Application of SDDAB in improving the extraction efficiency of PEG / sulfate aqueous two-phase system

Abstract

The application discloses application of SDDAB in improving the extraction performance of a PEG / sulfate aqueous two-phase system (ATPS), and specifically uses sulfobetaine surfactant 3-sulfopropyl dodecyl dimethyl betaine (SDDAB) to improve the extraction performance of a polyethylene glycol (PEG) / sulfate aqueous two-phase system for biological small molecules, and uses PEG 400 / (NH4)2SO4, PEG 400 / Na2SO4, PEG 4000 / (NH4)2SO4 and PEG 4000 / Na2SO4 aqueous two-phase systems with SDDAB as an additive to extract L-tryptophan (L-Trp) and vanillin (Van); effectively solve the problem of low extraction efficiency of the ATPS in extracting L-Trp and Van; and simultaneously realize the back extraction of L-Trp and Van to the lower phase of the ATPS by secondary back extraction, and has a high total recovery rate.

Description

Technical Field

[0001] This invention belongs to the field of separation technology, specifically relating to the application of SDDAB in improving the extraction efficiency of PEG / sulfate aqueous two-phase systems. Background Technology

[0002] Aqueous two-phase systems (ATPS) are a novel green separation system with broad application prospects in extraction and separation. Some natural substances extracted from biomass can be used as secondary fuels, binders, dispersants, emulsifiers, chelating agents, and colorants; some have high nutritional value, or can prevent and treat diseases and promote health; and some can be further converted into aromatic chemicals, high-octane fuels, and carbon fibers. For example, phycocyanin extracted from spirulina has been widely used in food processing, cosmetics, and nutritional products. However, traditional separation techniques (filtration or centrifugation, precipitation, salting out, electrophoresis, ultrafiltration and freeze-drying, ion exchange, affinity chromatography, etc.) suffer from drawbacks in extracting these substances, including complex processes, high costs, low efficiency, low yields, and difficulty in scale-up.

[0003] Extraction using aqueous two-phase systems formed by two polymers or polymer and salt revealed high system viscosity, difficulty in phase separation within a short time, and low selectivity and sensitivity. Aqueous two-phase systems formed using positive and negative ionic surfactants exhibited very narrow two-phase regions, unsuitable for large-scale separation and analysis. Introducing surfactants as additives into PEG / salt or ionic liquid / surfactant aqueous two-phase systems reduces viscosity and interfacial tension, increases phase separation rate, and accelerates mass transfer during extraction. For example, adding 0.05 wt% sodium bis(2-ethylhexyl)sulfosuccinate (AOT) as an additive to ATPS formed by PEG 4000 or PEG 6000 and K2HPO4 or H3PO4 can affect the two-phase region of the phase diagram (Chemical Engineering Communications, https: / / doi.org / 10.1080 / 00986445,2020,1805439); 7 wt% sodium dodecyl sulfate (SDS) as an additive can reduce the temperature sensitivity of [Emim]Cl / TX-100ATPS and increase the phase separation rate of the system (Journal of Molecular Biology). Liquids, 2018, 256, 372–379; The presence of hexadecyltrimethylammonium bromide (CTAB) promotes the dye to enter the lower phase of PEG1500-6000 or polyethylene oxide-block polypropylene oxide-block polyethylene oxide copolymer (L35) / sodium citrate ATPS, thereby achieving selective separation of the dye (Fluid Phase Equilibria, 2018, 478, 14-22).

[0004] Tryptophan (L-Trp) is an important precursor for auxin synthesis in plants and is also one of the essential amino acids for humans. Vanillin has a vanilla bean aroma and a rich milky fragrance, and plays a role in enhancing and fixing aromas, and is widely used in cosmetics, tobacco, confectionery, and baked goods industries. Therefore, utilizing an aqueous two-phase system to improve the extraction performance of L-Trp and Van has practical application value. When L-Trp was extracted using 20wt% PEG 4000 / 10wt% Na2C2H4O6 ATPS, the partition coefficient of L-Trp was found to be 3.53. After introducing 5wt% [C4Clim]Cl as an additive into this ATPS, the partition coefficient of L-Trp was 2.31 (Fluid Phase Equilibria, 2021, 535, 112971). In ATPS containing PEG400 / salts (NaH2PO4, K2HPO4, K3PO4, K2CO3, K3C5H5O7), the partition coefficient of L-Trp reached as high as 8. Choline chloride ([N... 111(2OH) When Cl was introduced into the system as a weak salting-out agent, the partition coefficient of L-Trp decreased to below 1 (Food and Bioproducts Processing, 2020, 124, 48-56). When Van was extracted using 35 wt% maltodextrin / 35 wt% acetonitrile (ACN) ATPS, the Van partition coefficient was only 12.4 (Fluid Phase Equilibria 2018, 476, 179-185); in ATPS containing 40-50 wt% ACN / 10-20 wt% carbohydrates (sucrose, maltose, glucose, mannose, xylose, arabinose, and fructose), the Van partition coefficient was in the range of 3-10 (Separation and Purification Technology, 2013, 104, 106–113); and in 15 wt% K3PO4 / 25 wt% ionic liquids ([C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C7mim]Cl, [C... 10In the [amim]Cl and [amim]Cl) system, the partition coefficient of Van ranges from 3 to 50 (Separation and Purification Technology, 2010, 75, 39-47); in the 18wt% dextran (DEX) 10000 / 30wt% ACN system, the partition coefficient of Van can reach 11.26 at 25℃ (Separation and Purification Technology, 2014, 136, 74-80); when extracting Van using the 40wt% ACN / 30wt% sorbitol system, the partition coefficient of Van can reach 67 (Separation and Purification Technology, 2014, 124, 54–60). In summary, the extraction efficiency of the above substances using ATPS is low. Furthermore, L-Trp and Van are enriched in the upper phase of the above ATPS, and there is currently no effective method to recover L-Trp and Van to the lower phase of the aqueous two-phase system via back-extraction. Summary of the Invention

[0005] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides the application of SDDAB in improving the extraction efficiency of PEG / sulfate aqueous two-phase systems. This invention is the first to utilize the sulfobetaine surfactant 3-sulfopropyldodecyldimethylbetaine (SDDAB) to enhance the extraction efficiency of small biomolecules in polyethylene glycol (PEG) / sulfate aqueous two-phase systems. Specifically, it utilizes PEG 400 / (NH4)2SO4, PEG 400 / Na2SO4, PEG 4000 / (NH4)2SO4, and PEG 4000 / Na2SO4 aqueous two-phase systems with SDDAB as an additive to extract L-tryptophan (L-Trp) and vanillin (Van); effectively solving the problem of low extraction efficiency in ATPS extraction of tryptophan (L-Trp) and vanillin (Van).

[0006] Technical solution: In order to achieve the above objectives, the present invention provides the application of SDDAB in improving the extraction efficiency of L-tryptophan (L-Trp) or vanillin (Van) in a PEG / sulfate aqueous two-phase system.

[0007] The application process is as follows: L-Trp or Van is added to the PEG / sulfate aqueous two-phase system containing SDDAB, mixed, and then centrifuged to completely separate the two phases.

[0008] The structural formulas of the surfactant and extraction substance of the present invention are as follows:

[0009] The structural formulas for CTAB (n=14) and DTAB (n=10) are:

[0010] The structural formula of SDS is:

[0011] The structural formula of SDBS is:

[0012] The structural formula for AOT is:

[0013] The structural formula of TX-100 is:

[0014] The structural formula for SDDAB is:

[0015] The structural formula for L-Trp is:

[0016] The structural formula for Van is:

[0017] The PEG / sulfate aqueous two-phase system is any one of PEG 400 / (NH4)2SO4, PEG 400 / Na2SO4, PEG4000 / (NH4)2SO4 and PEG 4000 / Na2SO4 aqueous two-phase systems.

[0018] Further, in the system, the PEG / sulfate (ATPS) is, for L-Trp, any one of 25wt% PEG 400 / 18wt% (NH4)2SO4, 20wt% PEG 400 / 18wt% (NH4)2SO4, 20wt% PEG 400 / 10wt% Na2SO4, 20wt% PEG 4000 / 10wt% (NH4)2SO4, and 20wt% PEG4000 / 10wt% Na2SO4; for Van, the PEG / sulfate aqueous two-phase system is any one of 25wt% PEG 400 / 18wt% (NH4)2SO4, 20wt% PEG400 / 18wt% (NH4)2SO4, and 20wt% PEG4000 / 10wt% Na2SO4.

[0019] The pH value of the PEG / sulfate is 3-9.

[0020] Preferably, the pH value of the PEG / sulfate is 7.

[0021] The concentration of SDAB in the system is 1-7 wt%.

[0022] Preferably, the SDAB concentration is 5 wt%.

[0023] Furthermore, L-Trp and Van were extracted from the top-back phase of the PEG / sulfate aqueous two-phase system. The method for top-back extraction of L-Trp and Van from 25wt% PEG400 / 18wt% (NH4)2SO4 / 5wt% SDAB ATPS is as follows: Take the upper phase of this ATPS, add NaH2PO4·2H2O, NaCl, and H2O, mix well, equilibrate, and centrifuge to completely separate the two phases, forming ATPS. 反1 Then take the upper phase, add NaH2PO4·2H2O and H2O, mix well, equilibrate, and centrifuge to completely separate the two phases, forming ATPS. 反2 .

[0024] This invention uses a specific method to further enrich L-Trp and Van on the upper phase of ATPS. Based on this, the upper phase is taken, and salt and water are added to form a new aqueous two-phase system (i.e., a back-extraction system). This allows L-Trp and Van to reach the lower phase of the aqueous two-phase system, achieving the purpose of further separation, purification, and recycling of L-Trp and Van.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] 1. This invention is the first to introduce the zwitterionic surfactant SDDAB as an additive in an aqueous two-phase system, and it was found that the introduction of SDDAB can significantly improve the partition coefficients of L-Trp and Van in ATPS. For example, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, 5wt% SDDAB can increase the partition coefficient of L-Trp from 8.5 to 16.5, and the partition coefficient of Van from 89.3 to 291.0.

[0027] 2. This invention constructs an ATPS mixture with 27.8 wt% ATPS upper phase / 26 wt% NaH2PO4 / 4.6 wt% NaCl / 41.6 wt% H2O. 反1 61wt% ATPS 反1 ATPS of the upper phase / 18.6wt% NaH2PO4 / 24.4wt% H2O 反2 L-Trp and Van were extracted from a mixture of 25 wt% PEG 400 / 18 wt% (NH4)2SO4 / 5 wt% SDAB ATPS using top-to-back extraction. After two extractions, the overall recovery of L-Trp reached 74.0%, and the overall recovery of Van reached 66.8%. Attached Figure Description

[0028] Figure 1 The figure shows the effect of SDAB on the extraction of L-Trp by 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS.

[0029] Figure 2 The figure shows the effect of pH change on the extraction of L-Trp from 25wt% PEG 400 / 18wt% (NH4)2SO4 / SDDAB ATPS.

[0030] Figure 3 The figure shows the effect of SDAB on the extraction of L-Trp by different components using PEG / sulfate ATPS.

[0031] Figure 4 The figure shows the effect of SDAB on the extraction of Van by 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS.

[0032] Figure 5 The figure shows the effect of pH change on the extraction of Van from 25wt% PEG 400 / 18wt% (NH4)2SO4 / SDDAB ATPS.

[0033] Figure 6 The figure shows the effect of SDDAB on the extraction of Van by different components using PEG / sulfate ATPS. Detailed Implementation

[0034] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials can be obtained commercially, and the sources of the reagents are shown in Table 1.

[0035] Table 1. Reagent Sources and Specifications

[0036]

[0037]

[0038] In the following examples, phosphate buffer solution was used as the solvent for the preparation of L-Trp solution and Van solution.

[0039] Taking the preparation of L-Trp solution and Van solution as an example, the specific steps are as follows:

[0040] Prepare 0.4 mM L-Trp solution and 20 mM Van solution in 20 mM phosphate buffer solution at pH 7.0.

[0041] L-Trp exhibits a characteristic absorption peak at 279 nm. Using the lower phase of ATPS as a solvent, a series of L-Trp solutions with varying concentrations were prepared, and their absorbance at 279 nm was measured to obtain a standard curve. Then, ATPS containing 0.4 mM L-Trp was prepared, mixed in a constant-temperature mixer at 25 °C for 15 min, equilibrated for 30 min, and centrifuged for 5 min to ensure complete phase separation. Finally, the volume of the lower phase of ATPS was determined, and the concentration of L-Trp in the lower phase was obtained using the standard curve, allowing the calculation of the L-Trp partition coefficient.

[0042] Van exhibits a characteristic absorption peak at 280 nm. Using the lower phase of ATPS as a solvent, a series of Van solutions with gradient concentrations were prepared, and their absorbance at 280 nm was measured to obtain a standard curve. Then, ATPS containing 20 mM Van was prepared, mixed in a constant-temperature mixer at 25 °C for 15 min, equilibrated for 30 min, and centrifuged for 5 min to ensure complete separation of the two phases. Finally, the volume of the lower phase of ATPS was determined, and the concentration of Van in the lower phase was obtained using the standard curve, and the partition coefficient of Van was calculated.

[0043] The formula for calculating the allocation coefficient is as follows:

[0044] K = (C 总 V 总 -C 下 V 下 ) / C 下 V 下

[0045] Where K is the distribution coefficient of the substance; C 总 : Total concentration of substances (tryptophan or vanillin) in ATPS; V 总 : Total volume of ATPS; C 下 : Concentration of substances (tryptophan or vanillin) in the lower phase of ATPS; V 下 : Volume of the lower phase of ATPS.

[0046] The formula for calculating the recovery rate is as follows:

[0047] First-step back-extraction extraction rate: E 反1 =[C 反1 bottom V 反1 bottom / (C 反1 bottom V 反1 bottom +C 反1 top V 反1 top )]*100%

[0048] Second-step reverse extraction yield: E 反2 =[C 反2 bottom V 反2 bottom / (C 反2 bottom V 反2 bottom +C 反2 top V 反2 top )]*100%

[0049] Total extraction rate by back-extraction: E 反 =E 反1 +(100%-E) 反1 )*E 反2

[0050] Among them, E 反 : Extraction rate of back extraction; C 反1 top and C 反2 top ATPS 反1 and ATPS 反2 The total concentration of substances in the upper phase; C 反1 bottom and C 反2 bottom ATPS 反1 and ATPS 反2 Total concentration of substances in the lower phase; V 反1 top and V 反2 top ATPS 反1 and ATPS 反2 Volume of the upper phase; V 反1 bottom and V 反2 bottom ATPS 反1 and ATPS 反2 Volume of the lower phase. ATPS 反1 and ATPS 反2 For detailed definitions, please refer to Examples 5 and 6.

[0051] Example 1

[0052] Effect of SDDAB on L-Trp extraction by 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS:

[0053] (1) Weigh 0.18g (NH4)2SO4, 0.25g PEG 400 and 0.4mM L-Trp solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substances are completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 279nm. Calculate the concentration of L-Trp in the lower phase according to the standard curve.

[0054] (2) Weigh 0.18g (NH4)2SO4, 0.25g PEG 400, 0.01g SDDAB and 0.4mM L-Trp solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substances are completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 279nm. Calculate the concentration of L-Trp in the lower phase according to the standard curve.

[0055] (3) Change SDAB to 0.03g, 0.05g, and 0.07g, and repeat step (2).

[0056] (4) Change the pH of the system to 3 and 9 respectively, and repeat steps (1), (2) and (3).

[0057] Based on the formula for calculating the partition coefficient, the partition coefficient of L-Trp in each system is calculated, and the results are as follows: Figure 1 As shown. Figure 1 The results showed that at pH=7, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no SDAB addition, 1wt% SDAB as an additive had almost no effect on the partition coefficient of L-Trp; 3wt% SDAB as an additive increased the partition coefficient of L-Trp from 8.5 to 12.8; 5wt% SDAB as an additive increased the partition coefficient of L-Trp to 16.5; and 7wt% SDAB as an additive increased the partition coefficient of L-Trp to 22.3.

[0058] It was found that in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, SDAB as an additive can improve the partition coefficient of L-Trp. The partition coefficient further increases with increasing concentration. Subsequent experiments chose 5wt% SDAB primarily because 7wt% SDAB is not easily soluble in some systems.

[0059] Furthermore, changing the pH of the system to 3 and 9 yielded the following results: Figure 2 As shown, by Figure 2 The results show that at pH=3, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no SDAB addition, the partition coefficient of L-Trp increased slightly from 7.6 to 8.6 when 1wt% SDAB was added; the partition coefficient of L-Trp increased to 12.9 when 3wt% SDAB was added; the partition coefficient of L-Trp increased to 17.5 when 5wt% SDAB was added; and the partition coefficient of L-Trp increased to 23.4 when 7wt% SDAB was added. At pH 9, in 25 wt% PEG 400 / 18 wt% (NH4)2SO4 ATPS, compared to without SDAB, the partition coefficient of L-Trp increased slightly from 7.6 to 8.6 with 1 wt% SDAB as an additive; increased to 12.4 with 3 wt% SDAB; increased to 16.1 with 5 wt% SDAB; and increased to 21.4 with 7 wt% SDAB. Changes in pH had almost no effect on the partitioning of L-Trp.

[0060] Example 2

[0061] Effect of SDDAB on L-Trp extraction with different PEG / sulfate ATPS components:

[0062] (1) Weigh 0.18g (NH4)2SO4, 0.20g PEG 400, 0.05g SDDAB and 0.4mM L-Trp solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substances are completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 279nm. Calculate the concentration of L-Trp in the lower phase according to the standard curve.

[0063] (2) Change the aqueous two-phase system to 20wt%PEG400 / 10wt%Na2SO4, 20wt%PEG4000 / 10wt%(NH4)2SO4 and 20wt%PEG4000 / 10wt%Na2SO4, and repeat step (1).

[0064] Based on the formula for calculating the partition coefficient, the partition coefficient of L-Trp in each system is calculated, and the results are as follows: Figure 3 As shown. Figure 3The results show that in 20wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, 5wt% SDAB can increase the partition coefficient of L-Trp from 3.7 to 8.1; in 20wt% PEG 400 / 10wt% Na2SO4 ATPS, 5wt% SDAB can increase the partition coefficient of L-Trp from 6.8 to 20.2; in 20wt% PEG4000 / 10wt% (NH4)2SO4 ATPS, 5wt% SDAB can slightly increase the partition coefficient of L-Trp from 5.1 to 5.8; and in 20wt% PEG 4000 / 10wt% Na2SO4 ATPS, 5wt% SDAB can increase the partition coefficient of L-Trp from 6.4 to 12.4.

[0065] It can be observed that in the above five PEG / sulfate aqueous two-phase systems (including 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS in Example 1), 5wt% SDAB as an additive can improve the partition coefficient of L-Trp.

[0066] Example 3

[0067] Effect of SDDAB on the extraction of Van by 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS:

[0068] (1) Weigh 0.18g (NH4)2SO4, 0.25g PEG 400 and 20mM Van solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substance is completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 280nm. Calculate the concentration of Van in the lower phase according to the standard curve.

[0069] (2) Weigh 0.18g (NH4)2SO4, 0.25g PEG 400, 0.01g SDDAB and 20mM Van solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substances are completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 280nm. Calculate the concentration of Van in the lower phase according to the standard curve.

[0070] (3) Change SDAB to 0.03g, 0.05g, and 0.07g, and repeat step (2).

[0071] (4) Change the pH of the system to 3 and 9 respectively, and repeat steps (1), (2) and (3).

[0072] Based on the formula for calculating the partition coefficient, the partition coefficient of Van in each system is calculated, and the results are as follows: Figure 4 As shown. Figure 4 The results show that at pH=7, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, when 1wt% SDDAB is used as an additive, the partition coefficient of Van increases from 89.3 to 136.3; when 3wt% SDDAB is used as an additive, the partition coefficient of Van increases to 195.3; when 5wt% SDDAB is used as an additive, the partition coefficient of Van increases to 291.0; and when 7wt% SDDAB is used as an additive, the partition coefficient of Van increases to 497.8.

[0073] It was found that in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, SDDAB as an additive significantly improved the partition coefficient of Van. The partition coefficient further increased with increasing concentration. Subsequent experiments selected 5wt% SDDAB primarily because 7wt% SDDAB was not easily soluble in some systems.

[0074] Furthermore, changing the pH of the system to 3 and 9 yielded the following results: Figure 5 As shown, by Figure 5 The results show that at pH=3, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no SDAB addition, the partition coefficient of Van increased from 75.7 to 137.1 when 1wt% SDAB was added; the partition coefficient of Van increased to 301.6 when 5wt% SDAB was added; and the partition coefficient of Van increased to 489.5 when 7wt% SDAB was added. At pH=9, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no SDAB addition, the partition coefficient of Van increased from 84.0 to 136.4 when 1wt% SDAB was added; the partition coefficient of Van increased to 281.1 when 5wt% SDAB was added; and the partition coefficient of Van increased to 483.9 when 7wt% SDAB was added. Changes in pH had almost no effect on Van partitioning.

[0075] Example 4

[0076] Effect of SDDAB on the extraction of Van by different PEG / sulfate ATPS components:

[0077] (1) Weigh 0.18g (NH4)2SO4, 0.20g PEG400, 0.05g SDDAB and 20mM Van solution (total 1g) into a 1.5ml centrifuge tube, mix thoroughly, and after the solid substances are completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, centrifuge for 5min, separate the phases, take the lower phase, and detect the absorbance value of the lower phase of ATPS at 280nm. Calculate the concentration of Van in the lower phase according to the standard curve.

[0078] (2) Change the aqueous two-phase system to 20wt% PEG 400 / 10wt% Na2SO4, 20wt% PEG 4000 / 10wt% (NH4)2SO4 and 20wt% PEG 4000 / 10wt% Na2SO4, and repeat step (1).

[0079] Based on the formula for calculating the partition coefficient, the partition coefficient of Van in each system is calculated, and the results are as follows: Figure 6 As shown. Figure 6 In 20wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, 5wt% SDAB can increase the partition coefficient of Van from 18.9 to 115.7; in 20wt% PEG 400 / 10wt% Na2SO4 ATPS, 5wt% SDAB can decrease the partition coefficient of Van from 57.2 to 12.8; in 20wt% PEG 4000 / 10wt% (NH4)2SO4 ATPS, 5wt% SDAB can decrease the partition coefficient of Van from 39.9 to 22.8; and in 20wt% PEG 4000 / 10wt% Na2SO4 ATPS, 5wt% SDAB can increase the partition coefficient of Van from 51.4 to 133.4.

[0080] It can be found that in the above-mentioned PEG / sulfate aqueous two-phase systems, the addition of 5 wt% SDAB as an additive can significantly improve the partition coefficient of Van in 25 wt% PEG400 / 18 wt% (NH4)2SO4ATPS (Example 3), 20 wt% PEG 400 / 18 wt% (NH4)2SO4ATPS and 20 wt% PEG 4000 / 10 wt% Na2SO4ATPS.

[0081] Example 5

[0082] L-Trp was extracted from 25 wt% PEG 400 / 18 wt% (NH4)2SO4 ATPS enriched with L-Trp via top-back extraction:

[0083] (1) Weigh 2.224g of the upper phase of ATPS with a SDAB concentration of 5wt% in Example 1 (i.e., the upper phase after centrifugation of 25wt% PEG 400 / 18wt% (NH4)2SO4 / 5wt% SDAB ATPS in step 2 of Example 1), 2.08g of NaH2PO4·2H2O, 0.368g of NaCl and H2O (total 8g) into a 10ml centrifuge tube, mix thoroughly, and after the solid matter is completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, and centrifuge for 5min to obtain ATPS. 反1 The ATPS phases were separated, and the upper phase was used for later use. The absorbance values ​​of both phases were measured at 279 nm, and the concentration of L-Trp in both phases was calculated based on the standard curve. Combined with the volumes of the upper and lower phases, the concentration of L-Trp in the ATPS phase could be calculated. 反1 Recovery rate of the lower phase.

[0084] (2) Weigh 0.61g of the upper phase of the aqueous two-phase system from step (1), 0.186g of NaH2PO4·2H2O, and 0.244g of H2O into a 1.5ml centrifuge tube. Mix thoroughly until the solids are completely dissolved. Then, mix in a 25℃ constant temperature mixer for 15min, equilibrate for 30min, and centrifuge for 5min to obtain ATPS. 反2 The ATPS was separated into upper and lower phases, and the absorbance values ​​of the upper and lower phases were measured at 279 nm. The concentration of L-Trp in the upper and lower phases was calculated based on the standard curve. Combined with the volumes of the upper and lower phases, the concentration of L-Trp in the ATPS was calculated. 反2 The recovery rates of the lower phase and the total recovery rates of the two back-extractions are shown in Table 2.

[0085] Table 2 Composition and back-extraction efficiency of L-Trp back-extraction system

[0086]

[0087] Table 2 shows that the total extraction yield can reach 74.0% when L-Trp is extracted from the top-to-back mixture of 25wt% PEG 400 / 18wt% (NH4)2SO4 / 5wt% SDAB ATPS.

[0088] Example 6

[0089] Van was extracted from a sample enriched with 25 wt% PEG 400 / 18 wt% (NH4)2SO4 ATPS via top-reverse extraction.

[0090] (1) Weigh 2.224g of the upper phase of ATPS with a SDAB concentration of 5wt% in Example 3 (i.e., the upper phase after centrifugation of 25wt% PEG400 / 18wt% (NH4)2SO4 / 5wt% SDAB ATPS in step 2 of Example 3), 2.08g of NaH2PO4·2H2O, 0.368g of NaCl and H2O (total 8g) into a 10ml centrifuge tube, mix thoroughly, and after the solid matter is completely dissolved, mix in a constant temperature mixer at 25℃ for 15min, equilibrate for 30min, and centrifuge for 5min to obtain ATPS. 反1 The upper phase was separated and used for later use. The absorbance values ​​of the upper and lower phases of this ATPS were measured at 280 nm. The concentration of Van in the upper and lower phases was calculated according to the standard curve. Combined with the volume of the upper and lower phases, the recovery rate of Van in this ATPS lower phase can be calculated.

[0091] (2) Weigh 0.61g of the upper phase of the aqueous two-phase system from step (1), 0.186g of NaH2PO4·2H2O, and 0.244g of H2O into a 1.5ml centrifuge tube. Mix thoroughly until the solids are completely dissolved. Then, mix in a 25℃ constant temperature mixer for 15min, equilibrate for 30min, and centrifuge for 5min to obtain ATPS. 反2 The ATPS was separated into upper and lower phases, and the absorbance values ​​of the upper and lower phases were measured at 280 nm. The concentration of Van in the upper and lower phases was calculated based on the standard curve. Combined with the volumes of the upper and lower phases, the recovery rate of Van in the lower phase of this ATPS and the total recovery rate of the two back-extractions were calculated. The results are shown in Table 3.

[0092] Table 3 Composition and back-extraction efficiency of the Van back-extraction system

[0093]

[0094]

[0095] Table 3 shows that the total extraction rate of Van can reach 66.8% when back-extracting from the top phase of 25wt% PEG 400 / 18wt% (NH4)2SO4 / 5wt% SDAB ATPS. As demonstrated in Examples 5 and 6, this invention utilizes a two-stage back-extraction process to back-extract L-Trp and Van to the bottom phase of ATPS, achieving a high total recovery rate. Example 7

[0096] Effect of surfactant type on L-Trp extraction by 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS:

[0097] The method is the same as step (2) of Example 1, except that 0.05g SDAB is replaced with 0.05g DTAB, AOT and TX-100.

[0098] Based on the formula for calculating the partition coefficient, the partition coefficient of L-Trp in each system was calculated, and the results are shown in Table 4.

[0099] Table 4. Effect of surfactant type on partition coefficient of L-Trp in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS

[0100]

[0101] Table 4 shows that at pH 7, in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no surfactant addition, the partition coefficient of L-Trp decreased from 8.5 to 1.0 when 5wt% DTAB was used as an additive; the partition coefficient of L-Trp increased to 12.6 when 5wt% AOT was used as an additive; and the partition coefficient of L-Trp increased to 10.3 when 5wt% TX-100 was used as an additive. All of these are significantly lower than the partition coefficient of 16.5 when 5wt% SDAB was used as an additive. It can be found that in 25wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, SDAB as an additive has the best effect on improving the partition coefficient of L-Trp.

[0102] Example 8

[0103] The effect of surfactant type on the extraction of Van by 20wt% PEG 400 / 18wt% (NH4)2SO4 ATPS:

[0104] The method is the same as step (2) of Example 3, except that 0.05g SDAB is replaced with 0.05g DTAB, SDS, SDBS, AOT and TX-100.

[0105] Based on the formula for calculating the distribution coefficient, the distribution coefficient of Van in each system was calculated, and the results are shown in Table 5.

[0106] Table 5. Effect of surfactant type on the partition coefficient of Van in 20wt% PEG 400 / 18wt% (NH4)2SO4 ATPS

[0107]

[0108] Table 5 shows that at pH 7, in 20wt% PEG 400 / 18wt% (NH4)2SO4 ATPS, compared with no surfactant addition, the partition coefficient of Van decreased from 18.9 to 1.3 when 5wt% DTAB was added; the partition coefficient of Van increased to 69.4 when 5wt% SDS was added; the partition coefficient of Van increased to 72.4 when 5wt% SDBS was added; the partition coefficient of Van increased to 56.4 when 5wt% AOT was added; and the partition coefficient of Van increased to 81.5 when 5wt% TX-100 was added. All of these values ​​are significantly lower than the partition coefficient of Van (115.7) when 5wt% SDDAB was added. It can be found that in 20wt% PEG400 / 18wt% (NH4)2SO4 ATPS, SDDAB as an additive has the best effect on improving the partition coefficient of Van.

[0109] Examples 7 and 8 reveal that different types of surfactants have significantly different effects on the partition coefficients of L-Trp and Van. The cationic surfactant DTAB significantly reduced the partition coefficients of L-Trp and Van, causing them to no longer be primarily enriched in the PEG-rich phase of ATPS, but rather uniformly dispersed within ATPS. Anionic surfactants (SDS, SDBS, and AOT) and the nonionic surfactant TX-100 increased the partition coefficients of L-Trp and Van to some extent, while the amphoteric surfactant SDDAB significantly increased the partition coefficients of L-Trp and Van, further enriching them in the upper phase of ATPS. Among these, SDDAB showed the best effect in increasing the partition coefficients of L-Trp and Van.

Claims

1. Application of SDDAB in improving the efficiency of PEG / sulfate aqueous two-phase system in extracting L-tryptophan (L-Trp) or vanillin (Van), wherein the PEG / sulfate aqueous two-phase system is any one of PEG 400 / (NH4)2SO4, PEG 400 / Na2SO4, PEG 4000 / (NH4)2SO4 and PEG 4000 / Na2SO4 aqueous two-phase system, and the concentration of SDDAB in the system is 5wt%; for L-Trp, the PEG / sulfate aqueous two-phase system is any one of 25wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 10wt% Na2SO4, 20wt% PEG 4000 / 10wt%(NH4)2SO4 and 20wt% PEG 4000 / 10wt% Na2SO4; for Van, the PEG / sulfate aqueous two-phase system is any one of 25wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 18wt%(NH4)2SO4 and 20wt% PEG 4000 / 10wt% Na2SO4.

2. Use according to claim 1, characterized in that, The process of the application is as follows: L-Trp or Van is added to the PEG / sulfate aqueous two-phase system containing SDDAB, mixed well, centrifuged, and the two phases are completely separated.

3. Use according to claim 1 or 2, characterized in that, The pH value of the PEG / sulfate is 3-9.

4. Use according to claim 1 or 2, characterized in that, L-Trp or Van is extracted from the upper phase of the PEG / sulfate aqueous two-phase system.

2. The application of SDDAB in improving the efficiency of PEG / sulfate aqueous two-phase system in extracting L-tryptophan (L-Trp) or vanillin (Van), wherein the PEG / sulfate aqueous two-phase system is any one of PEG 400 / (NH4)2SO4, PEG 400 / Na2SO4, PEG 4000 / (NH4)2SO4 and PEG 4000 / Na2SO4 aqueous two-phase system, and the concentration of SDDAB in the system is 5wt%; for L-Trp, the PEG / sulfate aqueous two-phase system is any one of 25wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 10wt% Na2SO4, 20wt% PEG 4000 / 10wt%(NH4)2SO4 and 20wt% PEG 4000 / 10wt% Na2SO4; for Van, the PEG / sulfate aqueous two-phase system is any one of 25wt% PEG 400 / 18wt%(NH4)2SO4, 20wt% PEG 400 / 18wt%(NH4)2SO4 and 20wt% PEG 4000 / 10wt% Na2SO4. The process of the application is as follows: L-Trp or Van is added to the PEG / sulfate aqueous two-phase system containing SDDAB, mixed well, centrifuged, and the two phases are completely separated. The pH value of the PEG / sulfate is 3-9. L-Trp or Van 5. Use according to claim 4, characterized in that, The process of counter-extracting L-Trp or Van from the upper phase of the PEG / sulfate two-water-phase system is as follows: taking the upper phase of the two-phase complete separation, adding NaH2PO4*2H2O, NaCl, H2O, mixing, balancing, and then centrifuging to completely separate the two phases to form ATPS 反1 ; then taking the upper phase, adding NaH2PO4*2H2O, H2O, mixing, balancing, and then centrifuging to completely separate the two phases to form ATPS 反2 .

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