A method for extracting manninotriose, raffinose and stachyose from camellia honey

Abstract

The application discloses a method for extracting manninotriose, raffinose and stachyose from oil tea honey, belongs to the technical field of component extraction and separation, and is used for oil tea honey, and comprises the following steps: (1) pretreating the oil tea honey; (2) carrying out decolorization and desalination on the pretreated oil tea honey; (3) carrying out stachyose separation based on molecular exclusion on the oil tea honey after decolorization and desalination; (4) carrying out resin column loading by using a boric acid type strong acid cation exchange resin, and obtaining raffinose and manninotriose by using gradient elution; and (5) carrying out vacuum concentration and freeze drying on the obtained stachyose, raffinose and manninotriose. The application can simultaneously obtain manninotriose, raffinose and stachyose from oil tea honey through the same process, reduces or avoids the use of a large amount of toxic organic solvents, and solves the technical bottleneck that homologous oligosaccharides are difficult to separate by combining the "ion exchange chromatography" series process.

Description

Technical Field

[0001] This application belongs to the field of component extraction and separation technology, and more specifically, it relates to a method for extracting mannotriose, raffinose and stachyose from camellia honey. Background Technology

[0002] Oligosaccharides such as raffinose and stachyose are important prebiotics, while mannotriose also has potential probiotic and immunomodulatory activities, both of which have important biological activities for maintaining gut health.

[0003] There are three main methods for obtaining raffinose, stachyose, and mannotriose in existing technologies. The first is through chemical synthesis, but this method is complex, has low yield, high cost, and is prone to producing byproducts, making it unsuitable for large-scale production in the food industry. The second method utilizes microbial fermentation, which involves fermentation with specific microorganisms. However, this method suffers from complex fermentation broth composition, difficulty in separation and purification, and potential biosafety risks. The third method involves extraction from natural plants: raffinose and stachyose can be extracted from legumes such as soybeans and lupins, but the extraction process typically involves steps such as organic solvent defatting, alcohol precipitation, and multiple column chromatography, resulting in complex processes and a high risk of organic solvent residue. Mannotriose is currently mainly obtained through small-scale extraction from plants such as mannitol and asparagus, or through enzymatic synthesis, which is extremely costly.

[0004] Meanwhile, the aforementioned existing technologies have several significant core shortcomings: First, there is no existing method for simultaneously extracting the three monomers, mannose, raffinose, and stachyose. Second, since raffinose and mannose both have a molecular weight of 504 Da and similar structures, conventional gel filtration, activated carbon column chromatography, or ordinary chromatography cannot effectively separate them. In other words, existing technologies cannot separate simultaneous isomers or sugars of the same molecular weight, and cannot obtain their respective monomers. Third, traditional technologies typically require the use of large amounts of organic solvents, such as acetonitrile and pyridine, which are unsuitable for large-scale production in the food industry.

[0005] Therefore, the applicant focuses on solving the above problems and proposes the technical solution of this application. Summary of the Invention

[0006] The purpose of this application is to provide a method for extracting mannose, raffinose and stachyose from camellia honey. This method can simultaneously obtain mannose, raffinose and stachyose from camellia honey through the same process, and reduces or avoids the use of large amounts of toxic organic solvents. It also solves the technical bottleneck of separating oligosaccharides of the same amount by combining a tandem process of ion exchange chromatography.

[0007] To achieve the above objectives, this application employs the following technical solution:

[0008] The method for extracting mannose, raffinose, and stachyose from camellia honey as described in this application includes the following steps:

[0009] (1) Pre-treatment of camellia oil honey;

[0010] (2) Decolorize and desalinate the pretreated camellia honey;

[0011] (3) Stachyose separation based on molecular exclusion was performed on the decolorized and desalted camellia honey;

[0012] (4) A boric acid-type strong acid cation exchange resin was used for resin packing, and raffinose and mannotriose were obtained by gradient elution.

[0013] (5) The obtained stachyose, raffinose and mannitol were concentrated under reduced pressure and freeze-dried.

[0014] In this application, the method for pretreating camellia honey in step (1) is as follows: mix camellia honey and deionized water in a mass ratio of 1:0.5 to 1:2 and stir thoroughly to dissolve; after stirring and dissolving, obtain a diluted solution, filter the diluted solution through a 0.45μm or 0.22μm microfiltration membrane, and collect the camellia honey filtrate.

[0015] In this application, the method for decolorizing and desalting the pretreated camellia honey in step (2) is as follows: the camellia honey filtrate collected in step (1) is passed through a chromatography column packed with activated carbon or macroporous adsorption resin at a flow rate of 1-5 BV / h for decolorization treatment, and the decolorized effluent is collected; the collected decolorized effluent is passed through a mixed column of anion and cation exchange resins to obtain a syrup solution.

[0016] In this application, the method for separating stachyose from decolorized and desalted camellia honey based on molecular exclusion in step (3) is as follows: the syrup solution obtained in step (2) is passed through a nanofiltration membrane with a molecular weight cutoff of 800 Da to elute the stachyose and obtain a stachyose solution, as well as a mixture of raffinose and mannotriose.

[0017] In this application, the boric acid-type strong acid cation exchange resin in step (4) is converted using the following method: First, select a 200-400 mesh Dowex 50WX8 resin matrix with a crosslinking degree of 8%, or an H+ type Amberlite IR-120 resin matrix, or a 001×7 (732) strong acid cation exchange resin. Wash thoroughly with deionized water until neutral. Then, soak and stir with 2 to 3 times the resin volume of 0.5M boric acid solution with a pH of 5.1 for 12 hours, changing the boric acid solution twice during the process. Next, wash away the free boric acid with deionized water until the conductivity of the effluent is <50 μS / cm.

[0018] In this application, the resin packing in step (4) includes: using a wet packing method to pack the chromatographic column, soaking and swelling the boric acid-type strong acid cation exchange resin in deionized water, then slowly pouring it into the column, while simultaneously flushing downwards with deionized water at a flow rate of 2 to 3 times the bed volume / h, so that the boric acid-type strong acid cation exchange resin settles naturally and evenly, avoiding air bubbles; after packing, equilibrate with 2 times the column volume of 0.1M boric acid solution until the pH of the effluent stabilizes at 5.0 to 5.5.

[0019] In this application, the gradient elution in step (4) includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05M boric acid aqueous solution with pH 5.0-5.2; mobile phase B is a 0.4M boric acid aqueous solution with pH 5.2-5.5; the obtained mixture of raffinose and mannotriose is prepared into a 20%-30% aqueous solution, injected into the column, and gradient elution is adopted. Raffinose is initially eluted with mobile phase A containing low concentration of boric acid, and then mannotriose is eluted with mobile phase B containing high concentration of boric acid.

[0020] In this application, the method for vacuum concentration and freeze-drying of the obtained stachyose, raffinose and mannotriose in step (5) is as follows: vacuum concentration is carried out at -0.09MPa to -0.095MPa and 40℃ to 45℃ to 1 / 10 to 1 / 5; the concentrate is transferred to a freeze-drying bottle, pre-frozen at -50℃ for 4h, and then dried at 0.1mbar to 0.2mbar vacuum for 48h; white powder products of stachyose, raffinose and mannotriose are obtained, weighed, sealed and stored in the dark.

[0021] In this application, the chromatographic column has a specification of φ2.6cm×100cm, and the flow rate is 1mL / min during the equilibration process using 2 column volumes of 0.1M boric acid solution after packing.

[0022] Compared with the prior art, the beneficial effects of this application are:

[0023] 1. This application is the first to attempt the simultaneous extraction of three oligosaccharide monomers from camellia honey. It not only obtains stachyose, but also overcomes the technical difficulty of separating raffinose and mannotriose due to their similar molecular weights. This helps to fill the gap in the field of obtaining the above three monosaccharides from camellia honey.

[0024] 2. The three monosaccharides obtained in this application have a purity of over 94%, and are characterized by high product purity and good quality. They can be used as analytical standards or high-purity functional food ingredients.

[0025] 3. The main separation process used in this application is water-based, with Prep-HPLC only used in the separation of raffinose and mannotriose. Food-grade ethanol can be used instead of acetonitrile, which helps to significantly reduce the toxicity introduced by solvents during the separation process and ensures the greenness and safety of the process. Furthermore, when using ion exchange chromatography, the participation of organic solvents can be completely avoided, achieving all-aqueous green production.

[0026] 4. Camellia oil honey is often discarded due to its unique taste and rapid crystallization. This application can turn camellia oil honey into a valuable resource, providing a new way to utilize camellia oil honey for high-value purposes, and has significant economic and social benefits.

[0027] 5. Both gel filtration and nanofiltration membranes used in this application can be industrialized and mass-produced, and can be linearly scaled up and are suitable for the production of high value-added products. Detailed Implementation

[0028] The technical solutions described in this application will be further described and illustrated below with reference to the embodiments.

[0029] Camellia oleifera honey naturally contains 1.44% mannose, 6.92% raffinose and 7.85% stachyose. The core method of this application is to first separate stachyose by utilizing molecular weight differences, and then separate raffinose and mannose monomers by utilizing structural or polarity differences, thereby achieving the purpose of simultaneously extracting three monosaccharides from camellia oleifera honey.

[0030] Example 1

[0031] 1. Pretreatment of camellia oil honey

[0032] First, take 500g of camellia oil honey, add 500mL of deionized water, and stir in a 40℃ water bath until completely dissolved.

[0033] After the camellia oil and honey have dissolved in deionized water, the resulting diluted solution is filtered through a 0.45 μm mixed cellulose ester microfiltration membrane to remove mechanical impurities and large molecular colloids. After this step, approximately 950 mL of filtrate can be collected.

[0034] 2. Decolorization and desalination treatment

[0035] After obtaining the filtrate, the filtrate was decolorized by passing it through a chromatography column packed with 200 mL of activated carbon at a flow rate of 2 BV / h, and the eluent was collected. The size of the chromatography column was φ2.5 cm × 40 cm.

[0036] The effluent was then passed through a chromatography column packed with 500 mL of mixed ion exchange resin to obtain a desalination solution. The mixed ion exchange resin chromatography column used a 1:1 volume ratio of 001×7 strong acidic cation exchange resin and D301 weakly basic anion exchange resin. This process and structure effectively removes organic acids, organic salts, and residual pigments. The conductivity of the desalination solution was 32 μS / cm, and the pH was 6.8.

[0037] The obtained desalted solution was treated and concentrated under reduced pressure at 50°C until the solid content was about 20%, yielding about 600 mL of syrup solution.

[0038] 3. Separation of stachyose based on size exclusion

[0039] The obtained syrup solution was separated using a nanofiltration membrane system. The membrane was constructed using a nanofiltration membrane module with a molecular weight cutoff of 800 Da and an area of ​​0.2 m². 2 The operating pressure is 1.5 MPa and the temperature is 25℃.

[0040] Collect the retentate to obtain approximately 180 mL of crude stachyose solution; collect the permeate for later use. The permeate contains raffinose, mannotriose, and monosaccharides.

[0041] 4. Separation of raffinose and mannotriose monomers from the permeate.

[0042] (1) The permeate is concentrated by nanofiltration to obtain a mixed component.

[0043] The permeate was passed through a nanofiltration membrane module with a molecular weight cutoff of 504 Da, and the pressure during operation was 1.8 MPa. Approximately 250 mL of retentate was collected to obtain a mixed component containing raffinose and mannotriose.

[0044] HPLC analysis revealed that the mass ratio of raffinose to mannotriose in the mixture was approximately 1:0.4, and the total sugar purity was approximately 82%.

[0045] (2) Preparation of boric acid type strong acid cation exchange resin

[0046] Take H +200g of type 200 to 400 mesh Dowex 50WX8 strong acid cation exchange resin was used and repeatedly washed with deionized water until neutral.

[0047] Dissolve boric acid in deionized water to obtain a 0.5M boric acid solution with a pH of 5.1. Add 500 mL of the solution to a Dowex 50WX8 strong acid cation exchange resin, soak and mechanically stir for 12 hours, replacing the solution twice at the same time intervals.

[0048] After soaking and stirring, the boric acid solution is removed, and the Dowex 50WX8 strong acid cation exchange resin is rinsed with plasma water until the conductivity of the effluent is <50 μS / cm, thus obtaining a boric acid-type strong acid cation exchange resin. This boric acid-type strong acid cation exchange resin can be used for direct column packing.

[0049] (3) Column installation and balancing

[0050] After obtaining the boric acid-type strong acid cation exchange resin, it was packed into a φ2.6cm×100cm glass chromatographic column using a wet packing method. The packing steps are as follows: First, about 1 / 3 volume of deionized water was added to the column, and the resin was slowly poured in while simultaneously flushing downwards at a flow rate of 2 times the bed volume / h to allow the resin to settle naturally and evenly, avoiding the formation of air bubbles. The final column height was approximately 90cm, and the column volume was approximately 480mL. The column was equilibrated with 2 column volumes (approximately 960mL) of 0.1M boric acid solution at a flow rate of 1mL / min until the pH of the effluent stabilized at 5.0–5.5.

[0051] (4) Sample preparation and loading

[0052] The mixed components containing raffinose and mannose were concentrated at 45°C to approximately 25% solids, yielding about 25 mL of concentrate containing approximately 6.2 g of the mixed sugars. The concentrate was injected entirely into the top of the column using a syringe, the outlet was opened, and the column wall was flushed with 5 mL of 0.05 M boric acid solution after the liquid level had dropped to the surface of the resin bed. Gradient elution was then initiated.

[0053] (5) Gradient elution and fraction collection

[0054] The system includes mobile phase A and mobile phase B. Mobile phase A is a 0.05M aqueous solution of boric acid with a pH of 5.1, which can be finely adjusted using dilute NaOH. Mobile phase B is a 0.04M aqueous solution of boric acid with a pH of 5.3.

[0055] Elution was performed according to the gradient program described in Table 1, with a flow rate of 1.0 mL / min during elution and a flow rate of 1.5 mL / min for the last 10 minutes of the equilibration phase. One 10 mL tube was collected every 10 minutes using an automated fraction collector, for a total of 240 tubes.

[0056] Table 1 Gradient elution program

[0057] 0 100 0 1.0 0～60 100→100 0→0 1.0 60～120 100→0 0→100 1.0 (Linear Gradient) 120～180 0 100 1.0 (Isocratically eluted mannitol) 180～210 0→100 100→0 1.5 (Fast Balancing) 210～240 100 0 1.0 (Rebalancing)

[0058] Take 50 μL from each collection tube and determine the total sugar content using the phenol-sulfuric acid method, then plot the elution curve. Based on the retention time of the standard (pre-calibrated with pure raffinose and pure mannotriose), combine tubes 5–7 (corresponding to elution times of 40–70 min) as the raffinose fraction, and combine tubes 14–16 (corresponding to elution times of 130–160 min) as the mannotriose fraction.

[0059] 5. Post-processing of products

[0060] (1) The collected 180 mL stachyose solution, 300 mL raffinose component, and 300 mL mannotriose component were placed in a rotary evaporator and concentrated under reduced pressure at 45 °C and -0.09 MPa to 1 / 8 to 1 / 10 of the original volume to obtain 20 mL stachyose concentrate, 35 mL raffinose concentrate, and mannotriose concentrate.

[0061] (2) Remove boric acid from raffinose concentrate and mannotriose concentrate.

[0062] Since both raffinose and mannotriose components contain boric acid, boron removal treatment is required. The boron removal process is as follows: The concentrated two components are passed separately through a φ2.6cm×60cm mixed ion exchange resin column packed with 001×7H⁺ and D301OH⁻ type resins in a 1:1 volume ratio and with a resin volume of 400mL, at a flow rate of 2mL / min. The eluent is collected, and the column is rinsed with 50mL of deionized water. The eluents are combined. Boric acid is detected using curcumin test paper, confirming that no residue is present.

[0063] (3) Freeze-drying

[0064] The obtained boron-free raffinose solution, mannotriose solution and stachyose solution were transferred to freeze-drying bottles, pre-frozen at -50℃ for 4 h, and then freeze-dried at 0.15 mbar vacuum for 48 h.

[0065] (4) Product yield and purity

[0066] After freeze-drying, three white powder products were obtained, including 2.7 g of mannotriose, 28.35 g of raffinose, and 32.27 g of stachyose. The HPLC purity of mannotriose was 94.9%, that of raffinose was 95.6%, and that of stachyose was 94.2%. The HPLC purity of mannotriose was determined under the following chromatographic conditions: amino column, acetonitrile:water = 70:30, flow rate 1 mL / min, and ELSD detection.

[0067] 6. Resin Regeneration

[0068] The boric acid ion exchange column used in this application can be regenerated with 2 column volumes of 0.5M boric acid solution at a flow rate of 1 mL / min, and then washed with deionized water until neutral. It can be reused at least 20 times without significant decrease in separation efficiency.

[0069] Finally, although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for extracting mannotriose, raffinose, and stachyose from camellia oil honey, characterized in that, The method includes the following steps: (1) Pre-treatment of camellia oil honey; (2) Decolorize and desalinate the pretreated camellia honey; (3) Stachyose separation based on molecular exclusion was performed on the decolorized and desalted camellia honey; (4) A boric acid-type strong acid cation exchange resin was used for resin packing, and raffinose and mannotriose were obtained by gradient elution. (5) The obtained stachyose, raffinose and mannitol were concentrated under reduced pressure and freeze-dried.

2. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 1, characterized in that, The method for pre-treating camellia honey in step (1) is as follows: mix camellia honey and deionized water in a mass ratio of 1:0.5 to 1:2 and stir thoroughly to dissolve; after stirring and dissolving, a diluted solution is obtained, and the diluted solution is filtered through a 0.45μm or 0.22μm microfiltration membrane to collect the camellia honey filtrate.

3. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 2, characterized in that, The method for decolorizing and desalting the pretreated camellia honey in step (2) is as follows: the camellia honey filtrate collected in step (1) is passed through a chromatography column packed with activated carbon or macroporous adsorption resin at a flow rate of 1-5 BV / h for decolorization treatment, and the decolorized effluent is collected; the collected decolorized effluent is passed through a mixed column of anion and cation exchange resins to obtain a syrup solution.

4. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 3, characterized in that, The method for separating stachyose from decolorized and desalted camellia honey based on molecular exclusion in step (3) is as follows: the syrup solution obtained in step (2) is passed through a nanofiltration membrane with a molecular weight cutoff of 800 Da to elute the stachyose and obtain a stachyose solution, as well as a mixture of raffinose and mannotriose.

5. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 4, characterized in that, The boric acid-type strong acid cation exchange resin in step (4) is converted using the following method: First, a 200-400 mesh Dowex 50WX8 resin matrix with a crosslinking degree of 8% or H is selected. + The AmberliteIR-120 resin matrix or 001×7 (732) strong acid cation resin was thoroughly washed with deionized water until neutral, and then soaked and stirred in 0.5M boric acid solution with pH 5.1 at 2 to 3 times the resin volume for 12 hours, during which the boric acid solution was replaced twice; then the free boric acid was washed away with deionized water until the conductivity of the effluent was <50μS / cm.

6. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 5, characterized in that, The resin packing in step (4) includes: using a wet packing method, soaking and swelling the boric acid-type strong acid cation exchange resin in deionized water, then slowly pouring it into the column, while simultaneously flushing downwards with deionized water at a flow rate of 2-3 times the bed volume / h, so that the boric acid-type strong acid cation exchange resin settles naturally and evenly, avoiding air bubbles; after packing, equilibrate with 2 times the column volume of 0.1M boric acid solution until the pH of the effluent stabilizes at 5.0-5.

5.

7. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 5, characterized in that, The gradient elution in step (4) includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05M boric acid aqueous solution with pH 5.0-5.2; mobile phase B is a 0.4M boric acid aqueous solution with pH 5.2-5.5; the obtained raffinose and mannotriose mixture is prepared into a 20%-30% aqueous solution, injected through the column, and gradient elution is adopted. Raffinose is initially eluted with mobile phase A with low concentration of boric acid, and then mannotriose is eluted with mobile phase B with high concentration of boric acid.

8. The method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 7, characterized in that, The method for vacuum concentration and freeze-drying of the obtained stachyose, raffinose and mannotriose in (5) is as follows: vacuum concentration is carried out at -0.09MPa to -0.095MPa and 40℃ to 45℃ to 1 / 10 to 1 / 5; the concentrate is transferred to a freeze-drying bottle and pre-frozen at -50℃ for 4 hours, and then dried at a vacuum of 0.1mbar to 0.2mbar for 48 hours; white powder products of stachyose, raffinose and mannotriose are obtained, weighed, sealed and stored in the dark.

9. A method for extracting mannotriose, raffinose, and stachyose from camellia honey according to claim 6, characterized in that, The chromatographic column has a specification of φ2.6cm×100cm. During the equilibration process after packing with 2 column volumes of 0.1M boric acid solution, the flow rate is 1mL / min.