CN113462591A - Lactobacillus plantarum 550 with constipation relieving and sleep aiding functions and application thereof - Google Patents
Lactobacillus plantarum 550 with constipation relieving and sleep aiding functions and application thereof Download PDFInfo
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- CN113462591A CN113462591A CN202110654202.8A CN202110654202A CN113462591A CN 113462591 A CN113462591 A CN 113462591A CN 202110654202 A CN202110654202 A CN 202110654202A CN 113462591 A CN113462591 A CN 113462591A
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- lactobacillus plantarum
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Abstract
The invention discloses lactobacillus plantarum 550 with functions of relieving constipation and helping sleep and application thereof, and belongs to the technical field of biology. The Lactobacillus plantarum 550(Lactobacillus plantarum 550) provided by the invention is preserved in the China center for type culture Collection in 2007 12 and 21 months, with the preservation number being CCTCC NO: m207202. The lactobacillus plantarum 550 provided by the invention is applied to the preparation of functional foods and medicines for relieving constipation, or assisting sleep, or/and reducing cholesterol, or/and inhibiting pathogenic bacteria, or/and degrading nitrite. The lactobacillus plantarum 550 grows well on MRS agar culture medium, grows rapidly, has strong acid production capacity, has good gastric acid and bile salt tolerance, and can realize field planting in intestinal tract. The lactobacillus plantarum 550 has good laxative effect; strong capacity of producing gamma-aminobutyric acid and function of reducing cholesterol in vitro; the inhibitor has good inhibition effect on common pathogenic bacteria; can quickly degrade nitrite and has good safety.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to lactobacillus plantarum 550 with functions of relieving constipation and assisting sleep and an application thereof.
Background
Constipation (Constipation) is a common clinical symptom, and chronic Constipation is the main symptom. Chronic constipation is mainly caused by gastrointestinal dysfunction and is manifested by difficulty in defecation and/or reduction in defecation frequency and dry and hard stools. The difficult defecation comprises the difficult defecation, incomplete defecation, anorectal blockage, time-consuming defecation and the need of auxiliary defecation; a decrease in the number of bowel movements means fewer than 3 bowel movements per week. The course of chronic constipation is at least 6 months. Long-term constipation can affect the life quality and mental health of patients and cause mental and psychological stress to the patients, such as insomnia, depression and the like. In recent years, the incidence of constipation has rapidly increased with increasing social stress, food over-processing, unreasonable dietary structure, psychopsychological factors and irregular work and rest habits, and human health has been seriously threatened. A domestic survey shows that the prevalence rate of adult chronic constipation is 4.0-10.0%. An epidemiological survey result aiming at 16078 adult chronic constipation patients in 5 regions shows that the prevalence rates of Beijing, Shanghai, Xian, Wuhan and Guangzhou functional constipation are 4.0%, 7.0%, 6.0%, 7.0% and 6.0% respectively, and the difference of the prevalence rates among the regions has statistical significance (ZHao, Y.F., et al. animal pharmacy & Therapeutics 34.8(2011): 1020-1029). In addition, the prevalence rate of constipation increases with age, and the prevalence rate of chronic constipation in people over 70 years old reaches 23.0%, 38.0% over 80 years old, and even 80.0% in elderly people under long-term care. The seventh census data in 2021 showed 26402 million people at age 60 and older, accounting for 18.70%, with 19064 million people at age 65 and older, accounting for 13.50%. Therefore, with the increase of the aging population in China, the constipation population tends to rise year by year. In addition, most relevant research results in China show that the prevalence rate of chronic constipation of women is higher than that of men (1.22: 1: 4.56:1) (Chinese consensus on chronic constipation experts (2019, Guangzhou)).
Long-term constipation can cause intestinal wall injury, hemorrhoids and anal fissure, and toxin accumulation in the intestinal tract can easily cause cytopathy, thereby causing colorectal cancer; especially for elderly patients with constipation, the pressure in abdominal cavity is too high to induce insomnia, acute myocardial infarction, cardiovascular and cerebrovascular diseases, arteriosclerosis, etc.; in addition, constipation is also important in the occurrence of hepatic encephalopathy, breast diseases, Alzheimer's disease, dysmenorrhea, urinary tract infection, depression, etc.
In the prior art, the medicaments for treating constipation mainly comprise Qihuang constipation relieving soft capsules, salt cathartic, shakeding and the like, and although the medicaments have quick curative effect, serious cathartic colon and medicament dependence side effects can be generated if the medicaments are taken for a long time. Therefore, it is necessary to provide a product which does not cause drug dependence or adverse reaction of patients, and can relieve constipation and help sleep. In recent years, an important means for treating constipation can be achieved by adjusting the microecology of the gastrointestinal tract to create a healthy intestinal micro-ecological environment (Caoheaifeng. the application effect of the microecological preparation in treating chronic constipation patients [ J ] modern diagnosis and treatment, 2019 (9): 1467-. Gamma-aminobutyric acid (GABA) is an important neurotransmitter, and research shows that the GABA has the effects of helping sleep, stabilizing, resisting convulsion, improving senile dementia, enhancing brain activity nerve cells and the like (Yamatsu, A., et al. Effect of oral gamma-aminobutyric acid (GABA) administration on sheet and its adsorption in humans, food Science and Biotechnology 25.2 (2016;) and acid 551), and is recognized as a new resource food by the Chinese health department in 2009. Among probiotics which have been disclosed so far to be capable of relieving intestinal constipation, for example, CN107099492A discloses a lactobacillus plantarum YS4 and its application in preparing food for preventing constipation; CN109234182A discloses Lactobacillus plantarum ZJUF T34 and application thereof, the strain has the capacity of tolerating high-content bile salt, is beneficial to survival and propagation in intestinal tracts, obviously improves the water content in mouse excrement, and has the potential of relieving constipation; CN 106834187A discloses that Bifidobacterium bifidum CGMCC NO.13632 is used for preparing a pharmaceutical composition and a fermented food for relieving constipation, the strain shortens the first-particle stool-blacking time, has the effect of improving the intestinal propulsion rate, and simultaneously has no toxic or side effect of a medicine for treating constipation. However, the existing probiotics strain inventions for relieving constipation do not have research reports on the capability of producing gamma-aminobutyric acid at the same time, and especially along with the deep basic research on the regulation of intestinal tracts and brains by a flora-intestinal-brain axis, the probiotics strain with the functions of relieving constipation, helping sleep and the like has important significance on human health.
Disclosure of Invention
One of the purposes of the invention is to provide lactobacillus plantarum 550, which has high biological activity, can tolerate gastric acid and bile salt, and can relieve constipation, help sleep, reduce cholesterol and inhibit pathogenic microorganisms.
The second object of the present invention is to provide an application of the lactobacillus plantarum 550.
It is a further object of the present invention to provide a composition comprising the lactobacillus plantarum 550.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the Lactobacillus plantarum 550(Lactobacillus plantarum 550) provided by the invention is preserved in the China center for type culture Collection in 2007 12, 14 th month, with the preservation number being CCTCC NO: m207202.
The lactobacillus plantarum 550 of the invention is separated from farmyard pickles in Qingcheng mountains of Yangtze river of rural township of China; the growth is good on an MRS agar culture medium, the bacterial colony is circular, medium in size, milky white, convex upwards, relatively neat in edges, moist and easy to pick; positive after gram staining.
The Lactobacillus plantarum 550 provided by the invention is sent to a strain collection center of a microorganism resource platform of Sichuan province for 16S rRNA identification, and the measured 16S rRNA sequence is compared with NCBI BLAST, so that the similarity of the sequence and Lactobacillus plantarum in Genebank is 99%, and the strain can be preliminarily identified as Lactobacillus plantarum. The 16S rRNA identification sequence of the strain is shown as SEQ ID NO. 1.
The lactobacillus plantarum 550 disclosed by the invention is rapid in growth and large in bacterial mass, the OD600 reaches 5.6 in 8 hours, and the highest OD600 reaches 9.305 in 18 hours. The lactobacillus plantarum 550 has strong acid production capacity, the acid production is rapid in the initial fermentation period, the acid production is 0.97g/100g in 8 hours and is 1.51g/100g in 24 hours, and the acid production tends to be gentle with the increase of time.
The lactobacillus plantarum 550 provided by the invention is applied to preparing functional food for relieving constipation, or/and helping sleep, or/and reducing cholesterol, or/and inhibiting pathogenic bacteria, or/and degrading nitrite.
The tolerance test of the lactobacillus plantarum 550 to gastric acid and bile salt shows that the lactobacillus plantarum 550 has higher survival rate under the acidic condition; when the concentration of bile salt reaches 3.0g/L, the survival rate of the strain reaches 76.00 percent. The lactobacillus plantarum 550 has good gastric acid and bile salt tolerance and is suitable for oral administration.
In some embodiments of the invention, the functional food is a functional food for relieving chronic constipation.
Animal experiments show that the lactobacillus plantarum 550 can obviously shorten the defecation time of mice and increase the defecation amount, and the ink propulsion rate is obviously higher than that of a model control group, so that the lactobacillus plantarum has the function of relaxing bowels. After the strain is orally administered to the mice for 30 days, no adverse effect on the growth of the mice is seen, and the safety is good.
The lactobacillus plantarum 550 has strong capacity of producing gamma-aminobutyric acid, and the yield of the lactobacillus plantarum is up to 429mg/kg through High Performance Liquid Chromatography (HPLC) detection. The lactobacillus plantarum 550 of the present invention plays a sleep-aiding role by producing gamma-aminobutyric acid.
In some embodiments of the invention, the pathogenic bacteria comprise at least one of klebsiella, staphylococcus aureus, bacillus cereus, and bacillus atrophaeus.
The bacteriostasis test shows that the diameter of the bacteriostasis zone of the lactobacillus plantarum 550 to the Klebsiella DNL03 is (10.91 +/-0.31) mm, the diameter of the bacteriostasis zone of the Staphylococcus aureus CMCC26003 is (8.98 +/-0.03) mm, the diameter of the bacteriostasis zone of the Bacillus cereus CMCC63303 is (20.15 +/-0.15) mm, and the diameter of the bacteriostasis zone of the Bacillus atrophaeus ATCC9372 is (21.83 +/-0.10) mm. The lactobacillus plantarum 550 has good inhibition effect on common pathogenic bacteria of klebsiella, staphylococcus aureus, bacillus cereus and bacillus atrophaeus.
The lactobacillus plantarum 550 has good cholesterol removal performance, and experiments show that the lactobacillus plantarum 550 has the cholesterol removal rate of 66.81% and has strong cholesterol reducing capacity in vitro.
Lactobacillus plantarum 550 is capable of rapidly degrading nitrite, and 3 hours is capable of degrading 30mg/kg of nitrite to less than 1 mg/kg.
In some embodiments of the present invention, the functional food comprises fruit and vegetable beverage, or/and kimchi, or/and solid beverage, or/and pill, or/and tablet.
The lactobacillus plantarum 550 provided by the invention is applied to preparation of medicines for relieving constipation, or/and helping sleep, or/and reducing cholesterol, or/and inhibiting pathogenic bacteria, or/and degrading nitrite.
In some embodiments of the invention, the agent is an agent that relieves chronic constipation.
In some embodiments of the invention, the pathogenic bacteria include at least one of the pathogens Klebsiella, Staphylococcus aureus, Bacillus cereus, and Bacillus atrophaeus.
In some embodiments of the invention, the medicament is an oral medicament.
The pharmaceutical composition provided by the invention comprises lactobacillus plantarum 550 and a pharmaceutically acceptable carrier.
As used herein, a "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
Compared with the prior art, the invention has the following beneficial effects:
the lactobacillus plantarum 550 disclosed by the invention grows well on an MRS agar culture medium, is rapid in growth and strong in acid production capacity, produces gamma-aminobutyric acid, has good gastric acid and bile salt tolerance, and is suitable for oral administration.
Experiments show that the lactobacillus plantarum 550 has good effects of relaxing bowels and helping sleep; strong cholesterol-reducing effect in vitro; the inhibitor has good inhibition effect on common pathogenic bacteria of Klebsiella, Staphylococcus aureus, Bacillus cereus and Bacillus atrophaeus; can rapidly degrade nitrite. Animal experiments show that the lactobacillus plantarum 550 disclosed by the invention has good safety.
Drawings
FIG. 1 is a colony morphology diagram of Lactobacillus plantarum 550 on MRS agar medium;
FIG. 2 is a morphological diagram (10 x 1000 times) of a gram-stained Lactobacillus plantarum 550;
FIG. 3 is a scanning electron micrograph of Lactobacillus plantarum 550;
FIG. 4 is a bacteriostatic circle diagram of Lactobacillus plantarum 550 against common pathogenic bacteria; wherein A is a Klebsiella DNL03 bacteriostatic map, B is a Staphylococcus aureus CMCC26003 bacteriostatic map, C is a Bacillus cereus CMCC63303 bacteriostatic map, and D is a Bacillus atrophaeus ATCC9372 bacteriostatic map;
FIG. 5 is a graph showing the growth of Lactobacillus plantarum 550 strain;
FIG. 6 is a graph of acid production by Lactobacillus plantarum 550;
FIG. 7 is a diagram of the tolerance of Lactobacillus plantarum 550 to various gastric acids;
FIG. 8 is a diagram showing the tolerance of Lactobacillus plantarum 550 to various concentrations of bile salts;
FIG. 9 is a HPLC detection spectrum of a gamma-aminobutyric acid standard substance;
FIG. 10 is an HPLC detection spectrum of the fermented supernatant of Lactobacillus plantarum 550.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following are media involved in the examples of the present invention:
MRS medium (lactobacillus plantarum 550): 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 801.0 g/L of tween, K2HPO4·7H2O2.0 g/L, anhydrous sodium acetate 5g/L, triammonium citrate 2.0g/L, MgSO4·7H2O 0.2g/L，MnSO4·H2O0.038 g/L (agar powder 15g/L as solid medium).
LB medium (klebsiella, bacillus cereus, bacillus atrophaeus, staphylococcus aureus): 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, and adjusting the pH value to 7.2 +/-0.2 (20 g/L of agar powder is added as a solid culture medium).
Example 1 isolation, screening and molecular biological identification of Lactobacillus plantarum 550
1. Material preparation
Samples were collected from farmyard pickles in the city of city, Yangtze river.
The collected sample was cut into pieces, and 1g of the cut pieces was weighed and placed in 9mL of sterile physiological saline. After fully shaking and mixing uniformly, the mixture is diluted by 10 times of gradient and spread in MRS solid culture medium, and cultured for 48h at 37 ℃. And (4) observing by naked eyes, picking single colonies with different shapes and sizes in the culture medium, and repeatedly streaking, purifying and culturing. The strain is primarily determined to be lactic acid bacteria by gram staining and calcium dissolving methods, and the purified strain 45% glycerol is stored in a refrigerator at minus 80 ℃ for standby.
2. And (3) identifying the strain in molecular biology: the purified strain is sent to a strain collection center of a microorganism resource platform of Sichuan province for 16S rRNA identification, and the measured 16S rRNA sequence is compared with an NCBI BLAST database, so that the similarity of the sequence and the Lactobacillus plantarum in Genebank is 99 percent, and the strain can be preliminarily identified as the Lactobacillus plantarum. The 16S rRNA identification sequence of the strain is shown as SEQ ID NO:1 and is named as Lactobacillus plantarum 550(Lactobacillus plantarum 550).
3. Morphological observation
The colony morphology of the strain is observed after the purified lactobacillus plantarum 550 is streaked on an MRS agar medium and is inversely cultured for 48 hours at 37 ℃, and the result is shown in the attached figure 1: the strain grows well on an MRS agar culture medium, and the bacterial colony is circular, medium in size, milky white, upwards convex, relatively neat in edge, moist and easy to pick. The gram stain is positive, the shape under the microscope is shown in figure 2, and the shape under the transmission electron microscope is shown in figure 3.
4. Bacteriostasis test
Inhibition of the growth of klebsiella DNL03, staphylococcus aureus CMCC26003, bacillus cereus CMCC63303 and bacillus atrophaeus ATCC 9372: pouring 10mL of water agar culture medium in a sterile plate, placing an Oxford cup after cooling and solidifying, respectively adding indicator bacterium suspensions (Klebsiella DNL03, Staphylococcus aureus CMCC26003, Bacillus cereus CMCC63303 and Bacillus atrophaeus ATCC9372) into agar culture medium which is cooled to 50 ℃ and correspondingly grows indicator bacteria, and enabling the concentration of the indicator bacteria to be 106Mixing CFU/mL, pouring onto water agar, solidifying, and taking out Oxford cup with forceps to form holemu.L of Lactobacillus plantarum 550 samples were added to each well, spread for 30min, and incubated at 37 ℃ for 24 h. Observing whether a bacteriostatic circle appears around the culture hole, measuring the diameter of the bacteriostatic circle by using a vernier caliper, recording the diameter of the bacteriostatic circle, and finally evaluating bacteriostatic activity according to the existence and the size of the bacteriostatic circle.
As shown in FIG. 4, the inhibition zone diameter of Lactobacillus plantarum 550 against Klebsiella DNL03 is (10.91. + -. 0.31) mm (FIG. 4A), the inhibition zone diameter of Staphylococcus aureus CMCC26003 is (8.98. + -. 0.03) mm (FIG. 4B), the inhibition zone diameter of Bacillus cereus CMCC63303 is (20.15. + -. 0.15) mm (FIG. 4C), and the inhibition zone diameter of Bacillus atrophaeus ATCC9372 is (21.83. + -. 0.10) mm (FIG. 4D). The results show that the lactobacillus plantarum 550 has a good inhibition effect on common pathogenic bacteria of klebsiella, staphylococcus aureus, bacillus cereus and bacillus atrophaeus.
Example 2 lactobacillus plantarum 550 growth ability test.
Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice. Inoculating the activated 550 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, uniformly mixing, and subpackaging into sterile test tubes (18mm multiplied by 180mm test tubes) according to 8 ml/tube; standing and culturing the subpackaged lactobacillus plantarum 550 bacterial liquid in a constant-temperature incubator at 37 ℃, measuring the absorbance value OD600 of the bacterial liquid by taking 3 test tubes, and calculating the average value; measuring the absorbance value OD600 of the bacterial liquid at 550 by taking 3 test tubes at regular intervals, and calculating the average value; and drawing a growth curve by taking time as an abscissa and an absorbance value OD600 as an ordinate.
The growth curve of the lactobacillus plantarum 550 strain is shown in fig. 5, and as can be seen from fig. 5, the strain grows rapidly and the bacterial size is large, the OD600 reaches 5.6 in 8h, and reaches the highest 9.305 in 18h, which indicates that the lactobacillus plantarum 550 can grow rapidly.
Example 3 lactobacillus plantarum 550 acidogenic capacity test.
Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice. Inoculating the activated 550 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, uniformly mixing, and subpackaging into sterile test tubes (18mm multiplied by 180mm test tubes) according to 8 ml/tube. Placing the split 550 bacterial liquid in a constant-temperature incubator at 37 ℃ for standing culture, taking 3 test tubes to measure the total acid of the bacterial liquid, and calculating the average value; and (3) measuring the total acid of the bacterial liquid by taking 3 test tubes at regular intervals, and drawing an acid production curve by taking the time as an abscissa and the acid production amount as an ordinate.
The acid production curve is shown in figure 6. As can be seen from FIG. 6, the acid production of the strain at the initial fermentation stage is rapid, the acid production is 0.97g/100g in 8 hours and is maximum in 1.51g/100g in 24 hours, and the acid production tends to be flat with the increase of time, which indicates that the acid production capacity of the Lactobacillus plantarum 550 is strong.
Example 4 Lactobacillus plantarum 550 antibiotic susceptibility test
The tolerance of Lactobacillus plantarum 550 to tetracycline (30. mu.g/tablet), erythromycin (15. mu.g/tablet), chloramphenicol (30. mu.g/tablet), ampicillin (10. mu.g/tablet), penicillin G (10 IU/tablet), and vancomycin (30. mu.g/tablet) was investigated by the filter paper method. Inoculating activated Lactobacillus plantarum 550 into MRS solid culture medium cooled to about 45 deg.C to obtain final concentration of 106CFU/mL, mixing well, pouring into a plate, coagulating, placing into an antibiotic filter paper, culturing at 37 ℃ for 24h, according to the Performance Standards for antibiotic Suadaptability Testing; CLSI Document M100-S25, decision criteria evaluate the sensitivity of lactic acid bacteria to different antibiotics.
TABLE 1 results of antibiotic susceptibility of Lactobacillus plantarum 550
| Antibiotic | Paper sheet content | Sensitivity of the |
| Erythromycin | ||
| 15 mu g/tablet | | |
| Tetracycline derivatives | ||
| 30 mug/ | S | |
| Chloromycetin | ||
| 30 mug/ | S | |
| Ampicillin | ||
| 10 mug/tablet | | |
| Penicillin G | ||
| 10 IU/tablet | I | |
| Rifampicin | 5 IU/ | S |
| Vancomycin | ||
| 30 mug/ | R | |
| Gentamicin | ||
| 10 IU/tablet | R |
Note that: s represents sensitivity, I represents moderate sensitivity, and R represents insensitivity test results show that the lactobacillus plantarum 550 provided by the invention is sensitive to common antibiotics.
Example 5 Lactobacillus plantarum 550 test for gastric acid and bile salt tolerance
1. Tolerance to gastric acid
The tolerance of lactobacillus plantarum 550 of the present invention to gastric acid was examined by simulating the conditions of gastric acid. Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice. Inoculating the activated 550 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, and performing static culture in a constant temperature incubator at 37 ℃ for 15 h. Centrifuging the cultured 550 bacterial liquid at 5000rpm for 10min to collect thallus, and shaking the thallus with sterile physiological saline; adding the uniformly shaken bacterial liquid into sterile physiological saline with pH values of 3.0, 3.5, 4.0, 5.0 and 6.0 according to the addition amount of 10 percent, taking the sterile physiological saline with pH value of 6.0 as a control, and incubating for 2 hours in a constant temperature incubator at 37 ℃. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterilized normal saline, beating and uniformly mixing, detecting the number of the lactic acid bacteria, counting the detected live lactic acid bacteria, and calculating the survival rate, wherein the survival rate of the bacterial strain is multiplied by 100 percent by a test group/a control group.
The test results are shown in FIG. 7: the survival rates of the strain at the pH values of 4.0 and 3.5 are 91.96% and 79.90% respectively, and the survival rate is high; the survival rate at pH 3.0 was still 58.79%. Generally, the pH of gastric juice after eating is around 3.5, indicating that the Lactobacillus plantarum strain 550 can tolerate gastric acid after eating.
2. Tolerance to bile salts
The lactobacillus plantarum 550 strain is inoculated into MRS liquid culture medium according to the inoculation amount of 5%, activated and cultured for 24 hours at 37 ℃, and activated twice continuously. Inoculating the activated 550 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, and performing static culture in a constant temperature incubator at 37 ℃ for 15 h. And centrifuging the cultured bacterial liquid at 5000rpm for 10min to collect thalli, and shaking the thalli uniformly by using sterile physiological saline.
The uniformly shaken bacterial liquid is added into MRS culture media with cholate concentrations of 1.0g/L, 2.0g/L, 3.0g/L and 0.0g/L (initial bacterial liquid) according to the addition of 10 percent, and the cholate concentration of 0.0g/L is taken as a control group. Then incubated in a 37 ℃ incubator for 3 h. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterile normal saline, beating and uniformly mixing, and detecting the number of lactic acid bacteria; counting the detected live lactobacillus, and calculating the survival rate according to the following formula:
the survival rate (%) of the strain was 100% for the test group/control group.
The 550 strain bile salt tolerance data is shown in figure 8: when the concentration of the bile salts is 1.0g/L and 2.0g/L, the survival rate of the strain is 96.57 percent and 80.57 percent respectively, but when the concentration of the bile salts reaches 3.0g/L, the survival rate of the strain still reaches 76.00 percent. The concentration of the bile salts in the intestinal tract is 0.3-3.0 g/L, which indicates that 550 strains can tolerate the bile salts in the intestinal tract.
Example 6 degradation Properties of Lactobacillus plantarum 550 to nitrous acid
Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice; inoculating the activated bacterial liquid into an MRS liquid culture medium containing sodium nitrite according to the inoculation amount of 5%, standing and culturing at 37 ℃ in a constant-temperature incubator, and sampling every 3 hours to determine the content of the sodium nitrite (the sodium nitrite is determined according to GB 5009.33-2016).
The lactobacillus plantarum 550 has nitrite degradation data shown in table 2.
TABLE 2 Lactobacillus plantarum 550 strain nitrite degradation experimental data
| Incubation time (h) | 0 | 3 | 6 |
| Sodium nitrite content (mg/kg) | 30 | <1 | <1 |
As can be seen from Table 2, the strain can rapidly degrade nitrite, and 30mg/kg of nitrite can be degraded to less than 1mg/kg in 3 hours, which indicates that the Lactobacillus plantarum 550 strain has excellent nitrite degradation performance and can degrade nitrite in vivo in the gastrointestinal tract.
Example 7 Lactobacillus plantarum 550 Cholesterol removal Properties
MRS culture solution containing cholesterol (MRS-CHOL culture medium) and MRS culture solution containing no cholesterol are prepared.
MRS-CHOL Medium: the culture medium consists of MRS culture solution, sodium thioglycolate, bile salt and cholesterol, wherein the concentration of the sodium thioglycolate in the MRS-CHOL culture solution is 2g/L, the concentration of the bile salt in the MRSO-CHOL culture solution is 0.2 weight part, and the concentration of the cholesterol is 150 mu g/mL.
Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice. Respectively inoculating the activated bacterial liquid into MRS-CHOL culture solution (experimental group) and MRS culture solution (control group) without cholesterol according to the inoculation amount of 5%, culturing at the constant temperature of 37 ℃ for 32h, measuring the cholesterol content in supernatant by using an o-phthalaldehyde color developing agent every 8h (the culture solution is centrifuged at 5000r/min, 4 ℃ and 10min), and calculating the cholesterol removal capacity, wherein the cholesterol removal capacity is the difference between the cholesterol of the control group and the cholesterol of the experimental group/the cholesterol content of the control group. The test results are shown in table 3.
TABLE 3 experiment of cholesterol-removing ability of Lactobacillus plantarum 550 Strain
| Incubation time (h) | 0 | 8 | 16 | 24 | 32 |
| Cholesterol content (μ g/mL) | 150 | 119.77 | 80.74 | 57.22 | 49.78 |
| Cholesterol removal Rate (%) | / | 20.15 | 46.17 | 61.85 | 66.81 |
The results in table 3 show that lactobacillus plantarum 550 has a cholesterol removal rate of 66.81%, and has a strong cholesterol-lowering capacity in vitro.
Example 8 relieving action of Lactobacillus plantarum 550 on Constipation in mice
1. Reagent: compound diphenoxylate tablets, guangxi hefeng pharmaceutical industry, llc; gum arabic powder, activated carbon (powder), (chengdu dragon chemical reagent factory).
2. And (3) testing a sample: lactobacillus plantarum 550 bacterial powder (100 hundred million CFU/g, batch number: 20160519) from Szechthy Biotech, Inc.
3. Animals: KM mice, 18-22g, were obtained from the animal center of the institute of Chinese medicine and sciences, Sichuan province (SPF grade). SPF grade experimental animal environment: room temperature: 18-22 ℃, relative humidity: 40-70 percent.
4. Dose grouping and test sample administration time: the experimental animal is provided with three administration groups, a blank control group and a model control group. Taking 10 times of the human recommended amount as a small dose administration group, wherein the medium dose administration group and the large dose administration group are respectively 20 times and 30 times of the human recommended amount; the corresponding mice were dosed at 0.08g/kg, 0.16g/kg, 0.24g/kg, respectively, with the test samples administered for 30 days.
5. Small intestine exercise experiment
And (3) orally gavage and administering the model-making medicine compound diphenoxylate, establishing a mouse small intestine peristalsis inhibition model, calculating the ink propulsion rate of the small intestine within a certain time, and judging the gastrointestinal peristalsis function of the model mouse.
After 30 days of sample administration, each group of mice was fasted for 16 hours without water deprivation. The compound diphenoxylate (5mg/kg-BW) is administrated to the model control group and the administration group by intragastric administration, and distilled water is supplied to the blank control group to establish a constipation model.
After 0.5 hour of administering the compound diphenoxylate, the administration group was administered with ink (containing 5% of activated carbon powder and 10% of gum arabic) containing the corresponding test sample, and the blank control group and the model control group were administered with ink for intragastric administration, respectively.
After 25 minutes, the animal is immediately killed by removing the cervical vertebra, the abdominal cavity is opened to separate mesentery, the intestinal canal with the upper end from the pylorus and the lower end to the ileocecal part is cut and placed on a tray, the small intestine is gently pulled into a straight line, the length of the intestinal canal is measured as the total length of the small intestine, and the length from the pylorus to the front edge of ink is measured as the ink propelling length. The ink propulsion rate was calculated as follows:
the ink propulsion rate (%) is ink propulsion length (cm)/total small intestine length (cm) × 100%
Statistics were performed by means of pairwise comparisons of mean values between test and control groups. On the premise that the model is established, when the ink propulsion rate of the mouse in the test sample group is obviously higher than that of the control group of the model, the positive result of the experiment can be judged.
The experimental results are shown in table 4, the ink propulsion rate of the mice is obviously reduced (P is less than 0.01) when the model control group is compared with the blank control group, and the model is established. The ink propulsion rate of the three dose groups of 550 bacterial powder is obviously higher than that of the model control group (P <0.01), and the experimental result is positive.
TABLE 4 influence of Lactobacillus plantarum 550 bacterial powder on the intestinal motility of constipation model mice
| Group of | Dosage form | Animal number (only) | Ink push Rate (%) |
| Blank control group | --- | 10 | 80.02±5.42 |
| Model control group | --- | 11 | 46.84±6.82** |
| 550 fungus powder |
10× | 10 | 57.60±7.85## |
| 550 fungus powder |
20× | 10 | 60.85±9.65## |
| 550 fungus powder |
30× | 11 | 59.34.±11.12## |
Note: p compared to blank control<0.01; compared with the model control group,##P<0.01。
6. measurement of defecation time, fecal particle count and fecal weight
The compound diphenoxylate as a model drug is given through oral gavage, a mouse constipation model is established, the defecation time of the first defecation grain of the mouse, the number of the defecation grains within 6 hours and the defecation weight are measured, and the defecation condition of the model mouse is reflected.
After 30 days of sample administration, each group of mice was fasted for 16 hours without water deprivation. The model control group and the administration group are administered with compound diphenoxylate (10mg/kg-BW) by intragastric administration, and the blank control group is administered with distilled water to establish a constipation model.
After 0.5 hour of administering the compound diphenoxylate, the administration group was administered with ink (containing 5% of activated carbon powder and 10% of gum arabic) containing the corresponding test sample, and the blank control group and the model control group were administered with ink for intragastric administration, respectively. Animals were all housed in a single cage and were fed with normal drinking water.
Starting from the ink filling, the time for discharging black feces of the first grain of each animal, the number of the discharged black feces in 6 hours and the weight of the discharged black feces are recorded.
Statistics were performed by means of pairwise comparisons of mean values between test and control groups. On the premise that a small intestine constipation model is established, the first grain defecation time of a mouse of a test sample group is obviously shorter than that of a model control group, and the positive result of the index can be judged; the number of the black defecation grains in 6 hours is obviously higher than that of the model control group, and the positive result of the index can be judged; the weight of the defecation is obviously higher than that of the model control group within 6 hours, and the positive result of the index can be judged.
And (4) judging a result: the result of any one of the weight and the grain number of the excrement in 6 hours is positive, and the result of any one of the small intestine movement experiment and the excrement discharging time is positive, so that the result of the experiment can be judged to be positive.
The results of the experiment are shown in table 5: compared with a blank control group, the model control group obviously prolongs the defecation time of the mice, obviously reduces the number of defecation granules, obviously lightens the defecation weight (P is less than 0.01), and establishes a model.
From the aspect of defecation time, the small, medium and large dose groups of the lactobacillus plantarum 550 bacterial powder can obviously shorten the black defecation time of mice (P <0.01), and the result can be judged to be positive. From the number of the defecation granules, the number of the defecation granules of the 550 bacterial powder medium-dose group and the large-dose group is obviously higher than that of the model control group (P <0.05 and P <0.01), and the result can be judged to be positive. From the aspect of defecation weight, the defecation weight of the lactobacillus plantarum 550 powder in the dose group and the large dose group is obviously higher than that of the model control group (P <0.05 and P <0.01), and the result can be judged to be positive.
TABLE 5 influence of Lactobacillus plantarum 550 bacterial powder on defecation time, grain number and weight of constipation model mice
Note: p compared to blank control group<0.05，**P<0.01; compared with the model control group,#P<0.05，##P<0.01。
5. small knot
After the mice are orally given with three doses of small, medium and large lactobacillus plantarum 550 bacterial powder samples respectively for 30 days, no adverse effect of the samples on the growth of the mice is seen. In the small intestine movement experiment, compared with a blank control group, the ink propulsion rate of the mouse is obviously reduced (P is less than 0.01), and the model is established. The ink propulsion rate of the 550 bacterial powder low dose group, the medium dose group and the high dose group is obviously higher than that of the model control group (P <0.01), and the experimental result is positive. In the defecation time, defecation grain number and defecation weight experiments, compared with a blank control group, the model control group obviously prolongs the defecation time of the mice, obviously reduces the defecation grain number, obviously lightens the defecation weight (P is less than 0.01), and establishes a model. The small, medium and large dose groups of 550 lactobacillus powder can obviously shorten the black defecation time of mice (P <0.01) from the defecation time, and the result can be judged to be positive. From the number of the defecation granules, the number of the defecation granules of the 550 bacterial powder medium-dose group and the large-dose group is obviously higher than that of the model control group (P <0.05 and P <0.01), and the result can be judged to be positive. From the aspect of defecation weight, the defecation weight of the 550 bacterial powder in the dose group and the large dose group is obviously higher than that of the model control group (P <0.05 and P <0.01), and the result can be judged to be positive. The lactobacillus plantarum 550 can be judged to have positive defecation result in both high dose and low dose, has good dose-effect relationship and has the function of defecation.
EXAMPLE 9 determination of the production of Gamma-aminobutyric acid (GABA) by Lactobacillus plantarum 550
1. Sample preparation
Inoculating lactobacillus plantarum 550 strain strains into an MRS liquid culture medium according to the inoculation amount of 5%, performing activation culture at 37 ℃ for 24 hours, and continuously activating twice; inoculating the activated bacterial liquid into MRS liquid culture medium containing 2g/L glutamic acid or sodium glutamate according to the inoculation amount of 5%, and performing static culture in a constant temperature incubator at 37 ℃ for 24h to obtain lactobacillus plantarum 550 fermentation liquid.
Weighing 1g of lactobacillus plantarum 550 fermentation liquor, adding 10ml of extracting solution (absolute ethyl alcohol: water 4: 1; V/V), standing for 5min after 30min of ultrasonic treatment, transferring the supernatant into a 25ml volumetric flask, repeatedly extracting once, combining the supernatants into the 25ml volumetric flask, and fixing the volume to 25ml by using the extracting solution. And (3) taking 1ml of sample to be subjected to derivatization, adding 0.2ml of sodium bicarbonate solution and 0.4ml of dansyl chloride derivatization reagent into the sample in a test tube, uniformly mixing the sample and the reagent, performing derivatization reaction in a water bath at 70 ℃ for 20min, and filtering the mixture by using a 0.45-micron water-phase filter membrane to be detected.
2. Chromatographic conditions
A chromatographic column: c18250mm × 4.6 mm; the grain diameter is 5 mu m; column temperature: 30 ℃; sample introduction amount: 50 mu L of the solution; flow rate: 1 ml/min; detection wavelength: 436 nm; the mobile phase is acetonitrile: sodium acetate (35: 65); the elution mode is isocratic.
3. Calibration curve and sample measurement calculation
Preparing 2ppm, 5ppm, 10ppm, 20ppm and 50ppm gamma-aminobutyric acid concentration standard solution respectively, carrying out chromatographic analysis according to the method, drawing a standard curve and a regression equation by using a peak area-standard solution concentration drawing method, wherein the regression equation is as follows:
f(x)＝403.876*x-418.660
wherein the linear correlation coefficient R is 0.9995268.
According to the detection result, the gamma-aminobutyric acid standard HPLC detection map is shown in FIG. 9, the gamma-aminobutyric acid HPLC detection map is shown in FIG. 10, the gamma-aminobutyric acid HPLC detection map of the fermentation supernatant of the lactobacillus plantarum 550 is obtained, according to comparison and analysis of spectrogram overlapping technology, the gamma-aminobutyric acid produced by the lactobacillus plantarum 550 is determined to be consistent with the standard, and according to calculation of a regression equation, the content of the gamma-aminobutyric acid in the fermentation liquid of the lactobacillus plantarum 550 is 429mg/kg, and the yield is high. The gamma-aminobutyric acid plays a role in aiding sleep through the action of the intestinal-cerebral axis, which shows that the lactobacillus plantarum 550 plays a role in aiding sleep through the production of the gamma-aminobutyric acid.
Application example 1: fruit and vegetable beverage containing lactobacillus plantarum 550 of the invention
Fresh vegetables and fruits are selected, washed, juiced, subjected to high-temperature instantaneous sterilization, and subjected to high-temperature heat sterilization at the temperature of 120-140 ℃ for 3-7 seconds, and then rapidly cooled to 20-37 ℃. Then inoculating the lactobacillus plantarum 550 bacterial powder prepared by the invention to ensure that the concentration reaches 107More than CFU/mL, and refrigerating and storing at the temperature of 4 ℃, thus obtaining the fruit and vegetable beverage containing the lactobacillus plantarum 550 live bacteria.
Application example 2: probiotic pickle containing lactobacillus plantarum 550 prepared by using the strain
Cleaning fresh vegetables, adding into 4-5 times of drinking water, adding edible glucose 1% of total volume and edible sodium chloride 0.4-0.6% of total volume, inoculating into the lactobacillus plantarum 550 powder to make its concentration reach 107And fermenting for 4-10h at room temperature by using CFU/mL or more, thus obtaining the probiotic pickle containing the lactobacillus plantarum 550 of the invention.
Application example 3: preparation of pills by using Lactobacillus plantarum 550 of the present invention
34.2 parts by weight (more than or equal to 300 hundred million CFU/g) of the lactobacillus plantarum 550 powder, 47.5 parts by weight of microcrystalline cellulose, 1.7 parts by weight of lecithin, 3.0 parts by weight of polyvinylpyrrolidone, 8.0 parts by weight of calcium carbonate, 2.5 parts by weight of selenium-enriched yeast and 2.0 parts by weight of magnesium stearate are respectively weighed and mixed with 1.1 parts by weight of ethanol uniformly, and then the conventional amount of refined honey is added to prepare the pill.
Application example 4: tablets of dietary supplement prepared using lactobacillus plantarum 550 of the present invention
Respectively weighing 30 parts by weight of lactose, 18.3 parts by weight of starch, 5 parts by weight of microcrystalline cellulose, 10 parts by weight of maltodextrin, 9 parts by weight of glucose and 1 part by weight of inulin, uniformly mixing, granulating into wet granules by adopting a wet 20-mesh screen with 30% alcohol, drying for 3.5 hours at 55 ℃, granulating by adopting a 20-mesh screen, adding 25.7 parts by weight of lactobacillus plantarum 550 powder (more than or equal to 300 hundred million CFU/g) and 1 part by weight of magnesium stearate, uniformly mixing, and tabletting by using a rotary tablet press to obtain the tablet of the active lactobacillus plantarum 550 dietary supplement.
Application example 5: preparation of solid beverage by using lactobacillus plantarum 550 of the invention
Respectively weighing 33 parts of lactobacillus plantarum 550 powder (more than or equal to 300 hundred million CFU/g), 12 parts of maltodextrin, 11 parts of sorbitol, 10 parts of galacto-oligosaccharide, 10 parts of corn peptide, 5 parts of soybean peptide, 4 parts of xylo-oligosaccharide, 4 parts of fructo-oligosaccharide, 3 parts of selenium-enriched yeast, 2 parts of sucralose, 2 parts of malic acid, 2 parts of citric acid, 1 part of glutathione and 1 parts of vitamin C, sieving the mixture with a 40-mesh sieve, uniformly mixing the mixture by using a packaging machine, and carrying out back sealing and packaging by using a screw to prepare 2 g/bag of probiotic solid beverage.
Finally, it should be noted that: the above embodiments are only preferred embodiments of the present invention to illustrate the technical solutions of the present invention, but not to limit the technical solutions, and certainly not to limit the patent scope of the present invention; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention; that is, the technical problems to be solved by the present invention, which are not substantially changed or supplemented by the spirit and the concept of the main body of the present invention, are still consistent with the present invention and shall be included in the scope of the present invention; in addition, the technical scheme of the invention is directly or indirectly applied to other related technical fields, and the technical scheme is included in the patent protection scope of the invention.
SEQUENCE LISTING
<110> Sichuan high-fortune Biotech Co., Ltd
<120> lactobacillus plantarum 550 with constipation relieving and sleep aiding functions and application thereof
<130> 20210529
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1441
<212> DNA
<213> Lactobacillus plantarum
<400> 1
tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc atgatttaca tttgagtgag 60
tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa 120
acagatgcta ataccgcata acaacttgga ccgcatggtc cgagcttgaa agatggcttt 180
ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta acggctcacc 240
atggcaatga tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg 300
cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 360
agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa 420
catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc acggctaact 480
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt attgggcgta 540
aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa ccgaagaagt 600
gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 660
gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 720
acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta gtccataccg 780
taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 840
aagcattccg cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc 900
cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 960
tgacatacta tgcaaatcta agagattaga cgttcccctt cggggacatg gatacaggtg 1020
gtgcatggtt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttatt atcagttgcc agcattaagt tgggcactct ggtgagactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggatggtaca acgagttgcg aactcgcgag agtaagctaa tctcttaaag 1260
ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccatgagagt ttgtaacacc caaagtcggt ggggtaacct tttaggaacc agccgcctaa 1440
g 1441
Claims (10)
1. Lactobacillus plantarum 550 with constipation relieving and sleep aiding functions is characterized in that the preservation number is CCTCC NO: m207202.
2. Use of lactobacillus plantarum 550 according to claim 1 for the preparation of a functional food for constipation relief, or/and sleep aid, or/and cholesterol reduction, or/and pathogen suppression, or/and nitrite degradation.
3. The use according to claim 2, wherein the functional food is a functional food for relieving chronic constipation.
4. The use according to claim 2, wherein the pathogenic bacteria comprise at least one of klebsiella, staphylococcus aureus, bacillus cereus, bacillus atrophaeus.
5. The use according to any one of claims 2 to 4, wherein the functional food comprises a fruit and vegetable drink, or/and kimchi, or/and a solid drink, or/and a pill, or/and a tablet.
6. Use of lactobacillus plantarum 550 according to claim 1 for the preparation of a medicament for constipation relief, or/and sleep aid, or/and cholesterol reduction, or/and pathogen suppression, or/and nitrite degradation.
7. The use of claim 6, wherein the medicament is a medicament for relieving chronic constipation.
8. The use of claim 6, wherein the pathogenic bacteria comprise at least one of Klebsiella, Staphylococcus aureus, Bacillus cereus, and Bacillus atrophaeus.
9. The use according to any one of claims 6 to 8, wherein the medicament is an oral medicament.
10. A pharmaceutical composition comprising lactobacillus plantarum 550 and a pharmaceutically acceptable carrier.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115161250A (en) * | 2022-09-06 | 2022-10-11 | 广东益可维生物技术有限公司 | Lactobacillus plantarum with functions of improving sleep, strengthening brain and improving intelligence and application thereof |
| CN115161250B (en) * | 2022-09-06 | 2022-12-13 | 广东益可维生物技术有限公司 | Lactobacillus plantarum with functions of improving sleep, strengthening brain and improving intelligence and application thereof |
| CN115521889A (en) * | 2022-10-30 | 2022-12-27 | 江南大学 | Lactobacillus plantarum WL02 capable of producing gamma-aminobutyric acid and application thereof |
| CN115521889B (en) * | 2022-10-30 | 2024-05-28 | 江南大学 | Lactobacillus plantarum WL02 for producing gamma-aminobutyric acid and application thereof |
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