CN103966139A - Lactobacillus brevis capable of producing gamma-aminobutyric acid at high yield in Sichuan pickle vegetables - Google Patents
Lactobacillus brevis capable of producing gamma-aminobutyric acid at high yield in Sichuan pickle vegetables Download PDFInfo
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- CN103966139A CN103966139A CN201410205013.2A CN201410205013A CN103966139A CN 103966139 A CN103966139 A CN 103966139A CN 201410205013 A CN201410205013 A CN 201410205013A CN 103966139 A CN103966139 A CN 103966139A
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- lactobacillus brevis
- aminobutyric acid
- short lactobacillus
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- gaba
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Abstract
The invention provides a lactobacillus brevis capable of producing gamma-aminobutyric acid at high yield in Sichuan pickle vegetables and the lactobacillus brevis can be used for solving the problem of low GABA production capability of the existing lactobacillus brevis. The bacterial are separated from Sichuan pickle vegetables and have the capability of producing the gamma-aminobutyric acid in a culture medium containing sodium glutamate, and belong to the lactobacillus brevis; the collection number of the bacterial strain is CGMCC NO.9029. The lactobacillus brevis is inoculated to irradiation-sterilized coarse rice powder so that the content of the gamma-aminobutyric acid in the coarse rice powder is increased by 5 times.
Description
technical field:
The present invention the invention belongs to technical field of food biotechnology, relates to the short lactobacillus of a kind of highly producing gamma-aminobutyric acid in pickles.
background technology:
γ-aminobutyric acid (γ-aminobutyric acid is called for short GABA) is a kind of bioactive nonprotein amino acid that has, and is inhibitory neurotransmitter important in mammalian central nervous system.Clinical study shows to take in GABA or analogue can be hypotensive, anticancer growth, irritation cancer apoptosis, all right tranquilizing and allaying excitement, diuresis.The relative disease that the food of GABA can effectively improve depression, insomnia, schizophrenia and drink for a long time and cause is rich in absorption.
Milk-acid bacteria is that a class can utilize carbohydrate fermentation to produce the general name of the bacterium of a large amount of lactic acid.Milk-acid bacteria is widely used in food, medicine and other fields.It can give food special local flavor in food fermentation process, improves the quality of food and the characteristic of nutrition.Milk-acid bacteria is the main source of probiotic bacterium, through scientific research, confirm that many milk-acid bacterias are the intrinsic probiotic bacteriums of human intestinal, there is regulating intestinal canal flora, reduce serum cholesterol, regulate blood pressure, anti-oxidant, anticancer and strengthen the different physiological roles characteristics such as host immune power.In whole food, GABA content is lower, and traditional zymotic pickles contain abundant Resource of lactic bacteria database.
summary of the invention:
The object of this invention is to provide a kind of from Sichuan spontaneous fermentation pickles water the isolated short lactobacillus with high yield GABA ability.
A kind of short lactobacillus of highly producing gamma-aminobutyric acid in Pickles, Sichuan Style, this bacterium is located away from Pickles, Sichuan Style, has in the ability containing producing γ-aminobutyric acid in the liquid nutrient medium of Sodium Glutamate, belongs to short lactobacillus (Lactobacillus brevis).
Technological operation step is:
The acquisition of short lactobacillus of the present invention: the pickles water of fetching laboratory is diluted to suitable gradient by stroke-physiological saline solution, gets 1mL diluent in sterile petri dish, topples over solid medium and mixes, be cultured to and occur a large amount of single bacterium colonies, picking colony, repeatedly streak culture, carry out purifying.
Short lactobacillus provided by the invention is from numbering: p-14, deposit number is CGMCC NO.9029, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address on April 9th, 2014: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Short lactobacillus of the present invention has the GABA of producing ability: short lactobacillus p-14 are seeded in liquid nutrient medium, 30 ℃ of fermentations, adopt thin-layer chromatography qualitative detection, it has the GABA of producing ability, measures GABA content in fermented liquid reached 501mg/100g with automatic analyzer for amino acids.
Short lactobacillus of the present invention is seeded in the coarse rice powder of irradiation sterilization, and 30 ℃ of fermentations 3 days, make GABA content in coarse rice powder reach 64.7mg/100g, than fermented brown rice powder not, have improved 5 times.
The present invention produces GABA short lactobacillus and has good stability, cultivates for continuous 20 times, and proterties is stable, still has high yield GABA ability.
Short lactobacillus of the present invention p-4 separation, from Sichuan traditional natural fermentation pickled vegetable water, without crossing artificial mutagenic treatment, belongs to generally recognized as safe (Generally Recognized As Safe, GRAS) bacterial classification, can be used for food-processing.
embodiment:
Below the specific embodiment of the invention is made a detailed explanation.
The separation method of high yield GABA short lactobacillus of the present invention, adopts the mixed bacterium method of gradient dilution separated from Sichuan spontaneous fermentation pickles water.Comprise the following steps: 1mL pickles water is carried out to gradient dilution by 9mL stroke-physiological saline solution, and being diluted to pickles water concentration is 10
-6-10
-8mL/mL, get 1mL diluent in sterile petri dish, topple over solid medium and mix, after culture medium solidifying, be placed in 30 ℃ of incubators and cultivate 48-96 hours, there are a large amount of bacterial colonys, picking has single bacterium colony of different characteristics, the purifying of ruling on isolation medium, and further cultivate, pure bacterial strain obtained.The bacterial strain of purifying adopts 2 kinds of mode preservations,
with liquid nutrient medium, at 30 ℃, cultivate after 24-36 hours, in cultured liquid nutrient medium, add glycerine, making glycerine final concentration is 20%(w/v) ,-20 ℃ are frozen, every switching in 1 year once;
bacterial classification percutaneous puncture-inoculation, in semisolid medium, is cultivated after 48 hours for 30 ℃, be put in 4 ℃ of refrigerations, every switching in 4 months once.
Short lactobacillus of the present invention is seeded on solid medium, cultivates after 48 hours for 30 ℃, observes colony characteristics, and bacterium colony is large, circular, projection, oyster white, and surface wettability is smooth, and neat in edge, with short lactobacillus plesiomorphism; By examining under a microscope cell characteristic after gramstaining, Gram-positive, cell is rod-short, without gemma.Catalase test is negative, 15 ℃ of growths, do not grow for 45 ℃, sodium chloride tolerance scope is 0-6%(v/w), can utilize pectinose, fructose, semi-lactosi, glucose, maltose, xylose fermentation for producing acid, can slowly utilize melibiose to produce acid, unfermentable amygdaloside, cellobiose, polychrom, lactose, N.F,USP MANNITOL, seminose, melizitose, raffinose, rhamnosyl, ribose, saligenin, sorbyl alcohol, sucrose, trehalose.Its physiological and biochemical property is the most similar to short lactobacillus (L.brevis), but also there is some difference, and it can not utilize polychrom, ribose, raffinose to produce acid, and the short lactobacillus reference culture L.brevis DSM20054 above three classes sugar products that can ferments are sour.In the present invention, short lactobacillus P-14 can not utilize ribose to produce acid, and this character is different from product GABA short lactobacillus L.brevis CCTCCM 208054.In the present invention, short lactobacillus can not utilize seminose fermentation, is that CCTCCM 209132 is different from high yield GABA short lactobacillus L2 preserving number.Extract after DNA, with bacterium universal primer 27F, 1492R amplifies its 16S rDNA, delivers to the order-checking of the English Weihe River prompt base (Shanghai) trade Co., Ltd, and sequence length is 1434bp, with short lactobacillus (L.brevis) similarity up to more than 99%.Identify that this bacterial strain is short lactobacillus (L.brevis).In short lactobacillus of the present invention and Genbank, short lactobacillus similarity is more than 99%, and short lactobacillus L.brevis CCTCCM 208054 and short lactobacillus CCTCCM 209132 are 97% with short lactobacillus similarity.
High yield GABA short lactobacillus of the present invention produces GABA qualitative detection, adopt thin layer chromatography, concrete steps comprise: the thalline of the appropriate refrigeration of picking is inoculated in 5mL liquid nutrient medium, 30 ℃ of standing for fermentation 48 hours, get 1 μ L fermented liquid point sample on silica gel thin-layer chromatography plate, take concentration as 0.2%(w/w) GABA standardized solution, the liquid nutrient medium that is added with 2% (w/w) GABA be contrast.After exhibition layer, in 105 ℃ of colour developing 5min, take pictures.Observe thin-layer chromatography result, high yield GABA short lactobacillus fermented liquid of the present invention has the band with the identical chromatography distance of GABA mark product.
High yield GABA short lactobacillus of the present invention produces GABA detection by quantitative, adopts automatic analyzer for amino acids (fork clip nurse S-433D type).The thalline streak inoculation of the appropriate refrigeration of picking, to solid medium, waits to grow single bacterium colony, and single bacterium colony thalline of picking is inoculated in 100mL liquid nutrient medium to be cultivated 10 hours, makes viable bacteria body number and is about 10
8individual seed fermentation liquid, by seed fermentation liquid with 2%(v/v) inoculum size be seeded in the triangular flask that 250mL is equipped with 100mL liquid nutrient medium, 30 ℃ of standing for fermentation 72 hours, gained fermented liquid is in the centrifugal 10min of 10000 * g, get supernatant liquor and with automatic analyzer for amino acids, analyze its amino acid composition after 0.22 μ m membrane filtration, take nonvaccinated liquid nutrient medium as contrast.Result shows that in short lactobacillus fermented liquid of the present invention, GABA content has reached 501mg/100g
After short lactobacillus activation of the present invention, be inoculated in liquid nutrient medium, 30 ℃ of cultivations, OD600 is 1.1-1.2, the centrifugal 5min of 6000 * g collects thalline, with after stroke-physiological saline solution washing, make the dense bacteria suspension that OD600 is 10 left and right, add 3mL to (coarse rice powder: sterilized water=1: 2 in 20g irradiation sterilization coarse rice powder; W/v), 30 ℃ of standing for fermentation 3 three days, to inoculate irradiation coarse rice powder, do not compare, after freeze-drying, with automatic analyzer for amino acids, analyze its GABA content, in inoculation fermentation coarse rice powder, GABA content is 65mg/100g, not inoculating GABA content in coarse rice powder is 11mg/100g, and GABA content has improved 5 times.
Short lactobacillus deposit number in foregoing invention is: CGMCC NO.9029, depositary institution: be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date: on April 9th, 2014.
liquid culture based formulas in the present invention is: peptone 10g, beef powder 5g, yeast powder 4g, glucose 20g, Sodium Glutamate 10g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, Triammonium citrate 2g, magnesium sulfate 0.2g, manganous sulfate 0.05g, tween 80 1mL, distilled water 1000mL, pH6.2 left and right.
Solid medium in the present invention is that liquid nutrient medium adds 1.7-2.0%(w/v) agar.
In the present invention, semisolid medium is that liquid nutrient medium adds 1(w/v) agar.
In the present invention, thin-layer chromatography developing agent forms: propyl carbinol: Glacial acetic acid: water (v: v: v=4: 1: 3), contains 0.4% triketohydrindene hydrate in developing agent.
Claims (1)
1. a kind of short lactobacillus of highly producing gamma-aminobutyric acid in Pickles, Sichuan Style, it is characterized in that this bacterium is located away from Pickles, Sichuan Style, have in the ability containing producing γ-aminobutyric acid in the substratum of Sodium Glutamate, belong to short lactobacillus (Lactobacillus brevis), bacterial strain preserving number: CGMCC NO.9029.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907672A (en) * | 2016-06-07 | 2016-08-31 | 广西多得乐生物科技有限公司 | Gamma-aminobutyric acid strain, screening method thereof and method for preparing pickled Chinese cabbage with same |
| CN108034599A (en) * | 2017-12-06 | 2018-05-15 | 江南大学 | One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid for being derived from brewed spirit system |
| CN111778191A (en) * | 2020-07-17 | 2020-10-16 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101724587A (en) * | 2009-09-10 | 2010-06-09 | 浙江师范大学 | Lactobacillus brevis L2 bacterial strain of high yield gamma-aminobutyrique and screening method and applications thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101724587A (en) * | 2009-09-10 | 2010-06-09 | 浙江师范大学 | Lactobacillus brevis L2 bacterial strain of high yield gamma-aminobutyrique and screening method and applications thereof |
Non-Patent Citations (1)
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| 董玲等: "四川泡菜中产γ-氨基丁酸微生物的分离及鉴定", 《食品与发酵科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907672A (en) * | 2016-06-07 | 2016-08-31 | 广西多得乐生物科技有限公司 | Gamma-aminobutyric acid strain, screening method thereof and method for preparing pickled Chinese cabbage with same |
| CN108034599A (en) * | 2017-12-06 | 2018-05-15 | 江南大学 | One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid for being derived from brewed spirit system |
| CN111778191A (en) * | 2020-07-17 | 2020-10-16 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
| CN111778191B (en) * | 2020-07-17 | 2021-05-14 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
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