A method for reducing the content of chloropropanols in arachidonic acid oil

Abstract

The present application relates to the technical field of biological oil fermentation, and particularly relates to a method for reducing the content of chloropropanol in arachidonic acid oil. The method for reducing the content of chloropropanol in arachidonic acid oil provided by the present application comprises: controlling the content of carbon source in the fermentation liquor to be greater than 0 g / L in the whole fermentation cycle of microorganism fermentation for producing arachidonic acid oil, and controlling the content of carbon source in the fermentation liquor to be 1.4-2.5 g / L in the end stage of fermentation; the end stage of fermentation is the last 1 / 4-1 / 7 stage of the total fermentation time. The method can effectively reduce the content of chloropropanol in arachidonic acid oil, and can promote the yield of arachidonic acid oil of microorganism and the increase of the content of ARA and triglyceride in the oil, and moreover, the reduction of the content of chloropropanol in refined oil simplifies the post-processing process of oil, reduces the processing cost and difficulty, and can better meet the requirements of special fields such as infant formula food for oil raw materials.

Description

Technical Field

[0001] This invention relates to the field of bio-oil fermentation technology, and in particular to a method for reducing the chloropropanol content in arachidonic acid oil. Background Technology

[0002] With technological advancements and improved living standards, food safety has become an increasingly important issue. As early as the 1970s, chloropropanols were recognized worldwide as harmful substances formed during food processing. Studies have found that chloropropanols can inhibit male hormone production, reduce fertility, and are carcinogenic. This harmful substance is not intentionally added and poses a significant threat to human health; therefore, researching its formation mechanism and control technologies is of great importance.

[0003] There are several pathways for the formation of chloropropanol, including: chloride ions directly replacing carboxyl groups to form chloropropanol; chloride ions replacing the hydroxyl groups of diglycerides and monoglycerides to form chloropropanol esters, which are then further hydrolyzed to form chloropropanol; or diglycerides first forming glycidyl esters, which then undergo ring-opening in the presence of chloride ions to form chloropropanol esters, which are then further hydrolyzed to form chloropropanol, etc.

[0004] The main sources of chloropropanol in oils and fats include: natural occurrence in raw materials, generation during raw material storage, washing with chlorinated water, formation during processing, migration from food packaging materials, formation during food storage, and generation after cooking of food and oils. Therefore, there are three main ways to reduce the content of chloropropanol and its fatty acid esters in oils and fats: reducing the content of key reactants in raw materials, improving oil refining processes, and removing 3-MCPD and related substances from refined products. Previously, applicants have controlled the content of chloropropanol in oils and fats by controlling endogenous generation and reducing exogenous generation. For example, patent applications CN201710874273.2, CN201710874016.9, and CN202110730304.3 successfully reduced the chloropropanol content in the final oil and fat products by adding lipase inhibitors to inhibit the activity of intracellular lipases, using macroporous resin adsorption, and modifying the deodorization process. However, some application requirements and regulations restrict the addition of lipase inhibitors, and using adsorption to reduce chloropropanol content increases the investment and cost of process equipment. Low-temperature deodorization processes also have high requirements for the composition of oils and fats. Therefore, new methods for reducing chloropropanol content in oils and fats still need to be developed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for reducing the chloropropanol content in arachidonic acid oil.

[0006] Arachidonic acid (ARA) oil is a bio-triglyceride commonly produced by microbial fermentation, with *Morchella alpineensis* being a frequently used fermenting microorganism. Early microbial fermentation processes for producing ARA oil typically did not involve carbon source replenishment throughout the fermentation cycle (e.g., patent application CN101560529A). Therefore, at the end of fermentation, the carbon source content in the fermentation broth might be 0 g / L or close to 0 g / L, but this was generally considered not to affect the yield of ARA oil. In recent years, in order to further improve the fermentation efficiency of arachidonic acid oil, the current arachidonic acid oil fermentation production process adds carbon source in the middle and late stages of fermentation. However, it has been found that the yield of arachidonic acid oil will not continue to increase significantly after the feeding reaches a certain level. Considering that the growth and reproduction rate of microorganisms will decrease due to aging at the end of fermentation, and considering production costs, feeding is usually stopped at the end of fermentation. As a result, the carbon source in the fermentation liquid at the end of fermentation (especially near the end of fermentation) is generally maintained at a concentration level of depletion or close to 0 g / L (e.g., patent application CN102925502A; Zheng Zhida et al., Feeding process for arachidonic acid production by fermentation of *Morchella alpina* based on pH control, Food Science, 2016, 37(17), 145-149; Nie Zhikui et al., Feeding process for arachidonic acid oil production by fermentation of *Morchella alpina*, Bioprocessing, 2015, 13(4)). Moreover, it is generally believed that in the later stages of fermentation, the ability of the microorganisms to consume carbon sources decreases. Depleting the carbon sources at the right time is beneficial to maintaining the balance between oil and ARA production, thereby obtaining a relatively high ARA yield.

[0007] In some basic studies on ARA fermentation in the prior art, residual sugar during fermentation is controlled within a relatively high range, including the control of residual sugar at 5-20 g / L as described in CN103571896A. However, this is merely a means to study the fermentation curve of *Morchella alpineensis*, analyzing the upper limits of production and sugar consumption while ensuring sufficient carbon source. However, the methods used to study strain performance are significantly different from actual production processes and do not need to consider problems that may arise during actual industrial production. After obtaining the aforementioned key indicators of the strain, process optimization is then carried out to improve efficiency and save costs. Prior to this application, the fermentation production processes of *Morchella alpineensis* known and understood by the applicant in this field all require the depletion of residual sugar and maintenance for a period of time to maintain a balance between oil and ARA yield.

[0008] In summary, in existing arachidonic acid oil fermentation production processes, the carbon source at the end of fermentation is usually controlled at a very low level, either exhausted or nearly exhausted.

[0009] During a chance adjustment of the fermentation process, the applicant unexpectedly discovered that by controlling the carbon source at a relatively high level at the end of fermentation, rather than depleting or nearly depleting the carbon source, the content of 3-MCPD in the refined oil was significantly lower than that of conventional processes. Further comparative studies revealed that if the carbon source in the fermentation broth is insufficient to maintain the balance of cell growth at the end of fermentation or even throughout the entire fermentation cycle, such as when the carbon source content is extremely low or depleted, the content of 3-MCPD in the refined oil will increase. The reason for this phenomenon is speculated to be that while conventional sugar control processes do not affect ARA accumulation, they cannot provide sufficient glucose to sustain microbial metabolic activities at the end of fermentation. This leads to microorganisms utilizing fatty acids more to maintain essential life functions, and some of the triglyceride-based fatty acids synthesized in the early stages are broken down into diglycerides and other forms. The refining process of ARA oil inevitably involves high-temperature conditions during heating and holding steps, especially the deodorization step, which requires even higher temperatures. Therefore, diglycerides, as a key precursor to 3-MCPD formation, readily generate 3-MCPD during refining. However, the pathways and precursors involved in 3-MCPD formation are diverse, and it cannot be ruled out that carbon source control during fermentation affects other related metabolic pathways or precursors of 3-MCPD. Based on these findings, the applicant is attempting to develop methods to reduce endogenous 3-MCPD formation from the perspective of fermentation control.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] This invention provides a method for reducing the chloropropanol content in arachidonic acid oil, the method comprising: controlling the carbon source content in the fermentation broth to be greater than 0 g / L throughout the entire fermentation cycle of microbial fermentation to produce arachidonic acid oil, and controlling the carbon source content in the fermentation broth to be 1.4-2.5 g / L at the end of the fermentation period;

[0012] The carbon source described in this invention is an exogenous carbon source, which is also known as the fermentation indicator "residual sugar" as commonly understood by those skilled in the art.

[0013] The final stage of fermentation refers to the last 1 / 4 to 1 / 7 of the total fermentation time.

[0014] In this invention, the final stage of fermentation includes the fermentation endpoint.

[0015] This invention reveals that controlling the carbon source content at the end of fermentation not only affects the chloropropanol content in refined oil but also influences the yield, ARA content, and triglyceride content of arachidonic acid oil. To ensure ARA production efficiency, it is necessary to simultaneously achieve low chloropropanol content and high oil and ARA yield. Controlling the carbon source content in the fermentation broth at 1.4-2.5 g / L (preferably based on glucose content) at the end of fermentation (including the fermentation endpoint), while ensuring that the carbon source is not depleted throughout the fermentation cycle, can reduce the content of chloropropanol precursors (e.g., diglycerides) in the oil and the chloropropanol content in the refined oil, while ensuring that the oil yield, ARA, and triglyceride content are not only not adversely affected but even improved to some extent. Conversely, when the carbon source content is higher than the above range, the chloropropanol content in the refined oil increases significantly, and the ARA and triglyceride contents in the arachidonic acid oil decrease significantly; when it is lower than the above range, the chloropropanol content in the refined oil increases significantly.

[0016] Preferably, the carbon source content in the fermentation broth is controlled to be ≥1 g / L throughout the entire fermentation cycle of microbial fermentation for producing arachidonic acid oil. More preferably, the carbon source content in the fermentation broth is controlled to be ≥1.4 g / L throughout the entire fermentation cycle of microbial fermentation for producing arachidonic acid oil.

[0017] In this invention, chloropropanol is preferably 3-chloropropanol.

[0018] In this invention, the microorganism used for fermentation to produce arachidonic acid oil is preferably Mortierella alpine.

[0019] In some embodiments of the present invention, the microorganism used for fermentation to produce arachidonic acid oil is Mortierella alpine strain Y16.

[0020] Mortierella alpine Y16 was deposited at the China Center for Type Culture Collection (CCTCC) on July 2, 2015, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2015421. This strain has been disclosed in patent CN105586275B.

[0021] It is worth noting that the method for reducing the chloropropanol content in arachidonic acid oil provided by the present invention has no special limitations on the *Morchella alpina* strain used for fermentation production of arachidonic acid oil. Other strains, such as *Morchella alpina* CCTCC NO: M2013419 disclosed in patent CN103571896B, can also be applied to the method of the present invention for reducing the chloropropanol content in arachidonic acid oil and achieve the expected results.

[0022] In addition to controlling the carbon source content at the end of fermentation, this invention further discovers that controlling the carbon source within a certain range during the middle of fermentation can further reduce the content of chloropropanol and its precursors in the oil and is more conducive to ensuring the yield of arachidonic acid oil and the content of ARA and triglycerides in it.

[0023] Preferably, the method further includes controlling the carbon source content in the fermentation broth to be 4-15 g / L during the mid-stage of microbial fermentation to produce arachidonic acid oil.

[0024] The middle stage of fermentation is defined as the period from 1 / 15 to 1 / 8 of the total fermentation time after the start of fermentation until the beginning of the end stage of fermentation.

[0025] Furthermore, the method also includes: controlling the carbon source content in the fermentation broth to be 20-40 g / L during the initial fermentation stage of microbial fermentation to produce arachidonic acid oil;

[0026] The initial fermentation period refers to the first 1 / 15 to 1 / 8 of the total fermentation time, excluding the time point at the beginning of the middle fermentation period.

[0027] In the early stages of fermentation, the carbon source content in the fermentation broth is affected by the initial carbon source content in the fermentation medium and the timing of feeding. Controlling the carbon source content within the aforementioned range in the early stages of fermentation can further promote a reduction in the content of chloropropanol and its precursors in the oil, and an increase in the yield of arachidonic acid oil and the content of ARA and triglycerides.

[0028] In this invention, the entire fermentation stage consists of an initial fermentation stage, a middle fermentation stage, and a final fermentation stage, which are consecutive and do not overlap.

[0029] In this invention, the carbon source content is calculated based on the glucose content in the fermentation broth.

[0030] In this invention, the carbon source is one or more selected from glucose, sucrose, and starch. When using carbon sources other than glucose or mixed carbon sources, those skilled in the art can convert them into glucose content based on the molecular composition and stoichiometry of the carbon source.

[0031] When the carbon source content in the fermentation broth is lower than the required range, it is necessary to replenish the carbon source content to bring it back within the required range.

[0032] For feeding methods, commonly used intermittent feeding or feeding-flow feeding methods can be adopted, or a combination of the two methods can be used. Intermittent feeding methods include, but are not limited to, pulse feeding; feeding-flow feeding methods include, but are not limited to, constant-speed feeding, exponential-rate feeding, and variable-speed feeding.

[0033] Preferably, the carbon source content is controlled at the end and middle of fermentation by intermittent feeding and / or fed-batch feeding.

[0034] More preferably, the carbon source content is controlled at the end of fermentation by fed-batch feeding, and the carbon source content is controlled in the middle of fermentation by fed-batch feeding or a combination of intermittent feeding and fed-batch feeding.

[0035] Using fed-batch feeding at the end of fermentation to control carbon source content can make the carbon source concentration more stable, which is more conducive to reducing the impact of changes in the composition of substances in the fermentation system on cell metabolism, and thus helps to further reduce the chloropropanol content of oil.

[0036] No carbon source is added during the initial stage of fermentation. The carbon source content is preferably controlled by controlling the initial carbon source content in the fermentation medium.

[0037] Preferably, the initial carbon source content in the fermentation medium is 20-40 g / L based on the glucose content.

[0038] In some embodiments of the present invention, the method includes: supplementing the carbon source and controlling the carbon source content in the microbial fermentation production of arachidonic acid oil using the following method:

[0039] In the early stage of fermentation: the initial carbon source content is 20-40g / L. No carbon source is added from the start of fermentation until 1 / 15-1 / 8 of the total fermentation time is reached.

[0040] Mid-fermentation stage: Feeding begins when the total fermentation time is 1 / 15 to 1 / 8. Carbon source is added using a fed-batch method, or carbon source is added first using an intermittent feeding method and then using a fed-batch method. The carbon source content in the fermentation broth is controlled at 4-15 g / L based on the glucose content, until the end of the fermentation stage begins.

[0041] Late fermentation stage: Starting from the last 1 / 4 to 1 / 7 of the total fermentation time, carbon source is added by fed-batch feeding to control the carbon source content in the fermentation broth at 1.4-2.5 g / L until the end of fermentation.

[0042] Preferably, the fermentation medium comprises the following components: 20-40 g / L glucose and 10-30 g / L yeast extract.

[0043] The fermentation process is carried out at a temperature of 20-30℃ (preferably 23-28℃).

[0044] During the fermentation process, the aeration rate should be controlled at 0.5-2.5 vvm, the stirring speed at 50-250 rpm, and the tank pressure at 0.02-0.1 MPa.

[0045] In the above fermentation culture, the inoculum size is 4-8%.

[0046] Prior to fermentation culture, the process also includes steps of activating and / or seeding the fermentation strain.

[0047] In some embodiments of the present invention, the fermentation strain is first inoculated into PDA activated solid medium for primary seed culture, and then the primary seed is inoculated into seed medium containing 70-90 g / L glucose and 5-20 g / L yeast extract for secondary seed culture to obtain seed liquid. The seed liquid is then inoculated into fermentation medium at an inoculation rate of 4-8% for fermentation culture.

[0048] The optimal temperature for culturing seeds at each stage is 23-28℃. The optimal culturing time for primary seeds is 5-8 days; the optimal culturing time for secondary seeds is 36-60 hours.

[0049] This invention also provides a method for producing arachidonic acid oil by microbial fermentation, which is the same as the method described above for reducing the chloropropanol content in arachidonic acid oil.

[0050] The beneficial effects of this invention are as follows: The fermentation production method provided by this invention can effectively reduce the chloropropanol content in arachidonic acid oil, while simultaneously promoting the yield of arachidonic acid oil by microorganisms and increasing the content of ARA and triglycerides in the oil, without significantly increasing the fermentation production cost. Furthermore, the reduction of chloropropanol content in refined oil simplifies the post-processing of the oil, reducing post-processing costs and difficulty, and better meeting the requirements of special fields such as infant formula for oil raw materials. Compared with existing methods for reducing chloropropanol content in oils, the method of this invention has lower costs, less dependence on special equipment, and is more suitable for industrial-scale production. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is a graph showing the glucose consumption curve in Comparative Example 1 of the present invention. Detailed Implementation

[0053] 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. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0054] The PDA culture medium slant formulation used in the following examples is as follows: 200 g / L potato, 20 g / L glucose, 20 g / L agar, 0.5 g / L KH2PO4, 0.25 g / L MgSO4, natural pH.

[0055] In the following embodiments, glucose detection and replenishment are achieved through an online detection-controlled automatic feeding system, with a fermentation scale of 5m³. 3 .

[0056] Example 1

[0057] This embodiment uses *Morchella alpineensis* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil, and provides a method for reducing the chloropropanol content in arachidonic acid oil. The specific method is as follows:

[0058] 1. Primary seed culture: The *Morchella alpineensis* strain Y16, stored at -80℃, was inoculated onto an activated medium slant (PDA medium slant) and cultured at 28℃ until it was fully colonized with mycelium. The culture time was 7 days.

[0059] 2. Secondary seed culture: Use an inoculation spatula to pick up block-shaped primary seed PDA slant and inoculate it into a shake flask medium containing 80 g / L glucose and 10 g / L yeast extract. Incubate at 25℃ and 100 rpm for 48 h until white spherical mycelia appear.

[0060] 3. Fermentation tank culture: After continuing multi-stage seed culture with the secondary seed culture at a 5% inoculation rate (2.5L), finally inoculate with a 5% inoculation rate into a 2.5m fermenter. 3 Fermentation was carried out in a fermenter with an initial fermentation medium containing 30 g / L glucose and 20 g / L yeast extract.

[0061] Fermentation tank parameter settings: fermentation temperature is 25℃, natural fermentation without pH control, stirring speed is 220 rpm, aeration rate is 2vvm (L / L.min), which means that the amount of air required per minute per liter of fermentation liquid is 2L, tank pressure is 0.1Mpa, and fermentation time is 9 days.

[0062] The specific processes for sugar supplementation and control during fermentation are as follows:

[0063] During the initial stage of fermentation: no sugar is added from the start of fermentation until 16 hours into fermentation;

[0064] Mid-fermentation stage: Sugar supplementation begins after 16 hours of fermentation, using a 50% glucose solution in a fed-batch manner, with the supplementation rate controlled at 0.2-0.6 minutes per minute. 3 / h, to maintain the glucose concentration in the fermentation broth at 6-10g / L; specifically, before 16-120h of fermentation, at 0.6m 3 Sugar was fed in at a rate of 0.3 mg / L per hour to maintain a glucose concentration of 10 g / L in the fermentation broth. Before 120-170 hours of fermentation, sugar was added at a rate of 0.3 mg / L per hour. 3 The glucose concentration in the fermentation broth was maintained at 6 g / L by feeding sugar at a rate of / h.

[0065] Final stage of fermentation: Begin after 170 hours of fermentation, using 0.1m... 3 A 50% glucose solution was added to the fermentation broth at a rate of 1 h to maintain the glucose concentration at 2.0 ± 0.1 g / L until the fermentation ended after 216 h.

[0066] Example 2

[0067] This embodiment uses *Morchella esculenta* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. Based on this, a method for reducing the chloropropanol content in arachidonic acid oil is provided. The specific method differs from the method in Example 1 only slightly in the method of sugar supplementation. During the middle stage of fermentation, a combination of intermittent pulsed sugar supplementation and fed-batch sugar supplementation is adopted.

[0068] Mid-fermentation stage: Sugar supplementation begins after 16 hours of fermentation. A 50% glucose solution is added to the fermentation broth all at once, bringing the glucose concentration to 15 g / L. After 72 hours, a continuous feeding process with a 50% glucose solution begins, with the feeding rate controlled at 0.3-0.5 minutes per minute. 3 / h, so that the glucose concentration in the fermentation broth is maintained at 6-10g / L.

[0069] Example 3

[0070] This embodiment uses *Morchella esculenta* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. Based on this, a method for reducing the chloropropanol content in arachidonic acid oil is provided. The specific method differs from the method in Example 1 only in the sugar supplementation and sugar control processes during fermentation, as detailed below:

[0071] During the initial stage of fermentation: no sugar is added from the start of fermentation until 24 hours into fermentation;

[0072] Mid-fermentation stage: Sugar supplementation begins after 24 hours of fermentation, using a 50% glucose solution in a fed-batch manner, with the supplementation rate controlled at 0.2-0.6 minutes per minute. 3 / h, so that the glucose concentration in the fermentation broth is maintained at 6-10g / L until fermentation reaches 165h;

[0073] Final stage of fermentation: After 165 hours of fermentation, the 50% glucose solution was diluted with 0.1 ml of water. 3 Feed was added at a rate of 1 / h to maintain the glucose concentration in the fermentation broth at 1.5±0.1g / L until fermentation ended after 216h.

[0074] Example 4

[0075] This embodiment uses *Morchella esculenta* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. Based on this, a method for reducing the chloropropanol content in arachidonic acid oil is provided. The specific method differs from the method in Example 1 only in that the total fermentation time is 192 hours, the initial fermentation medium is different, and the sugar supplementation and sugar control processes during fermentation are different, as detailed below:

[0076] The initial fermentation medium contained 35 g / L glucose and 25 g / L yeast extract;

[0077] The specific processes for sugar supplementation and control during fermentation are as follows:

[0078] During the initial stage of fermentation: no sugar is added from the start of fermentation until 14 hours into fermentation;

[0079] Mid-fermentation stage: Sugar supplementation begins after 14 hours of fermentation, using a 50% glucose solution in a fed-batch manner, with the supplementation rate controlled at 0.2-0.7 minutes per minute. 3 / h, so that the glucose concentration in the fermentation broth is maintained at 6-10g / L until 165h of fermentation;

[0080] Final stage of fermentation: After 165 hours of fermentation, the 50% glucose solution was diluted with 0.1 ml of water. 3 Feed was added at a rate of 1 h to control the glucose concentration in the fermentation broth at 2.4 ± 0.1 g / L until fermentation ended after 192 h.

[0081] Example 5

[0082] This embodiment uses *Morchella esculenta* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. Based on this, a method for reducing the chloropropanol content in arachidonic acid oil is provided. The specific method differs from the method in Example 1 only in the sugar supplementation and sugar control processes during fermentation, as detailed below:

[0083] During the initial stage of fermentation (from the start of fermentation to less than 16 hours), no sugar is added.

[0084] Mid-fermentation stage: Sugar supplementation begins after 16 hours of fermentation, using a 50% glucose solution in a fed-batch manner, with the supplementation rate controlled at 0.2-0.6 minutes per minute. 3 / h, so that the glucose concentration in the fermentation broth is maintained at 6-10g / L until 150h of fermentation;

[0085] Final stage of fermentation: After 150 hours of fermentation, the 50% glucose solution was diluted with 0.1 ml of water. 3 Feed was added at a rate of 1 h to control the glucose concentration in the fermentation broth at 2.0 ± 0.1 g / L until the fermentation ended after 216 h.

[0086] Comparative Example 1

[0087] This comparative example uses *Morchella alpineensis* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. The specific method differs from that of Example 1 only in the following aspects:

[0088] The processes for adding and controlling sugar at the end of fermentation differ, as detailed below:

[0089] Final stage of fermentation: No further sugar replenishment was performed starting at 170 hours of fermentation until the end of 216 hours. Testing showed that the glucose concentration in the fermentation broth had decreased to 0.2 g / L at 186 hours. Figure 1 ).

[0090] Comparative Example 2

[0091] This comparative example uses *Morchella alpineensis* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. The specific method differs from that of Example 1 only in the sugar supplementation and sugar control processes at the end of fermentation, as detailed below:

[0092] Final stage of fermentation: Starting at 170h, the sugar supplementation and control process in the middle stage of fermentation of Example 1 is still followed, and the glucose concentration in the final stage of fermentation is maintained at 3±0.1g / L until the end of fermentation at 216h.

[0093] Comparative Example 3

[0094] This comparative example uses *Morchella alpineensis* strain Y16 as the fermentation strain for the fermentation production of arachidonic acid oil. Based on this, a method for reducing the chloropropanol content in arachidonic acid oil is provided. The specific method differs from the method in Example 1 only in the sugar supplementation and sugar control processes at the end of fermentation, as detailed below:

[0095] Final stage of fermentation: Begin fermentation at 170 hours, according to 0.05m... 3The sugar replenishment rate of / h kept the glucose concentration in the fermentation broth at 0.9±0.1g / L until the fermentation ended after 216h.

[0096] Experimental Example: Post-processing of Arachidonic Acid Oil and Detection of Chloropropanol Content

[0097] The arachidonic acid oils produced in the above embodiments and comparative examples underwent post-treatment, and the specific methods are as follows:

[0098] ARA crude oil: After fermentation, the fermentation broth is separated by plate and frame filtration to obtain Alpine Monascus mycelium (filter cake, i.e., wet mycelium). The mycelium is then crushed by a pulverizer. The wet mycelium is dried using a fluidized bed dryer to obtain dry mycelium. The dry mycelium is then extracted with n-hexane to obtain arachidonic acid (ARA) crude oil.

[0099] Refining process: The crude ARA oil was refined using the refining process described in Comparative Example 1 of patent application CN114574282A. The refined ARA oil was obtained through solvent removal, degumming, alkali refining, decolorization, and deodorization. This process is the most conventional refining process in the field.

[0100] The contents of ARA and triglycerides (TGA) in arachidonic acid oil were determined using the national standard GB26400-2001.

[0101] The content of 3-chloropropanol and triglycerides in refined oils was detected using the AOCS standard method. With the advancement of detection methods and instruments, the detection limit of 3-chloropropanol using GC-MS-MS is now significantly lower than that of the previously used GC-MS method (detection limit of 0.1-1 ppm). This invention uses GC-MS-MS to detect 3-chloropropanol.

[0102] The test results of arachidonic acid oil and its refined oil produced in each embodiment and comparative example are shown in Table 1 and Table 2, respectively.

[0103] Table 1. Detection of arachidonic acid in oils before refining

[0104] Example 1 14.5g / L 46.6％ 97.1％ Example 2 15.6g / L 50.7％ 96.8％ Example 3 14.2g / L 47.0％ 96.5％ Example 4 13.3g / L 48.6％ 96.5％ Example 5 8.4g / L 40.0％ 96.2％ Comparative Example 1 14.3g / L 47.7％ 93.6％ Comparative Example 2 14.0g / L 45.1％ 96.3％ Comparative Example 3 14.5g / L 46.1％ 94.5％

[0105] Table 2. Detection of Indicators in Refined Arachidonic Acid Oil

[0106] Example 1 0.08 97.0 Example 2 0.06 96.5 Example 3 0.08 96.4 Example 4 0.07 96.1 Example 5 0.1 96.0 Comparative Example 1 0.84 93.2 Comparative Example 2 0.57 95.4 Comparative Example 3 0.22 94.2

[0107] The above results indicate that fermentation using the methods of Examples 1-4 can achieve high levels of ARA yield, ARA content, and triglyceride content. Furthermore, the 3-MCPD content in the refined oil can be controlled below 0.1 ppm. However, stopping sugar supplementation at the end of fermentation, reducing the sugar content in the fermentation broth to 0 (Comparative Example 1), or controlling the sugar content too low at the end of fermentation (Comparative Example 3), leads to a significant increase in the 3-MCPD content in the refined oil. Similarly, using high sugar content control at the end of fermentation (Comparative Example 2) also leads to a significant increase in the 3-MCPD content in the refined oil. Moreover, high sugar control does not further increase oil yield and ARA content, but also increases fermentation production costs. Lowering the sugar content control prematurely during fermentation leads to a significant decrease in ARA yield (Example 5).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for reducing the chloropropanol content in arachidonic acid oil, characterized in that, The method includes: controlling the carbon source content in the fermentation broth to be greater than 0 g / L throughout the entire fermentation cycle of microbial fermentation to produce arachidonic acid oil; not supplementing the carbon source in the early stage of fermentation, and controlling the carbon source content by controlling the initial carbon source content in the fermentation medium, which is 20-40 g / L based on glucose content; controlling the carbon source content in the fermentation broth to be 4-15 g / L in the middle stage of fermentation; and controlling the carbon source content in the fermentation broth to be 1.4-2.5 g / L in the late stage of fermentation. The initial fermentation period refers to the first 1 / 15 to 1 / 8 of the total fermentation time, excluding the time point at the beginning of the middle fermentation period; The middle stage of fermentation is defined as the period from 1 / 15 to 1 / 8 of the total fermentation time after the start of fermentation to before the start of the end stage of fermentation. The final stage of fermentation refers to the last 1 / 4 to 1 / 7 of the total fermentation time. The carbon source content is calculated based on the glucose content in the fermentation broth; The microorganism is *Morchella alpineensis* ( Mortierella alpine ).

2. The method according to claim 1, characterized in that, The entire fermentation process consists of an initial fermentation stage, a middle fermentation stage, and a final fermentation stage. These three stages are consecutive and do not overlap.

3. The method according to claim 1, characterized in that, The carbon source is one or more selected from glucose, sucrose, and starch.

4. The method according to any one of claims 1 to 3, characterized in that, Carbon source content control during the late and middle stages of fermentation is achieved through intermittent feeding and / or fed-batch feeding.

5. The method according to claim 4, characterized in that, Carbon source content control at the end of fermentation is achieved through fed-batch feeding, while carbon source content control in the middle of fermentation is achieved through fed-batch feeding or a combination of intermittent feeding and fed-batch feeding.

6. The method according to any one of claims 1 to 3, 5, characterized in that, The method includes: supplementing and controlling the carbon source content for the microbial fermentation production of arachidonic acid oil using the following methods: In the early stage of fermentation: the initial carbon source content is 20-40 g / L based on the glucose content. No carbon source is added from the start of fermentation until 1 / 15-1 / 8 of the total fermentation time is reached. Mid-fermentation stage: When fermentation has reached 1 / 15 to 1 / 8 of the total time, feeding begins. Carbon source is added using a fed-batch method, or carbon source is added first using an intermittent feeding method and then using a fed-batch method. The carbon source content in the fermentation broth is controlled at 4-15 g / L based on the glucose content, until the end of fermentation begins. Late fermentation stage: Starting from the last 1 / 4 to 1 / 7 of the total fermentation time, carbon source is added by fed-batch feeding to control the carbon source content in the fermentation broth at 1.4-2.5 g / L until the end of fermentation.

7. The method according to claim 6, characterized in that, The fermentation medium comprises the following components: glucose 20-40 g / L and yeast extract 10-30 g / L.

8. The method according to claim 6, characterized in that, The fermentation process takes place at a temperature of 20-30℃.

9. The method according to claim 6, characterized in that, During fermentation, the aeration rate should be controlled at 0.5-2.5 vvm, the stirring speed at 50-250 rpm, and the tank pressure at 0.02-0.1 MPa.

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