Schizochytrium limacinum and application thereof in production of fatty acid in specific proportion

Abstract

The invention relates to the technical field of microorganisms, in particular to schizochytrium limacinum and application of the schizochytrium limacinum in production of fatty acid in a specific proportion. A strain for producing the schizochytrium limacinum with a specific fatty acid proportion is obtained through screening, the content of C18: 1 in the bio-oil obtained through fermentation production of the schizochytrium limacinum is high, the proportion of n-3, n-6 and n-9 is in an advantage mass ratio, the bio-oil can be directly used for production of fatty acid supplements or related products in the subsequent production process, other kinds of grease do not need to be blended, and the production cost is low. The method is simple in process, easy to control the quality of the raw materials, simple in formula, capable of effectively improving the production efficiency and quality of the fatty acid supplement or related products and high in application value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a schizochytrium and application thereof in producing fatty acids in a specific ratio. Background Art

[0002] Docosahexaenoic acid (DHA) is an omega-3 long-chain polyunsaturated fatty acid that plays an important role in the development of the human brain, nerves, vision, and cardiovascular nutrition support. It is especially important for the development of the fetus and infant. Because it is difficult for the human body to synthesize it on its own, DHA generally requires additional supplementation.

[0003] At present, DHA products are mainly derived from deep-sea fish, marine microalgae and other marine organisms, which are called fish oil DHA and algae oil DHA respectively. Fish oil DHA is relatively cheap and has a long history of consumption, but it has disadvantages such as marine pollution, the production process level has a great impact on quality differences, and it cannot meet the needs of vegetarians. Algae oil DHA adopts microbial fermentation, which has the advantages of not being restricted by resources, food safety and quality control, traceability, the oil produced is in natural form, and the human body has a higher absorption and utilization rate. With the continuous increase in the market demand for DHA, relying solely on marine fish oil as a production source can no longer meet the wide demand. Nutritional enhancers such as algae oil DHA are widely used in the field of health food and are in a stage of continuous expansion. With the continuous deepening of basic research and application exploration, people are more and more aware of the nutritional, health care, disease prevention and other functions of nutrients such as algae oil DHA. At the same time, benefiting from the continuous improvement of the application technology level, these nutrients can be added to more healthy foods, such as liquid milk, yogurt, snacks, etc. At present, European and American countries have widely used algae oil DHA in food and health products. Infant formula foods (infant formula milk powder and infant complementary foods) with added algae oil DHA have been launched in many countries. In addition, fortified foods such as canned fish, lactic acid beverages, fish sausages, bread, and candy with added DHA have also appeared in the European and American markets.

[0004] Schizochytrium sp. is one of the representative species for industrial fermentation production of DHA due to its high biomass, high oil content and easy cultivation. Schizochytrium sp. can produce up to 70% of total fatty acids (TFA) by cell weight, of which DHA accounts for 25-50% of TFA. In recent years, DHA algae oil produced by Schizochytrium sp. has also been approved as a new resource food, and its food safety has been widely certified.

[0005] The existing technology for DHA algae oil research focuses on n-3, n-6 and n-9 fatty acids (n-3 and n-6 ​​are polyunsaturated fatty acids, n-9 is a monounsaturated fatty acid). n-3 fatty acids include α-linoleic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), etc. They are essential fatty acids that the human body cannot synthesize on its own (essential fatty acids), and have the effects of promoting heart health and regulating anti-inflammatory processes. n-6 fatty acids are also essential fatty acids, and like n-3, they play an important role in maintaining human health. They include linoleic acid (LA), γ-linolenic acid (GLA), arachidonic acid (ARA) and docosapentaenoic acid (DPAn-6). The two series of fatty acids, n-3 and n-6, exhibit completely different properties. n-3 fatty acids regulate anti-inflammatory processes, while n-6 fatty acids regulate pro-inflammatory processes. The two regulate different functions of the immune system. If the content is unbalanced, various diseases will occur. n-9 fatty acid is a monounsaturated fatty acid. Because it has only one double bond, it can be naturally synthesized by the human body. However, studies have shown that consuming more n-9 fatty acids in the diet instead of some saturated fatty acids is still beneficial to health, such as reducing inflammation and making insulin more sensitive. In summary, n-3 / 6 / 9 fatty acids have different benefits and functions, and they need to be kept within a certain balance range. Therefore, in terms of diet, a balanced intake of n-3 / 6 / 9 fatty acids is essential for health. Insufficient or unbalanced intake may lead to the inability to maintain functional balance and cause various chronic diseases. For the general population, the ideal level of n-6 / n-3 fatty acid intake should be 1~2:1. Regarding the intake principle of monounsaturated fatty acid MUFA, authoritative organizations at home and abroad recommend that while controlling the energy supply ratio of total fat and saturated fatty acids, the appropriate intake of n-6 and n-3 fatty acids should be met, and the remaining fat energy is provided by MUFA, that is, under these principles, the energy supply ratio of MUFA can be increased as much as possible, and the most important MUFA is n-9 fatty acid.

[0006] At present, the fatty acid supplements on the market gradually tend to have n-3 / 6 / 9 fatty acids in a dominant ratio to ensure the balance between lipid nutrients in the body, but the n-3 / 6 / 9 fatty acids in the DHA algae oil naturally produced by Schizochytrium are often in an unbalanced state. Although the prior art attempts to regulate the ratio of n-3 / 6 / 9 fatty acids in the fermentation production stage, such as by regulating the fermentation process or by molecular biological means, it is often difficult to achieve the regulation of the three unsaturated fatty acids by bioengineering means because the fatty acids themselves are relatively complex. Therefore, existing fatty acid supplement products often use formula oils to mix oils from other sources to make the ratio of n-3 / 6 / 9 fatty acids reach a dominant ratio. For example, patent CN106819154A obtains a formula oil raw material with a more reasonable fatty acid ratio by using the ratio of flaxseed, sunflower kernels and other oil crops rich in unsaturated fatty acids; for example, CN104894176B uses iodoacetamide to regulate the DPA / DHA ratio in Schizochytrium oil. However, with the increasing demands of the application side for controlling the quality of raw materials and pursuing simple formulas, the method of formulating oils is gradually failing to meet the current market requirements. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a schizochytrium and an application thereof in producing fatty acids in a specific ratio.

[0008] In the first aspect, the present invention studies and screens a Schizochytrium that produces fatty acids in a specific ratio, and biologically preserves the same. The preservation information is as follows: Deposit number: CCTCC NO: M 2024961; Classification name: Schizochytrium sp. CABIO-A-2-VI; depository: China Center for Type Culture Collection; deposit address: Wuhan University, Wuhan, China; deposit date: May 15, 2024.

[0009] In a second aspect, the present invention provides a bacterial agent, which includes the Schizochytrium described above, or a fermentation product thereof.

[0010] In a third aspect, the present invention provides a product, comprising a fermentation product of the Schizochytrium.

[0011] The fermentation product of the present invention can be the total product of Schizochytrium after fermentation (including the bacteria itself), or the bio-oil obtained by fermentation.

[0012] Furthermore, the fermentation product in the product is preferably bio-oil obtained by fermentation of Schizochytrium.

[0013] Furthermore, the product is food, medicine, nutritional supplement or feed.

[0014] For example, the food may be dairy products, bread, nutrition bars, functional drinks or vegetarian meat, etc., and the nutritional supplement may be a fatty acid supplement.

[0015] Furthermore, the mass ratio of n-3, n-6 and n-9 in the bio-oil is (2-4): (1-1.5): 1. The present invention generally considers that the mass ratio of n-3, n-6 and n-9 is close to the range of 4:1:1 to 2:1:1 to meet the fatty acid supplementation advantage ratio, and the bio-oil fermented by Schizochytrium provided by the present invention is naturally close to the advantage ratio range. In actual production, due to the fluctuation of the content of different components, the mass ratio of n-6 and n-9 is difficult to strictly comply with 1:1, and it is generally believed that it belongs to the advantage ratio range when it is in the range of (1-1.5): 1.

[0016] Furthermore, the content of DHA in the bio-oil is not less than 40%.

[0017] Furthermore, the content of C18:1n-9 in the bio-oil is not less than 13%.

[0018] In a fourth aspect, the present invention provides a method for producing bio-oil, comprising: using the Schizochytrium CABIO-A-2-VI or the bacterial agent to ferment and produce bio-oil.

[0019] Furthermore, the fermentation production includes: seed culture and fermentation culture; The seed culture conditions include: 28-30° C., 1-3 days; The fermentation culture conditions include: 28-30° C., and culture for 4-6 days.

[0020] In a fifth aspect, the present invention provides the use of the Schizochytrium or the bacterial agent in producing bio-oil; The mass ratio of n-3, n-6 and n-9 in the bio-oil is (2-4): (1-1.5): 1.

[0021] Furthermore, the mass ratio of n-3, n-6 and n-9 in the bio-oil is (2~4):(1.03~1.33):1.

[0022] Furthermore, the content of DHA in the bio-oil is not less than 40%.

[0023] Furthermore, the content of C18:1n-9 in the bio-oil is not less than 13%.

[0024] The present invention further provides the use of the Schizochytrium or the bacterial agent in improving the production efficiency and quality of bio-oil products.

[0025] In a sixth aspect, the present invention provides a method for screening Schizochytrium, comprising: performing atmospheric pressure and room temperature plasma mutagenesis on Schizochytrium.

[0026] The present invention has the following beneficial effects: The present invention studies and obtains a strain of Schizochytrium, in which the mass ratio of n-3, n-6 and n-9 in the bio-oil obtained by fermentation of the Schizochytrium is naturally a fatty acid dominant ratio. In the actual production process, it is not necessary to optimize the fatty acid ratio by adjusting a complex fermentation method, nor is it necessary to deploy other types of oils to optimize the fatty acid ratio. The Schizochytrium provided by the present invention can effectively improve the production efficiency of fatty acid supplements or related products, reduce production costs, and improve product quality, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is an example diagram of the Raman spectrum detection results provided in Example 1 of the present invention.

[0029] Figure 2 This is an example diagram of the Raman spectrum detection results provided in Example 1 of the present invention.

[0030] Figure 3 This is an example diagram of the Raman spectrum detection results provided in Example 1 of the present invention.

[0031] Figure 4 This is an example diagram of the Raman spectrum detection results provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional experimental methods in the art, and the experiments can be performed with reference to conventional experimental manuals in the art or kit instructions.

[0034] Unless otherwise mentioned, the experimental materials involved in the following examples can be purchased commercially.

[0035] The n-3, n-6 and n-9 fatty acids described in the following examples are omega-3, omega-6 and omega-9 fatty acids.

[0036] Example 1 The present invention provides a high-throughput fermentation method of Schizochytrium, comprising the following process: 1. Starting strain A Schizochytrium strain isolated from seawater and subjected to mutagenesis screening was used as the starting strain, and the deposit number of the strain is CCTCC NO: M2019990 (disclosed in patent 201911399018.2).

[0037] 2. Mutagenesis The Schizochytrium was subjected to gas-liquid phase plasma (GLPP) mutagenesis (with a lethality of more than 90%) to obtain a large number of induced single colonies.

[0038] 3. Fermentation The single colony obtained after mutagenesis was aseptically picked with a sterilized toothpick into a 48-well plate containing seed culture medium, with 1 ml of liquid in each well, culture conditions at 28°C, 230 rpm, and culture for 1 day. The single colony was cultured in an expanded well plate to form a seed solution, which was inoculated into a 48-well plate containing fermentation medium, with 1.5 ml of liquid in each well, an inoculation volume of 75 μL, culture conditions at 28°C, 220 rpm, and culture for 5 days.

[0039] The seed culture medium used includes: 4g glucose, 3g sodium glutamate, 0.6g yeast extract, 0.6g potassium dihydrogen phosphate, 0.8g anhydrous magnesium sulfate, 2g sodium chloride, 0.03g calcium chloride, 0.1g vitamin mixture (100ppm vitamin B1, 100ppm vitamin B2, 100ppm vitamin B5, 100ppm biotin in the fermentation broth), 100mL tap water; 2% agar is added to the solid culture medium.

[0040] The fermentation medium used includes: 20 g of sodium glutamate, 5 g of yeast extract powder, 25 g of sea crystal, and 1000 mL of tap water.

[0041] Glucose is sterilized separately, sterilization conditions: 121℃, 30min.

[0042] 4. High-throughput detection The fatty acid composition of the fermentation broth was determined using Raman spectrometry.

[0043] Raman detection first collects Raman spectra of hundreds of fermentation broth samples with known fatty acid composition (detected by HPLC), pre-processes the spectra, and performs PLS regression analysis on all Raman spectra based on offline chromatographic analysis values ​​to establish a prediction model. The accuracy of the model is more than 90% after external experimental verification. Figure 1-Figure 4 for example) 5. In the process of screening Schizochytrium with high EPA content, the present invention unexpectedly obtained a Schizochytrium strain whose fatty acid produced had a high C18:1 ratio and a ratio that was very consistent with the dominant ratio of n-3, n-6 and n-9 fatty acids. The present invention further deposited the strain biologically, and the deposit information is as follows: Deposit number: CCTCC NO: M 2024961; Classification name: Schizochytrium sp. CABIO-A-2-VI; depository: China Center for Type Culture Collection; deposit address: Wuhan University, Wuhan, China; deposit date: May 15, 2024.

[0044] The present invention further ferments the Schizochytrium, and the fermentation method is as follows: Pick a single colony to a 48-well plate containing seed culture medium (one colony per well for expansion), with 1 mL of liquid in each well, culture conditions at 28°C, 220 rpm, and culture for 2 days. The single colony is cultured in the expansion well plate to form a seed solution, which is then inoculated into a 24-well plate containing fermentation medium, with 1.5 ml of liquid in each well, 100 μL of inoculation, and culture conditions at 28°C, 220 rpm, and culture for 5 days. The remaining seed solution in part of the expansion well plate is mixed with 30% glycerol in a 1:1 ratio, and an airtight sealing film is attached to the plate and placed in a -24°C refrigerator as a simple strain storage. The other part is tested for content.

[0045] Seed culture medium: 4g glucose, 3g monosodium glutamate, 0.6g yeast extract, 0.6g potassium dihydrogen phosphate, 0.8g anhydrous magnesium sulfate, 2g sodium chloride, 0.03g calcium chloride, 0.1g vitamin mixture (100ppm vitamin B1, 100ppm vitamin B2, 100ppm vitamin B5, 100ppm biotin in the fermentation broth), 100mL tap water; add 2% agar to the solid culture medium.

[0046] Fermentation medium: 30 g of monosodium glutamate, 9 g of yeast extract powder, 2.1 g of potassium dihydrogen phosphate, 5 g of magnesium sulfate, 2.2 g of sodium chloride, 4.4 g of anhydrous sodium sulfate, 0.13 g of sodium bicarbonate, 0.8 g of potassium chloride, and 1000 mL of tap water.

[0047] After fermentation, the bacterial sludge was collected, freeze-dried and the fatty acid composition was detected by gas chromatography. The fatty acid ratio of the final product (bio-oil) was as follows: Table 1 Comparison of fatty acid composition of dried cells of multiple strains (fatty acid content unit: w / w%)

[0048] Table 2 Comparison of n-3, n-6 and n-9 contents in dry cells of different strains (fatty acid content unit: w / w%)

[0049] Among them, strain 2 is the aforementioned starting strain, and strain 1 and strain 3 are other strains induced at the same time.

[0050] From the results, the Schizochytrium obtained by mutation of the present invention has a higher C18:1 ratio, and the ratio of n-3, n-6 and n-9 fatty acids is about 3.2:1.17:1, which is consistent with the dominant ratio of n-3, n-6 and n-9.

[0051] 6. Multi-batch fermentation The present invention further conducts three batches of fermentation on Schizochytrium CABIO-A-2-VI. The fermentation method is the same as step 5. After the bio-oil (algae oil) is extracted, the fatty acid is detected by gas chromatography, and the results shown in the following table are obtained: Table 3 Different fatty acid ratios of three batches of bio-oil (fatty acid content unit w / v%)

[0052] Table 4 n-3, n-6 and n-9 contents in three batches of bio-oil (fatty acid content unit w / v%)

[0053] The fermentation method used in the present invention does not use special stress or regulation means, and the ratios of n-3, n-6 and n-9 of the three batches of bio-oils are 3.53:1.24:1; 3.24:1.03:1 and 3.65:1.33:1 respectively. It can be seen that the Schizochytrium provided by the present invention can stably produce bio-oils with a dominant ratio that satisfies fatty acid supplementation. However, general DHA algae oil needs to be blended with sunflower oil to meet the dominant ratios of n-3, n-6 and n-9.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Schizochytrium Schizochytrium sp. ) CABIO-A-2-VI, characterized in that, The deposit number is CCTCCNO: M 2024961.

2. A bacterial agent, characterized in that The bacterial agent comprises the Schizochytrium according to claim 1, or a fermentation product thereof.

3. A product, characterized in that: The product comprises the fermentation product of the Schizochytrium CABIO-A-2-VI according to claim 1; the product is food, medicine, nutritional supplement or feed.

4. The product according to claim 3, characterized in that The fermentation product is the bio-oil obtained by fermenting the Schizochytrium sp.; the mass ratio of n-3, n-6 and n-9 in the bio-oil is (2-4): (1-1.5): 1; and the content of DHA in the bio-oil is not less than 40%.

5. A method for producing bio-oil, characterized in that: include: The bio-oil is produced by fermentation using the Schizochytrium CABIO-A-2-VI described in claim 1 or the bacterial agent described in claim 2.

6. Use of the Schizochytrium described in claim 1 or the bacterial agent described in claim 2 in the production of bio-oil; the mass ratio of n-3, n-6 and n-9 in the bio-oil is (2~4): (1~1.5):

1.

7. The use according to claim 6, characterized in that: The content of DHA in the bio-oil is not less than 40%.

8. Use of the Schizochytrium described in claim 1 or the bacterial agent described in claim 2 in improving the production efficiency and quality of bio-oil products.

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