CN117720594A - Method for preparing 2' -fucosyllactose by continuous crystallization - Google Patents
Method for preparing 2' -fucosyllactose by continuous crystallization Download PDFInfo
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- CN117720594A CN117720594A CN202311796718.1A CN202311796718A CN117720594A CN 117720594 A CN117720594 A CN 117720594A CN 202311796718 A CN202311796718 A CN 202311796718A CN 117720594 A CN117720594 A CN 117720594A
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- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 title claims abstract description 104
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Abstract
The invention relates to the technical field of oligosaccharide production, in particular to a method for preparing 2' -fucosyllactose by continuous crystallization, which comprises the following steps: filtering the microbial fermentation broth containing 2' -fucosyllactose to obtain a clear liquid; step two: decolorizing the clear liquid, and filtering to obtain feed liquid; step three: exchanging the feed liquid with ions; step four: concentrating the feed liquid in vacuum; step five: continuously concentrating and crystallizing the concentrated solution through a pre-crystallization system to obtain a saturated crystallization solution; adding the supersaturated crystallization solution and acetic acid into a crystallization device, and cooling and crystallizing to obtain a coarse product of 2' -fucosyl lactose crystals; step six: centrifuging the coarse product of the 2'-fucosyllactose crystal to obtain 2' -fucosyllactose crystal; step seven: purifying the 2'-fucosyllactose crystal to obtain the 2' -fucosyllactose product. The invention improves the crystallization yield of the 2'-fucosyllactose by continuous crystallization and improves the purity of the 2' -fucosyllactose product.
Description
Technical Field
The invention relates to the technical field of oligosaccharide production, in particular to a method for preparing 2' -fucosyllactose by continuous crystallization.
Background
Breast milk oligosaccharide (human milk oligosaccharides, HMOs) is the third largest solid component next to fat and lactose in breast milk, is an important efficacy factor in breast milk, has important biological functions, and plays an important role in the production and development of newborns. Comprehensive data from in vitro and in vivo experiments demonstrate that HMOs can reduce microbial infection by modifying host epithelial cell surface glycoforms and indirectly affect the infant immune system by modulating immune responses; furthermore, HMOs have proven to be essential nutrients for brain development, especially in the first months after birth of newborns.
Breast milk oligosaccharides consist of five basic monosaccharides, D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (L-Fuc, fuc) and sialic acid (Sia), all of which are linked to lactose by different glycosidic linkages, thus forming different structures of HMOs. 2'-fucosyllactose (2' -FL) is the most abundant HMOs in human milk, accounting for about 30% of the total HMOsThe sub-formula is C 18 H 32 O 15 The molecular weight is 488.44, is easy to dissolve in water, and has optical rotation and hygroscopicity. In terms of structural composition, 2' -FL is composed of a molecule of lactose at the reducing end and a molecule of fucose at the non-reducing end in succession under the action of alpha-1, 2 glycosidic bonds, whereas the lactose structure is composed of a molecule of glucose and a molecule of galactose in succession through beta-1, 4 glycosidic bonds. The 2' -fucosyllactose has remarkable influence on the regulation of intestinal flora, the adhesion resistance of pathogenic bacteria, the regulation of immune function and brain development, and has very important significance on the promotion of skeletal development.
The microbial fermentation method is a method for realizing industrial production of 2'-fucosyllactose and has economical efficiency and high efficiency, but the separation and purification of high-purity 2' -fucosyllactose from fermentation liquor is a key difficult problem due to the complex components of the fermentation liquor. In the prior art, the 2' -fucosyllactose is purified by adopting a method of ion exchange times for many times or using acetic acid to promote crystallization, the process of ion exchange for many times is complex, and as the active sites of the ion exchange resin are gradually occupied or blocked in the adsorption and elution processes, the purification efficiency is difficult to be ensured, and continuous purification cannot be realized; the method of using acetic acid to promote crystallization is difficult to improve the crystal yield and the crystal purity simultaneously on the premise of higher concentration of fucosyl lactose.
Disclosure of Invention
Aiming at the technical problems of low purification efficiency and low purification purity of 2' -fucosyllactose in the prior art of separating and purifying 2' -fucosyllactose from microbial fermentation liquor, the invention provides a method for preparing 2' -fucosyllactose by continuous crystallization, which comprises the steps of sequentially filtering, decoloring, ion-exchanging and vacuum concentrating fermentation liquor containing 2' -fucosyllactose to form supersaturated crystallization solution in a pre-crystallization system, adding the supersaturated crystallization solution and acetic acid into a crystallization device for cooling and crystallizing, and improving the crystallization yield and the crystal purity of the 2' -fucosyllactose by continuous crystallization.
The technical scheme of the invention is as follows:
a method for preparing 2' -fucosyllactose by continuous crystallization, comprising the steps of:
step one: sequentially filtering, decoloring and ion-exchanging the microbial fermentation broth containing 2' -fucosyllactose to obtain a feed liquid with conductivity less than or equal to 50us/cm and light transmittance more than or equal to 95%;
step two: concentrating the feed liquid obtained in the step one in vacuum to obtain a concentrated solution with the mass percent of total solid matters of 70-75%;
step three: the concentrated solution is subjected to pre-crystallization treatment, and the method comprises the following steps:
s1, flash evaporating the concentrated solution at the temperature of less than or equal to 70 ℃ and under the vacuum pressure of-0.08-0.09 MPa to obtain concentrated syrup with the mass percent of total solid matters of 70-85%;
s2, cooling the concentrated syrup to 50-65 ℃ in the stirring process to obtain pre-crystallized massecuite, wherein the stirring speed is 8-10r/min; mixing the upper layer part of the pre-crystallized massecuite with the concentrated solution obtained in the step two to form new concentrated solution, and mixing the rest of the pre-crystallized massecuite with the concentrated syrup obtained in the step S1 to form new concentrated syrup;
step four: repeating the first to third steps until the mass percentage of the total solids in the residual pre-crystallized massecuite reaches 80-85%;
step five: mixing the pre-crystallized massecuite with the mass percentage of the total solid reaching 80-85% with acetic acid with the volume of 1-2 times, and cooling and crystallizing to obtain a coarse product of 2' -fucosyl lactose crystals;
step six: centrifuging the coarse product of the 2'-fucosyllactose crystal obtained in the step five to obtain the 2' -fucosyllactose crystal;
step seven: and (3) purifying the 2'-fucosyllactose crystal obtained in the step (six) to obtain a 2' -fucosyllactose product.
In the first step, a filtering membrane is adopted for filtering, and the filtering membrane is a ceramic membrane and/or an ultrafiltration membrane. The filtering membrane is adopted to filter the microbial fermentation broth so as to remove microbial thalli in the microbial fermentation broth. Wherein the aperture of the ceramic membrane is 0.1-2um, and the aperture of the ultrafiltration membrane is 1000-10000Da.
Further, in the first step, the powder carbon is used for decoloring, the consumption of the powder carbon is 1-10%, the consumption of the powder carbon refers to the mass percent of the powder carbon to the dry basis of the solid, the decoloring temperature is 55-60 ℃, the decoloring time is 40-60min, the light transmittance of the microbial fermentation broth after decoloring is more than 80%, and the conductivity is 5000-7000us/cm. Macromolecules such as proteins, pigments and the like can be removed through decolorization; and (3) filtering the microbial fermentation broth by adopting a plate frame after decoloring.
Further, in the first step, a continuous ion exchange system is used for ion exchange. The pH value of the material liquid subjected to ion exchange is 3-6, and the components in the material liquid are relatively stable and are not easy to degrade. Anion exchange resin and cation exchange resin are used for ion exchange, and can be used in single column or mixed bed.
Further, in the second step, the temperature of vacuum concentration is less than or equal to 70 ℃, and the vacuum pressure of vacuum concentration is-0.08 to-0.09 MPa. Vacuum concentration may be performed in a multi-effect vacuum falling film evaporator. The concentration of the material liquid subjected to ion exchange in the multi-effect vacuum falling film evaporator can improve the concentration efficiency, and continuous feeding and discharging are realized.
In the fifth step, the initial temperature of the cooling crystallization is 50-65 ℃, the cooling rate is 0.05-0.3 ℃/h, and the discharging temperature is 25-35 ℃. According to the invention, the crude product of the 2'-fucosyllactose crystal is obtained by adding acetic acid for cooling crystallization, and the solubility of the 2' -fucosyllactose in water is reduced in the cooling process, so that the growth of the crystal is promoted.
In the sixth step, 2' -fucosyllactose crystals contain 2' -fucosyllactose, difucosyllactose and D-lactose, wherein the content of 2' -fucosyllactose is more than or equal to 98%, the content of difucosyllactose DFL is less than or equal to 1%, and the content of D-lactose is less than or equal to 1%.
Further, in the seventh step, the purification includes performing secondary ion exchange, secondary concentration and drying on the 2' -fucosyllactose crystal obtained in the sixth step in sequence.
The invention has the beneficial effects that:
the method for preparing the 2'-fucosyllactose by continuous crystallization comprises the steps of sequentially filtering and decoloring a microbial fermentation liquid containing the 2' -fucosyllactose, removing macromolecules such as microbial thalli, proteins and pigments in the microbial fermentation liquid, reducing the viscosity of the microbial fermentation liquid, improving the ion exchange efficiency in the subsequent steps and prolonging the service life of an ion exchange device; the decolorized and filtered feed liquid is subjected to ion exchange, so that ionic impurities in the feed liquid are removed, and the filtering property of the feed liquid is improved.
According to the invention, a method of continuous concentration crystallization and acetic acid cooling crystallization is adopted, and pre-crystallized massecuite with low crystal content is subjected to flash evaporation and cooling stirring circulation, so that the controlled supersaturation continuous vacuum concentration crystallization operation can be realized, and the yield of 2' -fucosyllactose is improved; acetic acid is added in the cooling process, the solubility of the 2' -fucosyl lactose in water is reduced due to the addition of the acetic acid and the reduction of the temperature, and the acetic acid is kept in a liquid state in the cooling crystallization process, so that the influence of the crystal growth process on the viscosity of the crude product of the 2' -fucosyl lactose crystal is reduced, and the crude product of the 2' -fucosyl lactose crystal is easier to separate by centrifugation. According to the invention, through regulating and controlling the technological parameters of cooling crystallization, the formation of larger-size crystals containing 2' -fucosyllactose is promoted, so that the 2' -fucosyllactose can still produce crystals at a lower discharge temperature under the premise of higher concentration, and the purity of the 2' -fucosyllactose in the crystals is improved while the yield of the crystals is improved.
The invention also purifies the 2' -fucosyllactose crystal to remove the organic solvent residue in the 2' -fucosyllactose crystal, and the obtained 2' -fucosyllactose product meets the application requirements of infant food and has larger industrial production and application values.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The microbial fermentation broths containing 2' -fucosyllactose used in examples 1-2 and comparative example 1 were obtained by fermentation with E.coli K-12 MG1655 BLBYZT6, from Bolborona Co., ltd, having a purity of 70.5%, and E.coli K-12 MG1655 BLBYZT6 was disclosed in invention patent CN 117089503B.
Example 1
A 2' -fucosyllactose product, the method of preparation comprising the steps of:
step one: firstly, filtering a microbial fermentation broth containing 2' -fucosyllactose by using ceramic membrane filtering equipment with the pore diameter of 0.1 mu m, and removing microbial thalli in the microbial fermentation broth to obtain filtrate; and then filtering the filtrate by using an ultrafiltration membrane with the aperture of 2000Da to remove macromolecules, thereby obtaining clear liquid.
Step two: and (3) decoloring the clear liquid obtained in the step (I) by using 8% of powdered carbon, wherein the decoloring temperature is 60 ℃, the decoloring time is 40min, and then filtering by using a plate frame to obtain the feed liquid with the light transmittance of 80.5%.
Step three: and (3) carrying out ion exchange on the feed liquid obtained in the step (II) by adopting a continuous ion exchange system, wherein the continuous ion exchange system uses D001 cation resin and D301-F anion resin with the cation-anion exchange resin ratio of 1:1.2. The conductivity of the material liquid after the ion exchange treatment is 7.2 mu s/cm, the light transmittance is 99.1%, and the pH value is 4.7.
Step four: and (3) carrying out vacuum concentration on the material liquid subjected to ion exchange by adopting a multi-effect vacuum falling film evaporator, wherein the temperature of the vacuum concentration is 50 ℃, and the vacuum pressure is-0.09 MPa, so as to obtain a concentrated solution with the mass percent of total solid matters of 73.5%.
Step five: the concentrated solution is subjected to pre-crystallization treatment, and the method comprises the following steps:
s1: pumping the concentrated solution into a feed inlet at the upper end of a flash tank from a discharge port of a multi-effect vacuum falling film evaporator, and carrying out flash evaporation and concentration in the flash tank to obtain concentrated syrup with the total solid content of 81%, wherein the vacuum pressure in the flash tank is controlled at-0.085 MPa, and the temperature is 65 ℃.
S2: discharging concentrated syrup from a discharge port at the lower end of the flash tank, and allowing the concentrated syrup to enter a pre-crystallization main tank provided with a stirrer from a feed port at the lower end of the pre-crystallization main tank through a connecting pipeline provided with a material preheater, wherein the temperature is reduced to 60 ℃ in the stirring process, and the pre-crystallization massecuite is obtained, and the stirring speed is 9r/min. Under the stirring action, the pre-crystallized massecuite in the pre-crystallization main tank flows at a low speed, is pushed to the upper end of the pre-crystallization main tank in a laminar flow mode, and when the pre-crystallized massecuite in the pre-crystallization main tank reaches 95% of the liquid level of the pre-crystallization main tank, a circulating pump at the upper part of the pre-crystallization main tank is started, the upper part of the pre-crystallized massecuite is pumped out from a discharge port at the upper end of the pre-crystallization main tank, is mixed with the newly obtained concentrated solution in the step four, and then is fed into a flash tank together with a feed port at the lower end of the flash tank for flash evaporation and concentration, and the residual pre-crystallized massecuite is left in the pre-crystallization main tank and is mixed with concentrated syrup newly entering the pre-crystallization main tank.
Step six: repeating the above operation and detecting the mass percent of the total solid matters of the syrup in the pre-crystallization main tank until the mass percent of the total solid matters of the syrup in the pre-crystallization main tank reaches 83 percent.
Step seven: pumping the pre-crystallized massecuite containing part of crystals in the pre-crystallization main tank into a horizontal cooling crystallization tank from a discharge hole at the lower end of the pre-crystallization main tank by using a discharge pump at the bottom of the pre-crystallization main tank, wherein 1 time of acetic acid with the purity of 95% by volume is added into the horizontal cooling crystallization tank in advance and preheated at the temperature of 60 ℃. And (3) carrying out program cooling crystallization after uniformly mixing the pre-crystallized massecuite and acetic acid in the horizontal cooling crystallization tank, wherein the initial temperature of the program cooling crystallization is 60 ℃, the cooling rate is 0.1 ℃/h, the discharging temperature of the 2'-fucosyl lactose crystal crude product is 35 ℃, and the temperature is kept for 10 hours after the 2' -fucosyl lactose crystal crude product is discharged.
Step eight: and (3) centrifuging the coarse product of the 2'-fucosyllactose crystal obtained in the step seven to obtain the 2' -fucosyllactose crystal. The crystal purity of the 2' -fucosyllactose crystal was measured by using a liquid chromatograph amino column, the crystal yield was calculated and the crystal size distribution was measured, and the measurement results are shown in table 1.
Step nine: and (3) adding water to dissolve the 2' -fucosyllactose crystals obtained in the step (eight) to prepare a feed liquid with the mass percentage of total solid matters of 30%. The prepared feed solution was ion exchanged with D001 cationic resin and D301-F anionic resin in a volume ratio of 1:1.5, and discharged at a flow rate of 2 BV/h. Concentrating the ion exchange discharged material to obtain secondary concentrated feed liquid with solid content of 60% by using a multi-effect vacuum falling film evaporator, and vacuum spray drying the obtained secondary concentrated feed liquid under negative pressure of-1.0X100 Pa at 150-160deg.C and exhaust temperature of 95-105deg.C. And drying to obtain a 2'-fucosyllactose powder product, wherein the 2' -fucosyllactose powder product comprises the following components in percentage by mass: 2' -fucosyllactose: 99.12%, DFL:0.12%, lactose: 0.76%.
Example 2
A 2' -fucosyllactose product, the method of preparation comprising the steps of:
step one: firstly, filtering a microbial fermentation broth containing 2' -fucosyllactose by using ceramic membrane filtering equipment with the pore diameter of 0.2 mu m, and removing microbial thalli in the microbial fermentation broth to obtain filtrate; and then filtering the filtrate by using an ultrafiltration membrane with the aperture of 5000Da to remove macromolecules, thereby obtaining clear liquid.
Step two: and (3) decoloring the clear liquid obtained in the step (I) by using 10% powdered carbon, wherein the decoloring temperature is 60 ℃, the decoloring time is 60min, and then carrying out plate and frame filtration to obtain the feed liquid with the light transmittance of 84.5%.
Step three: and (3) carrying out ion exchange on the feed liquid obtained in the step (II) by adopting a continuous ion exchange system, wherein the continuous ion exchange system uses D001 cation resin and D301-F anion resin with the cation-anion exchange resin ratio of 1:2. The conductivity of the material liquid after the ion exchange treatment is 5.2 mu s/cm, the light transmittance is 99.5%, and the pH is 5.6.
Step four: and (3) carrying out vacuum concentration on the material liquid subjected to ion exchange by adopting a multi-effect vacuum falling film evaporator, wherein the vacuum concentration temperature is 50 ℃, and the vacuum pressure is-0.09 MPa, so as to obtain a concentrated solution with the mass percent of total solid matters of 73.5%.
Step five: the concentrated solution is subjected to pre-crystallization treatment, and the method comprises the following steps:
s1: pumping the concentrated solution into a feed inlet at the upper end of a flash tank from a discharge port of a multi-effect vacuum falling film evaporator, carrying out flash evaporation concentration in the flash tank, controlling the vacuum pressure in the flash tank at-0.09 MPa and the temperature at 60 ℃, and obtaining concentrated syrup with the mass percent of total solids of 83%.
S2: discharging the concentrated syrup obtained after flash evaporation and concentration from a discharge port at the lower end of a flash evaporation tank, and introducing the concentrated syrup into a pre-crystallization main tank provided with a stirrer through a feed port at the lower end of the pre-crystallization main tank by a connecting pipe, wherein the stirring speed is 10r/min, and cooling to 50 ℃ in the stirring process. Under the stirring action, the pre-crystallized massecuite in the pre-crystallization main tank flows at a low speed, is pushed to the upper end of the pre-crystallization main tank in a laminar flow mode, and when the pre-crystallized massecuite in the pre-crystallization main tank reaches 94% of the liquid level of the pre-crystallization main tank, a circulating pump at the upper part of the pre-crystallization main tank is started, the upper part of the pre-crystallized massecuite is pumped out from a discharge port at the upper end of the pre-crystallization main tank, and is mixed with the newly obtained concentrated solution in the step four, and then is fed into a flash tank together with a feed port at the lower end of the flash tank for flash evaporation and concentration, and the residual pre-crystallized massecuite is left in the pre-crystallization main tank and is mixed with concentrated syrup newly entering the pre-crystallization main tank.
Step six: repeating the above operation and detecting the mass percent of the total solid matters of the syrup in the pre-crystallization main tank until the mass percent of the total solid matters of the syrup in the pre-crystallization main tank reaches 85 percent.
Step seven: pumping the pre-crystallized massecuite containing part of crystals in the pre-crystallization main tank into a horizontal cooling crystallization tank from a discharge hole at the lower end of the pre-crystallization main tank by using a discharge pump at the bottom of the pre-crystallization main tank, wherein 1.2 times of acetic acid with 99% purity by volume is added into the horizontal cooling crystallization tank in advance and preheated at 50 ℃. And (3) carrying out program cooling crystallization after uniformly mixing the pre-crystallized massecuite and acetic acid in the horizontal cooling crystallization tank, wherein the initial temperature of the program cooling crystallization is 50 ℃, the cooling rate is 0.2 ℃/h, the discharging temperature of the 2'-fucosyl lactose crystal crude product is 25 ℃, and the temperature is kept for 10 hours after the 2' -fucosyl lactose crystal crude product is discharged.
Step eight: and (3) centrifuging the coarse product of the 2'-fucosyllactose crystal obtained in the step seven to obtain the 2' -fucosyllactose crystal. The crystal purity of the 2' -fucosyllactose crystal was measured by using a liquid chromatograph amino column, the crystal yield was calculated and the crystal size distribution was measured, and the measurement results are shown in table 1.
Step nine: and (3) adding water to dissolve the 2' -fucosyllactose crystals obtained in the step (eight) to prepare a feed liquid with the mass percentage of total solid matters of 30%. The prepared feed solution was ion exchanged with D001 cationic resin and D301-F anionic resin in a volume ratio of 1:1.5, and discharged at a flow rate of 2 BV/h. Concentrating the ion exchange discharged material to obtain secondary concentrated feed liquid with solid content of 60% by using a multi-effect vacuum falling film evaporator, and vacuum spray drying the obtained secondary concentrated feed liquid under negative pressure of-1.0X100 Pa at 150-160deg.C and exhaust temperature of 95-105deg.C. And drying to obtain a 2'-fucosyllactose powder product, wherein the 2' -fucosyllactose powder product comprises the following components in percentage by mass: 2' -fucosyllactose: 99.01%, DFL:0.22%, lactose: 0.77%.
Comparative example 1
A 2' -fucosyllactose product, the method of preparation comprising the steps of:
step one: firstly, filtering a microbial fermentation broth containing 2' -fucosyllactose by using ceramic membrane filtering equipment with the pore diameter of 0.2 mu m, and removing microbial thalli in the microbial fermentation broth to obtain filtrate; and then filtering the filtrate by using an ultrafiltration membrane with the pore diameter of 3000Da to remove macromolecules, thereby obtaining clear liquid.
Step two: and (3) decoloring the clear liquid obtained in the step (I) by using 10% powdered carbon, wherein the decoloring temperature is 50 ℃, the decoloring time is 60min, and then carrying out plate and frame filtration to obtain the feed liquid with the light transmittance of 81.5%.
Step three: and (3) carrying out ion exchange on the feed liquid obtained in the step (II) by adopting a continuous ion exchange system, wherein the continuous ion exchange system uses D001 cation resin and D301-F anion resin with the cation-anion exchange resin ratio of 1:1.5. The conductivity of the material liquid after the ion exchange treatment is 6.7 mu s/cm, the light transmittance is 99.0%, and the pH is 5.1.
Step four: and (3) carrying out vacuum concentration on the material liquid subjected to ion exchange by adopting a multi-effect vacuum falling film evaporator, wherein the vacuum concentration temperature is 65 ℃, and the vacuum pressure is-0.085 MPa, so as to obtain the concentrated solution with the mass percent of total solid matters of 83.0%.
Step five: pumping the concentrated solution into a horizontal cooling crystallization tank, wherein 1 time of acetic acid with 99% purity by volume is added into the horizontal cooling crystallization tank in advance and preheated at 50 ℃. And (3) carrying out program cooling crystallization after the supersaturated crystallization solution in the horizontal cooling crystallization tank is uniformly mixed with acetic acid, wherein the initial temperature of the program cooling crystallization is 50 ℃, the cooling rate is 0.2 ℃/h, the discharging temperature of the 2'-fucosyl lactose crystal crude product is 25 ℃, and the temperature is kept for 10h after the 2' -fucosyl lactose crystal crude product is discharged.
Step six: and (3) centrifuging the coarse product of the 2'-fucosyllactose crystal obtained in the step seven to obtain the 2' -fucosyllactose crystal. The crystal purity of the 2' -fucosyllactose crystal was measured by using a liquid chromatograph amino column, the crystal yield was calculated and the crystal size distribution was measured, and the measurement results are shown in table 1.
Step seven: and D, adding water to dissolve the 2' -fucosyllactose crystals obtained in the step six to prepare a feed liquid with the mass percentage of total solid matters of 30%. The aqueous solution was ion exchanged with a D001 cationic resin and a D301-F anionic resin in a volume ratio of 1:1.5 and discharged at a flow rate of 2 BV/h. Concentrating the ion exchange discharged material to obtain secondary concentrated feed liquid with solid content of 60% by using a multi-effect vacuum falling film evaporator, and vacuum spray drying the obtained secondary concentrated feed liquid under negative pressure of-1.0X100 Pa at 150-160deg.C and exhaust temperature of 95-105deg.C. And drying to obtain a 2'-fucosyllactose powder product, wherein the 2' -fucosyllactose powder product comprises the following components in percentage by mass: 2' -fucosyllactose: 96.01%, DFL:2.22%, lactose: 1.77%.
TABLE 1 test results for crystals of 2' -fucosyllactose of examples 1-2 and comparative example 1
As can be seen from table 1, the preparation method disclosed in examples 1-2 adopts the method of the present invention to prepare 2'-fucosyllactose, and the crystal yield, crystal purity and crystal ratio of more than 100 mesh of the obtained crystals are all higher than those of the crystals obtained in comparative example 1, so that the preparation method disclosed in the present invention can significantly improve the crystal yield of 2' -fucosyllactose, increase the crystal size of 2'-fucosyllactose, and improve the crystal purity of 2' -fucosyllactose to more than 98%.
Although the present invention has been described in detail by way of the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. A method for preparing 2' -fucosyllactose by continuous crystallization, comprising the steps of:
step one: sequentially filtering, decoloring and ion-exchanging the microbial fermentation broth containing 2' -fucosyllactose to obtain a feed liquid with conductivity less than or equal to 50us/cm and light transmittance more than or equal to 95%;
step two: concentrating the feed liquid obtained in the step one in vacuum to obtain a concentrated solution with the mass percent of total solid matters of 70-75%;
step three: the concentrated solution is subjected to pre-crystallization treatment, and the method comprises the following steps:
s1, flash evaporating the concentrated solution at the temperature of less than or equal to 70 ℃ and under the vacuum pressure of-0.08-0.09 MPa to obtain concentrated syrup with the mass percent of total solid matters of 70-85%;
s2, cooling the concentrated syrup to 50-65 ℃ in the stirring process to obtain pre-crystallized massecuite, wherein the stirring speed is 8-10r/min; mixing the upper layer part of the pre-crystallized massecuite with the concentrated solution obtained in the step two to form new concentrated solution, and mixing the rest of the pre-crystallized massecuite with the concentrated syrup obtained in the step S1 to form new concentrated syrup;
step four: repeating the first to third steps until the mass percentage of the total solids in the residual pre-crystallized massecuite reaches 80-85%;
step five: mixing the pre-crystallized massecuite with the mass percentage of the total solid reaching 80-85% with acetic acid with the volume of 1-2 times, and cooling and crystallizing to obtain a coarse product of 2' -fucosyl lactose crystals;
step six: centrifuging the coarse product of the 2'-fucosyllactose crystal obtained in the step five to obtain the 2' -fucosyllactose crystal;
step seven: and (3) purifying the 2'-fucosyllactose crystal obtained in the step (six) to obtain a 2' -fucosyllactose product.
2. The method for preparing 2' -fucosyllactose by continuous crystallization according to claim 1, wherein in the first step, filtration is performed by using a filtration membrane, and the filtration membrane is a ceramic membrane and/or an ultrafiltration membrane.
3. The method for preparing 2' -fucosyllactose by continuous crystallization according to claim 1, wherein in the first step, powdered carbon is used for decolorization, the dosage of the powdered carbon is 1-10%, the decolorization temperature is 55-60 ℃, the decolorization time is 40-60min, the light transmittance of the decolorized microbial fermentation broth is more than 80%, and the electrical conductivity is 5000-7000us/cm.
4. A method for the continuous crystallization of 2' -fucosyllactose according to claim 1, wherein in step one.
5. The method for preparing 2' -fucosyllactose by continuous crystallization according to claim 1, wherein in the second step, the vacuum concentration temperature is less than or equal to 70 ℃ and the vacuum concentration vacuum pressure is-0.08 to-0.09 MPa.
6. The method for preparing 2' -fucosyllactose by continuous crystallization according to claim 1, wherein in the fifth step, the initial temperature of the cooling crystallization is 50-65 ℃, the cooling rate is 0.05-0.3 ℃/h, and the discharging temperature is 25-35 ℃.
7. The method for producing 2'-fucosyllactose by continuous crystallization according to claim 1, wherein in the step six, 2' -fucosyllactose crystals contain 2'-fucosyllactose, difucosyllactose and D-lactose, wherein the content of 2' -fucosyllactose is not less than 98%, the content of difucosyllactose DFL is not more than 1% and the content of D-lactose is not more than 1%.
8. The method for preparing 2'-fucosyllactose by continuous crystallization according to claim 1, wherein in the seventh step, the purification comprises subjecting the 2' -fucosyllactose crystals obtained in the sixth step to ion exchange, concentration and drying in sequence.
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