Production process and application of lactosucrose

Abstract

The invention discloses a production process and application of lactosucrose and relates to the technical field of lactosucrose preparation. According to the method, ion exchange resin is modified through polyethyleneimine, then beta-fructofuranosidase is loaded, then a lactosucrose synthesis reaction is conducted, polyethyleneimine contains a polar group amino and a hydrophobic group vinyl and has excellent adhesiveness and adsorbability, the specific surface area of the resin is greatly increased, the contact area of the resin and enzyme is increased, and the adsorption capacity of the resin is improved; the enzyme loading performance of the resin is improved, the enzyme activity is retained, byproducts in the lactosucrose production process are reduced, the synthesis reaction time is shortened, the stability is good, and the resin can be repeatedly used for multiple times. Meanwhile, the ultrafiltration and nanofiltration membrane separation technology greatly improves the purity of lactosucrose, has the advantages of simplicity in operation, high separation efficiency, no secondary pollution and low energy consumption, and avoids product loss caused by activated carbon adsorption.

Description

Technical Field

[0001] The invention relates to the technical field of lactofructo-oligosaccharide preparation, and in particular to a production process of lactofructo-oligosaccharide and application thereof. Background Art

[0002] Lactosucrose (LS) is a trisaccharide composed of glucose, galactose and fructose. Its flavor characteristics are similar to sucrose, and its sweetness is about 30% of sucrose. It is a natural sweetener that has the functions of improving intestinal flora, enhancing immunity, and promoting calcium absorption. Due to its low calories and difficulty in digestion and absorption by organisms, it has the effects of inhibiting fat accumulation and lowering cholesterol. It is a new type of functional oligosaccharide. It was listed in Japan's foods for specific health uses (FOSHU) in 2005 and is favored by consumers and many industries such as daily chemicals and medicine. According to statistics from Verified Market Research, the market size of lactosucrose reached US$343.5 million in 2021, and it is expected to grow at a rate of 6.2% from 2023 to 2030, reaching US$590.2 million in 2030.

[0003] However, the existing enzymatic method for producing oligosaccharides of lactofrucose is costly, the enzyme activity loss is large, the product purity is low, it contains many impurities, and it is difficult to separate and purify it later. In addition, the commonly used decolorizing agent activated carbon will adsorb some oligosaccharides in the micropores while adsorbing pigment substances, resulting in a decrease in product content. Therefore, the current industrial production is progressing slowly.

[0004] Therefore, how to obtain a production process of oligolactose and its application is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a production process of lactofructo-oligosaccharide and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A production process of lactofructo-oligosaccharide comprises the following steps:

[0008] (1) Pre-treat the ion exchange resin by soaking it in 0.5-1 mol / L hydrochloric acid for 6-8 hours, and then wash it with distilled water until it becomes neutral;

[0009] (2) adding the pretreated ion exchange resin to a polyethyleneimine solution and reacting the mixture under stirring to obtain a modified ion exchange resin;

[0010] (3) adding β-fructofuranosidase to the modified ion exchange resin and performing constant temperature adsorption. After the adsorption is completed, the supernatant is discarded, and the product is washed with a buffer until the protein cannot be detected, thereby obtaining an enzyme-loaded resin;

[0011] (4) adding sucrose and lactose to PBS buffer to obtain a mixed solution, adding enzyme-loaded resin to the mixed solution to perform a reaction to synthesize oligolactose to obtain a lactofructo-oligosaccharide reaction solution, removing the resin, and then placing the reaction solution in a boiling water bath for 10 min to inactivate the enzyme;

[0012] (5) Cooling to room temperature, performing ultrafiltration treatment, and then performing nanofiltration treatment on the obtained ultrafiltration permeate to obtain nanofiltration retentate, which is freeze-dried to obtain lactofructo-oligosaccharide.

[0013] Preferably, in step (2), the ion exchange resin is an ion exchange weakly alkaline anion exchange resin, the mass ratio of the ion exchange resin to polyethyleneimine is 1:3-4, the stirring rate is 800-1000 rpm, the reaction temperature is 35-40°C, and the reaction time is 6-8h.

[0014] The beneficial effects of the above technical solution are: the ionic charge force of the ion exchange resin has a better adsorption effect on the enzyme, and the weak alkalinity is not easy to inactivate β-fructofuranosidase.

[0015] The ion exchange resin is modified with polyethyleneimine, which contains polar amino groups and hydrophobic vinyl groups, and has excellent adhesion and adsorption properties, greatly increasing the specific surface area of ​​the resin, increasing the contact area between the resin and the enzyme, improving the enzyme loading performance of the resin, and retaining the activity of the enzyme.

[0016] Preferably, the activity U of β-fructofuranosidase in step (3) is the amount of enzyme required to catalyze the hydrolysis to produce 1 μmol glucose per minute at a temperature of 35°C and a pH of 6.5, and the unit is μmol / (mL·min).

[0017] Preferably, in step (3), the amount of β-fructofuranosidase added is 110-130 U / g modified ion exchange resin, the adsorption temperature is 25-40° C., the adsorption time is 6-10 h, and the reaction pH is 6-7.5.

[0018] Preferably, in step (4), the mass ratio of sucrose to lactose is 1:1, the mass fraction of sucrose in the mixed solution is 20-30%, and the mass fraction of lactose in the mixed solution is 20-30%.

[0019] Preferably, in step (4), the solid-liquid ratio of the enzyme-loaded resin to the mixed solution is 1:30-40 g / mL.

[0020] Preferably, in step (4), the reaction temperature is 30-45° C., the reaction time is 10-12 h, and the reaction pH is 6-8.

[0021] Preferably, the molecular weight cut-off of the ultrafiltration treatment in step (5) is 2500-3000 Da, and the operating pressure is 0.2-0.25 MPa.

[0022] Preferably, the molecular weight cut-off of the nanofiltration treatment in step (5) is 450-600 Da, and the operating pressure is 0.4-0.5 MPa.

[0023] As an inventive concept identical to the above technical solution, the present invention also requests protection for lactofructo-oligosaccharide prepared by the above production process.

[0024] As an inventive concept identical to the above technical solution, the present invention also requests protection for the application of the above lactofructo-oligosaccharide, wherein the application is for preparing medicines, health products or foods.

[0025] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a production process and application of oligosaccharide, which has the following beneficial effects:

[0026] (1) The present invention modifies the ion exchange resin with polyethyleneimine, then loads β-fructofuranosidase, and then performs a reaction to synthesize oligolactose. Polyethyleneimine contains polar amino groups and hydrophobic vinyl groups, has excellent adhesion and adsorption, greatly increases the specific surface area of ​​the resin, increases the contact area between the resin and the enzyme, improves the loading performance of the resin for the enzyme, retains the activity of the enzyme, reduces by-products in the process of producing oligolactose, shortens the synthesis reaction time, has good stability, and can be reused many times.

[0027] (2) The ultrafiltration and nanofiltration membrane separation technology of the present invention greatly improves the purity of oligolactose, has the advantages of simple operation, high separation efficiency, no secondary pollution, and low energy consumption, and avoids product loss caused by activated carbon adsorption.

[0028] (3) The present invention improves the utilization rate of raw materials, reduces the subsequent purification cost, and is suitable for large-scale industrial applications. DETAILED DESCRIPTION

[0030] The activity U of the β-fructofuranosidase used in the present invention is the amount of enzyme required to catalyze the hydrolysis to produce 1 μmol glucose per minute at a temperature of 35°C and a pH of 6.5, and the unit is μmol / (mL·min).

[0031] Example 1

[0032] A production process of lactofructo-oligosaccharide comprises the following steps:

[0033] (1) Pre-treat the ion exchange resin by soaking it in 1 mol / L hydrochloric acid for 8 h and then washing it with distilled water until it becomes neutral;

[0034] (2) adding the pretreated ion exchange resin to a polyethyleneimine solution and reacting the mixture under stirring to obtain a modified ion exchange resin;

[0035] (3) adding β-fructofuranosidase to the modified ion exchange resin and performing constant temperature adsorption. After the adsorption is completed, the supernatant is discarded, and the product is washed with a buffer until the protein cannot be detected, thereby obtaining an enzyme-loaded resin;

[0036] (4) adding sucrose and lactose to PBS buffer to obtain a mixed solution, adding enzyme-loaded resin to the mixed solution to perform a reaction to synthesize oligolactose to obtain a lactofructo-oligosaccharide reaction solution, removing the resin, and then placing the reaction solution in a boiling water bath for 10 min to inactivate the enzyme;

[0037] (5) cooling to room temperature, performing ultrafiltration treatment, and then performing nanofiltration treatment on the obtained ultrafiltration permeate to obtain a nanofiltration retentate, and freeze-drying to obtain lactofructo-oligosaccharide;

[0038] In step (2), the ion exchange resin is an ion exchange weakly alkaline anion exchange resin, the mass ratio of the ion exchange resin to polyethyleneimine is 1:3, the stirring rate is 800 rpm, the reaction temperature is 35° C., and the reaction time is 6 h;

[0039] In step (3), the amount of β-fructofuranosidase added is 110 U / g modified ion exchange resin, the adsorption temperature is 25° C., the adsorption time is 6 h, and the reaction pH is 6;

[0040] In step (4), the mass ratio of sucrose to lactose is 1:1, the mass fraction of sucrose in the mixed solution is 20%, and the mass fraction of lactose in the mixed solution is 20%;

[0041] In step (4), the solid-liquid ratio of the enzyme-loaded resin to the mixed solution is 1:30 g / mL;

[0042] In step (4), the reaction temperature is 30° C., the reaction time is 10 h, and the reaction pH is 6;

[0043] The molecular weight cut-off of the ultrafiltration treatment in step (5) is 2500Da and the operating pressure is 0.2Mpa;

[0044] The molecular weight cut-off of the nanofiltration treatment in step (5) is 450Da and the operating pressure is 0.4Mpa;

[0045] Example 2

[0046] A production process of lactofructo-oligosaccharide comprises the following steps:

[0047] (1) Pre-treat the ion exchange resin by soaking it in 1 mol / L hydrochloric acid for 8 h and then washing it with distilled water until it becomes neutral;

[0048] (2) adding the pretreated ion exchange resin to a polyethyleneimine solution and reacting the mixture under stirring to obtain a modified ion exchange resin;

[0049] (3) adding β-fructofuranosidase to the modified ion exchange resin and performing constant temperature adsorption. After the adsorption is completed, the supernatant is discarded, and the product is washed with a buffer until the protein cannot be detected, thereby obtaining an enzyme-loaded resin;

[0050] (4) adding sucrose and lactose to PBS buffer to obtain a mixed solution, adding enzyme-loaded resin to the mixed solution to perform a reaction to synthesize oligolactose to obtain a lactofructo-oligosaccharide reaction solution, removing the resin, and then placing the reaction solution in a boiling water bath for 10 min to inactivate the enzyme;

[0051] (5) cooling to room temperature, performing ultrafiltration treatment, and then performing nanofiltration treatment on the obtained ultrafiltration permeate to obtain a nanofiltration retentate, and freeze-drying to obtain lactofructo-oligosaccharide;

[0052] In step (2), the ion exchange resin is an ion exchange weakly alkaline anion exchange resin, the mass ratio of the ion exchange resin to polyethyleneimine is 1:4, the stirring rate is 1000 rpm, the reaction temperature is 40° C., and the reaction time is 8 h;

[0053] In step (3), the amount of β-fructofuranosidase added is 130 U / g modified ion exchange resin, the adsorption temperature is 40° C., the adsorption time is 10 h, and the reaction pH is 7.5;

[0054] In step (4), the mass ratio of sucrose to lactose is 1:1, the mass fraction of sucrose in the mixed solution is 30%, and the mass fraction of lactose in the mixed solution is 30%;

[0055] In step (4), the solid-liquid ratio of the enzyme-loaded resin to the mixed solution is 1:40 g / mL;

[0056] In step (4), the reaction temperature is 35° C., the reaction time is 10 h, and the reaction pH is 7;

[0057] The molecular weight cut-off of the ultrafiltration treatment in step (5) is 3000Da and the operating pressure is 0.25Mpa;

[0058] The molecular weight cut-off of the nanofiltration treatment in step (5) is 600Da, and the operating pressure is 0.5Mpa.

[0059] Example 3

[0060] A production process of lactofructo-oligosaccharide comprises the following steps:

[0061] (1) Pre-treat the ion exchange resin by soaking it in 1 mol / L hydrochloric acid for 8 h and then washing it with distilled water until it becomes neutral;

[0062] (2) adding the pretreated ion exchange resin to a polyethyleneimine solution and reacting the mixture under stirring to obtain a modified ion exchange resin;

[0063] (3) adding β-fructofuranosidase to the modified ion exchange resin and performing constant temperature adsorption. After the adsorption is completed, the supernatant is discarded, and the product is washed with a buffer until the protein cannot be detected, thereby obtaining an enzyme-loaded resin;

[0064] (4) adding sucrose and lactose to PBS buffer to obtain a mixed solution, adding enzyme-loaded resin to the mixed solution to perform a reaction to synthesize oligolactose to obtain a lactofructo-oligosaccharide reaction solution, removing the resin, and then placing the reaction solution in a boiling water bath for 10 min to inactivate the enzyme;

[0065] (5) cooling to room temperature, performing ultrafiltration treatment, and then performing nanofiltration treatment on the obtained ultrafiltration permeate to obtain a nanofiltration retentate, and freeze-drying to obtain lactofructo-oligosaccharide;

[0066] In step (2), the ion exchange resin is an ion exchange weakly alkaline anion exchange resin, the mass ratio of the ion exchange resin to polyethyleneimine is 1:3.5, the stirring rate is 1000 rpm, the reaction temperature is 40° C., and the reaction time is 8 h;

[0067] In step (3), the amount of β-fructofuranosidase added is 120 U / g modified ion exchange resin, the adsorption temperature is 35° C., the adsorption time is 8 h, and the reaction pH is 7;

[0068] In step (4), the mass ratio of sucrose to lactose is 1:1, the mass fraction of sucrose in the mixed solution is 20%, and the mass fraction of lactose in the mixed solution is 20%;

[0069] In step (4), the solid-liquid ratio of the enzyme-loaded resin to the mixed solution is 1:35 g / mL;

[0070] In step (4), the reaction temperature is 35° C., the reaction time is 10 h, and the reaction pH is 7;

[0071] The molecular weight cut-off of the ultrafiltration treatment in step (5) is 2800Da and the operating pressure is 0.25Mpa;

[0072] The molecular weight cut-off of the nanofiltration treatment in step (5) is 500Da, and the operating pressure is 0.5Mpa.

[0073] Comparative Example 1

[0074] Sucrose and lactose were added to PBS buffer to obtain a mixed solution, the mass ratio of sucrose to lactose was 1:1, the mass fraction of sucrose in the mixed solution was 20%, and the mass fraction of lactose in the mixed solution was 20%. β-Fructofuranosidase was added to the mixed solution, the amount of β-Fructofuranosidase was 600U / L mixed solution, and oligolactose was synthesized at a temperature of 35°C, a reaction time of 10h, and a reaction pH of 7 to obtain oligolactose reaction solution, which was then inactivated in a boiling water bath for 10min to obtain a crude lactofructo-oligosaccharide solution.

[0075] Comparative Example 2

[0076] Sucrose and lactose were added to PBS buffer to obtain a mixed solution, the mass ratio of sucrose to lactose was 1:1, the mass fraction of sucrose in the mixed solution was 20%, and the mass fraction of lactose in the mixed solution was 20%. β-Fructofuranosidase was added to the mixed solution, the amount of β-Fructofuranosidase was 500U / L mixed solution, and oligolactose was synthesized at a temperature of 35°C, a reaction time of 10h, and a reaction pH of 7 to obtain oligolactose reaction solution, which was then inactivated in a boiling water bath for 10min to obtain a crude lactofructo-oligosaccharide solution.

[0077] Comparative Example 3

[0078] Sucrose and lactose were added to PBS buffer to obtain a mixed solution, the mass ratio of sucrose to lactose was 1:1, the mass fraction of sucrose in the mixed solution was 20%, and the mass fraction of lactose in the mixed solution was 20%. β-Fructofuranosidase was added to the mixed solution, the amount of β-Fructofuranosidase was 500U / L mixed solution, and oligolactose was synthesized at a temperature of 35°C, a reaction time of 20h, and a reaction pH of 7 to obtain oligolactose reaction solution, which was then inactivated in a boiling water bath for 10min to obtain a crude lactofructo-oligosaccharide solution.

[0079] Experiment 1: Verification of the catalytic effect and stability of enzyme-loaded resin

[0080] 1.1 Catalytic effect

[0081] The oligolactose reaction liquid obtained in each embodiment and comparative example was tested for the content of oligolactose by RID-HPLC. The chromatographic conditions were acetonitrile: water = 75:25, flow rate 1 mL / min, and column temperature 30°C. The results are shown in Table 1. It can be seen that compared with the conventional method of directly adding β-fructofuranosidase, the present invention greatly shortens the synthesis reaction time while increasing the yield of oligolactose.

[0082] Table 1

[0083]

[0084]

[0085] 1.2 Stability Verification

[0086] Four groups of resins were set up, and the first to third groups were 0.1 g of the enzyme-loaded resins of Examples 1 to 3, respectively. The preparation method of the fourth group was as follows: directly adding β-fructofuranosidase to the ion exchange resin of unmodified polyethyleneimine for constant temperature adsorption, the addition amount of β-fructofuranosidase was 120 U / g ion exchange resin, the adsorption temperature was 35° C., the adsorption time was 8 h, and the reaction pH was 7; after the adsorption was completed, the supernatant was discarded, and the product was rinsed with buffer until no protein could be detected, to obtain 0.1 g of unmodified resin.

[0087] With 1 mol / L sucrose solution as substrate, 4 groups of resins were added respectively, and sucrose was hydrolyzed at 35°C and pH 6.5. After 1 hour of reaction, the reaction solution was removed, and the resin was rinsed with a phosphate buffer with a pH of 6.5 until sugar could not be detected. Then the substrate solution was added for the next reaction, and repeated 10 times in a row. After the first and last reactions were completed, the glucose content of the reaction solution was measured with a sugar meter, and the ratio of the last enzyme activity to the first was obtained by division, and the stability of the resin for repeated use was compared. The results are shown in Table 2. It can be seen from the analysis that the enzyme-loaded resin modified with polyethyleneimine in the present invention has excellent stability, can be reused many times, and maintains high enzyme activity.

[0088] Table 2

[0089] Group Relative enzyme activity (%) Example 1 91.3 Example 2 90.4 Example 3 93.8 Unmodified resin 24.6

[0090] Experiment 2: Verification of the effect of ultrafiltration and nanofiltration treatment

[0091] The main components of the liquid before ultrafiltration and the nanofiltration retentate in step (5) of each embodiment and the crude lactofructo-oligosaccharide liquid obtained in each comparative example were detected. The results are shown in Table 3:

[0092] Sugar detection method: Use high performance liquid chromatography to detect the product content. Filter the sample through a 0.45μm syringe filter into a liquid phase vial for testing. Chromatographic detection conditions: column temperature 30℃; mobile phase acetonitrile: water = 75:25; flow rate 1.0mL / min; injection volume 10μL; gas pressure 172.37kPa.

[0093] Protein concentration detection method: Use BCA protein concentration determination kit to determine the enzyme protein content in the feed solution.

[0094] Table 3

[0095]

[0096] Therefore, the present invention adopts a combination of ultrafiltration treatment and nanofiltration treatment, which greatly improves the purity of oligolactose, is simple to operate, has high separation efficiency, and reduces product loss.

[0097] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will comply with the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A production process of oligolactose, characterized in that: The following steps are involved: (1) Pre-treat the ion exchange resin by soaking it in 0.5-1 mol / L hydrochloric acid for 6-8 hours, and then wash it with distilled water until it becomes neutral; (2) adding the pretreated ion exchange resin to a polyethyleneimine solution and reacting the mixture under stirring to obtain a modified ion exchange resin; (3) adding β-fructofuranosidase to the modified ion exchange resin and performing constant temperature adsorption. After the adsorption is completed, the supernatant is discarded, and the product is washed with a buffer until the protein cannot be detected, thereby obtaining an enzyme-loaded resin; (4) adding sucrose and lactose to PBS buffer to obtain a mixed solution, adding enzyme-loaded resin to the mixed solution to perform a reaction to synthesize oligolactose to obtain a lactofructo-oligosaccharide reaction solution, removing the resin, and then placing the reaction solution in a boiling water bath for 10 min to inactivate the enzyme; (5) Cooling to room temperature, performing ultrafiltration treatment, and then performing nanofiltration treatment on the obtained ultrafiltration permeate to obtain nanofiltration retentate, which is freeze-dried to obtain lactofructo-oligosaccharide.

2. The production process of oligolactose according to claim 1, characterized in that: In step (2), the ion exchange resin is an ion exchange weakly alkaline anion exchange resin, the mass ratio of the ion exchange resin to polyethyleneimine is 1:3-4, the stirring rate is 800-1000 rpm, the reaction temperature is 35-40° C., and the reaction time is 6-8 hours.

3. The production process of oligolactose according to claim 1, characterized in that: In step (3), the amount of β-fructofuranosidase added is 110-130 U / g modified ion exchange resin, the adsorption temperature is 25-40° C., the adsorption time is 6-10 h, and the reaction pH is 6-7.

5.

4. The production process of oligolactose according to claim 1, characterized in that: In step (4), the mass ratio of sucrose to lactose is 1:1, the mass fraction of sucrose in the mixed solution is 20-30%, and the mass fraction of lactose in the mixed solution is 20-30%.

5. The production process of oligolactose according to claim 1, characterized in that: In step (4), the solid-liquid ratio of the enzyme-loaded resin to the mixed solution is 1:30-40 g / mL.

6. The production process of oligolactose according to claim 1, characterized in that: In step (4), the reaction temperature is 30-45° C., the reaction time is 10-12 h, and the reaction pH is 6-8.

7. The production process of oligolactose according to claim 1, characterized in that: The molecular weight cut-off of the ultrafiltration treatment in step (5) is 2500-3000Da, and the operating pressure is 0.2-0.25Mpa.

8. The production process of oligolactose according to claim 1, characterized in that: The molecular weight cut-off of the nanofiltration treatment in step (5) is 450-600Da, and the operating pressure is 0.4-0.5Mpa.

9. Lactofructo-oligosaccharide prepared by the production process according to any one of claims 1 to 8.

10. The use of lactofructooligosaccharide according to claim 9, characterized in that: The application is the preparation of medicines, health products or foods.

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