CN110447414B

CN110447414B - Method for increasing content of mogroside V in momordica grosvenori

Info

Publication number: CN110447414B
Application number: CN201910776404.2A
Authority: CN
Inventors: 张玉华; 兰玉; 莫雪燕
Original assignee: Guilin Layn Natural Ingredients Corp
Current assignee: Guilin Layn Natural Ingredients Corp
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2022-04-05
Anticipated expiration: 2039-08-22
Also published as: CN110447414A

Abstract

A method for increasing the content of mogroside V in Momordica grosvenori fruits comprises treating the Momordica grosvenori fruits with an inducer before picking, wherein the inducer is trehalose, methyl jasmonate or sucrose, or methyl jasmonate. According to the invention, the mogroside V inducer directly acts on young fruits of the momordica grosvenori, so that the generation of mogrol and low-glycosylation mogroside thereof can be promoted, the conversion and accumulation of mogroside V are promoted, the action efficiency of the inducer is improved, and the content of the mogroside after treatment exceeds 50-100% of that of the contrast; meanwhile, the invention does not need to treat the tissue culture seedlings with strict environmental requirements and other plants in the growing and developing period, reduces the frequency of fruit treatment, has simple and effective treatment method and is suitable for industrial production.

Description

Method for increasing content of mogroside V in momordica grosvenori

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the content of mogroside V in momordica grosvenori.

Background

The momordica grosvenori is a traditional medicine-food dual-purpose plant in China, contains various active ingredients, and contains a strong-sweetness non-sugar material triterpene ingredient, namely momordica grosvenori glycoside, which is a main effective ingredient, wherein the content of the momordica grosvenori glycoside V is the most abundant. In 1996, the product is approved as a food additive in China, can be used as a sweetener to partially or completely replace cane sugar and can be widely used in various beverage foods, particularly as substitute sugar for patients with obesity and diabetes. Research make internal disorder or usurp shows that mogroside as a functional high-efficiency natural sweetener has wide application prospect.

The mogroside in the momordica grosvenori belongs to cucurbitane triterpenoid glycoside compounds, mainly comprises mogroside IV, mogroside V, siamenoside I and the like, wherein the content of the mogroside V is the highest and accounts for about 0.4-0.5% of the weight of fresh fruits. The price of the sweet glycoside V extracts with the content of 50 percent and 90 percent in the market is respectively as high as 4000 yuan/kg and 20000 yuan/kg, so the sweet glycoside V extracts can not compete with sucrose in the fields of food, health care products and daily health products.

At present, two methods for improving the content of mogroside V are mainly adopted: firstly, the content of mogroside V is increased by an extraction and purification method, such as Chinese patent CN106967142B 'a method for simultaneously extracting mogroside V, mogroside VI and 11-O-based glycoside V', and the like; second, the content of mogroside V in luo han guo is increased by using emerging mogroside V inducers, such as:

chinese patent CN106718920B discloses a method for increasing mogroside V content, which comprises the following steps: step one, dissolving methyl jasmonate in an ethanol water solution with the volume fraction of 20% to prepare ethanol mother liquor of the methyl jasmonate with the concentration of 50 mmol/L; dissolving methyl jasmonate in ethanol to prepare a methyl jasmonate solution with the concentration of 50-400 mu mol/L; secondly, inoculating the grosvenor momordica tissue culture seedling into a solid culture medium, wherein an ethanol mother solution of methyl jasmonate is added into the solid culture medium when the solid culture medium is prepared, so that the final concentration of the methyl jasmonate is 50-400 mu mol/L; and step three, transplanting the tissue culture seedlings into a field, spraying methyl jasmonate solution once in the morning, in the middle of the day and at night respectively 50-60 days after pollination of the momordica grosvenori, and continuously spraying for 10 days.

Chinese patent CN108277197A discloses a method for increasing the content of mogroside V in suspension cells of Siraitia grosvenori, which comprises the step of adding 10-200 mg/L of yeast inducer and 50-260 mg/L of methyl jasmonate into 5-11 days of the culture cycle of the suspension cells of Siraitia grosvenori respectively, so as to increase the synthesis rate and yield of mogroside V.

However, the use of the inducer has certain defects, seedlings and plants cannot be directly acted on the momordica grosvenori fruits by treating the seedlings and the plants with the methyl jasmonate, and the action efficiency of the inducer is not high; meanwhile, the inducer can simultaneously improve the content of the dulcoside II or VI, and the action target is not specific.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the content of mogroside V, which obviously improves the synthesis and accumulation of the mogroside V in the ripening process of the fruits of the momordica grosvenori.

The purpose of the invention is realized by the following scheme:

the invention provides a method for improving mogroside V content in momordica grosvenori, which is characterized in that young fruits of momordica grosvenori are treated by using an inducer before picking, wherein the inducer is trehalose, methyl jasmonate or sucrose and methyl jasmonate.

Preferably, the concentration of the inducer is 100-700 mM of trehalose and 5-20 mM of methyl jasmonate;

preferably, the concentration of the inducer is 100-700 mM of sucrose and 5-20 mM of methyl jasmonate;

preferably, the inducer is formulated as follows: dissolving 3.5-4.8 g of MS powder in 1000 ml of water to prepare an MS culture solution, and preparing a dulcoside V inducer containing trehalose, methyl jasmonate or sucrose and methyl jasmonate by using the MS culture solution;

preferably, the inducer is treated by smearing or spraying;

preferably, the treatment time of the inducer is 5-15 days after pollination;

preferably, the treatment time of the inducer is from 5 days to 30 days after pollination, and the total treatment times are 2-6 times;

preferably, the picking time is 70-90 days after pollination.

The invention overcomes the following technical problems:

according to the invention, trehalose and methyl jasmonate or sucrose and methyl jasmonate are applied when the momordica grosvenori is planted to directly act on momordica grosvenori fruits so as to induce the high expression of key enzymes (such as cucurbitadienol synthetase and cytochrome P450 enzyme) genes in the synthesis route of momordica grosvenori glycoside V, improve the synthesis of cucurbitadienol, promote the glycosylation of cucurbitadienol and the conversion of low-glycosylated sweet glycoside (such as sweet glycoside II) to sweet glycoside V, and thus the content of momordica grosvenori glycoside V is rapidly increased in a short time. The invention directly acts the inducer on the momordica grosvenori fruits, does not cause the products to generate substances harmful to human bodies, and has the acting concentration in a safe and controllable range.

The invention has the beneficial effects that:

1. the invention does not need to process the tissue culture seedlings with strict environmental requirements, reduces the frequency of fruit processing, has simple and effective processing method and is suitable for industrial production.

2. The inducer provided by the invention does not need to act on seedlings and plants, but directly acts on the momordica grosvenori fruits, so that the action efficiency of the inducer is improved, and the content of the mogrosides in the treated momordica grosvenori fruits exceeds 50-100% of that of the control.

3. The inducer of the invention has specific target, can promote the accumulation and transformation of other low-glycosylation sweet glycosides (such as sweet glycoside II and sweet glycoside IV) to the sweet glycoside V in the high-glycosylation fructus momordicae, only improves the content of the sweet glycoside V, and does not simultaneously improve the content of the sweet glycoside II or VI.

Drawings

FIG. 1 HPLC chart of dulcoside V standard.

FIG. 2 is an analytical HPLC chart of the content of dulcoside in a control sample.

FIG. 3 is a graph of analytical HPLC of the sweet glycoside content of trehalose treated samples.

FIG. 4 analytical HPLC plot of the sweet glycoside content of trehalose/methyl jasmonate treated samples.

FIG. 5 analytical HPLC plot of the sweet glycoside content of sucrose treated samples.

FIG. 6 analytical HPLC plot of the sweet glycoside content of sucrose/methyl jasmonate treated samples.

FIG. 7 effect of sucrose/methyl jasmonate treatment on mogroside V content.

Wherein, SUC500 is sucrose alone, and SUC500/MJ10 is sucrose combined with methyl jasmonate. FIG. 8 effect of trehalose/methyl jasmonate treatment on mogroside V content.

Wherein Tre500 is trehalose used alone, and Tre500/MJ10 is trehalose used in combination with methyl jasmonate.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Example one

A conventional MS culture medium was used to prepare a dulcoside V inducer containing 500mM trehalose and 10mM methyl jasmonate. Before picking, the momordica grosvenori fruits are treated by an inducer in a smearing or spraying mode, wherein the treatment frequency is 4 times; and (4) harvesting ripe fruits, and detecting the content of mogroside V by adopting an HPLC method.

Example two

A conventional MS culture medium was used to prepare a dulcoside V inducer containing 500mM sucrose and 10mM methyl jasmonate. Before picking, the momordica grosvenori fruits are treated by an inducer in a smearing or spraying mode, wherein the treatment frequency is 4 times; and (4) harvesting ripe fruits, and detecting the content of mogroside V by adopting an HPLC method.

EXAMPLE III

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 3.5 g of MS powder (Sigma # M0404) in 1000 ml of water. A sweet glycoside V inducer containing 100mM trehalose and 5mM methyl jasmonate is prepared by MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

fruits on day 5 after pollination were treated with mogroside V inducer. The treatment method adopts smearing, and the treatment is carried out for 2 times between 5 days and 30 days after pollination.

III, harvesting

And (4) harvesting mature fruits 70 days after pollination, and detecting the content of mogroside V by adopting an HPLC method.

Example four

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.8 g of MS powder (Sigma # M0404) in 1000 ml of water. A sweet glycoside V inducer containing 700mM trehalose and 20mM methyl jasmonate is prepared by MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

fruits at day 15 after pollination were treated with mogroside V inducer. The treatment method adopts smearing or spraying, and the treatment is carried out for 6 times between 5 days and 30 days after pollination.

III, harvesting

And (5) harvesting mature fruits 90 days after pollination, and detecting the content of mogroside V by adopting an HPLC method.

EXAMPLE five

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 3.5 g of MS powder (Sigma # M0404) in 1000 ml of water. A sweet glycoside V inducer containing 100mM sucrose and 5mM methyl jasmonate is prepared by MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

EXAMPLE six

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.8 g of MS powder (Sigma # M0404) in 1000 ml of water. A sweet glycoside V inducer containing 700mM sucrose and 20mM methyl jasmonate is prepared by MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

EXAMPLE seven

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.4 g of MS powder (Sigma # M0404) in 1000 ml of water. A sweet glycoside V inducer containing 500mM trehalose and 10mM methyl jasmonate is prepared by MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

fruits on day 10 after pollination were treated with mogroside V inducer. The treatment method adopts spraying, and the treatment is carried out for 4 times between 5 days and 30 days after pollination.

III, harvesting

And (4) harvesting mature fruits 80 days after pollination, and detecting the mogroside V content by adopting an HPLC method.

Example eight

Firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.4 g of MS powder (Sigma # M0404) in 1000 ml of water. An inducer of dulcoside V containing 500mM sucrose and 10mM methyl jasmonate was prepared from MS culture medium.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

Comparative examples 1 to 4 for evaluating the combined action of trehalose or sucrose and methyl jasmonate

Comparative example 1

Compared with the example 7, the trehalose is not added, and other conditions are the same, and the specific conditions are as follows:

firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.4 g of MS powder (Sigma # M0404) in 1000 ml of water. Preparing sweet glycoside V inducer containing methyl jasmonate 10mM by using MS culture solution.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

Comparative example 2

Compared with the example 8, the method has the following specific conditions without adding sucrose:

firstly, preparing a mogroside V inducer:

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

Comparative example 3

Compared with example 7, maltose is used instead of trehalose, and other conditions are the same, specifically as follows:

firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.4 g of MS powder (Sigma # M0404) in 1000 ml of water. An inducer of dulcoside V containing 500mM maltose and 10mM methyl jasmonate was prepared from MS culture medium.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

Comparative example 4

Compared with example 8, lactose replaces sucrose, and other conditions are the same, specifically as follows:

firstly, preparing a mogroside V inducer:

MS culture was prepared by dissolving 4.4 g of MS powder (Sigma # M0404) in 1000 ml of water. An inducer of dulcoside V containing 500mM lactose and 10mM methyl jasmonate was prepared from MS culture medium.

II, inducing momordica grosvenori sweet glycoside V:

III, harvesting

Comparative examples 5-8 for evaluating action time and frequency of mogroside V inducer

Comparative example 5

Compared with example 7, the inducer has earlier action time, and other conditions are the same, and the specific conditions are as follows:

firstly, preparing a mogroside V inducer:

II, inducing momordica grosvenori sweet glycoside V:

treating with mogroside V inducer 3 days after pollination, spraying, and treating for 4 times between 5 days and 30 days after pollination.

III, harvesting

Comparative example 6

firstly, preparing a mogroside V inducer:

II, inducing momordica grosvenori sweet glycoside V:

treating with mogroside V inducer on day 1 after pollination, spraying, and treating for 4 times between 5 days and 30 days after pollination.

III, harvesting

Comparative example 7

Compared with the example 7, the treatment frequency between 10 days and 30 days is improved, and other conditions are the same, and the specific conditions are as follows:

firstly, preparing a mogroside V inducer:

II, inducing momordica grosvenori sweet glycoside V:

fruits on day 10 after pollination were treated with mogroside V inducer. The treatment method adopts spraying, and the treatment is carried out for 10 times between 5 days and 30 days after pollination.

III, harvesting

Comparative example 8

firstly, preparing a mogroside V inducer:

II, inducing momordica grosvenori sweet glycoside V:

fruits on day 10 after pollination were treated with mogroside V inducer. The treatment method adopts spraying, and the treatment is carried out for 15 times between 5 days and 30 days after pollination.

III, harvesting

Results and analysis

The content of mogroside V in the harvested momordica grosvenoris in examples 1-8 and comparative examples 1-8 is detected by an HPLC method, and the result is shown in table 1, wherein the content of mogroside V in table 1 refers to the percentage of the mogroside V content in the dry weight of the fruit content.

TABLE 1 results of measurement of mogroside V content in examples 1 to 8 and comparative examples 1 to 8

In example 7, with trehalose added (at 500mM) compared to comparative example 1, the amount of dulcoside after fruit treatment was increased by nearly 40% compared to the control (comparative example 1), respectively. In example 8, when sucrose (500 mM) was added, the content of dulcoside after the treatment of the fruits was increased by 46% compared to the control (comparative example 2), respectively. Comparative example 3 Using maltose instead of trehalose (at a concentration of 500mM), the difference in the content of sweet glycosides after fruit treatment was significant compared to example 7. Comparative example 4 Using lactose instead of sucrose, the amount of sweet glycosides after fruit treatment was approximately doubled compared to example 8. Therefore, the trehalose or sucrose and methyl jasmonate are considered to act together to induce the high expression of the key enzyme gene in the metabolic synthesis pathway of mogroside V, so that the conversion of other low-glycosylated mogrosides (such as mogroside II and mogroside IV) to the accumulated glycoside V of the mogroside V in the high-glycosylated mogroside V can be promoted, and the content of the mogroside V can be rapidly increased in a short period.

In comparative examples 5 to 6, the action time of the inducer is advanced, but the induction effect disappears, in comparative examples 7 to 8, the treatment frequency is increased in the following 30 days, and the mogroside content is not further increased, so that the action time and frequency of trehalose, methyl jasmonate or sucrose and methyl jasmonate are also not negligible.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for improving mogroside V content in Momordica grosvenori is characterized in that an inducer is used for treating young fruits of Momordica grosvenori before picking, wherein the inducer is 100-700 mM trehalose and 5-20 mM methyl jasmonate, or 100-700 mM sucrose and 5-20 mM methyl jasmonate; the treatment time of the inducer is 5-15 days after pollination; the total times of the inducer treatment are 2-6 times.

2. The method of claim 1, wherein the inducing agent is formulated as follows: dissolving 3.5-4.8 g of MS powder in 1000 ml of water to prepare an MS culture solution, and preparing a dulcoside V inducer containing trehalose, methyl jasmonate or sucrose and methyl jasmonate from the MS culture solution.

3. The method of claim 1, wherein the inducing agent is applied by spreading or spraying.

4. The method of claim 1, wherein the inducing agent is administered from about 5 days to about 30 days after pollination.

5. The method for increasing mogroside V content of claim 1, wherein the picking time is 70-90 days after pollination.