CN118307605A - Method for separating lactose-N-neotetraose from microbial fermentation liquid - Google Patents
Method for separating lactose-N-neotetraose from microbial fermentation liquid Download PDFInfo
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Abstract
The invention belongs to the technical field of separation and purification, and particularly relates to a method for separating lactose-N-neotetraose from microbial fermentation broth. The invention provides a method for separating lactose-N-neotetraose from microbial fermentation broth, which comprises the steps of filtering the fermentation broth, desalting to reduce the conductivity, and separating the components in a sample class by class in a form of classifying fast and slow components. The method can separate lactose-N-neotetraose with the purity of liquid phase reaching 99.9% from fermentation liquor with high efficiency and high yield, is far higher than the purity of the products sold in the market at present, and has the advantages of simple process, easy operation, large-scale continuous production, environmental protection and high degree of automation. Meanwhile, the lactose-N-new tetraose purified solution can be obtained into a high-purity lactose-N-new tetraose finished product only by corresponding drying, and decoloring and crystallizing operations are not needed.
Description
Technical Field
The invention belongs to the technical field of separation and purification, and particularly relates to a method for separating lactose-N-neotetraose from microbial fermentation broth.
Background
Lactose-N-neotetraose (Lacto-N-neotetraose, LNnT), a key human milk oligosaccharide (Human Milk Oligosaccharides, HMOs) component in breast milk, exhibits its versatile biological effects when used as a bioactive additive in infant formulas: enhancing host immune defense mechanism, reducing risk of pathogenic microorganism infection, promoting infant intestinal microflora maturation, improving intestinal health, and improving infant immunity. Microbial fermentation as a preferred strategy for the synthesis of LNnT presents several technical challenges, such as limitations in substrate conversion, the impact of complex metabolic pathways on target product synthesis, and problems with metabolic process intermediates and isomer content control.
Traditional LNnT production methods rely primarily on extraction and separation from breast milk, but such methods are limited by low yields and the complexity of the purification process. To overcome these limitations, the production strains are modified by gene editing technology to directly metabolize and synthesize LNnT during the growth process, thereby effectively improving the yield. However, although this approach solves the yield problem, the process of separating and purifying LNnT from the fermentation broth still faces the challenge of low purification efficiency.
The current methods for separating lactose-N-neotetraose from microbial fermentation broth are mainly divided into two types:
1. The lactose-N-neotetraose is filtered and separated by adopting a traditional membrane filtration method, but the method has very severe requirements on the effective filtration pore diameter of a filter membrane, and has poor filtration effect on small molecular substances, low yield, large loss and long time consumption;
2. The method for separating lactose-N-neotetraose by column chromatography is a common method used at present, and has high separation precision, good separation effect and continuous production; however, the existing column chromatography method for purifying lactose-N-neotetraose synthesized by microorganism metabolism still has the problem that the precursor lactose-N-trisaccharide and lactose-N-neotetraose isomer cannot be thoroughly removed.
For example: chinese patent application publication No. CN113966338 a provides a method for purifying lactose-N-neotetraose (LNnT) from fermentation broth, by nanofiltration followed by a Simulated Moving Bed (SMB), and then by crystallization to obtain high purity LNnT powder. The LNnT purity obtained after the SMB is about 80%, further crystallization is needed to improve the purity, the crystallization process consumes long time, the cooling precision in the crystallization process is strict, the process is complex, and the large-scale industrial production of the LNnT is not facilitated.
Disclosure of Invention
It is an object of a first aspect of the present invention to provide a method.
The object of the second aspect of the invention is to provide a use.
In order to achieve the above purpose of the invention, the invention adopts the technical proposal that
In a first aspect of the present invention, there is provided a process for the isolation of lacto-N-neotetraose from a microbial fermentation broth, characterized by the steps of:
1. performing solid-liquid separation on the microbial fermentation liquid to obtain filtrate;
2. Desalting the filtrate to reduce the conductivity;
3. and (3) feeding the liquid subjected to the desalting treatment into a sequential simulated moving bed chromatographic system, and separating and purifying to obtain the product.
Preferably, the microbial fermentation broth is a fermentation broth obtained by liquid fermentation using a modified Bacillus subtilis BP05 strain provided by university of Jiangnan laboratory (Dong Xiaomin. Metabolic engineering Bacillus subtilis lactoyl-N-neotetraose [ D ]. University of Jiangnan 2020.DOI: 10.27169/d.cnki.gwqgu.2020.000145.).
Preferably, the filtering of step 1) comprises the steps of:
a1 Removing insoluble solids through the filter membrane 1;
a2 The soluble macromolecules are removed by the filter membrane 2.
Preferably, the filtration membrane 1 has a filtration pore size of 0.1 to 1.2. Mu.m.
Preferably, the filtering membrane 1 comprises at least one of a ceramic membrane, a metal membrane and an organic polymer membrane.
Preferably, the filter membrane 1 comprises a ceramic membrane.
Preferably, the molecular retention of the filtration membrane 2 is 5-10 KD.
Preferably, the filtration membrane 2 comprises an organic polymer membrane.
Preferably, the conductivity of the liquid after the desalting treatment in the step 2) is less than or equal to 100 mu S/cm.
Preferably, the desalting comprises at least one of electrodialysis desalting and ion exchange chromatography column desalting.
Preferably, the desalting comprises electrodialysis desalting and ion exchange chromatography column desalting.
Preferably, the ion exchange chromatography column comprises at least one of a cation exchange chromatography column and an anion exchange chromatography column.
Preferably, the ion exchange chromatography column comprises a cation exchange chromatography column and an anion exchange chromatography column.
Preferably, the cation exchange chromatography column packing is a strong cation exchange resin.
Preferably, the anion exchange chromatography column packing is a strong anion exchange resin.
Preferably, the ion exchange chromatography column has a column diameter to height ratio of 1: (5-50); more preferably, it is 1: (10-20).
Preferably, the step 2) is carried out to obtain lactose-N-neotetraose concentrated mother liquor after desalting and before entering the sequential simulated moving bed chromatography.
Preferably, the sugar degree of the lactose-N-neotetraose concentrated mother solution is 20-40 degrees Bx; more preferably 25 to 30 Bx.
Preferably, the concentration of lactose-N-neotetraose in the lactose-N-neotetraose concentrated mother liquor is 20-120 g/L; more preferably 40 to 80g/L.
Preferably, the stationary phase filler of the chromatographic column of the sequential simulated moving bed chromatographic system in the step 3) is a metal chelating resin.
Preferably, the metal ion of the metal chelating resin comprises at least one of K +、Na+、Ca2+、Fe2+、Mg2+、Zn2+; further preferred is a K + chelating resin.
Preferably, the resin of the metal chelate resin comprises at least one of polystyrene resin, polypropylene resin, polyethylene resin, polyvinyl chloride resin, and ABS resin.
Preferably, the resin comprises a polystyrene resin.
Preferably, the sequential simulated moving bed chromatographic system is provided with n chromatographic columns which are connected in series and sequentially recorded as No. 1 column to No. n column, wherein the tail end discharge port of each chromatographic column can be connected with the top end feed inlet of other chromatographic columns through a circulating pump to form a serial loop.
Preferably, n is equal to or greater than 6, and more preferably, n=6.
Preferably, the single column volume of the chromatographic column is 30-50mL.
Preferably, the chromatographic columns are provided with a feed inlet, a water inlet, a discharge outlet and a water outlet.
Preferably, the chromatographic column has a thermal insulation device to keep the temperature constant.
Preferably, the temperature of the chromatographic column is 40-60 ℃; further preferably 45-55 ℃.
Preferably, the sequential simulated moving bed chromatography system removes lactose-N-neotetraose as a fast component and then removes lactose-N-neotetraose as a slow component.
Preferably, the sequential simulated moving bed chromatography system comprises the following stages:
s1, collecting and circulating multiple columns in series;
s2, single-column elution and sample injection circulation;
s3, separating and circulating the multi-column series connection.
Preferably, the stages have a sequence, each stage has a corresponding circulation system and operation time, when the operation time of the stage S1 arrives, the next stage S2 is entered, and when the operation time of the stage S2 arrives, the next stage S3 is entered.
Preferably, the time required for the collecting and circulating stage of the multi-column series connection in the step S1 is 10-100S; more preferably 70 to 95s.
Preferably, the number of the tandem chromatographic columns of the collection cycle with the multi-column tandem in the S1 is more than or equal to 2 and less than n.
Preferably, the number of the tandem chromatographic columns of the collection cycle of the multi-column tandem in the S1 is n-1.
Preferably, the time required for the single column elution and sample injection circulation stage in the step S2 is 20-60S; more preferably 40 to 50s.
Preferably, the time required for the separation cycle stage of the multi-column series in S3 is 200 to 300S, more preferably 210 to 250S.
Preferably, the number of columns in series of the separation cycle of the multi-column series in S3=n.
Preferably, the multi-column series separation cycle is used to collect the fast component.
Preferably, the single column elution and sample injection cycle stage is used to collect slow components.
Preferably, the multi-column serial collection cycle is used to separate enriched fast and slow components in the sequential simulated moving bed chromatography system.
According to a preferred embodiment of the present invention, the separation process of the sequential simulated moving bed chromatography specifically comprises: the columns S1 and 1-5 in the first period are connected in series, no sample is injected into the system, pure water is pumped into the system from the column 1, waste liquid flows out from the column 5, and the chromatographic column is rinsed; s2, removing the series connection of the No. 4 columns in the first period, and loading samples from the No. 4 columns; s3, columns 1 to 6 of the first period are connected in series, pure water is pumped into the column 1, the column 6 flows out of the waste liquid, no component is collected at the moment, fast and slow components in a sample pumped from the column 4 are enriched in two different areas, and the fast components are enriched in the column 4 to the column 6; the slow component is enriched in columns 1 to 3. When the second period starts, the SSMB valve is switched to a No. 2 column, the No. 2-6 columns are connected in series in the S1 step, pure water is pumped from the No. 2 column to separate fast components, and the fast components flow out of the No. 6 column and are collected; s2 in the second period, removing the series connection of the No. 2 column, loading the collected fast component from the No. 5 column, simultaneously injecting pure water into the No. 2 column for eluting, and separating the slow component in the No. 2 column; the second cycle of S3, columns 1-6 were serially connected, purified water was pumped from column 2, effluent from column 1, and the components were redistributed among 6 columns. When the third period starts, the SSMB valve is switched to a No. 3 column, the No. 3, the No. 4, the No. 5, the No. 6 and the No. 1 column are connected in series in the S1 step, pure water is pumped into the No. 3 column to separate fast components, and the fast components flow out of the No. 1 column and are collected; s2 in the third period, removing the separation of the No. 3 column, loading the collected fast component from the No. 6 column, simultaneously injecting pure water into the No. 3 column for eluting, and separating the slow component from the sample in the No. 3 column. And by analogy, continuously separating the pumped feed liquid in the SSMB separation cycle process, and carrying out targeted collection according to a lactose-N-new tetraose purification liquid outlet of the target substance.
In a second aspect of the invention there is provided the use of the method of the first aspect of the invention for the preparation and/or isolation and purification of lactose-N-neotetraose.
The beneficial effects of the invention are as follows:
The invention provides a method for separating lactose-N-new tetraose from microbial fermentation broth, which comprises the following steps of pre-treating the lactose-N-new tetraose microbial fermentation broth: (1) Filtering to remove insoluble substances and soluble macromolecular substances; (2) Desalting the filtrate to reduce the conductivity to a proper range (the conductivity is less than or equal to 100 mu S/cm), reducing the conductivity to a proper range value through pretreatment of fermentation liquor, and improving the separation precision of components in a chromatographic column; introducing the pretreated sugar mixed mother liquor into a sequential simulated moving bed, and separating the components in the pretreated sugar mixed mother liquor by class in a mode of classifying the components in the sugar mixed mother liquor according to the speed components, so that the recovery rate, namely the purity, of a target product is improved; the lactose-N-neotetraose with high purity and high recovery rate can be separated and purified from the fermentation mother liquor by combining the lactose-N-neotetraose with the fermentation mother liquor. The sequential simulated moving bed technology takes pure water as an eluent, and separates components in the pretreated saccharide mixed mother liquor after fast and slow component classification, wherein lactose-N-new tetraose is firstly subjected to impurity removal by fast component components, then subjected to impurity removal by slow component components and purified and enriched, thereby rapidly, effectively and highly accurately separating three components of lactose-N-new tetraose, lactose-N-new tetraose and lactose-N-trisaccharide. The high-purity lactose-N-neotetraose can be separated from lactose-N-neotetraose-containing saccharide mixed mother liquor obtained from microbial fermentation liquor, the purity of the lactose-N-neotetraose is as high as 99.9%, the purity is far higher than that of products sold in the market at present, and the method is simple, easy to operate, can realize large-scale continuous production, and has the advantages of high product purity, environmental protection and high degree of automation. Meanwhile, by strictly controlling the conductivity of the mixed mother liquor of saccharides, the continuous sequential simulated moving bed chromatographic separation system can be used for carrying out equipment amplification production, and can be used for preparing high-purity lactose-N-neotetraose on a large scale.
Drawings
FIG. 1 is a liquid phase diagram of lactose-N-neotetraose fermentation broth after desalting and conductivity-reducing in example 2, wherein ① red arrow indicates the front hetero-peak adjacent to lactose-N-neotetraose; ② The red arrow indicates the latter hetero-peak adjacent to lactose-N-neotetraose.
FIG. 2 is a schematic diagram of a Sequential Simulated Moving Bed (SSMB) chromatographic separation system according to example 2.
FIG. 3 is a schematic diagram of the five column series separation cycle stage of example 2.
FIG. 4 is a schematic diagram of the single column elution and feed cycle stages of example 2.
Fig. 5 is a schematic diagram of a six-column serial separation cycle stage in example 2, and the contents of the top-down red frames are respectively.
FIG. 6 is a schematic diagram showing the sequential simulated moving bed, after classifying according to the fast and slow components, for separating and purifying lactose-N-neotetraose class by class, wherein the red frame content from top to bottom is respectively the fast component and lactose-N-neotetraose as slow component as a whole.
FIG. 7 is a liquid phase diagram of the separation of lactose-N-neotetraose fermentation broth from a sequential simulated moving bed of the rapid and slow component classification of example 2.
FIG. 8 is a liquid phase diagram of high purity lactose-N-neotetraose obtained by separation and purification of the rapid and slow component classification sequence simulated moving bed in example 2.
FIG. 9 is a schematic diagram showing the separation and purification of lactose-N-neotetraose from the conventional sequential simulated moving bed according to the peak sequence in comparative example 1, wherein the contents of red frames from top to bottom are respectively that the conventional SSMB separates the first material according to the peak time and separates lactose-N-neotetraose.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
EXAMPLE 1 preparation of lactose-N-New tetraose microbial fermentation broth
1) A proper amount of bacillus subtilis BP05 stored in a glycerol LB freezing tube of a refrigerator at the temperature of minus 80 ℃ is taken, the strain is from a laboratory of Jiangnan university and is disclosed in the literature (Dong Xiaomin. Metabolic engineering modification of bacillus subtilis to produce lactoyl-N-neotetraose [ D ]. Jiangnan university, 2020.DOI: 10.27169/d.cnki.gwqgu.2020.000145.), and the strain is subjected to monoclone on LB solid medium plates and cultured for 24 hours at the temperature of 37 ℃ until larger single colonies are grown.
2) Single colonies were selected and inoculated into 150mL Erlenmeyer flasks containing 30mL of LB liquid medium, and cultured at 37℃and 200rpm for about 15-18 hours as primary seed liquid (OD 600 2-4). The primary seed liquid was transferred to a 1000mL Erlenmeyer flask containing 200mL of LB liquid medium at a 10% inoculum size, and cultured at 37℃and 200rpm for 6-8 hours (OD 600 4-6) to obtain a secondary seed liquid.
3) The secondary seed liquid is inoculated into a fermentation culture medium for fermentation at the inoculation amount of 10 percent, the temperature is 37+/-0.1 ℃, the pH value is maintained to 7.0+/-0.05 by using 30 percent phosphoric acid and 25 percent ammonia water, the stirring and the ventilation are regulated to control DO to 30+/-5 percent, and when the ventilation and the rotating speed reach the maximum and still can not maintain dissolved oxygen, the pressure can be increased to 0.04Mpa to ensure the dissolved oxygen (the pressure is maintained unchanged later).
4) When OD 600 is more than or equal to 60 (8-9 h), xylose with the final concentration of 18g/L is added for induction at one time, when the glucose concentration is lower than 5g/L, the glucose concentration is maintained at 5-10g/L by feeding a material-supplementing carbon source, and lactose can be stabilized in a certain range without detection.
5) And (3) lower tank standard: the fermentation is carried out for about 45-50 hours, the activity of the subsequent thalli is obviously reduced, the sugar consumption is slow, the new tetraose yield is basically not increased any more, and the new tetraose can be put into a tank, and the volume of each tank is 3-4L.
Example 2 SSMB separation System for fast and slow component grouping
1) Filtering to remove salt
Taking 20L of lactose-N-new tetraose microorganism fermentation liquor (prepared in example 1), centrifuging the fermentation liquor at 5000rpm for 15min, removing most insoluble substances such as thalli and the like through preliminary centrifugation, and collecting supernatant.
And (3) removing insoluble particles and residual thalli after centrifugation from supernatant obtained after centrifugation through a ceramic membrane (the pore diameter of the membrane is 0.22 mu m, the operating temperature is 50 ℃ and the operating pressure is 0.6 MPa), sequentially adding 0.5L pure water into residual fermentation liquor in equipment for top washing for 2 times, collecting filtrate and pure water washing liquid of the ceramic membrane, and combining to obtain ceramic membrane effluent.
The effluent of the ceramic membrane is subjected to organic membrane (MWCO is 5kDa, the operating temperature is 35 ℃, the operating pressure is 0.48 MPa) to remove most protein, and the residual fermentation liquor in the inner tube of the equipment is sequentially added with 0.5L of pure water for top washing for 2 times. Collecting filtrate of the organic film and pure water washing liquid, combining to obtain organic film effluent,
The organic film effluent is subjected to vacuum rotary evaporation concentration operation at 65 ℃ and the pressure of-0.5 MPa, and 40g/L lactose-N-neotetraose concentrated filtrate with the volume of 4.4 being about L is obtained.
And (3) preliminarily reducing the solution conductivity to about 1mS/cm by using a Bona experimental BONA-ED-18 electrodialysis instrument (voltage: 14V) to lactose-N-neotetraose concentrated filtrate, collecting electrodialysis desalination liquid, concentrating by rotary evaporation at the temperature of 60-65 ℃ and the pressure of-0.5 MPa, and obtaining the electrodialysis desalination liquid concentrate.
And (3) passing the electrodialysis desalination liquid concentrate through a D001 type strong cation exchange chromatographic column (column specification: 45cm 4.6cm, effective column volume of 500 ml) at room temperature to remove positively charged substances in the solution, maintaining the pump flow rate at 15ml/min, maintaining the loading sugar degree of the electrodialysis desalination liquid concentrate at 20 Bx, and collecting effluent.
Collecting and concentrating the effluent of the strong cation exchange chromatographic column (the temperature is 60-65 ℃ and the pressure is less than or equal to minus 0.5 MPa) until the sugar degree is about 20 Bx (pure water is added in the chromatographic process to carry out top washing, the concentration of the liquid is diluted and the concentration is needed to be re-concentrated), further removing the negatively charged substance in the solution through a D201 type strong anion exchange chromatographic column (the column specification is 45cm, 4.6cm and the effective column volume is 500 ml), maintaining the flow rate of a substance pump for 15ml/min, and collecting the effluent.
The conductance of lactose-N-new tetraose fermentation liquor after Jiang Yangjiang anion exchange chromatography is reduced to below 20 mu S/cm, and the liquid phase diagram of lactose-N-new tetraose fermentation liquor after desalting and conductance reduction is shown in figure 1; concentrating the lactose-N-new tetrasaccharide fermentation liquor after desalting and conductivity reducing (the temperature is 60-65 ℃ and the pressure is less than or equal to 0 MPa) until the sugar degree is about 30 degrees Bx, wherein the LNnT concentration is about 40g/L (detected by using a high performance liquid chromatograph), and the total volume is 1L.
2) SSMB loading
A Sequential Simulated Moving Bed (SSMB) chromatographic apparatus is purchased from the company of new materials of the technology, inc. of the blue dawn of Siam, model SSMB-K62B, the separation system is schematically shown in figure 2, pure water is used as the mobile phase in the whole process, the separation temperature is set to be 50 ℃, and the steps of SSMB separation are as follows:
s1, collecting and circulating the five columns in series;
s2, single-column elution and sample injection circulation;
S3, six columns are connected in series for separation circulation;
the above steps are repeated until equilibrium is reached (the materials in each chromatographic column in the system reach a uniform state, and the sugar value of the effluent liquid from the fast and slow component collecting ports is stable). Wherein, a schematic diagram of the separation cycle of five columns connected in series is shown in fig. 3; a schematic of single column elution and sample cycling is shown in figure 4; a schematic of a six column series collection cycle is shown in figure 5.
The specific process is as follows: the columns S1 and 1-5 in the first period are connected in series, no sample is injected into the system, pure water is pumped into the system from the column 1, waste liquid flows out from the column 5, and the chromatographic column is rinsed; s2, opening a sample injection valve at the position of a No. 4 column, loading a sample solution from the No. 4 column, and flowing out waste liquid from the No. 5 column in the first period; s3, columns 1 to 6 of the first period are connected in series, pure water is pumped into the column 1, the column 6 flows out of the waste liquid, no component is collected at the moment, fast and slow components in a sample pumped from the column 4 are enriched in two different areas, and the fast components are enriched in the column 4 to the column 6; the slow component is enriched in columns 1 to 3. When the second period starts, the SSMB valve is switched to a No. 2 column, the No. 2-6 columns are connected in series in the S1 step, pure water is pumped from the No. 2 column to separate fast components, and the fast components flow out of the No. 6 column and are collected; s2, removing the series connection of the No. 5 column in the second period, loading the collected fast component from the No. 5 column, discharging waste liquid from the No. 6 column, simultaneously injecting pure water into the No. 2 column for eluting, and separating the slow component in the No. 2 column; the second cycle of S3, 2- & gt 3- & gt 4- & gt 5- & gt 6- & gt No. 1 columns are connected in series, pure water is pumped from the No. 2 column, waste liquid flows out from the No. 1 column, and components are redistributed in the 6 chromatographic columns. When the third period starts, the SSMB valve is switched to a No. 3 column, the No. 3, the No. 4, the No. 5, the No. 6 and the No. 1 column are connected in series in the S1 step, pure water is pumped into the No. 3 column to separate fast components, and the fast components flow out of the No. 1 column and are collected; s2 in the third period, removing the separation of the No. 3 column, loading the collected fast component from the No. 6 column, discharging waste liquid from the No. 1 column, simultaneously injecting pure water into the No. 3 column for eluting, and separating the slow component from the sample in the No. 3 column. The third cycle of S3,1-6 columns are connected in series, pure water is pumped from the No. 3 column, waste liquid flows out from the No. 2 column, and the components are redistributed in 6 chromatographic columns. And by analogy, continuously separating the pumped feed liquid in the SSMB separation cycle process, and carrying out targeted collection according to a lactose-N-new tetraose purification liquid outlet of the target substance.
In the process, various components in the sample liquid are grouped into fast and slow components, the lactose-N-new tetraose sugar isomer and the lactose-N-new tetraose are integrally used as fast components for collection and collection, impurities are removed and enter the SSMB again, the position distance between the lactose-N-new tetraose sugar isomer and the lactose-N-new tetraose in the SSMB is gradually pulled through multiple cycles, peaks of the lactose-N-new tetraose sugar isomer and the lactose-N-new tetraose can be better distinguished, and the lactose-N-new tetraose is used as the slow component for separation and purification in the S2 stage, so that the previous impurities are removed. A schematic of the separation of the fast and slow components is shown in figure 6.
In this example, the sample filtered to remove salt in step 1) was subjected to two SSMB loadings, with the first loading system parameters set forth in table 1.
TABLE 1 sequential simulated moving bed chromatographic separation System parameters for removal of impurities after peaks for the first time
| 2) | Time(s) | 3) Flow rate of feed pump (mL/min) | 4) Flow rate of water inlet pump (mL/min) |
| 5)S1 | 94 | / | 3.6 |
| 6)S2 | 40 | 1.75 | 3.6 |
| 7)S3 | 210 | / | 3.6 |
After the first sample loading system is operated for 6 cycles, the balance is achieved, and the result in the balance state is that: the lactose-N-neotetraose is collected as a fast component at the S1, namely the outlet of the five-column series separation stage, and the fast component collection liquid contains partial pre-impurities besides the lactose-N-neotetraose component, wherein the ratio of the lactose-N-neotetraose to the pre-impurities is about 1: the liquid phase detection purity of the lactose-N-neotetraose is about 50%, and the yield is more than or equal to 91%. The collected slow component collection liquid mainly contains xylose, glucose, lactose and other impurities, and is discarded.
Concentrating the obtained rapid component collecting solution (temperature 65 deg.C, pressure-0.5 MPa), sugar degree about 30 degree Bx, and LNnT concentration about 150 g/L. The pre-impurities were removed by performing SSMB secondary loading and eluted with ultrapure water. The second loading system parameters are shown in Table 2.
TABLE 2 sequential simulated moving bed chromatographic separation System parameters for second time for removal of impurities before peaks
After the second loading system is operated for 6 cycles, the balance is achieved, and the result in the balance state is that: the lactose-N-neotetraose is collected as a slow component at the S2 single column eluting separation stage outflow port, and the collected mother liquor only contains the lactose-N-neotetraose component, the lactose-N-neotetraose liquid phase detection purity reaches 99.9 percent (figure 8), the yield is more than or equal to 91 percent (the calculation formula is) And collecting lactose-N-neotetraose purification mother liquor, and freezing or spray drying to obtain a high-purity white powdery finished product.
Comparative example 1 separation and purification of lactose-N-neotetraose by conventional SSMB
1) Filtering to remove salt
As in example 2.
2) SSMB loading
Loading SSMB, namely, pure water is used as a mobile phase in the whole process, the separation temperature is set to be 50 ℃, and the SSMB separation steps are as follows:
The procedure is as in example 2.
The conventional SSMB separation sequence is to separate one by one according to a liquid chromatogram of a sample, namely to separate one by one according to the peak time of a substance, and the result is that the purity of a finally obtained target product is not high, and operations such as crystallization and the like still need to be performed; and the final target product has high purity, and the obtained sample mother liquor can be directly dried to obtain the product. The specific separation pattern is shown in fig. 9.
In this comparative example, the sample filtered to remove salt in step 1) was subjected to two SSMB loadings, with the first loading system parameters set forth in table 3.
TABLE 3 sequential simulated moving bed chromatographic separation System parameters for first removal of impurities after peaks
After the first sample loading system is operated for 6 cycles, the balance is achieved, and the result in the balance state is that: separating one by one according to the peak-out time of the substance, removing the impurity whose peak-out time is positioned in front of lactose-N-neotetraose, at this time, the recovered mother liquor contains many other impurity components besides lactose-N-neotetraose, for example, xylose, glucose and lactose, etc., the content of lactose-N-neotetraose is about 10% -20%, the liquid phase detection purity of lactose-N-neotetraose is about 10%, and its yield is 80% (the calculation formula is) Wherein M Loading sample ＝100g、M Recovery of =80 g, i.e
Concentrating the mother solution obtained after the first sample loading (temperature 65 deg.C, pressure-0.5 MPa), sugar degree about 30 deg.Bx, LNnT concentration about 50 g/L. The SSMB is subjected to secondary loading, the separation is carried out one by one according to the peak time of the substances, lactose-N-neotetraose is separated as a first substance, and the separation is carried out by using ultrapure water. The second loading system parameters are shown in Table 4.
TABLE 4 sequential simulated moving bed chromatographic separation system parameters for the second separation of lactose-N-neotetraose
After the second loading system is operated for 6 cycles, the balance is achieved, and the result in the balance state is that: separating according to the peak time of the substances one by one, separating lactose-N-neotetraose as the first substance of the peak, wherein the mother liquor is lactose-N-neotetraose purified liquor, the lactose-N-neotetraose liquid phase detection purity is only 80% -85%, and the yield is 85% (the calculation formula is) Wherein M Loading sample ＝80g、M Recovery of =68g, i.e
The results show that: as shown in FIG. 9, the lactose-N-neotetraose obtained by the conventional SSMB has the liquid phase detection purity of 80% -85% and the yield of 56% -76.5%, and the obtained lactose-N-neotetraose purified solution still needs to be recrystallized for further purification.
In conclusion, the separation and purification effect of the SSMB on the LNnT can be effectively improved through the separation strategy of the fast and slow component groups.
Claims (10)
1. A method for separating lactose-N-neotetraose from a microbial fermentation broth, comprising the steps of:
1) Performing solid-liquid separation on the microbial fermentation liquid to obtain filtrate;
2) Desalting the filtrate to reduce the conductivity;
3) And (3) feeding the liquid subjected to the desalting treatment into a sequential simulated moving bed chromatographic system, and separating and purifying to obtain the product.
2. The method according to claim 1, characterized in that:
Removing the lactose-N-neotetraose as a fast component from the mixed material in the sequential simulated moving bed chromatography system, and removing the lactose-N-neotetraose as a slow component from the mixed material.
3. The method according to claim 1, wherein the filtering of step 1) comprises the steps of:
a1 Removing insoluble solids through the filter membrane 1;
a2 Removing the soluble macromolecules by the filtering membrane 2;
Preferably, the filter pore diameter of the filter membrane 1 is 0.1-1.2 μm;
Preferably, the filtering membrane 1 comprises at least one of a ceramic membrane, a metal membrane and an organic polymer membrane;
preferably, the filter membrane 1 comprises a ceramic membrane;
preferably, the molecular retention of the filtering membrane 2 is 5-10 KD;
preferably, the filtration membrane 2 comprises an organic polymer membrane;
preferably, the microbial fermentation broth comprises a fermentation mixed broth obtained by liquid fermentation of bacillus subtilis BP05 strain.
4. The method according to claim 1, characterized in that:
the conductivity of the liquid after the desalting treatment in the step 2) is less than or equal to 100 mu S/cm;
Preferably, the desalting comprises at least one of electrodialysis desalting and ion exchange chromatography column desalting;
preferably, the desalting comprises electrodialysis desalting and ion exchange chromatography column desalting;
preferably, the ion exchange chromatography column comprises at least one of a cation exchange chromatography column and an anion exchange chromatography column;
Preferably, the cation exchange chromatography column packing is a strong cation exchange resin;
Preferably, the anion exchange chromatography column packing is a strong anion exchange resin;
Preferably, the ion exchange chromatography column has a column diameter to height ratio of 1: (5-50); more preferably, it is 1: (10-20).
5. The method according to claim 4, wherein:
The liquid after the desalination treatment in the step 2) is concentrated to obtain lactose-N-new tetraose concentrated mother liquor before entering a sequential simulated moving bed chromatographic system;
preferably, the concentration of lactose-N-neotetraose in the lactose-N-neotetraose concentrated mother liquor is 20-120 g/L; more preferably, 40 to 80g/L;
Preferably, the sugar degree of the lactose-N-neotetraose concentrated mother solution is 20-40 degrees Bx; more preferably 25 to 30 Bx.
6. The method according to claim 1, characterized in that:
The stationary phase filler of the chromatographic column of the sequential simulated moving bed chromatographic system in the step 3) is metal chelate resin;
Preferably, the metal ion of the metal chelating resin comprises at least one of K +、Na+、Ca2+、Fe2+、Mg2+、Zn2+;
preferably, the resin of the metal chelate resin comprises at least one of polystyrene resin, polypropylene resin, polyethylene resin, polyvinyl chloride resin, and ABS resin.
7. The method according to claim 6, wherein:
the sequential simulated moving bed chromatographic system is provided with n chromatographic columns which are connected in series and sequentially marked as No.1 column to No. n column, wherein a discharge port at the tail end of each chromatographic column can be connected with a feed port at the top end of other chromatographic columns through a circulating pump to form a serial loop; preferably, n is not less than 3,
Preferably, n is greater than or equal to 6; further preferably, said n=6;
preferably, the single column volume of the chromatographic column is 30-50mL;
preferably, the chromatographic columns are provided with a feed inlet, a water inlet, a discharge outlet and a water outlet;
preferably, the chromatographic column has a thermal insulation device to keep the temperature constant;
Preferably, the temperature of the chromatographic column is 40-60 ℃; further preferably 45-55 ℃.
8. The method according to any one of claims 6-7, characterized in that:
The sequential simulated moving bed chromatography system comprises the following stages:
s1, collecting and circulating multiple columns in series;
s2, single-column elution and sample injection circulation;
S3, separating and circulating the multi-column series connection;
preferably, the stages have a sequence, each stage has a corresponding circulation system and operation time, when the operation time of the S1 stage arrives, the next stage S2 is entered, and when the operation time of the S2 stage arrives, the next stage S3 is entered;
Preferably, the time required for the collecting and circulating stage of the multi-column series connection in the step S1 is 10-100S; more preferably, 70 to 95s;
Preferably, the number of the serial chromatographic columns of the collection cycle with the multi-column serial connection in the S1 is more than or equal to 2 and less than or equal to n;
Preferably, the number of the tandem chromatographic columns of the collection cycle with the multi-column tandem in the S1 is n-1;
Preferably, the time required for the single column elution and sample injection circulation stage in the step S2 is 20-60S; more preferably, 40 to 50 seconds;
preferably, the time required for the separation cycle stage of the multi-column series connection in the step S3 is 200 to 300S, more preferably 210 to 250S;
preferably, the number of columns in series of the separation cycle of the multi-column series in S3=n.
9. The method according to claim 8, wherein:
the multi-column serial separation cycle is used for collecting fast components; and/or
The single column elution and sample injection circulation stage is used for collecting slow components; and/or
The multi-column serial collection cycle is used to separate enriched fast and slow components in the sequential simulated moving bed chromatography system.
10. Use of the method according to any one of claims 1-9 for the preparation and/or isolation and purification of lactose-N-neotetraose.
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