CN106987544A - One 28 plants of plant height proteolysis vigor Lactobacillus plantarum PE and the method that ginseng peptide is prepared using the bacterial strain - Google Patents
One 28 plants of plant height proteolysis vigor Lactobacillus plantarum PE and the method that ginseng peptide is prepared using the bacterial strain Download PDFInfo
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Abstract
One 28 plants of plant height proteolysis vigor Lactobacillus plantarum PE and the method that ginseng peptide is prepared using the bacterial strain, are related to microbial technology field.It is an object of the invention to provide the new method that a kind of utilization lactobacillus ferment zymolysis technique prepares functional bioactive peptide.28 plants of plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) PE of the present invention, is preserved in China typical culture collection center, deposit number is on March 6th, 2017：CCTCC NO:M2017087.Ginseng peptide product prepared by 28 plants of the Lactobacillus plantarum PE and its fermentation enzymolysis that the present invention is provided can serve as feature probiotics and functional raw material is applied to the fields such as food, health products and medicine, and the small molecule bioactive ginseng peptide obtained has increase significantly immunity and antifatigue effect.
Description
Technical field
The present invention relates to microbial technology field, and in particular to PE-28 plants of a plant height proteolysis vigor Lactobacillus plantarum
And the method that ginseng peptide is prepared using the bacterial strain.
Background technology
Biologically active peptide (Bioacti) is divided into dipeptides, small-molecular peptides and polypeptide, peptide, which is removed, has nutrition according to peptide chain length
Beyond function, also extensive physiological regulation function, such as hypotensive, opioid activity, antithrombotic, promotion are immunized and promote battalion
Support digesting and assimilating for material.Biologically active peptide not only has extensive activity and diversity, and with abundance, cost
Low, security is good, be easy to the advantage of industrialized production.In recent years, with the mechanism of action to biologically active peptide, physiologic effect with
And the further investigation of preparation method, people are from various lactoproteins, soybean protein, zein, fish and shellfish albumen, collagen egg
Isolated Several Active Peptides in the white enzymolysis product or fermented product for waiting food proteins.Some biologically active peptides are
Industrialized production is realized as healthy food, functional food and medicine, and obtains huge economic benefit, biologically active peptide should
It is wide with DEVELOPMENT PROSPECT.
At present, the research to active peptide is become increasingly active, and the type and quantity of in the market active peptide are continuously increased.Active peptide
Preparing mainly includes the methods such as protease hydrolyzed, genetic engineering, chemical synthesis and microbial fermentation.Sent out using food-grade microorganisms
Ferment enzymolysis activity peptide technology of preparing, such as lactobacillus-fermented lactoprotein prepares Antihypertensive Peptides, anti-oxidation peptide.
The content of the invention
Functional bioactive peptide is prepared it is an object of the invention to provide a kind of utilization lactobacillus ferment zymolysis technique
New method, based on this, the present invention provides PE-28 plants of a plant height proteolysis vigor Lactobacillus plantarum and prepares people using the bacterial strain
Join the method for peptide.
The present invention is as follows to solve the technical scheme that technical problem is used：
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention,
China typical culture collection center is preserved on March 6th, 2017, deposit number is：CCTCC NO:M2017087.
Present invention also offers use an above-mentioned plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus
Plantarum) the method for PE-28 plants of preparation ginseng peptides, this method comprises the following steps：
Step 1: dry ginseng's root is crushed, with 60% ethanol solution refluxing extraction twice, filtered filtration residue is with 80~100
DEG C hot water is extracted twice, and 0.5~1.5h is extracted every time, and merging filtrate after filtering, concentrated, centrifuged supernatant adds solid
(NH4)2SO4Precipitated, 4 DEG C stand overnight, then ginseng total protein freeze-dried powder is obtained through centrifugation, dialysis, lyophilized technique；
Step 2: ginseng total protein freeze-dried powder is made into ginseng protein culture medium, by Lactobacillus plantarum
PE-28 plants of (Lactobacillus plantarum) is inoculated in ginseng protein culture medium, the Lactobacillus plantarum
The inoculum concentration that PE-28 plants of (Lactobacillus plantarum) is the 3% of ginseng protein culture volume, and 37 DEG C stand training
15~25h is supported, ginseng peptide is obtained through centrifugation, concentration, lyophilized technique.
As preferred embodiment, step one, dry ginseng's root is crushed to 30 mesh.
As preferred embodiment, step one, dry ginseng's root chooses 5 years dry ginsengs' below by way of cultivation acquisition
Root.
As preferred embodiment, step one, the hot water temperature is 80 DEG C.
It is used as preferred embodiment, step one, (NH4)2SO4Concentration is 70%.
As preferred embodiment, in step 2, the preparation process of the ginseng protein culture medium is as follows：Weigh ginseng total
Protein freeze-dried powder 25g, glucose 20g, add water and are configured to 1000mL solution, 115 DEG C of sterilizing 20min, obtain ginseng protein culture
Base.
As preferred embodiment, in step 2, the condition of centrifugation is：6000rpm, 10min, 4 DEG C.
The ginseng peptide obtained using above-mentioned preparation method.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
Application in food, health products, medicine is prepared.
The beneficial effects of the invention are as follows：
The innovative point of the present invention is to provide one plant of lactic acid bacteria new strains-Lactobacillus plantarum that there is high protein to digest vigor
PE-28 plants of (Lactobacillus plantarum), and utilize the Lactobacillus plantarum (Lactobacillus
Plantarum) the method that PE-28 plants of fermentation enzymolysis ginseng proteins prepare feature ginseng peptide, is microbial fermentation enzymolysis preparation
Biologically active peptide provides a new way.Lactobacillus plantarum (Lactobacillus plantarum) PE- that the present invention is provided
Ginseng peptide product prepared by 28 plants and its fermentation enzymolysis can serve as feature probiotics and functional raw material be applied to food,
The field such as health products and medicine, the small molecule bioactive ginseng peptide obtained has increase significantly immunity and antifatigue work
With.
Brief description of the drawings
Fig. 1 composes for the SDS-PAGE of ammonium sulfate precipitation ginseng total protein.
Fig. 2 is the ginseng of the PE-28 plants of preparations of Lactobacillus plantarum (Lactobacillus plantarum) using the present invention
Total protein HPLC analyzes collection of illustrative plates.
Fig. 3 is the ginseng of the PE-28 plants of preparations of Lactobacillus plantarum (Lactobacillus plantarum) using the present invention
Peptide HPLC analyzes collection of illustrative plates.
Embodiment
The present invention is the one plant of newborn bar of lactic acid bacteria new strains-plant that there is high protein to digest vigor obtained through screening extensively
PE-28 plants of bacterium (Lactobacillus plantarum), and using ginseng protein as substrate, utilize Lactobacillus plantarum
(Lactobacillus plantarum) PE-28 plants of fermentation enzymolysis prepare small molecule bioactive ginseng peptide, and the small molecule is biological
Active ginseng peptide has increase significantly immunity and antifatigue effect, is exploitation healthy food and the important function of health food
The factor.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention,
It is preserved in China typical culture collection center on March 6th, 2017, abbreviation CCTCC, address is：Wuhan City, Hubei Province is military
In the school (Wuhan University's collection), deposit number is for prosperous No. 299 Wuhan Universitys of area's Bayi Road：CCTCC NO:M2017087.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention,
Obtained using northeast fermented pickled Chinese cabbage through culture purified repeatedly, 20% glycerine, -80 DEG C save backup.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
Through a variety of test for identification, Gram-positive, negative catalase test, indole test feminine gender, hydrogen sulfide production test are cloudy
Property, gelatin liquefaction test negative, hydrolysis starch negative and nitrate reduction test it is negative.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
Physio-biochemical characteristics it is as follows：Gram-positive, negative catalase, benzidine test is negative, and indole test is negative, acetyl
Carbinol methine experiment is positive；No motion of bacillus；Optimum growth temperature is 37~42 DEG C, and appropriate pH is 5.0~7.0；At 15 DEG C
It can be grown with 45 DEG C；It is resistant to 6.5%NaCl；Not hydrolysis starch, do not liquefy gelatin, does not produce hydrogen sulfide；Glucose fermentation is produced
Sour not aerogenesis；It is in uniform turbid growth in liquid MRS culture mediums, is long placed in the white precipitation of thalline.
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
API 50CH (French Mei Liai companies) qualification result it is as follows：Can be using ribose, galactolipin, glucose, fructose, xylose, sweet
Reveal sugar, mannitol, sorbierite, amarogentin, ursin, N- acetyl-gucosamine, seven leaf-alcohols, salicin, cellobiose, synanthrin,
Lactose, melibiose, sucrose, trehalose, sorbose, melezitose, raffinose, geraniol, gluconate；A Dong can not be utilized
Alcohol, arabinose, glycogen, dulcitol, maltose, inositol, Alpha-Methyl-D-Glucose glycosides, Alpha-Methyl-D-MANNOSE glycosides, starch,
Rhamnose, xylitol, D- turanoses, D-Tag, D- meters of threoses, gluconate, D-R alcohol, rock sugar, L- I
Primary sugar alcohol, 2- keto-D-gluconates salt, 5- keto-D-gluconate salt.
The bacterial strain PE-28 of the present invention can be defined as Lactobacillus plantarum through 16S rDNA sequence homology analysis
(Lactobacillus plantarum)。
PE-28 plants of the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
A kind of can be as active component in product is prepared application.Described product can be to utilize Lactobacillus plantarum
Food that PE-28 plants of (Lactobacillus plantarum), its fermentate or its fermentation broth extract are made, protect
Strong product or medicine etc..
Utilize plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) PE- of the present invention
The method that 28 plants of fermentation enzymolysis prepare ginseng peptide, comprises the following steps：
Step 1: dry ginseng's root is crushed, with 60% ethanol solution refluxing extraction twice, filtered filtration residue is with 80~100
DEG C hot water is extracted twice, and 0.5~1.5h is extracted every time, and merging filtrate after filtering, concentrated, centrifuged supernatant adds solid
(NH4)2SO4Precipitated, 4 DEG C stand overnight, then ginseng total protein freeze-dried powder is obtained through centrifugation, dialysis, lyophilized technique；
Step 2: ginseng total protein freeze-dried powder is made into ginseng protein culture medium, by Lactobacillus plantarum
PE-28 plants of (Lactobacillus plantarum) is inoculated in ginseng protein culture medium, the Lactobacillus plantarum
The inoculum concentration that PE-28 plants of (Lactobacillus plantarum) is the 3% of ginseng protein culture volume, and 37 DEG C stand training
15~25h is supported, ginseng peptide is obtained through centrifugation, concentration, lyophilized technique.
The present invention is described in further detail with reference to embodiments.
Test material used, unless otherwise specified, is bought by routine biochemistry reagent shop in following examples
Arrive.Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.
Solid BCP culture mediums：Solvent is water, 2.5g/L containing yeast extract, peptone 5g/L, glucose 5g/L, bromocresol purple
0.04g/L, agar 15g/L, pH are 7.0.
Liquid B CP culture mediums are added without agar with differing only in for solid BCP culture mediums.
Solid MRS culture mediums：Solvent is water, 10g/L containing peptone, beef extract 10g/L, yeast extract 5g/L, KH2PO42g/
L, sodium acetate 5g/L, sodium citrate 5g/L, MgSO4·7H2O 0.2g/L、MnSO4·4H2O0.05g/L, Tween-80 1mL/L,
Agar 15g/L, glucose 20g/L, pH are 6.6.
Liquid MRS culture mediums are added without agar with differing only in for solid MRS culture mediums.
The preparation of PE-28 plants of the Lactobacillus plantarum (Lactobacillus plantarum) of the present invention of embodiment 1
First, the separation of bacterial strain
1st, sample is drawn materials：Northeast fermented pickled Chinese cabbage.
Northeast fermented pickled Chinese cabbage 5g is taken, solid is ground in the pasty state with mortar, add 45mL physiological saline (for 10 times of dilutions), mix
It is even to take 0.5mL dilutions, doubling dilution is carried out to 10 with physiological saline again-3, take 100uL liquid (10-1、10-2、10-3) coating
Cultivated in solid BCP culture mediums (37 DEG C, 48~72h).The single bacterium colony of picking production yellow circle is inoculated in solid MRS culture mediums
Continue to cultivate (37 DEG C, 48h) on flat board, rule repeatedly culture purified (37 DEG C, 48h) through solid MRS culture medium flat plates, obtain many
Strain Pure cultured spawn.
2nd, by the strain of many plants of pure cultures of above-mentioned acquisition be inoculated in respectively in liquid MRS culture mediums culture (37 DEG C, 14~
18h), 20% glycerine is then added, is placed in -80 DEG C of refrigerators and preserves.
3rd, through Gram-positive, catalase test is negative, indole test is negative, hydrogen sulfide production test is negative,
Gelatin liquefaction test is negative, hydrolysis starch negative and nitrate reduction test feminine gender are screened from the strain of many plants of pure cultures
Go out plurality of plants lactobacillus, choose 1 plant therein and be named as Lactobacillus plantarum (Lactobacillus plantarum) PE-28
Strain.
2nd, the identification of bacterial strain
1st, the Physiology and biochemistry identification knot of PE-28 plants of Lactobacillus plantarum (Lactobacillus plantarum) of the invention
Fruit is as follows：
Gram-positive, negative catalase, benzidine test is negative, and indole test is negative, acetyl methyl carbinol examination
Test the positive；
No motion of bacillus；
Optimum growth temperature is 37~42 DEG C, and appropriate pH is 5.0~7.0；
It can be grown at 15 DEG C and 45 DEG C；
It is resistant to 6.5%NaCl；
Not hydrolysis starch, do not liquefy gelatin, does not produce hydrogen sulfide；
The sour not aerogenesis of glucose fermentation production；
It is in uniform turbid growth in liquid MRS culture mediums, is long placed in the white precipitation of thalline.
2nd, API 50CH (the French plums of PE-28 plants of Lactobacillus plantarum (Lactobacillus plantarum) of the invention
Li Ai companies) qualification result is as follows：
Ribose, galactolipin, glucose, fructose, xylose, mannose, mannitol, sorbierite, amarogentin, black bearberry can be utilized
Glycosides, N- acetyl-gucosamine, seven leaf-alcohols, salicin, cellobiose, synanthrin, lactose, melibiose, sucrose, trehalose, sorbose,
Melezitose, raffinose, geraniol, gluconate；
Can not using adonite, arabinose, glycogen, dulcitol, maltose, inositol, Alpha-Methyl-D-Glucose glycosides, α-
Methyl-Dmannose glycosides, starch, rhamnose, xylitol, D- turanoses, D-Tag, D- meters of threoses, gluconate, D- Ah
Draw primary sugar alcohol, rock sugar, L-arabinose alcohol, 2- keto-D-gluconates salt, 5- keto-D-gluconate salt.
3rd, 16S rDNA sequence homology analysis
(1) CTAB methods extract strain gene group DNA
The plurality of plants lactobacillus of separation is inoculated in liquid MRS culture mediums, 37 DEG C of culture 16h take 10mL nutrient solutions,
10000r/min centrifuges 10min, abandoning supernatant after centrifugation, with TE buffer solution for cleaning thalline 1 time；Thalline after cleaning is resuspended
In the TE buffer solutions for floating on 1.9mL, add 0.1mL mass fractions be 10% SDS solution, 10 μ L concentration be 20mg/mL egg
White enzyme K, the lysozyme that 5 μ L concentration are 50mg/mL, after mixing in 37 DEG C of water-bath water-bath 1h；After water-bath, add into system
The NaCl solution and 300 μ L mass fractions for entering 300 μ L 5M are 10% CTAB-NaCl solution, in 65 DEG C of water-bath after mixing
Middle water-bath 20min；After water-bath, added into system isometric chloroform-isoamyl alcohol solution (chloroformic solution and isoamyl alcohol
Volume ratio is 24:1) 10000r/min centrifugations 30min after, fully mixing；Aspirate supernatant is into new centrifuge tube, the body such as addition
(phenol solution, chloroformic solution, the volume ratio of isoamyl alcohol are 25 to long-pending phenol-chloroform-isoamyl alcohol:24:1), fully mix
10000r/min centrifuges 30min afterwards；Aspirate supernatant adds the isoamyl alcohol of 0.6 times of volume into new centrifuge tube ,-
2h is placed under conditions of 20 DEG C, then 10000r/min centrifuges 20min, abandoning supernatant；With the ethanol that volume fraction is 70%
Solution cleaning precipitation, then 10000r/min centrifugations 20min, places to ethanol and drains completely at ambient temperature；It will precipitate molten
In 20 μ LTE buffer solutions, 1 μ L RNase and the water-bath 1h in 37 DEG C of water-bath is added, strain gene group DNA is obtained, puts
In -20 DEG C of preservations.
(2) PCR is expanded
1. amplimer：According to the conservative region of the 16S rDNA sequences of lactic acid bacteria, using Jung-Hoon Yoon et al.
The primer of design, its sequence is as follows：
16sF：5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇；
16sR：5 ＇-AGAAAGGAGGTGATCCAGC-3 ＇.
2. PCR reaction systems are as shown in table 1：
Table 1
| System component | Reaction system |
| TaKaRa Ex Taq(5U/μL) | 0.25μL |
| 10×Ex Taq Buffer(Mg2+Plus)5μL | 5μL |
| DNTP Mixture (each 2.5mmol/L) | 4μL |
| Genomic DNA | 1μL |
| Forward primer (10 μm of ol/L) | 1μL |
| Reverse primer (10 μm of ol/L) | 1μL |
| The double distilled waters of sterilizing | 37.75μL |
| Cumulative volume | 50μL |
3. PCR amplification programs are as follows：
4. pcr amplification product is detected
Previously prepared mass fraction is 1% Ago-Gel (the ethidium bromide EB for adding 10mg/mL), PCR amplification knots
Shu Hou, the 6 × Loading buffer for drawing 5 μ LPCR amplified productions and 1 μ L are well mixed, and agarose is added to pipettor
Electrophoresis is carried out in the loading wells of gel slab.Target fragment length is about 1500bp, using DL2000 DNA Marker, with 1 ×
TAE is as electrophoresis liquid, and electrophoretic voltage is 100V, and electrophoresis time is 40min.
(3) analyze
By test it is pure after pcr amplification product be sent to Shanghai life work be sequenced；The 16S rDNA sequences for obtaining each bacterial strain are led to
The sequence information for crossing blast program and known bacterial strain in GenBank storehouses carries out sequence analysis analysis, identifies strain to be tested；With
It is that standard progress category kind is sorted out that 16S rDNA sequence homologies, which are more than 99%, then again by strain to be tested and the 16S of type strain
RDNA sequences utilize the Neighbor-Joining method phylogenetic tree constructions in the softwares of MEGA 4.0.16s rDNA sequences such as sequence
SEQ ID NO in list:Shown in 1.Based on above-mentioned analysis result it was determined that the bacterial strain PE-28 of the present invention is the newborn bar of plant
Bacterium (Lactobacillus plantarum).
4th, the preservation of bacterial strain
PE-28 plants of the Lactobacillus plantarum (Lactobacillus plantarum) of the present invention, is protected on March 6th, 2017
It is hidden in China typical culture collection center, abbreviation CCTCC, address is：No. 299 Wuhan of Wuhan City, Hubei Province Wuchang District Bayi Road
In institution of higher education (Wuhan University's collection), deposit number is：CCTCC NO:M2017087.
The prolease activity analysis of PE-28 plants of the Lactobacillus plantarum of embodiment 2 (Lactobacillus plantarum)
Using the prolease activity of o-phthalaldehyde method screening and assessment bacterial strain, o-phthalaldehyde method is that one kind is recognized extensively
Can protease hydrolysis activity assay method.Its principle is that occur hydrolysis by OPA and beta -mercaptoethanol, is released
A-amino acid is released, has strong absworption peak at ultraviolet wavelength 340nm, reflects the egg of lactic acid bacteria by determining a-amino acid content
Plain boiled water solution capacity of water.It is comprised the following steps that：
(1) preparation of reagents
The preparation of 100 μ g/mL tyrosine solutions：Tyrosine is dried to constant weight in 105 DEG C of baking ovens, constant weight is accurately weighed
Tyrosine 0.1000g, being gradually added 6mL, 1mol/L hydrochloric acid dissolves it, is settled to 100mL with 0.2mol/L hydrochloric acid, now
Tyrosine solution concentration is 1000 μ g/mL, then draws this solution 10mL, is settled to 100mL with 0.2mol/L hydrochloric acid, that is, is configured to
Concentration is 100 μ g/mL tyrosine solution, and this solution should be timely used after being made into or be put into time in refrigerator and preserved.
The preparation of OPA reagents：By 62.5mL, 0.1M sodium tetraborate solution, 6.25mL, 20%SDS solution, 100mg is adjacent
Phthalaldehyde, 5mL methanol solutions, 250 μ L beta -mercaptoethanols solution mixing is settled to 250mL, that is, is configured to OPA reagents, OPA
Reagent needs matching while using, and is kept in dark place.
(2) measure of standard curve
0,1mL, 2mL, 3mL, 4mL, 5mL tyrosine solution (concentration is 100 μ g/mL) are measured respectively, use ddH2O will be molten
Liquid is mended to 5mL, is well mixed, standby；The tyrosine solution of 150 μ L various concentrations is taken, 3mL OPA reagents is added, gently shakes
Uniformly, 2min is reacted at room temperature, OD is determined340Value；By abscissa of tyrosine concentration, OD340It is worth and makes standard curve for ordinate：
Y=0.0146X+0.0021 (R2=1).
(3) sample is determined
The PE-28 plants of amounts by 3% (v/v) of Lactobacillus plantarum (Lactobacillusplantarum) of the present invention are inoculated with
In 11% skimmed milk, 37 DEG C of culture 24h；1.25mL bacterium solutions are taken, 0.3mL ddH are added2O, 3mL, 0.75M trichloroacetic acid, are mixed
After closing uniformly, the static 10min of room temperature；6000r/min, 4 DEG C of centrifugation 6min, take supernatant standby；150 μ L of supernatant are taken, 3mL is added
OPA reagents, are gently shaken, room temperature condition arrest reaction 2min, and the absorbance of solution is determined at wavelength 340nm；Obtain
OD340Value is contrasted with standard curve, and obtained protease hydrolysis activity, also can be directly with OD equivalent to the amount of tyrosine340Value
Compare.
Lactobacillus plantarum (Lactobacillus plantarum) PE-28 of the present invention it can be seen from the result of table 2
The protease hydrolysis activity of strain is higher than other test strains (NC305, DM-12, NC301), and free aminoacid content can reach
137.48μg/mL。
The high proteolytic activity bacterial strain secondary screening result of table 2
| Bacterial strain | Free aminoacid content (μ g/mL) |
| PE-28 | 137.48 |
| NC305 | 10.04 |
| DM-12 | 0.97 |
| NC301 | 56.74 |
Embodiment 3 is digested using PE-28 plants of fermentations of Lactobacillus plantarum (Lactobacillus plantarum) of the present invention
Prepare ginseng peptide
1st, the extraction of ginseng total protein
Selection：The root of the dry ginseng obtained below by way of cultivation for 5 years.The root 2000g of less than 5 years cultivation dry ginsengs is weighed,
30 mesh are crushed to, adding 10~30 times of amounts, (the weight ratio of the root of dry ginseng and 60% ethanol solution is 1:60% second 10-30)
Alcoholic solution refluxing extraction 2 times, filtering, with 10~30 times of amounts, (the weight ratio of filter residue and 80 DEG C of hot water is 1 to filter residue:10-30) 80
DEG C hot water boils extraction 2 times, each 1h, filtering, merging filtrate, takes filtrate 1L to be concentrated into 100mL, centrifugation (4000rpm,
10min, 4 DEG C), supernatant adds solid (NH4)2SO4Precipitated, (NH4)2SO4Concentration is the 70% (quality hundred of ammonium sulfate
Divide content g/100g), 4 DEG C stand overnight, and through centrifugation (5000rpm, 30min, 4 DEG C), dialysis (6000~8000Da), freeze
Ginseng total protein freeze-dried powder.
2nd, the preparation of ginseng peptide
The preparation of culture medium：Ginseng total protein freeze-dried powder 25g, glucose 20g are weighed, adds water and is configured to 1000mL solution,
115 DEG C of sterilizing 20min, obtain ginseng protein culture medium；
Fermentation：Lactobacillus plantarum (the Lactobacillus of the present invention is accessed by ginseng protein medium body fraction 3%
Plantarum) PE-28 plants, 37 DEG C of quiescent culture 20h are concentrated through centrifuging (6000rpm, 10min, 4 DEG C), supernatant, are freezed to obtain people
Join peptide.
3rd, the SDS-PAGE electrophoretic analysis and the analysis of ginseng peptide of ginseng total protein
SDS-PAGE electrophoresis is carried out to ginseng total protein using ammonium sulfate precipitation experiment, its result is as shown in Figure 1.
4th, ginseng total protein and the high performance liquid chromatography of ginseng peptide (HPLC) analysis
The ginseng total protein of above-mentioned acquisition is centrifuged into 10min under the conditions of 5000rpm, 4 DEG C, the supernatant mistake after centrifugation
0.22 μm of miillpore filter carries out HPLC detections, with the mobile phase ratio gradient elution of table 3；Detection wavelength：280nm, column temperature：50 DEG C,
Flow velocity：0.5mL/min, chromatographic column：ZORBAX 300SB-C18, its HPLC collection of illustrative plates is as shown in Figure 2.
Similarly, the ginseng peptide zymotic fluid of above-mentioned acquisition is centrifuged into 10min under the conditions of 5000rpm, 4 DEG C, it is upper after centrifugation
Clear liquid crosses 0.22 μm of miillpore filter and carries out HPLC detections, with the mobile phase ratio gradient elution of table 3；Detection wavelength：280nm, column temperature：
50 DEG C, flow velocity：0.5mL/min, chromatographic column：ZORBAX 300SB-C18, its HPLC collection of illustrative plates is as shown in Figure 3.
As a result show：By Lactobacillus plantarum (Lactobacillus plantarum) PE-28 for being compared with the present invention
Strain fermentation digests the ginseng total protein HPLC collection of illustrative plates (Fig. 2) prepared and understood with ginseng peptide HPLC collection of illustrative plates (Fig. 3), obtained ginseng
Peptide occurs different small-molecular peptides peaks in different retention times：1.57/1.75/2.19/3.32/4.51/5.25/6.22/7.44.
Table 3
| Gradient elution (min) | 0.1% trifluoroacetic acid | The second eyeball of 0.01% trifluoroacetic acid+80% |
| 0.00 | 98 | 2 |
| 1.00 | 98 | 2 |
| 8.80 | 55 | 45 |
| 9.00 | 5 | 95 |
| 15.00 | 5 | 95 |
| 15.20 | 98 | 2 |
| 25.00 | 98 | 2 |
The invention discloses plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) PE-
28 plants, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, institute
There is similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention.
The product of the present invention is described by preferred embodiment, and related personnel can substantially not depart from present invention, essence
Product as described herein is modified in refreshing and scope or suitably change is with combining, to realize and apply the technology of the present invention.
SEQUENCE LISTING
<110>Ming Zhiyuan bio tech ltd of Jilin Province
<120>One PE-28 plants of plant height proteolysis vigor Lactobacillus plantarum and the method that ginseng peptide is prepared using the bacterial strain
<160>1
<210>1
<211>1472
<212>DNA
<213>Manually
<400>1
ACTTCACCCTAATCATCTGTCCCACCTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCG 60
ACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT 120
ATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTG 180
CAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCA 240
ACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT 300
TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTT 360
AATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG 420
ACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAAT 480
CTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAA 540
ACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGG 600
CCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCT 660
CCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCT 720
ACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTG 780
TTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCA 840
CTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGAC 900
TTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTAC 960
GTATTACCGCGGCTGCTGGCACGTAGTTAGCCGAGGCTTTCTGGTTAAATACCGTCAATA 1020
CCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACC 1080
CTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTAC 1140
TGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTC 1200
AGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCG 1260
CGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAG 1320
TTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCC 1380
CACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATC 1440
AATACCAGAGTTCGTTCGACTTGCATGATAGC 1472
Claims (10)
1. PE-28 plants of a plant height proteolysis vigor Lactobacillus plantarum (Lactobacillusplantarum), it is characterised in that
The bacterial strain is preserved in China typical culture collection center on March 6th, 2017, and deposit number is：CCTCC NO:
M2017087。
2. utilize the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus described in claim 1
Plantarum) the method for PE-28 plants of preparation ginseng peptides, it is characterised in that comprise the following steps：
Step 1: dry ginseng's root is crushed, with 60% ethanol solution refluxing extraction twice, filtered filtration residue is with 80~100 DEG C of heat
Water is extracted twice, and 0.5~1.5h is extracted every time, and merging filtrate after filtering, concentrated, centrifuged supernatant adds solid (NH4)2SO4Precipitated, 4 DEG C stand overnight, then ginseng total protein freeze-dried powder is obtained through centrifugation, dialysis, lyophilized technique；
Step 2: ginseng total protein freeze-dried powder is made into ginseng protein culture medium, by Lactobacillus plantarum
(Lactobacillusplantarum) PE-28 plants are inoculated in ginseng protein culture medium, the Lactobacillus plantarum
(Lactobacillusplantarum) PE-28 plants of inoculum concentration is the 3% of ginseng protein culture volume, 37 DEG C of quiescent cultures
15~25h, ginseng peptide is obtained through centrifugation, concentration, lyophilized technique.
3. preparation method according to claim 2, it is characterised in that step one, 30 mesh are crushed to by dry ginseng's root.
4. preparation method according to claim 2, it is characterised in that step one, dry ginseng's root is chosen to be led to for less than 5 years
Cross the root for the dry ginseng that cultivation is obtained.
5. preparation method according to claim 2, it is characterised in that step one, the hot water temperature is 80 DEG C.
6. preparation method according to claim 2, it is characterised in that step one, (NH4)2SO4Concentration is 70%.
7. preparation method according to claim 2, it is characterised in that in step 2, the system of the ginseng protein culture medium
Standby process is as follows：Ginseng total protein freeze-dried powder 25g, glucose 20g are weighed, adds water and is configured to 1000mL solution, 115 DEG C of sterilizings
20min, obtains ginseng protein culture medium.
8. preparation method according to claim 2, it is characterised in that in step 2, the condition of centrifugation is：6000rpm,
10min, 4 DEG C.
9. the ginseng peptide obtained using the preparation method described in any one in claim 2 to 8.
10. the plant height proteolysis vigor Lactobacillus plantarum (Lactobacillus plantarum) described in claim 1
The PE-28 plants of applications in food, health products, medicine is prepared.
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| CN110917330A (en) * | 2019-10-30 | 2020-03-27 | 通化力神保健品有限公司 | Ginseng small-molecule peptide graphene eye mask and preparation method thereof |
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