Fermentation process for producing neohesperidin by using yarrowia lipolytica

Abstract

The invention discloses a fermentation process for producing neohesperidin by using yarrowia lipolytica, and belongs to the technical field of biological fermentation. The invention aims to solve the technical problems of high chemical synthesis method cost, poor environmental protection property, extremely low biological fermentation yield, insufficient precursor metabolic flow supply, insufficient metabolic regulation optimization and the like in the existing neohesperidin production. According to the technical scheme, the fermentation technology for producing neohesperidin through yarrowia lipolytica is characterized in that a yarrowia lipolytica strain YO1 is adopted, through precise nitrogen source regulation and control in a seed culture stage and a gradient feeding strategy in a fermentation stage, and through cooperative control over key parameters such as the ventilation ratio, metabolic synergy of thallus growth and product synthesis is achieved. According to the process, the yield of neohesperidin is increased to 18.7-18.9 g / L, the industrial threshold value is broken through, and the requirements of high efficiency, low cost and environmental protection are met.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology and discloses a fermentation process for producing novel hesperidin by Yeast Extract. Background Technology

[0002] For understanding the technical content of this invention: Neohesperidin is a dihydroflavonoid compound with a chemical structure consisting of 3,5,7-trihydroxy-4'-methoxyflavonoid and rhamnosyl β-1,2-glucose. This compound is mainly found in Rutaceae plants such as sour orange, sweet orange, and trifoliate orange, and possesses antioxidant, lipid-lowering, and anti-atherosclerotic biological activities. Its derivative, neohesperidin dihydrochalcone (DHC), is widely used in the food and pharmaceutical industries as a low-calorie sweetener. The structural formula of neohesperidin is shown below:

[0003] Neohesperidin is currently mainly synthesized through natural extraction and naringin synthesis. The country of publication is China, publication number CN106432386B, publication date 2019.04.19. This document discloses a method for synthesizing neohesperidin using naringin as a raw material. The technology includes the following steps: (1) Hydrolysis: Naringin is dissolved in an alkaline aqueous solution, heated and pressurized for hydrolysis, cooled to room temperature, pH value is adjusted with acid, cooled to crystallize, filtered, and dried to obtain the hydrolysis product - root bark acetylphenyl-4'-β-neohesperidin (PN); (2) Condensation: PN is dissolved in an organic solvent, isovalin and histidine are added, reflux reaction is carried out, frozen to crystallize, filtered, and dried to obtain crude neohesperidin; (3) Refining: Crude neohesperidin is dissolved in a low-carbon alcohol aqueous solution by heating, cooled to crystallize, filtered, and the filter cake is dried to obtain refined neohesperidin. The aforementioned method yields novel hesperidin with high purity and low impurity content. However, this technology requires expensive hesperidin as a raw material, and the process necessitates catalysts and organic solvents, significantly increasing the cost of novel hesperidin and hindering large-scale industrial production. In contrast, bio-fermentation technology, utilizing synthetic biology techniques and microbial metabolic regulation, achieves highly efficient conversion. This aligns better with the concept of green manufacturing and demonstrates a significant cost advantage.

[0004] Relevant non-patent literature retrieved: The journal or book title is *Trends Biotechnol*, the article title is *What makes Yarrowialipolytica well suited for industry?*, volume number 2023 Feb;41(2):242-254, and the publication date is February 2023. This article discloses that *Yarrowialipolytica* possesses both natural and engineered characteristics that make it a good host for bioproduction in the chemical, fuel, food, and pharmaceutical industries. Research related to *Yarrowialipolytica* has shown a dramatic increase over the past decade, highlighting its importance and potential in bioproduction.

[0005] Relevant patent documents retrieved: This document, published in China (CN117987286A) on May 7, 2024, discloses a method for synthesizing neohesperidin and neohesperidin dihydrochalcone from *Yersinia lipolytica* using glycerol. The method involves transferring a flavonoid O-methyltransferase (YlOMT) from *Yersinia lipolytica* and other plant-derived flavonoid synthases into the *Yersinia lipolytica* yeast. The constructed chassis cells can synthesize neohesperidin de novo, and further hydrogenation of neohesperidin yields neohesperidin dihydrochalcone. The yield of neohesperidin prepared by this invention in a shake flask is 4.32 mg / L.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The production of neohesperidin cannot simultaneously meet the industrial demands of "high efficiency, low cost, and green environmental protection," and there is an urgent need to break through the bottleneck by optimizing microbial metabolic regulation and fermentation processes.

[0007] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: 1. In existing technologies, excessively high nitrogen sources (C / N ≤ 15) inhibit citrate lyase activity, leading to insufficient synthesis of acetyl-CoA and malonyl-CoA. However, excessively low nitrogen sources limit cell growth, preventing the formation of sufficient biomass to support product synthesis. Therefore, the primary challenge is to precisely control the C / N ratio in the culture medium and during the feeding process to ensure cell growth to the target density (OD600 above 100) while avoiding nitrogen inhibition of key enzymes.

[0008] 2. The feeding rate directly affects carbon source supply and metabolic flux distribution: feeding too slowly leads to insufficient carbon source and a lack of precursor synthesis raw materials; feeding too quickly causes carbon source accumulation, resulting in excessive metabolic load on the cells (such as the accumulation of byproducts like ethanol), which in turn inhibits neohesperidin synthesis. In this process, it is necessary to accurately determine the cell growth stage and the timing of the feeding rate increase. The dynamic relationship between cell density and metabolic state is complex, requiring extensive time-series sampling analysis to optimize a reasonable gradient.

[0009] 3. In seed tank culture, ammonia is not only a pH regulator, but its supply also affects the metabolic direction of the bacteria: continuous ammonia supply maintains a high-nitrogen environment, inhibiting the induction of enzymes related to precursor synthesis; cutting off ammonia too early will cause bacterial growth to stagnate and fail to reach the target biomass; cutting off ammonia too late will fail to effectively activate the metabolic shift. The determination of this "critical point" requires combining the bacterial growth curve and enzyme activity changes.

[0010] 4. The Yeast strain YO1 (CGMCC No. 34491) used in this invention needs to be stably produced under adjusted culture conditions (such as gradient feeding and ammonia control). However, it was found in the early stages that the strain had a stress response to environmental changes (such as accelerated feeding and ammonia cutoff). This invention overcomes difficulties such as precise control of strain adaptability and finally achieves efficient fermentation production of neohesperidin. Summary of the Invention

[0011] The purpose of this invention is to provide: A fermentation process for producing neohesperidin using Yersinia lipolytica, and related technologies, to solve the technical problems of existing neohesperidin production, such as high cost and poor environmental performance of chemical synthesis methods, extremely low yield of biological fermentation, insufficient supply of precursor metabolic flux, and insufficient optimization of metabolic regulation, or a combination thereof.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] The definition of the standard chemical term can be found in the reference "Genetic Engineering, Higher Education Press, August 1, 2013, 2nd edition".

[0015] Unless otherwise specified, conventional methods within the scope of the art, such as scale-up culture methods, fermentation culture methods, autoclaving methods, HPLC methods, etc., shall be used.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The term "Yersinia lipophila" as used in this article refers to a fungus belonging to the genus Yersinia that possesses a strong lipid metabolism capacity, particularly adept at synthesizing oils from various carbon sources (such as glucose, glycerol, and fatty acids). Therefore, it has garnered widespread attention and application in the field of industrial biotechnology. It can be used not only to produce microbial oils but also to synthesize products such as biodiesel and biosurfactants, and it also shows potential in degrading oily pollutants in the environment.

[0018] The term "neohesperidin" as used in this article refers to a naturally occurring flavonoid compound, primarily derived from the peel, pulp, or juice of citrus fruits (such as oranges, lemons, and grapefruits). It possesses various biological activities, including antioxidant, anti-inflammatory, and lipid-lowering effects, and has certain applications in the food, pharmaceutical, and cosmetic industries. For example, in the food industry, it can be added as a sweetener or functional ingredient; it has a high sweetness but low calorie content, making it suitable for the production of low-sugar foods.

[0019] The term "activated strain" as used in this article The term "feedback" as used in this article refers to an operational method in microbial fermentation, which involves supplementing the fermentation system with a certain amount of nutrients (such as carbon sources, nitrogen sources, vitamins, minerals, etc.) or other necessary components. The purpose is to maintain a favorable growth environment for microorganisms, prolong the fermentation cycle, and increase the yield of the target product. For example, in the fermentation of certain antibiotics, supplementing with carbon sources such as glucose can prevent substrate insufficiency that could lead to stunted cell growth, thereby increasing the amount of antibiotic synthesized.

[0020] The term "inoculum size" used in this article refers to the ratio (or absolute quantity) of the number of microorganisms inoculated into the culture medium to the volume of the culture medium during microbial culture or fermentation. It is one of the important factors affecting microbial growth and product synthesis. Too small an inoculum size will lead to slow cell growth and a prolonged fermentation cycle; too large an inoculum size may affect normal cell growth and the synthesis of the target product due to problems such as excessive consumption of nutrients and excessive accumulation of metabolic products. In practice, the appropriate inoculum size needs to be determined based on factors such as the characteristics of the microorganism and the purpose of the culture.

[0021] The term "substrate" as used in this article refers to the following: In biology and chemistry, substrate generally refers to a substance that participates in a biochemical or chemical reaction, and is the object of action of enzymes or catalysts. In microbial metabolism, substrate generally refers to the nutrients used by microorganisms for growth and reproduction, such as carbohydrates (glucose, sucrose), proteins, and fats. These substances are broken down or transformed through the metabolic processes of microorganisms, providing them with energy and raw materials for synthesizing their own substances.

[0022] The term "neohesperidin biosynthetic metabolic pathway" used in this article refers to the key steps in the synthesis of neohesperidin by microorganisms such as Yersinia lipophila. Figure 1 As shown, it can be broken down into the following core steps: (1) Synthesis of precursor substances: Citric acid cleavage: Starting from citric acid, acetyl-CoA is generated by catalysis of citric acid lyase. Malonyl-CoA generation: Acetyl-CoA combines with ATP and HCO3- - (2) Flavonoid skeleton construction: Chalcone synthase (CHS) catalysis: p-coumaric acid coenzyme A and malonyl coenzyme A are condensed by CHS to generate naringenin chalcone. Chalcone isomerase (CHI) catalysis: naringenin chalcone isomerizes by CHI to generate naringenin. (3) Glycosylation modification: Glycosyltransferase (UGT) catalysis: naringenin is converted into naringin by UGT, introducing glucose, rhamnosyl and other sugar units in sequence, first synthesizing naringin-7-O-glucoside, and finally converting into neohesperidin.

[0023] In a first aspect, the present invention provides a fermentation process for producing neohesperidin from *Yarrowia lipolytica*, the fermentation process comprising the following steps: S1. The activated lipophilic yeast strain YO1 with preservation number CGMCC No.34491 was inoculated into seed culture medium and cultured to obtain seed liquid; S2. Inoculate the seed culture medium with the seed solution, introduce ammonia water, and carry out expansion culture. When the OD600 value of the culture solution reaches 2.0, turn off the ammonia water and continue to culture until the OD600 value of the culture solution reaches 10-20 to obtain the expanded culture seed solution. S3. Inoculate the expanded culture seed liquid into the fermentation medium containing the substrate, adjust the pH with ammonia water, and ferment for 18-20 hours; S4. Feed the fermentation broth at a rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100-150, increase the feeding rate to 12 kg / h and continue fermentation to obtain a fermentation broth containing neohesperidin. The aeration ratio of the ammonia water mentioned in step S2 or step S3 is 0.4-0.8 vvm; The composition of the feed in step S4 is: 700-800 g / L glucose solution and / or 700-800 g / L glycerol solution.

[0024] The inoculation amount of the lipophilic yeast strain YO1 mentioned in technical feature step S1 is selected from 2%-5% v / v.

[0025] The preferred inoculation amount of the lipophilic yeast strain YO1 in technical feature step S1 is 2.0%-2.5%v / v, 2.5%-3.0%v / v, 3.0%-3.5%v / v, 3.5%-4.0%v / v, 4.0%-4.5%v / v, or 4.5%-5.0%v / v.

[0026] In particular, the inoculation amount of the lipophilic yeast strain YO1 in technical feature step S1 is further preferably 2.0% v / v.

[0027] The cultivation conditions in technical feature step S1 are: rotation speed 200-220 rpm, temperature 25-35℃, and cultivation time 24-48h.

[0028] The preferred cultivation conditions in technical feature step S1 are: rotation speed of 200-210 rpm or 210-220 rpm.

[0029] The cultivation conditions in technical feature step S1 are further preferred to be: rotation speed 220 rpm.

[0030] The preferred cultivation conditions in technical feature step S1 are: temperatures of 25-26℃, 26-27℃, 27-28℃, 28-29℃, 29-30℃, 30-31℃, 31-32℃, 33-34℃, or 34-35℃.

[0031] The cultivation conditions in technical feature step S1 are further preferred to be: temperature 30℃.

[0032] The preferred cultivation conditions in technical feature step S1 are: cultivation time of 24-25h, 25-26h, 26-27h, 27-28h, 28-29h, 29-30h, 30-31h, 31-32h, 33-34h, 34-35h, 35-36h, 36-37h, 37-38h, 38-39h, 39-40h, 40-41h, 41-42h, 42-43h, 43-44h, or 44-45h.

[0033] The cultivation conditions in technical feature step S1 are further optimized to include a cultivation time of 24 hours.

[0034] The seed liquid inoculation amount in technical feature step S2 is 10%-15% v / v.

[0035] The preferred inoculation amount of the seed liquid in technical feature step S2 is 10%-11%v / v, 11%-12%v / v, 12%-13%v / v, 13%-14%v / v, or 14%-15%v / v.

[0036] The seed liquid inoculation amount in technical feature step S2 is further preferably 10% v / v.

[0037] The ammonia water introduction mentioned in technical feature step S2 is: introducing 32% v / v ammonia water to maintain the pH at 5-6.

[0038] The preferred method for introducing ammonia water in technical feature step S2 is to introduce 32% v / v ammonia water and maintain the pH at 5.0-5.1, 5.1-5.2, 5.2-5.3, 5.3-5.4, 5.4-5.5, 5.5-5.6, 5.6-5.7, 5.7-5.8, 5.8-5.9 or 5.9-6.0.

[0039] The aeration ratio of the ammonia water in technical feature step S2 or step S3 is 0.4-0.5vvm, 0.5-0.6vvm, 0.6-0.7vvm or 0.7-0.8vvm.

[0040] In particular, the aeration ratio of the ammonia water in technical feature step S2 is preferably 0.4vvm.

[0041] In particular, the aeration ratio of the ammonia water in technical feature step S3 is selected as 0.8vvm.

[0042] The conditions for the scale-up culture described in technical feature step S2 or the fermentation culture described in step S3 are: rotation speed 100-200 rpm, temperature 25-35℃, and dissolved oxygen 30%-50%.

[0043] The preferred conditions for the scale-up culture described in technical feature step S2 or the fermentation culture described in step S3 are: rotation speed of 100-110 rpm, 110-120 rpm, 120-130 rpm, 130-140 rpm, 140-150 rpm, 150-160 rpm, 160-170 rpm, 170-180 rpm, 180-190 rpm, or 190-200 rpm.

[0044] The conditions for the scale-up culture described in step S2 or the fermentation culture described in step S3 are further preferably 150 rpm.

[0045] The preferred conditions for the scale-up culture described in step S2 or the fermentation culture described in step S3 are: temperatures of 25-26℃, 26-27℃, 27-28℃, 28-29℃, 29-30℃, 30-31℃, 31-32℃, 33-34℃, or 34-35℃.

[0046] The conditions for the scale-up culture described in step S2 or the fermentation culture described in step S3 are further preferably: a temperature of 30°C.

[0047] The preferred conditions for the scale-up culture described in technical feature step S2 or the fermentation culture described in step S3 are: dissolved oxygen 30%-31%, 31%-32%, 32%-33%, 33%-34%, 34-35%, 35%-36%, 36%-37%, 37%-38%, 38%-39%, 39%-40%, 40%-41%, 41%-42%, 42%-43%, 43%-44%, 44%-45%, 45%-46%, 46%-47%, 47%-48%, 48%-49%, or 49%-50%.

[0048] The conditions for the expanded culture described in technical feature step S2 or the fermentation culture described in step S3 are further preferably: dissolved oxygen 40%.

[0049] The continuous culture described in technical feature step S2 is as follows: continuous culture until the OD600 value of the culture medium reaches 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19 or 19-20.

[0050] The continuous culture described in technical feature step S2 is: continuous culture until the OD600 value of the culture medium reaches 10-11, 11-12, 12-13, 13-14 or 14-15.

[0051] The continuous culture described in technical feature step S2 is: continuous culture until the OD600 value of the culture medium reaches 15.

[0052] In particular, the ammonia water used in technical feature step S3 to adjust the pH is 32% v / v ammonia water to adjust the pH to 5-6.

[0053] The preferred method for adjusting pH using ammonia water in technical feature step S3 is to introduce 32% v / v ammonia water to maintain pH at 5.0-5.1, 5.1-5.2, 5.2-5.3, 5.3-5.4, 5.4-5.5, 5.5-5.6, 5.6-5.7, 5.7-5.8, 5.8-5.9, or 5.9-6.0.

[0054] The substrate mentioned in technical feature step S3 includes any one or more of glucose and glycerol.

[0055] The substrate described in technical feature step S3 is preferably glucose or glycerol.

[0056] The concentration of the substrate mentioned in technical feature step S3 is 20-40 g / L.

[0057] The concentration of the substrate in technical feature step S3 is preferably 20-21 g / L, 21-22 g / L, 22-23 g / L, 23-24 g / L, 24-25 g / L, 25-26 g / L, 26-27 g / L, 27-28 g / L, 28-29 g / L, 29-30 g / L, 30-31 g / L, 31-32 g / L, 32-33 g / L, 33-34 g / L, 34-35 g / L, 35-36 g / L, 36-37 g / L, 37-38 g / L, 38-39 g / L, or 39-40 g / L.

[0058] The concentration of the substrate in technical feature step S3 is further preferably 20 g / L.

[0059] The composition of the feed in technical feature step S4 is: 700 g / L glucose solution or 800 g / L glycerol solution.

[0060] The preferred composition of the feed in technical feature step S4 is a 700 g / L glucose solution with a pH of 6.0-7.0.

[0061] The preparation method of the 700g / L glucose solution is as follows: glucose is dissolved in pure water, the pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50℃ and kept warm for later use.

[0062] The 800g / L glycerol solution is a glycerol solution that is autoclaved and cooled to room temperature before use.

[0063] Among them, the technical feature step S4 is to feed the material at a feeding rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100-110, 110-120, 120-130, 130-140 or 140-150, the feeding rate is increased to 12 kg / h and the fermentation culture continues to obtain a fermentation broth containing neohesperidin.

[0064] In particular, the preferred technical feature step S4 is to feed the material at a feeding rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100, the feeding rate is increased to 12 kg / h, and fermentation continues to obtain a fermentation broth containing neohesperidin.

[0065] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred solution: introduce 32% v / v ammonia water to maintain the pH at 5-6. This solution not only solves the technical problem of "extremely low bio-fermentation yield in the production of new hesperidin", but also addresses the technical problem of "insufficient synthesis of precursors (acetyl-CoA, malonyl-CoA) due to imbalance between nitrogen supply and pH regulation (high nitrogen inhibits the activity of key enzymes or low nitrogen limits cell growth)".

[0066] The second preferred option is to further optimize the aeration ratio of the ammonia water in step S2 to 0.4 vvm. This technical solution not only solves the technical problem of "extremely low bio-fermentation yield in the production of new hesperidin", but also further solves the technical problem of "fluctuations in cell metabolism caused by unstable nitrogen source supply rate".

[0067] The third preferred option: The continuous culture mentioned in step S2 is: continuous culture until the OD600 value of the culture medium reaches 15. This technical solution, in addition to solving the technical problem of "extremely low bio-fermentation yield in the production of neohesperidin", further solves the technical problem of "low product synthesis efficiency caused by insufficient or excessive microbial biomass".

[0068] The fourth preferred solution: The pH adjustment method described in step S3 is to use 32% v / v ammonia water to adjust the pH to 5-6. This technical solution not only solves the technical problem of "extremely low bio-fermentation yield in the production of new hesperidin", but also further solves the technical problem of "metabolic pathway disorder caused by unstable pH and insufficient nitrogen source during fermentation (non-ammonia water regulators cannot simultaneously ensure nitrogen source supply, affecting precursor synthesis)".

[0069] The fifth preferred option is to further optimize the aeration ratio of the ammonia water in step S3 to 0.8 vvm. This technical solution not only solves the technical problem of "extremely low bio-fermentation yield in the production of new hesperidin", but also solves the technical problem of "poor synergy between energy metabolism and product synthesis caused by mismatch between dissolved oxygen and nitrogen supply during fermentation (insufficient dissolved oxygen inhibits aerobic metabolism, and uneven nitrogen supply affects precursor accumulation)".

[0070] The sixth preferred option: Step S4 is preferably fed at a rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100, the feeding rate is increased to 12 kg / h, and fermentation continues to obtain a fermentation broth containing neohesperidin. This technical solution, in addition to solving the technical problem of "extremely low bio-fermentation yield in neohesperidin production," further addresses the technical problem of "imbalance in carbon source metabolic flow distribution caused by mismatch between feeding rate and cell growth stage (too slow feeding results in precursor deficiency, while too fast feeding leads to byproduct accumulation and inhibition of synthesis)."

[0071] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: Compared with existing technologies, this invention has better technical effects in terms of neohesperidin yield, production efficiency, cost control, and environmental friendliness. According to experimental tests, this invention increases the yield of neohesperidin to over 18.7 g / L, significantly exceeding the yield threshold for industrial production.

[0072] Furthermore, based on the present invention: Based on the comparison of Examples 2-3 and Comparative Examples 1-13, this invention employs a combination of techniques including "specific strain CGMCC No. 34491 + precise nitrogen source regulation in the seed stage + gradient feeding strategy + aeration ratio in the fermentation stage," achieving high and stable yields of neohesperidin. For example, when using only single gradient feeding (Comparative Examples 2-3) or single nitrogen source regulation (Comparative Example 9), the yields were 17.1 g / L, 17.6 g / L, and 17.1 g / L, respectively. However, after combining the above techniques, the yields in Examples 2-3 reached 18.7-18.9 g / L, significantly higher than the sum of the effects of each individual technique, demonstrating a synergistic effect. This proves that multi-parameter synergistic regulation can more accurately guide metabolic flow to the neohesperidin synthesis pathway, avoiding metabolic imbalances caused by single optimization, and ultimately achieving efficient and stable industrial production. The combined technical effect is superior to the sum of the effects of each individual technique.

[0073] Preservation Instructions Preserved strain: Lipolysaccharidase strain YO1; Classification and nomenclature: Yersinia lipophila ( Yarrowia lipolytica ); Accession number: CGMCC No. 34491; Preservation date: May 9, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0074] Figure 1This is a flowchart of the metabolism of acetyl-CoA to neohesperidin.

[0075] Figure 2 This is the liquid phase diagram of neohesperidin.

[0076] Figure 3 This is the standard curve for the new hesperidin.

[0077] Figure 4 This is a graph showing the glucose fermentation yield.

[0078] Figure 5 This is a graph showing the yield of glycerol fermentation. Detailed Implementation

[0079] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0080] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0081] Basic Example 1 Determination of neohesperidin content: HPLC, conditions as follows: Chromatographic column: SHIMADZU Wondasil C18 column (150*4.6μm 5μm), No:5020-39001 (7# column); Temperature: 35℃; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Detection wavelength: 280nm Mobile phase A: Methanol; Mobile phase B: 0.1% aqueous phosphoric acid solution; The gradient elution procedure is shown in Table 1: Table 1

[0082] The chromatogram of neohesperidin is shown below. Figure 2 As shown.

[0083] The standard curve for neohesperidin is shown in Table 2. Figure 3 As shown: Table 2

[0084] Example 1 A lipophilic yeast strain, YO1, was deposited on May 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34491. The strain is stored at -80℃ for future use.

[0085] YPD liquid culture medium: contains 0.1 g / L yeast extract, 0.2 g / L peptone, and 20 g / L glucose. Preparation method: Weigh the yeast extract and peptone, dissolve them in ultrapure water to a final volume of 960 mL, autoclave, cool to room temperature, and then add 40 mL of sterilized 50% glucose solution.

[0086] Activation of YO1 strain of lipophilic yeast: Thaw the YO1 strain of lipophilic yeast that has been stored at low temperature, add it to YPD liquid medium at an inoculation rate of 2% v / v, and incubate it in a constant temperature shaker at 30℃ and 220rpm for 12-16h. Measure the OD600 value of the bacterial solution under a wavelength of 600nm in the ultraviolet light. If the absorbance value is between 6 and 8, the activated bacterial solution is obtained.

[0087] Example 2 1. Preparation of seed culture medium Seed culture medium: Contains 20 g / L glucose, 7.5 g / L ammonium sulfate, 14.4 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.4% v / v trace element stock solution, and 0.2% v / v vitamin stock solution. Preparation method: Weigh ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate, dissolve in pure water, adjust pH to 6.0, autoclave at 121℃ for 30 min, cool to room temperature, add trace element stock solution, vitamin stock solution, and glucose solution, mix well, and set aside. The glucose solution is obtained by dissolving glucose in pure water.

[0088] The vitamin stock solution contains 0.05 g / L biotin, 0.2 g / L para-aminobenzoic acid, 1 g / L niacin, 1 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 1 g / L thiamine hydrochloride, and 25 g / L inositol. The preparation method is as follows: Weigh biotin, para-aminobenzoic acid, niacin, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, and inositol, dissolve them in pure water, filter to remove bacteria, and set aside.

[0089] The trace element mother liquor contains 4.5 g / L calcium chloride dihydrate, 4.5 g / L zinc sulfate heptahydrate, 3 g / L ferrous sulfate heptahydrate, 1 g / L boric acid, 1 g / L manganese chloride tetrahydrate, 0.4 g / L sodium molybdate dihydrate, 0.3 g / L cobalt chloride hexahydrate, 0.1 g / L copper sulfate pentahydrate, 0.1 g / L potassium iodide, and 15 g / L ethylenediaminetetraacetic acid. The preparation method is as follows: Weigh calcium chloride dihydrate, zinc sulfate heptahydrate, ferrous sulfate heptahydrate, boric acid, manganese chloride tetrahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, potassium iodide, and ethylenediaminetetraacetic acid, add them to pure water, adjust the pH to 4, slowly dissolve, autoclave, and then cool to room temperature for later use.

[0090] 2. Shake-flask seed culture Add the seed culture medium to the Erlenmeyer flask, and inoculate the activated bacterial solution from Example 1 into the seed culture medium at an inoculation rate of 2% v / v. After culturing at 220 rpm and 30°C for 24 h on a shaker, the seed solution is obtained. 3. Seed tank expansion culture and induction (1) Add the seed culture medium to the seed tank and inoculate the seed solution into the seed tank containing the seed culture medium at an inoculation rate of 10% v / v.

[0091] (2) 32% v / v ammonia water was introduced into the seed tank to maintain the pH at 5-6, the aeration ratio at 0.4 vvm, the temperature at 30℃, the dissolved oxygen at 40%, and the rotation speed at 150 rpm for cultivation. When the OD600 value of the culture medium reached 2.0, the ammonia water was turned off, and the culture was continued until the OD600 value reached 15 to obtain the expanded culture seed medium.

[0092] 4. Fermentation culture (1) Add 15kg ammonium sulfate, 28.8kg potassium dihydrogen phosphate, 1kg magnesium sulfate heptahydrate, 0.25g biotin, 5g calcium pantothenate, 5g nicotinic acid, 125g inositol, 5g thiamine, 5g pyridoxine, 1g para-aminobenzoic acid, 37g zinc sulfate heptahydrate, 0.85g copper sulfate pentahydrate, 8.5g boric acid, 0.85g potassium iodide, 8.5g manganese chloride tetrahydrate, 2.5g cobalt chloride hexahydrate, 3.3g sodium molybdate dihydrate, 37.3g calcium chloride, 25g ferrous sulfate heptahydrate, and 128.2g EDTA to the fermenter. After adding water to a final volume of 2000L, autoclave the fermenter at 121℃ for 30min and cool it to room temperature for later use.

[0093] (2) Add 20 g / L of glucose to the fermenter through the feeding tank, and inoculate the expanded culture seed liquid into the fermenter containing the fermentation medium at an inoculation rate of 12% v / v. Adjust the pH to 5-6 with 32% v / v ammonia water, aeration ratio of 0.8 vvm, temperature of 30℃, dissolved oxygen of 40%, rotation speed of 150 rpm, and ferment for 18 h.

[0094] (3) After fermentation, feed is added at a rate of 8 kg / h, and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100 (i.e., fermentation for about 40 hours), the feed rate is increased to 12 kg / h, and fermentation is completed after 140 hours. Figure 4 This is a graph showing the glucose fermentation yield.

[0095] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0096] Example 3 The difference between Example 3 and Example 2 lies in "4. Fermentation Culture," the specific steps of which are as follows: (1) Add 15kg ammonium sulfate, 28.8kg potassium dihydrogen phosphate, 1kg magnesium sulfate heptahydrate, 0.25g biotin, 5g calcium pantothenate, 5g nicotinic acid, 125g inositol, 5g thiamine, 5g pyridoxine, 1g para-aminobenzoic acid, 37g zinc sulfate heptahydrate, 0.85g copper sulfate pentahydrate, 8.5g boric acid, 0.85g potassium iodide, 8.5g manganese chloride tetrahydrate, 2.5g cobalt chloride hexahydrate, 3.3g sodium molybdate dihydrate, 37.3g calcium chloride, 25g ferrous sulfate heptahydrate, 128.2g EDTA, and 40kg glycerol to the fermenter. After adding water to a final volume of 2000L, autoclave the fermenter at 121℃ for 30min and cool it to room temperature for later use.

[0097] (2) The expanded culture seed liquid was inoculated into a fermenter containing fermentation medium at an inoculation rate of 12% v / v. The pH was adjusted to 5-6 with 32% v / v ammonia water, the aeration ratio was 0.8 vvm, the temperature was 30℃, the dissolved oxygen was 40%, the rotation speed was 150 rpm, and the fermentation culture was carried out for 18h.

[0098] (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100 (i.e., fermentation for about 40 hours), the feeding rate is increased to 12 kg / h and fermentation continues for 140 hours to complete fermentation. Figure 5 This is a graph showing the yield of glycerol fermentation.

[0099] The feed composition is: 800g / L glycerol solution, autoclaved, cooled to room temperature and then ready for use.

[0100] Comparative Example 1 The only difference between Comparative Example 1 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is maintained at 8 kg / h and fermentation is completed after 140 h.

[0101] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0102] Comparative Example 2 The only difference between Comparative Example 2 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 10 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is maintained at 10 kg / h and fermentation is completed after 140 h.

[0103] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0104] Comparative Example 3 The only difference between Comparative Example 3 and Example 2 is that step (2) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 12 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is maintained at 12 kg / h and fermentation is completed after 140 h.

[0105] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0106] Comparative Example 4 The only difference between Comparative Example 4 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is increased to 12 kg / h and fermentation is completed after 140 h.

[0107] The feed composition is as follows: 600 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0108] Comparative Example 5 The only difference between Comparative Example 5 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 0.8 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is increased to 12 kg / h and fermentation is completed after 140 h.

[0109] The feed composition is as follows: 900 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0110] Comparative Example 6 The only difference between Comparative Example 6 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 0.6 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is increased to 12 kg / h and fermentation is completed after 140 h.

[0111] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0112] Comparative Example 7 The only difference between Comparative Example 7 and Example 2 is that step (3) in "4. Fermentation Culture" is different. The specific steps are as follows: (3) After fermentation, feed is added at a rate of 8 kg / h and fermentation continues (aeration ratio 1.0 vvm, temperature 30℃, dissolved oxygen 40%, rotation speed 150 rpm). When the OD600 value reaches 100, the feeding rate is increased to 12 kg / h and fermentation is completed after 140 h.

[0113] The feed composition is as follows: 700 g / L glucose solution, glucose is dissolved in pure water, pH is adjusted to 6.0-7.0 with 32% w / v sodium hydroxide solution, autoclaved, cooled to 50°C and kept warm for later use.

[0114] Comparative Example 8 The only difference between Comparative Example 8 and Example 2 is that step (2) in "3. Seed tank expansion culture and induction" is different. The specific steps are as follows: The seed culture was carried out in a seed tank at a temperature of 30℃ and a rotation speed of 150 rpm until the OD600 value reached 15, thus obtaining the expanded culture seed solution.

[0115] Comparative Example 9 The only difference between Comparative Example 9 and Example 2 is that step (2) in "3. Seed tank expansion culture and induction" is different. The specific steps are as follows: The seed tank was circulated with 32% v / v ammonia to maintain the pH at 5-6, with an aeration ratio of 0.4 vvm, a temperature of 30℃, dissolved oxygen of 40%, and a rotation speed of 150 rpm for incubation. Once the OD600 value of the culture medium reached 3.0, the ammonia was stopped, and incubation continued until the OD600 value reached 15, yielding the expanded seed culture medium.

[0116] Comparative Example 10 The only difference between Comparative Example 10 and Example 2 is the step (2) in "3. Seed tank expansion culture and induction". The specific steps are as follows: The seed tank was circulated with 32% v / v ammonia to maintain the pH at 5-6, with an aeration ratio of 0.4 vvm, a temperature of 30℃, dissolved oxygen of 40%, and a rotation speed of 150 rpm for incubation. Once the OD600 value of the culture medium reached 4.0, the ammonia was stopped, and incubation continued until the OD600 value reached 15, yielding the expanded seed culture medium.

[0117] Comparative Example 11 The only difference between Comparative Example 11 and Example 2 is that step (2) in "4. Fermentation Culture" is different. The specific steps are as follows: (1) Add 20 g / L of glucose through the feed tank, and inoculate the expanded culture seed liquid into the fermenter containing fermentation medium at an inoculation rate of 12% v / v. Adjust the pH to 5-6 with 32% v / v sodium hydroxide aqueous solution, aeration ratio of 0.8 vvm, temperature of 30℃, dissolved oxygen of 40%, rotation speed of 150 rpm, and ferment for 18-20 h.

[0118] Comparative Example 12 The recombinant strain ZYL-4 was prepared according to the method described in patent CN117987286A. The recombinant strain ZYL-4 was added to YPD liquid medium at an inoculation rate of 2% v / v and cultured in a constant temperature shaker at 30℃ and 220rpm for 12-16h. The OD600 value of the bacterial solution was measured at a wavelength of 600nm under ultraviolet light. The absorbance value was between 6 and 8, thus obtaining the activated recombinant strain ZYL-4 bacterial solution.

[0119] 1. Preparation of seed culture medium Same as Example 2.

[0120] 2. Shake-flask seed culture Add seed culture medium to Erlenmeyer flasks, and inoculate the activated recombinant strain ZYL-4 bacterial solution into the seed culture medium at an inoculation rate of 2% v / v. After culturing at 220 rpm and 30℃ for 24 h on a shaker, the seed culture of recombinant strain ZYL-4 is obtained. 3. Seed tank expansion culture and induction (1) Add seed culture medium to the seed tank, and inoculate the recombinant strain ZYL-4 seed solution into the seed tank containing seed culture medium at an inoculation rate of 10-15% v / v. In the seed tank, culture at 30℃ and 150 rpm until the OD600 value reaches 15 to obtain the expanded culture recombinant strain ZYL-4 seed solution.

[0121] 4. Fermentation culture (1) Add 15kg ammonium sulfate, 28.8kg potassium dihydrogen phosphate, 1kg magnesium sulfate heptahydrate, 0.25g biotin, 5g calcium pantothenate, 5g nicotinic acid, 125g inositol, 5g thiamine, 5g pyridoxine, 1g para-aminobenzoic acid, 37g zinc sulfate heptahydrate, 0.85g copper sulfate pentahydrate, 8.5g boric acid, 0.85g potassium iodide, 8.5g manganese chloride tetrahydrate, 2.5g cobalt chloride hexahydrate, 3.3g sodium molybdate dihydrate, 37.3g calcium chloride, 25g ferrous sulfate heptahydrate, and 128.2g EDTA to the fermenter. After adding water to a final volume of 2000L, autoclave the fermenter at 121℃ for 30min and cool it to room temperature for later use.

[0122] (2) Add 20 g / L of glucose to the fermenter through the feeding tank, and inoculate the seed liquid of the recombinant strain ZYL-4 at an inoculation rate of 12% v / v into the fermenter containing the fermentation medium. Fermentation is completed after 4 days of culture in the fermenter.

[0123] Comparative Example 13 The recombinant strain ZYL-4 was prepared according to the method described in patent CN117987286A. The recombinant strain ZYL-4 was added to YPD liquid medium at an inoculation rate of 2% v / v and cultured in a constant temperature shaker at 30℃ and 220rpm for 12-16h. The OD600 value of the bacterial solution was measured at a wavelength of 600nm under ultraviolet light. The absorbance value was between 6 and 8, thus obtaining the activated recombinant strain ZYL-4 bacterial solution.

[0124] The only difference between Comparative Example 13 and Example 2 is that the activated bacterial solution of Example 1 in Example 2 is replaced with the activated recombinant strain ZYL-4 bacterial solution.

[0125] Example 1 The fermentation broths prepared in Examples 2-3 or Comparative Examples 1-13 were subjected to the following operations: Fermentation was sonicated, and 0.5 mL was transferred to a 10 mL volumetric flask, diluted to volume with pure water, and sonicated to obtain a diluted solution. 2 mL of the diluted solution was taken, 2 mL of ethanol was added, and the mixture was sonicated for 30 min, centrifuged at 800 rpm for 10 min, and 1 mL of the supernatant was collected to prepare a liquid phase sample. The content of neohesperidin in each fermentation broth was determined using HPLC.

[0126] The results of the determination of neohesperidin content in the fermentation broths prepared in Examples 2-3 or Comparative Examples 1-13 are shown in Table 3: Table 3

[0127] Verification of technical effectiveness and / or analysis of technical problem solving This invention explores the optimal seed stress process by comparing different feed concentrations, feeding methods, and fermentation aeration ratios, as well as comparing environmental stress and stress timing. It also compares fermentation processes using ammonia, sodium hydroxide, and different carbon sources to discover the most suitable process for neohesperidin production. By optimizing the fermentation environment and production process, efficient neohesperidin production is achieved, with good economic and environmental benefits.

[0128] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fermentation process for producing neohesperidin using *Yarrowia lipolytica*, characterized in that, The fermentation process includes the following steps: S1. The activated lipophilic yeast strain YO1 with preservation number CGMCC No.34491 was inoculated into seed culture medium and cultured to obtain seed liquid; S2. Inoculate the seed culture medium with the seed solution, introduce ammonia water, and carry out expansion culture. When the OD600 value of the culture solution reaches 2.0, turn off the ammonia water and continue to culture until the OD600 value of the culture solution reaches 10-20 to obtain the expanded culture seed solution. S3. Inoculate the expanded culture seed liquid into the fermentation medium containing the substrate, adjust the pH with ammonia water, and ferment for 18-20 hours; S4. Feed the fermentation broth at a rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100-150, increase the feeding rate to 12 kg / h and continue fermentation to obtain a fermentation broth containing neohesperidin. The aeration ratio of the ammonia water mentioned in step S2 or step S3 is 0.4-0.8 vvm; The composition of the feed in step S4 is: 700-800 g / L glucose solution and / or 700-800 g / L glycerol solution.

2. The fermentation process according to claim 1, characterized in that, The inoculation amount of the lipophilic yeast strain YO1 mentioned in step S1 is 2%-5% v / v.

3. The fermentation process according to claim 1, characterized in that, The cultivation conditions described in step S1 are: rotation speed 200-220 rpm, temperature 25-35℃, and cultivation time 24-48h.

4. The fermentation process according to claim 1, characterized in that, The inoculation amount of the seed liquid mentioned in step S2 is 10%-15% v / v.

5. The fermentation process according to claim 1, characterized in that, The ammonia water introduced in step S2 refers to 32% v / v ammonia water, maintaining the pH at 5-6.

6. The fermentation process according to claim 5, characterized in that, The ammonia water aeration ratio in step S2 is 0.4vvm; the ammonia water aeration ratio in step S3 is 0.8vvm.

7. The fermentation process according to claim 1, characterized in that, The conditions for the scale-up culture described in step S2 or the fermentation culture described in step S3 are: rotation speed 100-200 rpm, temperature 25-35℃, dissolved oxygen 30%-50%.

8. The fermentation process according to claim 1, characterized in that, The continuous culture mentioned in step S2 is: continuous culture until the OD600 value of the culture medium reaches 15.

9. The fermentation process according to claim 1, characterized in that, The inoculation amount of the expanded culture seed solution mentioned in step S3 is 10%-15% v / v.

10. The fermentation process according to claim 1, characterized in that, In step S3, the pH is adjusted to 5-6 with 32% v / v ammonia.

11. The fermentation process according to claim 1, characterized in that, The substrate mentioned in step S3 includes any one or more of glucose and glycerol.

12. The fermentation process according to claim 1, characterized in that, The concentration of the substrate mentioned in step S3 is 20-40 g / L.

13. The fermentation process according to claim 1, characterized in that, The composition of the feed in step S4 is: 700 g / L glucose solution or 800 g / L glycerol solution.

14. The fermentation process according to claim 1, characterized in that, The composition of the feed in step S4 is: a 700 g / L glucose solution with pH 6.0-7.

0.

15. The fermentation process according to claim 1, characterized in that, Step S4 involves feeding the fermentation broth at a rate of 8 kg / h. When the OD600 value of the fermentation broth reaches 100, the feeding rate is increased to 12 kg / h, and fermentation continues to obtain a fermentation broth containing neohesperidin.

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