CN113248559B - Purification preparation method of siamenoside I - Google Patents

Abstract

The invention discloses a method for purifying and preparing high-purity natural sweetener Siamenoside I (Siamenoside I) from a biocatalytic reaction system. In particular to the removal of bacterial debris and protein, pigment removal and product purification preparation. The invention comprehensively applies a protein removing method and separation materials such as ion exchange resin, macroporous resin and the like, establishes a process for preparing high-quality siamenoside I from a biosynthesis system, and has the product yield of more than 70 percent. The purification preparation process only uses water and ethanol, has simple process, low cost and high safety, and is expected to be applied in industrialization.

Description

Purification preparation method of siamenoside I

Technical Field

The invention belongs to the technical field of biosynthesis, separation and purification of natural products, and particularly relates to biosynthesis, purification and preparation of mogroside I which is a natural sweetener of mogroside.

Background

After the 21 st century, with the increasing living standard of human beings, the number of people with three highs also shows explosive growth, wherein diabetes becomes a global chronic disease and occupies a large amount of medical resources. In recent years, people have come to appreciate the importance of low-sugar diets for health. Therefore, natural sweeteners such as stevioside, rubusoside, glycyrrhizic acid, rebaudioside a, mogroside and the like gradually enter the visual field of people, become substitute terms of the low-heat beverage additives and show extremely high commercial value.

Siamenoside I (siamenoside I) is a cucurbitane tetracyclic triterpenoid saponin derived from Momordica grosvenori, has a relative sweetness 460 and 563 times that of sucrose, tastes similar to sucrose, and has been identified by FDA as a "generally recognized as safe" (GRAS) ingredient. Currently, siamenoside I has become a hot compound for the development of new low-calorie sweeteners by many beverage and food companies. However, the natural content of siamenoside I is very low, less than 0.5%, and the scale production is difficult. The invention patent (CN112063678A) in the previous stage of the research group discloses a method for successfully biosynthesizing siamenoside I by adopting glycosyltransferase and metabolic flux to modify escherichia coli, and provides a feasible scheme for solving the problem of the source of siamenoside I. However, because of the particularity of the bacterial components, impurities such as proteins and water-soluble pigments are often incorporated into the purified and prepared siamenoside I, so that the quality of the product is difficult to control.

Aiming at the physicochemical properties of protein and pigment, the invention tries methods such as heating and boiling, activated carbon adsorption, ethanol precipitation, ion exchange, physical adsorption and the like, finally establishes a process for purifying and preparing high-quality siamenoside I, successfully removes the interference of the protein and the pigment, and provides a technology for the large-scale production of the siamenoside I.

Disclosure of Invention

The purification and preparation process of the siamenoside I obtained by biosynthesis is easily interfered by impurities such as bacterial protein, pigment and the like, and the siamenoside I with stable quality is difficult to obtain by conventional macroporous resin adsorption. Therefore, aiming at the characteristics of the protein and the pigment, the invention establishes a new purification and preparation process of siamenoside I, which is simple and convenient to operate, low in cost and suitable for large-scale application after systematically investigating a protein and pigment removal method.

The technical scheme of the invention is as follows:

1) constructing a biosynthetic system of siamenoside I according to the prior invention patent CN 112063678A;

2) investigating the adsorption condition of the activated carbon on the pigment and the protein in an aqueous solution or an ethanol/water mixed solution;

3) inspecting the adsorption condition of the nonionic macroporous resin on the pigment and the protein in an aqueous solution or an ethanol/water mixed solution;

4) inspecting the protein removal effect of boiling and ethanol precipitation;

5) investigating the effect of removing protein and pigment by the anion macroporous resin;

6) and establishing an optimal purification preparation process of the siamenoside I according to the process investigation result, and verifying. The method specifically comprises the following steps:

expanding a biosynthesis system of siamenoside I to 0.5-1L according to CN112063678A, after the reaction is finished, utilizing a dilute hydrochloric acid solution to adjust the pH value of a reaction liquid to 5.0-5.5, boiling or adding equal volume of ethanol, filtering to remove precipitates, adsorbing and removing proteins by using D101 macroporous resin, adjusting the pH value of a filtrate to about 7.0 by using a sodium hydroxide solution for color filtering, then removing pigments by using D900 macroporous weak-base anion exchange resin, and drying the collected eluent to obtain the high-quality siamenoside I.

Or after the reaction is finished, using a dilute hydrochloric acid solution to boil the pH value of the reaction liquid to 5.0-5.5, or adding equal volume of ethanol, filtering to remove precipitates, using a sodium hydroxide solution to adjust the pH value of the filtrate to about 7.0, then using D900 macroporous weak-base anion exchange resin to remove pigments, carrying out secondary adsorption on the collected eluent by using D101 macroporous resin, carrying out isocratic elution by using a 40% ethanol water solution, and drying the eluent to obtain the high-quality siamenoside I. The invention has the following advantages:

1) a process for preparing high-quality siamenoside I in a large scale from a biosynthesis system is established, and the method is theoretically suitable for the purification and preparation of other types of mogrosides;

2) toxic solvents are not used in the purification preparation process of the siamenoside I, so that the problems of process safety and product safety are avoided;

3) the D101 macroporous resin and the D900 macroporous weak-base anion exchange resin used in the purification preparation process of the siamenoside I can be recycled, so that the process cost is reduced;

4) the purification preparation process of siamenoside I established by the invention is simple to operate, good in repeatability and suitable for large-scale production.

Drawings

FIG. 1 is HPLC analysis of siamenoside I and mogroside IIIE in the reaction system

FIG. 2 shows adsorption/desorption rates of siamenoside I by different nonpolar macroporous resins

FIG. 3 shows the SDS-PAGE results of the purified siamenoside I

FIG. 4 shows HPLC detection results of pure siamenoside I

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 HPLC detection method of siamenoside I

The HPLC analysis of the siamenoside I sample provided by the invention has the following method parameters:

the instrument model is as follows: agilent 1260

A chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm)

Mobile phase A: water (containing 1 ‰ formic acid); mobile phase B: acetonitrile (containing 1 ‰ formic acid)

Column temperature: 30 ℃; flow rate: 1 mL/min; detection wavelength: 205 nm; sample introduction amount: 5 μ L

Elution gradient: 10% -90%; gradient time 25 min.

Under the above HPLC detection conditions, the retention times of mogroside IIIE and siamenoside I were 8.7min and 9.5min, respectively (FIG. 1).

Example 2 comparison of the biosynthesis of siamenoside I and the method for removing bacterial debris

A0.5L siamenoside I biosynthesis system is constructed by using a whole-cell biocatalyst in CN112063678A, wherein the concentration of a substrate mogroside IIIE is 15g/L, the concentration of sucrose is 500mg/mL, a reaction solution is an M9 culture medium with the pH value of 9.0, and the reaction time is 12 hours. After the reaction, the pH of the reaction solution was adjusted to 5.0 to 5.5 with dilute hydrochloric acid (0.1M) and divided into two equal portions (A and B). The reaction solution A was boiled for 5 minutes, and the protein and bacterial debris were removed by filtration. And adding the same amount of absolute ethyl alcohol into the reaction solution B, fully and uniformly mixing, standing for 5 minutes, and filtering to remove proteins and bacterial fragments. The obtained filtrate is yellowish and still contains protein through SDS-PAGE detection, and the two methods have no significant difference.

Example 3 adsorption examination of macroporous adsorbent resin

According to the literature report, the purification effect of five nonpolar macroporous resins D3520, HPD100, D101, HP20 and AB-8 on siamenoside I is examined. Firstly, respectively soaking macroporous resin in 95 percent ethanol solution for 8 hours, and washing the macroporous resin with distilled water until no ethanol smell exists; soaking in 5% NaOH solution for 4 hr, washing with distilled water to neutrality, soaking in 5% hydrochloric acid for 4 hr, and washing with distilled water to neutrality.

Weighing 5g of each of the five pretreated resins, placing the five pretreated resins in a shaking bottle, adding 40mL of boiled reaction liquid into each bottle, and oscillating and adsorbing for 12 hours at normal temperature. And (3) taking out the resin, carrying out oscillation desorption for 6 hours by using a 95% ethanol solution, measuring the contents of the siamenoside I in the reaction solution before and after adsorption and the desorption solution by using HPLC, and calculating the saturated adsorption amount, the adsorption and the desorption efficiency of each resin. As shown in FIG. 2, the adsorption rate and the resolution rate were significantly different from each other, although the 5 kinds of macroporous resins were able to adsorb siamenoside I. Wherein, the macroporous resin D101 has the highest adsorption rate to the siamenoside I, and the saturated adsorption amount reaches 59.2mg/g, and the desorption rate also reaches 92 percent. Therefore, macroporous resin D101 is selected as the purification material of siamenoside I. SDS-PAGE analysis showed that D101 substantially removed the protein but not the pigment from the solution. In contrast, siamenoside I in a siamenoside I solution containing 50% ethanol was not well adsorbed by the other macroporous resins described above.

Example 4 activated carbon depigmentation examination

Respectively standing and adsorbing the solution containing siamenoside I and the siamenoside I containing 50% ethanol obtained by resolving the D101 macroporous resin for 1 hour by using active carbon, and filtering to obtain colorless transparent solution. HPLC detection results show that the filtrate contains no siamenoside I, and the siamenoside I and the pigment are completely adsorbed by the activated carbon. Therefore, the removal of the pigment cannot be performed with activated carbon.

Example 5 comparison of anion exchange resin deproteinization and pigment removal methods

Since acidic or alkaline conditions are not favorable for stabilizing glycoside compounds, 2 reaction solutions obtained in example 2 were adjusted to pH 7.0 with sodium hydroxide solution. Under this pH condition, proteins and water-soluble pigments are negatively charged. Therefore, the invention selects D900 macroporous weak-base anion exchange resin and D201 macroporous strong-base styrene anion resin to remove protein and pigment.

Weighing 5g D900 column materials respectively, and completing the operation procedures of saturated NaCl soaking, deionized water cleaning, 3% HCl solution soaking, deionized water cleaning until the pH value is 6.0-7.0, 4% NaOH solution soaking, deionized water cleaning and the like according to the operation instructions so as to carry out column material pretreatment. Loading and eluting with water to obtain colorless transparent liquid containing siamenoside I, which is adsorbed on the column material but still contains very little protein. Meanwhile, the quality of the siamenoside I obtained by the D201 resin treated sample and the D900 resin treated sample is basically the same.

Example 6 combinatorial purification of siamenoside I1

50mL of the reaction solution A in example 2 was taken, the pH was adjusted to 5.0 with a dilute hydrochloric acid solution, and after boiling for 5min, the precipitate was removed by filtration to obtain a yellowish filter. Adjusting pH to 7.0 with dilute sodium hydroxide solution, loading to D900 macroporous weak base anion exchange resin, eluting with deionized water, collecting and mixing to obtain colorless solution containing siamenoside I. Loading the obtained product to macroporous resin D101, adsorbing for 12 hours, eluting by using 40% (v/v) ethanol solution, collecting the eluent containing siamenoside I, merging and drying to obtain the white siamenoside I solid. SDS-PAGE analysis showed that the siamenoside I product had no protein residue (FIG. 3); HPLC analysis indicated that the purity of siamenoside I was 96.5% (FIG. 4).

Example 7 combinatorial purification of siamenoside I2

50mL of the reaction solution A in example 2 was taken, the pH was adjusted to 5.0 with a dilute hydrochloric acid solution, and after boiling for 5min, the precipitate was removed by filtration to obtain a yellowish filter. Loading the obtained product onto macroporous resin D101, adsorbing for 12 hr, eluting with 40% (v/v) ethanol solution, collecting eluate containing siamenoside I, mixing, and adjusting pH to 7.0 with dilute sodium hydroxide solution. And then loading the sample to D900 macroporous weak-base anion exchange resin, eluting with deionized water, collecting and combining to obtain a colorless solution containing siamenoside I, and drying to obtain the siamenoside I white solid. SDS-PAGE and HPLC showed both protein and pigment removal (FIG. 3).

EXAMPLE 8 preparation of siamenoside I

When the reaction system was enlarged to 1L according to example 2 and siamenoside I was prepared according to the procedure described in example 6, 11.4g of a solid powder of high-purity siamenoside I free of proteins and pigments was obtained, the recovery rate of the sample was 76.53% and the purity was 96.4% (FIG. 3).

Claims (1)

1. A purification preparation method of siamenoside I is characterized by comprising the key steps of removing bacterial fragments, proteins and pigments, and the specific purification process is as follows:

1) adjusting the pH value of a whole-cell biosynthesis system for synthesizing siamenoside I by using mogroside IIIE as a substrate to 5.0-5.5 with diluted hydrochloric acid, boiling or adding absolute ethyl alcohol with the same volume, filtering or centrifuging to remove bacterial debris, and collecting reaction liquid;

2) adjusting the pH value of the reaction liquid collected in the step 1) to 7.0, removing pigments by adopting alkaline anion exchange resin, adsorbing the collected eluent by using nonpolar macroporous resin, eluting by using 40% ethanol water solution in volume fraction, and collecting the eluent;

3) or adsorbing the reaction liquid collected in the step 1) by using nonpolar macroporous resin, eluting by using 40% ethanol aqueous solution in volume fraction, collecting the eluent containing the siamenoside I, adjusting the pH to 7.0 by using a sodium hydroxide dilute solution, removing pigments by using alkaline anion exchange resin, wherein an elution solvent is deionized water, and collecting the eluent;

4) drying the eluent containing the siamenoside I finally collected in the steps 2) and 3) to obtain high-quality siamenoside I;

the nonpolar macroporous resin is D101 type macroporous resin;

the basic anion exchange resin is D900 macroporous weak-base anion exchange resin.

Images