method for preparing a microalgae culture product, which comprises inoculating the microalgae isolate of the present invention in a liquid medium, and culturing at a temperature of about 15 ° C to about 35 ° C under irradiation and aeration. This culture product was obtained. The present invention also provides a culture product obtained by the above method.

The "liquid medium" for culturing the microalgae isolate in the present invention may be any liquid medium base which allows the microalgae isolate to grow, multiply and produce 1,3-bismercaptoglyceride and/or fatty acid, for example, C. medium (containing 15mg per 100mL of _{Ca (NO 3) 2 .4H 2} O, KNO 10mg of β- glycerol disodium phosphate _3, 5mg of .5H ₂ O, 4mg of _{MgSO 4 .7H 2 O, 0.01 μ} g of B12,0.01 μ g vitamin biotin (biotin), 1 μ g of the thiophene amine HCl, 0.3mL of PIV metals (100mL each containing 100mg of Na ₂ EDTA.2H ₂ O, 19.6mg of FeCl ₃ .6H ₂ O , MnCl 3.6mg of ₂ .4H ₂ O, _1.04mg of ZnCl _2, 0.4 μ g of _{CoCl 2 .6H 2 O, 0.25 μ} g of Na ₂ MoO ₄ .2H ₂ O and water), 50mg of Tris and water) , BG-11 medium (containing 1,500 mg of NaNO ₃ , 40 mg of K ₂ HPO ₄ per 100 Ml, 75 mg of MgSO ₄ .7H ₂ O, 27.18 mg of CaCl ₂ , 6 mg of citric acid, 6 mg of ferric ammonium citrate, 1 mg) the Na Na ₂ .Mg.EDTA.2H2O, 20mg of ₂ CO _3, HBO MnCl _2.86mg to _{3, 1.181mg} of ₂ .4H ₂ O, _0.222mg of ZnSO ₄ .7H ₂ O, 0.39mg of Na ₂ MoO ₄ .2H ₂ O, 0.0718 mg of CuSO ₄ .5H ₂ O, 0.049 mg Co(NO ₃ ) ₂ .6H ₂ O and water) and MA medium (containing 10 mg of Ca(NO ₃ ) ₂ .4H ₂ O per 100 mL, 10 mg of KNO ₃ , 5 mg of NaNO ₃ , 4 mg of Na ₂ SO ₄ , 5mg of MgCl ₂ .6H ₂ O, β- disodium glycerol phosphate 10mg of .5H ₂ O, 0.5mg of Na ₂ EDTA.2H ₂ O, 0.05mg of FeCl ₃ .6H ₂ O, 0.5mg of MnCl _2. 4H ₂ O, 0.05 mg of ZnCl ₂ , 0.5 mg of CoCl ₂ .6H ₂ O, 0.08 mg of Na ₂ MoO ₄ .2H ₂ O, 2 mg of H ₃ BO ₃ and 50 mg of Bicine).

In the liquid medium of the present invention, NaOH may be added as an inducer for increasing the yield of the oil, and the final concentration of the added NaOH may be from about 0.5 mM to about 2 mM, preferably from about 0.5 mM to about 1.5 mM, more preferably. It can be about 1 mM.

Moreover, suitable conditions for culturing the microalgae isolate of the present invention are intended to allow conditions such as temperature, illumination, and culture time to allow the microalgae isolate to grow, propagate, and produce 1,3-bisguanidinoglyceride and/or fatty acid. Those skilled in the art can adjust the composition and culture conditions of the medium based on prior knowledge.

In an embodiment of the invention, the culture temperature may be from about 15 ° C to about 35 ° C, preferably from about 20 ° C to about 30 ° C; and the amount of illumination may be from about 100 lux to about 4,000 lux, preferably about 2,000. Lux.

By "venting" herein is meant the continuous passage of air into the liquid medium, and the aeration may be from about 0.05 vvm to about 1 vvm, preferably from about 0.1 vvm to about 0.5 vvm. The content of carbon dioxide in the air may be naturally adjusted to be about 0.04%, and may be additionally adjusted so that the content of carbon dioxide therein may be as high as about 0.1%, about 0.5%, about 1%, about 2%, about 3%, About 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

In the method for preparing a microalgae culture product of the present invention, the step of isolating the culture product may be included as needed, and the separation step may be a conventional method step such as centrifugation and/or filtration.

Since the microalgae culture product of the present invention is rich in 1,3-bismercaptoglyceride and/or fatty acid, it can be used as a raw material for obtaining 1,3-bismercaptoglyceride and/or fatty acid, and then used separately. For the production of healthy oils and / or biomass energy.

The "fatty acid" herein means a carboxylic acid compound having 8 to 30 carbon atoms and 0 to 6 unsaturated bonds, preferably a carboxylic acid compound having 12 to 20 carbon atoms and 0 to 5 unsaturated bonds. More preferably, it is a carboxylic acid compound having 16 to 18 carbon atoms and 0 to 3 unsaturated bonds.

The 1,3-dimercaptoglyceride and the fatty acid can be obtained by any extraction and separation methods well known in the art, such as Folch et al. (The Journal of Biological Chemistry, 1956, 23: 497-509.), Balasubramanian, etc. Methods of Human (Bioresource Technology, 2011, 102: 3396-3403.) and Sajilata et al. (Journal of Food Engineering, 2008, 84: 321-326). Briefly, the method may comprise pulverizing the cells in a manner such as grinding or ultrasonication, extracting the 1,3-bisguanidinoglyceride or fatty acid in the cells by a suitable solvent, and by, for example, HPLC. And/or the technique of ion exchange resin to obtain 1,3-bismercaptoglycerides or fatty acids.

All publications, patents and patent documents mentioned herein are hereby incorporated by reference in their entirety.

The following examples are provided to assist those skilled in the art in practicing the invention. </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Changes are still within the scope of the invention.

Example Materials and Methods 1. Medium formula

(1) C medium

Add 15 mg of Ca(NO ₃ ) ₂ in sequence. 4H ₂ O, 10 mg of KNO ₃ , 5 mg of β-glycerol phosphate disodium. 5H ₂ O, 4 mg of MgSO ₄ . 7H ₂ O, 0.01 μ g of vitamin B12,0.01 μ g of biotin (Biotin), 1 μ g of the thiophene amine HCl, PIV metal Tris 0.3mL of 50mg, followed by replenishment to the volume 100mL, pH was adjusted to Autoclave after 7.5. If it is 1.5% acacia solid medium, it needs to be sterilized by adding 15g of vegetable gum.

The PIV metal was prepared by sequentially adding 100 mg of Na ₂ EDTA. 2H ₂ O, 19.6 mg of FeCl ₃ . 6H ₂ O, 3.6 mg of MnCl ₂ . 4H ₂ O, 1.04mg of ZnCl _2, 0.4 μ g of CoCl _2. 6H ₂ O and 0.25 μ g of Na ₂ MoO _4. 2H ₂ O, followed by doubling the volume to 100 mL and autoclaving.

2 collecting algae, isolation and culture

Approximately 10 ml of the water sample of the cultured fish pond of Changhua, Changhua, Taiwan was placed in a 50 ml centrifuge tube, and about 30 ml of C culture was added, and the culture was carried out at 25 ° C. During the culture, the growth of the algae was observed by a microscope, and then an appropriate amount of the culture medium containing the algae was taken out, transferred to a plate medium, and cultured at 25 ° C. After the algae grows, a single algae species is taken and spread in the plate medium, and the above steps need to be repeated until the sieve is applied to the single algae body. For plate culture, a single algal bloom was applied to the C plate medium, and cultured at 25 ° C. In a large number of cultures, the freshly cultured single algae body is scraped from the plate and added to the C liquid medium, and the OD ₆₈₂ nm value of the culture solution after adding the algae is about 0.1 to 0.15, and the aeration culture is carried out at 25 ° C.

3. Oil staining analysis

20 μl of the cultured algae was mixed with 1 μl of Nile Red (0.1 mg/mL in dimethyl sulfoxide) for oil droplet staining, stained and allowed to stand at room temperature for 5 minutes, and then used for fluorescence. Observed by a microscope. (Chen, W. et al. , 2009. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae . Journal of Microbiological Methods 77: 41-47 and Huang, GH, et al. , 2009. Rapid screening method for Lipase production in alga based on Nile red fluorescence. Biomass and bioenergy 33:1386-1392).

4. Molecular identification of algae species

Extraction of genomic DNA: Freshly cultured algae were scraped from the plate, collected in a 2 ml microcentrifuge tube, rinsed with 200 μl EB (1 M NaCl, 70 mM Tris, 30 mM Na ₂ EDTA) solution, and centrifuged. Then, add 400 μl of EB solution to reconstitute the algae, then add appropriate amount of glass sand, shake with a percussive cell disrupter (Retsch® MM400) for about 5 minutes, repeat twice to homogenize the algae, and add 10 μl of RNAase. After 37 minutes at 37 ° C, 50 μl of 10% CTAB was sequentially added to 400 μl of phenol: chloroform: isoamyl alcohol (25:24:1) for 3 minutes, centrifuged at 13,000 rpm for 10 minutes at 4 ° C, and the supernatant was taken. In another new centrifuge tube, mix with 400 μl of phenol:chloroform:isoamyl alcohol (25:24:1) for 3 minutes, repeat the centrifugation process, transfer the supernatant to another new centrifuge tube, and add an equal volume of 2 - Acryl alcohol was mixed, placed at -30 ° C for more than 30 minutes, centrifuged at 13,000 rpm for 15 minutes at 4 ° C, the supernatant was removed, the precipitate was washed with 70% EtOH, air dried, and 50 μl ddH ₂ O Reconstitute this genomic DNA precipitate.

PCR amplification and sequencing analysis: using algal DNA as a PCR template, 18S rRNA and ITS region (including the back end of 18S ribosomal RNA, internal transcribed spacer 1, 5.8S ribosomal RNA, internal transcribed spacer 2 A primer set associated with the sequence of the front end of the 28S ribosomal RNA) is used to amplify its gene fragment. The PCR reaction solution was as follows: An appropriate amount of the gene DNA solution was used as a PCR template, and 8 μl of 10 mM dNTP, 10 μl of 10X PCR buffer, 10 pmole of 5 ' end primer and 3 ' end primer and 5 U Taq enzyme were used. The PCR reaction conditions were 95 ° C for 3 minutes; (95 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, 2 minutes 30 seconds) for a total of 30 cycles; 72 ° C, 10 minutes; and finally maintained at 4 ° C. 5 μl of the product was taken for electrophoresis. The PCR products were purified and sequenced with appropriate primers, and the sequence results were analyzed by sequence recombination and sequence similarity alignment with Vector NTI Suite 9 software (VNTI) and NCBI/Blastn.

5. Algal analysis

(1) Analysis of oil content of algae: Refer to the method of modifying Folch et al. (Folch, J. et al. , 1956. A simple method for the isolation and purification of total lipids from animal tissue. The Journal of biological Chemistry 23: 497-509), the process is to take 30mg freeze-dried algal flour (A value) to 2ml microcentrifuge tube, add about 2.0mL chloroform / methanol (v: v = 2: 1) and the right amount of large glass beads, The mixture was shaken for about 5 minutes with a percussive cell disrupter (Retsch® MM400) and repeated twice. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was taken to a disposable 15 ml centrifuge tube, and then about 2.0 mL of chloroform/methanol (v:v=2:1) was added to a 2 ml microcentrifuge tube, and then ultrasonically oscillated and After centrifugation, the supernatant was taken to a disposable 15 ml centrifuge tube until the extract was colorless. Add an equal volume of 145 mM NaCl solution to a 15 mL centrifuge tube containing the extract, mix well with a Ferris wheel, centrifuge at 4,500 rpm for 10 minutes, and remove the liquid from the glass vial to the weighed glass bottle (B value). in. The liquid in the glass bottle was air-dried overnight and weighed (C value), and the percentage (D value) of the oil content of the dried algae was calculated. Algae dry oil content calculation formula: (CB) / Ax100 = D%.

(2) Fatty acid pattern analysis method: A proper amount of dry algae was scraped and placed in a glass test tube, and 1 mL of solution 1 (NaOH 45 g, methanol 150 mL, and ddH ₂ O 150 mL) was added to shake the algae. Heat at 100 ° C for 5 minutes, then shake all the algae and continue heating for 25 minutes. 2 mL of solution 2 (6N HCl 325 mL and methanol 200 mL) was added, and the mixture was heated at 80 ° C for 10 minutes, and then rapidly cooled. 1.25 mL of Solution 3 (200 mL of hexane, 200 mL of butyl methyl ether) was added, and the mixture was slowly mixed for 10 minutes, and the lower layer liquid was aspirated by a glass pipette tip and discarded. The upper liquid was added to 3 mL of Solution 4 (NaOH 10.8 g, ddH ₂ O 900 mL), and after mixing for 5 minutes, the supernatant liquid was aspirated and analyzed for its fatty acid content by GC/MS (HP 5973 GC/MS System). For GC/MS analysis methods, reference is made to the method of Valencia, I. et al. , 2007 (Valencia, I. et al. , 2007. Development of dry fermented sausages rich in docosa hexaenoic acid with oil from the microalgae Schizochytrium sp.: Influence on nutritional properties, Sensorial quality and oxidation stability. Food Chemistry 104: 1087-1096), GC/Mass analysis conditions: capillary column: SP-2560, 75 m x 0.18 mm ID, 0.14 μm. Injection inlet temperature: Inj, 250 °C. Ion source temperature: FID, 250 °C. Column oven temperature: starting temperature 140 ° C, after 5 minutes, the temperature was raised to 240 ° C at a heating rate of 4 ° C / min for 2 minutes. Carrier gas: He. Column flow: 40 cm / sec @ 175 ° C. Injection: 1 μL. Split ratio: 1/100. Fatty acid standard: 37-Component FAME Mix (Cat. 18919-1 AMP, Sigma-Aldrich). After setting the conditions, analyze the standard and confirm the map correctly before performing sample analysis. The results of the analysis are compiled in a table for easy comparison.

(3) Analysis of oil composition: The extracted algal oil sample was analyzed by HPLC for its oil composition. HPLC analysis conditions: Separation column was Silica gel (4.6 mm id × 250 mm, particle size 5 μ m) manufactured by Merck, Germany; Solvent A: hexane; solvent B: hexane/ethyl acetate/isopropyl alcohol = 80:10:10 (v/v), solvent A/B=98:2 (v/v) at 0 minutes Linear increase to solvent A/B = 50:50 (v/v) at 8 minutes, linear increase to solvent A/B = 2:98 (v/v) at 8.5 minutes, same gradient for 15 minutes, linearity for 20 minutes Reduced to solvent A/B=98:2 (v/v); flow rate: 1.2 mL/min; evaporative light scattering detector (ELSD; Evaporative Light Scattering Detector) conditions; gas flow rate 2.6 L/min; evaporator temperature: 40 °C (Zhan Guojing et al., the production of 1,3-biguanide glycerol by transesterification of glycerol with vegetable oil using lipolytic enzyme. Taiwan Agrochemical and Food Science, 45:19-25 (2010)).

6. Analysis of culture characteristics of algae

(1) Test of culture temperature: the algae were scraped from the plate, suspended in an appropriate amount of C medium, and the OD ₆₈₂ nm value was measured. 2 ml of the algae solution was transferred to a 12-well culture dish and sealed with 10% carbon dioxide. The bags were incubated at different temperatures of 20 ° C, 30 ° C and 37 ° C, and then the OD ₆₈₂ nm values were measured on days 7 and 14 of the culture.

(2) Effect of different carbon dioxide concentrations on the growth of algae: adding algae culture To 1L of the culture flask containing C culture solution, air (0.04% carbon dioxide) and 5% carbon dioxide gas (95% air) were respectively introduced under the condition of 0.1 vvm, and cultured at 30 ° C for 14 days, and the comparison was different. Under the concentration of carbon dioxide, the difference in dry weight of algae and the growth of algae and oil production efficiency were recorded. Measurement of dry weight of algae: Take 100 mL of algae solution, centrifuge at 5,000 rpm for 20 minutes, remove the supernatant, suspend the precipitated algae with deionized water, wash away the remaining salts, and centrifuge at 5,000 rpm. After the minute, the supernatant was removed, and the algae collected by centrifugation were placed in a -80 ° C freezer for pre-cooling. After the pre-cooling process was completed, the freeze-drying machine was freeze-dried. After about 72 hours, the freeze-dried algae were weighed, the dry weight (WA) was recorded, and the algae oil content, fatty acid profile, and oil composition analysis were performed.

(Chisti, Y.2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26:126-131.)

(3) Induced culture of algal oil production: In the first stage, an appropriate amount of fresh algae solution was inoculated into a 1 L serum bottle containing 900 ml of C medium to have an initial OD ₆₉₀ nm value of 0.1 to 30 ° C, 0.5 vvm air and 2,000 The conditions of lux were cultured for 14 days. In the second stage, an oil production inducing factor (NaOH final concentration of 1 mM) was added to the medium, and cultured for 7 days under the same conditions to analyze the dry weight of the algae and the oil content of the algae (Nayak, M., et al. , 2013. Maximizing Biomass Productivity and CO ₂ Biofixation of Microalga, Scenedesmus sp. by Using Sodium Hydroxide. J. Microbiol. Biotechnol. 23: 1260-1268).

Example 1. Identification of algae strains

The fish pond water sample in Yongjing, Changhua, Taiwan was isolated and purified to obtain the FP-7MA strain. According to the 1,000X microscope, the algae existed in a non-clustered single algae body. The central part of the algae cell was slightly depressed, and both ends of the cell were blunt-type non-fine-pointed and curved in the same direction as a kidney shape, and the cell length was about 10 ~15μm, width is about 5~8μm (Fig. 1A). After staining with Nile Red, a large number of distinct and yellowish oil droplets were observed inside the algae by a fluorescence microscope, indicating that oil droplets could accumulate in the algae (Fig. 1B).

The 18S sequence of FP-7MA (SEQ ID NO: 1) was aligned with NCBI's nr database to obtain the top 4 most similar sequences from (1) Ankistrodesmus gracilis (Acession no. AB917098.1), similar The degree is 99%. (2) Ankistrodesmus gracilis (Acession no. Y16937.1) with a similarity of 99%. (3) Raphidocelis subcapitata SAG 12.81 (Acession no. KF673369.1) with a similarity of 99%. (4) Monoraphidium sp. FXY-10 (Acession no. JQ809706.1) with a similarity of 99%. The alignment of the above DNA sequences revealed that the FP-7MA strain may be Ankistrodesmus or Raphidocelis or Monoraphidium . The morphological characteristics of the FP-7MA strain were compared, and it was found to be similar to Raphidocelis subcapitata SAG 12.81, but it was significantly different from Ankistrodesmus gracilis and Monoraphidium sp. FXY-10.

The ITS sequence of FP-7MA (SEQ ID NO: 2) was aligned with NCBI's nr database to obtain the top 5 most similar sequences from (1) Nephrochlamys subsolitaria (Acession no. AB917131.1), similar The degree is 94%. (2) Scenedesmus regularis isolate DRL2 (Acession no. JX138999.1) with a similarity of 94%. (3) Monoraphidium sp. KMMCC 1531 (Acession no. JQ315786.1) with a similarity of 92%. (4) Scenedesmus sp. GUBIOTJT116 (Acession no. KF471115.1) with a similarity of 90%. (5) Ankistrodesmus sp. RS-2012 (Acession no. JX456463.1) with a similarity of 90%. The above ITS sequence alignment results showed that the similarity of FP-7MA strains to Nephrochlamys subsolitaria , Scenedesmus regularis isolate DRL2, Monoraphidium sp. KMMCC 1531, Scenedesmus sp. GUBIOTJT116 and Ankistrodesmus sp. RS-2012 was less than 95%. It is shown that the FP-7MA strain has a great difference from the above algae species; and the DNA sequence of the IMAP region of the Raphidocelis subcapitata SAG 12.81 has not been published in the public database, so the ITS sequence of the FP-7MA strain cannot be compared with it.

The FP-7MA strain has a kidney shape and exists in a non-clustered single algae, which is closer to Raphidocelis subcapitata SAG 12.81. The comprehensive DNA sequence and morphological characteristics are compared and the FP-7MA strain is initially identified as Raphidocelis sp. .

The FP-7MA strain was deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute of Taiwan Foundation on December 10, 2014. The deposit number is BCRC 980034; and the FP-7MA strain has also been based on the Budapest Treaty (Budapest). Treaty) was deposited on January 11, 2015 at the China Center For Type Culture Collection (CCTCC), Wuhan University, China, with the registration number CCTCC M 2015030.

Example 3: Analysis of culture characteristics of FP-7MA strain

(1) Test of culture temperature: 2 mL of algae solution (C medium) was transferred to a 12-well culture dish and placed in a sealed bag containing 10% carbon dioxide, and cultured at different temperatures of 20 ° C, 30 ° C and 37 ° C. The OD ₆₈₂ nm value was measured on days 7 and 14 of the culture. Figure 2 shows that the OD _682nm value of FP-7MA can be increased from 0.219 to 0.574 and 0.402 at 20 ° C and 30 ° C culture temperature, and the algae can grow continuously. In addition, under the temperature of 37 ° C, the OD _682nm value decreased from 0.219 to 0.096, and the growth of the algae was inhibited.

(2) Effects of different carbon dioxide concentrations on the growth of algae: The FP-7MA algae solution was incubated in a 1 L C-containing medium at 30 ° C, and air was introduced at 0.1 vvm (carbon dioxide content 0.04%). Or contain 5% carbon dioxide gas. After 14 days, the dry weight and oil content of the algae of the algae strain passing through the air and 5% carbon dioxide were measured. The results are shown in Table 1.

The results in Table 1 show that the dry weight of the algal body reached 1,584 mg/L under the condition of 5% carbon dioxide at the time of culture, which was 4.4 times the dry weight of the algae (360 mg/L) under air-passing conditions. The oil content of the algae decreased from 34.75% to 23.37% after the introduction of 5% carbon dioxide. The biomass yield, oil yield and carbon dioxide fixation rate under the condition of 5% carbon dioxide culture were up to 113.19 mg/L/day, 26.46 mg/L/day and 212.80 mg/L/day, respectively. The biomass yield, oil yield and carbon dioxide fixation rate under air conditions were 25.71 mg/L/day, 8.97 mg/L/day and 48.34 mg/L/day, respectively. Therefore, compared with air culture, FP-7MA algae solution can increase its biomass yield by 4.4 times, oil yield by 2.9 times and carbon dioxide fixation rate by 4.4 times, respectively. The results showed that the FP-7MA algae solution was cultured in C medium under carbon light and cultured with carbon dioxide, which significantly improved its biomass yield, algal oil yield and carbon dioxide fixation rate.

Secondly, the results in Table 2 show that the introduction of air and 5% carbon dioxide also affect the fatty acid composition of the algae strain, and found that the main component of the fatty acid composition is the carbon chain C16 and C18 fatty acids, which account for the total fatty acid content, respectively. 84.9% and 91.34%. After calculation, the DU (Dgree of Unsaturation) values are 110.8 and 112.38, respectively, which are less than 137, which is in line with the European Union's biodiesel standard (Ramos et al. , 2009. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour.Technol.100:261-268), it is suitable as a raw material for refining biodiesel. From the above results, it can be found that the introduction of 5% carbon dioxide gas helps the FP-7MA strain to produce quality diesel; while the FP-7MA strain has a carbon dioxide fixation rate of up to 212.80 mg/L/day, indicating that it has a reduced atmosphere. The potential of carbon dioxide.

In addition, after analyzing the oil composition of algae strains which were introduced with air or 5% carbon dioxide, it was found that the triglyceride (TAG) and 1,3-bisguanidinoglyceride (1,3-DAG) of the algae strain were cultured in air. The content of monoterpene glyceride (MAG) and free fatty acid (FA) was 49.08%, 48.56%, 2.14% and 4.96%, respectively; and the triglyceride (TAG) and 1, which were cultured with 5% carbon dioxide. The content of 3-bis-decyl glyceride (1,3-DAG) was 94.57% and 5.43%, respectively, and no monodecyl glyceride (MAG) and free fatty acid (FA) were detected (Table 3). From the above results, it is shown that the FP-7MA strain is composed of air or 5% carbon dioxide, and the oil composition of the strain is suitable as a raw material for producing diesel fuel. The FP-7MA strain has an air cultured 1,3-bisguanidinoglyceride (1,3-DAG) content of up to 48.56%. It is the only algae that can be synthesized to synthesize 1,3-bisguanidinoglyceride. Can be applied to the production of 1,3-bisguanidinoglyceride Kang fat.

(3) Induction culture of algal oil production: The FP-7MA strain was first cultured in C medium at 30 ° C for 14 days, and then cultured for 7 days in a medium containing 1 mM NaOH inducer to induce algal oil production. The results are shown in Table 4. The dry weight of the algae induced by 1 mM NaOH reached 240 mg/L, the algal oil content accounted for 54.5% of the dry weight of the algae; the dry weight of the algae in the control group was 230 mg/L, and the oil content of the algae accounted for The dry weight of the algae was 38.2%. Therefore, compared with the control group, the algal oil content in the algae treated with the 1 mM NaOH inducer increased by 1.43 times. This indicates that 1 mM NaOH can be used in the mass production of FP-7MA strain as an inducing factor for oil production to increase algal oil production.

in conclusion

The present invention firstly found a novel microalgae FP-7MA isolate which was initially identified as Raphidocelis sp. The algae of the algae has an oil content of 23.37% or more, and the oil content can be increased by 54.5% after being induced by 1 mM NaOH. The FP-7MA strain is cultured in air, and its 1,3-bisguanidinoglyceride content accounts for 48.56% of the oil composition. It is the only algae species that has been found to produce 1,3-bisguanidinoglyceride, so it can be applied to Production of 1,3-bisguanidinoglyceride health oil. The FP-7MA strain was cultured for 14 days under 5% CO ₂ , and the obtained biomass yield (113.19 mg/L/day) and oil yield (26.46 mg/L/day) were all compared with air culture. The method has high yield, and its oil composition is mainly triglyceride (94.57%), and the fatty acid composition is mainly C16~C18 fatty acid, and its DU value is 112.38. The above results show that FP-7MA strain is suitable for doing. In order to refine raw materials for biodiesel. The FP-7MA strain was cultured for 14 days under 5% CO ₂ , and the carbon dioxide fixation rate was 212.8 mg/L/day, which could be used as a carbon reduction tool. From the above results, Raphidocelis sp. FP-7MA strain is a novel microalgae, which can be used as a raw material for the production of 1,3-bisguanidinoglyceride health oil and biodiesel, and can also be used as a fixed carbon dioxide to reduce It is a tool for carbon dioxide in the atmosphere, so it plays an important role in the fields of biomass energy, healthy oils and carbon reduction.

【Biomaterial Storage】

Domestic registration information [please note according to the registration authority, date, number order]

Food Industry Development Research Institute, December 10, 2014, BCRC 980034

Foreign deposit information [please note according to the country, organization, date, number order]

China, China Typical Culture Collection Center, January 11, 2015, CCTCC M 2015030

<110> Food Industry Development Research Institute

<120> Microalgae and its use

<130> 18663

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1731

<212> DNA

<213> Raphidocelis sp.

<400> 1

<210> 2

<211> 709

<212> DNA

<213> Raphidocelis sp.

<400> 2

Claims (16)

A microalgae isolate, which is a strain of algae deposited under the No. BCRC 980034, deposited by the Food Industry Development Research Institute. A method for preparing a culture product of a microalgae, which comprises inoculating a microalgae isolate according to claim 1 in a liquid medium, and culturing at a temperature of about 15 ° C to about 35 ° C under irradiation and aeration to obtain the culture product. The method of claim 2, wherein the liquid medium further comprises from about 0.5 mM to about 2 mM NaOH. The method of claim 3, wherein the NaOH content of the liquid medium is about 1 mM. The method of any one of claims 2 to 4, wherein the temperature is from about 20 ° C to about 30 ° C. The method of any one of claims 2 to 4, wherein the amount of illumination is from about 100 lux to about 4,000 lux. The method of any one of claims 2 to 4, wherein the aeration amount is from about 0.05 vvm to about 1 vvm. The method of any one of claims 2 to 4, wherein the amount of carbon dioxide in the venting is from about 0.04% to about 10%. The method of claim 8, wherein the amount of carbon dioxide in the venting is about 5%. The method of any one of claims 2 to 4, further comprising the step of isolating the culture product. The method of any one of claims 2 to 4, which can be used to fix carbon dioxide. A microalgae culture product comprising the microalgae isolate of claim 1. A process for the preparation of 1,3-bismercaptoglyceride comprising the isolation of 1,3-dimercaptoglyceride from the culture of the microalgae of claim 12. A method of producing a fatty acid comprising isolating a fatty acid from the culture of the microalgae of claim 12. The method of claim 14, wherein the fatty acid has 8 to 30 carbon atoms and 0 to 6 unsaturated bonds. The method of claim 15, wherein the fatty acid has from 16 to 18 carbon atoms and from 0 to 3 unsaturated bonds.