CN110157645B - Lactobacillus salivarius Y4 and application thereof - Google Patents
Lactobacillus salivarius Y4 and application thereof Download PDFInfo
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- CN110157645B CN110157645B CN201910458213.1A CN201910458213A CN110157645B CN 110157645 B CN110157645 B CN 110157645B CN 201910458213 A CN201910458213 A CN 201910458213A CN 110157645 B CN110157645 B CN 110157645B
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- lactobacillus salivarius
- strain
- pathogenic bacteria
- lactobacillus
- salivarius
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Abstract
The invention provides lactobacillus salivarius Y4 and application thereof, wherein the lactobacillus salivarius Y4 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 17459; it can be used for inhibiting pathogenic bacteria including Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Listeria monocytogenes, etc.; it can also be used for preparing food, feed, etc. The lactobacillus salivarius Y4 has strong acid and bile salt resistance and rapid propagation in vitro culture, can be used for developing microecological preparations, can effectively resist the infection of various pathogenic bacteria, and has effective protection effect on animals.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus salivarius Y4 and application thereof.
Background
The Strain (Strain) is also called Strain, and means pure culture of different sources of the same microorganism, and each pure culture of microorganism isolated from nature can be called a Strain. For example, when culturing typhoid bacillus, it can be isolated from blood, bone marrow, feces and bile, and the best of them is bile, so called standard strain. Probiotics, a class of active microorganisms that are beneficial to the host, colonize the human body, have been widely used in the prevention and treatment of a variety of diseases, and their efficacy has strong evidence in certain clinical settings. For example, WO 2007/043933 describes the use of probiotics in the manufacture of food, feed products, dietary supplements, to control weight gain, prevent obesity, increase satiety, prolong satiety, reduce food intake, reduce fat deposition, improve energy metabolism, enhance insulin sensitivity, treat obesity and treat insulin resistance.
Lactobacillus salivarius is one of the important members of the genus lactobacillus, mostly one of the probiotics of animal body surfaces and internal mucosal surfaces. Probiotics existing on the surfaces of gastrointestinal tract mucous membranes, oral cavity respiratory tract mucous membranes and reproductive tract mucous membranes have double protection effects on the intestinal tract mucous membranes and the respiratory tract mucous membranes as living resident bacteria, and on one hand, the probiotics can be attached to the surfaces of the mucous membranes for permanent planting to maintain the microbial flora balance of the gastrointestinal tract mucous membranes and the respiratory tract mucous membranes; on the other hand, the probiotics can directly interact with the immune system of the host, especially the mucosal immune system of the host to induce mucosal immunity; can stimulate the development of immune central organs such as spleen, thymus, bursa of fabricius and the like, and can also promote macrophages to play an adjuvant role; by influencing the synthesis and secretion of cell factors and the like of a mucous membrane system, the response performance of T, B cells to antigen stimulation is enhanced, and the specific immunity effect is effectively enhanced; the resident probiotics on the surface of the mucous membrane can activate related lymphoid tissues in the mucous membrane, enhance sIgA biosynthesis, improve the immune function of the mucous membrane of the digestive tract and the mucous membrane of the respiratory tract, and exert the immunoregulation effect by inducing lymphocytes and macrophages to generate cytokines, thereby enhancing the immune function of the organism.
Disclosure of Invention
The lactobacillus salivarius Y4 is strong in acid resistance and bile salt resistance, is cultured in vitro and propagated quickly, can be used for development of microecological preparations, can effectively resist infection of various pathogenic bacteria, and has an effective protection effect on animals.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the strain is separated from feces of healthy children, inoculated and cultured by an MRS culture medium, separated and purified to obtain a lactobacillus salivarius (the strain number is Y4), and the microbial preservation number is CGMCC NO. 17459; the preservation date is as follows: 29 months 03, 2019; the classification is named as: lactobacillus salivarius (Lactobacillus salvarius); the preservation unit: china general microbiological culture Collection center; and (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
The present invention is mainly to isolate the above-mentioned Lactobacillus salivarius strains from stool samples from healthy children by the following method.
Directly inoculating the collected children feces on an MRS solid culture medium, selecting bacterial colonies with obvious characteristics, repeatedly carrying out loop-line separation until the bacterial colonies with consistent shapes and sizes are treated, carrying out gram staining, and carrying out microscopic observation on the shapes of bacteria; repeatedly inoculating and purifying to obtain the final product.
The strain judgment is carried out by an API identification system of French Meiriei company, and molecular biological identification is carried out, so that the classification status of the lactobacillus salivarius strain Y4 separated by the method belongs to the following categories: lactobacillus salivarius (Lactobacillus salvarius).
The bacterial colony of the strain on an MRS plate is hemispherical, the surface of the bacterial colony is smooth and moist, the bacterial colony is milky white, the diameter of the bacterial colony is about 1-2mm, and the bacterial colony is uniform in size; gram staining observation shows that the strain is gram-positive bacteria, and the thallus is rod-shaped and wide: 0.5-0.6um, long: 1.2-3.0 um; good growth was achieved in MRS liquid medium (pH 6.2) with uniform turbid growth, reaching the end of logarithmic growth for 8 hours at 37 ℃ and 120 rpm.
The lactobacillus salivarius Y4 has a good application effect in inhibiting pathogenic bacteria, and is applied to preparation of a pathogenic bacteria bacteriostatic agent, wherein the pathogenic bacteria comprise escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, listeria monocytogenes and the like. Especially, the inhibition effect on salmonella is the best. Meanwhile, the lactobacillus salivarius Y4 can also be applied to preparation of foods, feeds and the like.
The invention has the following beneficial effects:
the strain Y4 of the invention was identified as Lactobacillus salivarius by 16S rDNA. The lactobacillus salivarius has strong acid and bile salt resistance, and can grow in a culture solution with pH of 3 and a culture solution containing 0.35g/L sodium deoxycholate.
The strain is cultured in vitro and propagated quickly, and can be used for developing microecological preparation. The in vitro bacteriostatic test of the strain shows that the lactobacillus salivarius Y4 has obvious inhibiting effect on escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae and listeria monocytogenes, can effectively resist the infection of various pathogenic bacteria, and has effective protection effect on animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a colony morphology of Lactobacillus salivarius Y4 strain;
FIG. 2 is a molecular phylogenetic tree of Lactobacillus salivarius Y4 strain 16S rDNA;
FIG. 3 is a graph showing the growth curve and pH change of Lactobacillus salivarius Y4.
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 with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: screening and identification of lactobacillus salivarius
1. Isolation and screening of Lactobacillus salivarius Y4
Taking feces of healthy children as a sample, taking the feces sample in sterile physiological saline by using a sterile wooden stick, uniformly mixing, carrying out gradient dilution, carrying out pouring culture on an MRS culture medium (supplemented with X-gal) to screen a blue bacterial colony, carrying out purification for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as Y4. The MRS culture medium is a general culture medium for culturing lactobacillus and has the formula as follows: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 801 mL/L of tween-801, 2g/L of dipotassium phosphate, 5g/L of sodium acetate trihydrate, 2g/L of triammonium citrate, 0.58g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate and pH 6.2.
The dominant morphological characteristics of the colonies of the strain Y4 on the MRS plate are as follows: as shown in fig. 1. Colonies were round, smooth, blue on MRS medium with X-gal, and opaque. Gram stain positive, rod-shaped, broad: 0.5-0.6um, long: 1.2-3.0 um.
2. Determination and identification of strain by physiological and biochemical indexes
The biochemical map of the separated Y4 strain is identified by adopting a carbohydrate identification reagent strip of French Merrier. Performing species identification on the strain according to a biochemical map, placing the reagent strips respectively added with bacterial suspensions with specified concentration (the turbidity is equivalent to the bacterial suspension of 2 McFarland) in an incubator at 37 ℃ according to the specification of the identification reagent strips for incubation, recording the reaction result after incubation for 24 hours, and recording the fermentation reaction biochemical map according to the reaction result of 24 hours, wherein the strain biochemical map is as follows: xylose, glucose, mannose, sorbitol, esculin, saligenin, cellobiose, maltose, lactose, sucrose, trehalose, and raffinose are positive; glycerol, arabinose, rhamnose, mannitol, matsutake sugars were negative; the strain is judged by applying software of API identification system of French Merrill company, and the strain identification result is Lactobacillus salivarius.
3. 16S rDNA sequencing of Y4 Strain
Taking the DNA of the strain Y4 as a template, amplifying the 16S rDNA universal primer, and carrying out sequence determination on the amplified fragment; blast comparison shows that the 16S rDNA sequence of the strain sequence in a GenBank gene library has the highest homology and the homology rate is 99 percent. The 16S rDNA sequence of the existing Lactobacillus model strain in the RDP library is subjected to genetic evolution analysis through MEGA 4.0, as shown in the result of FIG. 2, the strain Y4 has the highest homology with Lactobacillus salivarius, and the strain Y4 is judged to be Lactobacillus salivarius.
The strain Y4 obtained by screening is preserved in the preservation unit: china general microbiological culture Collection center (CGMCC); the preservation date is as follows: 29 months 03, 2019; the preservation number of the Lactobacillus salivarius is CGMCC NO. 17459.
Example 2: growth assay for Lactobacillus salivarius Y4
Inoculating the activated Y4 bacterial liquid into the MRS liquid culture medium with the inoculation amount of 2% (about 107CFU/mL), culturing in a shaking table at 37 ℃, culturing at 220rpm for 24h, sampling at 0, 2, 4, 6, 7, 8, 10, 12 and 24h respectively, and then drawing a growth curve and a pH change graph by taking the culture time as an abscissa and taking the pH and OD600nm values as ordinate. As shown in FIG. 3, the experimental results show that Y4 is cultured for 2h, namely, the logarithmic growth phase is entered, and 8h is entered the stationary phase. The pH of the culture broth dropped to 3.86 after 24h of culture.
Example 3: tolerance determination of Lactobacillus salivarius Y4 to acid and bile salt
The activated Y4 bacterial liquid was inoculated into MRS liquid medium at pH 2.0, 2.5, 3.0, 3.5, 4.0, 6.2 at an inoculum size of 2% (about 107CFU/mL), cultured at 37 ℃ and 220rpm for 2 hours, diluted to an appropriate ratio, plated, and cultured for 24 hours for counting. The results of acid tolerance of Lactobacillus salivarius Y4 are shown in Table 1.
Treating the strain by the same method, inoculating the strain solution into culture medium with sodium deoxycholate concentration of 0%, 0.20%, 0.25%, 0.30% and 0.35%, culturing at 37 deg.C and 220rpm for 3 hr, diluting at appropriate ratio, plating, and culturing for 24 hr for counting. The results of the tolerance of Lactobacillus salivarius Y4 to deoxycholic acid sodium salt are shown in Table 1.
TABLE 1 acid tolerance results for Lactobacillus salivarius Y4
TABLE 2 results of tolerance of Lactobacillus salivarius Y4 to deoxycholic acid sodium salt
As can be seen from tables 1 and 2, the pH of the Lactobacillus salivarius Y4 strain at pH 2.5 is the critical point of acid tolerance, the critical point of normal growth of the strain is not affected at pH 4.0, the strain grows at pH 2.5-4.0, but the growth speed is slower than normal; the tolerance critical point of the lactobacillus salivarius Y4 strain to deoxycholic acid sodium salt is more than 0.35%, and the critical range of normal growth of the strain is not influenced when the concentration of bile salt is 0-0.3%.
Example 4: in vitro bacteriostatic test of Lactobacillus salivarius Y4 strain
In vitro bacteriostatic test, the bacteriostatic activity of the probiotics is determined by an oxford cup method. Lactobacillus salivarius Y4 preserved by glycerol is streaked and inoculated to MRS solid culture medium for 24h at 37 ℃, then colonies are selected and inoculated to MRS liquid culture medium for overnight culture, and Lactobacillus salivarius Y4 bacterial suspension cultured overnight at 37 ℃ is taken as bacteriostatic liquid for later use. Inoculating Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Listeria monocytogenes preserved in glycerol into a TSB solid culture medium by streaking, culturing at 37 ℃ for 24h, selecting colonies, inoculating into a TSB liquid culture medium, culturing until OD600 is 1.0, and treating with physiological saline 1: 103, diluting, marking 200uL of diluted bacterial suspension into a TSB solid culture medium in a rice-shaped manner, and airing for later use.
Taking MRS liquid culture medium as a blank control, uniformly placing 3 Oxford cups on each TSB flat plate on average, adding 200uL of antibacterial liquid into each Oxford cup, culturing for 24h in a constant-temperature incubator at 37 ℃, observing the antibacterial ring condition of the bottom of the Oxford cup, and determining the diameter of the antibacterial ring. The results of the experiment are shown in table 3.
TABLE 3 bacteriostatic results of Lactobacillus salivarius Y4 against 6 pathogenic bacteria
As can be seen from Table 3, Lactobacillus salivarius Y4 has certain inhibitory effects on Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Listeria monocytogenes, and Lactobacillus salivarius Y4 has the best inhibitory effect on Salmonella.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. Lactobacillus salivarius Y4, wherein the Lactobacillus salivarius Y4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 17459.
2. The lactobacillus salivarius Y4 as claimed in claim 1 wherein the medium for culturing the lactobacillus salivarius is MRS solid medium with pH 6.2.
3. Lactobacillus salivarius Y4 as claimed in claim 1 wherein the Lactobacillus salivarius has a cultivation temperature of 37 ℃.
4. Use of lactobacillus salivarius Y4 as claimed in claim 1 in the manufacture of a pathogenic bacteria bacteriostatic agent.
5. The use of claim 4, wherein the pathogenic bacteria are Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Listeria monocytogenes.
6. Use of lactobacillus salivarius Y4 as claimed in claim 1 in the preparation of food and feed.
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| CN113388550B (en) * | 2021-07-16 | 2023-05-05 | 新希望六和股份有限公司 | Lactobacillus salivarius NHE-LsE33 and application thereof |
| CN115927045B (en) * | 2022-07-13 | 2023-10-20 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactobacillus salivarius 069 with cholesterol reducing and liver injury relieving functions caused by hyperlipidemia and application thereof |
| CN115216434B (en) * | 2022-09-14 | 2023-06-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Lactobacillus salivarius strain and application thereof |
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