CN106906165A - A kind of Lactobacillus plantarum and its application in the food for preparing Constipation - Google Patents
A kind of Lactobacillus plantarum and its application in the food for preparing Constipation Download PDFInfo
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- CN106906165A CN106906165A CN201710278401.7A CN201710278401A CN106906165A CN 106906165 A CN106906165 A CN 106906165A CN 201710278401 A CN201710278401 A CN 201710278401A CN 106906165 A CN106906165 A CN 106906165A
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- lactobacillus plantarum
- constipation
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- lactobacillus
- activated carbon
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
It is CCTCC NO the invention discloses deposit number：The Lactobacillus plantarum YS1 (Lactobacillus plantarum YS1) of M2016747 and its application in the food for preparing Constipation, not only expand the range of application of Lactobacillus plantarum YS1, improve its application value, and prevention to constipation brings new hope.
Description
Technical field
The invention belongs to microbial technology field, it is related to a kind of Lactobacillus plantarum and its application in food is prepared.
Background technology
Yak yogurt is a kind of spontaneous fermentation dairy products in Qinghai-Tibet Tibetan area, contains abundant nutritional ingredient, tool
There are anti-oxidant, reduction cholesterol, regulation immunity.Due to special spontaneous fermentation condition, including fermentation raw material breast, hair
Ferment temperature, fermentation time, fermentation vessel, particularly special fermentative microorganism composition, cause yak yogurt to have flavour
And quality.
Constipation state can be considered at when defecation frequency is less than 3 times weekly, when number of times is considered as seriously less than 1 time
Constipation state, be it is a kind of threaten health, particularly there is the complicated symptom of considerable influence to colon health.Constipation is usual
Disease is not considered as, majority of case can be by diet and the regulation Constipation of custom.
The content of the invention
It is an object of the invention to carry out separation identification to the microorganism in yak yogurt, and its activity and application are carried out
Research.
Through research, the present invention provides following technical scheme：
1. Lactobacillus plantarum YS1 (Lactobacillus plantarum YS1), deposit number is CCTCC NO：
M2016747。
2. applications of the Lactobacillus plantarum YS1 in the food for preparing Constipation.
The present invention has carried out separating identification to the microorganism in the yak yogurt of Yushu district, Qinghai Tibetan autonomous prefecture, will wherein one
Lactobacillus plantarum (Lactobacillus plantarum) is named as YS1, and Chinese Typical Representative is preserved on December 14th, 2016
Culture collection (abbreviation CCTCC, address：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road), deposit number
It is CCTCC NO：M2016747.
Activated carbon inducing constipation model mice result of study shows that the anti-hydrochloric acid in gastric juice and cholate ability of Lactobacillus plantarum YS1 are strong
In lactobacillus bulgaricus, the Mouse Weight decline that constipation is caused can be suppressed, stool weight, granule number and water content decline,
Propulsion rate of the activated carbon in small intestine can be improved simultaneously, reduce the time of the first grain melena of discharge, moreover it is possible to make the blood of mice with constipation
Clearing stomach therbligs (MTL), gastrin (Gas), Endothelin (ET), acetylcholinesterase (AChE), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) and vasoactive
Peptide (VIP) serum levels are improved, and growth hormone release inhibiting hormone (SS) level declines.RT-PCR experimental results further show, Lactobacillus plantarum
YS1 can raise c-Kit, SCF, GDNF mRNA expression in mice with constipation small intestine, lower TRPV1, NOS mRNA expression.
The Lactobacillus plantarum YS1 of high concentration shows preferably effect, and is significantly better than the lactobacillus bulgaricus of same concentration.These realities
Testing result proves, Lactobacillus plantarum YS1 can effectively suppress constipation, can be used in preparing the food of Constipation.
The beneficial effects of the present invention are：The invention provides a kind of Lactobacillus plantarum YS1, it can effectively suppress just
It is secret, and effect is significant can be used in preparing the food of Constipation better than conventional lactobacillus bulgaricus, not only expand plant
The range of application of thing lactobacillus YS1, improves its application value, and prevention to constipation brings new hope.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of the genomic DNA of Lactobacillus plantarum YS1, and wherein M is λ DNA/Hind III
Marker, 1 is the genomic DNA of Lactobacillus plantarum YS1.
Fig. 2 is the 16S rDNA genetic fragment PCR amplifications of Lactobacillus plantarum YS1, and wherein M is for DNA Marker, C
Negative control, 1 is the 16S rDNA genetic fragment pcr amplification products of Lactobacillus plantarum YS1.
Fig. 3 is influences of the Lactobacillus plantarum YS1 to activated carbon inducing constipation Mouse Weight.
Fig. 4 is influences of the Lactobacillus plantarum YS1 to the small mouse's head grain melena efflux time of activated carbon inducing constipation.
Fig. 5 is Lactobacillus plantarum YS1 to c-Kit and SCF mRNA expression in activated carbon inducing constipation mouse small intestine tissue
Influence.
Fig. 6 is Lactobacillus plantarum YS1 to TRPV1, GDNF and NOS mRNA in activated carbon inducing constipation mouse small intestine tissue
The influence of expression.
In Fig. 3 to Fig. 6, LB represents lactobacillus bulgaricus, and LP-YS1 represents Lactobacillus plantarum YS1.
Specific embodiment
In order that the object, technical solutions and advantages of the present invention are clearer, below in conjunction with accompanying drawing to of the invention excellent
Embodiment is selected to be described in detail.
The separation and identification of embodiment 1, Lactobacillus plantarum YS1
First, lactic acid bacteria isolates and purifies
Pick up from 10 parts of the spontaneous fermentation yak yogurt sample of Yushu district, Qinghai Tibetan autonomous prefecture herdsman family, every part of sample nothing
After bacterium glass rod is fully stirred evenly, draw 50mL and be put into sterile centrifugation tube, be placed in and laboratory is taken back in food sampling box, 4 DEG C of refrigerators
Preserve.
Every part of yak yogurt sample takes 1mL, and 10 times of gradient dilutions to 10 are made with sterile saline-7.Take 10-5、10-6、
10-7Three each 1mL dilutions of dilution factor respectively at equipped with 15mL MRS culture mediums (containing the CaCO that mass ratio is 5%3) plate
In, mix, put 30 DEG C of 72 ± 3h of insulating box culture.Three, each dilution factor work is parallel.After bacterium colony is formed, its form is observed special
Levy, the different bacterium colonies that picking has molten calcium circle are inoculated in degreasing milk medium respectively, put 30 DEG C of 24~48h of culture, treat that bacterial strain is given birth to
After length is good, streak inoculation puts 30 DEG C of culture 48h in MRS agar mediums；Above step is repeated at least three times, until obtaining
Pure bacterium colony.
2nd, the identification of Bacillus acidi lactici
Pure bacterial strain is rule on MRS solid mediums, 30 DEG C of culture 48h are put, with 10 times amplify sem observation bacterium colony sizes,
The appearance features such as shape, carry out Gram's staining, and the typical bacterial strain of picking form carries out catalase test.By gram sun
Property, the negative meningitidis strains and bacillus strain of catalase test, it is lactic acid bacteria to fix tentatively.
Lactic acid bacillus mycopremna is carried out growth temperature experiment (10 DEG C, 45 DEG C, 60 DEG C, 30min), the examination of pH value gradient respectively
Test, mobility test, catalase test, oxidase test, hydrogen sulfide production test, gelatin liquefaction experiment, nitrate reduction test, Yin
Diindyl experiment, the experiment of glucose aerogenesis, benzidine test, litmus milk tests, arginine produce ammonia test, casein decomposition run and
Various sugar alcohol fermentation tests etc..
Result is isolated from 10 parts of yak yogurt samples and is purified into 20 plants of Bacillus acidi lacticis, be Gram-positive,
Catalase test is negative.By morphological observation and physiological and biochemical test, this 20 plants of bacterial strains are initially identified as 7 kinds of lactic acid
Bacillus, number consecutively is Lb1~Lb7, wherein, numbering is Lb4 Bacillus acidi lactici is initially identified as Lactobacillus plantarum
(Lactobacillus plantarum) (table 1).
The numbering of table 1 is LbThe morphological feature and physiological and biochemical test result of 4 Bacillus acidi lactici
Note：+, it is positive；-, it is negative；+ w, weakly positive.
3rd, the identification of Lactobacillus plantarum
It is L to take numberingbSupernatant, collects thalline, according to bacterium base are abandoned in the bacteria suspension of 4 Bacillus acidi lactici Liquid Culture, centrifugation
Because a group DNA extraction kit (Beijing Suo Laibao scientific ＆ technical corporation) methods described extracts genomic DNA, enter row agarose gel electrophoresis
(gel mass concentration ratio 0.8%, voltage 80V, electrophoresis 80min), with GelRed nucleic acid staining dyes after, in gel imaging system
Middle observation (Fig. 1).The genomic DNA that will be extracted dilutes 200 times, and concentration purity testing is carried out with ultraviolet specrophotometer.Using
Primer 2 7F：5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID No.1) and 1492R：5’-
GGCTACCTTGTTACGACTT-3 ' (SEQ ID No.2) enters performing PCR amplification (Fig. 2) to 16S rDNA genetic fragments, and amplification is produced
Thing is sequenced by Sangon Biotech (Shanghai) Co., Ltd. and Beijing Liuhe Huada Genomics Technology Co., Ltd, and will
Sequencing result (SEQ ID No.3) is compared with correlated series in Genbank.Result shows that numbering is Lb4 Bacillus acidi lactici
16S rDNA gene fragment orders reached with the similitude of Lactobacillus plantarum strain NBRC 15891
99%.
Therefore, numbering is Lb4 Bacillus acidi lactici is accredited as Lactobacillus plantarum (Lactobacillus plantarum), will
It is named as YS1, and is preserved in China typical culture collection center (abbreviation CCTCC, address on December 14th, 2016：Lake
No. 299 Wuhan Universitys of Bayi Road of Bei Sheng wuchang, wuhan area), deposit number is CCTCC NO：M2016747.
The prevention effect of embodiment 2, Lactobacillus plantarum YS1 to activated carbon inducing mouse constipation
Gastrointestinal tract mucous surface is adhered to by activated carbon by mouse stomach activated carbon, cause in alimentary canal under water content
Drop, digestive juice is reduced, and triggers gastrointestinal motility to slow down to form constipation.In to the early-stage Study of lactic acid bacteria constipation preventive effect, hair
A person of good sense confirms that first grain melena defecation time, level of MTL, Gas, ET, SS, AChE, SP, VIP etc. all can be used as evaluation lactic acid bacterias
The index of constipation prevention effect；Correlation mRNA expression can be more true in detecting colon by further molecular biology experiment
The physiological function of the confirmation lactic acid bacteria for cutting.
Main material, instrument and the experimental animal that the present embodiment is used are as follows：Lactobacillus plantarum YS1 (preserving numbers：CCTCC
NO：M2016747), lactobacillus bulgaricus (Chinese industrial Microbiological Culture Collection administrative center)；MTL、Gas、ET、SS、
AChE, SP and VIP (Beijing pul great achievement bio tech ltd)；Trizol reagents, OligodT18、RNase、dNTP、MLV
Reverse transcriptase (Invitrogen companies of the U.S.)；ROX reference Dye and SYBR Premix Ex Taq II (Japan
TAKARA companies)；RT-PCR primer (Beijing Tiangeng biochemical technology Co., Ltd).D550 digital cameras (CANON)；
SevenEasy pH meters (Mettler Toledo Inc. of Switzerland)；(U.S. Bole is public for iMark ELIASAs, T100 grads PCRs instrument
Department).Seven week old female KM mouse are purchased from Medical University Of Chongqing's Experimental Animal Center (credit number：SYXK (Chongqing) 2012-
0001), raise in temperature and humidity control environment (25 ± 2 DEG C of temperature, relative humidity 50 ± 5%), light dark cycles are adjusted per 12h, carry
For mouse standard feed and drinking-water.
First, the anti-hydrochloric acid in gastric juice abilities of Lactobacillus plantarum YS1 and cholate tolerance are detected
Lactic acid bacteria will play probiotic action, it is necessary under the strong acidic condition of stomach and enteron aisle, arrive at (typically
Large intestine) it is colonized and plays its physiological function.Therefore, in order to study the potential probiotic action of lactic acid bacteria, external virtual mould is set up
Type, the anti-hydrochloric acid in gastric juice ability and cholate tolerance for detecting lactic acid bacteria are important detection means.
It is 3.0 to be dissolved in distilled water and adjust pH with the HCl of 1mol/L NaCl and pepsin, reaches the quality of NaCl
Than being 0.2%, the mass ratio of pepsin is 0.35%, and vacuum filter removal bacterium is standby as simulated gastric fluid.By plant breast
Cultivated after bacillus YS1 activation, taken nutrient solution 5mL, 3000r/min centrifugation 10min, collects thalline, with 5mL physiological saline weights
After outstanding, mixed at 1: 9 by volume with simulated gastric fluid, 37 DEG C of culture 3h determine Lactobacillus plantarum YS1 viable counts during 0h and 3h,
Survival rate (%)=3h viable count (CFU/mL)/0hs of the Lactobacillus plantarum YS1 in the simulated gastric fluids of pH 3.0 is calculated by formula to live
Bacterium number (CFU/mL) × 100.
It is 0.0,0.3%, 0.5%, 1.0% that Lactobacillus plantarum YS1 is inoculated in containing mass ratio by 2% inoculum concentration
The MRS-THIO culture mediums of bovine bile, 37 DEG C of culture 24h, are control with the blank cultures of non-inoculated plant lactobacillus YS1,
Mensuration absorbance value at 600nm, survival rates (%) of the Lactobacillus plantarum YS1 in cholate=contain bile salt culture-medium is calculated by formula
OD600/ blank cultures OD600×100。
Result is as shown in table 2, survival rates of the Lactobacillus plantarum YS1 in the simulated gastric fluids of pH 3.0 and in various concentrations cholate
Lactobacillus bulgaricus is above, and survival rates of the Lactobacillus plantarum YS1 in the simulated gastric fluids of pH 3.0 is bulgarian milk bar
About 2.5 times of bacterium, and the survival rate in various concentrations cholate is about 10 times of lactobacillus bulgaricus, illustrates Lactobacillus plantarum
YS1 has the anti-hydrochloric acid in gastric juice ability and cholate tolerance more stronger than lactobacillus bulgaricus.
The anti-hydrochloric acid in gastric juice abilities of the Lactobacillus plantarum YS1 of table 2 and cholate tolerance are detected
2nd, prevention effects of the Lactobacillus plantarum YS1 to activated carbon inducing mouse constipation
Choose the kunming mice 100 of body weight about 25g, be equally divided into 5 groups, be respectively normal group, constipation control group, protect plus
Leah lactobacillus treatment group, Lactobacillus plantarum YS1 low concentrations treatment group and high concentration treatment group.After adapting to raise 1 week, normal group
Diet is normally freely taken in constipation control group mice and is drunk water 2 weeks；Except normally freely taking in diet and drinking-water in this is 2 weeks
Outward, lactobacillus bulgaricus treatment group mouse is daily every the 1.0 × 10 of gavage 2mL9The lactobacillus bulgaricus of CFU/mL, plants
Thing lactobacillus YS1 low concentrations treatment group and high concentration treatment group mouse are daily every the 1 × 10 of difference gavage 2mL8CFU/mL's
Lactobacillus plantarum YS1 and 1 × 109The Lactobacillus plantarum YS1 of CFU/mL.After 2 weeks in addition to normal group mouse, other 4 groups of mouse are every
(temperature is 4 DEG C to the activated carbon water of day gavage 2mL, and 10% activated carbon is added into the water containing 10% gum arabic in mass ratio
Suspension is made in solution), while lactic acid bacteria treatment each group continues gavage lactic acid bacteria, continue 3 days.The daily morning determines in experimentation
When determine body weight, dietary intake, amount of drinking water, stool weight and the excrement humidity of all mouse.
1st, influences of the Lactobacillus plantarum YS1 to Mouse Weight and excrement
In experimentation the timing of daily morning determine the body weight of all mouse, dietary intake, amount of drinking water, stool weight and
Excrement humidity.
Changes of weight is the important indicator that mouse occurs constipation.As seen from Figure 3, in first 2 weeks each group mouse body weight
Normal growth state is all showed, there is no notable difference between each group；After activated carbon inducing constipation, not by activated carbon at
The normal group Mouse Weight of reason continues to increase, and declining occurs in remaining each group Mouse Weight, wherein the body of constipation control group mice
It is reduced by up to again, the body weight of Lactobacillus plantarum YS1 high concentration treatment group mouse is significantly higher than the guarantor of same concentration closest to normal group
Plus Leah lactobacillus treatment group, illustrate that Lactobacillus plantarum YS1 can suppress the Mouse Weight decline that constipation is caused, and effect is better than
Lactobacillus bulgaricus.
Defecation state most can significantly embody the degree of constipation, stool weight, granule number and water content in constipation state
It is important excrement state index, these index decreaseds show the exacerbation of constipation degree.As shown in table 3, the 1-14 days it is each
Stool weight, granule number and the water content for organizing mouse are not significantly different from；After activated carbon inducing constipation, the 15-17 days normal groups
The stool weight of mouse, granule number and water content highest, constipation control group are minimum, lactobacillus bulgaricus and Lactobacillus plantarum
YS1 can stool weight that significantly relief of constipation is caused, granule number and water content decline (P ＜ 0.05), but Lactobacillus plantarum
, closest to normal group, the guarantor for being significantly higher than same concentration adds for the stool weight of YS1 high concentration treatment group mouse, granule number and water content
Leah lactobacillus treatment group, illustrates that Lactobacillus plantarum YS1 can suppress the stool in mice weight that constipation is caused, granule number and aqueous
Amount declines, and effect is better than lactobacillus bulgaricus.
Influences of the Lactobacillus plantarum YS1 of table 3 to stool in mice
Note：Alphabetical different expression group differences are significantly (P ＜ 0.05).
2nd, influences of the Lactobacillus plantarum YS1 to activated carbon propulsion rate and first grain melena efflux time
To implementing jejunitas 24h including all mouse including normal group after 17th day gavage activated carbon water, but still give mouse
Drinking-water is freely taken in, the activated carbon water of every gavage 0.2mL of all mouse after 24h；Every group of half mouse (10) is real after 30min
Apply disconnected neck to put to death, take that mice plasma is standby, while taking propulsion rate of the small intestine observation activated carbon in mouse small intestine, propulsion rate (%)
=activated carbon advances length/total small intestinal length × 100；Every group of remaining 10 mouse continue to observe its first grain melena of discharge when
Between.
Constipation causes the reduction of intestines peristalsis frequency, and the time that excrement rests on enteron aisle increases, and causes harmful bacteria with excrement
For food is continuously bred, so as to threaten intestinal health, constipation is aggravated.Activated carbon propulsion in small intestine after activated carbon inducing mouse constipation
Length and activated carbon propulsion rate can be as the indexs for evaluating small intestine activity and constipation degree.As can be seen from Table 4, activated carbon is lured
After leading constipation, lactobacillus bulgaricus and Lactobacillus plantarum YS1 can promote propulsion length of the activated carbon in mouse small intestine,
But the activated carbon propulsion rate highest of Lactobacillus plantarum YS1 high concentration treatment groups, is significantly higher than the treatment of Lactobacillus plantarum YS1 low concentrations
Group and the lactobacillus bulgaricus treatment group (P ＜ 0.05) with concentration, illustrate that Lactobacillus plantarum YS1 can improve activated carbon small
Propulsion rate in mouse small intestine, and effect is better than lactobacillus bulgaricus.
The influence that the Lactobacillus plantarum YS1 of table 4 is advanced to mice with constipation intestines and stomach
Note：Alphabetical different expression group differences are significantly (P ＜ 0.05).
Constipation causes intestinal movement to slow down, and excrement holdup time in enteron aisle increases, and lengthens first grain melena efflux time,
Shorter first grain melena efflux time means that intestinal movement is more normal.As seen from Figure 4, the first grain melena discharge of normal group
Time is most short (88 ± 7min), and the first grain melena efflux time of constipation control group is (231 ± 33min) most long, Lactobacillus plantarum YS1
The first grain melena efflux time (132 ± 16min) of high concentration treatment group is considerably shorter than Lactobacillus plantarum YS1 low concentration treatment groups
(163 ± 22min) and the lactobacillus bulgaricus treatment group (175 ± 27min) (P ＜ 0.05) with concentration, illustrates plant breast bar
Bacterium YS1 can reduce the first grain melena efflux time of mice with constipation, add better than guarantor with good constipation mitigation, and effect
Leah lactobacillus.
3rd, influences of the Lactobacillus plantarum YS1 to mice serum MTL, Gas, ET, SS, AChE, SP and VIP level
The mice plasma of collection is taken, 4500rmp centrifugation 15min separate serum, by kit time-and-motion study mice serum
MTL, Gas, ET, SS, AChE, SP and VIP level.MTL can stimulate the generation of pepsin and promote intestinal movement；Gas energy
Promote gastrointestinal secretion and gastrointestinal motility, promote pylorus to relax, play a part of relief of constipation；ET is in antiotasis stability and dimension
Hold and play an important role in basic cardiovascular system；SS can stimulate intestinal movement；AChE can be with condition contraction of muscle and mucus point
Secrete, loosening all muscles can be made so as to advance excrement to discharge；SP is a kind of material for contributing to intestines peristalsis；VIP is kept in intestinal wall
In normal contents be also to stablize the important means of function of intestinal canal.
As can be seen from Table 5, the horizontal highest of MTL, Gas, ET, AChE, SP and VIP of normal group mice serum, SS levels
It is minimum；Constipation control group mice shows opposite trend；Lactobacillus bulgaricus and Lactobacillus plantarum YS1 can make above-mentioned
The serum levels of material are close to normal group level, but the effect of Lactobacillus plantarum YS1 is more notable, illustrate that Lactobacillus plantarum YS1 can
So that MT in serum L, Gas of mice with constipation, ET, SS, AChE, SP and VIP level are tried one's best, holding is normal, so that relief of constipation.
Influences of the Lactobacillus plantarum YS1 of table 5 to mice serum MTL, Gas, ET, SS, AChE, SP and VIP level
Note：Alphabetical different expression group differences are significantly (P ＜ 0.05).
4th, the influence that Lactobacillus plantarum YS1 is expressed mouse small intestine tissue mRNA
The small intestine of mouse is taken, colon's total serum IgE is extracted with RNAzol after crushing, be diluted to 1 μ g/ μ L；Take 2 μ L dilute
Total RNAs extraction liquid after releasing, sequentially adds oligodT18, RNase, dNTP, MLV enzyme of 1 μ L and the 5 × buffer of 10 μ L, 37
DEG C 120min, 99 DEG C of 4min, 4 DEG C of 3min synthesis cDNA；Then use primer described in table 2, RT-PCR amplification c-Kit, SCF,
The mRNA expression of TRPV1, GDNF and NOS, using housekeeping gene GAPDH as internal reference；Finally with containing the bromination that mass ratio is 1%
The agar electrophoresis of second ingot checks pcr amplification product.
Table 6RT-PCR primer sequences
ICC (Cajal interstitial cells) in constipation patient enteron aisle is less, and c-Kit is the Specific marker of ICC, is ICC
The key of propagation.Reproduction of the SCF concentration to ICC is extremely important, and ICC can not be grown in the environment of SCF does not exist.Document report
Road, the ICC contents in mice with constipation colon are less, and the c-Kit expressions in colon also decline.Lactobacillus plantarum
YS1 is shown in Fig. 5 to the influence that mouse small intestine tissue c-Kit and SCF mRNA are expressed, in constipation control group mice small intestine c-Kit and
SCF mRNA expression declines, and Lactobacillus plantarum YS1 can significantly raise c-Kit the and SCF mRNA expression in mouse small intestine, and
Acted on during high concentration more obvious.
TRPV1 it is verified that have a substantial connection with defecation, activation TRPV1 can trigger the release of neurotransmitter so that
Cause intestinal motility obstacle.TRPV1 expression increases are a notable phenomenons of damage of intestines, because gastrointestinal disorder causes enteron aisle to damage
Wound, makes constipation patient have TRPV1 higher to express.GDNF can adjust the function of gangliocyte, so as to contribute to reparation to receive
The enteron aisle of damage, can prevent constipation.NOS plays an important role to Gastrointestinal motility adjustment.The increase of NO can cause more serious colon to move
Power obstacle, control NOS can reduce the content of NO, be the feasible way for controlling constipation.Lactobacillus plantarum YS1 is to mouse small intestine group
Fig. 6 is shown in the influence for knitting middle TRPV1, GDNF and NOS mRNA expression, and there is normal group mouse highest GDNF mRNA to express and most
Low TRPV1, NOS mRNA expression, and the GDNF mRNA of constipation control group mice express minimum, TRPV1 and NOS mRNA tables
Up to highest, relative to constipation control group, lactobacillus bulgaricus and Lactobacillus plantarum YS1 can raise GDNF mRNA expression
Expressed with TRPV1, NOS mRNA is lowered, but the effect of Lactobacillus plantarum YS1 is substantially better than lactobacillus bulgaricus.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although by ginseng
According to the preferred embodiments of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can
To make various changes to it in the form and details, without departing from the present invention that appended claims are limited
Spirit and scope.
<110>The college of education of Chongqing second；Tan Fang
<120>A kind of Lactobacillus plantarum and its application in the food for preparing Constipation
<160> 15
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 2 7F
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 1492R
<400> 2
ggctaccttg ttacgactt 19
<210> 3
<211> 1472
<212> DNA
<213>Lactobacillus plantarum YS1（Lactobacillus plantarum YS1）
<220>
<223>16S rDNA genetic fragments
<400> 3
gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctgga ttgattggtg 60
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gcccagaagc gggggataac acctggaaac agatgctata ccgcataaca acttggaccg 180
catggtccga gnttgaaaga tggcttcggc tatcactttt ggatggtccc gcggcgtatt 240
agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc tgagagggta 300
atcggccaca ttgggactga gacacggcca aactcctacg ggaggcagca gtagggaatc 360
ttccacaatg gacgaagtct gatggagcaa cgccgcgtga gtgaaaaggg tttcggctcg 420
taaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480
taacagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540
ttgtccggat ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag 600
ccttcggctc aaccgaagaa gtgcatcgga aatgggaaac ttgagtgcag aagaggacag 660
tggaactcca ttgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 720
gctgtctggt ctgtaactga cgctgaggct cgaaagtatg gtagcaaaca ggattagata 780
ccctggtatc cataccgtaa acgatgaatg ctaagtgttg gagggtttcc gcccttcagt 840
gctgcagcta acgcattaag cattccgcct ggggagtacg gccgcaaggc tgaaactcaa 900
aggaattacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 960
gaaccttacc aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg 1020
acatggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttagtc 1080
ccgcacgagc gcaaccctta ttatcagttg ccagcattaa gttgggcact ctggtgagac 1140
tgccggtgac aaacggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200
gggctacaca cgtgctacaa tggatggtac aacagttgcg aactcgcgag agtaagctaa 1260
tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1320
tcgctagtaa tccggatcag catgccgcgg tgaatacgtt cccgggcctt gacacaccgc 1380
ccgtcacacc atgagagttt gtaacaccca aagtcggtgg ggtaaccttt taggaaccag 1440
ccgcctaagg gggacagatg attagggtga ag 1472
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification c-Kit mRNA expression
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agaccgaacg caactt 16
<210> 5
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification c-Kit mRNA expression
<400> 5
ggtgccatcc acttca 16
<210> 6
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification SCF mRNA expression
<400> 6
aaactggtgg cgaatc 16
<210> 7
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification SCF mRNA expression
<400> 7
cacgggtagc aagaac 16
<210> 8
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<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification TRPV1 mRNA expression
<400> 8
agcgagttca aagacccaga 20
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<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification TRPV1 mRNA expression
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ttctccacca agagggtcac 20
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<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification GDNF mRNA expression
<400> 10
ttttattcaa gccaccatc 19
<210> 11
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification GDNF mRNA expression
<400> 11
agcccaaacc caagtca 17
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<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification NOS mRNA expression
<400> 12
ccacatctgg caggatgaga a 21
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification NOS mRNA expression
<400> 13
aggcacagaa ctgagggtac a 21
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>The sense primer of RT-PCR amplification GAPDH mRNA expression
<400> 14
cggagtcaac ggatttggtc 20
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<211> 20
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<213>Artificial sequence
<220>
<223>The anti-sense primer of RT-PCR amplification GAPDH mRNA expression
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agccttctcc atggtcgtga 20
Claims (2)
1. Lactobacillus plantarum YS1 (Lactobacillus plantarum YS1), deposit number is CCTCC NO：M2016747.
2. applications of the Lactobacillus plantarum YS1 described in claim 1 in the food for preparing Constipation.
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| CN107099491A (en) * | 2017-07-05 | 2017-08-29 | 重庆第二师范学院 | Lactobacillus plantarum YS3 and its application in the food of Constipation is prepared |
| CN107267416A (en) * | 2017-07-05 | 2017-10-20 | 重庆第二师范学院 | Lactobacillus plantarum YS2 and its application in the food of Constipation is prepared |
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| CN109700033A (en) * | 2019-03-04 | 2019-05-03 | 山东环亿生物科技有限公司 | A kind of adjusting enteron aisle, probiotic composition of prevent constipation and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099491A (en) * | 2017-07-05 | 2017-08-29 | 重庆第二师范学院 | Lactobacillus plantarum YS3 and its application in the food of Constipation is prepared |
| CN107267416A (en) * | 2017-07-05 | 2017-10-20 | 重庆第二师范学院 | Lactobacillus plantarum YS2 and its application in the food of Constipation is prepared |
| CN107723256A (en) * | 2017-09-01 | 2018-02-23 | 杭州娃哈哈科技有限公司 | One lactobacillus plantarum new strains and its application |
| CN109700033A (en) * | 2019-03-04 | 2019-05-03 | 山东环亿生物科技有限公司 | A kind of adjusting enteron aisle, probiotic composition of prevent constipation and preparation method thereof |
| CN109700033B (en) * | 2019-03-04 | 2021-06-04 | 山东环亿生物科技有限公司 | Probiotic composition for regulating intestinal tract and preventing constipation and preparation method thereof |
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| CN106906165B (en) | 2020-07-24 |
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