A lipid obtained from a fermentation broth of a microbial cell, and a method of preparing and use thereof

Abstract

The application discloses a kind of lipids obtained from the fermentation broth of microbial cells and its preparation method and application, the prepared lipid peroxide value≤0.05 mmol / kg, p-anisidine value≤4.0, malondialdehyde≤0.10 mg / kg, total oxidation value TOTOX≤4.0, trans fatty acids are not detected, 0 ℃ low-temperature clear transparent, fishy smell sensory score≤1.0, shelf life≥24 months.

Description

Technical Field

[0001] This invention relates to the field of oil refining technology, specifically to a lipid obtained from the fermentation broth of microbial cells, its preparation method, and its application. Background Technology

[0002] With the development of food science and technology, various functional oils are increasingly entering people's lives, playing a significant role in improving people's nutrition and health. DHA-enriched microalgae oil, as a new resource food and a functional oil, is rich in DHA and has effects such as enhancing memory and thinking abilities, improving intelligence, and promoting brain development.

[0003] To produce qualified DHA microalgae oil and ensure its shelf life, the crude DHA microalgae oil needs to undergo degumming, deacidification, and deodorization to remove gums, free acids, heavy metal ions, etc. However, current oil refining processes often involve over-processing (cumbersome procedures and harsh reaction conditions), which not only wastes resources and energy but also causes the loss of beneficial micronutrients naturally present in the oil and generates byproducts harmful to human health, such as trans fatty acids and propylene glycol. Meanwhile, research shows that commercially available omega-3 supplements commonly suffer from oxidation problems, especially products with added flavorings to mask fishy odors, where the TOTOX value (total oxidation value = 2POV + p-AV) exceeds the standard by as much as 68%. Traditional refining processes (such as high-temperature deodorization) can remove free fatty acids, but often lead to the formation of trans fatty acids and the accumulation of oxidation byproducts. Summary of the Invention

[0004] The purpose of this invention is to provide a lipid obtained from the fermentation broth of microbial cells, its preparation method and application. By performing vacuum demulsification and oil extraction, low-temperature continuous alkali refining, low-temperature winterization crystallization, activated carbon adsorption and high-vacuum deodorization under an inert atmosphere, the lipid has the advantages of low fishy smell, low malondialdehyde content and low total oxidation value.

[0005] The specific technical solution of the present invention is as follows: A lipid obtained from the fermentation broth of microbial cells, having a peroxide value ≤0.05mmol / kg, p-anisidine value ≤4.0, total oxidation value ≤4.0, malondialdehyde content ≤0.10mg / kg, no detectable trans fatty acids, and a fishy odor sensory score ≤1.0; It is obtained by vacuum demulsification and oil extraction of microbial cell fermentation broth under inert atmosphere protection, low-temperature continuous alkali refining, low-temperature winterization crystallization, activated carbon adsorption and high-vacuum deodorization.

[0006] In some preferred embodiments, the microbial cells are at least one of algal cells, yeast cells, fungal cells, protozoan cells, or bacterial cells that produce polyunsaturated fatty acids; preferably, the microbial cells are derived from the genera *Morchella*, *Cryptocoryne*, or *Schizochytrida*; preferably, the microbial cells are derived from the order *Schizochytrida*; preferably, the microbial cells are derived from the genera *Chytridium* and / or *Schizochytridium*.

[0007] The above-mentioned lipid preparation method includes inactivating the fermentation broth of microbial cells, followed by vacuum demulsification and oil extraction under an inert atmosphere, low-temperature continuous alkali refining, low-temperature winterization crystallization, activated carbon adsorption, and high-vacuum deodorization. The demulsifier for vacuum demulsification and oil extraction is an inorganic salt and an organic acid. The inorganic salt is potassium chloride, sodium sulfate, magnesium sulfate, calcium chloride, EDTA-2Na, sodium chloride, magnesium chloride, and potassium sulfate. The organic acid is at least one selected from malic acid, citric acid, oxalic acid, and glacial acetic acid. The pH of the vacuum demulsification and oil extraction is 5.5 to 7.0. Preferably, the pH of the vacuum demulsification and oil extraction is 6.0 to 6.5. The system and alkali solution are neutralized at room temperature in the low-temperature continuous alkali refining process, and then the temperature is raised to 55~60℃ for separation; preferably, the heating rate of the low-temperature continuous alkali refining process is 1~2℃ / min. The crystallization temperature of low-temperature winterization crystallization is -5 to -1℃, and the crystallization time is 24 to 36 hours; preferably, the crystallization temperature of low-temperature winterization crystallization is -2℃; preferably, the cooling rate of low-temperature winterization crystallization is 3 to 5℃ / h; preferably, the stirring speed of low-temperature winterization crystallization is 5 to 10 rpm. The pressure of high vacuum deodorization is ≤80Pa, the temperature of high vacuum deodorization is 150~170℃, the time of high vacuum deodorization is 2~4h, and the steam flow rate of high vacuum deodorization is 5~20kg / h; preferably, the temperature of high vacuum deodorization is 160℃.

[0008] In some preferred embodiments, the vacuum demulsification and oil extraction operation is as follows: at -0.05 to -0.1 MPa, inorganic salts are first added, and the mixture is kept at 70 to 90°C for 1 to 2 hours. Then, organic acids are added, and the mixture is kept at 70 to 90°C for another 0.5 to 2 hours. The upper crude oil is then separated and collected. Preferably, the inorganic salt is a mixture of magnesium chloride and calcium chloride, preferably in a mass ratio of 1:0.8 to 1.2, and more preferably 1:1; Preferably, the amount of inorganic salt added is 1-10 wt% of the solid content of the system, and more preferably 6.5 wt%. Preferably, the organic acid is citric acid; Preferably, the amount of organic acid added is 1.0~2.0 wt% of the solid content of the system, and more preferably 1.3 wt%. Preferably, vacuum demulsification and oil extraction further includes adjusting the pH of the upper crude oil to 6.2±0.1 using organic acids.

[0009] In some preferred embodiments, the low-temperature continuous alkali refining operation is as follows: the upper crude oil obtained by vacuum demulsification is neutralized with alkali solution at room temperature, and then the temperature is raised to 50~60°C to separate and obtain light phase deacidified oil. Preferably, the alkali solution is added at a rate of 0.2~0.3t% of the excess alkali. Preferably, the alkaline solution is a 10 wt% NaOH solution; Preferably, the room temperature is 25°C.

[0010] In some preferred embodiments, vacuum demulsification and oil extraction further includes adding a first antioxidant to the system; Preferably, the first antioxidant is a mixture of sodium D-isoascorbate and ascorbate palmitate; preferably, the mass ratio of sodium D-isoascorbate and ascorbate palmitate is 36:1; Preferably, the amount of the first antioxidant added is 10 g / kg based on the solid content of the system; Preferably, vacuum demulsification and oil extraction further includes, after adding the first antioxidant, keeping the mixture at 70-90°C for at least 1-3 hours; Preferably, the vacuum demulsification and oil extraction process further includes diluting the system with water to a solid content of 12-22 wt%, preferably 15 wt%, before adding the first antioxidant.

[0011] In some preferred embodiments, inactivation is performed by continuous sterilization at 121°C; preferably, the continuous sterilization time is ≤1 min, more preferably 30~60 s; and / or, In some preferred embodiments, the above preparation method further includes homogenizing and disrupting the cell wall of the inactivated fermentation broth before vacuum demulsification and oil extraction. Specifically, the inactivated fermentation broth is cooled to 80°C under an inert atmosphere and then homogenized and disrupted at 200 bar.

[0012] In some preferred embodiments, the process between low-temperature continuous alkali refining and low-temperature winterization crystallization further includes: washing with deionized water at 85~90°C, followed by vacuum drying at 80~85°C.

[0013] In some preferred embodiments, the specific operation of activated carbon adsorption is as follows: the product after low-temperature winterization crystallization is filtered, then heated to 45°C, and activated carbon at 2wt% of the weight of the oil is added for adsorption for 1 hour, followed by filtration to remove the activated carbon; preferably, the iodine value of the activated carbon is ≥1000 mg / g, and the main pore size distribution is 2~5 nm. Preferably, the methylene blue adsorption value of the activated carbon is ≥20 g / 100 g, and the pH is alkaline.

[0014] In some preferred embodiments, a second antioxidant is added to the product after high-vacuum deodorization.

[0015] In some preferred embodiments, the above preparation method includes the following steps: (1) High temperature inactivation: The fermentation broth was inactivated at 121℃ for 30~60s to obtain inactivated fermentation broth; (2) Cooling and homogenization: Cool the inactivated fermentation broth to 80°C and then homogenize it at 200 bar to obtain a cell wall broken emulsion; (3) Vacuum demulsification and oil extraction: Dilute the cell wall broken emulsion to 12~22wt% of its solid content, then add 10g / kg of the first antioxidant, add inorganic salt first at -0.05~-0.1MPa, keep warm at 70~90℃ for 1~2h, then add organic acid, continue to keep warm for 0.5~2h, and separate and collect the upper crude oil; (4) Low-temperature continuous alkali refining: At room temperature, add alkali solution to the upper crude oil at an excess alkali amount of 0.2~0.3% to neutralize it, and then heat it to 50~60℃ at a heating rate of 1~2℃ / min to separate and obtain light phase deacidified oil; (5) Washing and drying: The light phase deacidified oil is washed with deionized water at 85~90℃ and then vacuum dried at 80~85℃ to obtain dried crude oil; (6) Low-temperature winterization crystallization: The dried crude oil is cooled to -5 to -1℃ at a cooling rate of 3~5℃ / h, and after 24~36h, the liquid phase is filtered and retained to obtain clear lipids; (7) Activated carbon adsorption: The clarified lipid was heated to 45°C, and 2wt% of activated carbon was added for adsorption for 1 hour. Then the activated carbon was removed by filtration to obtain the adsorbed lipid. (8) High vacuum deodorization: The adsorbed lipids are deodorized in a deodorization tower with a pressure ≤80Pa at 150~170℃ for 2~4h to obtain the deodorized lipids. The steam flow rate of high vacuum deodorization is 5~20kg / h. (9) Rapid cooling and anti-oxidation: Cool the deodorized lipids to ≤60℃ within 3~5 min, add the second oxidant, and the product is obtained; Steps (2) to (9) are carried out under an inert atmosphere.

[0016] A microbial inoculant comprising the above-mentioned lipids or lipids prepared by the above-mentioned method.

[0017] An article, cosmetic or pharmaceutical composition for use in animals or humans, comprising the above-mentioned lipids, lipids prepared by the above-mentioned method, or the above-mentioned microbial agents.

[0018] Preferably, the food is an additive, nutritional supplement, or animal feed for use in animal or human food.

[0019] Preferably, the animal feed is aquaculture feed, livestock feed, zoo animal feed, draft animal feed, or domestic animal feed.

[0020] According to the above technical solution, the present invention has at least the following beneficial effects: 1. This invention, through the synergistic effect of vacuum and inorganic salts in the vacuum demulsification and oil extraction stage, removes pro-oxidation metal ions in advance, cutting off the formation pathway of malondialdehyde (MDA) at its source. This avoids the later generation of MDA due to lipid oxidation, which is difficult to remove by conventional deodorization methods. The final measured MDA content is ≤0.10 mg / kg. Furthermore, after this step, the peroxide value of the intermediate product is approximately 0.2 mmol / kg, and the anisidine value is only 3.5, indicating that the primary oxidation degree and secondary oxidation degree of the intermediate product are extremely low.

[0021] 2. The crude oil obtained in the vacuum demulsification and oil extraction stage of this invention has a DHA content of >55%, which is basically at the level of the finished lipid product, indicating that the pretreatment and vacuum demulsification and oil extraction stages have basically completed the refining and concentration of lipids.

[0022] 3. In this invention, the deodorization process utilizes a combination of vacuum and low temperature (150~170℃) to avoid thermal isomerization of DHA, resulting in no detectable trans fatty acids in the finished lipid product. Furthermore, the DHA content in the finished lipid product (58~60wt%) is basically consistent with the content after vacuum demulsification and oil extraction, which indirectly reflects that the loss of DHA during alkali refining, deodorization, and other processes is minimal.

[0023] 4. This invention significantly reduces the content of saturated fatty acids (down to about 65% of the pre-wintering content) by using a specific low-temperature winterization crystallization temperature (-5~-1℃) and an extremely slow cooling rate (3~5℃ / h), thereby improving the low-temperature stability of the product while also achieving a low DHA loss rate and a high oil yield.

[0024] 5. The low-temperature continuous alkali refining stage of the present invention adopts a strategy of first mixing at low temperature (room temperature) and then separating at 55~60°C, which takes into account both a low degree of oxidation (POV about 1.4) and complete deacidification (light phase FFA=0.03%).

[0025] 6. In this invention, an inert atmosphere is used throughout the preparation process after the fermentation broth is inactivated, which significantly reduces the degree of oxidation in each step. The synergistic effect of the inert atmosphere and the optimization of each stage results in the final oil having excellent oxidation indicators: the final product peroxide value ≤0.05 mmol / kg, p-anisidine value ≤4.0, total oxidation value ≤4.0, which is far below the GOED voluntary standard limit (≤26), and oxidation induction time (90℃) ≥7.0h.

[0026] 7. Through the synergistic effect of low-temperature continuous alkali refining, activated carbon adsorption and high-vacuum deodorization, the sensory score of fishy odor is ≤1.0 and the color of the final product is ≤R0.6 / Y7.5.

[0027] 8. Green and environmentally friendly: No organic solvents are used throughout the process, and solids such as cell walls can be recycled and reused. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0029] It is important to note that, in the context of this invention, all figures disclosed herein are approximate values, regardless of whether words such as "approximately" or "about" are used. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%, etc. Whenever a figure with a value of N is disclosed, any figure having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% is explicitly disclosed, where "+ / -" indicates addition or subtraction. Whenever a lower limit, RL, and an upper limit, RU, of a numerical range are disclosed, any value within that disclosed range is explicitly disclosed. In particular, the following value is included within this range: R = RL + K * (RU - RL), where K is a variable increasing in 1% increments from 1% to 100%. Examples include: 1%, 2%, 3%, 4%, 5%, 50%, 51%, 52%, 95%, 96%, 97%, 98%, 99%, or 100%. Additionally, the numerical ranges defined herein by the two R numbers are specifically included. Unless otherwise specified, "%" in this invention refers to mass percentage or dimensionless percentage.

[0030] The terms "above" or "below" in this invention include the stated number.

[0031] The term "homogenization and cell disruption" as used in this invention includes, but is not limited to, devices and combinations thereof well-known to those skilled in the art capable of producing homogenization effects, such as Freund's cell crushers, ultrasonic disruptors, homogenizers, ball mills, rod mills, pebble mills, bead mills, high-pressure grinding rollers, vertical shaft impactors, industrial mixers, high-shear mixers, paddle mixers, and polytron homogenizers. In some embodiments, cells are passed through an optionally heated homogenizer / homogenizer. In some embodiments, homogenization and cell disruption may include homogenization 1 to 3 times using a homogenizer.

[0032] The "full-process nitrogen protection" mentioned in this invention does not mean that a large amount of nitrogen is forcibly introduced into the material in all stages. Its specific implementation methods include: (a) in high vacuum stages such as deodorization, nitrogen is used as a barrier gas for mechanical seals to prevent external air from seeping in, while the interior of the tower relies on a vacuum system to maintain an ultra-low pressure environment; (b) when connecting stages and breaking the vacuum, nitrogen is used as a vacuum breaking medium to replace air in contact with hot grease; (c) a slight positive pressure (exemplarily 0.01~0.05MPa) of nitrogen is maintained in the headspace of the feed tank, buffer tank, mixer, and each feed / discharge port.

[0033] The "solid content" mentioned in this invention refers to the total mass of lipids, cell walls, and other solids in the system during this step.

[0034] The term "trans fatty acids not detected" in this invention means that, when tested using the method specified in GB 5009.257-2016, the content of trans fatty acids is below the detection limit of 0.012%.

[0035] Testing methods for various parameters of the product: Moisture content: GB 5009.236-2016; Impurities: GB / T 15688-2024; Acid value (AV): GB 5009.229-2025; Peroxide value (POV): GB 5009.227-2023; Anisidine value (p-AV): GB / T 24304-2024; DHA content: GB 5009.168-2016; Saturated fatty acid content: A0AC 996.06; Unsaturated fatty acid content: A0AC 996.06; Color: Q / XMHS 0001S-2025; Fishy smell rating: Q / XMHS 0001S-2025; Malondialdehyde content: GB 5009.181-2016; Trans fatty acid content: GB 5009.257-2016; Oxidation induction time: Refer to GB / T 21121-2024; Oxidation rate: Accelerated oxidation experiment using a 60℃ constant temperature chamber.

[0036] Example 1: Lipids obtained from fermentation broth of microbial cells and their preparation method Step 1: Fermentation and High-Temperature Inactivation Heterotrophic fermentation was carried out in a fermenter using Schizochytrium sp. HS01, with a fermentation cycle of 6 days.

[0037] After fermentation, the fermentation broth is subjected to high-temperature continuous sterilization at 121°C for 45 seconds (heating medium is saturated steam) through a plate heat exchanger to obtain an inactivated fermentation broth. This step can completely kill microorganisms, inactivate endogenous lipoxygenase (LOX) activity, and cause irreversible denaturation and inactivation of the enzyme protein.

[0038] Step 2: Cooling and homogenization (high-pressure homogenizer, 200 bar) The inactivated fermentation broth was cooled to 80°C using a plate heat exchanger, and then subjected to physical homogenization and cell wall disruption twice using a high-pressure homogenizer (200 bar) to obtain a cell-wall-broken emulsion. In the above steps, heating to 80°C improves the fluidity of the lipids in the fermentation broth, makes the cell walls easier to break down, and reduces the viscosity of the fermentation broth.

[0039] Step 3: Vacuum demulsification and oil extraction The emulsion after cell wall disruption enters the demulsification reaction tank for the following operations.

[0040] (a) Dilution and Antioxidant The cell wall-breaking emulsion was diluted with deionized water to a solid content of 15 wt%. Then, the first antioxidant, sodium D-isoascorbate and ascorbate palmitate (mass ratio 36:1), was added. Based on the current solid content of the system (i.e., the mass of the cell wall-breaking liquid before dilution), the amount of the first antioxidant added was 10 g / kg. The reaction vessel was kept at 80°C for 2 hours.

[0041] (ii) Adding inorganic salts under vacuum conditions The mixing tank was evacuated to -0.05 MPa. An inorganic salt solution (25 wt% mass concentration, a mixture of magnesium chloride and calcium chloride in a 1:1 mass ratio) was added, with a salt content equivalent to 6.5 wt% of the system's solid content. The temperature was controlled at 80 ℃ and the stirring speed at 30 rpm. Nitrogen gas was then introduced and the mixture was rolled for 1 hour.

[0042] (iii) Adding organic acids to break the emulsion Add a citric acid solution (20wt% mass concentration) with an acid content equivalent to 1.3wt% of the solid content of the system, mix well, stop stirring immediately, and let stand for 1 hour.

[0043] (iv) Three-phase disc centrifuge After settling, the material was fed into a three-phase centrifuge at 6200 rpm, and the upper crude oil phase was collected. Then, a 15 wt% citric acid solution was slowly added under stirring to adjust the pH to 6.0 ± 0.1, followed by low-temperature continuous alkali refining. The physicochemical properties of the upper crude oil obtained from vacuum demulsification and extraction are shown in Table 1.

[0044] Table 1 Physicochemical properties of the upper crude oil

[0045] Step 4: Low-temperature continuous alkali refining After pH adjustment, the upper crude oil enters the low-temperature continuous alkali refining section, employing a "low-temperature mixing + heating separation" strategy: at room temperature, it is neutralized with 0.2% excess alkali and 10wt% NaOH solution (alkali solution) (in this embodiment, a high-shear mixer + static mixer is used for mixing). Then, the temperature is increased to 55℃ at a rate of 1.5℃ / min, and then it is centrifuged (in this embodiment, a disc separator is used for centrifugation at a speed of 6800 rpm). After separation, the light phase deacidified oil is taken, and its FFA is measured to be 0.05%, with the acid value decreasing from 2.5 mg KOH / g in the upper crude oil to 0.05 mg KOH / g.

[0046] Step 5: Continuous washing and drying The light phase deacidified oil after alkali refining enters the continuous water washing section: it is washed in two stages of countercurrent water at 90°C with deionized water, and then the oil phase is separated and retained by a disc separator.

[0047] The washed oil phase was dried in a vacuum dryer (drying temperature 85℃, absolute pressure 2kPa, residence time 60s, output moisture 0.05wt%) to obtain dried crude oil.

[0048] Step Six: Low-Temperature Winterization Crystallization The dried crude oil was cooled to -2°C at a rate of 3°C / h, and winterization crystallization was completed after 24 hours (with slow stirring at 10 rpm). After crystallization, the material was fed into a sealed plate and frame filter press for solid-liquid separation (filtration temperature -2°C, filter medium: filter cloth, filtration pressure: 0.3 MPa). The collected liquid phase was clear lipid. The physicochemical properties before and after low-temperature winterization crystallization are compared in Table 2.

[0049] Table 2 Comparison of physicochemical properties before and after low-temperature winterization crystallization in Example 1

[0050] Step 7: Activated carbon adsorption for decolorization and deodorization The clarified lipids were heated to 45°C, and 2 wt% of activated carbon by weight of the oil was added. The mixture was stirred and adsorbed for 1 hour under nitrogen protection. The adsorbed lipids were then filtered through an activated carbon filter. The color and odor scores before and after adsorption are shown in Table 3.

[0051] Table 3 Comparison of color and odor scores before and after activated carbon adsorption in Example 1

[0052] Step 8: High-vacuum deodorization The adsorbed lipids were deodorized for 3 hours in a deodorization tower at 160±1℃ under a pressure of 80 Pa to obtain deodorized lipids. The steam flow rate for high-vacuum deodorization was 20 kg / h. The comparison of the physicochemical properties before and after deodorization is shown in Table 4.

[0053] The steam used in high vacuum deodorization is clean and dry steam, which is produced through multi-stage purification: deionized water → superheated to 150-200℃ → steam dryness booster → filtered through a 0.22μm PTFE filter.

[0054] Table 4 Comparison of physicochemical properties before and after high vacuum deodorization in Example 1

[0055] Step Nine: Rapid Cooling and Antioxidant Treatment The deodorized lipid discharge temperature is about 160℃. It is cooled down by a rapid cooling system: a plate heat exchanger (heat recovery) and a plate heat exchanger (10℃ chilled water) are connected in series, which can cool it down to below 60℃ within 3-5 minutes.

[0056] Immediately after cooling, a second antioxidant was added. This second antioxidant was a mixture of vitamin E (mixed tocopherols) and ascorbate palmitate. The amount of vitamin E added was 1.6 kg / t, and the content of ascorbate palmitate was 0.2 kg / t. The physicochemical properties of the final lipid product are shown in Table 5.

[0057] Table 5 Physicochemical properties of the finished lipid product from Example 1

[0058] Example 2 (Optimization of demulsification and oil extraction parameters) The difference between Example 2 and Example 1 is that the mass concentration of magnesium chloride and calcium chloride solutions was increased to 40%, while all other conditions remained the same. Table 6 shows a comparison of the physicochemical properties of Example 1 and Example 2 after vacuum demulsification and oil extraction, and Table 7 shows a comparison of the key physicochemical properties of the final lipid product.

[0059] Table 6 Comparison of physicochemical properties of Example 1 and Example 2 after vacuum demulsification and oil extraction

[0060] Table 7 Comparison of key physicochemical properties of the final lipid products from Example 1 and Example 2

[0061] Compared to Example 1, Example 2 increased the mass concentration of magnesium chloride and calcium chloride solution to 40 wt%, thereby increasing the yield of the upper crude oil obtained in the demulsification and oil extraction stage to 96%, reducing the peroxide value to 0.1 mmol / kg, the anisidine value to 2.0, and increasing the DHA content to 57.9%. In the final product lipid, the peroxide value, anisidine value, and malondialdehyde content all decreased.

[0062] Example 3 (Comparison of different alkali refining temperatures) To verify the advantages of the "25℃ mixing + heating to 55-60℃" strategy in low-temperature continuous alkali refining, the other preparation steps and parameter conditions, except for the temperature program of low-temperature continuous alkali refining, are as in Example 1. The results of different alkali refining methods are compared in Table 8.

[0063] Table 8. Impact of different low-temperature continuous alkali refining methods on the results of the low-temperature continuous alkali refining section.

[0064] Example 4 (Comparison of different winterization temperatures) The purpose of this embodiment is to investigate the effects of different winterization temperatures on the loss rate of saturated fatty acids and DHA, the yield of oil, and the low-temperature stability of clarified lipids. Except for the winterization temperature, other preparation steps and parameters are the same as in Example 1, and the test results are shown in Table 9.

[0065] Table 9. Effects of different winterization temperatures on the results of the low-temperature winterization crystallization section.

[0066] Example 5 (Verification of the effect of nitrogen purging protection throughout the process) The purpose of this embodiment is to investigate the effect of inert atmosphere protection on the peroxide value of the final product. Except for whether nitrogen protection is used, the other preparation steps and parameter conditions are the same as in Example 1, and the test results are shown in Table 10.

[0067] Table 10. Effect of inert atmosphere on lipid peroxide value of final product

[0068] Example 6 (Accelerated Oxidation Experiment) The purpose of this embodiment is to investigate the effect of adding different second oxidants on the oxidation rate of the finished lipids. Except for the different second oxidants, the other preparation steps and parameters are the same as in Example 1. The oxidation rate is tested by an accelerated oxidation experiment in a 60℃ constant temperature oven. The final results are shown in Table 11.

[0069] Table 11. Effect of the selection of the second oxidant on the oxidation rate of the final product.

[0070] The shelf life at 25°C is estimated to be ≥24 months.

[0071] Example 7 (Reproducibility Verification in Three Batches) The reproducibility of the sample was verified in three batches according to the preparation method in Example 1, and the results are shown in Table 12.

[0072] Table 12 Reproducibility verification of three batches in Example 1

Claims

1. A lipid obtained from the fermentation broth of microbial cells, characterized in that, Its peroxide value is ≤0.05mmol / kg, p-anisidine value is ≤4.0, total oxidation value is ≤4.0, malondialdehyde content is ≤0.10mg / kg, trans fatty acids are not detected and the fishy smell sensory score is ≤1.0; It is obtained by inactivating the fermentation broth of microbial cells, followed by vacuum demulsification and oil extraction under an inert atmosphere, low-temperature continuous alkali refining, low-temperature winterization crystallization, activated carbon adsorption, and high-vacuum deodorization.

2. The lipid according to claim 1, characterized in that, The microbial cells are at least one of the following: algal cells, yeast cells, fungal cells, protozoan cells, or bacterial cells that produce polyunsaturated fatty acids; preferably, the microbial cells are from the genera *Morchella*, *Cryptocoryne*, or *Schizochytrida*; preferably, the microbial cells are from the order *Schizochytrida*; preferably, the microbial cells are from the genera *Chytridium* and / or *Schizochytridium*.

3. The method for preparing lipids according to claim 1 or 2, characterized in that, This includes inactivating the fermentation broth from microbial cells, followed by vacuum demulsification and oil extraction under an inert atmosphere, low-temperature continuous alkali refining, low-temperature winterization crystallization, activated carbon adsorption, and high-vacuum deodorization; The demulsifier used in the vacuum demulsification oil extraction is an inorganic salt and an organic acid. The inorganic salt is potassium chloride, sodium sulfate, magnesium sulfate, calcium chloride, EDTA-2Na, sodium chloride, magnesium chloride, and potassium sulfate. The organic acid is at least one selected from malic acid, citric acid, oxalic acid, and glacial acetic acid. The pH of the vacuum demulsification oil extraction is 4.0 to 10.

0. Preferably, the pH of the vacuum demulsification oil extraction is 5.5 to 8.

5. The low-temperature continuous alkali refining process involves neutralizing the system with the alkali solution at room temperature, followed by heating to 55-60°C for separation; preferably, the heating rate of the low-temperature continuous alkali refining process is 1-2°C / min. The crystallization temperature of the low-temperature winterization crystallization is -5 to -1℃, and the crystallization time of the low-temperature winterization crystallization is 24 to 36 hours; preferably, the crystallization temperature of the low-temperature winterization crystallization is -2℃; preferably, the cooling rate of the low-temperature winterization crystallization is 3 to 5℃ / h; preferably, the stirring speed of the low-temperature winterization crystallization is 5 to 10 rpm. The high vacuum deodorization pressure is ≤80Pa, the high vacuum deodorization temperature is 150~170℃, the high vacuum deodorization time is 2~4h, and the high vacuum deodorization steam flow rate is 5~20kg / h; preferably, the high vacuum deodorization temperature is 160℃.

4. The preparation method according to claim 3, characterized in that, The vacuum demulsification and oil extraction operation is as follows: under -0.05~-0.1MPa, first add the inorganic salt, keep warm at 70~90℃ for 1~2h, then add the organic acid, continue to keep warm for 0.5~2h, and separate and collect the upper crude oil. Preferably, the inorganic salt is a mixture of magnesium chloride and calcium chloride, preferably in a mass ratio of 1:0.8 to 1.2, and more preferably 1:1; Preferably, the amount of inorganic salt added is 1-10 wt% of the solid content of the system, and more preferably 6.5 wt%. Preferably, the organic acid is citric acid; Preferably, the amount of organic acid added is 1.0~2.0 wt% of the solid content of the system, more preferably 1.3 wt%. Preferably, the vacuum demulsification and oil extraction further includes adjusting the pH of the upper crude oil to 6.2±0.1 using an organic acid.

5. The preparation method according to claim 3, characterized in that, The operation of the low-temperature continuous alkali refining is as follows: the upper crude oil obtained by vacuum demulsification is neutralized with alkali solution at room temperature, and then the temperature is raised to 50~60℃ to separate and obtain light phase deacidified oil. Preferably, the alkaline solution is added at a concentration of 0.2-0.3% of the excess alkali. Preferably, the alkaline solution is a 10 wt% NaOH solution; Preferably, the room temperature is 25°C.

6. The preparation method according to claim 3 or 4, characterized in that, The vacuum demulsification and oil extraction also includes adding a first antioxidant to the system; Preferably, the first antioxidant is a mixture of sodium D-isoascorbate and ascorbate palmitate; preferably, the mass ratio of sodium D-isoascorbate to ascorbate palmitate is 36:1; Preferably, the amount of the first antioxidant added is 10 g / kg based on the solid content of the system; Preferably, the vacuum demulsification and oil extraction further includes, after adding the first antioxidant, keeping the mixture at 70-90°C for at least 1-3 hours; Preferably, the vacuum demulsification and oil extraction further includes diluting the system with water to a solid content of 12-22 wt%, preferably 15 wt%, before adding the first antioxidant.

7. The preparation method according to claim 3, characterized in that, The inactivation is a continuous sterilization treatment at 121°C; preferably, the continuous sterilization treatment time is ≤1 min, more preferably 30~60 s; and / or, The method also includes homogenizing and disrupting the cell walls of the inactivated fermentation broth before the vacuum demulsification and oil extraction. Specifically, the inactivated fermentation broth is cooled to 80°C under an inert atmosphere, and then homogenized and disrupted at 200 bar; and / or, The process between the low-temperature continuous alkali refining and the low-temperature winterization crystallization further includes: washing with deionized water at 85-90°C, followed by vacuum drying at 80-85°C; and / or, The specific operation of activated carbon adsorption is as follows: the product after low-temperature winterization crystallization is filtered, then heated to 45°C, and activated carbon at 2wt% of the oil weight is added for adsorption for 1 hour, followed by filtration to remove the activated carbon; preferably, the activated carbon has an iodine value ≥1000 mg / g and a main pore size distribution of 2~5μm; and / or, After the high-vacuum deodorization, a second antioxidant is added to the product.

8. The preparation method according to claim 3, characterized in that, Includes the following steps: (1) High temperature inactivation: The fermentation broth was inactivated at 121℃ for 30~60s to obtain inactivated fermentation broth; (2) Cooling and homogenization: The inactivated fermentation broth was cooled to 80°C and then homogenized at 200 bar to obtain a cell wall broken emulsion; (3) Vacuum demulsification and oil extraction: Dilute the cell wall broken emulsion to 12-22 wt% of its solid content, then add 10 g / kg of the first antioxidant, add the inorganic salt first at -0.05--0.1 MPa, keep warm at 70-90℃ for 1-2 h, then add the organic acid, continue to keep warm for 0.5-2 h, and separate and collect the upper crude oil; (4) Low-temperature continuous alkali refining: Add alkali solution to the upper crude oil at an excess alkali amount of 0.2~0.3% to neutralize it, and then heat it to 50~60℃ at a heating rate of 1~2℃ / min to separate and obtain light phase deacidified oil; (5) Washing and drying: The light phase deacidified oil is washed with deionized water at 85~90℃ and then vacuum dried at 80~85℃ to obtain dried crude oil; (6) Low-temperature winterization crystallization: The dried crude oil is cooled to -5 to -1℃ at a cooling rate of 3~5℃ / h, and after 24~36h, the liquid phase is filtered and retained to obtain clear lipids; (7) Activated carbon adsorption: The clarified lipid is heated to 45°C, and 2wt% of activated carbon by weight of the lipid is added for adsorption for 1 hour. Then the activated carbon is removed by filtration to obtain the adsorbed lipid. (8) High vacuum deodorization: The adsorbed lipids are deodorized in a deodorization tower with a pressure ≤80Pa at 150~170℃ for 2~4h to obtain deodorized lipids. The steam flow rate of the high vacuum deodorization is 5~20kg / h. (9) Rapid cooling and anti-oxidation: Cool the deodorized lipids to ≤60℃ within 3~5 min, add a second oxidant, and the product is obtained; Steps (2) to (9) are carried out under an inert atmosphere.

9. A microbial inoculant, characterized in that, The lipids include those described in claim 1 or 2, or those prepared by any one of claims 3-8.

10. A product, cosmetic, or pharmaceutical composition for use in animals or humans, characterized in that, Includes the lipids described in claim 1 or 2, the lipids prepared by the method described in any one of claims 3-8, or the microbial agent described in claim 9; Preferably, the food is an additive, nutritional supplement, or animal feed for use in animal or human food. Preferably, the animal feed is aquaculture feed, livestock feed, zoo animal feed, draft animal feed, or domestic animal feed.

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