阅读说明：本技术 一种季也蒙毕赤酵母及分泌的胞外蛋白的应用 (Pichia guilliermondii and application of secreted extracellular protein ) 是由 王鸿江 谢海峰 谢期林 胡云岭 于 2019-04-03 设计创作，主要内容包括：本发明为一种季也蒙毕赤酵母及分泌的胞外蛋白的应用,涉及微生物技术领域,具体涉及一种利用季也蒙毕赤酵母高效合成赛门苷I的方法；所述的应用通过季也蒙毕赤酵母(<I>Meyerozyma guilliermondii</I>)的分泌的胞外蛋白实现催化功能；该胞外蛋白具有较强的催化活性,具有催化效率高,反应条件温和,特异性强等特点,有望为高效合成赛门苷I的理想催化剂,拓宽赛门苷I的来源。(The invention relates to application of pichia guilliermondii and secreted extracellular protein, relates to the technical field of microorganisms, and particularly relates to a method for efficiently synthesizing siamenoside I by using pichia guilliermondii; the application is realized by using pichia guilliermondii ( Meyerozyma guilliermondii ) The secreted extracellular protein realizes a catalytic function; the extracellular protein has the characteristics of strong catalytic activity, high catalytic efficiency, mild reaction conditions, strong specificity and the like, is expected to be an ideal catalyst for efficiently synthesizing the siamenoside I, and widens the source of the siamenoside I.)

1. Pichia guilliermondii (A. guilliermondii)Meyerozyma guilliermondii) The application of the compound serving as a catalyst in the preparation of siamenoside I.

2. The use according to claim 1, characterized in that said use is made of pichia guilliermondii (pichia guilliermondii) (pichia guilliermondii)) (Meyerozyma guilliermondii) The secreted extracellular protein of (a) realizes a catalytic function.

3. An application as claimed in claim 2, characterized in that the application is implemented as follows:

culturing the pichia guilliermondii to obtain fermentation liquor; centrifuging the fermentation liquor, collecting thalli sediment and supernatant,

and concentrating the supernatant to obtain the extracellular protein.

4. The use according to claim 3, wherein the supernatant concentration method is: refrigerating the supernatant at 4 deg.C for 5h, adding ammonium sulfate to adjust the saturation to 65%, and centrifuging at 4000rpm for 15 min; obtaining precipitate which is extracellular protein.

5. A preparation method of a composition prepared from Pichia guilliermondiiMeyerozyma guilliermondii) The produced cellA process for preparing siamenoside I from exoproteins, characterized in that extracellular proteins obtained in claim 2 or 3 are mixed at 0.01-10g/L, mogroside V at 0.1-100 g/L and pH7.2 in 0.2M phosphate buffer, and 25^oC, reacting for 5-72 hours under the stirring of 200rpm, and converting the mogroside V into the siamenoside I by the extracellular protein.

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to a method for efficiently synthesizing siamenoside I by using pichia guilliermondii.

Background

The momordica grosvenori (Siraitia grosvenorii) is a medicinal material in Guangxi province, has strict requirements on the habitat, is mainly vertically distributed in a subtropical sloping area with the altitude of 250-1000 m, and is mainly provided with moss, orchids, tea-oil trees, bamboo trees and other plants in the distribution area; the fructus momordicae is required to grow in a region with large day-night temperature difference and humid environment, the optimal temperature is 25-30 ℃, and the relative humidity of air is 70-85%; the glass is pleasant but not resistant to strong light and frost-resistant; is suitable for being cultivated in the loose and moist red yellow loam with good drainage and rich organic matters. The Guilin has the largest planting area of Lingui, Yongfu, Xingan, resource, Longsheng and Lingchuan, and is also the largest production base of the momordica grosvenori in China. The momordica grosvenori has the effects of cooling blood, relieving summer heat, smoothing intestine, relaxing bowels, reducing phlegm, relieving cough, soothing stomach, clearing lung, promoting fluid production, quenching thirst and the like, is clinically used for treating constipation due to intestinal dryness, hypertension and respiratory system diseases, has a certain antibacterial effect and also has a good curative effect on digestive system diseases. At present, the traditional Chinese medicines which are prepared by taking the momordica grosvenori as the main raw material comprise momordica grosvenori throat tablets, cough-relieving and phlegm-eliminating mixtures, momordica grosvenori cough-relieving dew, cough-relieving and asthma-relieving tablets, momordica grosvenori cough-relieving agents and the like (Li Shuang and the like, 2003). The fructus Siraitiae Grosvenorii glycoside has high sweetness, low calorific value, and good water solubility and heat stability. It is non-toxic, safe to eat and has no peculiar smell, so that it can be extensively used in the food and health-care product industry. In recent years, momordica grosvenori is made into foods and health-care products such as juice powder, fruit cakes, persimmon cake soup, bread, fruit herbal tea, monocrystal rock sugar and the like; the momordica grosvenori is very popular in foreign markets, and is approved as a food additive in Japan, Korea, England, Singapore and other countries, and especially, products such as high-grade throat-smoothing sugar, momordica grosvenori juice and the like made of the momordica grosvenori are very popular among people in Japan.

At present, cucurbitane triterpenoids extracted and identified from momordica grosvenori fruits comprise the following components: mogroside IV, mogroside V, mogroside IV A, mogrol I, benzoic acid, mogroside III, mogroside IIA 1, siamenoside I, 11-oxo-mogroside V, mogroside II E, mogroside III E, and mogroside A (Yang Xiuwei et al, 2008). The components are the main sweet components of the momordica grosvenori, and the total content of the momordica grosvenori in dried momordica grosvenori is 3.755-3.858%. The momordica grosvenori contains a plurality of saponin components, and more than 30 cucurbitane-type triterpenoid saponins are separated and identified from the momordica grosvenori, wherein mogrosides IV, V and siamenoside I are 3 components with the highest sweetness in the momordica grosvenori sweet glycosides reported so far, and are 392, 425 and 563 times of the sweetness of sucrose respectively. Siamenoside I is the sweet component of cucurbitane triterpenoid glycoside, and the sweetness of siamenoside I (0.01% concentration) is 563 times that of 5% sucrose.

In addition, in vitro experiments show that siamenoside has an inhibitory effect on maltase, and Suzuki and other researches find that the mogroside extract (containing mogroside V, IV, III, siamenoside I and the like) has a strong inhibitory effect on postprandial blood sugar of a single-dose oral maltose rat, and IC ₅₀14,12,10 and 1.6mM respectively. In the test of virus inhibition effect of mogroside by Konoshima et al, Japanese scholars found that siamenoside I can inhibit the growth of virus. The siamenoside I also has a strong function of reducing blood sugar and blood pressure. Yasushi A. Suzuki et al investigated the effect of crude extract of Momordica grosvenori on postprandial increase in blood glucose concentration, and found that the crude extract of Momordica grosvenori administered orally 3 minutes before rats took maltose significantly inhibited the increase in plasma glucose concentration, but did not inhibit glucose intake. The test result shows that the mogroside I has the inhibition capacity on maltase.

However, the production and preparation of siamenoside I are mainly obtained by plant extraction, but the plant content is extremely low, which severely restricts the large-scale application of siamenoside I, and the literature reports the method for producing siamenoside I by using plant tissue culture method in the last decade, and the siamenoside I is generally produced by adding inducer to induce plant cells. At present, no report on the preparation and production of a large amount of siamenoside I is published.

Disclosure of Invention

The invention aims to provide a new application of Pichia guilliermondii (Meyerozyma guilliermondii), the strain can produce extracellular protein, the extracellular protein has the function of efficiently catalyzing and synthesizing siamenoside I, and a new idea is provided for large-scale production of the siamenoside I.

The invention aims to provide a method for efficiently synthesizing siamenoside I by using pichia guilliermondii. The method can efficiently improve the synthesis of the mogroside I, has the characteristics of quick response, low cost, simple operation, convenient implementation, no toxicity, no residue and the like of products, meets the requirement of large-scale production of the mogroside I, and has strong practicability and popularization value.

The application of Pichia guilliermondii (Meyerozyma guilliermondii) as catalyst in preparing siamenoside I is provided.

The application realizes the catalytic function through the secreted extracellular protein of the pichia guilliermondii (Meyerozyma guilliermondii).

The application of pichia guilliermondii (Meyerozyma guilliermondii) in producing siamenoside I is characterized in that the application is realized according to the following steps:

step 1: streaking and inoculating Pichia guilliermondii (Meyerozyma guilliermondii) on a fresh slant culture medium, and culturing at 25 ℃ for 48h to obtain an activated slant strain;

step 2: selecting 1 ring of the activated slant strain obtained in the step 1 by using an inoculating ring, inoculating the activated slant strain into a 100mL triangular flask filled with 20mL seed culture medium, and performing shake cultivation for 48 hours at 25 ℃ and 200rpm to obtain a seed solution; then inoculating the seed solution into a 500mL triangular flask filled with 200mL liquid fermentation medium according to the inoculation amount of 2-10% by volume percentage, and carrying out shake cultivation at 25 ℃ and 200rpm for 48h to obtain fermentation liquor;

and step 3: and (3) centrifuging the fermentation liquor obtained in the step (2), collecting the bacterial precipitation and the supernatant, and purifying and concentrating the supernatant to obtain the extracellular protein.

And 4, step 4: using the extracellular protein of the pichia guilliermondii (Meyerozyma guilliermondii) obtained in the step 3 as a catalyst to prepare the siamenoside I. The reaction formula is as follows:

the use of (1), wherein the slant culture medium is used in the step (1)

Comprises 10g of soluble starch, 10g of glucose, 10g of peptone, 5g of yeast extract, 20g of agar and 1000mL of water, and has natural pH value.

The application is characterized in that in the step (2), the liquid fermentation medium comprises 20g of glucose, 20g of peptone, 10g of yeast extract,K₂HPO₄0.2g、MgSO₄·7H₂O 0.2g、Na₂CO₃0.5g and 1000mL of water, and the pH value is natural.

The application is characterized in that in the step (3), the method for purifying and concentrating the supernatant comprises the following steps: refrigerating the supernatant at 4 deg.C for 5h, adding ammonium sulfate to adjust the saturation to 65%, and centrifuging at 4000rpm for 15 min; obtaining precipitate which is extracellular protein.

A method for preparing siamenoside I by using extracellular protein produced by Pichia guilliermondii (Meyerozyma guilliermondii) is characterized in that 0.01-10g/L of extracellular protein, 0.1-100 g/L of mogroside V and 0.2M of phosphate buffer solution with pH of 7.2 are mixed, and the mixture is reacted for 5-72 hours at 25 ℃ under the stirring of 200 rpm.

The method is characterized in that the extracellular protein converts mogroside V into siamenoside I.

The pichia guilliermondii (Meyerozyma guilliermondii) has the following characteristics:

(1) individual morphology: the thallus is in an elliptical shape.

(2) Colony characteristics on solid medium plates: the colony is round, has wrinkled middle and is milky white.

(3) Growing at proper temperature of 25-30 deg.c and pH value of 6.0-7.0, and aerobic growth.

The solid culture medium consists of 10g of soluble starch, 10g of glucose, 10g of peptone, 5g of yeast extract, 20g of agar and 1000mL of water, and the pH value is natural.

The solid culture medium is sterilized at 121 deg.C under 0.1MPa for 15min before use.

The strain is subjected to comprehensive colony morphology, thallus morphological characteristic observation and molecular biological sequencing results to determine the Pichia guilliermondii (Meyerozyma guilliermondii).

Advantageous effects

1. The invention discovers that the symbiotic bacteria of the momordica grosvenori is the pichia guilliermondii (Meyerozymeguilliermondii) for the first time, and discovers that the culture of the symbiotic bacteria of the momordica grosvenori can convert the momordica grosvenori V into the siamenoside I for the first time. Furthermore, the pichia guilliermondii is cultured to obtain extracellular protein, the extracellular protein has strong catalytic activity, can specifically catalyze mogroside V into siamenoside I, has the characteristics of high catalytic efficiency, mild reaction conditions, good specificity and the like, is expected to be an ideal catalyst for efficiently synthesizing the siamenoside I, and widens the source of the siamenoside I.

2. The inventor tests ginsenoside Rb1, crocin I and oroxin B, the compounds glucose and mogroside V are 1-6 linked, and also tests fructus momordicae I, fructus momordicae IV, fructus momordicae II E, mogroside III E and mogroside A, but the experimental result shows that: only mogroside V can be catalyzed to siamenoside I, others are not.

Drawings

FIG. 1 is a colony map of Pichia guilliermondii (Meyerozyma guilliermondii) of the present invention;

FIG. 2 catalytic production of extracellular protein from Pichia guilliermondii (Meyerozyma guilliermondii) of example 4

Phylogenin I results;

FIG. 3 is a graph of HPLC peak times for different Luo Han Guo extracts;

FIG. 4 is a report of identification of Pichia guilliermondii (Meyerozyma guilliermondii);

FIG. 5 is a carbon spectrum of siamenoside I;

FIG. 6 is a hydrogen spectrum of siamenoside I.

Detailed Description

The present invention is further described in detail below with reference to examples to enable those skilled in the art to practice the invention with reference to the descriptions.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

Pichia guilliermondii (Meyerozyma guilliermondii) was previously isolated from the symbiotic bacteria of Momordica grosvenori, purchased from the China center for Industrial culture Collection of microorganisms, and verified to be identical.