CN117264819A - Lactobacillus plantarum YYS-B1 with hyaluronidase inhibition activity and application thereof - Google Patents
Lactobacillus plantarum YYS-B1 with hyaluronidase inhibition activity and application thereof Download PDFInfo
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- CN117264819A CN117264819A CN202311208718.5A CN202311208718A CN117264819A CN 117264819 A CN117264819 A CN 117264819A CN 202311208718 A CN202311208718 A CN 202311208718A CN 117264819 A CN117264819 A CN 117264819A
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- lactobacillus plantarum
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Abstract
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum YYS-B1 with hyaluronidase inhibition activity and application thereof. Lactobacillus plantarum (Lactiplantibacillus plantarum) YYS-B1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.27599 in 2023, month 06 and 12. The lactobacillus plantarum YYS-B1 has good acid resistance and bile salt resistance, and can smoothly reach intestinal tracts to exert the effect of probiotics; the hyaluronic acid gel has outstanding antiallergic, anti-inflammatory and antioxidant capabilities, is particularly high in hyaluronidase inhibition capability and strong in free radical removal capability, has a good inhibition effect on inflammatory reactions caused by allergy, can be applied to development of antiallergic foods, health-care products, cosmetics and other products, and has important application value.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum YYS-B1 with hyaluronidase inhibition activity and application thereof.
Background
Allergy is a complex immunological disease, and its symptoms include asthma, urticaria, food allergy, and inflammatory reactions such as rhinitis and dermatitis, which seriously affect the quality of life of patients. Current allergy treatment methods have side effects and risks, and therefore, a need exists to seek safe and effective antiallergic strategies.
In recent years, research on probiotics beneficial to human health has been attracting attention. Studies have shown that: probiotics exert antiallergic effect by inhibiting hyaluronidase activity, which is an enzyme capable of hydrolyzing hyaluronic acid, and excessive expression can cause excessive decomposition of hyaluronic acid to induce anaphylaxis. Inhibition of hyaluronidase can inhibit allergic reaction, plays an important role in type I and type IV allergic reaction, and has a certain degree of anti-inflammatory effect. Some natural products and compounds have been found to have an inhibitory effect on hyaluronidase activity, but have limited application. The existing research proves that: the probiotics such as lactobacillus plantarum can effectively inhibit the activity of hyaluronidase, reduce the IgE level and relieve allergic symptoms.
Raven et al have demonstrated that probiotics have hyaluronidase inhibiting effects, and lactobacillus plantarum (Lactiplantibacillus plantarum) and lactobacillus rhamnosus (Lactobacillus rhamnosus) have good inhibitory effects, and can also reduce IgE levels in serum.
In addition, in allergic diseases, an inflammatory reaction is often accompanied; the probiotics with anti-inflammatory activity can reduce allergic inflammatory reaction, regulate the balance of immune system, promote tissue repair and protect mucous membrane barrier. Through these mechanisms, probiotics can reduce the release of inflammatory mediators, inhibit the activation and migration of inflammatory cells, thereby reducing the inflammatory response caused by allergy, helping to alleviate symptoms and improve the quality of life of patients.
The Chinese invention patent with the application number of CN201510613663 and the publication date of 2017, 08 and 05 discloses that: the highest hyaluronidase inhibition rate of the agilawood extract with the sensitivity-relieving and anti-allergic effects can reach 92.0% when the addition amount reaches 8.0 mg/mL; the DPPH free radical clearance rate reaches 78.0 percent.
At present, few probiotics have been reported for inhibiting hyaluronidase, and few probiotics have been reported for simultaneously resisting allergy, relieving allergic inflammation and reducing oxidative damage caused by allergy and inflammatory reaction.
Disclosure of Invention
In order to solve the defects in the prior art mentioned in the background art, the invention provides lactobacillus plantarum YYS-B1, which has the following technical scheme:
the lactobacillus plantarum YYS-B1 provided by the invention is derived from tuber mustard of the Xiamen city of Fujian, and is classified and named as: lactobacillus plantarum YYS-B1, latin Wen Xueming: lactiplantibacillus plantarum, deposited on the China general microbiological culture Collection center, accession number: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation number: CGMCC No.27599.
Wherein the strain has the following properties: has hyaluronidase inhibiting ability; lowering the level of lge; acid resistance and bile salt resistance; scavenging free radical DPPH; scavenging free radical ABTS; reducing the release of inflammatory mediators.
Thus, it has the following functions: can inhibit hyaluronidase and reduce IgE level, and has antiallergic effect; can inhibit inflammatory reaction caused by allergy, and has anti-inflammatory effect; can prevent oxidative damage caused by anaphylaxis, and has antioxidant capacity; the product has good acid resistance and bile salt resistance, can be applied to the preparation of probiotic products and antiallergic or anti-inflammatory products, and provides new directions and more possibilities for the development of the antiallergic or anti-inflammatory products.
The invention provides application of lactobacillus plantarum YYS-B1 in preparation of a probiotic product.
The invention provides application of lactobacillus plantarum YYS-B1 in preparing an antiallergic product.
The invention provides application of lactobacillus plantarum YYS-B1 in preparing an anti-inflammatory product.
The invention provides application of lactobacillus plantarum YYS-B1 in preparation of an antioxidant product.
The invention also provides freeze-dried powder, which comprises the components of the lactobacillus plantarum YYS-B1.
In one embodiment, the viable count of the Lactobacillus plantarum YYS-B1 is not less than 1×10 11 CFU/g。
The invention also provides a microbial inoculum, the components of which comprise the freeze-dried powder and/or the components of which comprise the lactobacillus plantarum YYS-B1.
In one embodiment, the microbial agent further comprises other ingredients that are currently available for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-binding agents, integration agents, thickening agents, and inclusion agents.
The invention provides application of lactobacillus plantarum YYS-B1 in preparing functional products.
In one embodiment, the functional products include foods, health products, cosmetics, and medicines.
In one embodiment, the functional product comprises at least one of the following actions:
(1) Has hyaluronidase inhibiting ability;
(2) Lowering the level of lge;
(3) Acid resistance and bile salt resistance;
(4) Scavenging free radical DPPH;
(5) Scavenging free radical ABTS;
(6) Reducing the release of inflammatory mediators.
Based on the above, compared with the prior art, the lactobacillus plantarum YYS-B1 provided by the invention has the following beneficial effects:
the lactobacillus plantarum YYS-B1 has outstanding antiallergic, anti-inflammatory and antioxidant capabilities, is particularly high in hyaluronidase inhibition capability and strong in free radical removal capability, has anti-inflammatory activity, has the antiallergic capability, has good inhibition effect on inflammatory response caused by allergy, and can effectively relieve allergic response and oxidative damage of organisms caused by inflammatory response. In addition, the microbial agent has good acid resistance and bile salt resistance, can smoothly reach intestinal tracts to exert the effect of probiotics, can be applied to preparing products such as probiotic products, antiallergic products or anti-inflammatory products, has no side effect as natural probiotics, is safer and healthier, provides good microbial resources for developing antiallergic or anti-inflammatory products (foods, health-care products, cosmetics and the like), and provides new directions and more possibilities for product development.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that other drawings can be obtained from them without inventive effort for a person skilled in the art; the positional relationships described in the drawings in the following description are based on the orientation of the elements shown in the drawings unless otherwise specified.
FIG. 1 is a physical diagram of a calcium-dissolving ring in the separation process of lactobacillus plantarum YYS-B1;
FIG. 2 is a physical diagram of colony morphology of Lactobacillus plantarum YYS-B1;
FIG. 3 is a graph showing the result of gram staining of Lactobacillus plantarum YYS-B1;
FIG. 4 is an agarose gel electrophoresis chart of the 16S rRNA amplification product of Lactobacillus plantarum YYS-B1;
FIG. 5 is a graph showing the effect of Lactobacillus plantarum YYS-B1 on the activity of mouse serum and liver superoxide dismutase (SOD);
FIG. 6 is a graph showing the effect of Lactobacillus plantarum YYS-B1 on mouse serum and liver glutathione peroxidase (GSH-PX) activity;
FIG. 7 is a schematic diagram of a phylogenetic tree of Lactobacillus plantarum YYS-B1 based on the 16S rRNA sequence
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention; the technical features designed in the different embodiments of the invention described below can be combined with each other as long as they do not conflict with each other; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the description of the present invention, it should be noted that all terms used in the present invention (including technical terms and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs and are not to be construed as limiting the present invention; it will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The invention provides lactobacillus plantarum YYS-B1:
latin Wen Xueming of Lactobacillus plantarum YYS-B1: lactiplantibacillus plantarum, deposited on the China general microbiological culture Collection center, accession number: CGMCC No.27599.
The source is as follows: separating to Fujian Xiamen Wujiang hot pickled mustard tuber;
colony morphology: the surface of the bacterial colony is convex, milky white, circular and smooth;
the strain has the following characteristics: has hyaluronidase inhibiting ability; lowering the level of lge; acid resistance and bile salt resistance; scavenging free radical DPPH; scavenging free radical ABTS; reducing the release of inflammatory mediators.
Thus, it has the following functions: can inhibit hyaluronidase and reduce IgE level, and has antiallergic effect; can inhibit inflammatory reaction caused by allergy, and has anti-inflammatory effect; can prevent oxidative damage caused by anaphylaxis, and has antioxidant capacity; the product has good acid resistance and bile salt resistance, can be applied to the preparation of probiotic products and antiallergic or anti-inflammatory products, and provides new directions and more possibilities for the development of the antiallergic or anti-inflammatory products.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1: isolation of Lactobacillus plantarum YYS-B1
Aseptically sampling from the Wujiang hot pickled mustard tuber, taking 5g of the Wujiang hot pickled mustard tuber sample, placing the sample into an asepsis homogenizing bag, marking, adding 45ml of 0.85% physiological saline, and fully beating and uniformly mixing to obtain the sample.
Then 100 mu L of sample is sucked for 10 times serial gradient dilution, and the dilution times are respectively sucked for 10 times -4 、10 -5 、10 -6 Is coated on a sample 100. Mu.L containing 2.5% CaCO 3 Is cultured in an inverted manner at 37℃for 24 hours on the MRS plate. The colony with good growth vigor and large calcium dissolving ring is picked up (see figure 1), and repeatedly separated and purified by a plate streak separation method until a single colony is obtained, wherein the colony is in the shape of protruding surface of the colony, milky white, round and smooth in surface (see figure 2), the separated strain is named as YYS-B1, numbered, and glycerol is added for preservation in a fungus bank at-80 ℃.
Example 2: identification of Lactobacillus plantarum YYS-B1
2.1 physiological Biochemical assay
The screened and purified strain YYS-B1 was subjected to gram staining (see FIG. 3) and catalase test, and its physiological and biochemical indexes were measured, and the test results were compared with "Berger's System bacteriology Manual eighth edition" to carry out preliminary determination of the strain. The test shows that the gram staining of the screening strain YYS-B1 is purple and positive. The cell shape is rod-shaped, and is negative to contact enzyme, and no spore is formed.
5.2 16S rRNA identification
YYS-B1DNA was extracted according to the instructions of the bacterial gene DNA extraction kit, and PCR amplification of the 16S rRNA gene was performed using the DNA as a template.
Wherein, the universal primers are used as the amplification primers:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.1)。
1492R：5'-TACGGCTACCTTGTTACGACTT-3'(SEQ ID NO.2)。
the PCR reaction system is as follows: DNA 2. Mu.L, 27F 2. Mu.L, 1492R 2. Mu.L, premix Ex Taq 25. Mu.L, ddH 2 O 19μL。
PCR reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; finally, the extension is carried out for 5min at 72 ℃.
YYS-B1 16S rRNA amplified fragment after DNA genome extraction (see FIG. 4). The PCR amplified product was then sent to DNA sequencing (Optimus engineering, praeparata). Sequencing sequence results similar sequences were searched in NCBI database using Blast software, and the sequences were aligned with the 16S rRNA gene sequences of related species obtained from the gene library, and phylogenetic tree was constructed using Mega 11.0 software (see FIG. 7).
The 16S rRNA gene sequence was determined as follows:
TTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTGC(SEQ ID NO.3)。
the bacterial strain is identified as lactobacillus plantarum (Lactiplantibacillus plantarum) according to the colony and thallus morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and other data comprehensive analysis, and reference is made to the eighth edition of Bojk system bacteriology handbook.
Example 3: lactobacillus plantarum YYS-B1 hyaluronidase inhibition assay
3.1 hyaluronidase in vitro inhibition assay
Lactobacillus plantarum YYS-B1 strain was activated to second generation, reference was made to the Elson-Morgan improvement method. 0.1mL of 2.5 mmol/L CaCl was added to the tube 2， Adding 0.5mL of 600U/mL hyaluronidase, and treating at 37deg.C for 20min; adding 0.5mL of the bacterial suspension, treating for 20min at 37 ℃, adding 0.5mL of 0.5mg/mL sodium hyaluronate solution, and treating for 30min at 37 ℃; standing at room temperature for 5min, adding 0.5mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20mL of Na at 1.0 mol/L) 2 CO 3 Solution, as prepared) and 0.1mL of 0.4 mol/L NaOH solution, boiling water for 15min, and immediately ice-bathing for 5min; 1mL of an Escherichia reagent (0.8 g of p-dimethylaminobenzaldehyde is weighed and dissolved in 15mL of concentrated hydrochloric acid and 15mL of absolute ethyl alcohol, which are prepared in situ) is added, color development is performed at normal temperature for 20min, and the absorbance is measured at a wavelength of 530 nm.
The inhibition rate calculation mode is as follows:
inhibition ratio = [ (a-B) - (C-D) ]/(a-B) ×100%;
wherein A is the absorbance of the control group (acetic acid buffer solution replaces bacterial suspension), B is the absorbance of the control blank group (acetic acid buffer solution replaces enzyme solution and bacterial suspension), C is the absorbance of the sample group, and D is the absorbance of the sample blank group (acetic acid buffer solution replaces enzyme solution).
3.2 preparation of solutions
4.8mL of solution A (0.2 mol/L acetic acid 11.55mL glacial acetic acid in 1L distilled water), 45.2mL of solution B (0.2 mol/L sodium acetate 16.4g anhydrous sodium acetate or 27.2g sodium acetate trihydrate in 1L distilled water) and mixed and diluted to 100mL to prepare an acetic acid buffer solution with pH=5.6; acetylacetone solution: 50mL of 1.0 mL/L sodium carbonate solution and 3.5mL of acetylacetone solution were mixed well (ready-to-use). P-DAB color developer: 0.8g of p-dimethylaminobenzaldehyde is dissolved in 15mL of concentrated hydrochloric acid and 15mL of absolute ethyl alcohol and uniformly mixed.
MRS liquid medium: 20.0g of glucose, 10.0g of tryptone, 10.0g of beef extract, 5.0g of yeast extract, 1.0mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 2.0g of ammonium citrate, 5.0g of anhydrous sodium acetate, 0.5g of magnesium sulfate, 0.25g of manganese sulfate monohydrate, 1L of deionized water and pH=6.5 (1.5 percent of agar is added as MRS solid culture medium).
In this experiment, hyaluronic acid was used as a substrate, an enzyme reaction was performed under specific conditions, and a change in activity thereof was measured. According to the experimental result, by absorbance OD 530nm The detection shows that YYS-B1 fermentation obviously reduces the activity of hyaluronidase, and the inhibition rate can reach 76.2%.
The lactobacillus plantarum YYS-B1 provided by the invention has excellent hyaluronidase inhibition capability and can be applied to preparing antiallergic products.
Example 4: lactobacillus plantarum YYS-B1 DPPH free radical clearance determination test
4.1 preparation of solutions
DPPH solution: 0.002g of DPPH is dissolved in 50mL of ethanol each time and stored in a dark place. It is diluted with ethanol to absorbance of about 0.7 before use.
4.2 detection analysis of samples
Activating Lactobacillus plantarum YYS-B1 to second generation, centrifuging at 8000r/min for 10min, discarding supernatant, and re-suspending thallus with sterile PBS to give thallus concentration of 1×10 8 cfu/mL. The specific tests are divided into A, B, C groups, the dosages of all reactants are shown in Table 1, all reactants are sequentially added into a standard 96-well plate, incubated for 30min at room temperature under the dark condition, and the absorbance at 517nm is measured.
Wherein, the calculation formula of the clearance rate of the lactobacillus plantarum YYS-B1 to DPPH free radicals is as follows:
inhibition ratio = [1- (a-C)/B ] ×100%;
in the formula, A is the absorbance of the sample group, B is the absorbance of the blank group, and C is the absorbance of the control group.
TABLE 1
| Experimental group | Dilution DPPH (mu L) | Absolute ethyl alcohol (mu L) | Sample to be measured (mu L) |
| A | 100 | 0 | 100 |
| B | 0 | 100 | 100 |
| C | 100 | 100 | 0 |
By at wavelength OD 517nm Detecting a detection sample to obtain: YYS-B1 has a DPPH free radical scavenging rate of 86.2% + -1.3. The lactobacillus plantarum YYS-B1 provided by the invention has good DPPH free radical scavenging effect and can be applied toAnd (5) developing an antioxidant product.
Example 5: lactobacillus plantarum YYS-B1 ABTS free radical clearance assay
5.1 preparation of solutions
ABTS stock (7 mmol/L): ABTS 0.0384g was weighed and the volume was fixed with distilled water to 10mL. K (K) 2 S 2 O 8 Stock solution (2.45 mmol/L): weighing K 2 S 2 O 8 0.0066g, and distilled water to a volume of 10mL. ABTS working fluid: ABTS stock solution and K 2 S 2 O 8 Stock solution 1:1, mixing evenly, standing for 12-16h, and preparing the ABTS working solution.
5.2 detection analysis of samples
Activating Lactobacillus plantarum YYS-B1 to second generation, centrifuging at 8000r/min for 10min, discarding supernatant, and re-suspending thallus with sterile PBS to give thallus concentration of 1×10 8 cfu/mL. Taking a proper amount of ABTS working solution, diluting to OD by using PBS solution 734nm 0.7. The specific experiments are divided into A, B, C groups, the dosages of all reactants are shown in Table 2, all reactants are sequentially added into a standard 96-well plate, incubated for 6min at room temperature under the dark condition, and the absorbance at 734nm is measured. The formula of the free radical clearance of the plant lactobacillus YYS-B1 to the ABTS is as follows:
inhibition ratio = [1- (a-C)/B ] ×100%;
wherein A is the absorbance of the sample group, B is the absorbance of the blank group, and C is the absorbance of the control group.
TABLE 2
| Experimental group | ABTS working fluid (mu L) | PBS(μL) | Sample to be measured (mu L) |
| A | 100 | 0 | 100 |
| B | 0 | 100 | 100 |
| C | 100 | 100 | 0 |
By at wavelength OD 734nm The detection sample detects YYS-B1 to achieve 77.3% +/-1.4 of ABTS free radical clearance. The lactobacillus plantarum YYS-B1 provided by the invention has good ABTS free radical scavenging effect, and can be applied to the development of antioxidant products.
Example 6: preparation method of lactobacillus plantarum YYS-B1 bacterial powder
6.1 preparation of the Material used
The culture medium and lyoprotectant used in this example were as follows:
the seed culture medium comprises the following components in percentage by mass: 3% glucose, 2% peptone, 1.5% yeast extract, 1% beef extract, 0.5% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.06% manganese sulfate, 0.1% tween 80 and the balance water, wherein the pH value is 6.8;
the fermentation medium comprises the following components in percentage by mass: 2.3% glucose, 2.5% soy peptone, 2% yeast extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% tween 80 and the balance water;
the freeze-drying protective agent comprises the following components in percentage by mass: 10% of trehalose, 8% of skim milk powder, 0.5% of glycerol and the balance of water.
6.2 preparation process:
the preparation process of the freeze-dried powder comprises the following steps:
s1, inoculating lactobacillus plantarum YYS-B1 into a test tube filled with a seed culture medium for activation, and culturing in a constant-temperature incubator for 20 hours to obtain primary seed liquid.
S2, inoculating the first-stage seed liquid into a triangular flask filled with a seed culture medium, wherein the inoculation amount is 3% (mass percent), and culturing in a constant-temperature incubator for 18 hours to obtain the second-stage seed liquid.
S3, inoculating the secondary seed liquid into a triangular flask filled with a seed culture medium, wherein the inoculation amount is 5% (mass percent), and culturing in a constant-temperature incubator for 16 hours to obtain the tertiary seed liquid.
S4, inoculating the three-level seed liquid into a fermentation tank filled with a fermentation culture medium, wherein the inoculum size is 5% (mass percent), the stirring speed is preferably 50r/min, the fermentation initial pH is 6.0, and the culture is carried out at 37 ℃ for 8 hours.
S5, the viable count of the liquid to be fermented is more than or equal to 2.9X10 9 At cfu/mL, fermentation was stopped, and the cells were collected by centrifugation (8000 r/min,15 min).
S6, emulsifying and embedding thalli and a freeze-drying protective agent according to a mass ratio of 1:0.6, wherein the emulsifying temperature is 20 ℃, and the emulsifying time is 20min;
s7, pre-freezing the emulsion for 6 hours, vacuum freeze-drying for 30 hours at minus 30 ℃, wherein the vacuum degree is 0.2mbar, and crushing to obtain the lactobacillus plantarum YYS-B1 bacterial powder, wherein the viable count is 2.9x10 11 cfu/g。
Example 7: effect of Lactobacillus plantarum YYS-B1 on allergic and inflammatory responses in mice
7.1 Experimental preparation and procedure
40 BALB/c male mice were used as subjects, and were divided into 4 groups: normal control group, SEB+OVA model group, model control group, lactobacillus plantarum YYS-B1 group.
In addition to the normal control group, the other groups were administered by intraperitoneal injection at days 0, 3, 9, 11 and 14 with 0.2mL of sterile saline containing 10. Mu.g of Staphylococcus aureus enterotoxin B (SEB) and 20. Mu.g of Ovalbumin (OVA), respectively. After the day 14 of the day, the time period,model group 0.2mL of sterile saline containing 10 μg of Staphylococcus aureus enterotoxin B (SEB) and 20 μg of Ovalbumin (OVA); the model control group is injected with 0.2mL of sterile physiological saline; lactobacillus plantarum YYS-B1 lyophilized powder of Lactobacillus plantarum YYS-B1 obtained in example 6 is added with 0.9% physiological saline to prepare 2% fungus powder suspension with concentration of 5.8X10 9 cfu/mL, administered once daily for 7 consecutive days, 0.2mL.
7.1 test procedure
The total lge levels in mouse serum were determined using ELI SA, an indicator commonly used for the assessment of allergic reactions (test results are shown in table 3).
The amount of inflammatory factors such as IL-4 and I FN-gamma in the serum of mice was measured by ELI SA (the test results are shown in Table 4).
In addition, the content of diamine oxidase (DAO) in the serum of mice was determined, which is closely related to allergic reaction (the test results are shown in table 3).
Table 3 Lactobacillus plantarum YYS-B1 treatment and control mice serum I gE and DAO comparison (n=10, mean.+ -. SD)
Table 4 Lactobacillus plantarum YYS-B1 treated and control mice were compared (n=10, mean.+ -. SD)
7.3 test results:
the SEB+OVA experimental group showed significantly higher IgE levels compared to the normal control group, and the difference between groups was statistically significant (P < 0.05). This indicates that: the method is used for successfully inducing the food allergy of the mice.
After gavage of lactobacillus plantarum YYS-B1, ie was significantly reduced compared to the experimental group (P < 0.05). IL-4 is a cytokine that significantly increases IL-4 levels in the allergic body and decreases IFN-gamma levels. In this study, after the gastric lavage of Lactobacillus plantarum YYS-B1, the level of I FN-gamma in the serum of sensitized mice was significantly increased, while the level of IL-4 was significantly decreased.
The experimental results show that:
lactobacillus plantarum YYS-B1 can down-regulate elevated Th2 cytokines (i.e., reduce the release of inflammatory mediators), correct Th1/Th2 imbalance and relieve allergic symptoms. The experiment shows that the lactobacillus plantarum YYS-B1 can reduce allergic reaction of the allergic mice IgE and inflammatory reaction caused by allergy.
Example 8: lactobacillus plantarum YYS-B1 for measuring activity of mouse serum and liver superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)
8.1 Experimental raw materials and Processes
Healthy BALB/c male mice were randomly divided into experimental and control groups of 20 mice each. The lactobacillus plantarum YYS-B1 powder obtained in example 6 is added with 0.9 percent of physiological saline to prepare 2 percent of powder suspension with the concentration of 5.8x10 9 cfu/mL, 0.2mL of mice in the experimental group, and 0.9% normal saline in the control group in equal amount. Treatment was continued for 4 weeks, once daily. After the end of the experiment, mouse serum and liver tissue samples were taken. And (3) measuring related indexes by using an SOD and GSH-PX activity measuring kit.
8.1 test results:
the results of the serum SOD activity measurement of mice are shown in figures 5 and 6, and compared with a control group, the serum and liver SOD activity and GSH-PX activity of the mice in the experimental group are obviously improved, which shows that after the mice eat lactobacillus plantarum YYS-B1 bacterial powder, the SOD activity and GSH-PX activity of the organisms can be obviously improved, and the antioxidant capacity of the organisms is enhanced.
Example 9: acid resistance and bile salt resistance test of Lactobacillus plantarum YYS-B1
9.1 simulated gastric acid resistance test
0.2g NaC L and 0.35g pepsin are weighed and dissolved in a proper amount of distilled water to prepare 3 parts, the pH values are respectively adjusted to 1.5, 2.5 and 3.5 by 1.0 mol/L hydrochloric acid, and the volume is fixed to the scale by a 100mL volumetric flask. The Lactobacillus plantarum YYS-B1 bacterial powder obtained in example 6 is respectively inoculated into simulated artificial gastric acid solutions with different pH values according to the inoculation amount of 1% (w/v), the bacterial powder is subjected to water bath constant temperature treatment at 37 ℃ for 0, 2 and 4 hours, then respectively subjected to gradient dilution by 10 times, 100 mu L of bacterial liquid is taken out after uniform oscillation, the bacterial liquid is poured into a plate, and the bacterial liquid is cultured at 37 ℃ for 24 hours, and the number of YYS-B1 viable bacteria is calculated, and the measured results are shown in Table 5.
TABLE 5
As can be seen from table 5:
in simulated artificial gastric juice with different pH values, the number of the viable bacteria of the lactobacillus plantarum YYS-B1 is different, and the survival rate is also improved continuously along with the increase of the pH value. In simulated artificial gastric fluid at the same pH, the number of viable bacteria decreases with increasing treatment time.
However, in simulated artificial gastric juice with different pH values, the number of viable bacteria is kept at a higher level after 2 hours of treatment, which shows that YYS-B1 can still have higher survival number after digestion by human gastric juice and has excellent tolerance to gastric acid.
9.2 simulation of intestinal juice bile salt tolerance test
The lactobacillus plantarum YYS-B1 freeze-dried powder obtained in example 6 is respectively inoculated into pig gall salt solution with mass concentration of 0.03g/mL, 0.3g/mL and 0.5g/mL according to the inoculation amount of 1% (w/v), the pig gall salt solution is subjected to constant temperature treatment for 0, 2 and 4 hours in a water bath at 37 ℃, then respectively subjected to 10-time gradient dilution, 100 mu L of bacterial liquid is taken after uniform oscillation, and after 24 hours of culture at 37 ℃, YYS-B1 viable count is calculated, and the result is shown in Table 6.
TABLE 6
As can be seen from table 6:
the lactobacillus plantarum YYS-B1 has different viable counts and survival rates under the condition of simulating different initial mass concentrations of the bile salts of the artificial intestinal juice. Along with the increase of the mass concentration of the simulated intestinal juice bile salt, the initial viable count is gradually reduced, and the survival rate is gradually reduced as the treatment time is longer.
However, after the treatment for 2 hours, the human body has higher survival number after the closest bile salt concentration (the bile salt concentration in the small intestine of the human body is generally 0.03-0.3 g/mL), which indicates that YYS-B1 can still have more living bacteria after being digested by the stomach and the intestines in the human body. The lactobacillus plantarum YYS-B1 provided by the invention has excellent gastric acid and bile salt tolerance, can smoothly reach intestinal tracts, regulates intestinal flora and plays a probiotic effect.
Example 10: preparation of probiotic products by using Lactobacillus plantarum YYS-B1 of the invention
10.1 probiotic products
The probiotic product is a composition of lactobacillus plantarum YYS-B1 and other ingredients; in the product, the viable count of the lactobacillus plantarum YYS-B1 is not less than 1 multiplied by 10 8 CFU/mL or 1X10 8 CFU/g。
The ingredients include, but are not limited to, one or more of prebiotics, fillers, acidulants, solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, and filter aids.
It should be noted that: the probiotic product may be in the form of a solid beverage, a liquid beverage, a tableted confection, a granule, a capsule, a tablet, a pill or an oral liquid, etc. of a type of food suitable for and designed for human consumption.
10.2 probiotic product implementation examples of solid beverages:
in the embodiment, a probiotic product (lactobacillus plantarum YYS-B1 solid beverage) with a solid beverage dosage form is provided, and the probiotic product comprises, by weight, 10 parts of active fungus powder, 24.8 parts of xylitol, 24 parts of whole milk powder, 12 parts of fermented mulberry powder, 23 parts of maltodextrin, 5.6 parts of galacto-oligosaccharide, 0.1 part of fructo-oligosaccharide and 0.5 part of anhydrous citric acid.
The preparation method of the solid beverage comprises the following steps:
s1, culturing the lactobacillus plantarum YYS-B1 strain with a liquid culture solution, collecting and washing thalli, adding auxiliary materials (the protective agent in the embodiment 8), and drying to prepare active bacterial powder;
s2, uniformly mixing active bacterial powder, xylitol, whole milk powder, fermented mulberry powder, maltodextrin, galactooligosaccharide, fructooligosaccharide and anhydrous citric acid to obtain lactobacillus plantarum YYS-B1 with the viable count of not less than 1 multiplied by 10 8 CFU/g solid beverage.
It should be noted that: lactobacillus plantarum YYS-B1 lyophilized powder or other forms of Lactobacillus plantarum YYS-B1 can be added to food, and the ingredients can be existing food auxiliary materials and additives, including but not limited to those described in the examples.
In summary, the lactobacillus plantarum YYS-B1 provided by the invention comprises the following properties, effects and applications:
1. from the aspect of action mechanism, the lactobacillus plantarum YYS-B1 has the following actions:
(1) Has hyaluronidase inhibiting ability; the apparent efficacy is mainly antiallergic;
(2) Lowering the level of lge;
(3) Acid resistance and bile salt resistance;
(4) Scavenging free radical DPPH; the display effect is mainly antioxidation;
(5) Scavenging free radical ABTS; the display effect is mainly antioxidation;
(6) Reducing the release of inflammatory mediators; for example, the use of the bacterium can reduce the content of proinflammatory factor IL-4 and increase the content of anti-inflammatory factor IFN-gamma to inhibit the reaction and proliferation of Th2 cells, thereby reducing the release of cytokines secreted by the Th2 cells (namely inflammatory mediators) and reducing IgE antibody production, and playing an anti-inflammatory role.
2. From a functional characteristic point of view, it has the following functional characteristics:
the anti-allergic, anti-inflammatory and antioxidant effects are outstanding, the anti-allergic and antioxidant agent has the anti-allergic capability, has a good inhibition effect on inflammatory reaction caused by allergy, and can effectively relieve oxidative damage of organisms caused by allergic reaction and inflammatory reaction.
Lactobacillus plantarum YYS-B1 shows excellent gastric acid and bile salt tolerance, can smoothly reach the intestinal tract, regulate intestinal flora and exert the effect of probiotics.
3. Compared with the prior art, the invention has the advantages that:
at present, no oral probiotic strain having both hyaluronidase inhibiting, antiallergic and anti-inflammatory effects has been reported. Unlike the existing probiotic antiallergic preparation on the market, the lactobacillus plantarum YYS-B1 has the advantages that:
(1) The hyaluronidase inhibition rate is high;
(2) Can prevent and inhibit inflammatory reaction caused by allergy;
(3) Has excellent antioxidant capacity, and can effectively relieve oxidative damage of organism caused by anaphylaxis and inflammatory reaction.
In summary, the strain of the present invention can be used alone to effectively achieve antiallergic, antiinflammatory and antioxidant effects:
antiallergic: the lactobacillus plantarum YYS-B1 has outstanding antiallergic performance, and has a hyaluronidase inhibition rate of 76.2% over most other strains on the market, and can reduce the medium released by mast cells, thereby reducing anaphylactic reaction.
Antioxidant and anti-inflammatory:
the results of the antioxidant experiments show that: the strain has radical scavenging rates DPPH and ABTS of 86.2% and 77.3%, respectively. The mouse experiment shows that: the strain has the capabilities of resisting oxidization and inflammation, and has important significance for preventing and reducing allergy and inflammation.
In conclusion, the lactobacillus plantarum YYS-B1 has outstanding antiallergic, anti-inflammatory and antioxidant capabilities, particularly has very high hyaluronidase inhibition capability and very strong free radical removal capability, has anti-inflammatory activity, has antiallergic capability, has very good inhibition effect on inflammatory response caused by allergy, and can effectively relieve anaphylactic reaction and oxidative damage of organisms caused by inflammatory response. In addition, the microbial agent has good acid resistance and bile salt resistance, can smoothly reach intestinal tracts to exert the effect of probiotics, can be applied to preparing products such as probiotic products, antiallergic products or anti-inflammatory products, has no side effect as natural probiotics, is safer and healthier, provides good microbial resources for developing antiallergic or anti-inflammatory products (foods, health-care products, cosmetics and the like), and provides new directions and more possibilities for product development.
4. Lactobacillus plantarum YYS-B1 is applicable, depending on its role and properties, in functional products comprising at least one of the following roles:
(1) Has hyaluronidase inhibiting ability (mainly antiallergic effect);
(2) Lowering the level of lge;
(3) Acid resistance and bile salt resistance;
(4) Scavenging free radical DPPH (antioxidant);
(5) Scavenging free radical ABTS (antioxidant);
(6) Reducing the release of inflammatory mediators.
Among them, specifically described is: the bacteria YYS-B1 with the functions of (1) - (6) can be applied to antioxidant products, antiallergic products, anti-inflammatory products, and probiotic functional products, and has the obvious effects of resisting oxidation, inflammation, allergy and the like; it may also have a mechanism of action based on (1) - (6), yielding other apparent efficacy. Wherein the functional product includes, but is not limited to, food, health products, cosmetics or medicines.
It should be noted that:
(1) Definition:
the term "food" as used herein is broad and includes human foods and drinks. In certain embodiments, the food product employs dosage forms suitable for and designed for human consumption, including but not limited to solid beverages, liquid beverages, tabletted candies, granules, capsules, tablets, pills, or oral liquids, and the like.
"DPPH" is free radical 2, 2-biphenyl-1-picrylhydrazyl, "ABTS" is free radical 2,2' -biazino-bis-3-ethylbenzothiazoline-6-sulfonic acid.
"IgE" refers to immunoglobulin, which is an antibody of human body, which exists in blood and is the least amount of immunoglobulin in normal human serum.
The "OD" value is an abbreviation of optical density (also called absorbance), and the difference between the energy of light passing through the object to be measured and the energy of light passing through the object to be measured is the energy absorbed by the object to be measured. "ODx" is an optical density value measured when the wavelength is set to x nm, and is a standard indicator for tracking the microorganism density in a liquid culture, and is usually used to indicate the cell density of a cell. Among these, the "OD" value determination method is prior art, and the principle and method thereof will not be described here.
The experimental procedures referred to in the examples of the present invention are conventional in the art, and reagents or instruments referred to are commercially available from regular sources, unless explicitly indicated otherwise.
In addition, it should be understood by those skilled in the art that although many problems exist in the prior art, each embodiment or technical solution of the present invention may be modified in only one or several respects, without having to solve all technical problems listed in the prior art or the background art at the same time. Those skilled in the art will understand that nothing in one claim should be taken as a limitation on that claim.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the 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 scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. Lactobacillus plantarum YYS-B1 with hyaluronidase inhibitory activity is characterized in that: the preservation number is CGMCC No.27599.
2. Use of lactobacillus plantarum YYS-B1 as claimed in claim 1 for the preparation of a probiotic product.
3. Use of lactobacillus plantarum YYS-B1 as claimed in claim 1 for the manufacture of an antiallergic product.
4. Use of lactobacillus plantarum YYS-B1 as claimed in claim 1 for the manufacture of an anti-inflammatory product.
5. Use of lactobacillus plantarum YYS-B1 as claimed in claim 1 for the manufacture of an antioxidant product.
6. A lyophilized powder, characterized in that: a component comprising lactobacillus plantarum YYS-B1 as claimed in claim 1.
7. A microbial inoculum, characterized in that: the components of the microbial inoculum comprise the freeze-dried powder as claimed in claim 6, and/or the components of the microbial inoculum comprise the lactobacillus plantarum YYS-B1 as claimed in claim 1.
8. Use of lactobacillus plantarum YYS-B1 as claimed in claim 1 for the manufacture of functional products.
9. The use according to claim 8, characterized in that: the functional products comprise foods, health products, cosmetics and medicines.
10. The use according to claim 8, characterized in that: the functional product comprises at least one of the following functions:
(1) Has hyaluronidase inhibiting ability;
(2) Lowering IgE levels;
(3) Acid resistance and bile salt resistance;
(4) Scavenging free radical DPPH;
(5) Scavenging free radical ABTS;
(6) Reducing the release of inflammatory mediators.
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