Schizochytrium sp. for producing high-content and high-clarity oil and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a kind of schizochytrium sp. producing high content, high clarity oil and application thereof. A schizochytrium strain with high quality after winterization is found in the screening process of schizochytrium, and the DHA content of the oil produced by fermentation is much higher than that of other strains. The schizochytrium provided by the present application is particularly suitable for preparing winterization products. After winterization treatment, a considerable amount of DHA is preserved in soft fat, and the quality of the winterization oil product is high, with high economic value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a Schizochytrium sp. producing high-content and high-clarity oil and application thereof. 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, and is particularly important for the development of fetuses and infants. Since the human body cannot synthesize DHA itself, DHA generally needs to be supplemented. At present, DHA products mainly come from deep-sea fish, marine microalgae and other marine organisms, which are called fish oil DHA and algal oil DHA, respectively. Algal oil DHA is generally produced by microbial fermentation, and the produced oil is in a natural form. Moreover, it is not limited by resources, has the advantages of food safety and quality controllability, traceability, and higher human body absorption and utilization rate, and the market scale of algal oil DHA is increasing.

[0003] 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 25-40% of TFA. DHA algal oil produced by Schizochytrium sp. has been recognized as a resource food, and its food safety has also been widely certified.

[0004] The oil produced by Schizochytrium sp. belongs to single-cell oil (SCO), and the oil is accumulated in the cell and belongs to mixed oil, which is mainly composed of triacylglycerol (TAG) and free fatty acid (FFA). The oil can be divided into neutral lipid (about 70%), polar lipid (about 3%) and glycolipid (about 20%). Among them, the main component of neutral lipid is triacylglycerol (93%), and a small amount of diacylglycerol (DAG) and monoacylglycerol (MAG) are also included. TAG is composed of a glycerol carbon skeleton esterified by three fatty acids, and the type depends not only on the composition of fatty acids, but also on the position distribution of fatty acids on the glycerol carbon skeleton.

[0005] In the process of extracting DHA algal oil, in order to obtain oil and fat which can keep clear and good fluidity at normal temperature and high DHA content, it is often necessary to remove high melting point triglycerides (including saturated fatty acid residues) and other impurities to achieve the enrichment of DHA through winterization treatment. Winterization treatment is also called winterization fractionation technology, which principle is that under certain temperature conditions, the oil and fat is separated into solid and liquid phases by using the difference of melting point and solubility of various triglycerides. Due to the different types and combinations of fatty acids constituting the triglycerides, the melting points (or freezing points) of different triglycerides are different. Generally speaking, the longer the carbon chain of the fatty acid constituting the triglyceride, the higher the unsaturation, the lower the melting point (or freezing point) of the triglyceride, and vice versa. The most commonly used oil and fat winterization fractionation technology at present is dry fractionation. The oil and fat is completely melted into liquid oil without adding any solvent, and then slowly cooled to a certain temperature while low-speed stirring, so that the solid fat components in the oil and fat are crystallized and precipitated, and then the solid and liquid phases are separated.

[0006] According to gas phase analysis, in addition to DHA and DPA, the DHA algal oil produced by the current commonly used Schizochytrium sp. also contains a large amount of palmitic acid (C16:0, PA), which accounts for 20-40%. In the winterization process, palmitic acid generally crystallizes and precipitates first, but due to the random distribution of palmitic acid, part of DHA is also taken out during winterization, so that the increase of DHA content in soft fat is limited, and the winterization effect is limited. In view of this phenomenon, it is the most effective to improve it from the source, that is, the strain itself, but due to the randomness of mutagenesis and the obstacles of detection and screening, it is difficult to obtain a strain with special TAG structure. SUMMARY

[0007] In order to solve the problems existing in the prior art, the present application provides a Schizochytrium sp. producing high content and high clarity oil and its application.

[0008] In the process of breeding and domestication of the strain, the present application uses deep well plate for high-throughput culture of Schizochytrium sp., detects the DHA content in the fermentation broth by near-infrared after fermentation, and selects the strain. Further through high-throughput fermentation screening, it is found that the DHA content in the supernatant of the oil produced by a strain under frozen conditions is significantly increased. Through the experiment of verifying the fatty acid composition and content and distribution, it is found that the SSD type triglyceride produced by the strain is less (this type of triglyceride is easy to take DHA into the hard fat precipitate), which reduces the loss of DHA in the supernatant during cold storage, so that the DHA content obtained by enrichment in the supernatant is high, that is, the oil produced by the strain has good winterization properties.

[0009] In a first aspect, the present application provides a Schizochytrium sp. CABIO-02, which is biologically preserved, and the preservation information is as follows:

[0010] The preservation number is CCTCC NO: M 2015716, the classification name is Schizochytrium sp. CABIO-02, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the postal code is 430072, and the preservation date is November 30, 2015.

[0011] The present application further provides a microbial agent, which comprises the Schizochytrium sp. CABIO-02 or a fermentation product thereof.

[0012] The fermentation product can comprise the Schizochytrium sp. CABIO-02 or can not comprise the Schizochytrium sp. CABIO-02.

[0013] The present application further provides a product, which comprises the Schizochytrium sp. CABIO-02 or the microbial agent, and the product is food, feed, cosmetics or medicine.

[0014] In a second aspect, the present application provides an application of the Schizochytrium sp. CABIO-02 or the microbial agent in preparing functional lipids.

[0015] Further, the functional lipids are winterized oil.

[0016] Further, the mass percentage of DHA in the functional lipids is not less than 40%, the mass percentage of PPD type triglyceride is not higher than 6%, the mass percentage of SSD type triglyceride is not higher than 10%, and the mass percentage of palmitic acid is not higher than 18%.

[0017] Further, the mass percentage of DHA in the functional lipids is not less than 45%, the mass percentage of PPD type triglyceride is not higher than 5.5%, the mass percentage of SSD type triglyceride is not higher than 8%, and the mass percentage of palmitic acid is not higher than 18%. In a more preferable condition, the mass percentage of DHA is not less than 47%.

[0018] The PPD type triglyceride is dipalmitoyl-docosahexaenoyl glyceride (without specific order). The SSD type triglyceride refers to a triglyceride with two saturated fatty acids and one docosahexaenoic acid connected on a glycerol skeleton, wherein the saturated fatty acid can be the aforementioned palmitic acid, or a saturated fatty acid such as stearic acid, C14:0, C17:0, etc., without specific position, and the two saturated fatty acids can be the same or different, and palmitic acid and stearic acid are commonly seen in the algal oil.

[0019] In a third aspect, the present application provides a functional lipid, wherein the mass percentage of DHA is not less than 40%, the mass percentage of PPD type triglyceride is not higher than 6%, the mass percentage of SSD type triglyceride is not higher than 10%, and the mass percentage of palmitic acid is not higher than 18%. In a more preferred condition, the mass percentage of DHA is not less than 45%; further, in a more preferred condition, the mass percentage of DHA is not less than 47%.

[0020] In a fourth aspect, the present application provides a preparation method of winterized oil, comprising: using the Schizochytrium sp. CABIO-02 or the bacterial agent for fermentation, oil extraction and winterization treatment.

[0021] Further, the winterization treatment comprises: filtering after settling at 4-10℃.

[0022] The existing screening methods include deep-well plate fermentation and screening of high-yield DHA Schizochytrium sp. by combining OD and fluorescence intensity indicators, but these methods do not consider the product quality after winterization treatment.

[0023] The present application also provides a method for screening Schizochytrium sp., comprising:

[0024] Fermentation culture, cell wall breaking, oil extraction, winterization treatment and DHA detection.

[0025] Further, the fermentation culture comprises:

[0026] The multi-well plate is used for seed culture, expansion culture, fermentation culture and preservation of Schizochytrium sp.

[0027] Further, the conditions of the seed culture and the fermentation culture comprise: culturing at 26-30℃ and 180-260rpm.

[0028] The present application uses a multi-well plate for the whole culture of Schizochytrium sp., which has shorter time consumption, longer preservation time, better strain revival, and is more convenient and fast in operation compared with the method of preparing a large amount of glycerol tube and transferring a large amount of different single colony seed liquid.

[0029] Further, the DHA detection adopts near infrared spectroscopy.

[0030] The near infrared spectroscopy has the advantages of rapidity, non-destructiveness, high efficiency and low cost, and is widely applied. It is an organic combination of near infrared equipment, chemometrics software and near infrared model. The near infrared technology is quantitative analysis based on the position and intensity of spectral absorption peaks generated by chemical components in a sample. The key technology of the NIR spectrum detection is to establish a quantitative function relationship between the two. The basic process includes: first, collect representative samples (the composition and variation range are close to the samples to be analyzed), and then use standard chemical methods to determine the chemical composition of the samples, then collect the spectral data of the samples, and then use chemometrics software to regress the spectral data and the chemical analysis data to determine the function relationship, and then establish a near infrared model; when analyzing the samples, first scan the sample to be measured, and then use the established near infrared model to calculate the component content of the sample to be measured according to the spectral data of the sample to be measured.

[0031] The present application has the following advantages:

[0032] The present application finds that the DHA content in the original fermentation product of a strain is not obviously superior to other strains, but the DHA content after winterization is much higher than that of other screening strains of the Schizochytrium sp. CABIO-02. The oil produced by the Schizochytrium sp. CABIO-02 is beneficial to obtain winterized oil with higher DHA content and more stable quality, and the yield of the winterized oil is considerable. The Schizochytrium sp. CABIO-02 provided by the present application can be used for the preparation of high-quality winterized oil, and has high economic value. BRIEF DESCRIPTION OF DRAWINGS

[0033] 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 for those skilled in the art, other drawings can also be obtained without creative labor.

[0034] Figure 1 is a schematic diagram of the verification result of the near infrared prediction model provided by the embodiment 1 of the present application. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] Example 1

[0037] 1. This invention first provides a method for fermentation culture of microorganisms, specifically including the following process:

[0038] Aseptically pick up single colonies from each sample tube and from acclimatized and mutagenized cultures using a sterile toothpick and transfer them to 48-well plates containing seed culture medium (one single colony per well for expansion). Each well contains 1 mL of medium, and the culture conditions are 28°C, 220 rpm, for 2 days. The resulting seed culture from the expanded wells is then inoculated into 24-well plates containing fermentation medium, with each well containing 1.5 mL of medium and an inoculum size of 100 μL. The culture conditions are 28°C, 220 rpm, for 5 days. A portion of the remaining seed culture from the expanded wells is mixed 1:1 with 30% glycerol, sealed with an airtight film, and stored at -24°C as a simple bacterial strain. The remaining portion is used for content analysis.

[0039] Seed culture medium: 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, 0.1g metal ion mixture, 100mL tap water; 2% agar is added to solid culture medium.

[0040] Fermentation medium: 30g sodium glutamate, 9g yeast extract, 2.1g potassium dihydrogen phosphate, 5g magnesium sulfate, 2.2g sodium chloride, 4.4g anhydrous sodium sulfate, 0.13g sodium bicarbonate, 0.8g potassium chloride, and 1000mL tap water.

[0041] 2. Near-infrared detection screening

[0042] The DHA content in the fermentation broth samples was then detected using near-infrared spectroscopy. Near-infrared spectroscopy involved collecting spectra from hundreds of fermentation broth samples with known DHA content (detected by GC), preprocessing the spectra, and then performing PLS regression analysis on all data based on offline chromatographic analysis values ​​to establish a predictive model (R² = 0.96). External experimental verification showed an accuracy exceeding 90%.

[0043] Near-infrared prediction models such as Figure 1 As shown, the left figure represents the internal validation results, and the right figure represents the external validation results.

[0044] The DHA content results of some samples are shown in Table 1:

[0045] Table 1 DHA content test results

[0046] Strain 1 Strain 2 Strain 3 CABIO-02 DHA content % 42.10 48.32 45.26 45.37

[0047] 3、The present application selects several strains with a content higher than 40%, and further carries out conventional fermentation experiments in a 1L fermenter (the same as the fermentation culture process in Example 2 in CN201911399018.2, and after fermentation, near-infrared is used to detect the fermentation liquid indicators, and the test results are shown in Table 2:

[0048] Table 2 DHA content test results

[0049] Strain 1 Strain 2 Strain 3 CABIO-02 DHA content % 42.34 50.3 47.82 48.73

[0050] At the same time, alkaline protease is added to the fermentation liquid (addition ratio 0.08%), 180rpm, 50℃ enzyme hydrolysis for 24h. After enzyme hydrolysis, centrifugation (5000r / min, 3min) is used to separate the enzyme hydrolysate into three layers of oil, water and solid impurities, and after separation of the oil, the sample is placed in a 0℃ refrigerator for cold storage.

[0051] During the cold storage process, it is found that the oil appears to be stratified, and the oil with fluidity in the upper layer is determined by gas chromatography.

[0052] The gas chromatography method is as follows:

[0053] Chromatographic column: DB-23 (30m x 0.25mm x 0.25um); flow control: constant pressure mode 12.3psi; injector temperature: 250℃; detector temperature: 280℃; programmed temperature: 90℃ for 1min, 9℃ / min to 240℃, 240℃ for 5min, 3℃ / min to 250℃, 250℃ for 4min; split mode: split ratio 50:1.

[0054] The DHA content in the oil is shown in Table 3

[0055] Table 3 DHA content test results

[0056] Strain 1 Strain 2 Strain 3 CABIO-02 DHA content % 43.63 48.05 49.57 54.85

[0057] As can be seen from Tables 1-3, the DHA content of the CABIO-02 strain in the fermentation liquid test after screening and scale-up culture is not very outstanding compared with other strains, but the DHA oil produced by it shows a significant increase in DHA content after long-term cold storage.

[0058] The application further studies the fatty acid distribution of the oils and fats by mass spectrometry, and the mass spectrometry is as follows:

[0059] Chromatographic system: Agilent 1290; chromatographic column: Agilent C18 column (100*2.1mm, 2.6um); A phase: water: methanol volume ratio = 1:2 (containing 5mM ammonium acetate); B phase: isopropanol (containing 5mM ammonium acetate), gradient elution condition: 0.5min, 20% B phase; 1.5min 40% B phase; 3min 60% B phase; 13min 98% B phase, 13.1min 20% B phase, 17min 20% B phase.

[0060] Mass spectrometry system: AB Sciex QTRAP 4500, APCI ion source, positive ion mode, the mass number range of mass spectrometry collection is m / z 100-1200, and the mass spectrometry conditions are as follows:

[0061] Positive ion mode: Curtain Gas: 30psi; Ion Source Gas1: 55psi; Ion Source Gas2: 55psi; IonSpray Voltage: 5000.00V, DP: 75V, CE: 35V; Temperature: 600℃;

[0062] Specific ion collection by multiple reaction monitoring, parent ion (m / z) 824.7, daughter ion 551.6.

[0063] The mass spectrometry results after analysis and addition are shown in the following table, and the results of tables 1-4 can show that the content of C16:0 and PPP of theultrabacterium CABIO-02 in several strains does not have a significant feature, but the content of PPD and PSD type triglyceride in CABIO-02 is obviously lower than that of other strains, and the total SSD type glyceride is also obviously lower than that of other strains, which reduces the loss of precipitated DHA in the refrigeration process of the oil produced by the ultrabacterium CABIO-02, so that the DHA content measured by soft fat (supernatant) is high, that is, the oil produced by the strain has good winterization properties.

[0064] Table 4 Comparison of contents of different types of fatty acids

[0065] Strain 1 Strain 2 Strain 3 CABIO-02 C16:0 content % 28.4 16.4 23.0 17.5 PPP content ‰ 63.43 0.52 37.76 1.05 PPD content ‰ 170.07 110.81 99.93 50.59 PSD content ‰ (S stands for C18:0) 32.85 13.17 15.84 10.99 SSD ‰ (S stands for saturated) 215.11 152.80 123.81 65.57

[0066] The PPP in the table is tri-saturated palmitic acid glyceride, the PPD is dipalmitoyl-docosahexaenoyl glyceride (without specific order), the PSD is palmitoyl-stearoyl-docosahexaenoyl glyceride (without specific order), and the SSD is a triglyceride with two saturated fatty acids and one docosahexaenoic acid connected on the glycerol skeleton. The content in the table is mass percentage, and the same in other tables.

[0067] The saturated fatty acid with the highest content in the DHA oil produced by the Schizochytrium is C16:0. Generally speaking, the lower the content of C16:0, the easier the winterization, and the more stable the quality of the winterized oil. However, the present study found that the fatty acid distribution of the triglyceride is also important for the winterized oil product. The lower the content of SSD (especially the content of PPD), the easier the DHA is retained in the soft fat. Under the condition that the quality of the winterized oil is guaranteed, the enrichment efficiency of DHA is higher, and the yield of soft fat is also higher.

[0068] 4. Application of winterized oil: The strain 2 and the Schizochytrium CABIO-02 are further fermented according to the 50L scale in the example 4 of CN201911399018.2, and after the oil is extracted, the winterized oil is obtained by the conventional method at-10℃ for 72h. The winterized oil sample is subjected to the freezing test, and is not turbid at 0℃ for 5.5h, which meets the requirement of the oil freezing test. The index results of the winterized oil are as follows.

[0069] Table 5 Comparison of winterized oil quality of strain 2, 3 and the present strain (Schizochytrium CABIO-02)

[0070]

[0071] In order to further investigate the stability of the winterized oil, the freezing time is further prolonged to 72h on the basis of the 0℃ freezing test, and the clear state is investigated.

[0072] Table 6 Stability test results of winterized oil

[0073] Sample Winterized oil from Strain 2 Winterized oil from CABIO-02 Winterized oil from Strain 3 Condition Clear Clear Turbid

[0074] From the results of tables 5 and 6, although the original DHA content of the strain 2 is higher, the dispersion of the main saturated fatty acid and the distribution of DHA and the saturated fatty acid make it not dominant in the yield and enrichment rate of soft fat in the application of winterization. The DHA content, enrichment rate and soft fat yield of the winterized oil of the strain 3 are all lower, and the winterization stability is poor. The Schizochytrium CABIO-02 provided by the present application is more suitable for preparing high-quality winterized oil.

[0075] In summary, only the Schizochytrium sp. fermentation product that meets the high DHA content, low PPD type triglyceride content, low SSD type triglyceride content and low palmitic acid content is most suitable for preparing high-quality winterized oil. The strain provided by the study has a low SSD type triglyceride content, but the palmitic acid content reaches about 30%, and a large amount of stearin needs to be removed during winterization, and the yield of natural winterized olein is low.

[0076] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; 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. CABIO-02, characterized in that, The Schizochytrium sp. has a preservation number of CCTCC NO: M 2015716.

2. An inoculant characterized in that, The microbial agent comprises the Schizochytrium sp. CABIO-02 of claim 1.

3. Use of the Schizochytrium sp. CABIO-02 of claim 1 or the microbial agent of claim 2 in the preparation of functional lipids. The functional lipids are winterized oils, the mass percentage of DHA in the functional lipids is not less than 40%, the mass percentage of PPD type triglyceride is not higher than 6%, the mass percentage of SSD type triglyceride is not higher than 10%, and the mass percentage of palmitic acid is not higher than 18%.

4. A method of preparing winterized oil, characterized by, The method comprises: The fermentation, extraction of oil and fat, and winterization are performed by using the Schizochytrium sp. CABIO-02 of claim 1 or the microbial agent of claim 2. The winterized oils have a mass percentage of DHA of not less than 40%, a mass percentage of PPD type triglyceride of not higher than 6%, a mass percentage of SSD type triglyceride of not higher than 10%, and a mass percentage of palmitic acid of not higher than 18%.

5. The preparation method according to claim 4, characterized in that, The winterization comprises: after settling at 4~10℃, filtering.

Images