Schizochytrium sp. and its use in the production of specific ratios of fatty acids

Abstract

The present application relates to the field of microbial technology, and particularly relates to a schizochytrium and application thereof in production of specific proportion of fatty acid. The present application screens a schizochytrium producing specific proportion of fatty acid, the content of C18:1 in the bio-oil produced by fermentation of the schizochytrium is higher, and the proportion of n-3, n-6 and n-9 is in a dominant mass ratio, which can be directly used in the production of fatty acid supplement or related products in the subsequent production process, without the need of blending other kinds of oil, the quality of raw materials is easier to control, the formula is simpler, the production efficiency and quality of the fatty acid supplement or related products can be effectively improved, and the application value is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a Schizochytrium and its application in producing specific ratio fatty acids. BACKGROUND

[0002] Docosahexaenoic acid (DHA) is an omega-3 long-chain polyunsaturated fatty acid, which plays an important role in the development of human brain, nerves, vision, and cardiovascular nutrition support, especially for the development of fetuses and infants. DHA generally needs to be supplemented because the human body cannot synthesize it by itself.

[0003] At present, DHA products mainly come from marine organisms such as deep-sea fish and marine microalgae, which are called fish oil DHA and algal oil DHA, respectively. Fish oil DHA has a relatively low price and a long history of consumption, but it has disadvantages such as marine pollution, large differences in product quality due to production process level, and inability to meet the needs of vegetarians. Algal oil DHA uses microbial fermentation, which has the advantages of not being limited by resources, food safety and controllable quality, traceability, natural form of produced oil, and higher human absorption and utilization rate. With the increasing demand for DHA in the market, relying only on marine fish oil as a source of production cannot meet the extensive demand. Nutritional supplements such as algal oil DHA are widely used in the field of healthy foods and are in the stage of continuous development. With the continuous deepening of basic research and application exploration, people's understanding of the nutritional, health, and disease prevention effects of nutrients such as algal oil DHA is increasing, and the application technology level is continuously improving. These nutrients can be added to more healthy foods, such as liquid milk, yogurt, and snack foods. At present, algal oil DHA has been widely used in foods and health products in European and American countries. Infant formula foods (infant formula milk powder and infant complementary foods) containing algal oil DHA have been marketed in many countries. In addition, there are also fortified foods such as fish cans, lactic acid beverages, fish sausage, bread, and candies containing DHA on the market in European and American countries.

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

[0005] The prior art researches on DHA algal oil focus on n-3, n-6 and n-9 fatty acids (n-3 and n-6 are polyunsaturated fatty acids, and n-9 is monounsaturated fatty acid), n-3 fatty acids including a-linoleic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the like, which are essential fatty acids that cannot be synthesized by the human body (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 play an important role in maintaining human health as n-3. They include linoleic acid (LA), gamma-linolenic acid (GLA), arachidonic acid (ARA) and docosapentaenoic acid (DPAn-6). n-3 and n-6 fatty acids exhibit completely different properties, n-3 fatty acids regulate anti-inflammatory processes, and n-6 fatty acids regulate pro-inflammatory processes, both of which regulate different functions of the immune system, and an unbalanced content can lead to various diseases. n-9 fatty acids are monounsaturated fatty acids, which can be naturally synthesized by the human body because they have only one double bond, but research shows that more dietary intake of n-9 fatty acids instead of some saturated fatty acids is still beneficial to health, for example, reducing inflammation and making insulin sensitivity higher. In summary, n-3 / 6 / 9 fatty acids have different benefits and functions and need to be ensured within a certain balance range. Therefore, in terms of diet, balanced intake of n-3 / 6 / 9 fatty acids is crucial to health, and deficiency or imbalance can lead to an inability to maintain functional balance and trigger various chronic diseases. For the general population, the ideal level of n-6 / n-3 fatty acid intake ratio should be 1-2:1. Regarding the intake principle of monounsaturated fatty acids (MUFA), authoritative organizations at home and abroad recommend that while controlling the energy supply ratio of total fat and saturated fatty acids, appropriate intake of n-6 and n-3 fatty acids is met, and the remaining fat energy is provided by MUFA, that is, under these principles, the energy supply ratio of MUFA can be as high as possible, and the most important MUFA is n-9 fatty acid.

[0006] The fatty acid supplements on the market are gradually inclined to n-3 / 6 / 9 fatty acids in a dominant proportion to ensure the balance between lipid nutrients in the body, but the n-3 / 6 / 9 fatty acids in the DHA algal oil produced by Schizochytrium naturally are often in an unbalanced state. Although the prior art attempts to regulate the n-3 / 6 / 9 fatty acid ratio during the fermentation production stage, for example, by regulating the fermentation process or using molecular biology methods for adjustment, it is difficult to regulate the three kinds of unsaturated fatty acids by means of bioengineering due to the complexity of fatty acids. Therefore, the existing fatty acid supplement products often use formula oil to mix other sources of oil to make the n-3 / 6 / 9 fatty acid ratio reach a dominant proportion, for example, patent CN106819154A obtains a formula oil raw material with a more reasonable fatty acid ratio by matching flaxseed, sunflower kernel 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 demand for raw material quality and the pursuit of simple formula at the application end, the formula oil method gradually becomes unsatisfactory to the current market requirements. SUMMARY

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

[0008] In the first aspect, the present application screens a Schizochytrium for producing specific ratio fatty acids, and the Schizochytrium is biologically preserved, and the preservation information is as follows:

[0009] Preservation number: CCTCC NO: M 2024961; classification name: Schizochytrium sp. CABIO-A-2-VI; preservation unit: China Center for Type Culture Collection; preservation address: Wuhan, China. Wuhan University; preservation date: May 15, 2024.

[0010] In the second aspect, the present application provides a microbial agent, which comprises the Schizochytrium or a fermentation product thereof.

[0011] In the third aspect, the present application provides a product, which comprises a fermentation product of the Schizochytrium.

[0012] The fermentation product of the present application can be the total product of the Schizochytrium after fermentation (including the bacterial body itself), or a biological oil obtained by fermentation.

[0013] Further, the fermentation product in the product is preferably a biological oil obtained by fermentation of the Schizochytrium.

[0014] Further, the product is a food, a medicine, a nutritional supplement or a feed.

[0015] For example, the food can be dairy products, bread, nutrition bars, functional beverages or vegetarian meat, etc., and the nutritional supplement can be a fatty acid supplement.

[0016] Further, the mass ratio of n-3, n-6 and n-9 in the biological oil is (2-4):(1-1.5):1. It is generally considered 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 advantage ratio of fatty acid supplement, and the biological oil produced by the Schizochytrium fermentation provided by the application is naturally close to the advantage ratio range. In actual production, due to the fluctuation of the content of different components, it is difficult to strictly comply with the mass ratio of 1:1 of n-6 and n-9, and it is generally considered that the range of (1-1.5):1 belongs to the advantage ratio range.

[0017] Further, the content of DHA in the biological oil is not less than 40%.

[0018] Further, the content of C18:1n-9 in the biological oil is not less than 13%.

[0019] In a fourth aspect, the application provides a method for producing biological oil, comprising: using the Schizochytrium sp. CABIO-A-2-VI or the bacterial agent to produce biological oil.

[0020] Further, the fermentation production comprises seed culture and fermentation culture.

[0021] The seed culture conditions comprise 28-30℃, and the culture time is 1-3 days.

[0022] The fermentation culture conditions comprise 28-30℃, and the culture time is 4-6 days.

[0023] In a fifth aspect, the application provides the use of the Schizochytrium sp. or the bacterial agent in the production of biological oil.

[0024] The mass ratio of n-3, n-6 and n-9 in the biological oil is (2-4):(1-1.5):1.

[0025] Further, the mass ratio of n-3, n-6 and n-9 in the biological oil is (2-4):(1.03-1.33):1.

[0026] Further, the content of DHA in the biological oil is not less than 40%.

[0027] Further, the content of C18:1n-9 in the biological oil is not less than 13%.

[0028] The application further provides the use of the Schizochytrium sp. or the bacterial agent in improving the production efficiency and quality of biological oil products.

[0029] In a sixth aspect, the present application provides a method for screening a Schizochytrium sp., comprising: subjecting the Schizochytrium sp. to atmospheric and room temperature plasma mutagenesis.

[0030] The present application has the following beneficial effects:

[0031] The present application provides a Schizochytrium sp., and the mass ratio of n-3, n-6 and n-9 in the bio-oil obtained by fermentation of the Schizochytrium sp. is the natural dominant proportion of fatty acids. In the actual production process, it is not necessary to adjust the fatty acid proportion by a complex fermentation method, nor is it necessary to adjust other types of oil to optimize the fatty acid proportion. The Schizochytrium sp. provided by the present application can effectively improve the production efficiency of fatty acid supplements or related products, reduce the production cost, improve the product quality, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0033] Figure 1 is an example of Raman spectrum detection result provided by the present application embodiment 1.

[0034] Figure 2 is an example of Raman spectrum detection result provided by the present application embodiment 1.

[0035] Figure 3 is an example of Raman spectrum detection result provided by the present application embodiment 1.

[0036] Figure 4 is an example of Raman spectrum detection result provided by the present application embodiment 1. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely in the following combined with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0038] The experimental methods involved in the following embodiments are all conventional experimental methods in the art if not specifically mentioned, and the experiments can be carried out by referring to the conventional experimental manuals or reagent kit instructions in the art.

[0039] The experimental materials involved in the following examples, if not specifically mentioned, can be purchased on the market.

[0040] The n-3, n-6 and n-9 fatty acids described in the following examples are also Omega-3, Omega-6 and Omega-9 fatty acids.

[0041] Example 1

[0042] The present application provides a high-throughput fermentation method of Schizochytrium sp., comprising the following process:

[0043] 1. Starting strain

[0044] A strain of Schizochytrium sp. obtained by mutagenesis screening after separation from seawater is used as the starting strain, and the preservation number of the strain is CCTCC NO: M2019990 (disclosed in patent 201911399018.2).

[0045] 2. Mutagenesis

[0046] The Schizochytrium sp. is subjected to gas-liquid phase plasma (GLPP) mutagenesis (lethality rate is more than 90%), and a large number of mutagenized single colonies are obtained.

[0047] 3. Fermentation

[0048] The single colonies obtained by mutagenesis are aseptically picked with sterilized toothpicks into 48-well plates containing seed culture medium, each well is filled with 1 ml of liquid, and the culture conditions are 28℃, 230 rpm, and the culture is carried out for 1 day. The single colonies are expanded in the well plate to form a seed liquid, which is inoculated into a 48-well plate containing fermentation medium, each well is filled with 1.5 ml of liquid, the inoculation amount is 75 μL, and the culture conditions are 28℃, 220 rpm, and the culture is carried out for 5 days.

[0049] The seed culture medium used includes: glucose 4g, sodium glutamate 3g, yeast extract 0.6g, potassium dihydrogen phosphate 0.6g, anhydrous magnesium sulfate 0.8g, sodium chloride 2g, calcium chloride 0.03g, vitamin mixture 0.1g (100ppm vitamin B1, 100ppm vitamin B2, 100ppm vitamin B5, 100ppm biotin in fermentation broth), tap water 100mL; the solid culture medium additionally contains 2% agar.

[0050] The fermentation medium used includes: sodium glutamate 20g, yeast extract 5g, seawater crystal 25g, tap water 1000mL.

[0051] Glucose is sterilized alone, and the sterilization conditions are: 121℃, 30min.

[0052] 4. High-throughput detection

[0053] The fermentation broth is subjected to fatty acid composition determination using a Raman spectrometer.

[0054] The Raman detection is first performed by collecting Raman spectra of hundreds of fermentation broth (determined by HPLC) samples with known fatty acid composition, pre-processing the spectra, performing PLS regression analysis on all Raman spectra according to off-line chromatographic analysis values, establishing a prediction model, and verifying the accuracy through external experiments, with an accuracy of over 90%. Figures 1-4 For example

[0055] 5. In the process of screening Schizochytrium sp. with high EPA content, a Schizochytrium sp. with a high proportion of C18:1 in the produced fatty acids and a proportion extremely consistent with the dominant proportions of n-3, n-6 and n-9 fatty acids is unexpectedly obtained. The present application further biologically preserves it, and the preservation information is as follows:

[0056] Preservation number: CCTCC NO: M 2024961; classification name: Schizochytrium Schizochytrium sp. CABIO-A-2-VI; preservation unit: China Center for Type Culture Collection; preservation address: Wuhan, China, Wuhan University; preservation date: May 15, 2024.

[0057] The present application further ferments the Schizochytrium sp., and the fermentation method is as follows:

[0058] The single colony is picked into a 48-well plate containing seed culture medium (one single colony per well for expansion), with a liquid volume of 1 mL per well, and the culture conditions are 28°C and 220 rpm, and the culture is performed for 2 days. The single colony is expanded in the well plate to form a seed liquid, which is inoculated into a 24-well plate containing fermentation medium, with a liquid volume of 1.5 mL per well and an inoculation amount of 100 μL. The culture conditions are 28°C and 220 rpm, and the culture is performed for 5 days. Part of the remaining seed liquid in the expanded well plate is mixed with 30% glycerol at a ratio of 1:1, sealed with a gas-impermeable sealing film and placed in a -24°C refrigerator as a simple strain preservation, and another part is subjected to content detection.

[0059] Seed culture medium: glucose 4g, sodium glutamate 3g, yeast extract 0.6g, potassium dihydrogen phosphate 0.6g, anhydrous magnesium sulfate 0.8g, sodium chloride 2g, calcium chloride 0.03g, vitamin mixture 0.1g (100ppm vitamin B1, 100ppm vitamin B2, 100ppm vitamin B5, 100ppm biotin in fermentation broth), tap water 100mL; solid culture medium additionally contains 2% agar.

[0060] Fermentation medium: sodium glutamate 30g, yeast extract 9g, potassium dihydrogen phosphate 2.1g, magnesium sulfate 5g, sodium chloride 2.2g, anhydrous sodium sulfate 4.4g, sodium bicarbonate 0.13g, potassium chloride 0.8g, tap water 1000mL.

[0061] After fermentation, the slurry is collected, and the fatty acid composition is detected by gas chromatography after freeze-drying, and the fatty acid ratio in the final product (bio-oil) is as follows:

[0062] Table 1 Comparison of fatty acid compositions of dry bacterial bodies of multiple strains (fatty acid content unit w / w%)

[0063]

[0064] Table 2 Comparison of n-3, n-6 and n-9 contents of dry bacterial bodies of different strains (fatty acid content unit w / w%)

[0065]

[0066] Among them, strain 2 is the aforementioned starting strain, and strain 1 and strain 3 are other strains obtained by simultaneous mutagenesis.

[0067] From the results, the Schizochytrium obtained by mutagenesis in the application has a higher C18:1 ratio, and the n-3, n-6 and n-9 fatty acid ratio is about 3.2:1.17:1, which meets the n-3, n-6 and n-9 dominant ratio.

[0068] 6. Multiple batches of fermentation

[0069] The application further carries out three batches of fermentation on Schizochytrium CABIO-A-2-VI, and the fermentation method and step 5 are the same, and after extracting bio-oil (algal oil), gas chromatography is used for fatty acid detection, and the following table shows the results:

[0070] Table 3 Different fatty acid ratios of three batches of bio-oil (fatty acid content unit w / v%)

[0071]

[0072] Table 4 n-3, n-6 and n-9 contents in three batches of bio-oil (fatty acid content unit w / v%)

[0073]

[0074] The fermentation method used in the application does not use special stress and control means, and the n-3, n-6 and n-9 ratios of the three batches of bio-oil obtained 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 application can stably produce bio-oil that meets the dominant ratio of fatty acid supplement. Generally, DHA algal oil needs to be mixed with sunflower oil to meet the dominant n-3, n-6 and n-9 ratio.

[0075] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A Schizochytrium sp. (Aurantiochytrium Schizochytrium sp. ) CABIO-A-2-VI, characterized in that, The preservation number is CCTCC NO: M 2024961.

2. An inoculant characterized in that, The bacterial agent comprises the Schizochytrium of claim 1.

3. A product characterized by, The product comprises the fermentation product of the Schizochytrium of claim 1. The fermentation product is a bio-oil obtained by fermentation of the Schizochytrium; the mass ratio of n-3 fatty acid, n-6 fatty acid and n-9 fatty acid in the bio-oil is (2-4):(1-1.5):1; the content of DHA in the bio-oil is not less than 40%. The product is food, medicine, nutritional supplement or feed.

4. A method of producing a bio-oil, characterized by, Comprise: The bio-oil is produced by fermentation of the Schizochytrium of claim 1 or the bacterial agent of claim 2.

5. The Schizochytrium of claim 1 or the bacterial agent of claim 2 is used in production of bio-oil; the mass ratio of n-3 fatty acid, n-6 fatty acid and n-9 fatty acid in the bio-oil is (2-4):(1-1.5):

1.

6. Use according to claim 5, characterized in that, The content of DHA in the bio-oil is not less than 40%.

7. The Schizochytrium of claim 1 or the bacterial agent of claim 2 is used in improving the production efficiency and quality of bio-oil product.

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