CN106434482B - The lactobacillus plantarum SG5 of one plant of production γ-aminobutyric acid - Google Patents
The lactobacillus plantarum SG5 of one plant of production γ-aminobutyric acid Download PDFInfo
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Abstract
The present invention provides a kind of lactobacillus plantarum SG5 for producing γ-aminobutyric acid, lactobacillus plantarum SG5 is deposited in Guangdong Province's Culture Collection, preservation address is Guangdong Microbes Inst, and the deposit date is on March 16th, 2016, deposit number was GDMCC No:60020；The content of fermentation GABA is carried out up to 59 μ g/mL using the bacterium, GABA has blood pressure lowering, promotes growth hormone secretion and reduces the healthcare function of the multiple beneficials such as cholesterol, the health food rich in γ-aminobutyric acid to develop new provides foundation, the lactobacillus plantarum SG5 is applied to the exploitation of food, health care product, is with a wide range of applications.
Description
Technical field
The present invention relates to microorganisms technical fields, more particularly, to a kind of lactobacillus plantarum for producing γ-aminobutyric acid
SG5。
Background technique
γ-aminobutyric acid (γ-aminobutyric acid, abbreviation GABA) is a kind of suppression of mammalian nervous maincenter
Property mediator processed has and adjusts blood pressure, promote ataraxy, promote brain blood flow, promote brain vigor, trophic nerve cell, increase
Growth hormone secretion, promotes multiple efficacies such as alcohol metabolism (sobering up) at strong liver benefit kidney.The official approval GABA of the Ministry of Public Health in 2009
For new resource food, the raw material for being enriched with GABA can be applied to production functional food, be reduced by diet and preventing hypertension.
The acquisition methods of GABA have chemical synthesis and bioanalysis.Chemical synthesis higher cost, easy residual chemicals in product, no
Belong to natural products, using limitation of having ready conditions.Bioanalysis is mainly obtained from plant and microbial fermentation solution, relative to plant
The at high cost of concentration method, low yield and the big disadvantage of limitation, microbe fermentation method more have superiority, it is not by space, environment
With the limitation of resource, there are the remarkable advantages such as at low cost, no chemical residues and yield height, be the ideal of enriching gamma-aminobutyric
Approach.
The microorganism of the isolated production γ-aminobutyric acid of the prior art is mostly fungi, and fungi growth is more slow, cannot
Meet production needs.
Summary of the invention
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, a kind of gamma-amino is provided
The lactobacillus plantarum SG5 of butyric acid.
Second purpose of the invention is to provide the microorganism formulation containing the lactobacillus plantarum SG5.
The purpose of the present invention is what is be achieved by the following technical programs:
A kind of lactobacillus plantarum SG5 producing γ-aminobutyric acid, lactobacillus plantarum SG5 are deposited in Guangdong Province's microbial bacteria
Kind collection, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst, preservation
Date is on March 16th, 2016, and deposit number is GDMCC No:60020.
The identification of bacterial strain colonial morphology: bacterium colony is smaller on MRS culture medium, round, median rise, and surface wettability is smooth, no
It is transparent, milky, neat in edge；Gram-positive bacteria, pairs of or chain.Strain morphology is in the rod-short of both ends blunt circle, length
It is different, no gemma, cell size be (0.5~1) μ m (2~3) μm, the bacterial strain be facultative anaerobic bacteria, API50 qualification result with
The ID of lactobacillus plantarum (Lactobacillus plantarum) is 99.9%.
The 16S rDNA sequence of the lactobacillus plantarum SG5 is as shown in SEQ ID NO:1.
Lactobacillus plantarum SG5 of the present invention can be grown on MRS culture medium, and wherein the optimum formula of culture medium is 20g/L ferment
Female cream, 20g/L glucose, 10g/L sodium succinate, 5mg/mL Pidolidone, 1mg/L phosphopyridoxal pyridoxal phosphate and 1mg/mL Ca2+。
30 DEG C of the optimum growth temperature of lactobacillus plantarum SG5, the pH value for being most suitable for growing environment is 6.8；It can high yield γ-ammonia
The best fermentation time of base butyric acid is 48h, and thallus inoculum concentration is 2%.
γ-aminobutyric acid has the function of reducing blood pressure as the new function factor, and therefore, the present invention also provides one kind
Microorganism formulation containing the lactobacillus plantarum SG5.
The present invention also provides application of the lactobacillus plantarum SG5 in terms of producing γ-aminobutyric acid.
Specifically, the application is 30 DEG C of culture 48h by lactobacillus plantarum SG5 in TYG culture medium, pH value 6.8,
The inoculum concentration of the lactobacillus plantarum SG5 is 2% (× 106cfu/mL)。
More specifically, the group of the TYG culture medium, which becomes, contains 20g/L yeast extract, 20g/L grape in every liter of culture medium
Sugar, 10g/L sodium succinate, 5mg/mL Pidolidone, 1mg/L phosphopyridoxal pyridoxal phosphate and 1mg/mL Ca2+。
Compared with prior art, the invention has the following advantages:
The present invention provides a kind of lactobacillus plantarum SG5 for producing γ-aminobutyric acid, lactobacillus plantarum SG5 is deposited in extensively
East saves Culture Collection, and preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province microorganism
Research institute, the deposit date is on March 16th, 2016, deposit number was GDMCC No:60020；Fermentation GABA is carried out using the bacterium
Content there is blood pressure lowering up to 59 μ g/mL, GABA, promote growth hormone secretion and reduce the health care of the multiple beneficials such as cholesterol
Function, the health food rich in γ-aminobutyric acid to develop new provide foundation, and the lactobacillus plantarum SG5 is applied to food
The exploitation of product, health care product, is with a wide range of applications.
Detailed description of the invention
Fig. 1 is the lactobacillus phylogenetic tree established based on 16SrDNA gene order.
Fig. 2 is fermentation liquid ultra performance liquid chromatography-mass spectrogram；Wherein, the peak figure of A-GABA standard items；B-GABA standard
Product molecular mass map；C-fermentation broth sample ultra performance liquid chromatography figure；D-fermentation broth sample molecular mass map.
Fig. 3 is bacterium colony shape of the SG5 on MRS culture medium (A) and lactic acid bacteria isolation medium (containing bromocresol green) plate (B)
State figure.
Fig. 4 is the Gram-stained cellular morphology of bacterial strain SG5.
Fig. 5 is that SG5 thallus projects electron microscope.
Specific embodiment
The contents of the present invention are further illustrated with specific embodiment with reference to the accompanying drawings of the specification, but should not be construed as to this
The limitation of invention.Without departing from the spirit and substance of the case in the present invention, to simple made by the method for the present invention, step or condition
Modifications or substitutions all belong to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is art technology
Conventional means known to personnel.
Separation, screening and the identification of 1 bacterial strain of embodiment
Strain isolation: fresh complete mulberry leaf are chosen and are first rinsed well with tap water, use sterile water in sterile super-clean bench
It rinses and with aseptic filter paper suck dry moisture, shreds again；The mulberry leaf shredded are placed in MRSG fluid nutrient medium (to add in MRS culture medium
Enter 2%v/v, the sodium glutamate of 1mg/mL) in, it is chosen in 30 DEG C of constant incubator culture 2d to grow thallus around mulberry leaf fragment
Taking thallus, repeatedly scribing line isolates and purifies on MRS solid medium；Bacterial strain after purification is cultivated in slant medium (MRS),
It chooses single colonie and is inoculated in fermentation medium TYG and do thin-layer qualitative after 30 DEG C of culture 48h, obtained strains known to Preliminary Identification separation
γ-aminobutyric acid can be produced.
Negative control: in order to check whether the degerming of mulberry leaf surface is thorough, last time aseptic water washing liquid is taken to be coated on MRS
It compares, separately compares the mulberry leaf material after sterile water wash on MRS solid medium, in identical on solid medium
Under the conditions of cultivate.
By being repeated several times, the plating medium of control is grown without any bacterium colony, it was demonstrated that and the degerming of mulberry leaf surface is thorough, point
From to bacterium be bacterium in mulberry leaf, rather than the epiphyte on plant surface.
The screening of bacterial strain: using functional activity substance-γ-aminobutyric acid as index, bacterial strain is optimized using Responds Surface Methodology
Fermentation condition.For the separating obtained bacterial strain that -80 DEG C are saved 30 DEG C in MRS fluid nutrient medium, 120r/min cultivates 16h, activation
Twice, it is inoculated in TYG fermentation medium and is enriched with GABA.According to experiment of single factor result to 4 principal elements (concentration of substrate,
Fermentation temperature, fermentation time) response surface optimization experiment is carried out, experimental model is established, it is excellent using response surface if experimental model is significant
Change test method, the response surface experiment of final design Three factors-levels, with concentration of substrate, fermentation temperature and fermentation time 3
Factor is independent variable, and GABA yield is response, determines optimal fermentation condition.With Berthelot colorimetric method or HPLC method to hair
The GABA quantitative analysis being enriched in zymotic fluid, it is final to determine high yield GABA bacterial strain (referred to as SG).
Bacterial strain Molecular Identification: Molecular Identification is carried out to the high yield GABA bacterial strain that screening obtains, sequentially includes the following steps: extraction
The genomic DNA of bacterial strain carries out PCR amplification by template of genomic DNA using the universal primer of bacterial 16 S rDNA.Then
It using plastic recovery kit recovery purifying PCR product, cloned, converted later, the bacterium colony of screening positive clone is trained through expanding
Commission Guangzhou Hua Da gene technology Co., Ltd is sequenced after supporting.
The 16S rDNA sequence length of bacterial strain is 1371bp known to sequencing, and sequence will be sequenced as shown in SEQ ID NO:1
As a result sequence analysis is carried out with the 16S rDNA sequence in GenBank, then with software building phylogenetic tree (such as Fig. 1 institute
Show), to determine the race relation of bacterial strain.Homology analysis the result shows that, the sequence and Lactobacillus plantarum
The homology of (lactobacillus plantarum) belongs to lactobacillus (Lactobacillus) up to 99%, therefore by the bacterial strain of high yield GABA,
For lactobacillus plantarum (Lactobacillus plantarum).
The identification of bacterial strain colonial morphology: bacterium colony is smaller on MRS culture medium, round, median rise, and surface wettability is smooth, no
It is transparent, milky, neat in edge；In lactic acid bacteria isolation medium (containing bromocresol green) plate, 30 DEG C of culture 48h occur round
Milky white or yellow color colonies, surrounding media become yellow person and primarily determine as lactic acid bacteria (as shown in Figure 3).Thalli morphology is blue through leather
After Albert'stain Albert with microscope in 100 times oil under the microscope and take pictures.As shown in Figure 4, it is known that it is gram-positive bacteria, in pairs
Or chain.Strain cell is observed through transmission electron microscope (TEM, Tenai 12, Holland), sees Fig. 5, and form is short in both ends blunt circle
Rod-shaped, different in size, no gemma, cell size is (0.5~1) μ m (2~3) μm.
Bacterium colony Physiology and biochemistry identification: referring to " Berger bacterial identification manual " (the 9th edition), " common bacteria system identification hand
Volume " and France biology Mei Liai API identification systems identify that lactobacillus plantarum, the results are shown in Table 1:
The API 50CHL testing result of 1 bacterial strain SG-5 of table
Note: " ﹣ " indicates negative, and " ﹢ " indicates positive.
It is verified again by 1 result of table, ID and the Lactobacillus plantarum (plant of lactobacillus plantarum SG5API
Lactobacillus) it is 99%, it is named as lactobacillus plantarum SG5, lactobacillus plantarum SG5 is deposited in Guangdong Province microorganism fungus kind
Collection (GDMCC), preservation address are Guangdong Microbes Inst, and the deposit date is on March 16th, 2016, deposit numbers
For GDMCC No:60020.
The culture of 2 bacterial strain of embodiment
Lactobacillus plantarum SG5 can be grown on MRS culture medium, and MRS culture medium (1000mL) is by 20g yeast extract, 20g grape
Sugar, 10g sodium succinate, 5mg/mL Pidolidone (0.5%v/v), 1mg phosphopyridoxal pyridoxal phosphate (growth factor) and 1mg/mL Ca2+
It is formed with the distilled water of surplus, pH value 6.8~7.0 is adjusted, in 121 DEG C of high pressure sterilization 20min.
1, the activation of lactobacillus plantarum SG5: sterile working in superclean bench.With oese picking lactobacillus plantarum
SG5 is crossed on MRS solid medium with oese, carries out culture 2d in 30 DEG C of incubators.
2, isolated lactobacillus plantarum SG5 the preparation of lactobacillus plantarum SG5 fermented sample: is inoculated in MRS liquid
In culture medium, make 3 repetitions, be placed in 150r/min, in 30 DEG C of constant-temperature shaking incubators, 16~18h of culture is taken out, as seed
Liquid；Seed liquor is inoculated into the triangular flask equipped with 50mL TYG fluid nutrient medium by 2% inoculum concentration, is placed in 150r/min
2d is cultivated in 30 DEG C of constant-temperature shaking incubators, obtaining fermentation liquid, (the optimized optimum growth temperature for obtaining lactobacillus plantarum SG5 is 30
DEG C, 6.8) pH value of the most suitable growth environment is.
3, qualitative and quantitative detection: GABA is carried out to GABA in fermentation broth sample with ultra high efficiency liquid phase-Mass Spectrometry
Standard items are purchased to Sigma company, ultra performance liquid chromatography-mass spectral analysis figure and the standard items GABA phase of streptococcus acidi lactici fermented solution sample
Compare and sees Fig. 2.
The appearance time of standard items GABA is in 0.4~0.6min, relative molecular mass as seen from Figure 2
104.0707；Detecting fermentation broth sample GABA relative molecular mass is 104.1072, the relative molecular mass with standard items GABA
Very close, the appearance time of fermentation broth sample is slightly wider compared with standard specimen, and there are one small miscellaneous peaks to occur, it may be possible to by
The interference of other matrix.The result, which confirms, contains GABA in fermentation liquid.
4, high performance liquid chromatography (HPLC) measures fermentation liquid GABA content
After the fermentation liquid boiling water bath 10min of SG5,10min is centrifuged with 12000r/min, draws supernatant with microsyringe
The concentration that liquid measures γ-aminobutyric acid in five sample fermentation liquids of primary dcreening operation with high performance liquid chromatography (HPLC) method respectively is (excellent
Change lactobacillus plantarum SG5 produce γ-aminobutyric acid optimal liquid fermentation time be 48h, the content of GABA is up to 59 μ g/mL).
SEQUENCE LISTING
<110>Agricultural University Of South China
The lactobacillus plantarum SG5 of<120>one plants of production γ-aminobutyric acids
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1371
<212> DNA
<213>the 16S rDNA sequence of lactobacillus plantarum SG5
<400> 1
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 60
gcgattccga cttcatgtag gcgagttgca gcctacaatc cgaactgaga atggctttaa 120
gagattagct tactctcgcg agttcgcaac tcgttgtacc atccattgta gcacgtgtgt 180
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 240
ccggcagtct caccagagtg cccaacttaa tgctggcaac tgataataag ggttgcgctc 300
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 360
atccatgtcc ccgaagggaa cgtctaatct cttagatttg catagtatgt caagacctgg 420
taaggttctt cgcgtagctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 480
tcaattcctt tgagtttcag ccttgcggcc gtactcccca ggcggaatgc ttaatgcgtt 540
agctgcagca ctgaagggcg gaaaccctcc aacacttagc attcatcgtt tacggtatgg 600
actaccaggg tatctaatcc tgtttgctac ccatactttc gagcctcagc gtcagttaca 660
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 720
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcacttcttc 780
ggttgagccg aaggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 840
ataaatccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 900
gtggctttct ggttaaatac cgtcaatacc tgaacagtta ctctcagata tgttcttctt 960
taacaacaga gttttacgag ccgaaaccct tcttcactca cgcggcgttg ctccatcaga 1020
ctttcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1080
cagtcccaat gtggccgatt accctctcag gtcggctacg tatcattgcc atggtgagcc 1140
gttaccccac catctagcta atacgccgcg ggaccatcca aaagtgatag ccgaagccat 1200
ctttcaagct cggaccatgc ggtccaagtt gttatgcggt attagcatct gtttccaggt 1260
gttatccccc gcttctgggc aggtttccca cgtgttactc accagttcgc cactcagtca 1320
aatgtaaatc atgatgcaag caccaatcaa taccagagtt cgttcgactg c 1371
Claims (4)
1. a kind of lactobacillus plantarum SG5 for producing γ-aminobutyric acid, which is characterized in that lactobacillus plantarum SG5 is deposited in Guangdong Province
Culture Collection, preservation address are Guangdong Microbes Inst, and the deposit date is on March 16th, 2016, preservations
Number is GDMCC No:60020.
2. the lactobacillus plantarum SG5 according to claim 1 for producing γ-aminobutyric acid, which is characterized in that the lactobacillus plantarum
The 16S rDNA sequence of SG5 is as shown in SEQ ID NO:1.
3. the lactobacillus plantarum SG5 according to claim 1 for producing γ-aminobutyric acid, which is characterized in that the lactobacillus plantarum
SG5 is gram-positive bacteria, and bacterium colony is rounded on MRS culture medium, median rise, and surface wettability is smooth, opaque, milky white
Color, neat in edge.
4. the microorganism formulation containing any one of claims 1 to 3 lactobacillus plantarum SG5.
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| CN106900872B (en) * | 2017-03-27 | 2020-04-14 | 华南农业大学 | Preparation method of mulberry milk containing gamma-aminobutyric acid |
| 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 |
| CN114836343B (en) * | 2022-04-19 | 2024-03-15 | 河北科技大学 | Lactobacillus plantarum P10 capable of high-yielding gammA-Aminobutyric acid and antagonizing harmful bacteria and application thereof |
| CN115372494B (en) * | 2022-04-24 | 2023-10-24 | 浙江省农业科学院 | Method for determining gamma-aminobutyric acid in fermentation liquor |
| 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 |
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| CN106434482A (en) | 2017-02-22 |
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