CN109694829A - A kind of selenium-rich saccharomyces cerevisiae, selenium-rich richness lycopene saccharomyces cerevisiae and preparation method thereof - Google Patents
A kind of selenium-rich saccharomyces cerevisiae, selenium-rich richness lycopene saccharomyces cerevisiae and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of selenium-rich saccharomyces cerevisiaes, selenium-rich richness lycopene saccharomyces cerevisiae and preparation method thereof, are related to lycopene fermentation technique.The method of preparation selenium-rich saccharomyces cerevisiae disclosed by the invention, includes the steps that adding surfactant into the fermentation medium for be inoculated with saccharomyces cerevisiae；It can effectively improve the selenium element content of yeast using the preparation method.
Description
Technical field
The present invention relates to lycopene fermentation technical fields, in particular to rich kind of a kind of selenium-rich saccharomyces cerevisiae, selenium-rich
Lycopene saccharomyces cerevisiae and preparation method thereof.
Background technique
Lycopene is a kind of natural pigment contained in plant.It is primarily present in the ripening fruits of plant of Solanaceae tomato
In.It is most one of the powerful antioxidant being found in the plant of nature at present.Science proof, the intracorporal singlet oxygen of people
It is the arch-criminal for encroaching on human body self immune system with oxygen radical.The effect of lycopene removing free radical, outclass other
Carotenoid and vitamin E, singlet-oxygen quenching rate constant are 100 times of vitamin E.It can with effectively preventing because
Aging, various diseases caused by immunity degradation.Therefore, its concern by countries in the world expert.Selenium (Se) is by world health
Tissue and Chinese Medical Association are set to the third-largest micronutrient health care element that must be mended after iodine, zinc 21 century, it has human body
Anti-oxidant, anti-canceration, improves the effects of immunity at toxin expelling.
Currently, the selenium conversion capability of common yeast is weaker, need to obtain superior strain by screening domestication, but screen domestication
The manpower that takes much time is needed, it is rare to have been reported that raising especially for the engineered strain built by gene technology
Engineering strain selenium element contains quantifier elimination.
The form of the most of selenomethionine of organic selenium exists in yeast, but few logical in the prior art
The expression contents of methionine in raising yeast are crossed to improve the content of organic selenium.
CN106566779A constructs the recombinant Saccharomyces cerevisiae of production lycopene, but restructuring yeast strains only produce tomato red
Element, product is single, and nutrition is single, and the utilization of resources is insufficient, lack in resulting lycopene product selenium element or its content compared with
It is low.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
It is an object of the present invention to provide a kind of methods for preparing Se-enriched yeast can effectively be mentioned using this method
The selenium element content of high yeast.
It is another object of the present invention to provide a kind of methods for preparing selenium-rich richness lycopene saccharomyces cerevisiae, using this
Preparation method contains the yeast of lycopene and organic selenium while being made.
Another object of the present invention is to provide a kind of selenium-rich saccharomyces cerevisiae, which contains higher selenium element and contain
Amount.
Another object of the present invention is to provide a kind of yeast of selenium-rich richness lycopene, in the yeast containing compared with
The selenium element and lycopene of high-content.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of methods for preparing selenium-rich saccharomyces cerevisiae comprising following steps:
Fermentation step: selenium-rich auxiliary agent is added into the fermentation medium for be inoculated with saccharomyces cerevisiae.
Further, in some embodiments of the present invention, the selenium-rich auxiliary agent be dodecyl sodium sulfate (SDS) or
Person's Sodium Cyclamate (honey element).
The preparation method changes the permeability of yeast cells using selenium-rich auxiliary agent, enables the inorganic selenium element in culture medium
Cell membrane is more passed through, intracellular, formation organic selenium is entered, and then while producing lycopene, in product
Organic selenium also correspondinglys increase.
Meanwhile selenium in vivo largely can be in conjunction with methionine, inventors have found that SDS (dodecyl sodium sulfonate is added
Sodium) or Sodium Cyclamate as organic sulfur, can promote the expression quantity of yeast enhancing methionine, further increase selenium with
The Percentage bound of methionine, and then improve organic selenium content.
Further, in some embodiments of the present invention, the 0-30h after being inoculated with the saccharomyces cerevisiae adds institute
State selenium-rich auxiliary agent.
Further, in some embodiments of the present invention, it is 0.1- that the additive amount of the selenium-rich auxiliary agent, which is additive amount,
5g/L。
Preferably, in some embodiments of the present invention, the additive amount of the selenium-rich auxiliary agent is 0.25g/L-0.5g/
L。
Further, in some embodiments of the present invention, the addition manner of the selenium-rich auxiliary agent be added at one time,
It is added portionwise or flows and add.
Being added portionwise in fact can be in two batches or three batches.In two batches plus time of addition be 10h, 30h, points three
Criticizing the time being added is 0h, 15h, 30h.
Further, in some embodiments of the present invention, during the fermentation, ultrasonic wave is carried out in 12-36h
Processing.
In order to further increase the amount that selenium enters cell, is handled using the ultrasonic wave of some strength, cell can be improved
Permeability, reinforce selenium element and enter intracellular, improve the content of organic selenium.
Further, in some embodiments of the present invention, the condition of the ultrasonication is as follows:
Frequency: 20-25KHz, power: 400-600W, interval time: each 3s is spaced 4s, ultrasonication total time
Are as follows: 90s-210s.
Ultrasound condition is one of the factor for influencing yeast cells permeability of cell membrane, only just using suitable ultrasound condition
The permeability of cell membrane that saccharomyces cerevisiae can be improved, the inorganic selenium element be conducive in environment are entered into the cell, have been synthesized
Machine selenium improves organic selenium content.
Further, in some embodiments of the present invention, selenium is added into the fermentation medium in 12-36h
Salt.
Further, in some embodiments of the present invention, the additive amount of the selenium salt is 20-200mg/L.
Preferably, in some embodiments of the present invention, the additive amount of the selenium salt is preferred 25-50mg/L.
Further, in some embodiments of the present invention, the deposit number of above-mentioned saccharomyces cerevisiae is CGMCC
NO.13013。
The Wine brewing yeast strain is added selenium element during culture yeasts, and selenium has been absorbed and utilized in when yeast growth, makes
Selenium is converted into Biological Selenium in conjunction with the intracorporal protein of yeast and polysaccharide organic, so that it is right to eliminate chemical selenium (such as sodium selenite)
The toxicity and stomach of human body stimulate, and enable that selenium is more efficient, be more safely absorbed by the body utilization.
The Wine brewing yeast strain is in September 19 in 2016 as being preserved in positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number: CGMCC NO.13013 is drawn
Fourth literature name: Saccharomyces cerevisiae.
The deposit number of the saccharomyces cerevisiae is CGMCC NO.13013, the restructuring yeast strains knockout gal1, gal7,
Gal10, ypl062w and rox1 gene, and include through 6 genetic fragments on yeast homologous recombination and integration to genome,
It is also further integrated into 1 genetic fragment on Yeast genome on the basis of it, and chooses the synthesis tomato of particular source combination
Functional gene crtE, crtB and crtI of red pigment, and specific yeast entogenous gene and foreign gene etc., it is whole through modularized design
It is bonded on gene knockout yeast strain genome, obtains the recombination for the high yield lycopene that deposit number is CGMCC No.13013
Bacterial strain.The preparation method of the bacterial strain can refer to that application No. is " CN201610969721.2 ", a kind of entitled " restructuring yeast strains
And its construction method and application " Chinese invention patent application.
It is another object of the present invention to provide a kind of method for preparing selenium-rich richness lycopene saccharomyces cerevisiae, packets
It includes: selenium-rich saccharomyces cerevisiae is prepared using the method for preparing Se-enriched yeast as described above；
Wherein, saccharomyces cerevisiae raw material used is the saccharomyces cerevisiae that deposit number is CGMCC NO.13013.
Using in yeast made from the preparation method while rich in lycopene and selenium element.
CGMCC NO.13013 saccharomyces cerevisiae is the gene constructed bacterial strain that can produce lycopene.Using the bacterial strain as original
Kind daughter bacteria, and the method for preparing Se-enriched yeast as described above is combined, so that the saccharomyces cerevisiae not only can produce lycopene,
Selenium element content also can be improved.
Preparation method of the invention for example adds suitable surface-active by the process conditions in optimization, change fermentation
The suitable ultrasonication condition etc. of agent, setting, makes the selenium conversion capability of restructuring yeast strains be greatly improved, is rich in
The lycopene product of selenium element especially organic selenium.
On the other hand, the present invention provides a kind of Se-enriched yeasts, by the method institute of above-mentioned preparation selenium-rich saccharomyces cerevisiae
It is made, or as obtained by the above-mentioned method for preparing selenium-enriched lycopene yeast.
On the other hand, it the present invention provides a kind of yeast of selenium-rich richness lycopene, is prepared by method as described above
It obtains.
There are also on the one hand, the present invention provides in a kind of yeast of selenium-rich richness lycopene, lycopene and organic selenium are same
When exist, the content of lycopene is not less than 3.5%；
Further, in some embodiments of the inventionly, the selenium-enriched lycopene yeast lycopene content not
Less than 4.6%.
In some embodiments of the present invention, which is not less than 302ppm.
Further, in some embodiments of the present invention, which is not small
In 412ppm.
Further, which is not less than 826ppm, and further, selenium element content is not
Less than 1133ppm, further, selenium element content is not less than 1530ppm, and further, selenium element content is not less than 2230ppm, into
One step, selenium element content are not less than 2490ppm, and further, selenium element content is not less than 2840ppm, further, selenium element content
Not less than 3020ppm, further, selenium element content is 3450ppm.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The method provided in this embodiment for preparing selenium-enriched lycopene product, specific as follows:
Strain used: saccharomyces cerevisiae, deposit number are CGMCC NO.13013.
1 actication of culture:
After strain carries out plate streaking in slant medium, the activation culture 24-36h in 30 DEG C of constant incubators.
Wherein, slant medium contains (g/L): glucose 20, yeast extract 10, peptone 20,20,121 DEG C of agar sterilizings
20min。
2 seed cultures:
Strain transfer after activation culture is into seed culture medium, and cultivation temperature is 30 DEG C, revolving speed 250rpm, culture 12
Seed liquor is used as after~16h.
Seed culture medium (g/L): glucose 20, yeast extract 10, peptone 20,50mg/L uracil, 121 DEG C of sterilizings
20min。
3 fermentations:
Activated seed liquor is accessed in fermentation medium and (is denoted as 0h), inoculum concentration is 5% (v/v), cultivation temperature
It is 30 DEG C, revolving speed 250rpm, cultivates 120h.
Fermentation medium (g/L): glucose 40, yeast extract 10, peptone 20,20mg/L uracil, 10g/L D- (+)-
Galactolipin, 121 DEG C of sterilizing 20min.
4 add selenium salt
Fermentation for 24 hours, toward the sodium selenite of fermentation medium addition 25mg/L, (added in i.e. every L fermentation medium
The sodium selenite of 100mg), continue to ferment.
5 add SDS
12h after being inoculated with seed liquor is added at one time SDS into fermentation medium by the amount of 0.25g/L, after supervention
Ferment.
6 ultrasonic treatments
20h after being inoculated with seed liquor, is ultrasonically treated, condition: frequency: 20KHz, power: 500W, when interval
Between: each 3s is spaced 4s, ultrasonication total time are as follows: 120s.
After 7 fermented and cultureds, the thallus obtained after fermentation is collected, will be dried after thallus filtering cleaning, obtain dry weight, i.e.,
Selenium-enriched lycopene product.
8 detections
The detection of 8.1 lycopenes
Precise 0.02g dry mycelium is extracted with tetrahydrofuran, is examined with high performance liquid chromatography after acid heat method broken wall
Survey yield of lycopene.
Wherein, high-efficiency liquid chromatography method for detecting is with reference to as follows:
(1) chromatographic condition:
Chromatographic column: Suplex PKB-100 (Supelco)；250 × 4.6mm, 5 μm；
Wavelength: 472nm；
Flow velocity: 0.5ml/min；
Sampling volume: 10 μ L；
Column temperature: 30 DEG C；
(2) mobile phase preparation and condition:
A phase: methanol
B phase: weighing 50mg BHT into 1L volumetric flask, and 20ml isopropanol is added to dissolve.0.2ml N, N- diisopropyl is added
Ethamine, 0.2% ammonium acetate solution of 25ml, 455ml acetonitrile, 450ml methanol, solution restore to room temperature, and extremely with methanol dilution
Scale.
Eluent gradient table
| Time (min) | Flow velocity (ml/min) | A% | B% |
| 0 | 0.5 | 0 | 100 |
| 32 | 0.5 | 16 | 84 |
| 50 | 0.5 | 16 | 84 |
| 57 | 0.5 | 0 | 100 |
The detection method of 8.2 selenium element contents
(1) selenium standard solution
The dry sodium selenite (excellent pure grade) of 2.19g is accurately weighed, is dissolved in distilled water, the HBr of 80mL 48% is added,
It is diluted to 1L with distilled water, the standard selenium for being prepared into the 1g/L containing selenium is equipped with the selenium titer of liquid or directly purchase 1g/L.
(2) treatments of the sample
0.1~0.2g dry mycelium accurately is weighed in 150mL high foot beaker, is added a small amount of water-wet sample, is added digestive juice (hydrogen peroxide
: perchloric acid: sulfuric acid=3: 3: 1), shaking up, cover surface plate after bubble is calmed down, set and digest on the electric furnace in draught cupboard to end
Point, if digestion not exclusively can it is slightly cold after add hydrogen peroxide, continue hot digestion to complete.Set cold rear a small amount of water flushing surface plate
And bottle wall, adding sodium hydroxide tune pH value are 7.0, are shaken up, and are 2.0~3.0 with formic acid tune pH value, 40% hydrochloric acid hydroxyl of 4mL is added
Then amine is settled to 50mL, do blank solution with method.
(3) the total selenium measurement of sample
It takes digestive juice 10mL in 125mL separatory funnel, separately takes 7 separatory funnels, be separately added into and digestive juice equivalent
Blank solution and standard selenium working solution 0.0,2.0,4.0,6.0,8.0,10.0mL, add appropriate amount of water, 5%EDTA-2Na are respectively added
Solution 4mL, 0.5% 3,3- diaminobenzidine solution 2mL shake up, and dark place reaction 30min are set, with ammonia spirit tune pH value
6.5~7.0, toluene 4mL, fierce shaking out 3min is added, stratification takes toluene layer filtrate in 1cm cuvette, in wave
Absorbance is measured at long 425nm.Standard curve is drawn with the absorbance of standard selenium working solution, finds and digests from standard curve
The corresponding Se content of the absorbance of liquid.
(4) sample inorganic selenium measures
It takes sample 1.0g or so dry mycelium in 50mL distilled water, is placed in ultrasonic vibration 30min in ultrasonic oscillation instrument, it is small
The slightly boiled 30min of fire, filtering, constant volume 50mL draw 10mL supernatant, and accurate addition toluene 4mL, fierce shaking out 3min are quiet
Layering is set, takes toluene layer filtrate in l cm cuvette, absorbance is measured at wavelength 425nm.With the suction of standard selenium working solution
Luminosity draws standard curve, and corresponding inorganic Se content is found from standard curve.
(5) sample organic selenium content measures
The content that total Se content of organic selenium content measurement subtracts inorganic selenium in sample can be obtained.
Embodiment 2-6
The method for preparing selenium-enriched lycopene product that embodiment 2-6 is provided parameter substantially the same manner as Example 1, different
It see the table below 1.
Table 1
Wherein, "-" represents is not ultrasonically treated in embodiment 6.
Embodiment 7
The present embodiment uses common saccharomyces cerevisiae, is purchased from Guangdong Huan Kai company, and saccharomyces cerevisiae reference culture ATC9763 is rich
The technique and parameter of selenium are same as Example 1.
Embodiment 8
The present embodiment uses common saccharomyces cerevisiae, is purchased from Guangdong Huan Kai company, and saccharomyces cerevisiae reference culture ATC9763 is rich
The technique and parameter of selenium are same as Example 1, the difference is that the present embodiment is not using ultrasonic treatment.
Comparative example 1
The method for preparing selenium-enriched lycopene product that this comparative example provides is substantially the same manner as Example 1, the difference is that with
Tween-80 replaces the SDS in embodiment 1.
Comparative example 2
The method for preparing selenium-enriched lycopene product that this comparative example provides is substantially the same manner as Example 1, the difference is that with
CTAB replaces the SDS in embodiment 1.
Comparative example 3
The method for preparing selenium-enriched lycopene product that this comparative example provides is substantially the same manner as Example 1, the difference is that this
The additive amount of comparative example inorganic selenium is 50mg/L, and the SDS of 0.05g/L is just added in 60h hours after being inoculated with seed liquor.
Comparative example 4
The method for preparing selenium-enriched lycopene product that this comparative example provides is substantially the same manner as Example 1, the difference is that this
Comparative example is 30mg/L in the additive amount of inorganic selenium, and SDS additive amount is 7g/L, and the power of ultrasonic treatment is 400w.
Comparative example 5
The method for preparing selenium-enriched lycopene product that this comparative example provides is substantially the same manner as Example 1, the difference is that this
Comparative example is not added with SDS, also without ultrasonic treatment.
Comparative example 6
This comparative example use common saccharomyces cerevisiae, be purchased from Huan Kai company, saccharomyces cerevisiae reference culture ATC9763, technique with
Embodiment 1 is identical, the difference is that this comparative example is not added with SDS also without ultrasonic treatment.
Comparative example 7
This comparative example use common saccharomyces cerevisiae, be purchased from Huan Kai company, saccharomyces cerevisiae reference culture ATC9763, technique with
Embodiment 1 is identical, the difference is that this comparative example SDS additive amount is 0.01g/L.
Testing result
By the lycopene of the offer in embodiment 1 and the detection method of selenium element, to embodiment 1-6, comparative example 1-5
Collection fermentation after obtained thallus detected, as a result such as the following table 2:
Table 2
Wherein ppm is the selenium amount contained in every kilogram of yeast product.
As can be seen that the lycopene content or selenium conversion ratio made from method using embodiment 1-6 in thallus are obvious
Higher than comparative example 1-5, comparison is equally apparently higher than using the selenium-rich of embodiment 7 and 8 content of common saccharomyces cerevisiae and selenium conversion ratio
Example 6 and comparative example 7, thus illustrate, can be improved kind using the preparation method of selenium-enriched lycopene provided in an embodiment of the present invention
Lycopene simultaneously, improves the content of selenium element.In addition, being compared by embodiment 1 and embodiment 7, comparative example 5 and comparative example 6 are compared,
It can be found that the deposit number of building is the saccharomyces cerevisiae of CGMCC NO.13013, there is better selenium-rich table than common saccharomyces cerevisiae
It is existing.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (12)
1. a kind of method for preparing selenium-rich saccharomyces cerevisiae, which is characterized in that itself the following steps are included:
Fermentation step: selenium-rich auxiliary agent is added into the fermentation medium for be inoculated with saccharomyces cerevisiae.
2. the method according to claim 1, wherein the selenium-rich auxiliary agent is dodecyl sodium sulfate or hexamethylene
Sulfamic acid sodium.
3. according to the method described in claim 2, it is characterized in that, the 0-30h after being inoculated with the saccharomyces cerevisiae adds institute
State selenium-rich auxiliary agent.
4. according to the method described in claim 3, it is characterized in that, the additive amount of the selenium-rich auxiliary agent is 0.1-5g/L；
Preferably, the additive amount of the selenium-rich auxiliary agent is 0.25g/L-0.5g/L.
5. method according to claim 1-4, which is characterized in that the addition manner of the selenium-rich auxiliary agent is primary
Property be added, be added portionwise or flow add.
6. method according to claim 1-4, which is characterized in that during the fermentation, carried out in 12-36h
Ultrasonication.
7. according to the method described in claim 6, it is characterized in that, the condition of the ultrasonication is as follows:
Frequency: 20-25KHz, power: 400-600W, interval time: each 3s is spaced 4s, ultrasonication total time are as follows:
90s-210s。
8. method according to claim 1-4, which is characterized in that during the fermentation, in 12-36h toward institute
State addition selenium salt in fermentation medium.
9. a kind of method for preparing selenium-rich richness lycopene saccharomyces cerevisiae, characterized in that it comprises: use claim 1-8
Selenium-rich saccharomyces cerevisiae is prepared in described in any item methods, and the deposit number of the saccharomyces cerevisiae is CGMCC NO.13013.
10. a kind of yeast of selenium-rich, which is characterized in that it is prepared by the described in any item methods of claim 1-8, or
It is prepared by method as claimed in claim 9.
11. a kind of yeast of selenium-rich richness lycopene, which is characterized in that organic selenium and lycopene are deposited simultaneously in the yeast
It is not less than 3.5% in the content of, lycopene.
12. the yeast of selenium-rich richness lycopene according to claim 11, which is characterized in that the organic selenium of the yeast contains
Amount is not less than 302ppm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628654A (en) * | 2019-10-17 | 2019-12-31 | 嘉必优生物技术（武汉）股份有限公司 | Saccharomyces cerevisiae capable of being used for feed and preparation method and application thereof |
| CN112715917A (en) * | 2020-12-24 | 2021-04-30 | 江苏大学 | Preparation method of soy sauce rich in active selenium |
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