CN103045517A - Culture method for producing gamma-aminobutyric acid lactic acid bacteria - Google Patents
Culture method for producing gamma-aminobutyric acid lactic acid bacteria Download PDFInfo
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- CN103045517A CN103045517A CN 201210586397 CN201210586397A CN103045517A CN 103045517 A CN103045517 A CN 103045517A CN 201210586397 CN201210586397 CN 201210586397 CN 201210586397 A CN201210586397 A CN 201210586397A CN 103045517 A CN103045517 A CN 103045517A
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- aminobutyric acid
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Abstract
The invention discloses a culture method for producing gamma-aminobutyric acid lactic acid bacteria, which comprises the following steps: at room temperature, taking lactobacillus plantarum slant spores and using sterile water to prepare the spore suspension liquid; accommodating the spore suspension liquid into a sterile quartz tank and performing intensive pulse light treatment; using the sterile water to dilute the spore suspension liquid after the intensive pulse light treatment, and coating the MRS culture medium with the diluted bacterial suspension for constant-temperature culture; selecting a single strain with the diameter of the calcium-dissolving zone of more than 0.5 mm and leading in a triangular flask accommodating the MRS culture medium for static culture; and leading the bacterial suspension of the MRS culture medium in a triangular flask internally accommodating 50 mL GYP fluid nutrient medium which contains sodium glutamate for static culture, centrifuging and taking the supernatant fluid to screen out the bacterial strain of which the yield is larger than the yield of the original bacterial strain gamma-aminobutyric acid. According to the invention, the method is simple, the operation is safe, the mutagenic efficiency is high, the positive variation rate of the mutant strain of the lactobacillus plantarum after mutagenesis is 5-30 percent, and the yield of the bacterial strain gamma-aminobutyric acid is high and improved by 1-8 times of the yield of the original bacterial strain.
Description
Technical field
The invention belongs to microorganism mutagenesis field, particularly the cultural method of a kind of milk-acid bacteria for the production of γ-aminobutyric acid.
Background technology
The pulse strong-light technology is to utilize the pulse engineering spark and special rare gas element fluorescent tube, excite strong white light with impulse form, spectral distribution is similar to sunlight, light intensity is equivalent to arrive the light source technology of the thousands of of earth surface sunlight intensity and even tens thousand of times, it utilizes instantaneous, high-intensity pulsed light energy to kill each quasi-microorganism, is widely applied to the various fields such as water treatment, air disinfection, food-processing, pharmacy, agricultural byproducts.
γ-aminobutyric acid has another name called 4-Aminobutanoicacid (4-aminobutyricacid), be called for short GABA, it is a kind of inhibition transmitter substance, have anxiety, hypotensive, treatment epilepsy, improve sleep, anti-ageing, regulate hormone secretion and the important physiologically active of neurotrophy, be widely used in food, medicine and chemical field.
Plant lactobacillus (Lactobacillus plantarum) has the γ-aminobutyric acid ability that generates, and therefore studies plant lactobacillus fermentation preparation and is rich in the γ-aminobutyric acid functional foodstuff and has practical guided significance.But the γ-aminobutyric acid output of plant lactobacillus is generally 0.5g/L～1.2g/L, even after carrying out medium optimization, γ-aminobutyric acid output is the highest can only to reach 5.8g/L, and output is lower, is difficult to realize suitability for industrialized production.
Summary of the invention
The technical problem to be solved in the present invention provides the cultural method of a kind of milk-acid bacteria for the production of γ-aminobutyric acid, and the bacterial strain γ-aminobutyric acid output that this cultural method obtains is high, can realize suitability for industrialized production.
Technical solution of the present invention is:
The cultural method of a kind of milk-acid bacteria for the production of γ-aminobutyric acid, its concrete steps are as follows:
1, gets plant breast bar and detect γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
2, get spore suspension 0.1 mL～0.3 mL and pack in the aseptic quartz cell, then carry out pulse strong-light and process 5s～300s;
3, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-1Doubly～10
-7Doubly, the bacteria suspension 100 μ L that get dilution are coated on the MRS substratum, under 28 ℃～32 ℃, and constant temperature culture 48h～72h;
4, the single strain access of choosing molten calcium loop diameter＞0.5mm is equipped with in the triangular flask of 50mLMRS substratum, under 28 ℃～32 ℃, leaves standstill and cultivates 48h～72h; The bacteria suspension of getting 1mL～3mL MRS substratum accesses in the triangular flask of GYP liquid nutrient medium that in-built 50mL contains Sodium Glutamate, the concentration of described GYP liquid nutrient medium Glutamic Acid sodium is 8g/L～12g/L, under 28 ℃～32 ℃, leave standstill and cultivate 48h～72h, centrifugal, get supernatant liquor and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the milk-acid bacteria for the production of γ-aminobutyric acid.
When carrying out the pulse strong-light processing, adopt high light surface disinfection experiment cabinet, the operating frequency 1Hz of described high light surface disinfection experiment cabinet～5 Hz, single output energy 100J～500J, input voltage is 200V～240V, output voltage 2800V.
Beneficial effect of the present invention:
Adopt the mode of pulse strong-light irradiation, plant lactobacillus is carried out mutagenesis, mutafacient system is simple, operational safety, and efficiency of inducing mutation is high; The mutant strain positive variation rate 5%～30% of plant lactobacillus after mutagenesis, bacterial strain γ-aminobutyric acid output is high, and the γ-aminobutyric acid production peak can reach 21.6g/L, than 1 times～18 times of original strain output increaseds.
Embodiment
Embodiment 1
1, preparation MRS substratum and GYP liquid nutrient medium, substratum is composed as follows:
MRS substratum: peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, glucose 20 g/L, anhydrous sodium acetate 5 g/L, dibasic ammonium citrate 2 g/L, KH
2PO
42 g/L, tween 80 1mL/L, MnSO
44H
2O 0. 25 g/L, MgSO
47H
2O 0. 58 g/L, pH6. 2～6. 4;
GYP liquid nutrient medium: glucose 10g/L, yeast extract paste 10g/L, peptone 5g/L, anhydrous sodium acetate 2g/L, MgSO
47H
2O 0. 02g/L, MnSO
44H
2O 0. 001g/L, FeSO
47H
2O 0. 001g/L, NaCl 0. 001g/L, pH6. 6～6. 8;
2, getting plant breast bar adopts HPLC to detect γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.2mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet (pulse strong-light sterilizer) and carry out pulse strong-light processing 300s, the operating frequency 1Hz of high light surface disinfection experiment cabinet, single output energy 500J, input voltage is 220V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-7Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 30 ℃, and constant temperature culture 48h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 30 ℃, leave standstill and cultivate 48h; The bacteria suspension access of getting the 2mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 10g/L, under 30 ℃, leave standstill and cultivate 48h, centrifugal, getting supernatant liquor adopts HPLC to detect, and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 15%～18%, the output of γ-aminobutyric acid are 5.2g/L～18.4g/L, and the γ-aminobutyric acid turnout improves 3 times～15 times than original strain.
High performance liquid chromatography (HPLC): the supernatant liquor after centrifugal with the trichoroacetic acid(TCA) dilution of 50g/L carries out o-phthalaldehyde(OPA) (OPA) column front derivation as test sample solution.Deriving method: draw 400 μ L borate buffers (pH value 10. 2,0. 4 mol/L), 80 μ L test sample solution and o-phthalaldehyde(OPA) derivative reagent 160 μ L mix, and at room temperature react 2 min.Chromatographic condition is as follows: and moving phase V (0. 02mol/L sodium-acetate buffer, pH=7.5): V (acetonitrile)=3:1; Chromatographic column is Sun-Fire
TMC
18Post (4. 6 mm * 250 mm, 5 μ m), detector is UV-detector, and detecting wavelength is 338 nm, and flow velocity is 0. 8 mL/min, elution time 12 min, sample size 20 μ L.
Embodiment 2
1, preparation MRS substratum and GYP liquid nutrient medium, substratum forms with embodiment 1;
2, getting plant breast bar adopts HPLC to detect (the HPLC detection method is with embodiment 1) γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.1mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet and carry out pulse strong-light processing 180s, the operating frequency 3Hz of high light surface disinfection experiment cabinet, single output energy 200J, input voltage is 200V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-5Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 28 ℃, and constant temperature culture 72h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 28 ℃, leave standstill and cultivate 72h; The bacteria suspension access of getting the 1mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 8g/L, under 28 ℃, leave standstill and cultivate 72h, centrifugal, getting supernatant liquor adopts HPLC to detect (the HPLC detection method is with embodiment 1), and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 8%～13%, the output of γ-aminobutyric acid are 6.8g/L～13.4g/L, and the γ-aminobutyric acid turnout improves 5 times～11 times than original strain.
Embodiment 3
1, preparation MRS substratum and GYP liquid nutrient medium, substratum forms with embodiment 1;
2, getting plant breast bar adopts HPLC to detect (the HPLC detection method is with embodiment 1) γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.1mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet and carry out pulse strong-light processing 240s, the operating frequency 2Hz of high light surface disinfection experiment cabinet, single output energy 300J, input voltage is 220V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-1Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 32 ℃, and constant temperature culture 48h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 32 ℃, leave standstill and cultivate 48h; The bacteria suspension access of getting the 3mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 12g/L, under 32 ℃, leave standstill and cultivate 48h, centrifugal, getting supernatant liquor adopts HPLC to detect (the HPLC detection method is with embodiment 1), and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 25%～30%, the output of γ-aminobutyric acid are 14.2g/L～21.6g/L, and the γ-aminobutyric acid turnout improves 11 times～18 times than original strain.
Embodiment 4
1, preparation MRS substratum and GYP liquid nutrient medium, substratum forms with embodiment 1;
2, getting plant breast bar adopts HPLC to detect (the HPLC detection method is with embodiment 1) γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.3mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet and carry out pulse strong-light processing 120s, the operating frequency 1Hz of high light surface disinfection experiment cabinet, single output energy 400J, input voltage is 220V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-3Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 30 ℃, and constant temperature culture 60h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 30 ℃, leave standstill and cultivate 60h; The bacteria suspension access of getting the 2mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 12g/L, under 30 ℃, leave standstill and cultivate 60h, centrifugal, getting supernatant liquor adopts HPLC to detect (the HPLC detection method is with embodiment 1), and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 22%～26%, the output of γ-aminobutyric acid are 10.2 g/L～14.6g/L, and the γ-aminobutyric acid turnout improves 8 times～12 times than original strain.
Embodiment 5
1, preparation MRS substratum and GYP liquid nutrient medium, substratum forms with embodiment 1;
2, getting plant breast bar adopts HPLC to detect (the HPLC detection method is with embodiment 1) γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.2mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet and carry out pulse strong-light processing 120s, the operating frequency 4Hz of high light surface disinfection experiment cabinet, single output energy 100J, input voltage is 220V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-6Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 28 ℃, and constant temperature culture 64h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 28 ℃, leave standstill and cultivate 64h; The bacteria suspension access of getting the 1mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 10g/L, under 28 ℃, leave standstill and cultivate 64h, centrifugal, getting supernatant liquor adopts HPLC to detect (the HPLC detection method is with embodiment 1), and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 12%～23%, the output of γ-aminobutyric acid are 14.8 g/L～21.6g/L, and the γ-aminobutyric acid turnout improves 12 times～18 times than original strain.
Embodiment 6
1, preparation MRS substratum and GYP liquid nutrient medium, substratum forms with embodiment 1;
2, getting plant breast bar adopts HPLC to detect (the HPLC detection method is with embodiment 1) γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
3, get spore suspension 0.3mL and pack aseptic quartz cell into (in 10mm * 10mm * 35mm), put into high light surface disinfection experiment cabinet and carry out pulse strong-light processing 5s, the operating frequency 1Hz of high light surface disinfection experiment cabinet, single output energy 500J, input voltage is 240V, output voltage 2800V;
4, the spore suspension of pulse strong-light being processed is diluted to 10 with sterilized water
-4Doubly, the spore suspension 100 μ L that get dilution are coated on the MRS substratum, under 30 ℃, and constant temperature culture 48h;
5, choose the above single strain access of molten calcium loop diameter 0.5mm and be equipped with in the 250mL triangular flask of 50mLMRS substratum, under 30 ℃, leave standstill and cultivate 48h; The bacteria suspension access of getting the 3mLMRS substratum is equipped with in the 250mL triangular flask of GYP liquid nutrient medium of Sodium Glutamate that 50mL is added with 8g/L, under 30 ℃, leave standstill and cultivate 48h, centrifugal, getting supernatant liquor adopts HPLC to detect (the HPLC detection method is with embodiment 1), and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the plant lactobacillus of highly producing gamma-aminobutyric acid; Mutant strain positive variation rate 16%～25%, the output of γ-aminobutyric acid are 14.4 g/L～20.0g/L, and the γ-aminobutyric acid turnout improves 12 times～16 times than original strain.
Claims (2)
1. cultural method for the production of the milk-acid bacteria of γ-aminobutyric acid, it is characterized in that: concrete steps are as follows:
1.1, get plant breast bar and detect γ-aminobutyric acid output as original strain γ-aminobutyric acid output; At room temperature, get this plant lactobacillus slant pore and make 10 with sterilized water
4～10
6The spore suspension of individual spore/mL;
1.2, get spore suspension 0.1 mL～0.3 mL and pack in the aseptic quartz cell, then carry out pulse strong-light and process 5s～300s;
1.3, spore suspension that pulse strong-light is processed is diluted to 10 with sterilized water
-1Doubly～10
-7Doubly, the bacteria suspension 100 μ L that get dilution are coated on the MRS substratum, under 28 ℃～32 ℃, and constant temperature culture 48h～72h;
1.4, the single strain access of choosing molten calcium loop diameter＞0.5mm is equipped with in the triangular flask of 50mLMRS substratum, under 28 ℃～32 ℃, leave standstill and cultivate 48h～72h; The bacteria suspension of getting 1mL～3mL MRS substratum accesses in the triangular flask of GYP liquid nutrient medium that in-built 50mL contains Sodium Glutamate, the concentration of described GYP liquid nutrient medium Glutamic Acid sodium is 8g/L～12g/L, under 28 ℃～32 ℃, leave standstill and cultivate 48h～72h, centrifugal, get supernatant liquor and filter out bacterial strain greater than original strain γ-aminobutyric acid output, be the milk-acid bacteria for the production of γ-aminobutyric acid.
2. the cultural method of the milk-acid bacteria for the production of γ-aminobutyric acid according to claim 1, it is characterized in that: when carrying out the pulse strong-light processing, adopt high light surface disinfection experiment cabinet, the operating frequency 1Hz of described high light surface disinfection experiment cabinet～5 Hz, single output energy 100J～500J, input voltage is 200V～240V, output voltage 2800V.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182171A (en) * | 2018-09-04 | 2019-01-11 | 湖南肯基因科技有限公司 | The mutagenic strain and its biological agent of highly producing gamma-aminobutyric acid |
| CN110791451A (en) * | 2019-11-25 | 2020-02-14 | 山东中科嘉亿生物工程有限公司 | Lactobacillus plantarum JYLP-326 and application thereof in improving sleep and product |
| CN115161250A (en) * | 2022-09-06 | 2022-10-11 | 广东益可维生物技术有限公司 | Lactobacillus plantarum with functions of improving sleep, strengthening brain and improving intelligence and application thereof |
-
2012
- 2012-12-30 CN CN 201210586397 patent/CN103045517A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182171A (en) * | 2018-09-04 | 2019-01-11 | 湖南肯基因科技有限公司 | The mutagenic strain and its biological agent of highly producing gamma-aminobutyric acid |
| CN109182171B (en) * | 2018-09-04 | 2021-06-29 | 湖南肯基因科技有限公司 | Mutagenic strain for high yield of gamma-aminobutyric acid and biological preparation thereof |
| CN110791451A (en) * | 2019-11-25 | 2020-02-14 | 山东中科嘉亿生物工程有限公司 | Lactobacillus plantarum JYLP-326 and application thereof in improving sleep and product |
| CN115161250A (en) * | 2022-09-06 | 2022-10-11 | 广东益可维生物技术有限公司 | Lactobacillus plantarum with functions of improving sleep, strengthening brain and improving intelligence and application thereof |
| CN115161250B (en) * | 2022-09-06 | 2022-12-13 | 广东益可维生物技术有限公司 | Lactobacillus plantarum with functions of improving sleep, strengthening brain and improving intelligence and application thereof |
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Application publication date: 20130417 |