CN109694829B - Selenium-rich saccharomyces cerevisiae, selenium-rich lycopene-rich saccharomyces cerevisiae and preparation method thereof - Google Patents

Abstract

The invention discloses a selenium-enriched Saccharomyces cerevisiae, a selenium-enriched lycopene-enriched Saccharomyces cerevisiae and a preparation method thereof, and relates to a lycopene fermentation technology. The method for preparing selenium-enriched Saccharomyces cerevisiae disclosed in the invention includes the step of adding a surfactant to the fermentation medium inoculated with Saccharomyces cerevisiae; the preparation method can effectively increase the selenium element content of the yeast.

Description

Selenium-enriched Saccharomyces cerevisiae, selenium-enriched lycopene-enriched Saccharomyces cerevisiae and preparation method thereof

technical field

The invention relates to the technical field of lycopene fermentation, in particular to a selenium-enriched Saccharomyces cerevisiae, a selenium-enriched lycopene-enriched Saccharomyces cerevisiae and a preparation method thereof.

Background technique

Lycopene is a natural pigment contained in plants. Mainly found in the ripe fruit of the nightshade tomato. It is currently one of the strongest antioxidants found in plants in nature. Science has proved that singlet oxygen and oxygen free radicals in the human body are the main culprits against the body's own immune system. Lycopene is far more effective in scavenging free radicals than other carotenoids and vitamin E, and its rate constant for quenching singlet oxygen is 100 times that of vitamin E. It can effectively prevent various diseases caused by aging and weakened immunity. Therefore, it has attracted the attention of experts from all over the world. Selenium (Se) has been designated by the World Health Organization and the Chinese Medical Association as the third most important micronutrient and health care element after iodine and zinc in the 21st century.

At present, the selenium conversion ability of common yeast is weak, and high-yielding strains need to be obtained through screening and domestication, but screening and domestication requires a lot of time and manpower, especially for the engineered strains that have been constructed by genetic technology, there are rare reports that improve the selenium element of genetically engineered strains content research.

Most of the organic selenium in yeast exists in the form of selenomethionine, but in the prior art, the content of organic selenium is rarely increased by increasing the expression content of methionine in yeast.

CN106566779A constructed a recombinant Saccharomyces cerevisiae for producing lycopene, but the recombinant yeast strain only produces lycopene, the product is single, the nutrition is single, the resource utilization is insufficient, and the obtained lycopene product lacks selenium element or its content is low.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method for preparing selenium-enriched yeast, by which the selenium content of the yeast can be effectively increased.

Another object of the present invention is to provide a method for preparing selenium-enriched lycopene-enriched Saccharomyces cerevisiae, and a yeast containing both lycopene and organic selenium is prepared by the preparation method.

Another object of the present invention is to provide a selenium-enriched Saccharomyces cerevisiae, which contains relatively high selenium content.

Another object of the present invention is to provide a selenium-enriched lycopene-enriched yeast, which contains relatively high content of selenium and lycopene.

The present invention is realized in this way:

In one aspect, the present invention provides a method for preparing selenium-enriched Saccharomyces cerevisiae, comprising the following steps:

Fermentation step: Add selenium-rich auxiliary to the fermentation medium inoculated with Saccharomyces cerevisiae.

Further, in some embodiments of the present invention, the selenium-enriched auxiliary agent is sodium dodecyl sulfonate (SDS) or sodium cyclamate (cyclamate).

The preparation method utilizes selenium-enriched additives to change the permeability of yeast cells, so that the inorganic selenium element in the medium can pass through the cell membrane more smoothly and enter into the cell to form organic selenium element, and while producing lycopene, the product The organic selenium element also increased accordingly.

At the same time, most of selenium will be combined with methionine in the body. The inventor found that adding SDS (sodium dodecyl sulfonate) or sodium cyclamate as organic sulfur can promote yeast to enhance methionine. The expression amount was further increased, the binding rate of selenium and methionine was further increased, and the content of organic selenium was further increased.

Further, in some embodiments of the present invention, the selenium-enriched adjuvant is added at 0-30 h after inoculation of the Saccharomyces cerevisiae.

Further, in some embodiments of the present invention, the addition amount of the selenium-enriched auxiliary agent is an addition amount of 0.1-5 g/L.

Preferably, in some embodiments of the present invention, the addition amount of the selenium-enriched auxiliary agent is 0.25g/L-0.5g/L.

Further, in some embodiments of the present invention, the addition method of the selenium-enriched auxiliary agent is one-time addition, batch addition or stream addition.

In fact, the batch addition can be divided into two batches or three batches. The time of adding in two batches is 10h, 30h, and the time of adding in three batches is 0h, 15h, 30h.

Further, in some embodiments of the present invention, during the fermentation process, ultrasonication is performed at 12-36 h.

In order to further increase the amount of selenium entering cells, a certain intensity of ultrasonic treatment can be used to improve the permeability of cells, enhance the entry of selenium into cells, and increase the content of organic selenium.

Further, in some embodiments of the present invention, the conditions of the ultrasonic treatment are as follows:

Frequency: 20-25KHz, power: 400-600W, interval time: 3s each time, interval 4s, total ultrasonic treatment time: 90s-210s.

Ultrasonic conditions are one of the factors that affect the permeability of yeast cell membranes. Only by using appropriate ultrasonic conditions can the cell membrane permeability of Saccharomyces cerevisiae be improved, which is conducive to the entry of inorganic selenium elements in the environment into cells and the synthesis of organic selenium. Increase organic selenium content.

Further, in some embodiments of the present invention, selenium salt is added to the fermentation medium at 12-36 h.

Further, in some embodiments of the present invention, the added amount of the selenium salt is 20-200 mg/L.

Preferably, in some embodiments of the present invention, the added amount of the selenium salt is preferably 25-50 mg/L.

Further, in some embodiments of the present invention, the deposit number of the above-mentioned Saccharomyces cerevisiae is CGMCC NO.13013.

The Saccharomyces cerevisiae strain adds selenium in the process of cultivating yeast, and the yeast absorbs and utilizes selenium during growth, so that selenium is organically combined with the protein and polysaccharide in the yeast and converted into biological selenium, thereby eliminating chemical selenium (such as sodium selenite) Toxic and side effects on the human body and gastrointestinal stimulation, so that selenium can be absorbed and utilized by the human body more efficiently and safely.

The strain of Saccharomyces cerevisiae was deposited in the General Microorganism Center (CGMCC) of China Microbial Culture Collection Management Committee (CGMCC) located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on September 19, 2016. Literary name: Saccharomyces cerevisiae.

The deposit number of the Saccharomyces cerevisiae is CGMCC NO.13013, the recombinant yeast strain knocks out the gal1, gal7, gal10, ypl062w and rox1 genes, and includes 6 gene fragments integrated into the genome through yeast homologous recombination. It is further integrated into a gene fragment on the yeast genome, and the functional genes crtE, crtB and crtI for synthesizing lycopene from a specific source combination, as well as specific yeast endogenous genes and exogenous genes, etc., are integrated by modular design. On the genome of the gene knockout yeast strain, a high-yielding lycopene-producing recombinant strain with the deposit number of CGMCC No. 13013 was obtained. For the preparation method of the strain, please refer to the Chinese invention patent application with the application number of "CN201610969721.2" and the title of "a recombinant yeast strain and its construction method and application".

Another object of the present invention is to provide a method for preparing selenium-enriched lycopene-enriched Saccharomyces cerevisiae, comprising: preparing selenium-enriched Saccharomyces cerevisiae by using the above-mentioned method for preparing selenium-enriched yeast;

Wherein, the raw material of Saccharomyces cerevisiae used is Saccharomyces cerevisiae with the deposit number of CGMCC NO.13013.

The yeast prepared by the preparation method is rich in lycopene and selenium at the same time.

CGMCC NO.13013 Saccharomyces cerevisiae is a genetically constructed strain capable of producing lycopene. Using the strain as the original seed bacteria, combined with the above-mentioned method for preparing selenium-enriched yeast, the Saccharomyces cerevisiae can not only produce lycopene, but also increase the content of selenium element.

The preparation method of the present invention greatly improves the selenium conversion ability of the recombinant yeast strain by optimizing and changing the process conditions in the fermentation, such as adding suitable surfactants, setting suitable ultrasonic treatment conditions, etc., and obtains selenium-rich elements, especially It is a lycopene product of organic selenium.

In another aspect, the present invention provides a selenium-enriched yeast, which is prepared by the above-mentioned method for preparing selenium-enriched Saccharomyces cerevisiae, or prepared by the above-mentioned method for preparing selenium-enriched lycopene yeast.

In another aspect, the present invention provides a selenium-enriched lycopene-enriched yeast prepared by the above-mentioned method.

In another aspect, the present invention provides a selenium-enriched lycopene-enriched yeast, wherein lycopene and organic selenium coexist, and the lycopene content is not less than 3.5%;

Further, in some embodiments of the present invention, the lycopene content of the selenium-enriched lycopene yeast is not less than 4.6%.

In some embodiments of the present invention, the selenium element content of the selenium-enriched lycopene yeast is not less than 302 ppm.

Further, in some embodiments of the present invention, the selenium content of the selenium-enriched lycopene yeast is not less than 412 ppm.

Further, the selenium-enriched lycopene yeast selenium content is not less than 826ppm, further, the selenium content is not less than 1133ppm, further, the selenium content is not less than 1530ppm, further, the selenium content is not less than 2230ppm, further, the selenium content is not less than 2230ppm. less than 2490ppm, further, the content of selenium element is not less than 2840ppm, further, the content of selenium element is not less than 3020ppm, further, the content of selenium element is 3450ppm.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

The method for preparing selenium-enriched lycopene products provided in this embodiment is as follows:

The strain used: Saccharomyces cerevisiae, the deposit number is CGMCC NO.13013.

1 strain activation:

After the strains were streaked in the slant medium, they were activated and cultured in a constant temperature incubator at 30°C for 24-36h.

Wherein, the slant medium contains (g/L): glucose 20, yeast extract 10, peptone 20, agar 20, sterilized at 121° C. for 20 min.

2 Seed Cultivation:

The activated and cultured strains are transferred to the seed medium, the culture temperature is 30° C., the rotational speed is 250 rpm, and the seed solution is used after culturing for 12-16 hours.

Seed medium (g/L): glucose 20, yeast extract 10, peptone 20, 50 mg/L uracil, sterilized at 121° C. for 20 min.

3 Fermentation:

The activated seed liquid was inserted into the fermentation medium (denoted as the 0th hour), the inoculation amount was 5% (v/v), the culture temperature was 30°C, the rotation speed was 250rpm, and the culture was performed for 120h.

Fermentation medium (g/L): glucose 40, yeast extract 10, peptone 20, 20 mg/L uracil, 10 g/L D-(+)-galactose, sterilized at 121° C. for 20 min.

4 Add selenium salt

At the 24th hour of fermentation, 25 mg/L of sodium selenite was added to the fermentation medium (ie, 100 mg of sodium selenite was added to each L of the fermentation medium), and the fermentation was continued.

5 Supplemental SDS

On the 12th hour after inoculation of the seed liquid, SDS was added to the fermentation medium at one time in an amount of 0.25 g/L, and the fermentation was continued.

6 Sonication

At the 20th hour after inoculation of the seed solution, ultrasonic treatment was performed. Conditions: frequency: 20KHz, power: 500W, interval time: 3s each time, interval 4s, and the total ultrasonic treatment time: 120s.

7 After the fermentation and culture, collect the bacterial cells obtained after fermentation, filter and clean the bacterial cells and then dry them to obtain dry weight, that is, a selenium-enriched lycopene product.

8 detection

8.1 Lycopene detection

Accurately weigh 0.02g of dry cells, break the wall by acid-heat method, extract with tetrahydrofuran, and detect the lycopene yield by high performance liquid chromatography.

Among them, the high-performance liquid chromatography detection method is referred to as follows:

(1) Chromatographic conditions:

Chromatographic column: Suplex PKB-100 (Supelco); 250×4.6mm, 5μm;

Wavelength: 472nm;

Flow rate: 0.5ml/min;

Injection volume: 10 μL;

Column temperature: 30℃;

(2) Mobile phase preparation and conditions:

Phase A: Methanol

Phase B: Weigh 50mg BHT into a 1L volumetric flask, add 20ml isopropanol to dissolve. 0.2 ml of N,N-diisopropylethylamine, 25 ml of 0.2% ammonium acetate solution, 455 ml of acetonitrile, 450 ml of methanol were added, the solution was returned to room temperature, and diluted to the mark with methanol.

Mobile Phase Gradient Table

time (min) Flow rate (ml/min) A% B% 0 0.5 0 100 32 0.5 16 84 50 0.5 16 84 57 0.5 0 100

8.2 Detection method of selenium content

(1) Selenium standard solution

Accurately weigh 2.19g of dry sodium selenite (superior pure), dissolve it in distilled water, add 80mL of 48% HBr, and dilute to 1L with distilled water to prepare a standard selenium preparation solution containing 1g/L of selenium or buy it directly 1g/L selenium standard solution.

(2) Sample digestion

Accurately weigh 0.1-0.2g of dry cells into a 150mL tall beaker, add a small amount of water to wet the sample, add digestive solution (hydrogen peroxide:perchloric acid:sulfuric acid=3:3:1), shake well, and add after the bubbles subside. Cover with a watch glass and put it on the electric furnace in the fume hood to digest to the end point. If the digestion is not complete, add hydrogen peroxide after cooling a little, and continue to heat and digest until it is complete. After cooling, rinse the watch glass and the bottle wall with a small amount of water, add sodium hydroxide to adjust the pH to 7.0, shake well, adjust the pH to 2.0-3.0 with formic acid, add 4 mL of 40% hydroxylamine hydrochloride, and then dilute to 50 mL, the same as method to make a blank solution.

(3) Determination of total selenium in samples

Put 10 mL of the detoxifying solution into a 125 mL separatory funnel, take another 7 separatory funnels, add the blank solution and standard selenium working solution 0.0, 2.0, 4.0, 6.0, 8.0, 10.0 mL of the same amount as the digestive solution respectively, add an appropriate amount of water, Add 4 mL of 5% EDTA-2Na solution and 2 mL of 0.5% 3,3-diaminobenzidine solution to each, shake well, leave to react in the dark for 30 min, adjust the pH to 6.5-7.0 with ammonia solution, add 4 mL of toluene, shake vigorously Extraction for 3 min, standing for stratification, taking the filtrate of the toluene layer in a 1 cm cuvette, and measuring the absorbance at a wavelength of 425 nm. Draw a standard curve with the absorbance of the standard selenium working solution, and find out the selenium content corresponding to the absorbance of the digestion solution from the standard curve.

(4) Determination of sample inorganic selenium

Take about 1.0g of dried bacterial cells of the sample into 50mL of distilled water, place it in an ultrasonic oscillator for ultrasonic vibration for 30min, slightly boil it for 30min, filter, make up to 50mL, absorb 10mL of supernatant, accurately add 4mL of toluene, and shake vigorously for extraction 3min, stand for stratification, take the toluene layer filtrate in a 1 cm cuvette, and measure the absorbance at a wavelength of 425 nm. Draw a standard curve with the absorbance of the standard selenium working solution, and find out the corresponding inorganic selenium content from the standard curve.

(5) Determination of organic selenium content in samples

The content of organic selenium can be obtained by subtracting the content of inorganic selenium in the sample from the measured total selenium content.

Examples 2-6

The methods for preparing selenium-enriched lycopene products provided in Examples 2-6 are basically the same as those in Example 1, and the different parameters are shown in Table 1 below.

Table 1

Wherein, "-" represents that no ultrasonic treatment was performed in Example 6.

Example 7

This embodiment adopts common Saccharomyces cerevisiae, purchased from Guangdong Huankai Company, Saccharomyces cerevisiae standard strain ATC9763, and the selenium-enriched process and parameters are the same as those in Example 1.

Example 8

This example uses common Saccharomyces cerevisiae, purchased from Guangdong Huankai Company, Saccharomyces cerevisiae standard strain ATC9763, the process and parameters of selenium enrichment are the same as those in Example 1, the difference is that ultrasonic treatment is not used in this example.

Comparative Example 1

The method for preparing the selenium-enriched lycopene product provided in this comparative example is basically the same as that in Example 1, except that Tween-80 is used instead of SDS in Example 1.

Comparative Example 2

The method for preparing the selenium-enriched lycopene product provided in this comparative example is basically the same as that in Example 1, except that CTAB is used instead of SDS in Example 1.

Comparative Example 3

The method for preparing selenium-enriched lycopene products provided in this comparative example is basically the same as that in Example 1, the difference is that the amount of inorganic selenium added in this comparative example is 50 mg/L, and 0.05 selenium is added at the 60th hour after inoculating the seed solution. g/L of SDS.

Comparative Example 4

The method for preparing selenium-enriched lycopene products provided in this comparative example is basically the same as that in Example 1, except that the addition amount of inorganic selenium in this comparative example is 30 mg/L, the addition amount of SDS is 7 g/L, and the power of ultrasonic treatment is 30 mg/L. is 400w.

Comparative Example 5

The method for preparing the selenium-enriched lycopene product provided in this comparative example is basically the same as that in Example 1, except that this comparative example does not add SDS, nor does it undergo ultrasonic treatment.

Comparative Example 6

This comparative example uses common Saccharomyces cerevisiae, purchased from Huankai Company, Saccharomyces cerevisiae standard strain ATC9763, the process is the same as that of Example 1, the difference is that this comparative example does not add SDS and does not undergo ultrasonic treatment.

Comparative Example 7

This comparative example adopts common Saccharomyces cerevisiae, purchased from Huankai Company, Saccharomyces cerevisiae standard strain ATC9763, the process is the same as that of Example 1, the difference is that the amount of SDS added in this comparative example is 0.01g/L.

Test results

According to the detection method of lycopene and selenium provided in Example 1, the thalli obtained after the collection and fermentation of Examples 1-6 and Comparative Examples 1-5 were detected, and the results were as follows in Table 2:

Table 2

Among them, ppm is the amount of selenium contained in each kilogram of yeast products.

It can be seen that the lycopene content or selenium conversion rate in the thalli obtained by the method of Example 1-6 is significantly higher than that of Comparative Example 1-5, and the selenium-rich content of Examples 7 and 8 of common Saccharomyces cerevisiae is also used. The conversion rate of selenium and selenium is significantly higher than that of comparative example 6 and comparative example 7, which shows that the preparation method of selenium-enriched lycopene provided by the embodiment of the present invention can improve lycopene and at the same time increase the content of selenium element. In addition, from the comparison of Example 1 and Example 7, and the comparison of Comparative Example 5 and Comparative Example 6, it can be found that the constructed Saccharomyces cerevisiae with a deposit number of CGMCC No. 13013 has better selenium enrichment performance than ordinary Saccharomyces cerevisiae.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. a method for preparing selenium-enriched Saccharomyces cerevisiae, is characterized in that, it comprises the following steps: Fermentation step: adding selenium-enriched auxiliary agent to the fermentation medium inoculated with Saccharomyces cerevisiae, the addition time of selenium-enriched auxiliary agent is 0-30h after inoculation of the Saccharomyces cerevisiae, and the addition amount of the selenium-enriched auxiliary agent is 0.1 -5g/L; the selenium-enriched auxiliary agent is sodium dodecyl sulfonate or sodium cyclamate; In the fermentation process, ultrasonic treatment is performed at 12-36 h; the conditions of the ultrasonic treatment are as follows: frequency: 20-25KHz, power: 400-600W, and the total ultrasonic treatment time is: 90s-210s; During the fermentation process, selenium salt is added to the fermentation medium at 12-36 h, and the addition amount is 20-50 mg/L. 2 . The method according to claim 1 , wherein the addition amount of the selenium-enriched auxiliary agent is 0.25g/L-0.5g/L. 3 . 3 . The method according to claim 1 , wherein the addition mode of the selenium-enriched auxiliary agent is one-time addition, batch addition or stream addition. 4 . 4 . The method according to claim 1 , wherein the interval time of the ultrasonic treatment is 3s each time, and the interval is 4s. 5 . 5. a method for preparing selenium-enriched lycopene-enriched Saccharomyces cerevisiae, characterized in that it comprises: using the method described in any one of claims 1-4 to prepare selenium-enriched Saccharomyces cerevisiae, the preservation number of the Saccharomyces cerevisiae It is CGMCC NO.13013. 6. A selenium-enriched lycopene-enriched yeast is characterized in that, its preparation method comprises the following steps: Fermentation step: adding selenium-enriched adjuvant to the fermentation medium inoculated with Saccharomyces cerevisiae, the addition time of selenium-enriched adjuvant is 0-30h after the inoculation of the saccharomyces cerevisiae, and the selenium-enriched adjuvant is added by flow or batch method, the addition amount of the selenium-enriched auxiliary agent is 0.5-1 g/L; the selenium-enriched auxiliary agent is sodium dodecyl sulfonate; During the fermentation process, selenium salt is added to the fermentation medium in the 12-24 h of fermentation, and the amount of selenium salt added is 30-50 mg/L; During the fermentation process, ultrasonic treatment was performed at 12-36 h; the ultrasonic treatment conditions were as follows: frequency: 20KHz, power: 400-500W, and total ultrasonic treatment time: 120s.