CN110699271A

CN110699271A - Lactobacillus plantarum CQPC02 and application thereof in preparation of food for improving constipation

Info

Publication number: CN110699271A
Application number: CN201810751933.2A
Authority: CN
Inventors: 赵欣; 杜木英; 周先容
Original assignee: Chongqing University of Education
Current assignee: Thankcome Biotechnology Suzhou Co ltd
Priority date: 2018-07-10
Filing date: 2018-07-10
Publication date: 2020-01-17
Anticipated expiration: 2038-07-10
Also published as: CN110699271B

Abstract

The invention discloses Lactobacillus plantarum CQPC02 with the preservation number of CGMCC NO.14491 and application thereof in preparing food for improving constipation, which not only expands the application range of the Lactobacillus plantarum CQPC02 and improves the application value thereof, but also brings new hope for improving the constipation.

Description

Lactobacillus plantarum CQPC02 and application thereof in preparation of food for improving constipation

Technical Field

The invention belongs to the technical field of microorganisms, and relates to lactobacillus plantarum and application thereof in preparation of food.

Background

The production process of Sichuan pickle comprises cleaning fresh pickle, sealing pickle in jar, and anaerobically fermenting soaked pickle in saline water. The pickle water contains abundant natural lactic acid bacteria, and plays a key role in forming the flavor and the quality of the pickle. It uses soluble components (mainly sugar and nitrogen-containing substances) to proliferate, generate acidic substances and metabolize flavor components, so that the pickle has unique sour and crisp taste. The microorganism contained in the pickle water mainly comprises lactobacillus plantarum, L.brevis, lactobacillus casei, fermentation yeast, lactobacillus acidophilus and the like. The difference in the types of lactic acid bacteria may be caused by factors such as regions, climate and production process habits. Certain lactic acid bacteria are also used as probiotics and have various benefits to human health, including improving constipation, colitis, weight loss, etc. In order to better utilize the microbial resources, more extensive separation and identification work should be carried out, abundant strain resources are accumulated, and abundant industrial probiotic species are developed.

Disclosure of Invention

The invention aims to separate and identify microorganisms in pickle water and research the activity and application of the microorganisms.

Through research, the invention provides the following technical scheme:

1. lactobacillus plantarum (Lactobacillus plantarum) CQPC02 with the preservation number of CGMCC NO. 14491.

2. Application of lactobacillus plantarum CQPC02 in preparing food for improving constipation.

Further, the food is a fermented beverage.

Further, the food product is fermented soymilk.

3. A beverage is prepared by fermenting Lactobacillus plantarum CQPC 02.

Further, the beverage is soy milk.

4. A method for preparing soybean milk by fermenting Lactobacillus plantarum CQPC02 comprises the following steps: soaking soybean in water 3 times of the soybean for 12 hr, grinding, filtering, and concentrating to 10%⁵CFU/mL inoculated with Lactobacillus plantarum CQPC02, fermented at 37 ℃ for 12 hours.

The invention separates and identifies the microorganisms in the pickle water, one of the Lactobacillus plantarum is named as CQPC02, and the Lactobacillus plantarum is preserved in China general microorganism center of microorganism culture preservation management committee (CGMCC for short, address: No.3 Xilu No.1 Beichen of the sunward area in Beijing city) in 8-4 months in 2017, and the preservation number is CGMCC NO. 14491.

The High Performance Liquid Chromatography (HPLC) assay showed that the total content of soy isoflavones in the Lactobacillus plantarum CQPC02 fermented soymilk (LP-CQPC02-FSM) was highest and contained more free glycosides (daidzein, glycitein, genistein) than the unfermented soymilk (U-FSM) and the Lactobacillus bulgaricus fermented soymilk (LB-FSM).

The results of the experiments of LP-CQPC02-FSM on charcoal-induced constipation mice show that: compared with a model group, the LP-CQPC02-FSM can promote intestinal peristalsis, improve the small intestine propulsion rate, shorten the first-grain black stool time, obviously improve the serum MTL (motilin), Gas (gastrin), ET (endothelin), AChE (acetylcholinesterase), SP (substance P) and VIP (vasoactive intestinal peptide) levels of a constipated mouse, obviously reduce the SS (somatostatin) level, increase the mRNA expression of c-Kit, SCF, GDNF and AQP9 in the small intestine of the constipated mouse, reduce the mRNA expression of TRPV1 and AQP3, has a good improvement effect on constipation, and is obviously superior to U-FSM and LB-FSM.

The invention has the beneficial effects that: the invention provides a lactobacillus plantarum CQPC02 which can be used for preparing food for improving constipation, such as fermented beverage (such as fermented soybean milk), can effectively inhibit constipation, has better effect than the commonly used lactobacillus bulgaricus, not only expands the application range of the lactobacillus plantarum CQPC02 and improves the application value thereof, but also brings new hope for improving constipation.

Drawings

FIG. 1 shows the colony morphology of Lactobacillus plantarum CQPC 02.

FIG. 2 shows the gram-stained Lactobacillus plantarum CQPC 02.

FIG. 3 is an agarose gel electrophoresis of the 16S rDNA PCR amplification product of Lactobacillus plantarum CQPC02, wherein M is a DNA molecular weight standard, 0 is a negative control, and 1 is Lactobacillus plantarum CQPC 02.

FIG. 4 is a HPLC method for determining soybean isoflavone content in soybean milk, wherein A is mixed standard solution, B is-FSM), C is LB-FSM, and D is LP-CQPC 02-FSM; the chromatographic peaks No. 1-6 are daidzin, glycitin, genistin, daidzein, glycitein and genistein in sequence.

FIG. 5 shows the first black stool time of the mice,^a-eindicating that there is a significant difference (p) between the groups<0.05)。

FIG. 6 is a histopathological section of mouse small intestine.

FIG. 7 shows c-The level of mRNA expression of Kit and SCF,^a-eindicating that there is a significant difference (p) between the groups<0.05)。

FIG. 8 is a graph of TRPV1 and GDNF mRNA expression levels in mouse small intestine,^a-eindicating that there is a significant difference (p) between the groups<0.05)。

FIG. 9 is a graph showing AQP3 and AQP9mRNA expression levels in mouse small intestine,^a-eindicating that there is a significant difference (p) between the groups<0.05)。

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

First, the separation and identification of Lactobacillus plantarum CQPC02

1. Experimental Material

The method comprises the steps of collecting 6 parts of pickle water obtained by natural fermentation of farmers in the southern shore area of Chongqing, respectively sucking 40mL of pickle water, putting the pickle water into a sterile centrifuge tube, putting the pickle water into a food sampling box, and storing the pickle water in a laboratory refrigerator at 4 ℃ for later use.

2. Separation and purification of lactic acid bacteria

Respectively taking 1mL of sauerkraut water sample, and performing 10-fold gradient dilution to 10 with sterile physiological saline water^-6Then take 10 out^-4、10^-5、10^-6The 3 gradients of bacteria solution 100 u L plate coating, 37 degrees C culture 24-48h, observed and recorded colony morphology. And selecting colonies with different forms on the plate for streaking separation, culturing at 37 ℃ for 48h, then selecting single colonies with different forms on the plate again for streaking separation, and repeating the steps for 2 to 3 times until pure single colonies with consistent forms are obtained.

The colony morphology of the strain CQPC02 is shown in FIG. 1, and the colony is mostly white or milky white, round in shape, neat in edge, and moist and smooth in surface.

3. Preliminary identification of lactic acid bacteria

The pure colonies on the plate were picked and inoculated in 5mL MRS liquid medium and cultured at 37 ℃ for 24 h. And (3) putting 1mL of the culture medium containing the bacteria into a sterile centrifuge tube, centrifuging for 10min at 4000r/min, removing an upper culture medium, suspending the thallus precipitate in sterile normal saline, performing gram stain microscopy, and preliminarily identifying the positive thallus precipitate as the lactobacillus.

The strain with the number of CQPC02 shows positive gram staining, and under 100 times of oil lens, the cell morphology of the strain is shown in figure 2, the cell morphology has long rods and short rods, and no budding is existed.

4. Lactic acid bacteria DNA extraction

Inoculating the purified suspected target strain into MRS broth, culturing at 37 ℃ for 18-24h, and extracting DNA by using a bacterial genome DNA extraction kit. The extracted DNA was stored in a freezer at-20 ℃ for further use.

5. PCR amplification and agarose gel electrophoresis detection of genome DNA

The extracted DNA was used to amplify 16S rDNA by PCR, wherein 1. mu.L of the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No.1), 1. mu.L of the reverse primer 1495R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID No.2), 12.5. mu.L of 2 XTaq plus Buffer, and 1. mu.L of the template DNA were used as the primer₂O make up the system to 25. mu.L. And sterile ultrapure water was used as a negative control instead of the template DNA. The amplification conditions were: 5min at 94 ℃; 29 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; finally, extension is carried out for 5min at 72 ℃. Then 5 mul of amplification product is taken to carry out agarose gel electrophoresis detection, the agarose concentration is 1.5%, the electrophoresis condition is 110V, and 45 min.

The agarose gel electrophoresis detection result of the 16S rDNA amplification product of the strain with the number of CQPC02 is shown in figure 3, and a lane of a negative control group has no band, which indicates that the strain is not polluted in the PCR amplification process; the lane of the strain numbered CQPC02 has a band of about 1500bp in length, corresponding to the expected length of the amplified fragment.

The 16S rDNA amplification product of the strain with the serial number of CQPC02 is subjected to sequencing by Beijing Okagaku Biotechnology Co., Ltd, and the sequence is shown as SEQ ID No. 3. The alignment analysis of the sequences using the BLAST (basic Local alignment search tool) program in NCBI revealed that the strain numbered CQPC02 was Lactobacillus plantarum (Lactobacillus plantarum) among lactic acid bacteria, which has 99% homology to known lactic acid bacteria in the Gene Bank database.

6. In vitro resistance screening of lactic acid bacteria

(1) Capacity to tolerate 0.3% bile salts

Adding pig bile salt into MRS-THIO culture medium (MRS broth containing 0.2% sodium thioglycolate) to make its concentration be 0.3%, and sterilizing at 121 deg.C for 15 min; inoculating activated 5mL strain into MRS-THIO culture medium containing no bile salt (0.0%) and MRS-THIO culture medium containing 0.3% bile salt (2% (v/v)), respectively, culturing at 37 deg.C for 24 hr with blank culture medium (MRS-THIO culture medium without inoculated strain), and respectively determining OD of the culture medium with different concentrations_600nmThe tolerance of the strain to bile salts was calculated according to equation (1):

the result shows that the survival rate of the strain with the number of CQPC02 in 0.3% of bile salt is 17.3 +/-0.19%, and the strain has stronger bile salt tolerance capability.

(2) Simulated gastric fluid resistance test

Preparing artificial gastric juice: consists of 0.2 percent of NaCl and 0.35 percent of pepsin, the pH value is adjusted to 3.0 by 1mol/L of HCl, and then the mixture is filtered and sterilized by a filter membrane with the pore diameter of 0.22 mu m for standby.

Sucking 5mL of cultured bacteria-containing culture medium in a super-clean workbench, centrifuging for 10min at 3000r/min in a 10mL sterile centrifuge tube, removing an upper layer culture medium, collecting thalli, adding equal volume (5mL) of sterile normal saline, uniformly mixing to prepare a bacterial suspension, then uniformly mixing 1mL of bacterial suspension with 9mL of artificial gastric juice with the pH of 3.0, treating 1mL of the mixed solution as the artificial gastric juice for 0h, and culturing the rest 9mL of the mixed solution in a constant-temperature water bath shaker (37 ℃, 150r/min) for 3 h. The samples of 0h and 3h are respectively diluted by 10 times of gradient, the viable count is determined by selecting proper gradient and adopting a plate coating method, the samples are cultured for 48h at 37 ℃ on an MRS solid culture medium, and the survival rate (%) is calculated according to a formula (2).

The result shows that the survival rate of the strain with the number of CQPC02 in artificial gastric juice with the pH value of 3.0 is 92.06 +/-6.91%, and the strain has stronger gastric acid resistance.

Secondly, lactobacillus plantarum CQPC02 fermented soymilk

Soaking semen glycines (1kg) in water 3 times the weight of semen glycines for 12 hr, grinding into slurry, filtering, and concentrating to 10% respectively⁵CFU/mL inoculated with Lactobacillus plantarum CQPC02 and Lactobacillus bulgaricus, fermented at 37 deg.C for 12h to obtain Lactobacillus plantarum CQPC02 fermented soybean milk (LP-CQPC02-FSM) and Lactobacillus bulgaricus fermented soybean milk (LB-FSM). Unfermented soymilk (U-FSM) was also used as a control.

Third, HPLC method is used for determining the content of soybean isoflavone in the soymilk

The soybean contains soybean isoflavone substances with intestinal tract benefiting function besides common components such as protein. After fermentation with lactic acid bacteria, the bound soy isoflavones present in the soy milk are converted to free soy isoflavones which are more readily digested and absorbed.

1. Chromatographic conditions

A chromatographic column: pntulis QS-C18 chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase A: acetonitrile; mobile phase B: 0.1% o phosphoric acid; detection wavelength: 254 nm; column temperature: 40 ℃; flow rate: 1 mL/min; sample introduction amount: 10 μ L. The gradient elution mobile phase ratios are shown in table 1.

TABLE 1 gradient elution mobile phase ratio

2. Preparation of Standard solutions

Respectively and precisely weighing 20mg of daidzin, 20mg of glycitin, 20mg of genistin, 20mg of daidzein, 20mg of glycitein and 20mg of genistein in a 20mL volumetric flask, dissolving with 80% methanol and diluting to scale to obtain a single standard stock solution.

Diluting each single standard stock solution by 6, 12, 24, 36 and 48 times respectively to prepare single standard solutions with series concentrations.

1mL of each of 6 single standard stock solutions is taken in a 10mL volumetric flask, and diluted to the scale with 80% methanol to serve as a mixed standard solution.

3. Experiment of system applicability

Precisely absorbing 6 single standard solutions, injecting into a liquid chromatograph, recording chromatogram, and determining retention time of daidzin, glycitin, genistin, daidzein, glycitein, and genistein as 16.899, 18.298, 26.582, 39.483, 41.325, and 48.525 min. And then precisely absorbing the mixed standard solution, injecting the mixed standard solution into a liquid chromatograph, recording a chromatogram (see fig. 4A), determining that the No.1 to No. 6 chromatographic peaks in the chromatogram are daidzin, glycitin, genistin, daidzein, glycitein and genistein in sequence according to retention time and the maximum absorption wavelength displayed by an ultraviolet spectrum, wherein the separation degree between adjacent peaks meets the requirement.

4. Preparation of test solution

Respectively taking U-FSM, LB-FSM and LP-CQPC02-FSM 2mL in a 50mL volumetric flask, adding 80% methanol to the near scale, performing ultrasonic treatment at 50 ℃ for 1h, diluting with 80% methanol to the scale, mixing uniformly, centrifuging at 9000r/min for 15min, taking supernatant, and filtering with a 0.45 mu m filter membrane to obtain a test solution.

5. Drawing of standard curve

Precisely absorbing 5 mu L of single standard solution with a series of concentrations respectively, injecting the single standard solution into a liquid chromatograph, recording a chromatogram, and performing linear regression on the sample amount (X) by using a peak area (Y). The regression equation for the 6 soybean isoflavone standards is shown in table 2.

TABLE 26 regression equation for soy isoflavone standards

6. Measurement of test solution

Precisely absorbing 5 mu L of the test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of 6 soybean isoflavones in the soybean milk by using the peak area according to a regression equation listed in a table 2. The chromatograms of U-FSM, LB-FSM and LP-CQPC02-FSM are shown in FIGS. 4B, 4C and 4D, respectively, and it can be seen that U-FSM and LB-FSM contain daidzin, glycitin, genistin, daidzein and genistein, and that LP-CQPC02-FSM contains glycitein in addition to the above five isoflavones. The results of the calculation of the total isoflavone content in U-FSM, LB-FSM and LP-CQPC02-FSM are shown in Table 3, and it can be seen that LP-CQPC02-FSM has the highest total isoflavone content and contains more free glycosides (daidzein, glycitein, genistein) and less bound glycosides (daidzin, glycitin, genistin) compared to U-FSM and LB-FSM. From these results, it was seen that lactobacillus plantarum CQPC02 produced more active soy isoflavones during the fermentation of soy milk than lactobacillus bulgaricus.

TABLE 3 Soy milk isoflavone content (. mu.g/mL)

Fourth, improving effect of lactobacillus plantarum CQPC02 fermented soymilk on constipation model mice

1. Laboratory animal

SPF grade 6 week old female Kunming mice, 50, were purchased from Chongqing university of medicine laboratory animal center. The animals were kept in a standardized laboratory at room temperature of 25 + -2 deg.C and relative humidity of 50 + -5% for 12h light/12 h dark, and the experiment was started after one week of acclimatization.

2. Experimental methods

One week after adaptive feeding of 50 mice, the mice were randomly divided into 5 groups of 10, namely a normal group, a model group, a U-FSM group, an LB-FSM group and an LP-CQPC02-FSM group. The whole experiment period is 9 days, the normal group and the model group are respectively irrigated with normal saline every day, and the U-FSM group, the LB-FSM group and the LP-CQPC02-FSM are irrigated with U-FSM, LB-FSM and LP-CQPC02-FSM for 4 times every day, and the total volume is 2 mL; from day 7 to day 9, the mice of each group were gavaged with 0.2mL of 10% charcoal ice water every day, except for the normal group.

3. Calculation of first-particle stool time and small intestine propulsion rate

After the gavage is completed on day 9, all mice are fasted and are not forbidden to be watered for 24 hours, all mice are gavaged with 0.2mL of 10% activated carbon ice water on day 10, then each group of mice is divided into two groups, and the time for discharging the first black excrement of 5 mice is observed from the start of the gavage of the activated carbon ice water; the remaining 5 mice were sacrificed after 30min in the gavage activated carbon ice water and plasma was collected for future use, the small intestine portion from the pylorus, down to the ileocecal portion was taken, the length of the small intestine and the advancing distance of activated carbon in the small intestine were measured, and the small intestine advancing rate was calculated according to equation (3).

The first black stool time is an important index for evaluating the severity of constipation, and when constipation symptoms occur, the intestinal peristalsis is slowed down, and the retention time of the feces in the intestinal tract is prolonged. The first black stool time of each group of mice is shown in fig. 5, and the first black stool time of the model group of mice is the longest and is significantly higher than that of the normal group of mice (p < 0.05); after the U-FSM, the LB-FSM and the LP-CQPC02-FSM are respectively gavaged, although the first-grain black stool time of the mice is higher than that of a normal group, the mice have significant difference compared with a model group (p is less than 0.05); the first black stool time of LP-CQPC02-FSM gavage mice was significantly lower (p <0.05) than the other two soymilk (U-FSM and LB-FSM).

The influence of the soymilk on the intestinal propulsion rate of the mice with constipation induced by the activated carbon is shown in table 4, and the lengths of the small intestines of the mice in each group have no significant difference, which indicates that the molding of the activated carbon does not influence the length of the small intestine; the rate of intestinal transit was lowest in the model group mice, significantly lower than in the normal group (p < 0.05); after the mice with constipation are subjected to gastric lavage by U-FSM, LB-FSM and LP-CQPC02-FSM, the intestinal propulsion rate is remarkably improved (p is less than 0.05) compared with that of a model group, wherein the intestinal propulsion rate of the LP-CQPC02-FSM group is closest to that of a normal group, which shows that the LP-CQPC02-FSM can promote intestinal peristalsis, accelerate the propelling speed of activated carbon in the intestines and reduce the retention time of the activated carbon in the intestines, so that the constipation is improved to a certain extent.

TABLE 4 influence of soymilk on the intestinal motility of mice with constipation induced by activated carbon

^a-eIndicating that there is a significant difference (p) between the groups<0.05)。

4. Observation of small intestine histopathology section

Taking mouse small intestine tissue of about 0.5cm, immediately placing the mouse small intestine tissue in 10% formalin solution for fixation for 48h, dehydrating, transparentizing, waxing, embedding, slicing, performing HE staining, and observing the morphological change of the tissue under an optical microscope.

When the villi of the small intestine are damaged, the peristalsis function of the intestine is influenced to different degrees, and the slow peristalsis of the intestine is one of factors causing constipation, so the integrity of the villi of the small intestine has significance for evaluating the constipation. As shown in FIG. 6, the histopathological section observation of the small intestine of each group of mice shows that the small intestine villi of the normal group of mice are arranged regularly and uniformly, and the phenomena of fracture or shrinkage do not exist, while the small intestine villi of the model group of mice are seriously fractured and shrunk, and the goblet cells are incomplete; although the intestinal villi of the LP-CQPC02-FSM group and the U-FSM group and the LB-FSM group are also shrunk and broken to some extent, the intestinal villi are obviously more complete than those of the model group mice, and the intestinal villi of the LP-CQPC02-FSM group mice are almost consistent with those of the normal group mice.

5. Determination of MTL, Gas, ET, SS, AChE, SP and VIP levels in serum

Mouse plasma was collected and centrifuged at 4000r/min at 4 ℃ for 10min, and the supernatant was collected and their levels in serum were determined separately and exactly according to the kit instructions for MTL, Gas, ET, SS, AChE, SP and VIP.

The measurement results of the serum MTL, Gas, ET, SS, AChE, SP and VIP levels of each group of mice are shown in the table 5, and compared with the other four groups, the serum MTL, Gas, ET, AChE, SP and VIP levels of the mice in the normal group are the highest, and the SS level is the lowest; the model group showed the opposite trend, with highest SS levels in serum and lowest MTL, Gas, ET, AChE, SP and VIP levels; compared with the model group, the serum levels of MTL, Gas, ET, AChE, SP and VIP in mice of the LP-CQPC02-FSM group are obviously improved (p <0.05), and the SS level is obviously reduced (p < 0.05).

TABLE 5 mouse serum MTL, Gas, ET, SS, AChE, SP and VIP levels (pg/mL)

Some constipation patients have been reported to have altered neurotransmitter levels (e.g., MTL, Gas, ET, SS, AChE, SP, and VIP). MTL is an important index for evaluating gastrointestinal motility in recent years, and most scholars consider that MTL can promote movement of various parts of the gastrointestinal tract, and the decrease of MTL release can reduce gastrointestinal motility. Gas is an important gastrointestinal hormone which can promote gastric secretion, increase gastrointestinal motility, accelerate gastric emptying and simultaneously promote pyloric sphincter relaxation. ET is a multifunctional peptide that plays an important role in cardiovascular and intestinal function. Ach (acetylcholine) is currently considered as one of two neurotransmitters playing an important role in intestinal motility, and plays a role in promoting gastrointestinal motility by binding to its receptor. In general, AChE levels are positively correlated with Ach levels. SP is an excitatory transmitter of gastrointestinal motor neurons. It strongly promotes contraction of smooth muscles of the digestive tract, stimulates secretion of water and electrolytes from the mucous membranes of the small intestine and colon, and promotes gastrointestinal motility. VIP stimulates intestinal motility, thereby promoting gastrointestinal motility. The above experimental results show that the levels of MTL, Gas, ET, AChE, SP and VIP in the serum of the model group mice are significantly lower than those of the normal group, while the LP-CQPC02-FSM group can significantly increase the levels of these neurotransmitters, indicating that constipation occurs in association with the decrease in the levels of MTL, Gas, ET, AChE, SP and VIP, and the LP-CQPC02-FSM can increase the levels of these neurotransmitters and play a role in relieving constipation.

SS inhibits the release of gastrointestinal hormones, and also reduces the rate of gastric emptying and smooth muscle contraction, all of which can lead to constipation. The above experimental results show that the model group has the highest SS level, and the LP-CQPC02-FSM group has significantly reduced SS level (p <0.05), which means that LP-CQPC02-FSM has certain improvement effect on constipation.

6. Measurement of C-Kit, SCF, TRPV1, GDNF, AQP3 and AQP9mRNA expression levels in the Small intestine

Extracted according to the instructions of Trizol (Invitrogen Co.)Total RNA in the small intestine was purified and concentrated using a ultramicro-spectrophotometer, and the RNA concentration in each sample was adjusted to the same level (1. mu.g/. mu.L). Then 1. mu.L of 1. mu.g/. mu.L RNA sample was taken, 1. mu.L (oligo) primer dT and 10. mu.L sterile ultrapure water were added, mixed, reacted at 65 ℃ for 5min, further added with 1. mu.L Riblolock RNase Inhibitor, 2. mu.L 100mM dNTP mix, 4. mu.L 5 × Reaction buffer and 1. mu.L reverse Aid M-mu/v RT, mixed well, and cDNA was synthesized at 42 ℃, 60min and 70 ℃ for 5 min. The target gene was then reverse transcribed and amplified (see Table 6 for primer sequences). The reaction conditions are as follows: denaturation at 95 ℃ for 15min, annealing at 60 ℃ for 1h, extension at 95 ℃ for 15min, for a total of 40 cycles. GAPDH as housekeeping gene, by 2^-ΔΔCTCalculating the relative expression amount of the target gene.

TABLE 6 primer sequences

The expression levels of c-Kit and SCF mRNA in the small intestine of each group of mice are shown in FIG. 7, the expression levels of c-Kit and SCF mRNA in the small intestine of normal group of mice are strongest, the expression levels of c-Kit and SCF in the opposite model group are weakest, the expression levels of c-Kit and SCF in the small intestine of mice can be up-regulated by soybean milk treatment, and the expression levels of c-Kit and SCF in the small intestine of LP-CQPC02-FSM are stronger than those in LB-FSM and U-FSM.

Cajal Cell (ICC) is a special mesenchymal cell, and researches show that the colon movement speed is slowed due to the quantity of the colon ICC, the change of cell morphology and the abnormal cell network structure, and further slow transit constipation can be caused. C-Kit is one of the ICC specific markers, SCF is a natural ligand for the C-Kit receptor. The ICC density in the small intestine of constipation sufferers was found to decrease and indicates that the decrease in ICC is associated with down-regulation of c-Kit gene expression and decreased expression of c-Kit protein and mRNA in the sigmoid colon. The results of the above experiments show that the c-Kit and SCF protein expression and mRNA expression are significantly increased in the small intestine of the mice of LP-CQPC02-FSM group (p <0.05), indicating that LP-CQPC02-FSM treatment can increase the ICC number of constipation mice.

The expression levels of TRPV1 and GDNF mRNA in small intestines of mice in each group are shown in FIG. 8, the GDNF expression intensity of the small intestines of mice in the normal group is strongest, and the expression of TRPV1 is weakest; after constipation induction, GDNF expression of small intestine of the model group mice is reduced, and TRPV1 expression is enhanced; LP-CQPC02-FSM, LB-FSM and U-FSM were all able to up-regulate GDNF expression and down-regulate TRPV1 expression in the constipation mouse small intestine, and LP-CQPC02-FSM was more able to up-and down-regulate than LB-FSM and U-FSM.

TRPV1 is closely associated with defecation and activation of TRPV1 can trigger neurotransmitter release, leading to intestinal dyskinesia. An increase in TRPV1 expression is an important manifestation of intestinal injury, and intestinal injury caused by gastrointestinal diseases can lead to an increase in TRPV1 expression in constipation patients. GDNF can regulate the function of ganglion cells, help repair damaged intestines and improve constipation. Constipation is associated with the enteric nervous system, resulting in muscle tension and impaired gastrointestinal motility. Modulation of the expression levels of TRPV1 and GDNF is one of the important mechanisms for relieving constipation.

The expression levels of AQP3 and AQP9mRNA in small intestines of mice in each group are shown in FIG. 9, the expression of AQP3 in the small intestines of mice in a normal group is the weakest, and the expression of AQP9 is the strongest; after constipation induction, the expression of AQP9 in the small intestine of the model group mice is reduced, and the expression of AQP3 is enhanced; LP-CQPC02-FSM, LB-FSM and U-FSM can up-regulate AQP9 expression and down-regulate AQP3 expression of constipation mouse small intestine, and LP-CQPC02-FSM has stronger up-regulation and down-regulation capability than LB-FSM and U-FSM.

AQP3 can promote the colon to excessively absorb water in the intestine, so that the colon mucosa can absorb a large amount of water in the intestine, and the intestinal tract is dried, thereby causing constipation. And AQP9 can promote secretion of colon mucus, keep intestinal mucosa lubricated, facilitate excretion of feces, and relieve constipation.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Chongqing second college of education

<120> Lactobacillus plantarum CQPC02 and application thereof in preparation of food for improving constipation

<160>17

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

agagtttgat cctggctcag 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ctacggctac cttgttacga 20

<210>3

<211>1421

<212>DNA

<213> Lactobacillus plantarum CQPC02(Lactobacillus plantarum CQPC02)

<400>3

ttaggcggtg gttctaaagg ttacccaccg actttgggtg ttaaaactct catggtgtga 60

cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 120

agcgattccg acttcatgta ggcgagttgc agcctacaat ccgaactgag aatggcttta 180

agagattagc ttactctcgc gagttcgcaa ctcgttgtac catccattgt agcacgtgtg 240

tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300

accggcagtc tcaccagagt gcccaactta atgctggcaa ctgataataa gggttgcgct 360

cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420

tatccatgtc cccgaaggga acgtctaatc tcttagattt gcatagtatg tcaagacctg 480

gtaaggttct tcgcgtagct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540

gtcaattcct ttgagtttca gccttgcggc cgtactcccc aggcggaatg cttaatgcgt 600

tagctgcagc actgaagggc ggaaaccctc caacacttag cattcatcgt ttacggtatg 660

gactaccagg gtatctaatc ctgtttgcta cccatacttt cgagcctcag cgtcagttac 720

agaccagaca gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta 780

cacatggagt tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttctt 840

cggttgagcc gaaggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc 900

aataaatccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960

cgtggctttc tggttaaata ccgtcaatac ctgaacagtt actctcagat atgttcttct 1020

ttaacaacag agttttacga gccgaaaccc ttcttcactc acgcggcgtt gctccatcag 1080

actttcgtcc attgtggaag attccctact gctgcctccc gtaggagttt gggccgtgtc 1140

tcagtcccaa tgtggccgat taccctctca ggtcggctac gtatcattgc catggtgagc 1200

cgttacccca ccatctagct aatacgccgc gggaccatcc aaaagtgata gccgaagcca 1260

tctttcaaac tcggaccatg cggtccaagt tgttatgcgg tattagcatc tgtttccagg 1320

tgttatcccc cgcttctggg caggtttccc acgtgttact caccagttcg ccactcactc 1380

aaatgtaaat catgatgcaa gcaccaatca ataccagagt c 1421

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

catagcccag gtaaagcaca at 22

<210>5

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gaacactcca gaatcgtcaa ctc 23

<210>6

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

tcagggacta cgctgcgaaa g 21

<210>7

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

aagagctggc agaccgactc a 21

<210>8

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ccggcttttt gggaagggt 19

<210>9

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gagacaggta ggtccatcca c 21

<210>10

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ggggtatgga gaagttggct ag 22

<210>11

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ctatgagaat gctgccgaaa a 21

<210>12

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cctctggaca cttggatatg at 22

<210>13

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gggacggggt tgttgtag 18

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ctcctgatta ttgtcattgc 20

<210>15

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

atccaccaga agttgttt 18

<210>16

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

tgcaccacca actgcttag 19

<210>17

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

gatgcaggga tgatgttc 18

Claims

2. Use of lactobacillus plantarum CQPC02 according to claim 1 for the preparation of a food product for improving constipation.

3. Use of lactobacillus plantarum CQPC02 in the preparation of a food product for improving constipation according to claim 2, wherein the food product is a fermented beverage.

4. Use of lactobacillus plantarum CQPC02 in the preparation of a food product for improving constipation according to claim 3, wherein the food product is fermented soymilk.

5. A beverage prepared by fermenting Lactobacillus plantarum CQPC02 defined in claim 1.

6. The beverage prepared by fermentation of lactobacillus plantarum CQPC02 according to claim 5, wherein the beverage is soy milk.

7. A method for preparing soybean milk by fermenting lactobacillus plantarum CQPC02 according to claim 1, comprising the steps of: soaking soybean in water 3 times of the soybean for 12 hr, grinding, filtering, and concentrating to 10%⁵CFU/mL inoculated with Lactobacillus plantarum CQPC02, fermented at 37 ℃ for 12 hours.