CN113088463A - Lactobacillus acidophilus with probiotic characteristics and application thereof - Google Patents
Lactobacillus acidophilus with probiotic characteristics and application thereof Download PDFInfo
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- CN113088463A CN113088463A CN202110285880.1A CN202110285880A CN113088463A CN 113088463 A CN113088463 A CN 113088463A CN 202110285880 A CN202110285880 A CN 202110285880A CN 113088463 A CN113088463 A CN 113088463A
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- lactobacillus acidophilus
- food
- lactobacillus
- acidophilus
- bacterial
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Abstract
The invention discloses lactobacillus acidophilus with probiotic characteristics and application thereof, and belongs to the technical field of microorganisms. Lactobacillus acidophilus of the present invention: (Lactobacillus acidophilus) The lactobacillus acidophilus LA85 with the preservation number of CGMCC No.21802 belongs to human lactic acid bacteria, has high capability of resisting gastrointestinal tract stress, has obvious inhibiting effect on conditional pathogenic bacteria, namely escherichia coli, salmonella and staphylococcus aureus, is sensitive to common antibiotic drugs, has no drug resistance, has high growth rate, short growth period and high viable count, and can effectively regulate the organismThe immune response of the body can be used for preparing immunoregulation medicaments and used in the field of food.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus acidophilus with probiotic characteristics and application thereof.
Background
The human gastrointestinal tract contains a large number of beneficial bacterial communities that play an important role in maintaining human health. The normal, stable, beneficial gut microflora has multiple biological functions, such as improving digestion and absorption of nutrients, regulating the balance of gut microflora, stabilizing the integrity of the gut barrier, protecting against the adhesion and invasion of gut pathogens, stimulating host cell and humoral immune responses. The most common method of influencing the composition of the intestinal flora is dietary probiotics, and for many years many kinds of microorganisms have been used, which mainly consist of lactic acid bacteria and bifidobacteria, but also including bacilli and fungi. Traditional probiotics, such as lactic acid bacteria and bifidobacteria, have positive effects on preventing diseases and promoting human health, and have higher theoretical research and product application values.
Probiotics refer to living microorganisms that, when ingested in sufficient quantities, provide a health benefit to the host. The mechanism of probiotic action and the application value of the probiotic in different fields are further known by researching the survival condition of the probiotics in simulated gastrointestinal tract, the inhibition capability to the growth of pathogenic bacteria, the sensitivity to medicaments and the adhesion capability to the gastrointestinal tract.
The lactobacillus is a kind of probiotics with the widest application, the longest time and the most varieties, wherein the lactobacillus is widely applied, the lactobacillus acidophilus is one of the strains which are extremely important, researched and developed in lactobacillus families, the lactobacillus acidophilus has wide historical application in the dairy product industry, can secrete a large amount of acidic substances such as lactic acid, acetic acid and the like, can grow and reproduce in gastric juice with stronger acidity, is called as a 'mobile disinfection vehicle' in the gastric environment, and has an important role in maintaining the physiological functions of the gastrointestinal tract. Lactobacillus acidophilus is a main commercial species of lactobacillus, is widely added into commercial yoghurt and dairy product formulas and dietary supplements with beneficial effects due to the flavor and the probiotic effect of the lactobacillus acidophilus, is one of the most commonly used dietary lactobacillus varieties, and has higher application value.
Disclosure of Invention
The invention aims to provide lactobacillus acidophilus LA85 with probiotic characteristics and application thereof.
The purpose of the invention is realized by the following technical scheme:
lactobacillus acidophilus LA85 with probiotic property is classified and named Lactobacillus acidophilus, and is preserved in China general microbiological culture Collection center (CGMCC) at the time of 2021 year 02 and 01 month, with the address of No. 3 of Beijing Corp-Yang-facing district North Chen West Lu No. 1, and the preservation number of CGMCC No. 21802.
The lactobacillus acidophilus LA85 has the following characteristics:
(1) colony morphology: round, light gray, opaque, small bacterial colony, irregular edge, and non-wetting surface protrusion;
(2) the shape of the thallus: gram-positive bacilli have short-rod and long-rod forms, and two ends are round and do not produce spores;
(3) the growth characteristics are as follows: the optimum pH value of the culture medium is 6.0, the optimum temperature is 37 ℃, the growth is fast, the logarithmic growth phase is started after 4 hours, and the stationary phase is started after 10 hours.
The lactobacillus acidophilus LA85 has the following probiotic properties:
(1) the survival rate of the artificial gastric juice treated in pH2.5 for 3h is 99.07%;
(2) the survival rate of the treated intestinal juice is 98.93 percent after being treated in the artificial intestinal juice with the pH value of 8.0 for 3 hours;
(3) the growth of common conditional pathogenic bacteria such as escherichia coli, salmonella and staphylococcus aureus is strongly inhibited;
(4) shows different degrees of sensitivity to common antibiotic drugs and has no drug resistance.
The lactobacillus acidophilus LA85 can regulate the level of immune-related cell factors in mice, has an immune regulation function, and can be used for preparing immune regulation medicaments.
An immunomodulatory drug comprising said Lactobacillus acidophilus LA 85.
The Lactobacillus acidophilus LA85 can also be used in food field, such as health food, food containing active lactobacillus or fermented food, etc. The health food includes but is not limited to fermented food or microecologics.
A food product comprising said Lactobacillus acidophilus LA 85.
The invention has the beneficial effects that: the lactobacillus acidophilus LA85 provided by the invention belongs to human lactic acid bacteria, has higher capability of resisting gastrointestinal tract stress, has obvious inhibiting effect on conditional pathogenic bacteria such as escherichia coli, salmonella and staphylococcus aureus, is sensitive to common antibiotic drugs, has no drug resistance, has high growth rate, short growth cycle and high viable count, and can effectively regulate the immune response of organisms.
Drawings
FIG. 1 is a colony morphology of Lactobacillus acidophilus LA85 on MRS plate medium.
FIG. 2 is a gram-stained bacterial morphology of Lactobacillus acidophilus LA 85.
FIG. 3 is a graph of the growth curve and pH of Lactobacillus acidophilus LA85 in MRS medium.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Details not described in the examples are known to the person skilled in the art.
Example 1 isolation, screening and characterization of Lactobacillus acidophilus LA85
(1) Isolation and screening of strains
The strain is separated from the excrement of a healthy human body, and the specific separation and screening method comprises the following steps:
taking a healthy human body excrement sample 1g to 9mL of physiological saline, fully oscillating and dispersing the sample, taking 100 mu L of the sample for gradient dilution, and selecting 10 mu L of the sample-1、10-2、10-3Uniformly coating 100 mu L of each bacterial liquid on an MRS plate, placing the MRS plate under an anaerobic condition, culturing the MRS plate at 37 ℃ for 36-48 h, selecting suspected bacterial colonies, and carrying out microscopic examination on the suspected bacterial coloniesAnd (5) inspecting, carrying out primary screening and purification culture.
MRS medium formula (g/L): 10 parts of peptone, 5 parts of beef extract powder, 5 parts of yeast powder, 20 parts of glucose, 5 parts of anhydrous sodium acetate, 2 parts of diammonium hydrogen citrate, 801 parts of tween and K parts of2 HPO 4 2、MgSO4 0.2、MnSO40.05, pH6.5, autoclaving at 121 deg.C for 20min, and adding 1.5% agar powder into solid MRS culture medium.
(2) Morphology and 16S rDNA identification of strains
The morphological identification includes colony morphology of the strain on a plate culture medium and thallus morphology under a microscope after gram staining.
The colony and thallus morphology of Lactobacillus acidophilus LA85 of the present invention are shown in FIGS. 1 and 2. The bacterial colony is round, light gray and opaque, the edge is neat, the diameter is about 1-2 mm, and the surface is dry and convex. The thallus is gram-positive bacilli and arranged singly or in pairs.
Carrying out liquid amplification on the screened target strains, collecting thalli, extracting genome DNA, and carrying out amplification culture on the selected target strains by adopting a universal primer 27F: AGAGTTTGATCCTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT amplified its 16S rDNA fragment, and PCR amplification products were detected by agarose gel electrophoresis. And (2) carrying out sequencing analysis on the obtained target fragment, carrying out comparison analysis on the sequencing result in an NCBI database, and identifying the obtained strain as Lactobacillus acidophilus, wherein the Lactobacillus acidophilus is classified and named as Lactobacillus acidophilus, and the strain is preserved in China general microbiological culture Collection center (CGMCC) on 2021, 02/01 and has the address: beijing, Chaoyang district, Xilu No. 1, Beijing, institute of microbiology, national academy of sciences, zip code 100101, accession No. CGMCC No.21802, and Strain No. LA 85.
Example 2 Lactobacillus acidophilus LA85 growth and pH curves
Inoculating activated Lactobacillus acidophilus LA85 to 10mL fresh MRS liquid culture medium at 2% (V/V), anaerobically culturing at 37 deg.C for 24 hr, sampling at fixed time, and determining OD of the culture solution600And pH, 3 replicates per sample, with incubation time as abscissa, OD600Or pH as ordinate, and the strain growth curve and pH curve were plotted (see FIG. 3).The lactobacillus acidophilus LA85 enters a logarithmic growth phase within 4 hours and enters a stationary phase within 10 hours, and the fermentation period is short. The pH curve slowly decreases from 0 to 10 hours, and the pH is maintained at about 4.0 after 10 hours.
Example 3 tolerance of Lactobacillus acidophilus LA85 to Artificial gastrointestinal fluids
The formula of the artificial gastric juice comprises: preparing 0.85% physiological saline, adjusting pH to 2.5 with dilute hydrochloric acid, adding 0.3% pepsin, dissolving completely, and filtering with microporous membrane for sterilization.
The formula of the artificial intestinal juice comprises: preparing 0.85% physiological saline, adjusting pH to 8.0 with sodium hydroxide, adding 0.1% trypsin, dissolving completely, and filtering with microporous membrane for sterilization.
Activating an experimental strain to a stable period, transferring the experimental strain into the prepared artificial gastric juice and artificial intestinal juice at a ratio of 2% (V/V), repeating each treatment for 3 groups, determining the number of viable bacteria in the treatment solution, then incubating the treatment solution at 37 ℃ for 3 hours, determining the number of viable bacteria in the treatment solution again, and calculating the survival rate of the strain by adopting a formula of survival rate (%) (logN3/logN0) multiplied by 100%, wherein N3 is the number of viable bacteria after the artificial gastric juice/intestinal juice is treated for 3 hours, and N0 is the number of viable bacteria after the artificial gastric juice/intestinal juice is treated for 0 hour. The results of the tolerance of Lactobacillus acidophilus LA85 in artificial gastric juice and artificial intestinal juice are shown in Table 1. As can be seen from Table 1, the strain maintains very high survival rate after being treated in artificial gastric juice and intestinal juice for 3 hours, which indicates that the strain has good capability of tolerating the gastrointestinal juice.
TABLE 1L acidophilus LA85 survival rate for artificial gastric juice and intestinal juice tolerance
Example 4 inhibitory Activity of Lactobacillus acidophilus LA85 on pathogenic bacteria
Preparing an indicator bacterium suspension: respectively activating Escherichia coli, Salmonella and Staphylococcus aureus on plate culture medium, culturing at 37 deg.C for 24 hr, selecting thallus Porphyrae to normal saline to prepare bacterial suspension, and adjusting bacterial liquid concentration to 108-1010CFU/mL。
Preparing lactobacillus acidophilus LA85 fermentation liquor: lactobacillus acidophilus LA85 was inoculated at 1% (V/V) into MRS liquid medium and cultured anaerobically at 37 ℃ for 12 h.
Bacteriostatic experiments: cooling MRS broth culture medium containing 1.5% agar to about 55 deg.C, mixing with indicator bacteria suspension at a certain ratio to make the number of viable bacteria of indicator bacteria be 106CFU/mL or 108CFU/mL order of magnitude, rapidly pouring into a plate with Oxford cup in advance, cooling and solidifying the culture medium, taking out the Oxford cup, and injecting 200 μ L Lactobacillus acidophilus LA85 fermentation liquid (viable count is 10) into each hole8CFU/mL order), the zone diameter was measured after overnight incubation at 37 ℃. The results are shown in Table 2.
TABLE 2 inhibitory Effect of Lactobacillus acidophilus LA85 fermentation broth on pathogenic bacteria
From the above table, when the concentration of the indicator bacterium is 106And at CFU/mL, the lactobacillus acidophilus LA85 has very strong inhibition effect on the growth of staphylococcus aureus, escherichia coli and salmonella, particularly has the strongest inhibition effect on the escherichia coli, and the diameter of an inhibition zone reaches 44 mm. When the concentration of the indicator bacterium is adjusted to 108At CFU/mL, Lactobacillus acidophilus LA85 still shows strong inhibition effect on three pathogenic bacteria.
Example 5 Lactobacillus acidophilus LA85 cell surface hydrophobicity assay
Culturing Lactobacillus acidophilus LA85 in MRS liquid culture medium overnight, centrifuging 20mL bacterial liquid, removing supernatant, collecting thallus, washing cells with PBS buffer solution for 2 times, resuspending with PBS, shaking thoroughly to disperse cells, and adjusting thallus concentration to OD600About 1.0, taking 15mL of bacterial liquid to a test tube, adding 5mL of dimethylbenzene, taking the bacterial liquid without the dimethylbenzene as a reference, fully oscillating, standing for 20min, layering, taking the lower aqueous phase, and determining OD600Calculating the hydrophobic power (%) of the strain (1-At/A0) multiplied by 100%, wherein At is the water phase OD after the bacterial liquid and the dimethylbenzene are mixed uniformly600Value A0 is the initial OD of the bacterial liquid600The value is obtained.
The results showed that Lactobacillus acidophilus strain LA85OD before and after xylene treatment of body600The values are 0.649 and 0.059 respectively, and the strain has 90.9% of para-xylene hydrophobic power. The bacterial strain is shown to have strong adhesiveness on the surface of the intestinal mucosa, which is beneficial to the bacterial strain to better exert the probiotic effect in the intestinal tract.
Example 6 evaluation of safety of Lactobacillus acidophilus LA85
(1) Evaluation of sensitivity to common drugs
Activating Lactobacillus acidophilus LA85 on MRS solid plate by streaking, selecting thallus Porphyrae, adding into physiological saline to prepare bacterial suspension, and adjusting the concentration of bacterial suspension to 108And (3) uniformly coating 100 mu L of bacterial suspension on an MRS solid plate by using a sterile cotton swab, orderly placing antibiotic susceptibility test paper sheets on the surface of the plate, culturing for 24-36h at 37 ℃ under an anaerobic condition, and measuring the diameter of a bacteriostatic circle by using a vernier caliper.
The results of the sensitivity of the strain to 17 commonly used antibiotics were obtained according to the evaluation criteria of the american society for clinical and laboratory standards CLSI, see table 3, and the sensitivity of lactobacillus acidophilus LA85 to these drugs, indicating that the strain is safe and potentially useful in biologicals or dietary supplements.
TABLE 3 susceptibility of Lactobacillus acidophilus LA85 to antibiotics
Note: s-is sensitive; i-moderate drug resistance.
(2) Hemolytic test
The strains were subjected to a hemolysis experiment using Streptococcus pyogenes as a control. After the streptococcus pyogenes is inoculated on a columbia agar plate for culture, a transparent hemolysis ring appears around a bacterial colony, and the hemolysis is type B (beta hemolysis); after lactobacillus acidophilus LA85 is inoculated in the culture medium, no hemolytic ring appears around the bacterial colony, so that the bacterial strain is judged to have no hemolysis and no hemolytic harm is caused.
Example 7 immunomodulating function of Lactobacillus acidophilus LA85
Experimental animals: SPF class BLAB/c, 6-8 week old young male mice.
Feeding animals: all experimental mice were housed in plastic cages, on a standard diet, with free access to water. Ambient temperature: 23 +/-1 ℃ and humidity of 55 +/-10 percent.
Animal grouping and probiotic intervention: the mice were randomly divided into 2 groups, a control group and a lactobacillus acidophilus LA85 intervention group, each group consisting of 12 mice. Control group: each mouse was gavaged with 100 μ L10% (w/v) skim milk powder per day; lactobacillus acidophilus LA85 dried group: each mouse was gavaged with 100 μ L of 10810% (w/v) skimmed milk powder of CFU Lactobacillus acidophilus LA85, and feeding for 14 days.
Preparation of immune tissue samples: after the experiment was completed in 14 days, the mice were sacrificed, the small intestine was removed, dissected into sections, fixed in 95% alcohol, and then the tissues were embedded in paraffin blocks for immunofluorescence detection.
Immunofluorescence assay secretory IgA cell number: the number of IgA secretory cells in a mouse small intestine tissue section is measured by a direct immunofluorescence method, a histological section is soaked in xylene, then is hydrated in ethanol, is incubated for 30min at room temperature by 1% bovine serum albumin confining liquid, then is incubated for 30min with an anti-IgA specific antibody marked by Fluorescein Isothiocyanate (FITC) in 0.01M PBS buffer solution at 37 ℃, is washed for 3 times by the PBS solution after the incubation is finished, the number of positive fluorescent cells in 10 visual fields is observed by a fluorescence microscope, and the experimental result is shown in Table 4.
TABLE 4 Effect of Lactobacillus acidophilus LA85 on mouse intestinal IgA cell production
Note: indicates that the difference was statistically significant (P <0.001) compared to the control group.
Serum cytokine detection: after the mice were sacrificed, blood was collected and coagulated overnight at 4 ℃, then serum was extracted from the blood by centrifugation at 2000rpm for 10min, and the contents of IL-12 and IFN-. gamma.in the serum of mice of different treatment groups were measured by ELISA kits, respectively, and the results are shown in Table 5.
TABLE 5 detection of IL-12 and IFN-y cytokines in mouse sera
Note: indicates that the difference was statistically significant (P <0.01) compared to the control group.
The results show that the number of IgA producing cells in the small intestine of the mice is increased after 8514 days of feeding with Lactobacillus acidophilus LA, and the IL-12 level in the serum of the mice is increased, and the IFN-gamma level is reduced. IgA in the gastrointestinal tract plays a positive role in protecting the mucus surface from pathogenic bacterial infections and carcinogenesis; IL-12 is a cytokine released by macrophages under the stimulation of bacteria and metabolites thereof, and mainly has the functions of enhancing the cellular immunity and playing a positive regulation role in anti-tumor immunity; IFN-gamma, a Th1 type cytokine, has multiple functions in the host, is mainly released in cellular immune responses, and is involved in anti-tumor and anti-infectious responses. In conclusion, after the lactobacillus acidophilus LA85 is fed to the mice, the level of the cell factors related to the immunity of the mice can be adjusted, and the immune adjustment effect is realized on the mice.
Example 8 preparation of a solid beverage containing Lactobacillus acidophilus LA85 probiotic
The probiotic solid beverage containing lactobacillus acidophilus is prepared from the following raw materials: inulin (43%), fructo-oligosaccharide (20%), skim milk powder, erythritol, maltodextrin, blueberry fruit powder, lactobacillus acidophilus, bifidobacterium lactis, lactobacillus plantarum and lactobacillus rhamnosus. The specific method for preparing the solid beverage comprises the following steps: weighing maltodextrin, and mixing the components in a ratio of 1: 4-1: putting the mixture into warm water at 70 ℃ in a weight ratio of 6, fully stirring the mixture until the mixture is completely dissolved, and then sieving the mixture through a 80-mesh sieve to obtain slurry for later use; weighing inulin, fructo-oligosaccharide, skim milk powder, erythritol and blueberry fruit powder, putting into a boiling granulator, setting the temperature at 65 ℃, the induced air frequency at 28Hz, and mixing uniformly for 20min to obtain a mixed material; change setting boilThe temperature of the boiling granulator is 55 ℃, the induced air frequency is 28Hz, the mixed slurry is sprayed into the mixed material in the boiling granulator under the conditions that the pressure is 0.1MPa and the flow rate is 80r/min, and the slurry spraying time is 50min until the material has a uniform granularity state, so that granules are obtained; changing the set temperature of the boiling granulator to 65 ℃, the induced air frequency to 25Hz, drying the granules for 20min, stopping heating, and collecting the materials after the temperature of the materials is reduced to below 50 ℃; mixing the above materials with Lactobacillus acidophilus, Lactobacillus bifidus, Lactobacillus plantarum, and Lactobacillus rhamnosus, and packaging. Viable bacteria detection is carried out on the viable bacteria amount of the solid beverage of 3 batches, and the result shows that the viable bacteria amount of the probiotic solid beverage is more than 1 x 1010CFU/g。
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A Lactobacillus acidophilus (A. acidophilus)Lactobacillus acidophilus) The method is characterized in that: the lactobacillus acidophilus is lactobacillus acidophilus LA85 with the preservation number of CGMCC No. 21802.
2. Use of lactobacillus acidophilus according to claim 1 in the food sector.
3. Use according to claim 2, characterized in that: the food field comprises health food, food containing active lactic acid bacteria or fermented food.
4. Use according to claim 2, characterized in that: the health food comprises a microecological preparation.
5. A food product characterized by: the food product comprising lactobacillus acidophilus according to claim 1.
6. Use of lactobacillus acidophilus according to claim 1 for the preparation of a medicament for immunomodulation.
7. An immunomodulatory drug, comprising: the immunomodulatory drug comprises lactobacillus acidophilus according to claim 1.
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| CN116019842B (en) * | 2023-02-20 | 2023-08-25 | 微康益生菌(苏州)股份有限公司 | Antibacterial new application of lactobacillus acidophilus LA85 and application of lactobacillus acidophilus LA85 in preparation of medicines for relieving EIEC diarrhea |
| CN116515684A (en) * | 2023-03-29 | 2023-08-01 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving hyperphosphatemia and application thereof |
| CN116515684B (en) * | 2023-03-29 | 2024-03-01 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving hyperphosphatemia and application thereof |
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