CN108048363A - A kind of method of selenium-rich lactobacillus reuteri high density fermentation - Google Patents
A kind of method of selenium-rich lactobacillus reuteri high density fermentation Download PDFInfo
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Abstract
The invention discloses a kind of methods of selenium-rich lactobacillus reuteri high density fermentation, the thalline of the selenium-rich lactobacillus reuteri of high density fermentation is obtained using the culture medium containing sucrose after improvement, add the ability that unit volume zymotic fluid digests and assimilates inorganic selenium, by two-stage selenium-supply selenium concentration in zymotic fluid is controlled to be in reduced levels simultaneously, fermentation process is utilized convenient for lactic acid bacteria, by the inorganic selenium in zymotic fluid, it is fully converted to the endobacillary Organic Selenium of lactic acid, additionally due to control the selenium concentration of fermentation process, thalli growth is not inhibited, higher viable bacteria concentration can be reached interior for 24 hours, solve the problems, such as that inorganic Se content is far above the tolerance of lactic acid bacteria and causes lactobacter growth slow in culture medium in traditional handicraft, the problem of gained thalline includes elemental selenium is avoided simultaneously.Therefore the advantages of selenium-rich lactobacillus reuteri zymotic fluid that the present invention obtains has both complete Organic Selenium and high viable count.In addition, the probiotics that the present invention finally obtains contains only Organic Selenium.
Description
Technical field
The invention belongs to a kind of field of biological product, more particularly to a kind of selenium-rich lactobacillus reuteri high density fermentation
Method.
Background technology
Selenium is a kind of element necessary to human body, it was determined that low selenium can cause Kaschin-Beck disease and Keshan disease, in addition
Selenium deficiency can potentially influence health in all various aspects.For a long time, provincialism selenium source scarcity drastically influences the strong of people
Health and living standard, exploitation selenium-rich function food can effectively alleviate this problem.Selenium mainly has with inorganic selenium salt, simply
Machine selenium compound and containing the forms such as selenoaminoacid exist.The toxicity of Organic Selenium is relatively low, wherein known contain there are two types of selenoaminoacids：
Selenizing methionine and selenizing cysteine.The amino acid of selenizing can be synthesized to by special assembling approach in specific proteins
Form selenizing albumen.According to world health organisation recommendations, daily selenium intake of being grown up should be controlled in 30-40 μ g, man's
Daily most suitable selenium intake is 60 μ g, and woman is 53 μ g.
There are mainly two types of selenium-rich products, and one kind is by spraying selenium to soil, improving the Se content of agricultural product, pass through the method
Substantial amounts of Se-rich farm products, such as the grain of selenium-rich, tea, veterinary antibiotics are developed；Another kind is that selenium-rich is added in into food
Additive, this kind of selenium-supply product include salt, Yoghourt, milk powder, probiotics of selenium-rich etc..
Many lactic acid bacterias have the function of to be enriched with Organic Selenium.Zhu Qin et al. patent " a kind of selenium-rich lactobacillus preparation and its
Production method " (CN201110319785.5) report lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus and streptococcus lactis
For selenium-rich, 5-15mg selenium can be enriched with per g of formulation.Chen Long etc. patent " a kind of selenium-rich lactobacillus preparation and its production method and
Using " (CN200510039258.3) report lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus and streptococcus lactis be used for
Selenium-rich can be enriched with 5-15mg selenium per g of formulation.Patent " a kind of selenium-rich lactobacillus preparation production method and its review lactic acid bacteria system
Agent " (CN201310736926.2) report lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, animal bifidobacteria, saliva
Liquid lactobacillus and streptococcus lactis can be enriched with 4-12mg selenium for selenium-rich per g of formulation.The Se content of this three patent reports is far high
In theoretical selenium-rich amount, show to bring the inorganic selenium of several times in its method into, prepared by lactobacillus preparation be inorganic selenium and organic
The mixture of selenium has no body of laws and shows the characteristics of Organic Selenium is less toxic." a kind of Muscle of chicken that improves is yellowish pink in patent by Qin Shunyi et al.
Report sandlwood saccharobacillus is for selenium-rich in selenium-rich lactobacillus preparation and its application " (CN201410819192.9), the breast per g of formulation
Sour bacterium number 109CFU, per g of formulation Se content 5.2-22.8 μ g, and nearly half is still inorganic selenium, although inorganic Se content is significantly
It reduces, but still is the mixture of inorganic selenium and Organic Selenium.
The culture medium for preparing selenium-rich lactobacillus both at home and abroad at present uses traditional MRS culture mediums substantially.Lactic acid bacteria thalline exists
In fermentation process, the inorganic selenium in zymotic fluid can be converted into the endobacillary Organic Selenium of lactic acid.In conventional method, the breast after fermentation
Sour bacteria concentration is generally below (0.8-5) × 109CFU/ml, and the concentration of sodium selenite added in incubation is in 5-120mg/
L, the concentration value are far above the tolerable concentration of lactic acid bacteria, cause lactobacter growth slow, and fermentation time usually requires more than 36h,
Time-consuming and terminal viable count is low, therefore selenium-rich cost is higher.In addition, remaining sodium selenite in zymotic fluid obtained by conventional method
It is high, it usually needs still to be remained in thalline by washing to remove most of sodium selenite, after washing higher in fermentation process
In not by the selenium monomer and sodium selenite of sorption enhanced, therefore gained thalline is mostly the mixture of inorganic selenium and Organic Selenium, influence
The use of thalline product.
Selenium product for the fungi medicament reported that patent lactobacillus-fermented density is low, obtained is inorganic selenium and Organic Selenium
The problem of mixture, inventor make every effort to research and develop a kind of raising fermentation density, reduce production cost, and the micro- life finally obtained
Selenium product contained by object preparation is the selenium-rich method of Organic Selenium.
The content of the invention
In view of this, it is an object of the invention to provide a kind of fermentation density height, production cost is relatively low, and finally obtain
Selenium product contained by microorganism formulation is the method for the selenium-rich lactobacillus reuteri high density fermentation of Organic Selenium.
In order to achieve the above objectives, the technical scheme is that：
A kind of method of selenium-rich lactobacillus reuteri high density fermentation, includes the following steps：
A kind of method of selenium-rich lactobacillus reuteri high density fermentation, includes the following steps：
(1) by lactobacillus reuteri from plating to seed culture medium in, 37 DEG C of Anaerobic culturel 12-18h, every liter of seed
Culture medium prescription includes following raw material：20g sucrose, 20g yeast extracts, 3.0g dipotassium hydrogen phosphates, 7.0g sodium acetates, 1.0g lemons
Sour ammonium, 0.2g magnesium sulfate, 0.18g manganese sulfates, the distilled water of 1.0g Tween 80s and surplus, with after phosphoric acid tune pH to 6.2 during configuration
Obtain seed culture medium；
(2) seed is inoculated by 3%-10% inoculum concentrations in fermentation medium, Anaerobic culturel 16-24h, every liter of fermentation training
Foster based formulas includes：80-150g sucrose, 80-150g yeast extracts, 2-7g ammonium citrates, 5-9g sodium acetates, 2-6g phosphoric acid hydrogen two
Potassium, 0.2-0.6g manganese sulfates, 0.5-0.8g magnesium sulfate and 1-3g Tween 80s, with being obtained after phosphoric acid tune pH to 5.5-6.2 during configuration
Fermentation medium；
(3) 4h is proceeded in step (2) fermentation, it is 1g/L sodium selenite solutions that concentration is added in every liter of fermentation medium
1.1-3.3ml；Fermentation proceeds to 12-16h, and every liter of fermentation medium adds concentration as 1g/L sodium selenite solution 1.1-3.3ml,
Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal zymotic fluid.
Further, step (4) is further included：Stabilizer is added in terminal zymotic fluid in step (3), selenium-rich sieve is made
Yi Shi lactobacillus nutrient solutions；
The stabilizer is pectin, gelatin, converted starch, quickly dissolving agar, lactoprotein, carboxymethylcellulose sodium, acidproof
A variety of combinations or one of which in CMC, propylene glycol alginate, xanthans, carragheen, guar gum, Arabic gum.
Further, the lactoprotein includes casein sodium and/or lactalbumin.
Further, in step (4), selenium-rich lactobacillus reuteri nutrient solution viable count is (0.8-5) × 1010CFU/
Ml, Se content are 1400-3000 μ g/L.
Further, step (4) is further included：Terminal zymotic fluid in step (3) is filtered, selenium-rich is obtained after filtering
The thalline of lactobacillus reuteri, then adds freeze drying protectant, and the thalline for being with the addition of freeze drying protectant is freeze-dried, cold
It is lyophilized it is dry after obtain selenium-rich lactobacillus reuteri active bacteria formulation；
The freeze drying protectant for glycerine, sorbierite, mannitol, sucrose, trehalose, polyvinylpyrrolidone, glucan,
A variety of combinations or one of which in milk powder, vitamin E.
Further, in step (4), measured by the method for plate culture count, selenium-rich lactobacillus reuteri active bacteria formulation
Viable count for (0.1-5) × 1012CFU/g, Se content is 126-260 μ g/g.
Further, in step (3), measured by the method for plate culture count, lactobacillus reuteri is in fermentation medium
During middle culture 18h, viable count is (0.8-5) × 1010CFU/ml；
After the completion of fermentation, the Se content of terminal zymotic fluid is 1200-3000 μ g/L, and thalline Se content is 126-260 μ g/g.
Further, in step (3), selenium-rich Luo Yishi breasts are obtained after terminal zymotic fluid is centrifuged, is freeze-dried
The thalline of bacillus, dry cell weight 9-12g/L.
A kind of method of selenium-rich lactobacillus reuteri high density fermentation of the present invention, is obtained using the culture medium containing sucrose after improvement
The thalline of the selenium-rich lactobacillus reuteri of high density fermentation is obtained, adds the energy that unit volume zymotic fluid digests and assimilates inorganic selenium
Power, while by two-stage selenium-supply selenium concentration in zymotic fluid is controlled to be in reduced levels, it, will convenient for lactic acid bacteria using fermentation process
Inorganic selenium in zymotic fluid is fully converted to the endobacillary Organic Selenium of lactic acid, additionally due to the selenium concentration of fermentation process is controlled,
Thalli growth is not inhibited, and can reach higher viable bacteria concentration interior for 24 hours, solves in traditional handicraft that inorganic selenium contains in culture medium
The problem of amount causes lactobacter growth slow far above the tolerance of lactic acid bacteria, while avoid gained thalline and include simple substance
The problem of selenium.Therefore the selenium-rich lactobacillus reuteri zymotic fluid that obtains of the present invention has both the excellent of complete Organic Selenium and high viable count
Point.
To sum up, the invention has the advantages that：
1st, the present invention may be such that final cell concentration than common MRS culture mediums using high density fermentation culture medium and technique
The cell concentration of acquisition greatly improves.
2nd, the present invention uses selenium-supply strategy stage by stage, and inorganic selenium in fermentation process can be overcome to inhibit thalli growth, and same
When ensure inorganic selenium 100% be converted into Organic Selenium, avoid obtained preparation simultaneously include inorganic selenium and Organic Selenium.
3rd, technique of the invention, can be direct to remove unconverted inorganic selenium without the washing thalline after the completion of final fermentation
Additive of water etc. is quoted as animal, without other lock out operation.
Specific embodiment
In order to further explain the technical solution of the present invention, the present invention is explained in detail below by specific embodiment
It states.
Strain source：Lactobacillus reuteri used in a kind of method of selenium-rich lactobacillus reuteri high density fermentation of the present invention
(Lactobacillus reuteri) derives from National Industrial Organism Depositary (preservation of bacteria strain number CICC 6118).This
For lactobacillus reuteri as selenium-rich object, which is available for the prebiotic of humans and animals by health ministry approval in invention
Bacterium.
Actication of culture：To the lyophilized bacterium powder of the lactobacillus reuteri of preservation, sterile water 2ml is added in, then in anaerobic environment
Middle accessed in MRS culture mediums (i.e. lactic acid bacteria culturing medium) tablet activates.MRS culture mediums composition is as follows：Peptone 10.0g,
Powdered beef 5.0g, dusty yeast 4.0g, glucose 20.0g, Tween 80 volume 1.0mL, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g,
Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, agar powder 15.0g and distilled water 1000mL.
It is prepared by seed：Colony inoculation is chosen to seed culture medium from the tablet of above-mentioned activation, and 37 DEG C of Anaerobic culturel 12-18h are obtained
To seed.Every liter of seed culture based formulas includes following raw material：20g sucrose, 20g yeast extracts, 3.0g dipotassium hydrogen phosphates, 7.0g
Sodium acetate, 1.0g ammonium citrates, 0.2g magnesium sulfate, 0.18g manganese sulfates, the distilled water of 1.0g Tween 80s and surplus, when configuration, are used
Seed culture medium is obtained after phosphoric acid tune pH to 6.2.
It should be noted that above-mentioned MRS culture mediums and seed culture medium are both needed to packing after 121 DEG C of high pressure sterilizations
Then 15min -20min is cooled down spare.
Stabilizer：Stabilizer used in the present invention is pectin, gelatin, converted starch, quickly dissolving agar, lactoprotein (casein
Sour sodium, lactalbumin), carboxymethylcellulose sodium (CMC), acidproof CMC, propylene glycol alginate, xanthans, carragheen, cluster bean
A variety of combinations or one of which in glue and Arabic gum.
Freeze drying protectant：Freeze drying protectant used in the present invention is glycerine, sorbierite, mannitol, sucrose, trehalose, poly-
A variety of combinations or one of which in ethylene ratio pyrrolidone, glucan, milk powder and vitamin E.
Embodiment 1：
(1) above-mentioned cultured seed is inoculated by the inoculum concentration of 10% (v/v) in high density fermentation culture medium, anaerobism
Culture is for 24 hours.Include following raw material per high-density fermentative medium formula in the present embodiment：150g sucrose, 150g yeast extracts,
7g ammonium citrates, 9g sodium acetates, 6g dipotassium hydrogen phosphates, 0.6g manganese sulfates, 0.8g magnesium sulfate and 3g Tween 80s, phosphoric acid during configuration
PH to 6.2 is adjusted, obtains fermentation medium.
(2) when step (1) fermentation proceeds to 4h, it is 1g/L's to add in concentration in every liter of fermentation medium (i.e. zymotic fluid)
Sodium selenite solution 3.3ml；When fermentation proceeds to 16h, every liter of fermentation medium (i.e. zymotic fluid) adds the Asia that concentration is 1g/L
Selenic acid sodium solution 3.3ml.Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal zymotic fluid；It should be noted that it is fermenting
In the process, the fermentation medium in step (2) is substantially the zymotic fluid containing lactobacillus reuteri.
It is measured by the method for plate culture count, lactobacillus reuteri (L.reuteri) is trained in high density fermentation culture medium
Viable count has reached 5 × 10 after supporting 18h10CFU/ml.When fermentation is completed, the Se content of terminal zymotic fluid is 3000 μ g/L, wet
The Se content of thalline is 260 μ g/g.Terminal zymotic fluid is centrifuged, after thalline centrifugation, aas determination supernatant
Liquid selenium concentration is 0, that is, shows that the inorganic selenium in zymotic fluid has been fully converted to the in vivo Organic Selenium of lactobacillus reuteri.Terminal is sent out
Zymotic fluid obtains the thalline of selenium-rich lactobacillus reuteri after centrifuging, being freeze-dried, which is 11g/L (i.e. every liter of ends
Select the thalline containing 11g dry weight meters in zymotic fluid)；
(3) carboxymethylcellulose sodium (CMC) 6g/L and trehalose 3g/L are added in the terminal zymotic fluid in step (2),
That is, added in every liter zymotic fluid and 3g trehaloses are added in 6g carboxymethylcellulose sodium (CMC) and every liter of zymotic fluid.Then obtain
The selenium-rich lactobacillus reuteri nutrient solution that must stablize in 4 DEG C of preservations, is provided with using.Nutrient solution viable count is 5 × 1010CFU/ml,
Se content is 3000 μ g/L.
Embodiment 2：
(1) above-mentioned cultured seed is inoculated by 3% (v/v) inoculum concentration in high density fermentation culture medium, anaerobism training
Support 20h.Include following raw material per high-density fermentative medium formula in the present embodiment：80g sucrose, 80g yeast extracts, 3g lemons
Lemon acid ammonium, 5g sodium acetates, 3g dipotassium hydrogen phosphates, 0.4g manganese sulfates, 0.6g magnesium sulfate, 2g Tween 80s and phosphoric acid tune pH to 5.5, obtain
To fermentation medium.
(2) 4h, every liter of fermentation medium (i.e. zymotic fluid) are proceeded in step (1) fermentation
It is middle to add in the sodium selenite solution 1.1ml that concentration is 1g/L；Fermentation proceeds to 16h, and every liter of fermentation medium (is sent out
Zymotic fluid) add the sodium selenite solution 1.97ml that concentration is 1g/L.Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal hair
Zymotic fluid；
It is measured by the method for plate culture count, L.reuteri cultivates 18h viable counts in high density fermentation culture medium and reaches
0.8 × 1010CFU/ml.When fermentation is completed, terminal zymotic fluid Se content is 1400 μ g/L, and thalline Se content is 155 μ g/g.
Terminal zymotic fluid is centrifuged, after thalline centrifugation, aas determination supernatant selenium concentration is 0.Terminal zymotic fluid
The thalline of selenium-rich lactobacillus reuteri is obtained after centrifuging, being freeze-dried, dry cell weight is 9g/L (i.e. every liter of terminal zymotic fluids
In the thalline containing 9g dry weight meters)；
(3) converted starch 10g/L and mannitol 5g/L is added in the terminal zymotic fluid in step (2), i.e. every liter of terminal
It is added in zymotic fluid and 5g mannitol is added in 10g converted starches and every liter of terminal zymotic fluid.Then obtain stable selenium-rich sieve
Yi Shi lactobacillus nutrient solutions in 4 DEG C of preservations, are provided with using.Nutrient solution viable count is 0.8 × 1010CFU/ml, Se content 1400
μg/L。
Embodiment 3：
(1) above-mentioned cultured seed is inoculated by 5% (v/v) inoculum concentration in high density fermentation culture medium, anaerobism training
Support 16h.Include following raw material per high-density fermentative medium formula in the present embodiment：100g sucrose, 100g yeast extracts, 5g
Ammonium citrate, 7g sodium acetates, 4g dipotassium hydrogen phosphates, 0.4g manganese sulfates, 0.7g magnesium sulfate and 2g Tween 80s, with phosphoric acid tune during configuration
PH to 6.2, obtains fermentation medium.
(2) proceed to 4h in step (1) fermentation, the Asia that concentration is 1g/L is added in every liter of fermentation medium (i.e. zymotic fluid)
Selenic acid sodium solution 3.3ml；Fermentation proceeds to 16h, and every liter of fermentation medium (i.e. zymotic fluid) adds the selenous acid that concentration is 1g/L
Sodium solution 2.6ml.Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal zymotic fluid；
It is measured by the method for plate culture count, L.reuteri cultivates 18h viable counts in high density fermentation culture medium and reaches
4.6 × 1010CFU/ml.When fermentation is completed, the Se content of terminal zymotic fluid is 2640 μ g/L, and the Se content of thalline is 220 μ
g/g.Terminal zymotic fluid is centrifuged, after thalline centrifugation, aas determination supernatant selenium concentration is 0.Terminal is sent out
Zymotic fluid obtains the thalline of selenium-rich lactobacillus reuteri after centrifuging, being freeze-dried, which is 12g/L (i.e. every liter of ends
Select the thalline containing 12g dry weight meters in zymotic fluid)；
(3) by the terminal filtering fermentation liquor in step (2), the wet thallus of selenium-rich lactobacillus reuteri, every gram of wet bacterium are obtained
Body adds the freeze drying protectant of 1ml, which is 25% milk power solution (i.e. 100g distilled water is dissolved with 25g milk powder),
Selenium-rich lactobacillus reuteri active bacteria formulation can be obtained after freeze-drying, said preparation is lyophilized bacterium powder.The viable count of lyophilized bacterium powder is
(0.1-5)×1012CFU/g, Se content are 126-260 μ g/g.Preferably, the viable count for freezing bacterium powder is 5 × 1012CFU/g,
Se content is 176 μ g/g.
Embodiment 4：
(1) above-mentioned cultured seed is inoculated by 10% (v/v) inoculum concentration in high density fermentation culture medium, anaerobism training
Support 18h.Include following raw material per high-density fermentative medium formula in the present embodiment：90g sucrose, 90g yeast extracts, 3g lemons
Lemon acid ammonium, 5g sodium acetates, 4g dipotassium hydrogen phosphates, 0.3g manganese sulfates, 0.6g magnesium sulfate and 2g Tween 80s, with phosphoric acid tune pH during configuration
To 5.5, fermentation medium is obtained.
(2) 4h is proceeded in step (1) fermentation, every liter of fermentation medium (i.e. zymotic fluid) adds in the sub- selenium that concentration is 1g/L
Acid sodium solution 1.8ml；Fermentation proceeds to 12h, and every liter of fermentation medium (i.e. zymotic fluid) adds the sodium selenite that concentration is 1g/L
Solution 2.6ml.Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal zymotic fluid；
It is measured by the method for plate culture count, L.reuteri cultivates 18h viable counts in high density fermentation culture medium and reaches
2.8 × 1010CFU/ml.When fermentation is completed, the Se content of terminal zymotic fluid is 1890 μ g/L, and the Se content of thalline is 189 μ
g/g.Terminal zymotic fluid is centrifuged, after thalline centrifugation, aas determination supernatant selenium concentration is 0.Terminal is sent out
Zymotic fluid obtains the thalline of selenium-rich lactobacillus reuteri after centrifuging, being freeze-dried, and dry cell weight is 10g/L (i.e. every liter of terminals
Thalline containing 10g dry weight meters in zymotic fluid)；
(3) by the terminal filtering fermentation liquor in step (2), the wet thallus of selenium-rich lactobacillus reuteri, every gram of wet bacterium are obtained
Body adds the freeze drying protectant of 1ml, the freeze drying protectant include 5% glycerite (i.e. 100g distilled water dissolved with 5g glycerine) and
The mixed liquor of 25% milk power solution (i.e. 100g distilled water is dissolved with 25g milk powder), can obtain selenium-rich Luo Yishi breasts after freeze-drying
Bacillus active bacteria formulation, said preparation are lyophilized bacterium powder, and the viable count for freezing bacterium powder is 4.5 × 1012CFU/g, Se content are 145.4 μ
g/g。
Embodiment 5：
(1) above-mentioned cultured seed is inoculated by 10% (v/v) inoculum concentration in high density fermentation culture medium, anaerobism training
It supports for 24 hours.Include following raw material per high-density fermentative medium formula in the present embodiment：90g sucrose, 90g yeast extracts, 3g lemons
Lemon acid ammonium, 5g sodium acetates, 4g dipotassium hydrogen phosphates, 0.3g manganese sulfates, 0.6g magnesium sulfate and 2g Tween 80s, with phosphoric acid tune pH during configuration
To 5.5, fermentation medium is obtained.
(2) 4h is proceeded in step (1) fermentation, every liter of fermentation medium (i.e. zymotic fluid) adds in the sub- selenium that concentration is 1g/L
Acid sodium solution 3.3ml；Fermentation proceeds to 12h, and every liter of fermentation medium (i.e. zymotic fluid) adds the sodium selenite that concentration is 1g/L
Solution 1.1ml.Fermentation is completed after fermentation proceeds to 20-24h, obtains terminal zymotic fluid；
It is measured by the method for plate culture count, L.reuteri cultivates 18h viable counts in high density fermentation culture medium and reaches
1.6 × 1010CFU/ml.When fermentation is completed, the Se content of terminal zymotic fluid is 2006 μ g/L, and the Se content of thalline is 200 μ
g/g.Terminal zymotic fluid is centrifuged, after thalline centrifugation, aas determination supernatant selenium concentration is 0.Terminal is sent out
Zymotic fluid obtains the thalline of selenium-rich lactobacillus reuteri after centrifuging, being freeze-dried, and dry cell weight is 10g/L (i.e. every liter of terminals
Thalline containing 10g dry weight meters in zymotic fluid)；
(3) by terminal filtering fermentation liquor in step (2), the wet thallus of selenium-rich lactobacillus reuteri, every gram of wet thallus are obtained
The freeze drying protectant of 1ml is added, which includes 2% trehalose (i.e. 100g distillation water dissolution 2g trehaloses) and 25%
Milk powder (i.e. 100g distillation water dissolution 25g milk powder), can obtain selenium-rich lactobacillus reuteri active bacteria formulation after freeze-drying., the system
Agent is lyophilized bacterium powder, and the viable count for freezing bacterium powder is 4.2 × 1012CFU/g, Se content are 157.5 μ g/g.
The method of selenium-rich lactobacillus reuteri high density fermentation proposed by the present invention is realized synchronous rich while fermentation
Selenium, selenium-rich lactobacillus reuteri nutrient solution and selenium-rich lactobacillus reuteri active bacteria formulation are respectively provided with higher viable count, best result
It is not corresponding up to 5 × 1010CFU/ml and 5 × 1012CFU/g, and wherein containing only Organic Selenium, organic selenium content highest is distinguished corresponding
Up to 3000 μ g/L and 176 μ g/g；Therefore prepared selenium-rich lactobacillus reuteri nutrient solution and selenium-rich lactobacillus reuteri are lived
Bacteria preparation is a kind of excellent selenium-supply probiotic composition, available for food and feed additive.
The method of selenium-rich lactobacillus reuteri high density fermentation proposed by the present invention, has the advantages that：
1st, the present invention may be such that final cell concentration than common MRS culture mediums using high density fermentation culture medium and technique
The cell concentration of acquisition improves nearly 10 times.
2nd, the present invention uses selenium-supply strategy stage by stage, and inorganic selenium in fermentation process can be overcome to inhibit thalli growth, and same
When ensure inorganic selenium 100% be converted into Organic Selenium, avoid obtained preparation simultaneously include inorganic selenium and Organic Selenium.
3rd, technique of the invention, can be direct to remove unconverted inorganic selenium without the washing thalline after the completion of final fermentation
Additive of water etc. is quoted as animal, without other lock out operation.
The method for product of above-described embodiment and the non-limiting present invention, any person of an ordinary skill in the technical field is to it
The appropriate variation or modification done all should be regarded as not departing from the patent category of the present invention.
Claims (8)
- A kind of 1. method of selenium-rich lactobacillus reuteri high density fermentation, which is characterized in that include the following steps：(1) by lactobacillus reuteri from plating to seed culture medium in, 37 DEG C of Anaerobic culturel 12-18h, every liter of seed culture Based formulas includes following raw material：20g sucrose, 20g yeast extracts, 3.0g dipotassium hydrogen phosphates, 7.0g sodium acetates, 1.0g citric acids Ammonium, 0.2g magnesium sulfate, 0.18g manganese sulfates, the distilled water of 1.0g Tween 80s and surplus, with after phosphoric acid tune pH to 6.2 during configuration To seed culture medium；(2) seed is inoculated by 3%-10% inoculum concentrations in fermentation medium, Anaerobic culturel 16-24h, every liter of fermentation medium Formula includes：80-150g sucrose, 80-150g yeast extracts, 2-7g ammonium citrates, 5-9g sodium acetates, 2-6g dipotassium hydrogen phosphates, 0.2-0.6g manganese sulfates, 0.5-0.8g magnesium sulfate and 1-3g Tween 80s, with being fermented after phosphoric acid tune pH to 5.5-6.2 during configuration Culture medium；(3) 4h is proceeded in step (2) fermentation, it is 1g/L sodium selenite solutions 1.1- that concentration is added in every liter of fermentation medium 3.3ml；Fermentation proceeds to 12-16h, and every liter of fermentation medium adds concentration as 1g/L sodium selenite solution 1.1-3.3ml, fermentation Fermentation is completed after proceeding to 20-24h, obtains terminal zymotic fluid.
- 2. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as described in claim 1, which is characterized in that further include Step (4)：Stabilizer is added in terminal zymotic fluid in step (3), selenium-rich lactobacillus reuteri nutrient solution is made；The stabilizer is pectin, gelatin, converted starch, quickly dissolving agar, lactoprotein, carboxymethylcellulose sodium, acidproof CMC, algae A variety of combinations or one of which in acid propylene glycol ester, xanthans, carragheen, guar gum, Arabic gum.
- 3. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as claimed in claim 2, it is characterised in that：The breast Albumen includes casein sodium and/or lactalbumin.
- 4. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as claimed in claim 2, which is characterized in that in step (4) in, selenium-rich lactobacillus reuteri nutrient solution viable count is (0.8-5) × 1010CFU/ml, Se content are 1400-3000 μ g/L.
- 5. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as described in claim 1, which is characterized in that further include Step (4)：Terminal zymotic fluid in step (3) is filtered, the thalline of selenium-rich lactobacillus reuteri is obtained after filtering, then Freeze drying protectant is added, the thalline for being with the addition of freeze drying protectant is freeze-dried, selenium-rich Luo Yishi is obtained after freeze-drying Living preparation of lactobacillus preparation；The freeze drying protectant is glycerine, sorbierite, mannitol, sucrose, trehalose, polyvinylpyrrolidone, glucan, milk A variety of combinations or one of which in powder, vitamin E.
- 6. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as claimed in claim 5, it is characterised in that：In step (4) in, measured by the method for plate culture count, the viable count of selenium-rich lactobacillus reuteri active bacteria formulation for (0.1-5) × 1012CFU/g, Se content are 126-260 μ g/g.
- 7. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as described in claim 1, it is characterised in that：In step (3) in, measured by the method for plate culture count, when lactobacillus reuteri cultivates 18h in the fermentation medium, viable count is (0.8-5)×1010CFU/ml；After the completion of fermentation, the Se content of terminal zymotic fluid is 1200-3000 μ g/L, and thalline Se content is 126-260 μ g/g.
- 8. a kind of method of selenium-rich lactobacillus reuteri high density fermentation as described in claim 1, it is characterised in that：In step (3) in, the thalline of selenium-rich lactobacillus reuteri, dry cell weight 9- are obtained after terminal zymotic fluid is centrifuged, is freeze-dried 12g/L。
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| CN111534434A (en) * | 2020-06-28 | 2020-08-14 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis |
| CN115161221A (en) * | 2022-05-26 | 2022-10-11 | 广州朴成生物科技有限公司 | Preparation method and application of selenium-rich lactobacillus gasseri agent for improving diarrhea of weaned piglets |
| CN115176866A (en) * | 2022-07-15 | 2022-10-14 | 西北工业大学 | Micro-ecological feed additive for enriching nano selenium and zinc oxide and preparation method thereof |
| CN116286471A (en) * | 2023-01-10 | 2023-06-23 | 华南农业大学 | Application of tricholoma matsutake extract in improving number of live bacteria of lactobacillus reuteri high-density culture |
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| CN110016443A (en) * | 2019-04-02 | 2019-07-16 | 山东大学 | A kind of lactobacillus reuteri and its application in Se-enriched egg production |
| CN110016443B (en) * | 2019-04-02 | 2022-09-16 | 山东大学 | Lactobacillus reuteri and application thereof in production of selenium-rich eggs |
| CN111534434A (en) * | 2020-06-28 | 2020-08-14 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis |
| CN115161221A (en) * | 2022-05-26 | 2022-10-11 | 广州朴成生物科技有限公司 | Preparation method and application of selenium-rich lactobacillus gasseri agent for improving diarrhea of weaned piglets |
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| CN115176866A (en) * | 2022-07-15 | 2022-10-14 | 西北工业大学 | Micro-ecological feed additive for enriching nano selenium and zinc oxide and preparation method thereof |
| CN115176866B (en) * | 2022-07-15 | 2023-08-22 | 西北工业大学 | Microecological feed additive for enriching nano selenium and zinc oxide and preparation method thereof |
| CN116286471A (en) * | 2023-01-10 | 2023-06-23 | 华南农业大学 | Application of tricholoma matsutake extract in improving number of live bacteria of lactobacillus reuteri high-density culture |
| CN116286471B (en) * | 2023-01-10 | 2023-11-28 | 华南农业大学 | Application of tricholoma matsutake extract in improving number of live bacteria of lactobacillus reuteri high-density culture |
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Application publication date: 20180518 |