Fermentation process capable of shortening fermentation period and efficiently synthesizing Ectoine

Abstract

The invention discloses a fermentation process for shortening a fermentation period and efficiently synthesizing Ectoine. The fermentation process comprises the following steps: screening and activating high-yield strains, preparing a fermentation culture medium, performing fermentation culture, performing sectional dissolved oxygen regulation and control, performing double-stage pH dynamic regulation and control, performing carbon-nitrogen ratio dynamic regulation based on metabolic flow analysis, performing osmotic pressure gradient induction in the later stage of fermentation, and extracting and purifying a product. According to the fermentation process for shortening the fermentation period and efficiently synthesizing the Ectoin, the expression quantity of key enzymes for synthesizing the Ectoin is increased by 80% or above compared with that of an original strain through the Halomonassp.ECT-09 strain bred through ultraviolet-nitrosoguanidine compound mutation, the basic yield reaches 35 g / L, a foundation is laid for high yield, and the method is suitable for industrial production. A glucose-trehalose mixed carbon source and a yeast extract-ammonium nitrate mixed nitrogen source are adopted, and an L-aspartic acid-L-asparagine mixed precursor is added, so that the metabolic pathway is shortened, and the loss of an intermediate product is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial metabolic engineering and fermentation technology, specifically a fermentation process for efficiently synthesizing ectoine with a shortened fermentation cycle. Background Technology

[0002] Ectoin, a compatible solute with "molecular chaperone" function, can maintain its spatial structure and functional stability by forming hydrogen bonds with biomolecules under extreme conditions, and is widely used in high-end cosmetics, biopharmaceuticals, and biopharmaceuticals. Currently, microbial fermentation is the mainstream production method for ectoin, but traditional processes have three major bottlenecks: traditional processes rely on the natural metabolic rhythm of the strains and do not precisely control the metabolic conversion between cell growth and product synthesis, resulting in fermentation cycles of 72-100 hours, low equipment turnover rate, and low production capacity per unit time.

[0003] Most processes use constant carbon-nitrogen ratio and constant osmotic pressure culture, which cannot meet the different nutritional needs of strains at different metabolic stages, and do not effectively activate the ectoin synthesis pathway, resulting in yields generally below 25 g / L.

[0004] In traditional extraction and purification processes, impurities are not completely removed, leading to fluctuations in product purity and making it difficult to consistently meet pharmaceutical-grade standards.

[0005] Existing improvement technologies have significant shortcomings: Chinese patent CN114807623A increases yield by knocking out byproduct synthesis genes through genetic engineering, but the construction cost of gene-edited strains is high, the approval process is complex, and there is a risk of environmental release; Chinese patent CN115216891A uses constant high osmotic pressure to induce ectoin synthesis, but this leads to premature cell aging, which shortens the product synthesis window; some processes attempt to optimize the carbon-nitrogen ratio, but do not combine metabolic flux analysis for dynamic adjustment, making it impossible to accurately match the real-time needs of the strain. Therefore, developing a multi-dimensional, precise regulation process based on metabolic mechanisms without genetic modification is key to overcoming the bottleneck in ectoin production. Summary of the Invention

[0006] (I) Technical Problem Solved In view of the shortcomings of existing technologies, this invention provides a method for efficiently synthesizing ectodiol with a shortened fermentation cycle.

[0007] The fermentation process of the product.

[0008] (II) Technical Solution

[0009] To achieve the above object, the application provides the following technical scheme: a fermentation process for shortening fermentation period and efficiently synthesizing ectoine, comprising the following steps: S1: high-yield strain screening and activation: isolating halophilic strains from high-salt environment samples, obtaining high-yield ectoine strains through ultraviolet-nitrosoguanidine compound mutagenesis, inoculating into seed culture medium after slope activation for expansion culture, and preparing seed liquid;

[0010] S2: fermentation medium preparation: the fermentation medium comprises 12-20 g / L of basic carbon source, 6-12 g / L of basic nitrogen source, 2-5 g / L of inorganic salt, 3-8 g / L of compatible solute precursor, 0.5-2 g / L of ectoine synthesis inducer, and pH regulator;

[0011] S3: fermentation culture: inoculating the seed liquid into the fermentation medium at a 5%-10% inoculation amount, and implementing multidimensional synergistic regulation in the fermentation tank: S3.1: segmented dissolved oxygen regulation: 65%-75% of dissolved oxygen for 0-10 h, 45%-55% of dissolved oxygen for 10-24 h, 35%-45% of dissolved oxygen for 24-36 h, and 25%-35% of dissolved oxygen for 36 h to the end;

[0012] S3.2: two-stage pH dynamic regulation: pH 7.3-7.6 for 0-10 h, and pH 6.7-7.1 for 10 h to the end;

[0013] S3.3: dynamic adjustment of carbon-nitrogen ratio based on metabolic flow analysis: through online monitoring of the concentration of key intermediate metabolites of the bacteria, the carbon source supplement liquid and the nitrogen source supplement liquid are added in real time to maintain the carbon-nitrogen ratio (C / N) at 4:1-6:1 in the bacterial growth period and 8:1-12:1 in the product synthesis period;

[0014] S3.4: osmotic pressure gradient induction in the late fermentation period: after 36 h, the osmotic pressure of the fermentation liquid is gradually increased from the initial 300-350 mOsm / kg to 500-600 mOsm / kg by gradient addition of sodium chloride solution; at the same time, the fermentation temperature is controlled at 30-35 DEG C, and the fermentation period is 38-52 h;

[0015] S4: product extraction and purification: after the fermentation is completed, high-purity ectoine is obtained through centrifugation, microfiltration, decolorization by macroporous resin, nanofiltration concentration, low-temperature crystallization and vacuum drying in sequence.

[0016] Preferably, the strain in S1 is a halomonas sp. ECT-09 strain of Halomonas, with a preservation number of CGMCC No. 26981, and the ultraviolet irradiation dose is 120-180 muW / cm2, and the time is 40-70 s

[0017] The concentration of nitroso guanidine is 0.1-0.3 mg / mL, the treatment time is 20-30 min, and the compound mutagenesis is selected and bred to obtain the key enzyme of the expression of the ecdysone synthesis, which is more than 80% higher than that of the original strain.

[0018] Preferably, the seed culture medium formula in S1 is: glucose 12 g / L, yeast extract 6 g / L, sodium chloride 22 g / L, potassium chloride 1.2 g / L, magnesium sulfate 0.6 g / L, vitamin B1 0.01 g / L, pH 7.3-7.5.

[0019] The culture conditions are 33-35℃, 200-240r / min oscillation culture for 10-14h, the seed liquid OD600 reaches 2.0-2.5, and the bacterial concentration is≥1.5×10 9 CFU / mL.

[0020] Preferably, the basic carbon source in S2 is glucose and trehalose mixed in a mass ratio of 3:1-5:1, and the basic nitrogen source is yeast extract and ammonium nitrate mixed in a mass ratio of 2:1-3:1; the inorganic salt is potassium dihydrogen phosphate 1.2-2.0 g / L, calcium chloride 0.6-1.2 g / L, zinc sulfate 0.05-0.15 g / L, and manganese sulfate 0.03-0.08 g / L.

[0021] The compatible solute precursor is L-aspartic acid and L-asparagine mixed in a mass ratio of 1:1-2:1; and the ecdysone synthesis inducer is N-acetyl proline and glycine betaine mixed in a mass ratio of 1:2-1:4.

[0022] Preferably, the osmotic pressure gradient induction in the late fermentation stage in S3 is: adding sodium chloride solution at 36h to make the osmotic pressure reach 380-420 mOsm / kg, increasing to 450-480 mOsm / kg at 42h, and increasing to 500-600 mOsm / kg at 48h; the mass concentration of the sodium chloride solution is 20%-30%.

[0023] Preferably, in S4, the centrifugal condition is 9000-11000r / min centrifugation for 18-25min; the microfiltration adopts 0.1μm ceramic membrane; the decolorization adopts X-5 type macroporous adsorption resin; the nanofiltration concentration adopts 300Da molecular weight cut-off nanofiltration membrane, and is concentrated to 1 / 6-1 / 10 of the original volume; the crystallization adopts an ethanol-isopropyl alcohol mixed system, and is placed at 2-6℃ for 15-20h; the vacuum drying condition is 55-65℃, vacuum degree 0.09-0.095MPa, and drying time 8-10h.

[0024] (Three) beneficial effects

[0025] Compared with the prior art, the present application provides a fermentation process for shortening the fermentation period and efficiently synthesizing ectoine, which has the following beneficial effects: 1. The fermentation process for shortening the fermentation period and efficiently synthesizing ectoine, by changing the oxygen demand characteristics of the strain at different stages, from 70%±5% of the growth period of the bacteria to 30%±5% of the product accumulation period, avoids metabolic arrest caused by insufficient dissolved oxygen or energy waste caused by excessive dissolved oxygen, so that the bacteria quickly complete the growth

[0026] The conversion to product synthesis is achieved by changing the pH from 7.3-7.6 in the growth period of the bacteria to 6.7-7.1 in the product synthesis period, which inhibits the excessive activity of the respiratory chain of the bacteria and directs the directional conversion of the carbon source to ectoine, thereby shortening the metabolic conversion time by 8-12h compared with the constant pH process.

[0027] 2. The fermentation process for shortening the fermentation period and efficiently synthesizing ectoine, by using the Halomonas sp. ECT-09 strain (CGMCC No. 26981) obtained by ultraviolet-nitroso guanidine complex mutagenesis, the expression amount of the key enzyme (ectA / ectB / ectC) for ectoine synthesis is increased by more than 80% compared with the original strain, and the basic yield reaches 35g / L, which lays a foundation for high yield. A mixed carbon source of glucose and trehalose, a mixed nitrogen source of yeast extract and ammonium nitrate, and an L-aspartic acid-L-asparagine mixed precursor are added to shorten the metabolic pathway and reduce the loss of intermediate products. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application, obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0029] In order to more clearly describe the technical solutions of the present application, the operation details, parameter control and result verification of the fermentation process will be described in detail below in conjunction with three specific embodiments (covering the production scenes of cosmetic-grade and pharmaceutical-grade ectoine). In all embodiments, the content of ectoine is detected by high performance liquid chromatography (HPLC) method, and the chromatographic conditions are as follows: C18 chromatographic column (250mm×4.6mm, 5μm), mobile phase is methanol-water (5:95, v / v), flow rate is 1.0mL / min, column temperature is 30℃, detection wavelength is 210nm, sample size is 20μL; the osmotic pressure is detected by an ice point osmometer (precision ±1mOsm / kg); the concentration of the bacteria is determined by blood cell counting plate combined with OD600 value (ultraviolet spectrophotometer, 600nm wavelength).

[0030] Example 1: Activation and seed culture of high-yield strains for large-scale production of cosmetic-grade ectoine (500L fermenter): Slant activation: Halomonassp.ECT-09 strain preserved at CGMCC (accession number No. 26981) was used. Under aseptic conditions, a small amount of cells was picked and streaked onto a slant culture medium (formulation: glucose).

[0031] The mixture consisted of 12 g / L yeast extract, 6 g / L sodium chloride, 22 g / L potassium chloride, 1.2 g / L magnesium sulfate, 0.6 g / L vitamin B1, 0.01 g / L agar, and 20 g / L agar (pH 7.4). It was incubated in a 34°C incubator for 24 hours until uniform, plump single colonies (1-2 mm in diameter, milky white, with neat edges) grew on the slant.

[0032] Primary seed culture: Pick 3-5 single colonies from the activated slant culture and inoculate them into a 2L Erlenmeyer flask containing 500mL of seed culture medium (formula same as slant culture medium, but with agar removed). Incubate at 34℃ and 220r / min in a shaker for 12h. Sampling and analysis: OD600 = 2.2, cell concentration 1.8 × 10⁻⁶. 9 A seed culture with a concentration of CFU / mL and no contamination by other microorganisms (Gram staining microscopy reveals Gram-negative short bacilli with uniform morphology) is considered a qualified Grade 1 seed culture.

[0033] Secondary seed culture: 500 mL of the primary seed culture was inoculated into a 10 L seed tank containing 5 L of seed culture medium. The temperature was controlled at 34 °C, the stirring speed at 200 rpm, and the aeration rate at 1.0 vvm (volume of air / volume of culture medium / min). The culture was incubated for 10 h, and samples were taken for analysis: OD600 = 2.4, cell concentration 2.1 × 10⁻⁶. 9 The CFU / mL and residual sugar concentration (glucose) of 1.2g / L are qualified and used as secondary seed liquid for fermentation tank inoculation.

[0034] Preparation and sterilization of fermentation medium: formula configuration: 500L fermentation medium was prepared according to the following formula (the actual amount of raw materials was calculated according to the working volume of 450L): glucose 72kg (160g / L), trehalose 18kg (40g / L, the mass ratio of glucose to trehalose 4:1), yeast extract 45kg (100g / L), ammonium nitrate 18kg (40g / L, the mass ratio of yeast extract to ammonium nitrate 2.5:1), potassium dihydrogen phosphate 0.54kg (1.2g / L), calcium chloride 0.27kg (0.6g / L), zinc sulfate 0.045kg (0.1g / L), manganese sulfate 0.0225kg (0.05g / L), L-aspartic acid 13.5kg (30g / L), L-asparagine 9kg (20g / L, the mass ratio of the two 1.5:1), N-acetyl proline 0.45kg (1g / L), glycine betaine 1.35kg (3g / L, the mass ratio of the two 1:3).

[0035] pH adjustment and sterilization: the above raw materials were sequentially added into a 500L fermenter, deionized water was added to 450L, stirring (150r / min) was started to fully dissolve the raw materials, 1mol / L sodium hydroxide solution was used to adjust the pH to 7.4, the feed port was closed, and the fermenter sterilization system was started: 121℃, 0.11MPa high pressure steam sterilization for 30min, after sterilization, cooling water was introduced to cool to 34℃, and the air filtration system (three

[0036] stage filtration: primary efficiency cotton core + medium efficiency filter core + 0.22μm microporous filter membrane) was started, sterile air was introduced into the fermenter, and the positive pressure in the tank (0.02-0.03MPa) was maintained, and standby.

[0037] Fermentation culture:

[0038] Inoculation: under sterile conditions, 5L of secondary seed liquid was introduced into 450L of fermentation medium through the inoculation port, the inoculation amount was 1.1% (volume ratio), stirring and aeration were started, and the fermentation stage was entered.

[0039] Segmented dissolved oxygen control:

[0040] 0-10h (bacterial growth period): control the dissolved oxygen at 70%±5%, adjust the stirring speed (250r / min) and aeration amount (1.2vvm) to achieve, sample every 2h to detect the dissolved oxygen, if the dissolved oxygen is lower than 65%, increase the stirring speed (increase 20r / min each time) or aeration amount (increase 0.1vvm each time), to ensure that the dissolved oxygen is stable in the target interval.

[0041] 10-24h (initial stage of product synthesis): control the dissolved oxygen at 50%±5%, reduce the stirring speed to 200r / min, reduce the aeration rate to 1.0vvm, detect the dissolved oxygen every 3h, and adjust the parameters according to the detection results.

[0042] 24-36h (middle stage of product synthesis): control the dissolved oxygen at 40%±5%, reduce the stirring speed to 150r / min, and reduce the aeration rate to 0.8vvm.

[0043] 36h to the end of fermentation (product accumulation period): control the dissolved oxygen at 30%±5%, reduce the stirring speed to 120r / min, and reduce the aeration rate to 0.6vvm.

[0044] Two-stage dynamic pH regulation: 0-10h: maintain pH 7.4±0.1, and adjust in real time through the automatic alkali supplement system (1mol / L sodium hydroxide solution) of the fermenter. When the pH is lower than 7.3, turn on the alkali supplement pump, and adjust the alkali supplement rate according to the pH drop amplitude (0.5-1.0L / h).

[0045] 10h to the end: switch the pH target value to 6.9±0.1, and when the pH is higher than 7.0, turn on the automatic acid supplement system (1mol / L hydrochloric acid solution), with an acid supplement rate of 0.3-0.8L / h, to ensure that the pH is stable in the target range.

[0046] Dynamic adjustment of carbon-nitrogen ratio based on metabolic flow analysis: online monitoring: automatically sample and detect every 4h through the HPLC online detection module matched with the fermenter

[0047] Concentrations of alpha-ketoglutaric acid and oxaloacetic acid in the fermentation broth: 0-10h (bacterial growth period): target alpha-ketoglutaric acid 0.8-1.2mmol / L, oxaloacetic acid 0.5-0.8mmol / L. When the alpha-ketoglutaric acid is detected to be lower than 0.8mmol / L (reflecting insufficient nitrogen source), turn on the nitrogen source supplement pump, with a flow rate of 0.6L / h; when the oxaloacetic acid is higher than 0.8mmol / L (reflecting excessive carbon source), reduce the carbon source supplement flow rate.

[0048] 10h to the end (product synthesis period): target alpha-ketoglutaric acid 0.3-0.6mmol / L, oxaloacetic acid 1.0-1.5mmol / L. When the alpha-ketoglutaric acid is higher than 0.6mmol / L (reflecting excessive nitrogen source), stop the nitrogen source supplement; when the oxaloacetic acid is lower than 1.0mmol / L (reflecting insufficient carbon source), increase the carbon source supplement flow rate (50% glucose-sucrose mixed solution, mass ratio 2:1) to 1.0L / h.

[0049] Carbon nitrogen ratio maintenance: By the above adjustment, the carbon nitrogen ratio (C / N) of the bacterial growth period is ensured to be stable at 5:1±0.5, and the product synthesis period is stable at 10:1±1.

[0050] Osmotic pressure gradient induction in the late fermentation period: 36h: The initial osmotic pressure of the fermentation broth was detected to be 340mOsm / kg, the sodium chloride solution (25% mass concentration) feeding pump was started, the feeding rate was 0.2L / h, and the feeding was continued for 2h, and the osmotic pressure was increased to 400mOsm / kg.

[0051] 42h: The feeding rate was adjusted to 0.15L / h, and the feeding was continued for 3h, and the osmotic pressure was increased to 460mOsm / kg.

[0052] 48h: The feeding rate was adjusted to 0.1L / h, and the feeding was continued for 2h, and the osmotic pressure was increased to 550mOsm / kg, and then the osmotic pressure was maintained until the end of the fermentation.

[0053] Fermentation endpoint determination: Sample every 8h to detect the content of ectoine: 48h: The content of ectoine was 58.2g / L.

[0054] 52h: The content of ectoine was 58.6g / L, and the difference between two consecutive detections was 0.4g / L (<0.5g / L), which was determined to be the end of fermentation, and the stirring and aeration were stopped, and the tank was discharged.

[0055] Product extraction and purification:

[0056] Centrifugal impurity removal: 450L of fermentation broth was transferred into a high-speed centrifuge (disc type, processing capacity 50L / h), the rotation speed was controlled at 10000r / min, the centrifugation time was 20min, and the supernatant (about 430L) was collected to remove the bacterial cells and large impurities (centrifugal residue about 20L, containing 70% water, which can be recycled as a bacterial fertilizer raw material).

[0057] Microfiltration: The supernatant was pumped into a ceramic membrane filtration system (0.1μm pore size, membrane area 5m²), the operating pressure was 0.2MPa, the temperature was 35℃, the filtration flow rate was 15L / h, and the microfiltration permeate (about 420L) was collected to remove colloidal particles and macromolecular proteins (retentate about 10L, which can be further extracted to recover ectoine).

[0058] Macroporous resin decolorization: Pump the microfiltration permeate into a chromatography column (diameter 50 cm, height 200 cm, resin loading 392.5 L) filled with X-5 type macroporous adsorption resin, control the temperature at 45°C, the flow rate at 1.5 BV / h (BV is the column volume, i.e. 392.5 L / h), the adsorption time at 2 h, collect the decolorized filtrate (about 415 L), and detect the color of the filtrate: colorless and transparent (platinum-cobalt colorimetry, colority ≤ 10 APHA), the decolorization rate is 95.2%.

[0059] Nanofiltration concentration: Pump the decolorized filtrate into a nanofiltration system (300 Da molecular weight cut-off, spiral membrane, membrane area 20 m²), operate at a pressure of 1.8 MPa and a temperature of 38°C, concentrate to 1 / 8 of the original volume (about 52 L), detect the ico-dextrin concentration in the concentrated solution: 450 g / L, the residual salt concentration (sodium chloride) < 5 g / L (conductivity detection, conductivity < 10 mS / cm).

[0060] Low-temperature crystallization: Transfer the 52 L concentrated solution into a crystallization tank, add 104 L of ethanol-isopropanol mixed solvent (volume ratio 2:1), start stirring (50 r / min) to make the mixture uniform, then pass in cooling water to reduce the temperature to 4°C, and stand for crystallization for 18 h, and a large amount of white needle-shaped crystals are observed to precipitate.

[0061] Vacuum drying: Filter the crystallization solution through a plate-and-frame filter press (0.22 μm filter cloth), collect the crystals (wet weight about 23 kg, water content 15%), transfer into a vacuum drying oven, control the temperature at 60°C, the vacuum degree at 0.092 MPa, and dry for 10 h to obtain 19.55 kg of dried ico-dextrin crystals.

[0062] Product detection: Take samples for quality analysis: purity 99.1% (HPLC method), moisture content 0.3% (Karl Fischer method), heavy metal (lead, mercury, arsenic) content < 0.1 mg / kg (atomic absorption spectrophotometry), which meets the cosmetic-grade ico-dextrin industry standard (QB / T 5814-2023).

[0063] Example 2: Production of pharmaceutical-grade ico-dextrin (100 L fermenter, purity ≥ 99.5%) Seed culture and fermentation control (key parameter adjustment) Seed culture: Same as Example 1, the OD600 of the secondary seed liquid is 2.3, the bacterial concentration is 2.0 x 10 9 CFU / mL.

[0064] Fermentation medium: On the basis of Example 1, remove N-acetyl proline (avoid potential residual

[0065] risk), only add glycine betaine 1.8 g / L, other ingredients remain unchanged, adjust the pH to 7.3.

[0066] Fermentation control:

[0067] Dissolved oxygen: 0-10h 75%±5%, 10-24h 55%±5%, 24-36h 45%±5%, 36h to end 35%±5%.

[0068] pH: 0-10h 7.5±0.1, 10h to end 6.8±0.1.

[0069] Carbon nitrogen ratio: 4:1 in cell growth period, 12:1 in product synthesis period, 0.3-0.5mmol / L of alpha-ketoglutarate and 1.2-1.5mmol / L of oxaloacetate are monitored on-line by HPLC.

[0070] Osmotic pressure: 36h 420mOsm / kg, 42h 480mOsm / kg, 48h 600mOsm / kg.

[0071] Fermentation end point: 61.8g / L of ectoine at 46h, 62.1g / L at 48h, fermentation is ended, fermentation period is 48h.

[0072] Extraction and purification (medical grade strengthening treatment): centrifugation at 11000r / min for 25min, supernatant is filtered again through 0.22μm microporous filter membrane (to remove possible residual microorganisms).

[0073] Decolorization: X-5 resin + activated carbon are used in series for decolorization (activated carbon is added in an amount of 0.5%, stirring at 45℃ for 1h), colority of the filtrate after decolorization is ≤5APHA.

[0074] Nanofiltration: concentrated to 1 / 10 of the original volume (about 9L), the concentrated solution is subjected to secondary nanofiltration through 0.1μm ceramic membrane, to ensure that the residual salt concentration is <1g / L (conductivity <2mS / cm).

[0075] Crystallization: ethanol-isopropanol volume ratio is 3:1, temperature is 2℃, crystallization time is 20h, to improve the purity of the crystal.

[0076] Drying: vacuum drying at 55℃, 0.095MPa for 8h, to obtain 4.9kg of ectoine crystal.

[0077] Detection: purity is 99.6%, moisture content is 0.2%, endotoxin content is <0.1EU / mg (limulus reagent method), which meets the medical grade raw material standard (USP45-NF40).

[0078] Example 3: Process stability verification (3 batches of repeated tests, 500L fermenter) To verify the stability of the process of the application, 3 batches of continuous production are carried out according to the parameters of Example 1, and the key indicators are recorded as follows:

[0079] Fermentation cycle (h)

[0080] Ectoine production

[0081] Amount (g / L)

[0082] Product purity

[0083] (%)

[0084] Carbon source utilization rate

[0085] (%)

[0086] Batch-to-batch production fluctuation

[0087] (%)

[0088] 1 52 58.6 99.1 92.3 -2 50 59.2 99.3 93.1 +1.023 51 58.9 99.2 92.7 +0.51The results show that the fermentation cycle of the three batches is stable at 50-52h, the ectoine production is >58g / L, the purity is >99%, the carbon source utilization rate is >92%, the batch-to-batch production fluctuation is <1.5%, the process stability is excellent, and meets the needs of industrial continuous production.

[0089] Comparative Example: Comparison between traditional process and process of the application (100L fermenter) In order to highlight the technical advantages of the application, a traditional process control group (using wild Halomonas strain, constant dissolved oxygen 30%, constant pH 7.2, no osmotic pressure induction, constant carbon-nitrogen ratio 6:1) is set up, and the comparison results are as follows:

[0090] Index Traditional process Process of the application (Example 2)

[0091] Improvement / shortening range

[0092] Fermentation cycle 84h 48h Shortened by 42.9% Ectoine production 22.5g / L 62.1g / L Improved by 176% Carbon source utilization rate 65.8% 94.2% Improved by 43.2% Product purity 97.3% 99.6% Improved by 2.3% Unit time productivity (g / L·h)

[0093] 0.27 1.29 Improved by 377.8% It can be seen from the comparison that the process of the application is significantly superior to the traditional process in terms of fermentation cycle, production, productivity and other core indicators, has obvious technical advantages, and has high industrial application value.

[0094] Although embodiments of the application have been shown and described, it would be obvious to those skilled in the art that

[0095] It is to be understood that the embodiments which have been described are merely illustrative of the principles and applications of the present application. Numerous modifications, changes, omissions, and substitutions, in the exact construction, arrangements and combination of parts, and other details, can be made by those skilled in the art without departing from the spirit and scope of the application, as set forth in the following claims, and equivalents thereof.

Claims

1. A fermentation process for efficiently synthesizing ectoine by shortening the fermentation period, characterized by, The method comprises the following steps: S1: high-yield strain screening and activation: a halophilic strain is isolated from a high-salt environment sample, a high-yield ectoine-producing strain is obtained through UV-nitrosoguanidine compound mutagenesis, and after activation on a slope, the strain is inoculated into a seed culture medium for expansion culture to prepare a seed liquid; S2: preparation of a fermentation medium: the fermentation medium comprises a basic carbon source 12-20 g / L, a basic nitrogen source 6-12 g / L, inorganic salts 2-5 g / L, a compatible solute precursor 3-8 g / L, an ectoine synthesis inducer 0.5-2 g / L, and a pH regulator; S3: fermentation culture: the seed liquid is inoculated into the fermentation medium at an inoculation amount of 5%-10%, and multi-dimensional synergistic regulation is implemented in a fermentation tank: S3.1: segmented dissolved oxygen regulation: 0-10 h, dissolved oxygen 65%-75%; 10-24 h, dissolved oxygen 45%-55%; 24-36 h, dissolved oxygen 35%-45%; 36 h to the end, dissolved oxygen 25%-35%; S3.2: two-stage dynamic pH regulation: 0-10 h, pH 7.3-7.6; 10 h to the end, pH 6.7-7.1; S3.3: dynamic adjustment of the carbon-nitrogen ratio based on metabolic flow analysis: through online monitoring of the concentration of key intermediate metabolites of the bacterial cells, carbon source supplement liquid and nitrogen source supplement liquid are added in real time to maintain the carbon-nitrogen ratio (C / N) at 4:1-6:1 in the bacterial growth period and 8:1-12:1 in the product synthesis period; S3.4: osmotic pressure gradient induction in the late fermentation period: after 36 h, sodium chloride solution is added in a gradient to gradually increase the osmotic pressure of the fermentation broth from the initial 300-350 mOsm / kg to 500-600 mOsm / kg; at the same time, the fermentation temperature is controlled at 30-35°C, and the fermentation period is 38-52 h; S4: product extraction and purification: after the fermentation is completed, the fermentation broth is sequentially subjected to centrifugation, microfiltration, decolorization by a macroporous resin, nanofiltration concentration, low-temperature crystallization, and vacuum drying to obtain high-purity ectoine.

2. The fermentation process for efficiently synthesizing ectoine by shortening fermentation period according to claim 1, characterized in that: The strain in S1 is a halomonas sp. ECT-09 strain of the Halomonas genus, with a preservation number of CGMCC No. 26981, which is obtained through compound mutagenesis of ultraviolet irradiation at a dose of 120-180 μW / cm² for 40-70 s and nitrosoguanidine at a concentration of 0.1-0.3 mg / mL for 20-30 min, The expression amount of key ectoine synthesis enzymes of the strain is increased by more than 80% compared with that of the original strain.

3. The fermentation process for efficiently synthesizing ectoine by shortening fermentation period according to claim 1, characterized in that: The seed culture medium formula in S1 is: glucose 12 g / L, yeast extract 6 g / L, sodium chloride 22 g / L, potassium chloride 1.2 g / L, magnesium sulfate 0.6 g / L, vitamin B1 0.01 g / L, pH 7.3-7.5; the culture conditions are 33-35°C, 200-240 r / min shaking culture for 10-14 h, the seed liquid OD600 reaches 2.0-2.5, and the bacterial concentration is ≥1.5×10 9 CFU / mL.

4. The fermentation process for efficient synthesis of ectoine with shortened fermentation period according to claim 1, characterized in that: The basic carbon source in S2 is a mixture of glucose and trehalose at a mass ratio of 3:1-5:1, and the basic nitrogen source is a mixture of yeast extract and ammonium nitrate at a mass ratio of 2:1-3:1; the inorganic salts are potassium dihydrogen phosphate 1.2-2.0 g / L, calcium chloride 0.6-1.2 g / L, zinc sulfate 0.05-0.15 g / L, and manganese sulfate 0.03-0.08 g / L; The compatible solute precursor is L-aspartic acid and L-asparagine at a mass ratio of 1:1-2:1; and the ectoine synthesis inducer is N-acetyl proline and glycine betaine at a mass ratio of 1:2-1:

4.

5. The fermentation process for efficient synthesis of ectoine with shortened fermentation period according to claim 1, characterized in that: In the S3, the carbon source supplementing solution is a glucose-sucrose mixed solution with a mass concentration of 40-60%, a mass ratio of 2:1, and the nitrogen source supplementing solution is a yeast extract solution with a mass concentration of 15-25%; the concentrations of alpha-ketoglutaric acid and oxalacetic acid are monitored on-line by high performance liquid chromatography.

6. The fermentation process for efficient synthesis of ectoine with shortened fermentation period according to claim 1, characterized in that: In the fermentation of the S3, the osmotic pressure gradient is induced by adding sodium chloride solution at 36h to reach 380-420mOsm / kg, at 42h to reach 450-480mOsm / kg, and at 48h to reach 500-600mOsm / kg; the mass concentration of the sodium chloride solution is 20-30%.

7. The fermentation process for efficient synthesis of ectoine with shortened fermentation period according to claim 1, characterized in that: In the S4, the centrifugation condition is 9000-11000r / min for 18-25min; The microfiltration adopts 0.1μm ceramic membrane; the decolorization adopts X-5 type macroporous adsorption resin; the nanofiltration concentration adopts 300Da molecular weight cut-off nanofiltration membrane, and is concentrated to 1 / 6-1 / 10 of the original volume; the crystallization adopts an ethanol-isopropyl alcohol mixed system, and is placed at 2-6℃ for 15-20h; the vacuum drying condition is 55-65℃, a vacuum degree of 0.09-0.095MPa, and a drying time of 8-10h. ​