CN107981360A - A kind of application of lactobacillus plantarum X7021 - Google Patents
A kind of application of lactobacillus plantarum X7021 Download PDFInfo
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- CN107981360A CN107981360A CN201711188280.3A CN201711188280A CN107981360A CN 107981360 A CN107981360 A CN 107981360A CN 201711188280 A CN201711188280 A CN 201711188280A CN 107981360 A CN107981360 A CN 107981360A
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- lactobacillus plantarum
- lactobacillus
- bacterium
- blood fat
- dna
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a kind of applications of lactobacillus plantarum X7021 in blood fat reducing food, health products, medicine or pharmaceutical composition is prepared；A kind of blood fat-reducing product is also disclosed, to reduce the lipids contents in serum；Also disclose a kind of applications of lactobacillus plantarum X7021 in enteral microecological formulation is prepared；Also disclose a kind of probiotics with good intestinal colonisation ability.Its advantage is：Lactobacillus plantarum X7021 has good intestinal colonisation ability and good lipid-lowering effect, has a vast market application value.
Description
Technical field
The invention belongs to functional food technical field, is answering on a kind of lactobacillus plantarum X7021 specifically
With.
Background technology
The food of lactobacillus-fermented is acknowledged as functional food.Lactic acid bacteria can also adjust micro- life of human body intestinal canal at the same time
State balances, and is a kind of cheap, feature-rich health drink.Due to survival ability of the different strain in enteron aisle not
Together, so it plays the lactic acid bacteria that the effective dose of prebiotic effect is also different, has good colonization ability only in enteron aisle,
The prebiotic function that lactic acid bacteria has in itself can preferably be played.Substantial amounts of research shows, probiotics after digestive system is entered, its
Viable count must reach 1 × l06Its prebiotic function of more than CFU/mL competence exertions.Therefore viable count in fresh probiotic composition
1 × 10 should be not less than7CFU/mL could compensate the loss of probiotics viable count when passing through human gastrointestinal tract.
Hyperlipidemia is primarily referred to as the too high levels of T-CHOL (TC) or triglycerides (TG) in blood plasma or serum.Generally
Increased with T-CHOL (TC) content and low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) drop
Low and/or triglycerides (TG) content increases as main feature.Lot of experiments and clinical research data show, hyperlipidemia
It is closely related with the generation of the disease such as atherosclerosis, coronary heart disease, cranial vascular disease, obesity, it is to cause atherosclerosis
One of important risk factor occurred with cardiovascular and cerebrovascular disease.With the continuous improvement of living standards of the people and changing for dietary structure
Become, the incidence of hyperlipidemia is in the trend risen year by year.Therefore, hyperlipidemia is actively prevented, reduces the hair of cardiovascular and cerebrovascular diseases
Sick rate is just particularly important.
The content of the invention
First purpose of the present invention is to provide a kind of lactobacillus plantarum X7021 and is preparing blood fat reducing food, health products, medicine
Application in thing or pharmaceutical composition；Second object of the present invention is to provide a kind of blood fat-reducing product, to reduce in serum
Lipids contents；Third object of the present invention is that providing a kind of lactobacillus plantarum X7021 is preparing enteral microecological formulation
In application；Fourth object of the present invention is to provide a kind of probiotics with good intestinal colonisation ability.
In order to achieve the above object, the present invention provides following technical solution：
First purpose of the present invention is to provide a kind of lactobacillus plantarum X7021 and is preparing blood fat reducing food, health products, medicine
Application in thing or pharmaceutical composition.
Second object of the present invention is to provide a kind of blood fat-reducing product, and the blood fat-reducing product is with lactobacillus plantarum
X7021 is active component, and the deposit number of the lactobacillus plantarum X7021 is CCTCC NO:M2015039.
According to the present invention, the blood fat-reducing product is using the lactobacillus plantarum X7021 bacterium solutions as active component.
According to the present invention, the blood fat-reducing product includes blood fat reducing food, health products, medicine or pharmaceutical composition
Third object of the present invention is to provide a kind of lactobacillus plantarum X7021 answering in enteral microecological formulation is prepared
With.
Fourth object of the present invention is to provide a kind of probiotics, has good intestinal colonisation ability, micro- life
For state preparation using lactobacillus plantarum X7021 as active component, the deposit number of the lactobacillus plantarum X7021 is CCTCC NO:
M2015039。
Further, the probiotics is using the lactobacillus plantarum X7021 bacterium solutions as active component.
Further, the lactobacillus plantarum X7021 bacterium solutions be by lactobacillus plantarum X7021 freeze bacterium powder pass through it is sterile
Normal saline dilution obtains.
Further, the lactobacillus plantarum X7021, which freezes bacterium powder, is prepared by freezing and freezing, and is contained
1.5×1011The viable count of CFU/g, has good environmental stability.
Preferably, the environment temperature that the lactobacillus plantarum X7021 freezes bacterium powder is 4 DEG C.
The beneficial effects of the invention are as follows：
1st, the vacuum-packed lyophilized bacterium powders of lactobacillus plantarum X7021 have good stable vigor at 4 DEG C and -20 DEG C, and plant
Thing lactobacillus X7021 has good intestinal colonisation ability, can be used for preparing probiotics, particularly intestinal microecology system
Agent；2nd, lactobacillus plantarum X7021 has good lipid-lowering effect, can be used for prepare blood fat reducing food, health products, medicine or
Pharmaceutical composition etc..Therefore main fermentation strains of the lactobacillus plantarum X7021 as food industry, has a vast market application
Value.
Brief description of the drawings
Fig. 1 is the clump count that lactobacillus plantarum X7021 preserves 1 to 12 month at room temperature.
Fig. 2 is the clump count that lactobacillus plantarum X7021 preserves 1 to 12 month under 4 DEG C of environment.
Fig. 3 is the clump count that lactobacillus plantarum X7021 preserves 1 to 12 month under -20 DEG C of environment.
Fig. 4 is the amplification curve diagram in the intestinal colonisation experiment of embodiment 4.Wherein, amplification curve is respectively from left to right：
7.68×10-3ng/μL、1.14×10-3ng/μL、6.1×10-5ng/μL、7.17×10-6ng/μL、9.45×10-7ng/μL。。
Fig. 5 is the quantitation curves of embodiment 4.
Fig. 6 is the result figure that the real-time fluorescence quantitative PCR of embodiment 4 detects the viable bacteria content in fecal specimens.
Fig. 7 is the viable bacteria content in fecal specimens after the real-time fluorescence quantitative PCR detection gavage fermented soybean milk of embodiment 4
Result figure.
Fig. 8 is the variation diagram of the TC contents in the serum of embodiment 5.
Fig. 9 is the variation diagram of the TG contents in the serum of embodiment 5.
Figure 10 is the variation diagram of the HDL-C contents in the serum of embodiment 5.
Figure 11 is the variation diagram of the LDL-G contents in the serum of embodiment 5.
Figure 12 is the variation diagram of superoxide dismutase (SOD) enzyme activity in the serum of embodiment 5.
Figure 13 is the variation diagram of glutathione peroxidase (GSH-Px) enzyme activity in the serum of embodiment 5.
Figure 14 is the variation diagram of catalase (CAT) enzyme activity in the serum of embodiment 5.
Figure 15 is the variation diagram of malonaldehyde (MDA) content in the serum of embodiment 5.
Figure 16 is the variation diagram of the insulin content in the serum of embodiment 5.
Figure 17 is the variation diagram of the leptin content in the serum of embodiment 5.
Figure 18 is the variation diagram of the adiponectin content in the serum of embodiment 5.
Figure 19 is the transcriptional level in the liver of embodiment 5 with lipid metabolism related gene.
Figure 20 be embodiment 5 lactobacillus plantarum X7021 in 3 bile salt hydrolases Multiple amino acid sequence alignment.
Figure 21 be embodiment 5 lactobacillus plantarum X7021 in 3 bile salt hydrolases transcriptional level.
Figure 22 is the genome loop graph of the lactobacillus plantarum X7021 of embodiment 5.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.It is to be understood that following embodiments are merely to illustrate this
Invention is not for restriction the scope of the present invention.The experimental method of actual conditions is not specified in the following example, usually according to normal
Rule condition carries out.
1st, the material and reagent of following embodiments
(1) formula of MRS culture mediums is：4.8g MRS (the extensive and profound in meaning star biotechnology Co., Ltd in Beijing) are weighed, are added
Enter in 100mL distilled water, dispensed after dissolving, 115 DEG C of autoclaving 20min are spare.
(2) formula of PBS buffer is：0.8%NaCl, 0.02%KH2PO4, 0.115%Na2HPO4W/v, pH 6.4；
2nd, the other materials of following embodiments and equipment are commercially available.
3rd, the bacterium source of the present embodiment：Lactobacillus plantarum Lactobacillus plantarum X7021 bacterial strains, also known as
Lactobacillus plantarum S1L12, is isolated from bean curd with odor thick gravy, has been stored in Chinese Typical Representative training on January 16th, 2015
Collection is supported, deposit number is CCTCC NO:M2015039.
The activation of 1 lactobacillus plantarum X7021 bacterial strains of embodiment
From the glycerol tube for preserving lactobacillus plantarum Lactobacillus plantarum X7021, picking goes out part
Bacterium solution, rules on MRS solid plates, and 37 DEG C of culture 36-48h, choose the single bacterium colony on tablet and be inoculated in 10mL liquid MRS cultures
In base, 37 DEG C of culture 18h, activate 18h, by culture medium at 4 DEG C by the switching of 2% inoculum concentration in 10mL liquid MRS culture mediums
The sterile PBS buffer washing thalline of 3000rpm centrifugation 5min, abandoning supernatant, addition and culture medium equivalent 2 times, Ran Houshou
Collect thalline and add isometric sterile PBS buffer, piping and druming mixes, as examination bacterium solution.
2 lactobacillus plantarum X7021 of embodiment freezes the preparation of bacterium powder
In aseptic operating platform, chosen from the glycerol tube of lactobacillus plantarum Lactobacillus plantarum X7021
A small amount of bacterium solution is taken out, is rule on MRS solid plates, 37 DEG C of culture 36-48h, choose the single bacterium colony on tablet and be inoculated in 10mL liquid
In body MRS culture mediums, in the fermentation tank for the culture medium containing MRS that 10L is inoculated in after 37 DEG C of Anaerobic culturel 18h, anaerobic fermentation 24h,
Then 6000rpm centrifuges 10min and collects thalline, is washed 2 times with sterile phosphate buffer (pH 6.4).The bacterium that centrifuges and will obtain
Mud is with containing 18-22% skimmed milk powers, 4-9% trehaloses, 2-4% sodium glutamates, 1-5% glycerine and 0.1-1% cysteines
The different weight ratio of the sterilized freeze drying protectant mixing and emulsifying of hydrochloride, bacterium mud and freeze drying protectant.Bacterium mud and protection
After agent fully suspends uniformly, emulsion mixture pre-freeze 4-10h below -45 DEG C, places into freeze dryer and freezes 20-26h, you can
To lyophilized bacterium powder.
When obtained bacterium mud and contain 20% skimmed milk power, 6% trehalose, 2.5% sodium glutamate, 2% glycerine with
The weight ratio of the sterilized freeze drying protectant mixing and emulsifying of 0.5% cysteine hydrochloride, bacterium mud and freeze drying protectant is 1:
2, after bacterium mud and protective agent fully suspend uniformly, emulsion mixture more than pre-freeze 4h, places into freeze dryer and freezes below -45 DEG C
24h, you can obtain lyophilized bacterium powder, the viable count in bacterium powder is determined to 1.5 × 1011CFU/g。
3 lactobacillus plantarum X7021 of embodiment divides equally stability test in the environment
The Lactobacillus plantarum X7021 bacterium powders that embodiment 2 is obtained are vacuum-packed, and are individually positioned in room
Temperature, in 4 DEG C and -20 DEG C of three environment.Take out 1.0g bacterium powders every month from each environment, three Duplicate Samples, use physiological saline
Dilution, rubbing method are coated in MRS solid plates, 37 DEG C of insulating box culture 18-24h, calculate single bacterium colony number.Varying environment temperature
Lyophilized bacterium powder viable bacteria number under degree is as shown in Figs. 1-3.
As shown in Figure 1, the clump count that lactobacillus plantarum X7021 bacterium powders initially measure is 14.73 ± 0.45lg (CFU/g),
By the preservation of 1 to 12 month, at the 11st month and 12nd month, vigor was remarkably decreased, finally the bacterium at 12nd month
It is 9.68 ± 0.24lg (CFU/g) that powder, which has great-hearted clump count,.The result shows that lactobacillus plantarum X7021 bacterium powders are unsuitable for room
Middle benefit gas is placed for a long time.
As shown in Fig. 2, lactobacillus plantarum X7021 bacterium powders are positioned over viable count under 4 DEG C of environmental conditions by initial 14.03
13.20 ± the 0.27lg (CFU/g) of ± 0.04lg (CFU/g) to 12nd month, colony counts living illustrate plant without being remarkably decreased
Lactobacillus X7021 bacterium powders having good stability under 4 DEG C of environment.
As shown in figure 3, lactobacillus plantarum X7021 bacterium powders be positioned over -20 DEG C of environmental condition viable counts by most initial 14.03 ±
0.04lg (CFU/g), bacterium powder stability when preserving to 12nd month is changed into 11.69 ± 0.05lg (CFU/g), under vigor slightly has
Adjust but without significant changes.It is not more low better to illustrate lactobacillus plantarum X7021 bacterium powders storage temperature, and most suitable storage temperature is 4
℃。
Conclusion：The most suitable storage temperature of lactobacillus plantarum X7021 bacterium powders is 4 DEG C；Lactobacillus plantarum X7021 bacterium powders are 4
Preserved under DEG C environment, be conducive to store for a long time.
The intestinal colonisation ability test of 4 lactobacillus plantarum X7021 of embodiment
1st, the preparation of gavage bacterium solution
Lactobacillus plantarum Lactobacillus plantarum X7021 bacterium powder of the section Example 2 without embedding is weighed,
Set respectively according to table 1, be diluted to required bacteria concentration respectively with sterile saline, obtain the gavage bacterium solution of bacterial strain.
1 gavage liquid of table is set
| Gavage liquid title | Bacterium is dense (CFU/mL) in gavage liquid | Remarks |
| X7021 high concentrations | 1.0×1010 | Experimental group A |
| X7021 low concentrations | 5.0×108 | Experimental group B |
| Control | 0 | Control group C |
2nd, the preparation of gavage soya-bean milk
According to the bacterial strain activation method in embodiment 1, by Lactobacillus plantarum X7021 in 37 DEG C of cultures
18h, ferments 9h in 10mL sterilizes soya-bean milk by the switching of 5% inoculum concentration, then carries out gavage with fermented soybean milk, compare not ferment
Soya-bean milk.
3rd, the preparation of experimental animal
(1) animal house condition is：20 ± 2 DEG C of temperature is kept, humidity 50 ± 5%, changes a bedding and padding in every 3 days.Control 12h light
According to the circulation standard of 12h dark, 7 points of every morning turns on light.Rat freers drinking-water and free choice feeding during the experiment, body
Weight detection in every 3 days is once.Before the experiment, the laundering period of one week is first given, basal feed is fed in the laundering period.
(2) the male SD systems rat 30 that weight is 180-200g is only randomly divided into 5 groups, every group 10, numbering is A respectively
Group, B groups, C groups, D groups, E groups.
4th, animal gavage liquid method
The weight of every rat is weighed before gavage is carried out, is carried out by the dosage of 1mL/100g (bacterium solution volume/mouse weight)
Gavage, 9 points of every morning of quantitatively gavage 1 time, continuous gavage 7 days.Wherein, A groups gavage bacteria containing amount is 1.0 × 1010CFU/mL's
Lactobacillus plantarum X7021 high concentration bacterium solutions, B group gavages bacteria containing amount are 5.0 × 108The lactobacillus plantarum of CFU/mL
Lactobacillus plantarum X7021 low concentration bacterium solutions, C group gavage sterile salines, D groups are fermented soybean milk group, E
It is non-fermented soybean milk group to organize, normal to feed conventional feed and drinking water during gavage in addition to gavage is pinpointed.
5th, the collection of animal wastes
Stop gavage 1,3,5,7,9 and 11d gathers the fecal specimens of each group rat respectively, every group of sampling quantity is 10g, sampling
It is loaded on immediately in sterile 5mL EP pipes afterwards, in -40 DEG C of preservations.
6th, the bacterial content in real-time fluorescence quantitative PCR (Real-time PCR) measure fecal specimens
(1) extraction of lactobacillus plantarum X7021 full-length genomes
Picking goes out part bacterium solution from the glycerol tube of preservation lactobacillus plantarum X7021, and 10mL liquid is inoculated in by 2% (v/v)
In body MRS culture mediums after 37 DEG C of culture 18h, 12,000rpm centrifugation 10min collect thalline, using gram-positive bacterium gene
The full-length genome of the group small extraction reagent kits of DNA (Shanghai Jierui Biology Engineering Co., Ltd) extracting lactobacillus plantarum X7021.
(2) lactobacillus plantarum X7021 species-specific primers design
There are intergenic region (ISR) between full-length genome 16S-23S rRNA, the conservative degree in this region is low, in different strain
Between have great changeability, therefore lactic acid bacteria not of the same race can be identified and be distinguished according to this.
Used primer is as follows：
p514：5'-tggatcacctcctttcta-3, SEQ ID NO:1；
p675：5'-gtgcgccctttattaactt-3, SEQ ID NO:2；
Full-length genome ISR regions are expanded, and further design species-specific primer.
Amplification condition is：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 45 DEG C of annealing 30s, 72 DEG C of extension 60s, 30 are followed
Ring, amplification system are as shown in table 2.Amplified production is separated by electrophoresis with nucleic acid gel, there are 2 fragments of size in product, is used
2 large fragments in gel are carried out glue reclaim by plastic recovery kit, and the sequence for purifying recycling post-fragment is measured, and are ordered
Entitled 12L.
2 ISR PCR reaction systems of table
| Title | Volume (μ L) |
| Taq PCR Mastermix | 25 |
| p514 | 1 |
| p675 | 1 |
| DNA profiling | 2 |
| dd H2O | 21 |
The sequence fragment of 12L by sequencing is inputted into ncbi database, operation blastn programs are compared, select
The region of similarity minimum is respectively designated as 12S as species specificity fragment in 12L sequences, and according to the species specificity piece of bacterium
Section designs species-specific primer using software Primer premier 5.0 and synthesizes, as shown in table 3：
3 species-specific primer of table
(3) design of primer
Species-specific primer shown in table 3 is expanded and recycled to species specificity fragment, method is same as above.Then,
Obtained DNA fragmentation 12S will be recycled with pEASY-T1Simple cloning vectors (Tiangeng biochemical technology Co., Ltd) in 25 DEG C of bars
Insulation connection 10min under part, obtains connection liquid, and linked system is as shown in table 4.5 μ L connection liquid are taken to be added to 50 μ L Escherichia coli
In DH5 α competent cells (Tiangeng biochemical technology Co., Ltd), slight piping and druming mixes, after placing 30min on ice, in 42 DEG C of water
Heat shock 90s in bath, cools down in ice immediately after, adds 900 μ L LB fluid nutrient mediums, 1h is cultivated under the conditions of 37 DEG C,
Supernatant is abandoned after 3000rpm centrifugations 2min, is coated on after thalline piping and druming is mixed on the LB tablets containing kanamycins, in 37 DEG C of inversions
Culture, using the species-specific primer of table 3, identifies positive transformant by bacterium colony PCR and send sequencing.The reactant of bacterium colony PCR
System and amplification condition are same as above, wherein, the primer in reaction system uses 12Sf and 12Sr in table 3.
It will be inoculated with by the sequencing post-fragment sequence positive transformant identical with 12S sequences into the liquid containing kanamycins
Cultivated in LB, with the pEASY- of Plasmid Miniprep Kit (Shanghai Jierui Biology Engineering Co., Ltd) extracting restructuring
T1Simple cloning vectors, as the outer standard items of Real-time PCR quantitatively, are stored in -20 DEG C.Use ultramicron nucleic acid
The recombinant vector concentration that protein assay measure is extracted, and according to formula：Vector copies (copies/ μ L)=carrier sample
Concentration/(base number × 324) × 6 × 1014, calculate the copy number of the recombinant vector extracted.
4 linked system of table
| Title | Volume (μ L) |
| pEasy T1Simple cloning vector | 1 |
| Purpose fragment | 4 |
Since Real-time PCR products length is preferably between 100-150bp, and 500bp should not be exceeded, therefore
Need the primer of design Real-time PCR.The sequence fragment of 12L by sequencing is inputted into ncbi database, operation
Blastn programs are compared, and select the region of similarity minimum in 12S sequences as Real-time PCR purpose fragments, divide
12Q is not named as, and Real-time PCR primers are designed using software Primer premier 5.0 according to 12Q sequences and are closed
Into as shown in table 5：
5 Real-time PCR primers of table
(4) amplification curve in the intestinal colonisation experiment of lactobacillus plantarum X7021
Real-time PCR systems are as shown in table 6.Real-time PCR reactions are carried out by following condition, all fluorescence are determined
Amount PCR, which is reacted on ABI StepOne, to carry out.
PCR reaction conditions：Two-step method is reacted, 95 DEG C of pre-degeneration 15min；95 DEG C of denaturation 10s, 57 DEG C of annealing extension 30s, if
Put 40 circulations.
6 Real-time PCR reaction systems of table
| Title | Volume (μ L) |
| 2×SuperRealPreMix Plus | 10 |
| Forward primer | 0.3 |
| Reverse primer | 0.3 |
| DNA profiling | 1 |
| 50×ROX Reference Dye△ | 2 |
| RNase-free ddH2O | 6.4 |
Lactobacillus plantarum X7021 carries out the fluorescent quantitation amplification curve shown after Real-time PCR, as shown in Figure 4.
Wherein, in Fig. 4, DNA profiling concentration from left to right is respectively 7.68 × 10-3ng/μL、1.14×10-3ng/μL、
6.1×10-5ng/μL、7.17×10-6ng/μL、9.45×10-7ng/μL。
Conclusion：With the increase of period, its fluorescence intensity gradually strengthens the template of different copy numbers.Passing through one section of index
Curve ascendant trend is minimum and tend to be parallel after the amplification phase, occurs " platform effect ", exponential amplification phase template copy numbers with it is glimmering
The correspondence of light accumulated value forms the quantitative basis of this two plants of bacterium.Under same fluorescence intensity, template copy numbers are more, institute
Corresponding period is smaller.
(5) sensitivity experiment of the real-time fluorescence quantitative PCR primer of lactobacillus plantarum X7021
From amplification curve diagram as it can be seen that template number is less than 102Copies/ μ L still have feature linearity curve, illustrate the Real-time
PCR has preferable sensitivity.
(6) foundation of quantitation curves
1) outer standard items are subjected to l04-108Being serially diluted again, forms it into 102-106Copies/ μ L, utilize Real-
Time PCR measure the Ct values under different copy numbers to make standard curve, as shown in table 7.
The corresponding Ct values of 7 various concentrations template of table
| Template concentrations (ng/ μ L) | Ct values |
| 9.45×10-7 | 29.88 |
| 7.17×10-6 | 27.29 |
| 6.1×10-5 | 24.55 |
| 1.14×10-3 | 20.81 |
| 7.68×10-3 | 18.36 |
2) data processing and calculating
Using the logarithm of different templates copy number as abscissa, to reach the initial cycle of fluorescence threshold in PCR reaction process
Number (Ct) obtains the quantitation curves of lactobacillus plantarum X7021 for ordinate, as shown in figure 5, being lactobacillus plantarum X7021's
Quantitative criterion, linear equation y=-2.9533x+37.53, and linear relationship numerical value R2=0.9957.In use, for needing
Quantitative sample, measures the Ct values of lactobacillus plantarum X7021 in sample to be tested genome every time, is according to respective standard curve
The content of bacterium in fecal specimens can be calculated.
3) result of the test
From amplification curve diagram as it can be seen that template number is less than 102Copies/ μ L still have feature linearity curve, illustrate the Real-time
PCR has preferable sensitivity.Quantitative standard curve R2=0.9957, illustrate that linear relationship is preferable, in certain dynamic range
The interior ability with accurate quantification.
(7) the intestinal colonisation ability measure of lactobacillus plantarum X7021, is detected in fecal specimens using Real-time PCR
Viable bacteria content
Bacterial genomes DNA in interior extracting excrement when 12 is small after fecal specimens collection, with excrement extracting genome DNA reagent
Full-length genome in box extraction excrement, according to kit, additionally book is operated, and the genome after extraction is placed in -20 DEG C of guarantors
Deposit.The lactobacillus plantarum X7021 genes in fecal specimens genome are quantified by Real-time PCR reactions, are reacted
System is with method as shown in " amplification curve in the intestinal colonisation experiment of step (4) lactobacillus plantarum X7021 ".Amplification finishes
Afterwards, solubility curve analysis is carried out, reacts quantitation curves of the obtained Ct values according to this plant of bacterium, you can calculate excrement sample
The amount of lactobacillus plantarum X7021 in product.Experimental result is as shown in Figure 6 and Figure 7.
Intestinal colonisation ability explanation：Prebiotic function is played to the lactic acid bacteria for enabling intake internal, it survives in enteron aisle
The minimum of amount will reach about 106-8CFU/g intestines are tolerant (bacterial content of the gavage after one day).From fig. 6 it can be seen that high concentration
Group gavage liquid after 1-3 days, meets 106-8CFU/g, and from after gavage liquid 3 days, bacterial content gradually reduces；Low concentration group gavage liquid 1
Meet 106-8CFU/g after it, from after gavage liquid 1 day, bacterial content gradually reduces；Saline control group is equal from gavage liquid
Less than 104CFU/g.It can be seen from figure 7 that fermented soybean milk group gavage liquid meets 106-8CFU/g after 1-11 days, from gavage liquid
Risen after 11 days, bacterial content gradually reduces；104CFU/g-105CFU/g is in after soya-bean milk group gavage liquid.
Experimental result：(1) bacterial content of high concentration group is higher all than low concentration group, and explanation will make lactic acid bacteria energy in enteron aisle
Play potential prebiotic function, it is necessary to take in the bacterium of sufficient dosage, survival ability is relatively low in enteron aisle especially for those
Strain.(2) using fermented soybean milk as carrier when, effect is better than being used as carrier using physiological saline.In gavage the 2nd day, plant breast bar
The amount of bacterium X7021 reaches maximum, is 8.4lg (CFU/g).Illustrate that the soyabean oligosaccharides in soya-bean milk may advantageously facilitate intestinal colonisation
Ability.
5 lactobacillus plantarum X7021 reducing blood lipid zooperies of embodiment
1st, the bacterial strain of the Lactobacillus plantarum X7021 activated in embodiment 1 is transferred by 5% inoculum concentration
37 DEG C of quiescent culture 9h, spare in 10mL soya-bean milk.
2nd, experimental animal prepares
Kunming mice 60,20 ± 2g, half male and half female.Experimental situation adapt to 1 week after, start feed high lipid food, one
The total cholesterol level detected after month in blood determines whether hyperlipidemia model succeeds.Choose 50 successful hyperlipidemia animals of modeling
It is randomly divided into 5 groups：
I groups：Lactobacillus plantarum X7021 low dose groups, daily gavage 108CFU/mL bacterium solutions；
II groups：Lactobacillus plantarum X7021 high dose groups, daily gavage 1010CFU/mL bacterium solutions；
III groups：Lactobacillus plantarum X7021 fermented soybean milk groups, daily gavage fermented soybean milk；
IV groups：Soya-bean milk group, daily gavage soya-bean milk；
V groups：High fat group, daily gavage physiological saline.
Daily often only according to 10mL/kg dosage gavages, in experimentation, weigh weekly and record weight, and according to weight
Adjust given low.High lipid food is fed during gavage, continues gavage 4 weeks.The free diet of each group mouse, drinking-water.
3rd, T-CHOL TC in kit (Bioengineering Research Institute is built up in Nanjing) detection blood, triglycerides TG, highly dense
Spend lipoprotein cholesterol HDL-C, the changes of contents of low density lipoprotein cholesterol LDL-C.
Docking takes blood weekly, and 3000 × g, centrifuges 10min, collects upper serum, and the detection in serum is detected with kit
Before gavage, first week after gavage, second week, the 3rd week, T-CHOL TC, triglycerides TG, high density in the serum of 4th week
The changes of contents of lipoprotein cholesterol HDL-C, low density lipoprotein cholesterol LDL-C.TC, TG, HDL-C, LDL-C testing result
Respectively as shown in Fig. 8, Fig. 9, Figure 10, Figure 11.
The results show：(1) compared with gavage physiological saline and soya-bean milk group, in gavage bacterium powder and fermented soybean milk group serum
TC, TG content decline after a week, and have conspicuousness reduction in second week, the 3rd week, 4th week.Wherein, 1010CFU/mL groups
TC values decline 30.6%, the TG values of fermented soybean milk group decline 49.0%.
(2) compared with gavage soya-bean milk group, HDL-C starts to raise after a week and has with aobvious in gavage fermented soybean milk group serum
Sex differernce is write, and also has conspicuousness rise in 4th week.In 4th week, two groups of gavage low concentration and high-concentration bacterial powder compared with physiology
Brine group also has the rising of conspicuousness.Wherein, 108It is the most obvious that CFU/mL groups HDL-C rises 46.7%.
(3) compared with gavage soya-bean milk group, LDL-C begins to decline and has and has after a week in gavage high-concentration bacterial powder group serum
Significant difference.Though gavage low concentration bacterium powder and fermented soybean milk group LDL-C have decline, not significantly, high in 4th week, gavage
Concentration bacterium powder group has the reduction of conspicuousness compared with physiological saline group.Wherein, 108It is the brightest that the LDL-C of CFU/mL groups declines 16.7%
It is aobvious.
Conclusion：Gavage lactobacillus plantarum X7021 has obvious blood fat reducing function.
4th, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), peroxide in kit detection serum
Change the content of hydrogen enzyme (CAT) enzyme activity and malonaldehyde (MDA)
Docking takes blood weekly, 3000 × g, centrifuges 10min, collects upper serum, detects after gavage in last week serum
Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) enzyme activity and peroxide
Compound malonaldehyde (MDA) content.Testing result is respectively as shown in Figure 12, Figure 13, Figure 14, Figure 15.
The results show：Lactobacillus plantarum X7021 and its fermentation after soya-bean milk can significantly increased SOD, GSH-Px and CAT work
Power, reduces MDA contents.Wherein, SOD enzyme activities 108CFU/mL、1010CFU/mL, fermented soybean milk group and soya-bean milk group, physiological saline
Group has significant difference (P<0.05)；GSH-Px, CAT enzyme activity fermented soybean milk enzyme activity highest, have compared with soya-bean milk group
Significant difference (P<0.05)；MDA contents, 1010CFU/mL and fermented soybean milk group are minimum, have compared with soya-bean milk group and physiological saline
There is significant difference.
Conclusion：Lactobacillus plantarum X7021 has the potential ability for reducing oxidativestress damage.
5th, kit detection serum insulin (INS), leptin (LEP), the content of adiponectin (ADPN)
Glucose tolerance test：Animal fasting for solids but not liquids 12h, tail vein take blood (0min) to measure blood glucose, give animal gavage
2g/kg body weight dose glucose, measures blood glucose value in 30min, 60min, 90min, 120min respectively.Measure pancreas islet in serum
Element, leptin, the content of adiponectin.Testing result is respectively as shown in Figure 16, Figure 17, Figure 18.
The results show：After gavage 4 weeks, conspicuousness change does not occur for the Serum Leptin Levels and adiponectin of control group and experimental group
Change, but leptin level fermented soybean milk and high dose bacterium powder group have the raising of conspicuousness compared with non-fermented soybean milk group.
Conclusion：Lactobacillus plantarum X7021 has potential blood fat reducing function.
In conclusion the vacuum-packed lyophilized bacterium powders of lactobacillus plantarum X7021 of the present invention are respectively provided with different temperatures
It is good to stablize vigor, especially preserved under 4 DEG C of environment, be conducive to store for a long time；Meanwhile lactobacillus plantarum X7021 is with good
Good intestinal colonisation effect and good lipid-lowering effect.Therefore main fermentations of the lactobacillus plantarum X7021 as food industry
Bacterial strain, has a vast market application value.
6th, the transcriptional level in fluorescence quantitative PCR detection liver with Genes Associated with Lipid Metabolism
Liver total RNA is extracted according to animal total RNA extraction reagent box (Shanghai Jierui Biology Engineering Co., Ltd) specification,
Reverse transcription obtains cDNA, and the relative expression of gene in table 7 is quantified using Bio-Rad ABI StepOne Real-time PCR instruments
Amount, 2-ΔΔCtAnalyze data.PCR primer sequence design such as table 8 below.Real-time PCR systems such as table 6.Carried out by following condition
Real-time PCR react, and all quantitative fluorescent PCRs are reacted on ABI StepOne and carried out.PCR reaction conditions：Two-step method is anti-
Should, 95 DEG C of pre-degeneration 15min；95 DEG C of denaturation 10s, 60 DEG C of annealing extension 30s, set 40 circulations.
8 real-time quantitative PCR primer table of table
The results show：RT-qPCR measures the expression of liver lipid metabolism gene, as shown in figure 19, with the high fat of physiological saline group
Model is as control, fermented soybean milk group LDL receptor (LDL receptor), PPAR (peroxisome proliferations
Activated receptor), PPAR δ, PPAR γ, PGC1 α (1 α of peroxisome proliferators activated receptor γ co-activator), gene point
It is not 1.61,2.77,3.63,2.91,2.8 times of control group, HMG-CoA reductase (- 3 methylglutaric acid lists of 3- hydroxyls
Acyl coenzyme A reductases), CYP7A1 (cholesterol 7- hydroxylases), SREBP-1c (cholesterol modulation element conjugated protein 1c) AAC
(acetyl-CoA carboxylase) gene expression is the 0.63 of control group, 0.68,0.8,0.54 times respectively, the base of Leptin (leptin)
Because expression is 3.15 times of control group.
Conclusion：Lactobacillus plantarum X7021 and its fermented soybean milk by adjust with the transcriptional level of lipid metabolism related gene come
Further improve the hyperlipidemic conditions of high fat diet induction.
7th, bile salt hydrolase (Bile Salt Hydrolase, BSH, EC 3.5.1.24) in lactobacillus plantarum X7021
Gene and transcriptional level analysis
The data for carrying out full-length genome to lactobacillus plantarum X7021 with Pacbio RSII high throughput sequencing technologies parse.
The related gene of bile salt hydrolase is found from full-length genome, finds there are 3 coding cholate hydrolysis in lactobacillus plantarum X7021
The gene of enzyme, have found the bile salt hydrolase nucleotide in lactobacillus plantarum ST-III and lactobacillus plantarum WCFS1 from NCBI
Sequence, is compared and analysis of amino acid sequence by software DNAMAN, obtains the conserved active site of prediction.
The bile salt hydrolase substrate glycodesoxycholic acid (GDCA) of 1g/L, 3g/L, 5g/L, training are added in MRS culture mediums
After supporting 24h, bacterium total serum IgE is extracted according to animal total RNA extraction reagent box (Shanghai Jierui Biology Engineering Co., Ltd) specification,
Reverse transcription obtains cDNA, utilizes the quantitative 3 bile salt hydrolase genes of Bio-Rad ABI StepOne Real-time PCR instruments
Relative expression quantity, 2-ΔΔCtAnalyze data.PCR primer sequence design such as table 9 below.Real-time PCR systems such as table 6.By following
Condition carries out Real-time PCR reactions, and all quantitative fluorescent PCRs are reacted on ABI StepOne and carried out.PCR reaction conditions：
Two-step method is reacted, 95 DEG C of pre-degeneration 15min；95 DEG C of denaturation 10s, 60 DEG C of annealing extension 30s, set 40 circulations.
9 bile salt hydrolase primer of table
The results show：Such as the genome loop graph that Figure 22 is lactobacillus plantarum X7021.Plant is found by annotation of gene function
There is the gene of three bile salt hydrolases of coding in lactobacillus X7021 genomes, length is respectively 1016bp, 986bp, 974bp,
Compared and analysis of amino acid sequence by DNAMAN, obtain the conserved active site of prediction：Cys2、Arg18、Asp21、
Asn175, Arg228, as shown in figure 20, as shown in figure 21, in the case of the sweet ammonia deoxidation cholate of addition various concentrations, bsh3
Transcriptional level with concentration increase and have the increase of conspicuousness, illustrate may bsh3 coding bile salt hydrolase degrade courage consolidate
Main function is played during alcohol.The base sequence of bsh3 is：
atgtgtactgccataacttatcaatcttataataattacttcggtagaaatttcgattatgaaatttcatacaatga
aatggttacgattacgcctagaaaatatccactagtatttcgtaaggtggagaacttagatcaccattatgcaataa
ttggaattactgctgatgtagaaagctatccactttactacgatgcgatgaatgaaaaaggcttgtgtattgcggga
ttaaattttgcaggttatgctgattataaaaaatatgatgctgataaagttaatatcacaccatttgaattaattcc
ttggttattgggacaattttcaagtgttagagaagtgaaaaagaacatacaaaaactaaacttggttaatattaatt
ttagtgaacaattaccattatcaccgctacattggttggttgctgataaacaggaatcgatagttattgaaagtgtt
aaagaaggactaaaaatttacgacaatccagtaggtgtgttaacaaacaatcctaattttgactaccaattatttaa
tttgaacaactatcgtgccttatcaaatagcacaccccaaaatagtttttcggaaaaagtggatttagatagttata
gtagaggaatgggcggactaggattacctggagacttgtcctcaatgtctagatttgtcagagccgcttttactaaa
ttaaactcgttgccgatgcagacagagagtggcagtgttagtcagtttttccatatactagggtctgtagaacaaca
aaaagggctatgtgaagttactgacggaaagtacgaatatacaatctattcttcttgttgtgatatgaacaagggag
tttattactatagaacttatgacaatagtcaaattaacagtgtcaatttaaaccatgagcacttggatacgactgaa
ttaatttcttatccattacgatcagaagcacaatactatgcagttaactaa(SEQ ID NO：47)
Conclusion：There are the gene of 3 coding bile salt hydrolases in lactobacillus plantarum X7021, contribute to the reducing blood lipid of X7021
Function.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art
For personnel, without departing from the technical principles of the invention, some improvement and modification can also be made, these improve and become
Type also should be regarded as protection scope of the present invention.
Sequence table
<110>East China University of Science
<120>A kind of application of lactobacillus plantarum X7021
<130> 171038
<141> 2017-11-24
<160> 47
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 1
tggatcacct cctttcta 18
<210> 2
<211> 19
<212> DNA
<213>Other information (Lactobacillus)
<400> 2
gtgcgccctt tattaactt 19
<210> 3
<211> 29
<212> DNA
<213>Other information (Lactobacillus)
<400> 3
caatgacgac taacgtcgtc aatggagaa 29
<210> 4
<211> 25
<212> DNA
<213>Other information (Lactobacillus)
<400> 4
cccttcttgt acacaccgcc cgtca 25
<210> 5
<211> 25
<212> DNA
<213>Other information (Lactobacillus)
<400> 5
aaaggaggtg atccagccgc aggtt 25
<210> 6
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 6
ccatgagagt ttgtaacacc 20
<210> 7
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 7
cctgccttcc caaaatgtgc 20
<210> 8
<211> 25
<212> DNA
<213>Other information (Lactobacillus)
<400> 8
cccttcttgt acacaccgcc cgtca 25
<210> 9
<211> 21
<212> DNA
<213>Other information (Lactobacillus)
<400> 9
gggctggcgg tagactggat c 21
<210> 10
<211> 22
<212> DNA
<213>Other information (Lactobacillus)
<400> 10
caatctgtcc agtacatgaa gc 22
<210> 11
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 11
gggcgtccct attcacttgt 20
<210> 12
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 12
gatgcccaga ggatcacgag 20
<210> 13
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 13
tgtgggaacg gtgacactta 20
<210> 14
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 14
cttcaaattt tgggcactca 20
<210> 15
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 15
agcacctccg aaagtacgtg 20
<210> 16
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 16
tcaggaatgg ccagcttgag 20
<210> 17
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 17
tcgagacaca tcgtttgagc 20
<210> 18
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 18
tcaaaaagtg catccagcag 20
<210> 19
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 19
ggtcatactc gcaggaaa 18
<210> 20
<211> 19
<212> DNA
<213>Other information (Lactobacillus)
<400> 20
agcaaattat agcagccac 19
<210> 21
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 21
tcagggctgc cagtttcg 18
<210> 22
<211> 24
<212> DNA
<213>Other information (Lactobacillus)
<400> 22
gcttttggca tactctgtga tctc 24
<210> 23
<211> 21
<212> DNA
<213>Other information (Lactobacillus)
<400> 23
aacgagatca gcgtgcatgt g 21
<210> 24
<211> 22
<212> DNA
<213>Other information (Lactobacillus)
<400> 24
tgaggaagag gctgctgaag tt 22
<210> 25
<211> 21
<212> DNA
<213>Other information (Lactobacillus)
<400> 25
acagagatgg tggctgatgt c 21
<210> 26
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 26
gatccccatg gcaatctg 18
<210> 27
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 27
ggatgctact gttgcaag 18
<210> 28
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 28
catgtacacc gtgatgtg 18
<210> 29
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 29
ggagccatgg attgcacatt 20
<210> 30
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 30
aggaaggctt ccagagagga 20
<210> 31
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 31
agacttggtc atggggacag 20
<210> 32
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 32
ggggagacat cagaaggaca 20
<210> 33
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 33
tgagagggcc aagcaaag 18
<210> 34
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 34
ataaatcaca cggcgctctt 20
<210> 35
<211> 21
<212> DNA
<213>Other information (Lactobacillus)
<400> 35
cggttcaaga atggcatcat c 21
<210> 36
<211> 19
<212> DNA
<213>Other information (Lactobacillus)
<400> 36
tcacacccac caccacgat 19
<210> 37
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 37
tgccagaggg aatagggaaa 20
<210> 38
<211> 25
<212> DNA
<213>Other information (Lactobacillus)
<400> 38
ctctcccatc cttacttaca aacca 25
<210> 39
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 39
tgttgtccct gtatgcctct 20
<210> 40
<211> 20
<212> DNA
<213>Other information (Lactobacillus)
<400> 40
taatgtcacg cacgatttcc 20
<210> 41
<211> 19
<212> DNA
<213>Other information (Lactobacillus)
<400> 41
ttgactttga gacccgtat 19
<210> 42
<211> 15
<212> DNA
<213>Other information (Lactobacillus)
<400> 42
ttcagttggc gaccc 15
<210> 43
<211> 17
<212> DNA
<213>Other information (Lactobacillus)
<400> 43
gaatcctttc ggtcctg 17
<210> 44
<211> 16
<212> DNA
<213>Other information (Lactobacillus)
<400> 44
gcgtcccgtt atcttg 16
<210> 45
<211> 18
<212> DNA
<213>Other information (Lactobacillus)
<400> 45
tagtatttcg taaggtgg 18
<210> 46
<211> 16
<212> DNA
<213>Other information (Lactobacillus)
<400> 46
tttcattcat cgcatc 16
<210> 47
<211> 975
<212> DNA
<213>Other information (Lactobacillus)
<400> 47
atgtgtactg ccataactta tcaatcttat aataattact tcggtagaaa tttcgattat 60
gaaatttcat acaatgaaat ggttacgatt acgcctagaa aatatccact agtatttcgt 120
aaggtggaga acttagatca ccattatgca ataattggaa ttactgctga tgtagaaagc 180
tatccacttt actacgatgc gatgaatgaa aaaggcttgt gtattgcggg attaaatttt 240
gcaggttatg ctgattataa aaaatatgat gctgataaag ttaatatcac accatttgaa 300
ttaattcctt ggttattggg acaattttca agtgttagag aagtgaaaaa gaacatacaa 360
aaactaaact tggttaatat taattttagt gaacaattac cattatcacc gctacattgg 420
ttggttgctg ataaacagga atcgatagtt attgaaagtg ttaaagaagg actaaaaatt 480
tacgacaatc cagtaggtgt gttaacaaac aatcctaatt ttgactacca attatttaat 540
ttgaacaact atcgtgcctt atcaaatagc acaccccaaa atagtttttc ggaaaaagtg 600
gatttagata gttatagtag aggaatgggc ggactaggat tacctggaga cttgtcctca 660
atgtctagat ttgtcagagc cgcttttact aaattaaact cgttgccgat gcagacagag 720
agtggcagtg ttagtcagtt tttccatata ctagggtctg tagaacaaca aaaagggcta 780
tgtgaagtta ctgacggaaa gtacgaatat acaatctatt cttcttgttg tgatatgaac 840
aagggagttt attactatag aacttatgac aatagtcaaa ttaacagtgt caatttaaac 900
catgagcact tggatacgac tgaattaatt tcttatccat tacgatcaga agcacaatac 960
tatgcagtta actaa 975
Claims (10)
- A kind of 1. applications of lactobacillus plantarum X7021 in blood fat reducing food, health products, medicine or pharmaceutical composition is prepared.
- A kind of 2. blood fat-reducing product, it is characterised in that the blood fat-reducing product using lactobacillus plantarum X7021 as active component, The deposit number of the lactobacillus plantarum X7021 is CCTCC NO:M2015039.
- 3. blood fat-reducing product as claimed in claim 2, it is characterised in that the blood fat-reducing product is with the lactobacillus plantarum X7021 bacterium solutions are active component.
- 4. blood fat-reducing product as claimed in claim 2, it is characterised in that the blood fat-reducing product includes blood fat reducing food, protects Strong product, medicine or pharmaceutical composition.
- A kind of 5. applications of lactobacillus plantarum X7021 in enteral microecological formulation is prepared.
- 6. a kind of probiotics, has good intestinal colonisation ability, the probiotics is with the lactobacillus plantarum X7021 is active component, and the deposit number of the lactobacillus plantarum X7021 is CCTCC NO:M2015039.
- 7. probiotics as claimed in claim 6, it is characterised in that the probiotics is with lactobacillus plantarum X7021 Bacterium solution is active component.
- 8. probiotics as claimed in claim 7, it is characterised in that the lactobacillus plantarum X7021 bacterium solutions are by planting Thing lactobacillus X7021 freezes bacterium powder and dilutes acquisition by sterile saline.
- 9. probiotics as claimed in claim 8, it is characterised in that the lactobacillus plantarum X7021, which freezes bacterium powder, is It is prepared by freezing and freezing, contains 1.5 × 1011The viable count of CFU/g.
- 10. probiotics as claimed in claim 7, it is characterised in that the lactobacillus plantarum X7021 freezes bacterium powder Environment temperature is 4 DEG C.
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| CN110638037A (en) * | 2019-08-14 | 2020-01-03 | 浙江黑金生物科技有限公司 | Application of lactobacillus plantarum X7022, black garlic enzyme and preparation method and application of black garlic enzyme |
| CN114752692A (en) * | 2022-03-29 | 2022-07-15 | 中国检验检疫科学研究院 | Primer probe combination for detecting lactobacillus plantarum, RPA detection kit and detection method thereof |
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