CN103898163A - Lactic acid bacteria elemental selenium product and production method thereof - Google Patents
Lactic acid bacteria elemental selenium product and production method thereof Download PDFInfo
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- CN103898163A CN103898163A CN201210569505.0A CN201210569505A CN103898163A CN 103898163 A CN103898163 A CN 103898163A CN 201210569505 A CN201210569505 A CN 201210569505A CN 103898163 A CN103898163 A CN 103898163A
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- selenite
- short lactobacillus
- simple substance
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Abstract
The invention discloses a lactic acid bacteria elemental selenium product and a production method thereof, and belongs to the field of food biotechnology. The invention discloses a method for obtaining the elemental selenium product by adding selenite into a lactobacillus brevis-containing system. The lactic acid bacteria elemental selenium product and the production method thereof have the following advantages: firstly, the elemental selenium product having food safety characteristics is provided, and secondly, the method applying lactic acid bacteria fermentation to produce the elemental selenium is provided.
Description
Technical field
The present invention relates to a kind of milk-acid bacteria simple substance selenium product and production method thereof, belong to technical field of food biotechnology.
Background technology
Selenium is a kind of nonmetal chemical element, can be used as the essential nutritive element of photochromics, electrolytic manganese industry catalyzer and animal body and the useful nutritive element of plant.For human or animal body, although selenium is enjoyed the good reputations such as " long-lived element ", " anticancer king ", " natural toxinicide ", and be that other materials are irreplaceable to healthy effect, high density selenium has strong toxicity simultaneously.Selenium is divided in occurring in nature existence form: inorganic selenium (4 valencys and 6 valencys), organoselenium and simple substance selenium (nanometer selenium).Under different existence forms, its utilization ratio and cytotoxicity have marked difference.Particularly, the relative inorganic selenium toxicity of organoselenium is lower, bioavailability is higher; With respect to organoselenium, the toxicity of nanometer selenium is lower and utilization ratio is better, is therefore a kind of well nutritious supplementary, has been widely used at exploitation or the food nutrition field tool of functional foodstuff.
Along with the development of nanotechnology, increasing investigator adopts chemical method to synthesize simple substance selenium (nanometer selenium), except environmental pollution, the consumption energy, adopt the synthetic simple substance selenium of chemical method cannot be used for field of food or nutrition field, limit the purposes of simple substance selenium.In recent years, the report that selenate is converted into simple substance selenium about microorganism is more and more, wherein relates to the multi-strain bacterias such as intestinal bacteria, subtilis, Pseudomonas fluorescens, Chromatium vinosum, desulfurization bacterium, Crimson rhodospirillum, Spherical red antibacterial.Due to the problem of bacterial strain security, above-mentioned bacterial strains there is no method and applies in food.
Summary of the invention
The invention provides the method for short lactobacillus application in simple substance selenium is produced.
The invention provides a kind of milk-acid bacteria simple substance selenium product, be short lactobacillus (
lactobacillus brevis) the simple substance selenium product that obtains of fermentation selenite.
Because simple substance selenium is adsorbed in the fermented liquid of thalline surface or liquid culture, described simple substance selenium product is: 1) short lactobacillus liquid culture obtains fermented liquid; 2) by 1) in fermented liquid obtain the bacterium mud that contains simple substance selenium after centrifugal; 3) by 2) in thalline separate and obtain simple substance selenium with simple substance selenium; 4) product that short lactobacillus obtains on the solid medium that contains selenite.
Described short lactobacillus is CGMCC1.579, ATCC367 or JLD715(CGMCC No. 6683) in any, CGMCC1.579 is purchased from CGMCC, ATCC367 is purchased from ATCC, JLD715, for gathering voluntarily, derives from traditional dairy products.
JLD715 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18th, 2012, and deposit number is CGMCC No. 6683, and suggestion Classification And Nomenclature is short lactobacillus
lactobacillus brevis, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Described selenite concentration is no more than the tolerant maximum concentration of short lactobacillus, and preferably selenite concentration is 0.01mM-20 mM.
Described selenite refers to and contains positive tetravalence selenium, wherein preferred Sodium Selenite.
The present invention also provides a kind of method of producing milk-acid bacteria simple substance selenium product, use short lactobacillus (
lactobacillus brevis) fermentation selenite.
Described culture system can be liquid culture system or solid culture system, the preferably conventional fermention medium of short lactobacillus, and for example MRS substratum, substratum composition is to be optimized, medium optimization process is routine techniques means.
The selenite joining day is before fermentation starts, lag phase, logarithmic phase or the arbitrary period in stationary phase of short lactobacillus, also can all add in each period a certain amount of selenite, concrete addition manner can be determined according to the experiment of limited number of time.
The present invention can adopt following steps to realize:
1) in short lactobacillus fermention medium, add 0.01mM-20 mM selenite, access short lactobacillus aerobic fermentation is cultivated;
2) collect above-mentioned fermented liquid.
Incubation time can be determined as the case may be, for example, be cultured to simple substance selenium transformation efficiency the highest, is selenite remaining in fermented liquid is measured, in the time finding that selenite concentration no longer reduces, be defined as fermentation termination, also can, according to the difference of fermentation motivation, determine different fermentation times.
The present invention also can obtain simple substance selenium product by the mode of solid culture, is that short lactobacillus is inoculated in to the solid medium that contains selenite, for example MRS plate culture medium.
In the fermented liquid that short lactobacillus fermentation selenite obtains, contain simple substance selenium, simple substance selenium is adsorbed on thalline surface or is distributed in fermented liquid, and simple substance selenium can separate by centrifugal mode and fermented liquid, in the centrifugal bacterium mud obtaining of fermented liquid, contains simple substance selenium.
Separate simple substance selenium and also can adopt additive method, simple substance selenium and thalline can be separated by enzyme process or chemical method.
Described short lactobacillus culture temperature is the conventional temperature (25-35 ℃) of short lactobacillus, aerobic, anaerobism all can, add after selenite, incubation time transforms simple substance selenium situation according to selenite or selenate to be determined.
Anaerobism is to operate in anaerobic culture box, has been filled with nitrogen in environment, and liquid culture system also can redden, but anaerobic condition hypothallus biomass is lower, and selenite low conversion rate is in aerobic condition.
The collection mode of described product simple substance selenium comprises collects short lactobacillus fermented liquid or centrifugal collection short lactobacillus bacterium mud, as prepares microbial inoculum, can adopt lyophilize or spray-dired method.
The MRS substratum that milk-acid bacteria is conventional is composed as follows: peptone 10.0g, beef powder 10.0g, yeast soak powder 5.0g, glucose 20.0g, Triammonium citrate 2.6g, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, MgSO
47H
2o 0.58g, MnSO
4h
2o 0.25g, tween-80 1mL, adjust pH is 6.1-6.4, adds water to 1 000mL, 121 ℃ of sterilizing 15min.
About the measuring method of selenite, adopt the method for 2,3-diaminonaphthalene, in darkroom, react, and adopt spectrophotofluorometer to measure.
In the application, the mM of unit is this area conventional unit, refers to mmol/L.
For prior art, tool of the present invention has the following advantages:
1) provide simple substance selenium product, can in food, apply;
2) provide a kind of milk-acid bacteria to produce the method for simple substance selenium.
Accompanying drawing explanation
Fig. 1 short lactobacillus JLD715 scanning electron microscope and EDS analyze
Fig. 2 short lactobacillus JLD715 scanning electron microscope and EDS analyze
The transformation efficiency of Fig. 3 short lactobacillus to 0.2mM, 0.5mM, 1 mM Sodium Selenite
Fig. 4 transmission electron microscope is found thalline surface adsorption simple substance selenium
Embodiment
Embodiment 1 short lactobacillus JLD715 transforms the characteristic of selenite
In conjunction with Figure of description 1, embodiment 1 is described.
Short lactobacillus JLD715 is inoculated in the MRS substratum that concentration of sodium selenite is 10mM, cultivates 5 days for 30 degrees Celsius, centrifugal collection bacterium mud, carries out vacuum freezedrying, obtains orange powder.Sample presentation carries out scanning electron microscope analysis, and EDS energy spectrum analysis is carried out in thalline surface, the results are shown in Figure 1, Fig. 2.Find that thalline surface exists C, O and Se(0 valency) element, in conjunction with bibliographical information, can assert that short lactobacillus JLD715 has produced simple substance selenium.By scanning electron microscope, in conjunction with EDS energy spectrum analysis, simple substance selenium is enriched in thalline surface.
The transformation efficiency of embodiment 2 short lactobacillus to 0.2mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 0.2 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 200 hours, residue selenite content is as shown in Figure 3.
The transformation efficiency of embodiment 3 short lactobacillus to 0.5mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 0.5 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, residue selenite content is as shown in Figure 3.
The transformation efficiency of embodiment 4 short lactobacillus to 1mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 1 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, residue selenite content is as shown in Figure 3.
The transformation efficiency of embodiment 5 short lactobacillus to 5mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 5mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 26.2%.
The transformation efficiency of embodiment 6 short lactobacillus to 20 mM Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 20 mM Sodium Selenites, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 17.2%.
The transformation efficiency of embodiment 7 short lactobacillus to 30 mM Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 30 mM Sodium Selenites, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 7.6%.
The transformation efficiency of embodiment 8 short lactobacillus to 50 mM Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 50 mM Sodium Selenites, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 1.3%.
It is simple substance selenium that embodiment 9 short lactobacillus ATCC367 transform Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus ATCC367.In MRS substratum after sterilizing, add after 10 mM Sodium Selenites, access immediately 10% short lactobacillus ATCC367,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 13.9%, fermented liquid presents orange.
It is simple substance selenium that embodiment 10 short lactobacillus CGMCC1.579 transform Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus CGMCC1.579.In MRS substratum after sterilizing, add after 10 mM Sodium Selenites, access immediately 10% short lactobacillus CGMCC1.579,30 ℃ of aerobic cultivations 250 hours, selenite degradation rate is 2.9%, fermented liquid presents orange.
The transformation efficiency of embodiment 11 short lactobacillus to 0.1 mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 0.1 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 100 hours, selenite degradation rate is 100%.
The transformation efficiency of embodiment 12 short lactobacillus to 0.01 mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 0.01 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 50 hours, selenite degradation rate is 100%.
The transformation efficiency of embodiment 13 short lactobacillus to 0.001 mM Sodium Selenite
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, add after 0.001 mM Sodium Selenite, access immediately 10% short lactobacillus JLD715,30 ℃ of aerobic cultivations 24 hours, selenite degradation rate is 100%.
The addition manner 1 of embodiment 14 Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, access 10% short lactobacillus JLD715, be aerobicly cultured to the logarithmic phase later stage, add 0.01 mM Sodium Selenite, continue to cultivate 24 hours, selenite degradation rate is 100%.
The addition manner 2 of embodiment 15 Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, access 10% short lactobacillus JLD715, be aerobicly cultured to the logarithmic phase later stage, add 2 mM Sodium Selenites, continue to cultivate 200 hours, selenite degradation rate is 61%.
The addition manner 3 of embodiment 16 Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, access 10% short lactobacillus JLD715, be aerobicly cultured to stationary phase, add 20 mM Sodium Selenites, continue to cultivate 240 hours, selenite degradation rate is 21%.
The addition manner 4 of embodiment 17 Sodium Selenites
Fermention medium adopts MRS substratum, and starting strain is short lactobacillus JLD715.In MRS substratum after sterilizing, access 10% short lactobacillus JLD715, lag phase adds 5mM Sodium Selenite, continues to cultivate 240 hours, and selenite degradation rate is 21.1%.
The extraction 1 of embodiment 18 simple substance selenium
Collect the fermented liquid that embodiment 5 short lactobacillus JLD715 fermentation selenites obtain, fermented liquid is centrifugal, obtain red precipitate, centrifuged supernatant is non-redness, can preliminary judgement simple substance selenium many in throw out, the throw out obtaining be the product that contains simple substance selenium, and precipitation is carried out to lyophilize processing, lyophilized powder after treatment carries out scanning electron microscope in conjunction with energy spectrum analysis, and concrete outcome is shown in Fig. 1, Fig. 2.
The extraction 2 of embodiment 19 simple substance selenium
Collect short lactobacillus JLD715 fermentation selenite and obtain red fermented liquid, contain simple substance selenium in fermented liquid, fermented liquid is simple substance selenium product, can be further refining to fermented liquid, and purifying simple substance selenium.
The solid fermentation of embodiment 20 simple substance selenium
The MRS nutrient agar that short lactobacillus JLD715 is applied to the selenite that contains 10mM, obtains red bacterium colony, and picking one ring carries out TEM (transmission electron microscope) analysis, and Fig. 4 is the simple substance selenium of thalline surface adsorption.
Embodiment 21
Short lactobacillus JLD715 is activated in MRS liquid nutrient medium to two generations, the MRS liquid fermentation medium that access contains 10 mM Sodium Selenites, is cultured to stationary phase, gets after the centrifugal physiological saline washing of 1mL, stepwise dilution is coated MRS solid medium, and short lactobacillus JLD715 viable count is 6.29 × 10
7.
Short lactobacillus JLD715 is activated in MRS liquid nutrient medium to two generations, the MRS liquid fermentation medium that access contains 20 mM Sodium Selenites, is cultured to stationary phase, gets after the centrifugal physiological saline washing of 1mL, stepwise dilution is coated MRS solid medium, and short lactobacillus JLD715 viable count is 7.5 × 10
5.
Claims (15)
1. short lactobacillus, for the production of simple substance selenium, is characterized in that, is that selenite is reduced to simple substance selenium by short lactobacillus.
2. method according to claim 1, is characterized in that, described selenite is Sodium Selenite.
3. method according to claim 1, is characterized in that, short lactobacillus is CGMCC1.579, any in ATCC367 or CGMCC NO.6683.
4. a milk-acid bacteria simple substance selenium product, is characterized in that, is the simple substance selenium product that short lactobacillus fermentation selenite obtains.
5. simple substance selenium product according to claim 4, is characterized in that, described short lactobacillus is CGMCC1.579, any in ATCC367 or CGMCC NO.6683.
6. simple substance selenium product according to claim 4, is characterized in that, described selenite concentration is no more than the tolerant maximum concentration of short lactobacillus.
7. simple substance selenium product according to claim 4, is characterized in that, described selenite is Sodium Selenite.
8. simple substance selenium product according to claim 4, is characterized in that, described product is: 1) short lactobacillus liquid culture obtains fermented liquid; 2) by 1) in fermented liquid obtain the bacterium mud that contains simple substance selenium after centrifugal; 3) by 2) in thalline separate and obtain simple substance selenium with simple substance selenium; 4) product that short lactobacillus obtains on solid medium.
9. simple substance selenium product according to claim 4, is characterized in that, is the fermented liquid that short lactobacillus fermentation selenite obtains.
10. simple substance selenium product according to claim 4, is characterized in that, be short lactobacillus under aerobic condition, the fermented liquid that the Sodium Selenite of fermentation 0.01mM-20 mM obtains.
Produce the method for milk-acid bacteria simple substance selenium product for 11. 1 kinds, it is characterized in that, with the short lactobacillus selenite that ferments.
12. according to method described in claim 11, it is characterized in that, described short lactobacillus is CGMCC1.579, any in ATCC367 or CGMCC NO.6683.
13. according to method described in claim 11, it is characterized in that, described selenite is Sodium Selenite.
14. according to method described in claim 11, it is characterized in that, described selenite concentration is no more than the tolerant maximum concentration of short lactobacillus.
15. according to method described in claim 11, it is characterized in that, step is as follows:
In short lactobacillus fermention medium, add 0.01mM-20 mM selenite, access short lactobacillus aerobic fermentation is cultivated;
Collect above-mentioned fermented liquid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774875A (en) * | 2015-01-29 | 2015-07-15 | 中国农业大学 | Method for preparing biological nanoselenium by using Rahnella aquatilis |
| CN111304115A (en) * | 2019-12-30 | 2020-06-19 | 江南大学 | Lactobacillus casei capable of highly producing 3 forms of organic selenium and application thereof |
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| WO2010114864A1 (en) * | 2009-04-01 | 2010-10-07 | Little Calumet Holdings, Llc | Probiotic oral dosage forms |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774875A (en) * | 2015-01-29 | 2015-07-15 | 中国农业大学 | Method for preparing biological nanoselenium by using Rahnella aquatilis |
| CN111304115A (en) * | 2019-12-30 | 2020-06-19 | 江南大学 | Lactobacillus casei capable of highly producing 3 forms of organic selenium and application thereof |
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