Application and method of bacillus pumilus in joint production of squalene and unsaturated fatty acid and obtained microbial oil

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to application and method of bacillus pumilus in joint production of squalene and unsaturated fatty acid and obtained microbial oil. A method for joint production of squalene and unsaturated fatty acid comprises the following steps: S1, respectively carrying out amplification culture on schizochytrium limacinum HS08 and bacillus pumilus to obtain a schizochytrium limacinum amplification culture and a bacillus amplification culture; wherein the preservation number of the schizochytrium limacinum HS08 is CGMCC (China General Microbiological Culture Collection Center) No.40902, and the preservation number of the bacillus pumilus is CGMCC No.1.1166; s2, one of the schizochytrium limacinum enlarged culture and the bacillus enlarged culture obtained in S1 is inoculated into a co-culture medium for fermentation; the bacillus pumilus and the schizochytrium limacinum HS08 are subjected to combined fermentation, so that the yield of microbial oil produced by microalgae fermentation is greatly increased, particularly the content of squalene and EPA is also greatly increased, the method is suitable for industrial large-scale production, and the production cost of squalene and EPA is reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and particularly relates to the use, method and resulting microbial oil of a short-lived Bacillus strain for the co-production of squalene and unsaturated fatty acids. Background Technology

[0002] Eicosapentaenoic acid (EPA) is an n-3 long-chain polyunsaturated fatty acid commonly found in deep-sea fish oil. It offers numerous health benefits, playing a vital role in regulating the immune system, preventing cardiovascular disease, regulating cholesterol metabolism, and neurotransmission. While some bacteria and lower eukaryotes can synthesize EPA de novo via the polyketide synthase pathway, humans lack the key lipases and dehydrogenases (especially 12 and 13 / n-3 dehydrogenases) required for EPA synthesis. Therefore, EPA and other long-chain polyunsaturated fatty acids are primarily obtained through dietary intake. However, in recent years, n-3 fatty acids from deep-sea fish oil have presented several problems, including the rapid depletion of marine resources, nuclear pollution, heavy metal pollution, fishy odor, and the rise of vegetarianism. Consequently, primary biomass producers such as marine microalgae have gradually come into focus. Marine microalgae are initial producers of many bioactive substances, including polyols, polypeptides, organic acids, carotenoids, fatty acids, phytosterols, hormones, and vitamins. Among marine microalgae, certain species such as Schizochytridium, Porphyra, green algae, Chlorella, and yeast have been proven to possess the ability to efficiently synthesize EPA. Schizochytridium and yeast, in particular, are excellent strains for industrial production due to their short growth cycles and ease of large-scale cultivation. Meanwhile, squalene, an important triterpenoid compound, possesses physiological functions such as antioxidant activity, immune enhancement, skin moisturizing, and fatigue relief, and is widely used in pharmaceuticals, cosmetics, and health products. Its traditional source is shark liver oil, but this also faces ecological damage and sustainability issues. The production capacity of active substances from a single strain is limited by its own metabolism. Therefore, utilizing a co-culture system of oil-producing microalgae and bacteria can achieve synergistic metabolism and improve the accumulation efficiency of the target product. By optimizing the carbon-to-nitrogen ratio, temperature, and dissolved oxygen conditions, the co-production capacity of polyunsaturated fatty acids and squalene can be further enhanced, providing a feasible pathway to replace traditional plant and animal sources. Summary of the Invention

[0003] In view of the problems existing in the prior art, the purpose of this invention is to provide a method and application of Bacillus pumilus for the co-production of squalene and unsaturated fatty acids, and the resulting microbial oil, so as to solve the problems of the prior art.

[0004] To achieve the above objectives, in a first aspect, a use of Bacillus pumilus for the co-production of squalene and unsaturated fatty acids is provided, wherein the Bacillus pumilus is used in conjunction with Schizochytrium to co-produce squalene and unsaturated fatty acids, and the preservation number of the Bacillus pumilus is CGMCC No. 1.1166.

[0005] Secondly, a method for the joint production of squalene and unsaturated fatty acids is provided, comprising the following steps: S1: Split-chrysogenum HS08 and the Bacillus subtilis described in claim 1 were cultured on a large scale to obtain a large-scale culture of Split-chrysogenum and a large-scale culture of Bacillus subtilis; wherein, the preservation number of Split-chrysogenum HS08 is CGMCC No.40902; S2: Inoculate either the Schizochytrium expanded culture or the Bacillus expanded culture obtained in S1 into a common culture medium for fermentation. The fermentation process is divided into different fermentation stages. In this process, the sugar concentration of the common culture medium is controlled during at least part of the fermentation period; In particular, the pH value of the common culture medium is controlled during at least part of the fermentation period; In this process, the temperature of the common culture medium is controlled during at least part of the fermentation period; In this process, the dissolved oxygen level of the common culture medium is controlled during at least part of the fermentation period; During this process, at certain times, another expansion culture, or ammonium sulfate and valine, is added to the common culture medium. The "another expansion culture" refers to, when S2 is first executed, if the earliest expansion culture inoculated into the common culture medium is a *Schizochytrium* expansion culture, then the "another expansion culture" refers to a *Bacillus* expansion culture; if the earliest expansion culture inoculated into the common culture medium is a *Bacillus* expansion culture, then the "another expansion culture" refers to a *Schizochytrium* expansion culture.

[0006] In some embodiments of the second aspect, in S1, the expanded culture includes the following steps: T1: Take one of the Split-Chrystropharia HS08 or the Bacillus pumilus and inoculate it into an activation medium to obtain an activated seed solution; T2: The activated seed solution obtained in T1 is inoculated into the primary seed culture medium and cultured to obtain the primary seed solution; T3: The primary seed culture obtained from T2 is inoculated into the secondary seed culture medium and cultured to obtain the secondary seed culture; T4: Transfer the secondary seed culture obtained in T3 to the initial culture medium for fermentation to obtain either the *Schizochytrium* expanded culture or the *Bacillus* expanded culture. That is, when *Schizochytrium* HS08 is selected in T1, a *Schizochytrium* expanded culture is obtained here; when *Bacillus pumilus* is selected in T1, a *Bacillus* expanded culture is obtained here. In S1, the expanded culture is performed at least twice in total, with at least one expansion culture of *Schizochytrium* selected in T1 and at least one expansion culture of *Bacillus pumilus* selected in T1, to obtain sufficient *Schizochytrium* and *Bacillus* expanded cultures.

[0007] In some embodiments of the second aspect, T1 specifically involves: taking the *Schizochytrium HS08* or the *Bacillus pumilus*, inoculating it into an activation culture medium, and culturing it in a shaker at 25-30°C and a rotation speed of 150-250 r / min for 12-24 hours to obtain an activated seed solution.

[0008] In some embodiments of the second aspect, in T1, the activation culture medium comprises: 40-60 parts by weight of glucose, 10-20 parts by weight of yeast extract, 10-15 parts by weight of anhydrous sodium sulfate, 0.1-0.2 parts by weight of anhydrous calcium chloride, 0.3-0.6 parts by weight of potassium chloride, 0.5-1.5 parts by weight of potassium dihydrogen phosphate, 2.0-4.0 parts by weight of ammonium sulfate, 1.0-4.0 parts by weight of zinc sulfate, and 2.0-6.0 parts by weight of magnesium sulfate. The activated culture medium contains, in parts by weight, 0.5-1.5 parts potassium sulfate; preferably, the activated culture medium consists of (unit g / L): 40-60 parts glucose, 10-20 parts yeast extract, 10-15 parts anhydrous sodium sulfate, 0.1-0.2 parts anhydrous calcium chloride, 0.3-0.6 parts potassium chloride, 0.5-1.5 parts potassium dihydrogen phosphate, 2.0-4.0 parts ammonium sulfate, 1.0-4.0 parts zinc sulfate, 2.0-6.0 parts magnesium sulfate, and 0.5-1.5 parts potassium sulfate; the pH is natural.

[0009] In some embodiments of the second aspect, T2 specifically involves: inoculating the activated seed solution obtained in T1 into a primary seed culture medium at an inoculation rate of 5-8 v / v % and culturing it in a shaker at 25-30°C and a rotation speed of 150-250 r / min for 24-48 h to obtain the primary seed solution.

[0010] In some embodiments of the second aspect, in T2, the primary seed culture medium comprises: 50-70 parts by weight of glucose, 8-12 parts by weight of yeast extract, 10-15 parts by weight of anhydrous sodium sulfate, 0.1-0.2 parts by weight of anhydrous calcium chloride, 0.3-0.6 parts by weight of potassium chloride, 0.5-1.5 parts by weight of potassium dihydrogen phosphate, 2.0-4.0 parts by weight of ammonium sulfate, 1.0-4.0 parts by weight of zinc sulfate, 2.0-6.0 parts by weight of magnesium sulfate, and 0.5-1.5 parts by weight of potassium sulfate. Preferably, the primary seed culture medium comprises (in g / L): glucose 50-70, yeast extract 8-12, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5, and natural pH.

[0011] In some embodiments of the second aspect, T3 specifically involves: inoculating the primary seed culture obtained in T2 into the secondary seed culture medium at an inoculation rate of 8-12 v / v % and culturing it in a shaker at 25-30°C and a rotation speed of 150-250 r / min for 24-48 h to obtain the secondary seed culture.

[0012] In some embodiments of the second aspect, in T3, the secondary seed culture medium comprises: 60-100 parts by weight of glucose, 5-10 parts by weight of yeast extract, 10-15 parts by weight of anhydrous sodium sulfate, 0.1-0.2 parts by weight of anhydrous calcium chloride, 0.3-0.6 parts by weight of potassium chloride, 0.5-1.5 parts by weight of potassium dihydrogen phosphate, 2.0-4.0 parts by weight of ammonium sulfate, 1.0-4.0 parts by weight of zinc sulfate, 2.0-6.0 parts by weight of magnesium sulfate, and 0.5-1.5 parts by weight of potassium sulfate. Preferably, the secondary seed culture medium comprises (in g / L): glucose 60-100, yeast extract 5-10, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5, and natural pH.

[0013] In some embodiments of the second aspect, T4 specifically involves: transferring the secondary seed culture obtained in T3 to a fermenter at an inoculum of 20-50 v / v%, and culturing it for 110-150 h at a temperature of 22-32°C and a pH of 2.0-8.0 using the initial culture medium to obtain the expanded culture of *Schizochytrium* or the expanded culture of *Bacillus pumilus*.

[0014] In some embodiments of the second aspect, in T4, the initial culture medium composition includes: 10-30 parts by weight of glucose, 5-20 parts by weight of yeast extract, 4.0-5.0 parts by weight of ammonium sulfate, 10-20 parts by weight of anhydrous sodium sulfate, 4.0-5.0 parts by weight of magnesium sulfate, and 6.0-10 parts by weight of potassium dihydrogen phosphate. Preferably, the initial culture medium composition (in g / L) is: 10-30 parts by weight of glucose, 5-20 parts by weight of yeast extract, 4.0-5.0 parts by weight of ammonium sulfate, 10-20 parts by weight of anhydrous sodium sulfate, 4.0-5.0 parts by weight of magnesium sulfate, and 6.0-10 parts by weight of potassium dihydrogen phosphate.

[0015] In some embodiments of the second aspect, in S2, the common culture medium comprises: 10-30 parts by weight of glucose, 5-20 parts by weight of yeast extract, 4.0-5.0 parts by weight of ammonium sulfate, 10-20 parts by weight of anhydrous sodium sulfate, 4.0-5.0 parts by weight of magnesium sulfate, and 6.0-10 parts by weight of potassium dihydrogen phosphate. Preferably, the common culture medium comprises (in g / L): 10-30 parts by weight of glucose, 5-20 parts by weight of yeast extract, 4.0-5.0 parts by weight of ammonium sulfate, 10-20 parts by weight of anhydrous sodium sulfate, 4.0-5.0 parts by weight of magnesium sulfate, and 6.0-10 parts by weight of potassium dihydrogen phosphate.

[0016] In some embodiments of the second aspect, S2 specifically involves: inoculating the *Schizochytrium* expansion culture into the common culture medium at an inoculum size of 18-20 v / v% to initiate fermentation; controlling the sugar concentration at 40-60 g / L for 0-72 h and at 5-15 g / L for 72-120 h; adjusting the pH during fermentation with a mixed solution of ammonia, succinic acid, and acetic acid, maintaining the pH at 5.0-6.0 for 0-48 h, 6.0-7.0 for 48-96 h, and 6.5-7.5 for 96-120 h; initially maintaining the fermentation temperature at 29°C, decreasing to 26°C after 48 h; initially controlling the dissolved oxygen at 80-100%, maintaining it at 60-80% for 48-72 h, above 50% for 72-96 h, and below 30% for 96-120 h; adding 0.05-0.25 v / v% of the *Bacillus* expansion culture after 72 h of fermentation, and continuing fermentation for 96 h... Add 0.05-0.25 v / v% of the Bacillus spores culture at h, and add ammonium sulfate and valine, and ferment for at least 120 h.

[0017] Thirdly, a microbial oil obtained by the method described in the second aspect is provided, containing squalene and eicosapentaenoic acid (EPA), wherein the squalene content is 2-27 w / w, for example, it can be 2 w / w, 3 w / w, 4 w / w, 5 w / w, 6 w / w, 7 w / w, 8 w / w, 9 w / w, 10 w / w, 11 w / w, 12 w / w, 13 w / w, 14 w / w, 15 w / w, 16 w / w, 17 w / w, 18 w / w, 19 w / w, 20 w / w, 21 w / w, 22 w / w, 23 w / w, 24 w / w, or 25 w / w; and the eicosapentaenoic acid content is 3-12 w / w, for example, it can be 3 w / w, 4 w / w, 5 w / w, 6 w / w, 7 w / w, 8 w / w, 9 w / w, 10 w / w, 11 w / w, 12 w / w, 13 w / w, 14 w / w, 15 w / w, 16 w / w, 17 w / w, 18 w / w, 19 w / w, 20 w / w, 21 w / w, 22 w / w, 23 w / w, 24 w / w, or 25 w / w; w / w%, 8 w / w%, 9 w / w%, 10 w / w%, 11 w / w% or 12 w / w%.

[0018] Compared with the prior art, a certain embodiment of the present invention includes at least one of the following beneficial effects: By co-fermenting Bacillus pumilus with Schizochytrium HS08, the yield of microbial oil produced by microalgae fermentation was significantly increased, especially the content of squalene and EPA, which was also greatly improved, making it suitable for large-scale industrial production and reducing the production cost of squalene and EPA.

[0019] Terminology Definition In the context of this invention, all figures disclosed herein are approximate values, regardless of whether the words "approximately" or "about" are used. Based on the disclosed figures, each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0020] The term "inoculation volume" refers to the ratio of the volume of seed culture transferred to the volume of culture medium after inoculation.

[0021] The terms “Bacillus” and “Bacillus pumilus” are used interchangeably throughout the text.

[0022] The *Schizochytridica* HS08 used in this invention belongs to the *Schizochytridica* family (…). Schizochytrium The specimen (sp.) with accession number CGMCC No.40902 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on October 26, 2023. Detailed Implementation

[0023] 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.

[0024] Split-chrysophylloides ( Schizochytrium sp.)HS08 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.40902.

[0025] Bacillus pumilus ( Bacillus pumilus Purchased from the China General Microbiological Culture Collection Center, accession number CGMCC 1.1166.

[0026] In the following comparative examples and embodiments, the scaling-up culture step includes the following steps: T1: Take one of the above-mentioned *Schizochytrium HS08* or *Bacillus pumilus* (here, take a glycerol tube of the corresponding strain from a -80℃ freezer), inoculate it into the activation medium, and culture it in a shaker at 200 r / min for 24 h at a shaking temperature of 28℃ to obtain the activated seed solution. T2: The activated seed solution obtained in T1 was inoculated into the primary seed culture medium at an inoculation rate of 5 v / v % and cultured on a shaker at 28℃ and 180 r / min for 48 h to obtain the primary seed solution. T3: The primary seed culture obtained from T2 was inoculated into the secondary seed culture medium at an inoculation rate of 10 v / v % and cultured in a shaker at 28°C and 180 r / min for 48 h to obtain the secondary seed culture. T4: Transfer the secondary seed culture obtained in T3 to a fermenter and ferment using the initial culture medium at a fermentation temperature of 22-32℃ and a fermentation pH of 2.0-8.0. During fermentation, adjust the pH using ammonia and 50% malic acid. Fermentation time is 110-150 h to obtain one of the Spicachytrids expanded culture or the Bacillus expanded culture.

[0027] in: In T1, the activated culture medium consists of the following components (in g / L): glucose 40-60, yeast extract 10-20, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5; pH is natural.

[0028] In T3, the secondary seed culture medium consists of the following components (in g / L): glucose 60-100, yeast extract 5-10, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5, and natural pH.

[0029] In T4, the initial culture medium composition is (unit g / L): glucose 10-30, yeast extract 5-20, ammonium sulfate 4.0-5.0, anhydrous sodium sulfate 10-20, magnesium sulfate 4.0-5.0, and potassium dihydrogen phosphate 6.0-10.

[0030] Here, the expansion culture shall be performed at least twice in total, wherein the expansion culture of T1 using Schizochytrium shall be performed at least once, and the expansion culture of T1 using Bacillus pumilus shall be performed at least once, in order to obtain sufficient Schizochytrium expansion culture and Bacillus pumilus expansion culture.

[0031] In the following examples and comparative examples, the 8-ton tank refers to an 8-ton fermentation tank. Before the start of the experiments described in the following examples and comparative examples, the 8-ton tanks in each example and comparative example had been prepared and filled with 4 tons of common culture medium. The composition of the common culture medium is (unit g / L): glucose 10-30, yeast extract 5-20, ammonium sulfate 4.0-5.0, anhydrous sodium sulfate 10-20, magnesium sulfate 4.0-5.0, and potassium dihydrogen phosphate 6.0-10.

[0032] Comparative Example 1 The above-described expansion culture steps were followed to obtain a large-scale culture of *Schizochytrium*. This culture was then inoculated into an 8-ton tank at a 20 v / v% inoculum rate to begin fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted using 25 w / w % ammonia and 50 w / w % malic acid, maintaining a range of 5.0-6.0. The fermentation temperature was 29℃, and dissolved oxygen was controlled at 50-100 v / v%. The fermentation broth was obtained after 120 h of fermentation.

[0033] Comparative Example 2 The Bacillus culture was expanded according to the above expansion culture steps and inoculated into an 8-ton tank at an inoculum size of 20 v / v% to start fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted with 25 w / w% ammonia and 50 w / w% malic acid to maintain a value of 5.0-6.0. The fermentation temperature was 29℃ and the dissolved oxygen was controlled at 50-100%. The fermentation broth was obtained after 120 h of fermentation.

[0034] Example 1 The above-described expansion culture steps yielded a *Schizochytrium* expansion culture, which was inoculated into an 8-ton tank at a 20 v / v % inoculum to begin fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted using 25 w / w% ammonia and 40 w / w% succinic acid, remaining at 5.0-6.0 from 0-72 h and 6.0-7.0 from 72-120 h. The fermentation temperature was 29℃, and the dissolved oxygen was controlled at 50-100%. After 48 h of fermentation, 0.1 v / v% of the *Bacillus* expansion culture obtained from the above expansion culture steps was added for further culturing. The fermentation broth was obtained after 120 h of fermentation.

[0035] Example 2 The above-described expansion culture steps yielded a *Schizochytrium* expansion culture, which was inoculated into an 8-ton tank at a 20 v / v% inoculum rate to begin fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted using 25 w / w% ammonia and 40 w / w% succinic acid, maintaining a pH of 5.0-6.0 from 0-72 h and 6.0-7.0 from 72-120 h. The initial fermentation temperature was 29℃, which was lowered to 25℃ after 48 h. The dissolved oxygen was initially controlled at 50-100%, maintained at 40-70% from 48-72 h, 30-50% from 72-96 h, and below 30% from 96-120 h. After 12 h of fermentation, 1.5 v / v% of the *Bacillus* expansion culture obtained from the above expansion culture steps was added, and the fermentation broth was obtained after 120 h of fermentation.

[0036] Example 3 The Bacillus culture was expanded according to the above expansion culture steps and inoculated into an 8-ton tank at an inoculum size of 1.5 v / v% to start fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted with a mixed solution of 25 w / w% ammonia and 15 w / w% succinic acid + 15 w / w% acetic acid, maintaining a pH of 5.0-6.0 from 0-48 h and 6.0-7.0 from 48-120 h. The initial fermentation temperature was 29℃, which was reduced to 25℃ after 48 h. The dissolved oxygen was initially controlled at 80-100%, maintained at 60-80% from 48-72 h, maintained above 50% from 72-96 h, and maintained at 30%-50% from 96-120 h. After 12 h of fermentation, 20 v / v% of the Schizochytrium culture obtained in the above expansion culture steps was added, and the fermentation broth was obtained after 120 h of fermentation.

[0037] Example 4 The *Schizochytrium* culture obtained according to the above expansion culture steps was inoculated into an 8-ton tank at an inoculum size of 20 v / v% to start fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted with a mixed solution of 25 w / w% ammonia and 15 w / w% succinic acid + 15 w / w% acetic acid, maintaining a pH of 5.0-6.0 from 0-72 h and 6.0-7.0 from 72-120 h. The initial fermentation temperature was 29℃, which was reduced to 26℃ after 48 h. The dissolved oxygen was initially controlled at 80-100%, maintained at 60-80% from 48-72 h, and maintained above 50% from 72-120 h. After 72 h of fermentation, 0.05 v / v% of the *Bacillus* culture obtained in the above expansion culture steps was added, followed by another 0.05 v / v% of the *Bacillus* culture obtained in the above expansion culture steps after 96 h. The fermentation broth was obtained after 120 h of fermentation.

[0038] Example 5 The *Schizochytrium* culture was obtained according to the above expansion culture steps and inoculated into an 8-ton tank at a 20 v / v% inoculum to begin fermentation. The sugar concentration was controlled at 40-60 g / L. During fermentation, the pH was adjusted using a mixed solution of 25 w / w% ammonia and 15 w / w% succinic acid + 15 w / w% acetic acid: 5.0-6.0 from 0-48 h, 6.5-7.5 from 48-96 h, and 6.0-7.0 from 96-120 h. The initial fermentation temperature was 29℃, decreasing to 26℃ after 48 h. Dissolved oxygen was initially controlled at 80-100%, maintained at 60-80% from 48-72 h, above 50% from 72-96 h, and 30%-50% from 96-120 h. After 72 h of fermentation, 0.05 v / v% of the *Bacillus* culture obtained in the above expansion culture steps was added, followed by 4 g / L ammonium sulfate and 3 g / L acetic acid at 96 h. The fermentation broth was obtained after fermenting g / L valine for 120 h.

[0039] Example 6 The above-described expansion culture steps yielded a large-scale culture of *Schizochytrium*, which was inoculated into an 8-ton tank at a 20 v / v% inoculum to begin fermentation. The sugar concentration was controlled at 40-60 g / L from 0-72 h and 5-15 g / L from 72-120 h. During fermentation, the pH was adjusted using a mixed solution of 25 w / w% ammonia and 15 w / w% succinic acid + 15 w / w% acetic acid: 5.0-6.0 from 0-48 h, 6.0-7.0 from 48-96 h, and 6.5-7.5 from 96-120 h. The initial fermentation temperature was 29℃, decreasing to 26℃ after 48 h. Dissolved oxygen was initially controlled at 80-100%, maintained at 60-80% from 48-72 h, above 50% from 72-96 h, and maintained at 96-120 h. h is controlled below 30%; after 72 hours of fermentation, 0.25 v / v% of the Bacillus spores culture obtained in the above expansion culture step is added, and then 0.25 v / v% of the Bacillus spores culture obtained in the above expansion culture step is added again after 96 hours. 4 g / L ammonium sulfate and 3 g / L valine are added, and the fermentation broth is obtained after 120 hours of fermentation.

[0040] The biomass and fat (microbial oil) content of the fermentation broth obtained in the above comparative examples and embodiments were tested. The squalene and EPA contents in the microbial oil were also tested: 4 mL of fermentation broth was accurately pipetted into a centrifuge at 6000 r / min for 4 min, the supernatant was discarded, and the broth was washed twice with distilled water. Afterward, it was placed in a freeze dryer and dried. The mass of the dried broth was then measured. Biomass = mass of freeze-dried algal powder / 4 * 1000; the total fat content was determined according to Method 1 of GB 5009.6-2016; the squalene content was determined according to the method in GB / T 43732-2024; and the EPA content was determined according to the normalization method in GB5009.168-2016. The results are shown in Table 1. Wherein, biomass refers to the dry weight of *Schizochytrium* cells in the fermentation broth; total fat refers to the fat content (w / w %) in the fermentation broth; squalene and EPA are relative to the total fat content (w / w %).

[0041] Table 1 shows the content of squalene, total fat, and EPA in Comparative Examples 1-2 and Examples 1-6.

[0042] Comparative Examples 1 and 2 show that when Schizochytrium and Bacillus were fermented separately, the squalene and EPA contents were low when only Schizochytrium was fermented, and almost no fat was produced when only Bacillus was fermented. The examples show that the biomass and squalene content were significantly increased after the two were mixed and cultured, indicating that the metabolites of Bacillus can provide Schizochytrium with promoting factors related to the squalene metabolic pathway. Subsequently, by gradually optimizing the timing and amount of Bacillus supplementation, and supplementing with a phased temperature, pH, dissolved oxygen, and glucose regulation strategy, exogenous factors were added, which ultimately significantly increased the biomass, fat, squalene, and EPA production of the cells.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of Bacillus pumilus for the joint production of squalene and unsaturated fatty acids, characterized in that, The Bacillus pumilus has a preservation number of CGMCC No. 1.1166. 2.A method for co-production of squalene and unsaturated fatty acid, comprising the following steps: S1: separately expanding the Schizochytrium sp. HS08 and the Bacillus pumilus of claim 1 to obtain the Schizochytrium sp. expanded culture and the Bacillus pumilus expanded culture; wherein the Schizochytrium sp. HS08 has a preservation number of CGMCC No. 40902; S2: inoculating one of the Schizochytrium sp. expanded culture or the Bacillus pumilus expanded culture obtained in S1 into a co-culture medium for fermentation, the fermentation process being divided into different fermentation time periods; wherein the sugar concentration of the co-culture medium is controlled in at least part of the fermentation time periods; wherein the pH value of the co-culture medium is controlled in at least part of the fermentation time periods; wherein the temperature of the co-culture medium is controlled in at least part of the fermentation time periods; wherein the dissolved oxygen level of the co-culture medium is controlled in at least part of the fermentation time periods; wherein the other expanded culture, or ammonium sulfate and valine, is added to the co-culture medium at some time.

3. The process for the joint production of squalene and unsaturated fatty acids according to claim 2, characterized by the fact that: In S1, the expanding of the Schizochytrium sp. HS08 and the Bacillus pumilus comprises the following steps: T1: inoculating one of the Schizochytrium sp. HS08 or the Bacillus pumilus into an activation culture medium for culture to obtain an activation seed solution; T2: inoculating the activation seed solution obtained in T1 into a first-stage seed culture medium for culture to obtain a first-stage seed solution; T3: inoculating the first-stage seed solution obtained in T2 into a second-stage seed culture medium for culture to obtain a second-stage seed solution; T4: transferring the second-stage seed solution obtained in T3 into an initial culture medium for fermentation to obtain one of the Schizochytrium sp. expanded culture or the Bacillus pumilus expanded culture. 4.The method for co-production of squalene and unsaturated fatty acid according to claim 3, wherein: T1 specifically refers to: inoculating the Schizochytrium sp. HS08 or the Bacillus pumilus into an activation culture medium for shake flask culture at 25-30℃ and a rotation speed of 150-250 r / min for 12-24 h to obtain an activation seed solution; the activation culture medium comprises: 40-60 parts by mass of glucose, 10-20 parts by mass of yeast extract, 10-15 parts by mass of anhydrous sodium sulfate, 0.1-0.2 parts by mass of anhydrous calcium chloride, 0.3-0.6 parts by mass of potassium chloride, 0.5-1.5 parts by mass of potassium dihydrogen phosphate, 2.0-4.0 parts by mass of ammonium sulfate, 1.0-4.0 parts by mass of zinc sulfate, 2.0-6.0 parts by mass of magnesium sulfate, and 0.5-1.5 parts by mass of potassium sulfate. 5.The method for co-production of squalene and unsaturated fatty acid according to claim 4, wherein: T2 specifically refers to: inoculating the activation seed solution obtained in T1 into a first-stage seed culture medium at an inoculation amount of 5-8 v / v % for shake flask culture at 25-30℃ and a rotation speed of 150-250 r / min for 24-48 h to obtain a first-stage seed solution; ​ The primary seed culture medium comprises: 50-70 parts by mass of glucose, 8-12 parts by mass of yeast extract, 10-15 parts by mass of anhydrous sodium sulfate, 0.1-0.2 parts by mass of anhydrous calcium chloride, 0.3-0.6 parts by mass of potassium chloride, 0.5-1.5 parts by mass of potassium dihydrogen phosphate, 2.0-4.0 parts by mass of ammonium sulfate, 1.0-4.0 parts by mass of zinc sulfate, 2.0-6.0 parts by mass of magnesium sulfate, and 0.5-1.5 parts by mass of potassium sulfate.

6. The method for jointly producing squalene and unsaturated fatty acids according to claim 5, wherein: T3 is specifically: inoculating the primary seed liquid obtained in T2 into a secondary seed culture medium at an inoculation amount of 8-12 v / v %, and culturing the secondary seed culture medium in a shaking incubator at 25-30 DEG C and a rotation speed of 150-250 r / min for 24-48 h to obtain a secondary seed liquid; The secondary seed culture medium comprises: 60-100 parts by mass of glucose, 5-10 parts by mass of yeast extract, 10-15 parts by mass of anhydrous sodium sulfate, 0.1-0.2 parts by mass of anhydrous calcium chloride, 0.3-0.6 parts by mass of potassium chloride, 0.5-1.5 parts by mass of potassium dihydrogen phosphate, 2.0-4.0 parts by mass of ammonium sulfate, 1.0-4.0 parts by mass of zinc sulfate, 2.0-6.0 parts by mass of magnesium sulfate, and 0.5-1.5 parts by mass of potassium sulfate.

7. The method for jointly producing squalene and unsaturated fatty acids according to claim 6, wherein: T4 is specifically: transferring the secondary seed liquid obtained in T3 into a fermenter at an inoculation amount of 20-50 v / v %, and culturing the secondary seed liquid in the fermenter under the conditions of an initial culture medium, a temperature of 22-32 DEG C, and a pH of 2.0-8.0 for 110-150 h to obtain the Schizochytrium sp. expanded culture or the Brevibacillus sp. expanded culture; The initial culture medium comprises: 10-30 parts by mass of glucose, 5-20 parts by mass of yeast extract, 4.0-5.0 parts by mass of ammonium sulfate, 10-20 parts by mass of anhydrous sodium sulfate, 4.0-5.0 parts by mass of magnesium sulfate, and 6.0-10 parts by mass of potassium dihydrogen phosphate.

8. The method for jointly producing squalene and unsaturated fatty acids according to any one of claims 2-7, wherein: In S2, the common culture medium comprises: 10-30 parts by mass of glucose, 5-20 parts by mass of yeast extract, 4.0-5.0 parts by mass of ammonium sulfate, 10-20 parts by mass of anhydrous sodium sulfate, 4.0-5.0 parts by mass of magnesium sulfate, and 6.0-10 parts by mass of potassium dihydrogen phosphate.

9. The process for the joint production of squalene and unsaturated fatty acids according to claim 8, characterized by the fact that: S2 is specifically: the Schizochytrium sp. expanded culture is inoculated into the common medium to start fermentation at an inoculation amount of 18-20 v / v%; the sugar concentration is controlled at 40-60 g / L for 0-72 h and at 5-15 g / L for 72-120 h; the pH is adjusted with a mixed solution of ammonia, succinic acid and acetic acid, and the pH is controlled at 5.0-6.0 for 0-48 h, at 6.0-7.0 for 48-96 h, and at 6.5-7.5 for 96-120 h; the fermentation temperature is initially 29℃, and is reduced to 26℃ at 48 h; the dissolved oxygen is initially controlled at 80-100%, at 60-80% for 48-72 h, at more than 50% for 72-96 h, and at less than 30% for 96-120 h; 0.05-0.25 v / v% of the Bacillus sp. expanded culture is supplemented after 72 h of fermentation, and 0.05-0.25 v / v% of the Bacillus sp. expanded culture is supplemented again at 96 h of fermentation; ammonium sulfate and valine are supplemented; and the fermentation is carried out for at least 120 h.

10. A microbial oil obtainable by the process of any one of claims 2 to 9, characterized in that, containing squalene and eicosapentaenoic acid, the content of the squalene being 2-27 w / w %, and the content of the eicosapentaenoic acid being 3-12 w / w %.

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