A high-efficiency extraction and purification method of organic selenium in lactic acid bacteria

Abstract

The application relates to the technical field of extraction and purification, and discloses a high-efficiency leaching and purification method of organic selenium of lactic acid bacteria, which comprises the following steps: mixing organic selenium-rich lactic acid bacteria substrate suspension, polyethylene glycol, isopropyl alcohol, disodium ethylenediaminetetraacetate, L-sodium ascorbate and lysozyme, and carrying out reaction under constant-temperature stirring condition to release organic selenium substances in lactic acid bacteria cells; pumping high-concentration phosphate aqueous solution into the mixture after cooling, collecting light liquid phase after centrifugal separation; pumping the light liquid phase into an ultrafiltration membrane assembly for transmembrane filtration separation, collecting membrane interception liquid and membrane dialysis liquid respectively; pumping the membrane dialysis liquid into a cation exchange resin chromatographic column after adjusting the pH value of the membrane dialysis liquid to be acidic to capture short-chain selenoamino acids, and then pumping ammonia solution into the chromatographic column to elute and collect eluate; uniformly mixing the membrane interception liquid and the eluate, and carrying out spray drying to obtain lactic acid bacteria organic selenium powder. The method improves the purity and extraction amount of the lactic acid bacteria organic selenium powder.

Description

Technical Field

[0001] This invention relates to the field of extraction and purification technology, specifically to a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells. Background Technology

[0002] Lactic acid bacteria can convert inorganic selenium in the environment into organic selenium through biotransformation. The organic selenium enriched inside the cells of lactic acid bacteria has a high absorption and utilization rate. In order to obtain and apply the organic selenium substances inside the cells of organic selenium-rich lactic acid bacteria, it is necessary to perform cell wall breaking extraction and subsequent impurity removal and purification operations on the organic selenium-rich lactic acid bacteria, and finally prepare lactic acid bacteria organic selenium powder.

[0003] Existing extraction and purification processes typically employ physical methods such as high-pressure homogenization to disrupt the cell walls of selenium-rich lactic acid bacteria. Subsequently, impurities are removed and products are enriched using centrifugation and membrane separation techniques. In actual production, physical methods such as high-pressure homogenization generate fluid shear forces and localized high temperatures. The energy input into the material during physical extraction can easily exceed the dissociation energy of the selenium-carbon bonds in the organic selenium molecules, causing the organic selenium bound to the polypeptide chains of the lactic acid bacteria host protein to break chemical bonds and degrade into inorganic selenium.

[0004] Meanwhile, during centrifugation after cell disruption, non-target macromolecular impurities released from the cells of selenium-rich lactic acid bacteria are difficult to settle and separate. These impurities easily induce interfacial emulsification at the interface between the light and heavy liquid phases, leading to material loss in the centrifugation equipment and increasing purification difficulty. Furthermore, the small physical size of free short-chain selenomethylselenocysteine ​​and short-chain selenomethionine molecules makes them prone to permeating the pores of the membrane separation component during ultrafiltration, resulting in loss with the waste liquid and a decrease in the purity of the final product (lactic acid bacteria organic selenium powder) and a reduction in the absolute extraction quality of total organic selenium. Therefore, this invention proposes a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells to address the shortcomings of existing technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells. This method solves the problems of existing extraction and purification processes, such as the difficulty in effectively settling and separating non-target macromolecular impurities, which easily leads to phase interface emulsification, and the easy loss of free short-chain selenoamino acids with waste liquid during membrane separation, resulting in a decrease in the purity of lactic acid bacteria organic selenium powder and the extraction quality of total organic selenium.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells includes the following steps:

[0008] A suspension of selenium-rich lactic acid bacteria substrate was placed in a reaction vessel, and polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate and lysozyme were added sequentially to obtain a mixture. The initial pH of the mixture was adjusted to 6.2-6.8, and the reaction was carried out under constant temperature and stirring conditions.

[0009] The mixture is cooled down, and then a high-concentration phosphate aqueous solution is pumped into the mixture until the total mass fraction of phosphate in the mixture reaches 15% to 20% and the pH value of the mixture drops to 4.2 to 4.5. The mixture is then kept warm and matured and centrifuged to separate the light liquid phase, solid cake layer and heavy liquid phase. The light liquid phase is collected.

[0010] The light liquid phase is pumped into the ultrafiltration membrane module, deionized water is added to the light liquid phase for perfiltration, and the resulting membrane retentate and membrane dialysate are collected separately.

[0011] Adjust the pH of the membrane dialysis solution to 2.0–2.5, pump it into a chromatography column packed with cation exchange resin, and then pump an ammonia solution into the chromatography column for elution. Collect the resulting eluent.

[0012] The membrane retentate and eluent were mixed evenly to obtain a mixture, which was then spray-dried to obtain lactic acid bacteria organic selenium powder.

[0013] By employing the above technical solution, a high total organic selenium extraction rate and purity are achieved through the use of polyethylene glycol and isopropanol combined with lysozyme for gentle cell disruption, the use of high-concentration phosphate aqueous solution combined with isopropanol to promote phase separation of non-target macromolecular impurities, and the use of acidic conditions combined with cation exchange resin to capture free short-chain selenoamino acids in the membrane dialysis solution. The specific reaction and separation mechanism is as follows:

[0014] Lysozyme disrupts the peptidoglycan structure of the cell wall of selenium-rich lactic acid bacteria through hydrolysis. Polyethylene glycol and isopropanol alter the osmotic pressure and surface tension of the cell membrane of selenium-rich lactic acid bacteria, assisting in cell wall rupture and releasing contents. Sodium L-ascorbate, as an antioxidant, provides a reducing environment, inhibiting the oxidation of target organic selenium in its free state. The composite cell disruption process is carried out under constant temperature and stirring conditions to avoid the energy provided by the process conditions exceeding the dissociation energy of the selenium-carbon bond. This inhibits the chemical bond breakage of target organic selenium bound to the polypeptide chain of lactic acid bacteria host protein and prevents the target organic selenium molecule from degrading into inorganic selenium, thus maintaining the integrity of its molecular structure.

[0015] A high-concentration phosphate aqueous solution is used as a buffer system to adjust the pH of the mixture to the range of 4.2 to 4.5. This pH value is close to the isoelectric point of the main non-target protein impurities in the lactic acid bacteria host, which reduces the net surface charge of the non-target protein impurities and weakens the electrostatic repulsion between particles. Isopropanol, as an antisolvent, reduces the dielectric constant of the aqueous phase under the salting-out effect, thereby reducing the solubility of non-target macromolecular impurities and promoting their dehydration and precipitation. The non-target macromolecular impurities aggregate under the neutralization of surface charge and dehydration to form a relatively dense solid cake layer. This solid cake layer is easily separated from the liquid phase during centrifugation, thereby removing most of the non-target macromolecular impurities from the system, improving the efficiency of subsequent separation steps and reducing the risk of phase interface emulsification.

[0016] In the ultrafiltration permeation step, the macromolecularly bound target organic selenium is retained in the retentate by the ultrafiltration membrane. Meanwhile, free short-chain selenomethylselenocysteine ​​and short-chain selenomethionine molecules, whose molecular size is smaller than the ultrafiltration membrane's molecular weight cutoff, permeate the ultrafiltration membrane with the water flow and enter the dialysate. Polyethylene glycol also enters the dialysate. The amino and carboxyl groups in the short-chain selenoamino acid molecules are ionizable functional groups. The pH of the dialysate is adjusted to 2.0 to 2.5 using hydrochloric acid solution. Under these acidic conditions, the amino functional groups of the short-chain selenoamino acids accept protons to form positively charged -NH groups. 3+ The carboxyl functional group exists mainly in an undissociated state. The positively charged short-chain selenoamino acids enter the chromatography column with the feed solution and are adsorbed by combining with the negatively charged functional groups (such as sulfonic acid groups) on the surface of the cation exchange resin through electrostatic attraction.

[0017] Polyethylene glycol (PEG) molecules do not contain ionizable groups and are electrically neutral in acidic environments. They do not bind to cation exchange resins and pass through the chromatography column with the water flow. Subsequently, an ammonia solution is pumped into the column, raising the pH level. This causes the carboxyl functional groups of the short-chain selenoamino acids to deprotonate and become negatively charged, while the amino functional groups return to their electrical neutrality. This results in a net negative charge on the short-chain selenoamino acid molecules, which electrostatically repel the negatively charged resin functional groups, causing desorption from the resin. The desorbed components flow out with the ammonia solution, forming the eluent. This process recovers low-molecular-weight organic selenium components from the dialysate and separates them from impurities such as PEG, improving the overall product recovery rate.

[0018] Preferably, the mixture contains 12%–18% polyethylene glycol, 8%–12% isopropanol, 0.04–0.08 mol / L disodium ethylenediaminetetraacetate, 0.015–0.03 mol / L sodium L-ascorbate, and 0.1%–0.5% lysozyme of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

[0019] By adopting the above technical solution, disodium ethylenediaminetetraacetate can chelate the metal cations present in the organic selenium-rich lactic acid bacteria substrate suspension, reducing their inhibitory effect on lysozyme activity. Setting the ratio of polyethylene glycol, isopropanol and lysozyme not only helps to effectively break the cell wall of organic selenium-rich lactic acid bacteria, but also provides suitable initial conditions for the subsequent antisolvent phase separation step.

[0020] Preferably, the temperature of the reactor is controlled at a constant 35-40°C, and the mixture is continuously stirred at a speed of 40-80 rpm for 1.5-2.5 hours; the initial pH value of the mixture is adjusted using a sodium hydroxide solution or hydrochloric acid solution with a concentration of 0.5-1.5 mol / L.

[0021] By adopting the above technical solution, controlling the temperature of the reactor and the initial pH of the mixture provides a suitable enzymatic reaction environment for lysozyme, which helps to hydrolyze the peptidoglycan structure of the cell wall, thereby more fully releasing the products in the cells of selenium-rich lactic acid bacteria.

[0022] Preferably, the preparation steps of the organic selenium-rich lactic acid bacteria substrate suspension include: putting the organic selenium-rich lactic acid bacteria substrate into a homogenizing tank equipped with a stirrer, adding deionized water, setting the rotation speed to 200-400 rpm and dispersing for 20-40 minutes to obtain an organic selenium-rich lactic acid bacteria substrate suspension with a dry weight concentration of 8%-20%.

[0023] By adopting the above technical solution, the solid organic selenium-rich lactic acid bacteria substrate is prepared into a suspension before the enzymatic hydrolysis reaction, which avoids bacterial aggregation and increases the contact area between the bacterial cells and lysozyme and polyethylene glycol, thereby improving the liquid phase mass transfer efficiency.

[0024] Preferably, the preparation steps of the high-concentration phosphate aqueous solution include: adding potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a mass ratio of 15:1 to 20:1 to deionized water, stirring at a temperature of 80 to 90°C until completely dissolved, to obtain a high-concentration phosphate aqueous solution with a total salt mass concentration of 40% to 45%.

[0025] By adopting the above technical solution, a high-concentration phosphate aqueous solution was prepared by using potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a specific mass ratio, thus constructing an acidic phosphate buffer system with high buffering capacity. By adding the high-concentration phosphate aqueous solution, the overall pH value of the mixture can be stabilized in the isoelectric point region of non-target protein impurities without excessive dilution of the mixture.

[0026] Preferably, cooling the mixture and then pumping a high-concentration phosphate aqueous solution into the mixture specifically includes: cooling at a rate of 0.8–1.5 °C / min to 12–18 °C while increasing the rotation speed to 120–180 rpm; after pumping the high-concentration phosphate aqueous solution, reducing the reactor rotation speed to 10–20 rpm and maintaining the temperature for 20–40 min.

[0027] By adopting the above technical solution, the cooling process changes the solubility parameters of the system. Increasing the rotation speed helps to quickly and uniformly mix the high-concentration phosphate aqueous solution with the mixture. After the pumping is completed, reducing the rotation speed can avoid the destruction of the formed non-target macromolecular impurity aggregates by high-speed shear force. Slow stirring provides favorable hydrodynamic conditions for the further aggregation of non-target macromolecular impurity particles precipitated by dehydration and the formation of solid phase cake.

[0028] Preferably, adding deionized water to the light liquid phase for perfiltration specifically includes: the total volume of deionized water added is 3 to 5 times the initial volume of the light liquid phase.

[0029] By adopting the above technical solution, adding deionized water as the dialysate to the ultrafiltration membrane module can more effectively elute small molecules, such as free short-chain selenoamino acids and salt ions, into the membrane dialysate, while mitigating the concentration polarization phenomenon on the ultrafiltration membrane surface and ensuring separation efficiency.

[0030] Preferably, adjusting the pH of the membrane dialysis solution to 2.0–2.5 and pumping it into a chromatography column packed with cation exchange resin specifically includes: adjusting the pH of the membrane dialysis solution with a hydrochloric acid solution of 0.5–1.5 mol / L; and pumping the pH-adjusted membrane dialysis solution into the chromatography column at a flow rate of 1.0–2.0 BV / h.

[0031] By adopting the above technical solution, controlling the sample loading flow rate can ensure that the short-chain selenoamino acid molecules in the liquid are in full contact with the resin, thereby being effectively adsorbed and reducing the penetration loss caused by excessive flow rate.

[0032] Preferably, during the elution process of pumping ammonia solution into the chromatography column, the concentration of the ammonia solution is 1.0–2.0 mol / L.

[0033] By adopting the above technical solution and using a high-concentration ammonia solution as the eluent, the pH environment inside the cation exchange resin can be rapidly changed, causing the adsorbed short-chain selenoamino acids to desorb quickly. This allows for the recovery of the target product within a small elution volume, resulting in an eluent with a relatively high concentration.

[0034] Preferably, spray drying of the mixture specifically includes: an inlet air temperature of 150-170°C and an outlet air temperature of 75-85°C.

[0035] By adopting the above technical solution and setting the inlet and outlet air temperatures, it is possible to control the heating temperature of the material during the drying process while ensuring rapid evaporation of moisture to obtain dry powder. This helps to reduce the thermal degradation of organic selenium by lactic acid bacteria, thereby ensuring the quality of the final product.

[0036] This invention provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells. It offers the following advantages:

[0037] 1. This invention uses polyethylene glycol and isopropanol in combination with lysozyme to react a suspension of organic selenium-rich lactic acid bacteria substrate under constant temperature and stirring conditions. At the same time, sodium L-ascorbate is added to provide a reducing environment. This mild cell disruption method replaces physical cell disruption, avoiding the input of energy exceeding the dissociation energy of selenium-carbon bonds into the mixture. This prevents the selenium-carbon bonds of the target organic selenium molecules inside the mixture from breaking and oxidizing, maintaining the integrity of the target organic selenium molecule structure, and increasing the retention of organic selenium in the lactic acid bacteria organic selenium powder.

[0038] 2. This invention pumps a high-concentration phosphate aqueous solution into the mixture after the cell wall disruption reaction and cooling, adjusting the pH value of the mixture to the isoelectric point range of the non-target protein impurities of the lactic acid bacteria host. Combined with the antisolvent dehydration effect of isopropanol in the mixture, the isoelectric point precipitation mechanism combined with the antisolvent dehydration mechanism causes the non-target macromolecular impurities in the mixture to lose their surface charge and hydration layer. The non-target macromolecular impurities aggregate after centrifugation and form a solid cake layer, which isolates the non-target macromolecular impurities from the target organic selenium in the light liquid phase, preventing emulsification at the interface between the light and heavy liquid phases.

[0039] 3. This invention lowers the pH of the collected membrane dialysis solution and pumps it into a chromatography column packed with cation exchange resin. This causes the short-chain selenoamino acids entering the membrane dialysis solution during dialysis to be protonated and positively charged. The short-chain selenoamino acids are then adsorbed by the cation exchange resin through electrostatic interaction. Ammonia solution is used to desorb the short-chain selenoamino acids and collect them in the eluent. The combination of pH adjustment and cation exchange chromatography enables the capture and recovery of short-chain selenoamino acids, reduces the loss of small molecule free target organic selenium during ultrafiltration separation, and improves the total yield of lactic acid bacteria organic selenium powder. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the light liquid phase turbidity test results of the present invention;

[0041] Figure 2 This is a schematic diagram of the heavy liquid phase chemical oxygen demand test results of the present invention;

[0042] Figure 3This is a schematic diagram comparing the test results of the integrity of the target organic selenium and the total extraction rate of the present invention. Among them, (a) is a sub-plot comparing the test results of the total organic selenium extraction rate, and (b) is a sub-plot comparing the test results of the ratio of inorganic selenium to total selenium mass.

[0043] Figure 4 This is a schematic diagram showing the comparison of non-target impurity protein residual rate test according to the present invention;

[0044] Figure 5 This is a schematic diagram showing the comparison of non-target nucleic acid residual rate testing according to the present invention;

[0045] Figure 6 This is a schematic diagram showing the purity test comparison of the lactic acid bacteria organic selenium powder of the present invention. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Preparation Examples 1-6:

[0048] Preparation Example 1:

[0049] This preparation example provides a method for preparing a high-concentration phosphate aqueous solution, including the following steps:

[0050] Weigh out 37.5g of potassium dihydrogen phosphate and 2.5g of dipotassium hydrogen phosphate. Add the potassium dihydrogen phosphate and dipotassium hydrogen phosphate to 60.0g of deionized water. Stir the mixture at 200rpm using a mechanical stirrer under heating conditions at 80℃ until the potassium dihydrogen phosphate and dipotassium hydrogen phosphate are completely dissolved. Then transfer the solution to an insulated storage tank at 80℃ for later use. This yields a high-concentration phosphate aqueous solution with a total salt concentration of 40% and a potassium dihydrogen phosphate to dipotassium hydrogen phosphate mass ratio of 15:1.

[0051] Preparation Example 2:

[0052] This preparation example provides a method for preparing a high-concentration phosphate aqueous solution, including the following steps:

[0053] Weigh 39.79g of potassium dihydrogen phosphate and 2.21g of dipotassium hydrogen phosphate. Add the potassium dihydrogen phosphate and dipotassium hydrogen phosphate to 58.0g of deionized water. Stir the mixture at 200rpm using a mechanical stirrer under heating conditions of 85℃ until the potassium dihydrogen phosphate and dipotassium hydrogen phosphate are completely dissolved. Then transfer the solution to an insulated storage tank at 85℃ for later use. This yields a high-concentration phosphate aqueous solution with a total salt concentration of 42% and a potassium dihydrogen phosphate to dipotassium hydrogen phosphate mass ratio of 18:1.

[0054] Preparation Example 3:

[0055] This preparation example provides a method for preparing a high-concentration phosphate aqueous solution, including the following steps:

[0056] Weigh 42.86g of potassium dihydrogen phosphate and 2.14g of dipotassium hydrogen phosphate. Add the potassium dihydrogen phosphate and dipotassium hydrogen phosphate to 55.0g of deionized water. Stir the mixture at 200rpm using a mechanical stirrer under heating conditions at 90℃ until the potassium dihydrogen phosphate and dipotassium hydrogen phosphate are completely dissolved. Then transfer the solution to a 90℃ insulated storage tank for later use. This yields a high-concentration phosphate aqueous solution with a total salt concentration of 45% and a potassium dihydrogen phosphate to dipotassium hydrogen phosphate mass ratio of 20:1.

[0057] Preparation Example 4:

[0058] This preparation example provides a method for preparing a suspension of selenium-rich lactic acid bacteria substrate, including the following steps:

[0059] Weigh 320.0g of organic selenium-rich lactic acid bacteria substrate and put it into a homogenizing tank equipped with a stirrer. Add 680.0g of deionized water to the homogenizing tank and start the stirrer of the homogenizing tank. Stir and disperse at 200rpm for 20min to obtain a lactic acid bacteria substrate suspension with a dry weight concentration of 8%.

[0060] Preparation Example 5:

[0061] This preparation example provides a method for preparing a suspension of selenium-rich lactic acid bacteria substrate, including the following steps:

[0062] Weigh 480.0g of organic selenium-rich lactic acid bacteria substrate and put it into a homogenizing tank equipped with a stirrer. Add 520.0g of deionized water to the homogenizing tank and start the stirrer of the homogenizing tank. Stir and disperse at 300rpm for 30min to obtain a lactic acid bacteria substrate suspension with a dry weight concentration of 15%.

[0063] Preparation Example 6:

[0064] This preparation example provides a method for preparing a suspension of selenium-rich lactic acid bacteria substrate, including the following steps:

[0065] Weigh 600.0g of organic selenium-rich lactic acid bacteria substrate and put it into a homogenizing tank equipped with a stirrer. Add 400.0g of deionized water to the homogenizing tank and start the stirrer of the homogenizing tank. Stir and disperse at 400rpm for 40min to obtain a lactic acid bacteria substrate suspension with a dry weight concentration of 20%.

[0066] Examples 1-4:

[0067] Example 1:

[0068] This embodiment provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells, comprising the following steps:

[0069] The organic selenium-rich lactic acid bacteria substrate suspension provided in Preparation Example 4 was placed in a reaction vessel equipped with a temperature control jacket and a constant-speed mechanical stirrer.

[0070] Polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme were added sequentially to a selenium-rich lactic acid bacteria substrate suspension. The mass fractions of polyethylene glycol and isopropanol in the reaction vessel were 12%, 0.04 mol / L, 0.015 mol / L, and 0.1% of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

[0071] Add a 0.5 mol / L sodium hydroxide solution or hydrochloric acid solution to the material in the reactor to adjust the initial pH value of the material in the reactor to 6.2.

[0072] Control the jacket water temperature of the reactor to keep the temperature of the material inside the reactor constant at 35℃, turn on the mechanical stirring device of the reactor, and continuously stir the material inside the reactor at a speed of 40 rpm for 1.5 hours.

[0073] Cooling water is introduced into the jacket of the reactor to reduce the temperature of the material inside the reactor to 12℃ at a cooling rate of 0.8℃ / min, and the speed of the mechanical agitator of the reactor is increased to 120rpm.

[0074] A high-concentration phosphate aqueous solution provided in Preparation Example 1, which is in a heat-preserving state, is pumped into the material in the reactor using a constant flow pump until the total mass fraction of phosphate in the material in the reactor reaches 15%, at which point the pH value of the material in the reactor drops to 4.5.

[0075] Stop the constant flow pump operation, reduce the speed of the mechanical stirrer in the reactor to 10 rpm, and keep the material in the reactor at 10 rpm for 20 minutes to allow phase separation to occur and generate a solid phase cake layer at the phase interface.

[0076] After phase separation, the material in the reactor is pumped into a three-phase horizontal screw centrifuge for centrifugal separation, and a light liquid phase, a solid cake layer and a heavy liquid phase are obtained. The light liquid phase is collected.

[0077] The light liquid phase was pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da. Deionized water was continuously added to the light liquid phase for constant volume perfiltration. The total volume of deionized water added for perfiltration was controlled to be 3 times the initial volume of the light liquid phase. The membrane retentate and membrane dialysis solution generated during the ultrafiltration process were collected separately.

[0078] The pH of the membrane dialysis buffer was adjusted to 2.0 using 0.5 mol / L hydrochloric acid solution. The pH-adjusted membrane dialysis buffer was then pumped into a chromatography column packed with 001×7 type strong acid styrene-based cation exchange resin at a flow rate of 1 BV / h. The chromatography column was rinsed with pure water, and then a 1.0 mol / L ammonia solution was pumped into the chromatography column for elution. The eluent containing free selenoamino acids was collected.

[0079] The membrane retentate and eluent are mixed in a mixing tank to obtain a mixture. The mixture is then pumped into a centrifugal spray drying tower. The inlet air temperature of the centrifugal spray drying tower is controlled at 150°C and the outlet air temperature is controlled at 75°C. The mixture is then spray dried to obtain lactic acid bacteria organic selenium powder.

[0080] Example 2:

[0081] This embodiment provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells, comprising the following steps:

[0082] The organic selenium-rich lactic acid bacteria substrate suspension provided in Preparation Example 5 was placed in a reaction vessel equipped with a temperature control jacket and a constant-speed mechanical stirrer.

[0083] Polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme were added sequentially to a selenium-rich lactic acid bacteria substrate suspension. The mass fractions of polyethylene glycol and isopropanol in the reaction vessel were 15%, 0.06 mol / L, 0.02 mol / L, and 0.3% of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

[0084] Add a 1.0 mol / L sodium hydroxide solution or hydrochloric acid solution dropwise to the material in the reactor to adjust the initial pH value of the material in the reactor to 6.5;

[0085] Control the jacket water temperature of the reactor to keep the temperature of the material inside the reactor constant at 37°C, turn on the mechanical stirring device of the reactor, and continuously stir the material inside the reactor at a speed of 60 rpm for 2.0 hours.

[0086] Cooling water is introduced into the jacket of the reactor to reduce the temperature of the material inside the reactor to 15℃ at a cooling rate of 1.0℃ / min, and the speed of the mechanical agitator of the reactor is increased to 150 rpm.

[0087] A high-concentration phosphate aqueous solution provided in Preparation Example 2, which is in a heat-preserving state, is pumped into the material in the reactor using a constant flow pump until the total mass fraction of phosphate in the material in the reactor reaches 18%. At this point, the pH value of the material in the reactor drops to 4.3.

[0088] Stop the constant flow pump operation, reduce the speed of the mechanical agitator in the reactor to 15 rpm, and keep the material in the reactor at 15 rpm for 30 minutes to allow phase separation to occur and generate a solid phase cake layer at the phase interface.

[0089] After phase separation, the material in the reactor is pumped into a three-phase horizontal screw centrifuge for centrifugal separation, and a light liquid phase, a solid cake layer and a heavy liquid phase are obtained. The light liquid phase is collected.

[0090] The light liquid phase was pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da. Deionized water was continuously added to the light liquid phase for constant volume perfiltration. The total volume of deionized water added for perfiltration was controlled to be 4 times the initial volume of the light liquid phase. The membrane retentate and membrane dialysis solution generated during the ultrafiltration process were collected separately.

[0091] The pH of the membrane dialysis buffer was adjusted to 2.2 using 1.0 mol / L hydrochloric acid solution. The pH-adjusted membrane dialysis buffer was then pumped into a chromatography column packed with 001×7 type strong acid styrene-based cation exchange resin at a flow rate of 1.5 BV / h. The chromatography column was rinsed with pure water, and then a 1.5 mol / L ammonia solution was pumped into the chromatography column for elution. The eluent containing free selenoamino acids was collected.

[0092] The membrane retentate and eluent are mixed in a mixing tank to obtain a mixture. The mixture is then pumped into a centrifugal spray drying tower. The inlet air temperature of the centrifugal spray drying tower is controlled at 160°C and the outlet air temperature is controlled at 80°C. The mixture is then spray dried to obtain lactic acid bacteria organic selenium powder.

[0093] Example 3:

[0094] This embodiment provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells, comprising the following steps:

[0095] The organic selenium-rich lactic acid bacteria substrate suspension provided in Preparation Example 6 was placed in a reaction vessel equipped with a temperature control jacket and a constant-speed mechanical stirrer.

[0096] Polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme were added sequentially to a selenium-rich lactic acid bacteria substrate suspension. The mass fractions of polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, and sodium L-ascorbate in the reaction vessel were 18%, 12%, 0.08 mol / L, 0.03 mol / L, and 0.5% of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

[0097] Add a 1.5 mol / L sodium hydroxide solution or hydrochloric acid solution to the material in the reactor to adjust the initial pH value of the material in the reactor to 6.8.

[0098] Control the jacket water temperature of the reactor to keep the temperature of the material inside the reactor constant at 40℃, turn on the mechanical stirring device of the reactor, and continuously stir the material inside the reactor at a speed of 80 rpm for 2.5 hours.

[0099] Cooling water is introduced into the jacket of the reactor to reduce the temperature of the material inside the reactor to 18°C ​​at a cooling rate of 1.5°C / min, and the speed of the mechanical agitator of the reactor is increased to 180 rpm.

[0100] A high-concentration phosphate aqueous solution provided in Preparation Example 3, which is in a heat-preserving state, is pumped into the material in the reactor using a constant flow pump until the total mass fraction of phosphate in the material in the reactor reaches 20%. At this point, the pH value of the material in the reactor drops to 4.2.

[0101] Stop the constant flow pump operation, reduce the speed of the mechanical agitator in the reactor to 20 rpm, and keep the material in the reactor at 20 rpm for 40 minutes to allow phase separation to occur and generate a solid phase cake layer at the phase interface.

[0102] After phase separation, the material in the reactor is pumped into a three-phase horizontal screw centrifuge for centrifugal separation, and a light liquid phase, a solid cake layer and a heavy liquid phase are obtained. The light liquid phase is collected.

[0103] The light liquid phase was pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da. Deionized water was continuously added to the light liquid phase for constant volume perfiltration. The total volume of deionized water added for perfiltration was controlled to be 5 times the initial volume of the light liquid phase. The membrane retentate and membrane dialysate generated during the ultrafiltration process were collected separately.

[0104] The pH of the membrane dialysis buffer was adjusted to 2.5 using 1.5 mol / L hydrochloric acid solution. The pH-adjusted membrane dialysis buffer was then pumped into a chromatography column packed with 001×7 type strong acid styrene-based cation exchange resin at a flow rate of 2 BV / h. The chromatography column was rinsed with pure water, and then a 2.0 mol / L ammonia solution was pumped into the chromatography column for elution. The eluent containing free selenoamino acids was collected.

[0105] The membrane retentate and eluent are mixed in a mixing tank to obtain a mixture. The mixture is then pumped into a centrifugal spray drying tower. The inlet air temperature of the centrifugal spray drying tower is controlled at 170℃ and the outlet air temperature is controlled at 85℃. The mixture is then spray dried to obtain lactic acid bacteria organic selenium powder.

[0106] Example 4:

[0107] This embodiment provides a highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells, comprising the following steps:

[0108] The organic selenium-rich lactic acid bacteria substrate suspension provided in Preparation Example 5 was placed in a reaction vessel equipped with a temperature control jacket and a constant-speed mechanical stirrer.

[0109] Polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme were added sequentially to a selenium-rich lactic acid bacteria substrate suspension to achieve the following concentrations: polyethylene glycol 18% by mass, isopropanol 8% by mass, disodium ethylenediaminetetraacetate 0.06 mol / L, sodium L-ascorbate 0.03 mol / L, and lysozyme 0.1% of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

[0110] Add a 1 mol / L sodium hydroxide solution or hydrochloric acid solution to the material in the reactor to adjust the initial pH value of the material in the reactor to 6.5.

[0111] Control the jacket water temperature of the reactor to keep the temperature of the material inside the reactor constant at 40℃, turn on the mechanical stirring device of the reactor, and continuously stir the material inside the reactor at a speed of 60 rpm for 1.5 hours.

[0112] Cooling water is introduced into the jacket of the reactor to reduce the temperature of the material inside the reactor to 12℃ at a cooling rate of 1.5℃ / min, and the speed of the mechanical agitator of the reactor is increased to 150 rpm.

[0113] A high-concentration phosphate aqueous solution provided in Preparation Example 2, which is in a heat-preserving state, is pumped into the material in the reactor using a constant flow pump until the total mass fraction of phosphate in the material in the reactor reaches 20%. At this point, the pH value of the material in the reactor drops to 4.3.

[0114] Stop the constant flow pump operation, reduce the speed of the mechanical agitator in the reactor to 10 rpm, and keep the material in the reactor at 10 rpm for 30 minutes to allow phase separation to occur and generate a solid phase cake layer at the phase interface.

[0115] After phase separation, the material in the reactor is pumped into a three-phase horizontal screw centrifuge for centrifugal separation, and a light liquid phase, a solid cake layer and a heavy liquid phase are obtained. The light liquid phase is collected.

[0116] The light liquid phase was pumped into an ultrafiltration membrane module with a molecular weight cutoff of 3000 Da. Deionized water was continuously added to the light liquid phase for constant volume perfiltration. The total volume of deionized water added for perfiltration was controlled to be 4 times the initial volume of the light liquid phase. The membrane retentate and membrane dialysis solution generated during the ultrafiltration process were collected separately.

[0117] The pH of the membrane dialysis solution was adjusted to 2.0 using hydrochloric acid solution. The pH-adjusted membrane dialysis solution was then pumped into a chromatography column packed with 001×7 type strong acid styrene-based cation exchange resin at a flow rate of 1 BV / h. The chromatography column was rinsed with pure water, and then a 2.0 mol / L ammonia solution was pumped into the chromatography column for elution. The eluent containing free selenoamino acids was collected.

[0118] The membrane retentate and eluent are mixed in a mixing tank to obtain a mixture. The mixture is then pumped into a centrifugal spray drying tower. The inlet air temperature of the centrifugal spray drying tower is controlled at 160°C and the outlet air temperature is controlled at 80°C. The mixture is then spray dried to obtain lactic acid bacteria organic selenium powder.

[0119] Comparative Examples 1-5:

[0120] Comparative Example 1:

[0121] Compared with Example 2, the difference is that the addition of polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme to the selenium-rich lactic acid bacteria substrate suspension, along with the subsequent incubation and stirring reaction steps, is replaced by directly using a high-pressure homogenizer to perform high-pressure mechanical homogenization and cell disruption of the selenium-rich lactic acid bacteria substrate suspension without adding any additives. All other steps are the same.

[0122] Comparative Example 2:

[0123] Compared with Example 2, the difference is that isopropanol is not added to the selenium-rich lactic acid bacteria substrate suspension, and the high-concentration phosphate aqueous solution provided in Preparation Example 2, which is in a state of heat preservation, is pumped into the material in the reactor by a constant flow pump, instead of pumping in a neutral phosphate buffer solution of the same concentration, so that the pH value of the material in the reactor is always maintained at 6.5 without decreasing. All other aspects are the same.

[0124] Comparative Example 3:

[0125] The difference from Example 2 is that isopropanol is not added to the selenium-rich lactic acid bacteria substrate suspension, but all other aspects are the same.

[0126] Comparative Example 4:

[0127] Compared with Example 2, the difference is that the high-concentration phosphate aqueous solution provided in Preparation Example 2, which is in a heat-preserving state, is pumped into the material in the reactor by a constant flow pump, instead of pumping in a neutral phosphate buffer solution of the same concentration, so that the pH value of the material in the reactor is always maintained at 6.5 without decreasing. All other aspects are the same.

[0128] Comparative Example 5:

[0129] Compared with Example 2, the difference is that the adsorption and ammonia elution steps of the chromatography column filled with 001×7 type strong acid styrene-based cation exchange resin for the membrane dialysis solution are omitted. The membrane dialysis solution is directly discharged as waste liquid, and only the membrane retentate is collected for spray drying. Everything else is the same.

[0130] Test Examples 1-4:

[0131] Test Example 1: Macroscopic hydrodynamic and thermodynamic solidification parameters of phase interfaces

[0132] Light and heavy liquid phase samples were collected from the separation by a three-phase horizontal screw centrifuge. Comparative Example 1 used a high-pressure homogenization and cell disruption process without a three-phase separation process. This test was not performed on Comparative Example 1. The phase separation front-end steps of Comparative Example 5 were exactly the same as those of Example 2. The test results were consistent with those of Example 2 and will not be repeated in this test example.

[0133] The turbidity value of the light liquid phase sample was determined using a portable turbidimeter. 50 mL of the light liquid phase sample was placed into the measuring cell of the portable turbidimeter, and the value displayed by the portable turbidimeter was recorded. The unit is NTU. Each group of light liquid phase samples was measured in parallel three times, and the average value was calculated as the final light liquid phase turbidity test result.

[0134] The chemical oxygen demand (COD) of heavy liquid phase samples was determined using the potassium dichromate method. 10 mL of the heavy liquid phase sample was taken, and potassium dichromate standard solution and sulfuric acid solution containing silver sulfate were added. The sample was heated and digested at 150℃ for 2 h. After cooling, titration was performed using ferrous ammonium sulfate standard titration solution. The volume consumed was recorded, and the COD value of the heavy liquid phase sample was calculated in mg / L. Each group of heavy liquid phase samples was measured in parallel three times, and the average value was used as the final COD test result.

[0135] Table 1. Test Results of Phase Interface Fluid Dynamics and Thermodynamics of Curing

[0136] Group Light liquid phase turbidity (NTU) Heavy liquid phase chemical oxygen demand (mg / L) Example 1 11.83 4362.4 Example 2 8.27 3815.9 Example 3 10.42 4128.5 Example 4 9.65 3971.2 Comparative Example 2 268.41 19542.8 Comparative Example 3 154.76 13280.3 Comparative Example 4 129.53 10457.6

[0137] Conclusions and Analysis:

[0138] According to Table 1 and referring to Figure 1 and Figure 2The data showed that the light liquid phase turbidity test results and heavy liquid phase chemical oxygen demand test results of Examples 1 to 4 remained at a low level. The test results of Comparative Examples 2 to 4 were higher than those of Examples 1 to 4. In Comparative Example 2, no isopropanol was added to the reaction system and no high-concentration phosphate aqueous solution was pumped in for pH adjustment. The light liquid phase turbidity and heavy liquid phase chemical oxygen demand increased, indicating that a large number of non-target macromolecular impurities could not be solidified and precipitated at the interface, resulting in the free dispersion of non-target macromolecular impurities in the light and heavy liquid phases and triggering interfacial emulsification.

[0139] Comparative Example 3 did not include isopropanol. The data changes showed that isopropanol, as an antisolvent, could reduce the dielectric constant of the aqueous phase and force non-target macromolecular impurities to dehydrate and crystallize at the interface between the light and heavy phases. The absence of isopropanol directly led to the failure of solid cake formation.

[0140] Comparative Example 4 used neutral phosphate buffer to maintain the pH of the material at 6.5. The data showed that under neutral conditions, the surface of non-target macromolecular impurities retained a net charge and an intact hydration layer, and the electrostatic repulsion between particles prevented the aggregation of non-target macromolecular impurity particles.

[0141] Examples 1 to 4 simultaneously introduce isopropanol and a high-concentration phosphate aqueous solution. Isopropanol's interfacial enrichment under the salting-out effect generates a strong antisolvent precipitation, while a phosphate buffer system lowers the macroscopic pH of the material system to 4.2-4.5, targeting the isoelectric point of the lactic acid bacteria host protein impurities. The isopropanol antisolvent precipitation mechanism and the pH isoelectric point-promoted precipitation mechanism are thermodynamically coupled, eliminating the electrostatic repulsion on the surface of non-target macromolecular impurities and disrupting the hydration layer. This causes irreversible rigid densification of the non-target macromolecular impurities at the interface between the light and heavy liquid phases, forming a mechanically stable solid cake layer. Most of the non-target macromolecular impurities are firmly bound and trapped within the solid cake layer, thus purifying both the light and heavy liquid phases. This solves the problems of interfacial flocculation and emulsification, and centrifuge runoff during industrial scale-up of the polymer two-phase extraction process.

[0142] Test Example 2: Verification of Specific Charge Capture of Free Small Molecule Organoselenium

[0143] Collect the membrane dialysis solution generated by the ultrafiltration membrane module in Examples 1 to 4. At the same time, an additional portion of the membrane dialysis solution was taken from the constant volume dialysis step of the preparation process in Example 2 as a control sample. No hydrochloric acid solution was added to the control sample, and the original pH value of the control sample was retained. The pH value of the control sample was 4.3.

[0144] The concentration of polyethylene glycol (PEG) in the membrane dialysate, control sample, and column effluent was determined using a high-performance liquid chromatography (HPLC) system equipped with an evaporative light scattering detector (EVS). Two mL of the test liquid was filtered through a 0.22 μm PTFE microporous membrane, and the filtered liquid was injected into the HPLC system. The peak areas were recorded and substituted into a pre-plotted PEG standard curve to calculate the PEG concentration (mg / mL). The ratio of the PEG mass in the column effluent to the total PEG mass in the column feed liquid was calculated to obtain the PEG breakthrough rate.

[0145] The concentration of free small-molecule organic selenium in the membrane dialysis solution, control sample, effluent from each chromatography column, and eluent was determined using liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). Two mL of the test liquid was filtered and injected into the instrument to determine the total concentration of selenomethylselenocysteine ​​and selenomethionine in the liquid, expressed in μg / mL. The ratio of the total mass of free small-molecule organic selenium in the eluent to the total mass of free small-molecule organic selenium in the column feed liquid was calculated to obtain the elution recovery rate of free small-molecule organic selenium.

[0146] Table 2. Distribution and concentration variation data of polyethylene glycol and free small molecule organose selenium in the chromatography column.

[0147] Group Concentration of polyethylene glycol in the column loading solution (mg / mL) Effluent polyethylene glycol concentration (mg / mL) Polyethylene glycol penetration rate (%) Concentration of free organic selenium in the column loading solution (μg / mL) Concentration of free organic selenium in the effluent (μg / mL) Concentration of free organic selenium in the eluent (μg / mL) Free organic selenium elution recovery rate (%) Example 1 14.28 14.15 99.09 26.54 0.81 185.32 96.95 Example 2 17.65 17.43 98.75 31.87 1.15 218.66 96.38 Example 3 20.31 20.12 99.06 36.42 1.34 243.51 96.32 Example 4 19.86 19.68 99.09 34.25 1.09 231.87 96.81 control sample 17.65 17.41 98.64 31.87 25.13 48.24 21.19

[0148] Conclusions and Analysis:

[0149] According to the data in Table 2, the membrane dialysis solutions of Examples 1 to 4 showed high polyethylene glycol penetration and high free organic selenium elution recovery rates in chromatography columns packed with 001×7 type strong acid styrene-based cation exchange resin.

[0150] Polyethylene glycol (PEG) is a nonionic homopolymer. PEG's molecular chain does not contain dissociable groups, and it is electrically neutral under extremely acidic conditions (pH 2.0 to 2.5). The sulfonic acid groups on the surface of the 001×7 type strongly acidic styrene-based cation exchange resin cannot adsorb and capture PEG through electrostatic interactions, causing PEG to directly penetrate the chromatography column with the water flow. Free selenomethylselenocysteine ​​and selenomethionine, among other small-molecule organic selenium compounds, are amphoteric compounds, containing both amino and carboxyl groups in their molecular structure.

[0151] In Examples 1 to 4, the pH of the membrane dialysis solution was adjusted to 2.0-2.5 using hydrochloric acid solution. At this point, the hydrogen ion concentration in the system was higher than the isoelectric point of the free small molecule organoselenium. The amino group of the free small molecule organoselenium underwent complete protonation and was converted into a positively charged ammonium ion, while the carboxyl group remained in an undissociated state. The positively charged free small molecule organoselenium was adsorbed into the inside of the chromatography column by electrostatic attraction and combined with the negatively charged sulfonic acid groups on the surface of the 001×7 type strong acid styrene-based cation exchange resin.

[0152] The subsequently pumped ammonia solution is alkaline, raising the pH value within the chromatography column. This causes the carboxyl groups of the free small-molecule organoselenium to dissociate and become negatively charged, while the amino groups lose protons and regain electroneutrality. The free small-molecule organoselenium as a whole becomes negatively charged, generating electrostatic repulsion with the sulfonic acid groups on the surface of the 001×7 type strong acid styrene-based cation exchange resin. Consequently, it detaches and flows out with the ammonia solution. In the control sample, no hydrochloric acid solution was added to adjust the pH value, which was maintained at 4.3, close to the isoelectric point region of the free small-molecule organoselenium. The net charge on the surface of the free small-molecule organoselenium tends to be 0, making it unable to form an effective electrostatic bond with the 001×7 type strong acid styrene-based cation exchange resin. This resulted in a high concentration of free organoselenium in the effluent of the control sample, leading to a decrease in the elution recovery rate. The test results demonstrate that the process of lowering the pH value and coupling the cation exchange resin can effectively separate the free small-molecule organoselenium from polyethylene glycol.

[0153] Test Example 3: Evaluation of Target Organic Selenium Integrity and Total Extraction Rate

[0154] The final product, organic selenium lactic acid bacteria powder, prepared in Examples 1 to 4, Comparative Example 1, and Comparative Example 5 was collected, and the corresponding organic selenium-rich lactic acid bacteria substrate suspensions were extracted during the preparation process of the above groups as initial reference samples.

[0155] The total organic selenium content and inorganic selenium content in lactic acid bacteria organic selenium powder and organic selenium-rich lactic acid bacteria substrate suspension were determined by high performance liquid chromatography-inductively coupled plasma mass spectrometry. 0.5 g of lactic acid bacteria organic selenium powder sample and an equal dry weight of organic selenium-rich lactic acid bacteria substrate suspension sample were weighed and added to a buffer solution containing protease for enzymatic extraction at 37 °C for 24 h to completely release the encapsulated selenium element.

[0156] After centrifugation and filtration through a microporous membrane, the extract was injected into a high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) instrument for analysis. The chromatographic peaks of organic selenium forms such as selenocysteine, selenomethylselenocysteine, and selenomethionine, as well as inorganic selenium forms such as selenite, were recorded in the chromatograms. The mass of each selenium form was calculated by substituting the peaks into the standard curve.

[0157] The total organic selenium extraction rate (%) is calculated by summing the masses of all organic selenium forms in the lactic acid bacteria organic selenium powder and dividing by the total mass of all organic selenium forms in the corresponding organic selenium-rich lactic acid bacteria substrate suspension. The mass of inorganic selenium forms such as selenite in the lactic acid bacteria organic selenium powder is also calculated by dividing by the total mass of all selenium forms in the lactic acid bacteria organic selenium powder.

[0158] Table 3. Test data on total organic selenium extraction rate and inorganic selenium percentage.

[0159] Group Total organic selenium extraction rate (%) Inorganic selenium as a percentage of total selenium (%) Example 1 88.62 0.84 Example 2 92.17 0.69 Example 3 89.45 0.77 Example 4 90.83 0.81 Comparative Example 1 68.31 19.56 Comparative Example 5 62.48 0.74

[0160] Conclusions and Analysis:

[0161] According to Table 3 and referring to Figure 3 The data showed that the total organic selenium extraction rate of Examples 1 to 4 was at a high level, while the ratio of inorganic selenium to total selenium mass remained at a low value. Comparative Example 1 used a high-pressure mechanical homogenizer to break the cell walls of lactic acid bacteria. The test results showed that the total organic selenium extraction rate of Comparative Example 1 decreased significantly, and the ratio of inorganic selenium to total selenium mass increased.

[0162] During the processing of Comparative Example 1, the high-pressure homogenizer generated strong fluid shear force and instantaneous local high temperature. The activation energy of the input material system exceeded the dissociation energy of the fragile selenium carbon bond in the target product of lactic acid bacteria. This caused irreversible chemical bond breakage and molecular shedding of the organic selenium originally bound to the polypeptide chain of lactic acid bacteria protein, which was converted into inorganic selenium forms such as selenite in large quantities.

[0163] Comparative Example 1 did not add sodium L-ascorbate to the system, thus lacking the protection of the exogenous reduction potential, which aggravated the oxidative degradation of selenium in the free state. Examples 1 to 4 used a chemical and biological enzymatic method of polyethylene glycol, isopropanol and lysozyme to gently break the cell wall, which achieved cell wall perforation and release of contents while maintaining the structural integrity of the target organic selenium molecule to the greatest extent.

[0164] Comparative Example 5 eliminated the step of pumping the membrane dialysis solution into a chromatography column packed with 001×7 type strong acid styrene-based cation exchange resin for adsorption and elution, and directly discharged the membrane dialysis solution. The test results showed that the total organic selenium extraction rate of Comparative Example 5 decreased. In the separation stage of the ultrafiltration membrane module with a molecular weight cutoff of 3000 Da, the large molecular bound organic selenium was retained by the membrane. However, the molecular physical size of the free short-chain selenoamino acids in the system was much smaller than the pore size of the ultrafiltration membrane, and they would penetrate the ultrafiltration membrane with the deionized water used for dialysis and enter the membrane dialysis solution.

[0165] In Comparative Example 5, the membrane dialysis solution was directly discarded, resulting in the complete loss of free short-chain selenoamino acids and causing a loss in the absolute mass of the material in the extraction system. Examples 1 to 4 introduced a cation exchange resin chromatography step at the end of the ultrafiltration process. By adjusting the material to extremely acidic conditions, the short-chain selenoamino acids were protonated and became positively charged through a charge reversal mechanism. The short-chain selenoamino acids that penetrated the ultrafiltration membrane were specifically captured by electrostatic attraction. They were then recovered without damage using ammonia elution solution and combined with the membrane retentate for drying. This blocked the loss path of the target product and improved the total organic selenium extraction rate.

[0166] Test Example 4: Evaluation of Non-Target Impurity Removal Rate and Final Product Purity

[0167] The final product, lactic acid bacteria organic selenium powder, prepared in Examples 1 to 4, Comparative Examples 2, 3 and 4 was collected.

[0168] The residual amount of non-target impurity protein in lactic acid bacteria organic selenium powder was determined by the diquinoline formic acid method. A specified mass of lactic acid bacteria organic selenium powder was weighed and dissolved in deionized water to prepare the test sample solution. Diquinoline formic acid working solution was added to the test sample solution, and the mixture was incubated at 37℃ for 30 min. The absorbance was measured at 562 nm using an ELISA reader. The absorbance was substituted into a pre-plotted bovine serum albumin standard curve to calculate the mass of non-target impurity protein. The mass of non-target impurity protein was divided by the total dry mass of the lactic acid bacteria organic selenium powder to calculate the residual rate of non-target impurity protein, in units of 1.

[0169] The residual amount of non-target nucleic acids in lactic acid bacteria organic selenium powder was determined by ultraviolet-visible spectrophotometry. The sample solution to be tested was injected into a quartz cuvette, and the absorbance was measured at a wavelength of 260 nm using an ultraviolet-visible spectrophotometer. The mass of non-target nucleic acids was calculated based on the absorbance. The mass of non-target nucleic acids was divided by the total dry mass of lactic acid bacteria organic selenium powder to calculate the residual rate of non-target nucleic acids, in units of 0.5%.

[0170] Based on the mass data of various organic selenium forms measured in Test Example 3, the purity of the lactic acid bacteria organic selenium powder is calculated by dividing the total mass of all organic selenium forms contained in the lactic acid bacteria organic selenium powder by the total dry mass of the lactic acid bacteria organic selenium powder, with the unit being mg / g.

[0171] Table 4. Results of Non-Target Impurity Removal Rate and Final Product Purity Test

[0172] Group Non-target impurity protein residue rate (%) Non-target nucleic acid residual rate (%) Purity (mg / g) of lactic acid bacteria organic selenium powder Example 1 3.12 1.25 19.45 Example 2 2.34 0.98 21.67 Example 3 2.87 1.14 20.38 Example 4 2.51 1.06 21.05 Comparative Example 2 18.45 6.72 6.84 Comparative Example 3 12.31 4.58 9.73 Comparative Example 4 14.62 5.21 8.45

[0173] Conclusions and Analysis:

[0174] According to Table 4 and referring to Figure 4 , Figure 5 and Figure 6 According to the data, the residual rates of non-target impurity proteins and non-target nucleic acids in the lactic acid bacteria organic selenium powders prepared in Examples 1 to 4 are in the low range, while the purity of the lactic acid bacteria organic selenium powders is in the high range.

[0175] The residual rates of non-target impurity proteins and non-target nucleic acids in Comparative Examples 2 to 4 increased significantly, and the purity of the lactic acid bacteria organic selenium powder decreased. In Comparative Example 2, no isopropanol was added to the reaction system and neutral phosphate buffer was pumped in by a constant flow pump. As a result, non-target macromolecular impurities in the system failed to precipitate. A large number of non-target macromolecular impurities remained in the light liquid phase and entered the spray drying stage, resulting in the final product being mixed with non-target macromolecular impurities.

[0176] In Comparative Example 3, a high concentration of acidic phosphate was added to the reaction system, but isopropanol was not added. The dielectric constant of the aqueous phase did not decrease, the hydration layer on the surface of the non-target macromolecular impurities was not removed, and the non-target macromolecular impurities could not crystallize and solidify at the interface between the light and heavy liquid phases.

[0177] In Comparative Example 4, isopropanol was added to the reaction system, but neutral phosphate buffer was pumped in. The pH value of the material in the reactor was maintained at 6.5. The pH value of the material system deviated from the isoelectric point region of the non-target macromolecular impurities. The surface of the non-target macromolecular impurities had a net charge, and the electrostatic repulsion between the particles hindered the aggregation of the non-target macromolecular impurities.

[0178] In Examples 1 to 4, isopropanol and a high-concentration phosphate aqueous solution were introduced simultaneously. The antisolvent dehydration precipitation mechanism caused by isopropanol and the isoelectric point precipitation mechanism caused by the acidic phosphate aqueous solution were superimposed and combined. The non-target macromolecular impurities simultaneously lost their surface charge and bound water. Under the centrifugal force field of the three-phase horizontal screw centrifuge, a dense solid cake layer was formed at the phase interface. The dense solid cake layer was discharged with the centrifuge slag discharge port. From the spatial distribution, the non-target macromolecular impurities were separated from the target organic selenium enriched in the light liquid phase, blocking the transfer path of the non-target macromolecular impurities to the final product and improving the purity of the lactic acid bacteria organic selenium powder.

Claims

1. A highly efficient extraction and purification method for organic selenium from lactic acid bacteria cells, characterized in that, Includes the following steps: A suspension of selenium-rich lactic acid bacteria substrate was placed in a reaction vessel, and polyethylene glycol, isopropanol, disodium ethylenediaminetetraacetate, sodium L-ascorbate, and lysozyme were added sequentially to obtain a mixture. The initial pH of the mixture was adjusted to 6.2–6.8, and the reaction was carried out under constant temperature and stirring conditions. The mixture is cooled, and then a high-concentration phosphate aqueous solution is pumped into the mixture until the total mass fraction of phosphate in the mixture reaches 15% to 20% and the pH value of the mixture drops to 4.2 to 4.

5. The mixture is then kept warm and matured, and centrifuged to separate a light liquid phase, a solid cake layer and a heavy liquid phase. The light liquid phase is collected. The light liquid phase is pumped into the ultrafiltration membrane module, deionized water is added to the light liquid phase for perfiltration, and the resulting membrane retentate and membrane dialysate are collected separately. The pH of the membrane dialysis solution is adjusted to 2.0-2.5 and pumped into a chromatography column packed with cation exchange resin. Then, an ammonia solution is pumped into the chromatography column for elution, and the resulting eluent is collected. The membrane retentate and the eluent are mixed evenly to obtain a mixture, which is then spray-dried to obtain lactic acid bacteria organic selenium powder.

2. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, The mixture contains polyethylene glycol at a mass fraction of 12%–18%, isopropanol at a mass fraction of 8%–12%, disodium ethylenediaminetetraacetate at a concentration of 0.04–0.08 mol / L, sodium L-ascorbate at a concentration of 0.015–0.03 mol / L, and lysozyme at a concentration of 0.1%–0.5% of the dry weight of the lactic acid bacteria in the selenium-rich lactic acid bacteria substrate suspension.

3. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, The temperature of the reactor is controlled to be constant at 35-40℃, and the mixture is continuously stirred at a speed of 40-80 rpm for 1.5-2.5 hours. The initial pH value of the mixture is adjusted using a sodium hydroxide solution or hydrochloric acid solution with a concentration of 0.5–1.5 mol / L.

4. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, The preparation steps of the selenium-rich lactic acid bacteria substrate suspension include: The organic selenium-rich lactic acid bacteria substrate was placed into a homogenizing tank equipped with a stirrer, deionized water was added, and the mixture was stirred and dispersed at a speed of 200-400 rpm for 20-40 minutes to obtain a suspension of the organic selenium-rich lactic acid bacteria substrate with a dry weight concentration of 8%-20%.

5. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, The preparation steps of the high-concentration phosphate aqueous solution include: Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were added to deionized water at a mass ratio of 15:1 to 20:1, and stirred at a temperature of 80 to 90°C until completely dissolved to obtain a high-concentration phosphate aqueous solution with a total salt mass concentration of 40% to 45%.

6. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, Cooling the mixture and then pumping a high-concentration phosphate aqueous solution into it specifically includes: The cooling rate is 0.8–1.5℃ / min, cooling down to 12–18℃, while simultaneously increasing the mechanical stirring speed of the reactor to 120–180 rpm; After the high-concentration phosphate aqueous solution is pumped in, the rotation speed of the reactor is reduced to 10-20 rpm, and the reactor is kept warm for 20-40 minutes.

7. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, The specific steps of adding deionized water to the light liquid phase for perfiltration include: The total volume of deionized water added is 3 to 5 times the initial volume of the light liquid phase.

8. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, Adjusting the pH of the membrane dialysis solution to 2.0–2.5 and pumping it into a chromatography column packed with cation exchange resin specifically includes: The pH value of the membrane dialysis solution was adjusted using a hydrochloric acid solution with a concentration of 0.5–1.5 mol / L. The membrane dialysis solution, after pH adjustment, is pumped into the chromatography column at a flow rate of 1.0–2.0 BV / h.

9. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, During the elution process of pumping the ammonia solution into the chromatography column, the concentration of the ammonia solution is 1.0–2.0 mol / L.

10. The efficient extraction and purification method for organic selenium from lactic acid bacteria cells according to claim 1, characterized in that, Spray drying of the mixture specifically includes: The mixture is pumped into a centrifugal spray drying tower. The inlet temperature of the centrifugal spray drying tower is 150-170℃, and the outlet temperature is 75-85℃.

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