CN103146599B - Lactobacillus plantarum producing high yield gamma-aminobutyric acid and application of lactobacillus plantarum - Google Patents
Lactobacillus plantarum producing high yield gamma-aminobutyric acid and application of lactobacillus plantarum Download PDFInfo
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Abstract
The invention provides a lactobacillus plantarum (L. plantarum) LW106 producing high yield gamma-aminobutyric acid. The L. plantarum LW106 is preserved in the Chinese common microorganism culture preservation management center, the address is culture preservation center, institute of microbiology, Chinese academy of science at No.1 institute on north star west road, Chaoyang District of Beijing, the postcode is 100101, the preservation number is CGMCC No.5655, and the preservation date is until December 26th in the year of 2011. According to the L. plantarum LW106, ferment liquor with the gamma-aminobutyric acid content of 20-25 g/L can be obtained through fermental cultivation, the total solid content meets requirements of spray drying, and through the spray drying, the mass fraction of a gamma-aminobutyric acid product is above 20%. The L. plantarum LW106 is from traditional fermented food, and the gamma-aminobutyric acid produced and prepared by means of the L. plantarum LW106 has no potential safety hazards and achieves a food safety level.
Description
(1) technical field
The present invention relates to the new bacterial strain of a strain highly producing gamma-aminobutyric acid---plant lactobacillus (Lactobacillus plantarum) LW106, and prepare the application in γ-aminobutyric acid in microorganism fermentation.
(2) background technology
Nervous physiology and medical research confirm that γ-aminobutyric acid is active substance important in human brain, but the accumulation of human body γ-aminobutyric acid becomes abnormal difficult with stress with advancing age.Therefore external source is supplemented γ-aminobutyric acid to make it maintain normal level especially very important for the health of insomnia, depression, high pressure, climacterium, elderly population to human body, studies to show to take in every day the natural γ-aminobutyric acid of 30 ~ 50mg and just can play very good health-care effect.Due to consumption pettiness, γ-aminobutyric acid also can be with other trophic function factor compatibility, and for bread and cheese and protective foods, therefore γ-aminobutyric acid has good market outlook.
The γ-aminobutyric acid that utilizes crude substance and adopt biotechnology to produce can be considered natural γ-aminobutyric acid, has better security and absorptivity compared with the synthetic γ-aminobutyric acid of chemical method.Therefore, high batching and the food containing γ-aminobutyric acid of Applied Biotechnology manufacture is the Main Means that meets γ-aminobutyric acid great market demand.Milk-acid bacteria, as the generally recognized as safe microorganism of one, can efficiently be prepared γ-aminobutyric acid by bio-transformation.Compared with animality milk-acid bacteria, plant lactobacillus has stronger acidproof and bile tolerance ability, is therefore more conducive to γ-aminobutyric acid biosynthesizing.Current separation screening short lactobacillus, Lactobacterium acidophilum, plant lactobacillus etc. produce the plant lactobacillus of γ-aminobutyric acid.
Plant lactobacillus is a kind of important plant lactobacillus, the distinctive lactobacillin of energy output in its fermenting process, there is natural anticorrosion effect, the acid mass-energy degraded heavy metal of its output, its β glucuroide has higher activity, have attenuation, plant lactobacillus also has the ability of synthesis of conjugated linoleic acid in addition, and within 2009, plant lactobacillus is approved for new resource food.Therefore plant lactobacillus is that a kind of good γ-aminobutyric acid is produced bacterial classification.Separate at present the plant lactobacillus that obtains many strains product γ-aminobutyric acids.The Liang Yan of China Agricultural University etc. screens the plant lactobacillus S35 of a strain product γ-aminobutyric acid, and output is 4.52g/L, and Fan Jie etc. screen the plant lactobacillus of a strain product γ-aminobutyric acid, and output is 4.98g/L; Zhejiang Normal University's Jiang's coltsfoot is separated to a lactobacillus plantarum, and γ-aminobutyric acid output is about 4g/L; The Ding Xing of Zhejiang Polytechnical University etc. is separated to a lactobacillus plantarum, and γ-aminobutyric acid output is 0.527g/L, but it is not high in its achievement in research, to produce GABA ability, seldom can be applied to suitability for industrialized production yet.Therefore development and the human health of the functional plants milk-acid bacteria of, screening high yield GABA from milk-acid bacteria to GABA industry all has great importance.
(3) summary of the invention
Product γ-aminobutyric acid plant lactobacillus for current seed selection produces the problems such as γ-aminobutyric acid ability is not strong, the invention provides lactic bacterium strains---plant lactobacillus (Lactobacillus plantarum) LW106 and application thereof that a strain utilizes the dull and stereotyped fast high-flux sieve method of solid double-layer from spontaneous fermentation pickles, to screen the highly producing gamma-aminobutyric acid obtaining.
The technical solution used in the present invention is:
Plant lactobacillus (Lactobacillus plantarum) LW106 of one strain highly producing gamma-aminobutyric acid, be preserved in Chinese common micro-organisms culture presevation administrative center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City DSMZ of Institute of Microorganism, Academia Sinica, postcode 100101, deposit number is: CGMCCNO.5655, preservation date is: on December 26th, 2011, Classification And Nomenclature is plant lactobacillus (Lactobacillus plantarum).
On this bacterial strain MRS solid medium, bacterium colony is oyster white, little, rounded, center projections, smooth surface; Under microscope, cell is rod-short, sometimes paired or one-tenth catenation; Gram-positive, amphimicrobian; This bacterial strain catalase, oxydase, glucose aerogenesis, arginine product ammonia, gelatine liquefication, Starch Hydrolysis, nitrate reductase, V.P. react all negative, and API reagent identifies that the homology of this bacterial strain and plant lactobacillus is 99.7%; The homology of this bacterial strain 16SrRNA sequence and plant lactobacillus reaches 99%.Above morphology, Physiology and biochemistry and this bacterial strain of 16SrRNA Sequence Identification are plant lactobacillus.
The invention still further relates to described plant lactobacillus LW106 and prepare the application in γ-aminobutyric acid in microorganism fermentation.
Concrete, described in be applied as: plant lactobacillus LW106 is seeded to the fermention medium that is applicable to plant lactobacillus containing Sodium Glutamate, carries out fermentation culture, after fermentation ends, in fermented liquid, obtain γ-aminobutyric acid.Conventionally before fermentation culture, the bacterial strain of preservation also needs activated cultivation and seed enlarged culturing.
Preferably, the fermentation of described microorganism is carried out at pH5.0 ~ 5.5,28 ~ 32 DEG C.
Concrete, utilize described plant lactobacillus LW106 carry out microorganism fermentation prepare the process of γ-aminobutyric acid can be as follows:
1. the appropriate puncture of test tube bacterial classification access of picking MRS liquid nutrient medium, 28 ~ 32 DEG C, activation culture 18 ~ 24h;
2. containing in the MRS liquid nutrient medium (first order seed substratum) of 1 ~ 5g/L Sodium Glutamate, by 1% ~ 5%(v/v) inoculum size inoculate activated nutrient solution, at 28 ~ 32 DEG C, leave standstill cultivation 18 ~ 24h as primary seed solution;
3. containing in the fermention medium (secondary seed medium) of 20 ~ 25g/L Sodium Glutamate, by 1% ~ 5%(v/v) inoculum size inoculation first order seed nutrient solution, at 28 ~ 32 DEG C, leave standstill cultivation 36 ~ 48h as secondary seed solution;
4. containing in the fermention medium of 40 ~ 50g/L Sodium Glutamate, by 1% ~ 5%(v/v) inoculum size inoculation secondary seed nutrient solution, in 28 ~ 32 DEG C, leave standstill cultivation 48 ~ 120h, in culturing process, use 65% phosphorus acid for adjusting pH, controlled fermentation pH is 5 ~ 5.5.
Described fermentative medium formula following (not comprising the Sodium Glutamate of interpolation): contain wheat 5 ~ 10g in every liter of substratum, wheat bran 15 ~ 20g, soyflour 5 ~ 10g, tomato juice 5 ~ 10mL, glossy ganoderma fermentation raffinate 10 ~ 50mL, yeast powder 5 ~ 10g, calcium chloride 0.1 ~ 0.3g, vitamin B6 0.3 ~ 0.5g, surplus is tap water, pH is 6 ~ 8; The compound method of substratum is: first wheat-flour, wheat bran are mixed with soyflour, after adding 1/3 ~ 1/2 water, add a certain amount of α-amylase, 85 DEG C of insulation 1 ~ 2h, add other compositions and water after being cooled to room temperature, regulating pH is 6.5 ~ 6.8,121 DEG C of sterilizing 20 ~ 30min.
Described glossy ganoderma fermentation raffinate is the fermented liquid of glossy ganoderma after liquid fermenting, removal mycelium, and fermented liquid exocellular polysaccharide content is about 0.3 ~ 0.5g/L.Concrete preparation method can be as follows:
(1) actication of culture: picking Ganderma lucidum strain is inoculated in PDA slant medium, in 28 ~ 30 DEG C of constant temperature culture 6 ~ 8d, puts 4 DEG C of refrigerators for subsequent use; PDA slant medium composition: potato 100 ~ 200g/L, glucose 10 ~ 50g/L, agar 10 ~ 20g/L, pH nature;
(2) seed enlarged culturing: the inclined-plane mycelium access liquid seed culture medium of activation, shaking culture 4d under 28 ~ 30 DEG C, 150 ~ 200r/min, obtains seed liquor; Liquid seed culture medium composition: potato 100 ~ 00g/L, glucose 10 ~ 50g/L, yeast powder 1 ~ 5g/L, potassium primary phosphate 1 ~ 5g/L, MgSO
47H
2o1 ~ 5g/L, pH nature;
(3) fermentation culture: cultured seed culture fluid is inoculated into liquid fermentation medium by 3 ~ 10% volume ratio, and at 28 ~ 39 DEG C, under 150 ~ 200r/min, shaking culture 6 ~ 8d, obtains fermented liquid; Liquid fermentation medium composition: glucose 20 ~ 50g/L, yeast powder 1 ~ 5g/L, peptone 0.5 ~ 2g/L, potassium primary phosphate 0.5 ~ 2g/L, MgSO
47H
2o0.1 ~ 2g/L, pH nature.
Above-mentioned gained fermented liquid is centrifugal or filter removal mycelium, obtains described glossy ganoderma fermentation raffinate.
Beneficial effect of the present invention is mainly reflected in: the new bacterial strain that the invention provides a strain highly producing gamma-aminobutyric acid, can obtain through fermentation culture the fermented liquid that alpha-aminobutyric acid content is 20 ~ 25g/L, total solids content reaches spray-dired requirement, spray-dried, the massfraction of product γ-aminobutyric acid is more than 20%.Plant lactobacillus LW106 of the present invention comes from traditional fermented food, utilizes the γ-aminobutyric acid of plant lactobacillus LW106 manufacture not have potential safety hazard, reaches food safety rank.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this: embodiment 1: from Pickles, Sichuan Style, screen plant lactobacillus LW106 bacterial strain
(1) substratum
The solid double-layer substratum of the milk-acid bacteria of fast high-flux primary dcreening operation highly producing gamma-aminobutyric acid: lower floor's culture medium prescription is: CaCO
320g/L, glucose 30g/L, yeast extract (powder) 5g/L, peptone 10g/L, dipotassium hydrogen phosphate 2g/L, citric acid diamino 2g/L, sodium-acetate 5g/L, magnesium sulfate 7H
2o0.5g/L, manganous sulfate 7H
2o0.2g/L, tween 80 0.1g/L, agarose 1 ~ 15g/L, surplus is water; Upper strata culture medium prescription is: Sodium Glutamate 50g/L, and tween 80 5g/L, pyridoxal phosphate 0.5g/L, phenol 0.15g/L, chlorine bleach liquor (available chlorine 0.25g/L), agarose 10 ~ 15g/L, surplus is water;
Sieve again MRS liquid nutrient medium: Sodium Glutamate 50 ~ 100g/L, glucose 30g/L, yeast extract (powder) 5g/L, peptone 10g/L, dipotassium hydrogen phosphate 2g/L, citric acid diamino 2g/L, sodium-acetate 5g/L, MgSO
47H
2o0.5g/L, MnSO
47H
2o0.2g/L, tween 80 0.1g/L, surplus is water.
(2) method
Get kimchi juice 0.5 ~ 5mL, carry out gradient dilution 10
-1~ 10
-6,get respectively 10
-4, 10
-5concentration 100 μ L coat primary dcreening operation substratum, cultivate 36 ~ 48h for 28 ~ 32 DEG C.Select large single bacterium colony, the separation and purification of the circle of blue-greenish colour hydrolysis around, it is the multiple sieve substratum of 5% ~ 10% Sodium Glutamate by isolated strains cut-in quality mark, cultivate 36 ~ 48h, get fermented liquid and utilize automatic analyzer for amino acids analysis confirmation γ-aminobutyric acid output for 28 ~ 32 DEG C.Finally, the isolated strains of highly producing gamma-aminobutyric acid is carried out to morphology, Physiology and biochemistry and 16s RNA qualification, screening lactobacillus classification.
Plant lactobacillus (L.plantarum) LW106 that screening obtains, bacterium colony oyster white, little, rounded, center projections, smooth surface; Under microscope, cell is rod-short, sometimes paired or one-tenth catenation; Atrichia, does not move, without spore; Gram-positive, amphimicrobian; API identifies that the homology of this bacterial strain and plant lactobacillus is 99.7%; The homology of 16S rRNA sequence and plant lactobacillus reaches 99%.
Embodiment 2: plant lactobacillus (L.plantarum) LW106 is for the method for the efficient preparation of γ-aminobutyric acid
(1) bacterial strain
Deposit number is: plant lactobacillus (Lactobacillus plantarum) LW106 of CGMCCNO.5655;
(2) culture medium prescription
Activation medium (MRS liquid nutrient medium): glucose 30g/L, yeast extract (powder) 5g/L, peptone 10g/L, dipotassium hydrogen phosphate 2g/L, citric acid diamino 2g/L, sodium-acetate 5g/L, MgSO
47H
2o0.5g/L, MnSO
47H
2o0.2g/L, tween 80 0.1g/L, agarose 10 ~ 15g/L, natural pH, surplus is tap water;
First order seed substratum: the MRS liquid nutrient medium that Sodium Glutamate massfraction is 3g/L, MRS liquid culture based formulas is the same;
Secondary seed medium: the fermention medium that Sodium Glutamate massfraction is 25g/L, every 1L fermentative medium formula: contain wheat 8g in every liter of substratum, wheat bran 18g, soyflour 8g, tomato juice 8mL, glossy ganoderma fermentation raffinate 30mL, yeast powder 8g, calcium chloride 0.2g, vitamin B6 0.5g, surplus is tap water, and pH is 6 ~ 8.The compound method of substratum is: first wheat-flour, wheat bran are mixed with soyflour, add that to add the high temperature resistant α-amylase of 1mL after 1/3 ~ 1/2 water (rich vertical, enzyme is lived as 20000U), 85 DEG C of insulation 1 ~ 2h, after being cooled to room temperature, add other compositions and water, regulating pH is 6.5 ~ 6.8,121 DEG C of sterilizing 20 ~ 30min.
Glossy ganoderma fermentation raffinate preparation process is as follows:
(1) actication of culture: No. 1 (TL-1) bacterial classification of picking Ganoderma lucidum is inoculated in PDA slant medium, in 28 DEG C of constant temperature culture 6d, puts 4 DEG C of refrigerators for subsequent use; PDA slant medium composition: potato 200g/L, glucose 20g/L, agar 20g/L, pH nature;
(2) seed enlarged culturing: the inclined-plane mycelium access liquid seed culture medium of activation, shaking culture 4d under 28 DEG C, 150r/min, obtains seed liquor; Liquid seed culture medium composition: potato 200g/L, glucose 20g/L, yeast powder 2g/L, potassium primary phosphate 3g/L, MgSO
47H
2o1.5g/L, pH nature;
(3) fermentation culture: cultured seed culture fluid is inoculated into liquid fermentation medium by 3% volume ratio, and at 28 DEG C, under 150r/min, shaking culture 6d, obtains fermented liquid; Liquid fermentation medium composition: glucose 35g/L, yeast powder 2g/L, peptone 1g/L, potassium primary phosphate 1g/L, MgSO
47H
2o0.5g/L, pH nature.
Above-mentioned gained fermented liquid is centrifugal or filter removal mycelium, obtains described glossy ganoderma fermentation raffinate.
(3) method
Picking proper amount of strains access MRS liquid nutrient medium from the MRS solid stab culture base of preservation, 28 ~ 32 DEG C leave standstill activation 20h;
In 250mL triangular flask, pack 100mL first order seed substratum into, get and leave standstill the activation culture liquid of cultivating 20h, after 5% inoculum size inoculation, 28 ~ 32 DEG C leave standstill cultivation 20h as primary seed solution;
In 6L tool plug triangular flask, pack 5L secondary seed medium into, get primary seed solution, after 5% inoculum size inoculation, 28 ~ 32 DEG C leave standstill cultivation 40h as secondary seed solution;
In 700L fermentor tank, pack 500L barley fermention medium into, after 3% inoculum size inoculation, 28 ~ 32 DEG C leave standstill cultivation 72h, use 65% phosphorus acid for adjusting pH in culturing process in culturing process, and controlled fermentation pH is 5.5.
This preparation method can obtain the fermented liquid of high gamma-aminobutyric acid content, detects to be through amino acid acid automatic analyser: 21.8g/L, Sodium Glutamate transformation efficiency is: 90%.Fermented liquid is through concentrated, adds the dry alpha-aminobutyric acid content that can obtain of W-Gum spraying at 20% ~ 25% powder-product.
Finally, it is also to be noted that, what more than enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (3)
- The plant lactobacillus of one strain highly producing gamma-aminobutyric acid ( lactobacillus plantarum) LW106, be preserved in Chinese common micro-organisms culture presevation administrative center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City DSMZ of Institute of Microorganism, Academia Sinica, postcode 100101, deposit number is: CGMCC NO.5655, preservation date is: on December 26th, 2011.
- 2. plant lactobacillus LW106 as claimed in claim 1 prepares the application in γ-aminobutyric acid in microorganism fermentation.
- 3. application as claimed in claim 2, is characterized in that the fermentation of described microorganism carries out at pH5.0 ~ 5.5,28 ~ 32 DEG C.
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| CN103320362B (en) * | 2013-07-03 | 2016-02-10 | 江南大学 | One strain is produced the bacterial strain of L-Glutamic decarboxylase and is produced the method for γ-aminobutyric acid with it |
| TWI510615B (en) * | 2014-09-26 | 2015-12-01 | Nature Vigour Co; Ltd | Solid medium for culturing ganoderma strains and application thereof |
| CN106434482B (en) * | 2016-10-28 | 2019-09-20 | 华南农业大学 | The lactobacillus plantarum SG5 of one plant of production γ-aminobutyric acid |
| CN106939288B (en) * | 2016-10-28 | 2020-02-04 | 华南农业大学 | Application of lactobacillus plantarum SG5 in production of gamma-aminobutyric acid |
| CN108893413A (en) * | 2018-07-20 | 2018-11-27 | 西北农林科技大学 | A kind of screening technique of conjugated linoleic acid production bacterium |
| 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 |
| CN109749914B (en) * | 2019-02-25 | 2021-12-14 | 山西农业大学 | Biological enhancement method for improving gamma-aminobutyric acid content of Shanxi mature vinegar |
| CN110747145A (en) * | 2019-11-27 | 2020-02-04 | 天津科技大学 | Lactobacillus for high yield of gamma-aminobutyric acid and isolated culture method and application thereof |
| CN111117926B (en) * | 2020-01-14 | 2021-07-09 | 华南农业大学 | Strain for high yield of gamma-aminobutyric acid and application of strain to preparation of gamma-aminobutyric acid-rich fruit juice |
| CN114933989A (en) * | 2022-05-18 | 2022-08-23 | 四川生力源生物工程有限公司 | Lactobacillus plantarum and fermentation medium thereof, mulberry leaf tea rich in gamma-aminobutyric acid and preparation method of mulberry leaf tea |
| CN115895974B (en) * | 2022-12-23 | 2024-02-13 | 闽榕茶业有限公司 | Lactobacillus plantarum rich in selenium and high in gamma-aminobutyric acid yield and application thereof |
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