TWI626313B - Method for increasing content of fructosylized mangiferin in plant extract - Google Patents

Abstract

A use of a lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for carrying out a biotransformation reaction of mangiferin and a glycosyl donor to generate fructosylized mangiferin. A specific embodiment of the use includes a method for increasing the content of fructosylated mangiferin in the extract of mango skin.

Description

Method for increasing content of fructosylized mangiferin in plant extract

The present invention relates to a method for increasing the content of fructosylated mangiferin in a mango peel extract using a deposit number BCRC 11697 ( Lactobacillus plantarum ), and a mangiferin using a deposit number BCRC 11697 lactic acid bacteria ( Lactobacillus plantarum ). Use of a biotransformation reaction with a glycosyl donor to produce fructosylated mangiferin.

Mangiferin is a carbanoside of tetrahydroxypyridone, which belongs to flavonoid flavonoids. It exists in a variety of plants, such as mango tree, almond tree, northeast gentian and timidum. Mangiferin molecular formula: C ₁₉ H ₁₈ O ₁₁ , molecular weight: 422.3. Mangiferin has a variety of biological activities and pharmacological effects, such as anti-oxidation, anti-tumor, immunoregulation, anti-diabetes, anti-inflammatory, etc. (Deng Jiagang, Yulan. Journal of Changchun University of Traditional Chinese Medicine, 2008, 24 (4): 463-464. ).

Although mangiferin has a wide range of pharmacological activities, its solubility is very poor, which severely limits the development of its preparations. In order to facilitate the application, the solubility and bioavailability of mangiferin need to be improved.

For natural active ingredients with complex structures, chemical synthesis for structural modification has the disadvantages of low yield, poor reaction specificity, and many by-products. In particular, some reactions are currently difficult to achieve using chemical means, but biotransformation technology can Make up for the lack of chemical synthesis. Cantagrel et al. (Publication number: FR 2882762 A1) glycosylated the mangiferin with a glucosyltransferase derived from two strains of Leuconostoc mesenteroides, where mangiferin is a glycosyl acceptor and sucrose is a glycosyl In vivo, glucosyl-β- (1,6) -mangiferin (CASRN: 908570-23-0) was obtained. When Leuconostoc mesenteroides NRRL B-1299 strain was used as a biocatalyst, the yield of glycosylation reaction was 25% under the conditions of 0.4g / L mangiferin and 40g / L sucrose; when Leuconostoc mesenteroides NRRL 521-F strain was used as a biocatalyst, Under the condition of 0.4g / L mangiferin and 40g / L sucrose, the yield of glycosylation reaction was 28%.

Chinese Patent Application Publication No. CN 102863484 A discloses a method for preparing fructosylized mangiferin, which method comprises adding a substance having fructosylase activity to a transformation solution containing mangiferin, and carrying out a biotransformation reaction to make mangiferin It is transformed into fructosylized mangiferin, and the transformed solution contains mangiferin and a glycosyl donor. The substance having fructosylase activity is a fermentation broth derived from Arthrobacter nicotianae and its deposit number is CCTCC M2010164.

In the aforementioned French Patent Application No. FR 2882762 A1 and Chinese Patent Application No. CN 102863484 A, purified mangiferin was used for fructosylation of mangiferin.

A main object of the present invention is to provide a method for directly carrying out a biotransformation reaction using a plant extract containing mangiferin as a substrate to convert the mangiferin into fructosylized mangiferin.

Another object of the present invention is to provide a suitable microorganism for carrying out a biotransformation reaction directly using a mangiferin-containing plant extract as a substrate to convert the mangiferin into fructosylated mangiferin.

Another object of the present invention is to provide a suitable microorganism and method for increasing the content of fructosylated mangiferin in the extract of mango skin.

A method for increasing the content of fructosylized mangiferin in a plant extract according to the present invention includes the following steps: providing a plant extract containing mangiferin; and adding a sugar-based donor to the plant extract, A biotransformation reaction is performed in the presence of a substance having fructosylase activity to convert the mangiferin to fructosylizing mangiferin, wherein the substance having fructosylase activity is passed through a lactic acid bacteria ( Lactobacillus plantarum ) having the deposit number BCRC 11697 Produced by fermentation.

Preferably, the plant extract is a mango peel extract.

Preferably, the glycosyl donor is sucrose or glucose. More preferably, the glycosyl donor is sucrose.

Preferably, the lactic acid bacteria ( Lactobacillus plantarum ) with the accession number of BCRC 11697 is added to the plant extract.

Preferably, a fermentation broth of the Lactobacillus plantarum deposited under the accession number BCRC 11697 is added to the plant extract.

Preferably, a fermentation supernatant of the lactic acid bacteria ( Lactobacillus plantarum ) deposited under the accession number BCRC 11697 is added to the plant extract.

Preferably, a fructosylase from a fermentation supernatant of the Lactobacillus plantarum deposited as BCRC 11697 is added to the plant extract.

Preferably, the biotransformation reaction is performed in a lactic acid bacteria culture medium at 20-40 ° C. for 0.5 to 96 hours without stirring or stirring speed not more than 400 rpm, and based on 100 mL of the culture medium, the glycosyl donor in an amount of 1-20 g, the amount of the extract of the plant is 1-60mL, and the amount of the lactic acid bacterium is ^{10 5 ~ 10 7 CFU / mL} .

The invention provides the use of a lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for carrying out a biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.

The invention provides the use of a fermentation broth with a deposit number of BCRC 11697 ( Lactobacillus plantarum ) for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.

The invention provides the use of a fermentation supernatant of lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.

The present invention provides the use of a fructosylase from a fermentation supernatant of a lactic acid bacterium ( Lactobacillus plantarum ) deposited under the deposit number BCRC 11697 for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylized mangiferin. .

Figure 1 shows the ultraviolet absorption spectra of fructosylated mangiferin standards at different wavelengths.

FIG. 2 is an ultraviolet light absorption spectrum of a supernatant liquid after centrifugal removal of lactic acid bacteria by Lactobacillus plantarum lactic acid bacteria transformation according to the present invention at different wavelengths.

Figure 3 is a liquid chromatography mass spectrometry (LC-MS) spectrum of a fructosylated mangiferin standard.

FIG. 4 is a LC-MS spectrum of a supernatant liquid after centrifugal removal of Lactobacillus plantarum lactic acid bacteria according to the method of the present invention.

Figure 5 shows the effect of different glycosyl donors on the conversion of mangiferin to the glycosylated mangiferin by the strain of the present invention.

FIG. 6 shows the comparison results of the sucrose glycosyl groups with different dosages (ratio) on the efficiency of glycosylated mangiferin converted from mangiferin by the strain of the present invention.

A preferred embodiment according to the present invention includes a method for increasing the content of fructosylated mangiferin in a mango peel extract, which mainly comprises adding a sugar-based donor to the mango peel extract, and A biotransformation reaction is performed in the presence of a substance having fructosyl converting enzyme activity to convert the mangiferin contained in the mango skin extract to fructosylized mangoside, wherein the substance having fructosyl converting enzyme activity is deposited It is produced by fermentation of Lactobacillus plantarum BCRC 11697. Through the above method, the mango skin can be more efficiently prepared into glycosylated mangiferin, which not only solves the source of mango skin when mango is made into fruit juice or dried mango or canned mango flesh when mango is in full production, which is beneficial to reducing environmental pollution. It also achieves the goal of using the whole fruit and improving the economic value of mango.

A method suitable for preparing a mango peel extract for use in the present invention includes (but is not limited to) the following steps: washing and pulverizing the mango peel; and then homogenizing the cellulase at room temperature to 50 ° C for 60-180 Minutes; drying at 60 ° C to remove moisture, and pulverizing into powder; mixing the powder with a polar solvent at a ratio of 1: 5 to 1: 20g / mL, and at room temperature to 50 ° C and preferably on ultrasonic With shaking, for example, at a power of 125 to 500 W / L, extraction is performed at 50 ° C. for 60 minutes, and the polar solvent is removed to obtain a peel extract containing mangiferin.

Suitable polar solvents such as water, C1-C3 alcohols, and mixtures thereof are preferably a mixture of ethanol and water, such as 50% (v / v) ethanol.

The aforementioned mango skin extract is a source of matrix material for transforming mangiferin by microorganisms according to the present invention, and the present invention uses cellulase or other similar enzymes capable of decomposing plant cell ridges to promote the extraction efficiency of intracellular materials. In addition, the extraction is supplemented by ultrasonic oscillation, which can further improve the extraction effect of flavonoids in mango skin. In the present invention, parameters such as the solvent ratio, ultrasonic power, and extraction time are studied to obtain a 50% (v / v) ethanol aqueous solution, ultrasonic power of 500 W / L, and extraction at 50 ° C for 60 minutes. A higher content of mango can be obtained. Mango peel extract rich in phenol and mangoside. In a preferred embodiment, the content of fructosylated mangiferin in the mango skin extract was quantitatively analyzed by HPLC as 18 μg / mL.

A suitable glycosyl donor for use in the present invention includes (but is not limited to) sucrose, glucose and fructose, with sucrose or glucose being preferred, and sucrose as optimal.

Preferably, in the biotransformation reaction of the method of the present invention, a fermentation supernatant of the lactic acid bacteria ( Lactobacillus plantarum ) with the accession number of BCRC 11697 is added to the mango skin extract. A suitable medium formula for lactic acid bacteria used for the BCRC 11697 includes: glucose 2 ~ 80g / L, peptone 5 ~ 50g / L, KH ₂ PO ₄ 0.4 ~ 4g / L, MgSO ₄ . 7H ₂ O 0.05 ~ 1.0g / L, MnSO ₄ H ₂ O 0.01 ~ 0.1g / L, lactic acid bacteria culture medium with pH 6 ~ 8.

The method of the present invention preferably further comprises purifying a product having an increased fructosylized mangiferin content obtained from the biotransformation reaction, for example, obtaining a water phase portion thereof by means of resin purification. This aqueous phase can be used directly or after concentration as an antioxidant, and it can be added to skin care products such as sunscreen or cosmetics. Alternatively, the fructosylized mangiferin can also be separated from the liquid portion by known purification methods to obtain a purified fructosylized mangiferin.

Examples Materials and Methods I. Materials and methods

1. Medium formula and strain culture

(1) Lactic acid bacteria medium (MRS medium)

MRS medium formulation: 1g Proteose peptone No. 3, 1g Beef extract, 0.5g Yeast extract, 2g glucose, 0.1g Tween 80 surfactant, 0.2 g of Ammonium citrate, 0.5 g of CH ₃ COONa, 10 mg of MgSO ₄ . 7H ₂ O, 5mg MnSO ₄ . H ₂ O, 0.2 g K ₂ HPO ₄ , and then make up the volume to 100 mL with water, adjust the pH to 6.5 and then autoclave. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 37 ° C, pH 6.5, and the culture was allowed to stand (0 rpm). When the OD ₆₀₀ of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.

(2) Bacterial medium (Nutrient medium)

Add 0.3 g of Beef extract and 0.5 g of peptone in sequence, then add water to make the volume reach 100 mL, adjust the pH to 7.0, and then autoclave. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 26 ° C., pH 7.0, and 100 rpm. When the OD ₆₀₀ of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.

(3) Yeast medium (YPD medium)

2g of peptone, 2g of glucose (Dextrose), and 1g of yeast extract (Yeast extract) were sequentially added, and then the water was replenished to a volume of 100 mL and then autoclaved. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 25 ° C., pH 7.0, and 100 rpm. When the OD ₆₀₀ of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.

(4) Filamentous fungal medium (malt extract)

2.0 g of glucose (Glucose), 1.0 g of peptone, and 2.0 g of malt extract were sequentially added, followed by rehydration to make the volume reach 100 mL, and then autoclaving. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. When the growth temperature is 24-30 ° C, pH 7.0, and 100 rpm, when the OD ₆₀₀ of the bacterial solution reaches 10, the bacterial cells are removed by centrifugation to obtain a supernatant.

2. Preparation of Mango Peel Extract

Wash and crush the mango skin, add cellulase at 2% (w / w), and hydrolyze at 50 ° C for 180 minutes, then dry it in an oven at 60 ° C to remove water, homogenize and pulverize it into mango skin powder The ultrasonic-assisted extraction process was carried out. The extraction conditions were 50% (v / v) ethanol, a material-liquid ratio of 1: 5, an ultrasonic power of 500 W / L, an ultrasonic frequency of 40 Hz, and an extraction temperature of 50 ° C. After extraction for 60 minutes, The ethanol was removed in a vacuum oven at 60 ° C to obtain a peel extract containing mangiferin.

3. Biotransformation reaction of fructosylated mango:

The bacterial solution, the mango peel extract, and an aqueous solution of a glycosyl donor were added to water or phosphate buffered saline (PBS), and a bioconversion reaction was performed at 37 ° C to convert mangiferin to fructosylated mangiferin The reaction time is 96 hours. The bacterial solution was added at 10% of the total conversion reaction solution volume, the mango peel extract was added at 10% of the total conversion reaction solution volume, and the glycosyl donor aqueous solution was added at 100 mL of the conversion reaction solution. Contains 0 to 20 grams of glycosyl (0 to 20 w / v%). The content of fructosylated mangiferin was analyzed by high performance liquid chromatography.

The above-mentioned steps of the biotransformation reaction are repeated except that the aforementioned bacterial solution is not added, and data of a control group used as a control is obtained.

4. Quantification of fructosylated mangiferin by high performance liquid chromatography (HPLC)

Weigh precisely 1 mg of fructosylated mangiferin (Fructosylated mangiferin) standard product, dissolved with 50% ethanol to make a reference solution containing 0.01 ~ 1mg in 1mL. The test sample is the liquid after the completion of the conversion reaction. Centrifuge at 8000 rpm for 5 minutes, take the supernatant, and apply 1 mL of the sample solution to a HPLC Atlantis T3 column (C18, 4.6 mm inner diameter × 250 mm, 5 μm particle size). The solution was acetonitrile: 1% glacial acetic acid solution (15:85), the reaction temperature was 25 ° C, the flow rate was set to 1.0 mL / min, the detector wavelength was set to 254 nm, and the injection sample volume was 20 μl. Aspirate the sample with a syringe and filter it through 0.22um PVDF for quantitative HPLC analysis (Guo Lingling, Zhang Tab, Liu Erwei, Han Lifeng, Wang Tao. 2013. Determination of mangiferin content in mango leaves. Journal of Tianjin University of Traditional Chinese Medicine, 32 ( 1): 43-45).

5. Identification of fructosylated mangiferin by liquid chromatography mass spectrometry (LC-MS)

A ZORBAX Eclipse XDB-C18 Narrow Bore RR column (2.1 x 150 mm, 3.5 μm) was used. The mobile phase was a 0.02% formic acid aqueous solution and a 0.02% formic acid methanol solution, respectively, and the flow rate was set to 0.2 ml / min. The dissolution process is as follows:

Micromass ZQ Mass Spectrometer with Electrospray Free Mass Spectrometer (eletrospray Ionozation, ESI-MS), the Cone voltage was 10V, 15V, 20V, 35V, 50V.

Results and discussion

1. Screening of potential strains that convert mangiferin to fructosylized mangiferin

Preliminary screening of fructosylase activity from lactic acid bacteria, bacteria, yeasts and filamentous fungi in the online catalog of biological resources of the Center for Biological Resources Conservation and Research (BCRC) of the Food Industry Development Research Institute (Hsinchu City, Taiwan) (β-fructofuranosidase, fructosyl transferase) or potential strains with tyrosinase inhibitory activity, there are 2 strains of bacteria, 1 strain of yeast, 10 strains of lactic acid bacteria, and 6 strains of filamentous fungi, 19 potential bacteria. The various strains were activated according to the aforementioned methods, biotransformation reactions were performed, and the fructosylated mangiferin content was analyzed by high performance liquid chromatography. The fruits are listed in Table 1. From the results in Table 1, we can see that four of them have potential, namely 2 bacteria Arthrobacter nicotiande BCRC 11219, Arthrobacter globiforms BCRC 10598, 1 yeast Saccharomyces cerevisiae BCRC 20855, and 1 lactic acid bacteria Lactobacillus plantarum BCRC 11697. Based on industrial applicability, GRAS strains and those with high fructosylated mangiferin content were used as targets. Therefore, Lactobacillus plantarum was selected as a potential strain for use in the present invention.

2. Analysis of the ability of different Lactobacillus plantarum strains to produce fructosylated mangiferin

Except L. plantarum strains were collected, respectively L.plantarum BCRC 11697, BCRC 12327, BCRC 14059, BCRC 15478, BCRC 17178, BCRC 17638, according to the method of activated each strain, biotransformation reactions, and high-performance liquid layer Analytical method to analyze the content of fructosylated mangiferin, the fruit is listed in Table II. As shown in Table 2, Lactobacillus plantarum BCRC 11697 has the best ability to transform to fructosylated mangiferin.

3. UV light absorption spectrum analysis results

Figure 1 and Figure 2 are UV absorption spectra of 500.0 g / mL standard solution of fructosylated mangiferin and liquid products obtained after bioconversion reaction with Lactobacillus plantarum BCRC 11697 at 200.0-400.0 nm. From Figs. 1 and 2, it can be seen that the peak shape of the absorption curve of the fructosylated mangiferin standard is the same as that of the liquid product obtained after the bioconversion reaction with Lactobacillus plantarum BCRC 11697, and all of them are at the wavelengths of 238, 254, 317, and 367 nm, respectively. There are absorption peaks, and the absorption peak at 254 nm is the strongest. Therefore, in the present invention, the measurement wavelength of the fructosylized mangiferin content is 254nm as the standard of fructosylized mangiferin and the liquid product obtained after the bioconversion reaction is performed.

4. LC-MS analysis results

3 and 4 are LC-MS spectra of a fructosylated mangiferin standard and a supernatant obtained by centrifuging a bacterial cell after bioconversion reaction with Lactobacillus plantarum BCRC 11697, respectively. Comparing the LC-MS spectra of FIG. 3 and FIG. 4, it can be seen that the transformant obtained after carrying out the biotransformation reaction by the method of the present invention does indeed contain fructosylated mangiferin.

5. Compare the effects of different glycosyl donors on the transformation of strains to fructosylated mangiferin

The results of the bioconversion reaction using Lactobacillus plantarum BCRC 11697 and different glycosyl donors (sucrose, glucose, fructose) in the aforementioned method are shown in FIG. 5. It can be seen that sucrose has the highest fructosylated mangiferin conversion efficiency. When the conversion time is within 24 hours, the fructosylated mangiferin content can reach a relatively high point of 35 μg / mL.

6. Explore the comparison of conversion efficiency of fructosylated mangiferin with different ratios of sucrose sugar groups

Figure 6 compares the effect of fructosylated mangiferin conversion efficiency with the addition of sucrose sugar groups at 0.5% to 20% (w / v). The control group was the result of transformation without the addition of Lactobacillus plantarum BCRC 11697 and the addition of only 0.5% glycosyl groups. The fructosylated mangiferin content was 25 μg / mL at 0 hours. From the results in FIG. 6, it can be known that when the sucrose sugar concentration is high (20%) and the low concentration (0.5%), the bioconversion reaction time is 6 hours, and the content of fructosylated mangiferin can reach a relatively high level. value. In addition, the average conversion efficiency of the 0.5% sucrose glycosyl group was better than other high-concentration groups. The conditions of 0.5% glycosyl and 6-hour transformation in Fig. 6 have the highest fructosylized mangiferin content, which can reach 57 μg / mL more than double the control group's 25 μg / mL.

Claims (15)

A method for increasing the content of fructosylized mangiferin in a plant extract, comprising the following steps: providing a plant extract containing mangiferin; and adding a glycosyl donor to the plant extract, in a plant having fructosylation A biotransformation reaction is performed in the presence of an enzyme-active substance to convert the mangiferin to fructosylized mangiferin, wherein the substance having fructosylase activity is fermented by a lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 produce. The method according to claim 1, wherein the plant extract is a mango peel extract. The method of claim 1, wherein the glycosyl donor is sucrose or glucose. The method of claim 1, wherein the glycosyl donor is sucrose. The method according to any one of claims 1 to 4, wherein the lactic acid bacteria ( Lactobacillus plantarum ) having the deposit number BCRC 11697 is added to the plant extract. The method according to any one of claims 1 to 4, wherein a fermentation broth of Lactobacillus plantarum having a deposit number of BCRC 11697 is added to the plant extract. The method according to any one of claims 1 to 4, wherein a fermentation supernatant of Lactobacillus plantarum having the deposit number BCRC 11697 is added to the plant extract. The method according to any one of claims 1 to 4, wherein a fructosylase from a fermentation supernatant of the lactic acid bacteria ( Lactobacillus plantarum ) deposited under the deposit number BCRC 11697 is added to the plant extract . The method according to claim 5, wherein the biotransformation reaction is performed in a lactic acid bacteria culture medium at 20-40 ° C and without stirring or the stirring speed is not more than 400 rpm for 0.5 to 96 hours, and based on 100 mL of the culture medium, the sugar The amount of the base donor is 1-20 g, the amount of the plant extract is 1-60 mL, and the amount of the lactic acid bacteria is 10 ⁵ to 10 ⁷ CFU / mL. The method of claim 6, wherein the biotransformation reaction is performed in a lactic acid bacteria culture medium at 20-40 ° C and without stirring or the stirring speed is not more than 400 rpm for 0.5 to 96 hours, and based on 100 mL of the culture medium, the sugar The amount of the base donor is 1-20 g, the amount of the plant extract is 1-60 mL, and the amount of the fermentation broth is 1-60 mL. The method of claim 7, wherein the biotransformation reaction is performed in a lactic acid bacteria culture medium at 20-40 ° C. for 0.5 to 96 hours without stirring or the stirring speed is not greater than 400 rpm, and based on 100 mL of the culture medium, the sugar The amount of the base donor is 1-20 g, the amount of the plant extract is 1-60 mL, and the amount of the fermentation supernatant is 1-60 mL. A use of a lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for carrying out a biotransformation reaction of mangiferin and a glycosyl donor to generate fructosylized mangiferin. A use of a fermentation broth with a deposit number of BCRC 11697 ( Lactobacillus plantarum ) for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin. A use of a fermentation supernatant of lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylized mangiferin. A use of fructosylase purified from a fermentation supernatant of a lactic acid bacteria ( Lactobacillus plantarum ) deposited under the accession number BCRC 11697 to perform a biotransformation reaction of mangiferin and a glycosyl donor to generate fructosylized mangiferin.