CN111944713A - Pediococcus acidilactici with excellent alcohol stress resistance and application thereof - Google Patents
Pediococcus acidilactici with excellent alcohol stress resistance and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/32—Alcohol-abuse
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
- A23V2400/413—Acidilactici
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to Pediococcus acidilactici with excellent alcohol stress resistance, which is named as follows: LTJ28, category name: pediococcus acidilactici, the preservation number is: CGMCC No.20087, preservation date: year 2020, 6 and 15 days, Xilu No. 1 Hospital No. 3, Kyoto, Chaoyang, the depository: china general microbiological culture Collection center. The Pediococcus acidilactici is derived from fermented grains in the traditional white spirit fermentation process of fermented food, and belongs to a well-known and safe strain which can be used for food. The pediococcus acidilactici has excellent alcohol stress resistance, is the dominant strain in the fermented grains of the white spirit, and has very strong alcohol stress resistance.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus acidilactici with excellent alcohol stress resistance and application thereof.
Background
Lactic acid bacteria are a general term for a group of bacteria capable of fermenting carbohydrates to produce lactic acid, and are gram-positive and mostly rod-shaped or spherical in shape. The good lactic acid bacteria required in the production and fermentation process have good fermentation performance and strong tolerance so as to ensure that microorganisms can live in the gastrointestinal tract of a human body after the fermented product is eaten, which is also the key point for the probiotic lactic acid bacteria to play the role.
Pediococcus acidilactici (Pediococcus acidilactici) is one of lactic acid bacteria, and bacteriocin produced by the Pediococcus acidilactici has an inhibiting effect on a plurality of gram-positive bacteria and also has an inhibiting effect on some gram-negative bacteria, so that the Pediococcus acidilactici shows a wide inhibiting spectrum. The bacteriocin produced by pediococcus acidilactici has heat resistance and salt tolerance. In addition, pediococcus acidilactici produces acid, regulates gastrointestinal flora, and maintains intestinal microecological balance. The product has antagonistic action on pathogenic microorganism in animal body, and can competitively inhibit pathogenic microorganism, enhance animal organism immunity, generate beneficial metabolite, activate acidic protease activity, participate in organism metabolism, and prevent harmful substance generation. The pediococcus acidilactici has better stress resistance and bacteriostatic property, and can be used as a candidate strain of probiotics.
Alcohol is an organic solvent that can cause changes in cell membrane permeability, loss of intracellular material, affect enzyme activity, etc. During the growth of the thallus, the over-high alcohol concentration will affect the physiological activity, metabolism and other aspects of the thallus to different degrees. Research shows that lactic acid bacteria are dominant bacteria in the fermentation process of white spirit, the acidity and the ethanol concentration in a fermentation system are increased along with the fermentation, the oxygen content is gradually reduced, most microorganisms cannot tolerate adverse conditions such as high acidity, high ethanol concentration and anaerobism and gradually die, and the lactic acid bacteria become the bacteria with absolute advantages. The ability of lactic acid bacteria cells to withstand environmental stresses, especially high concentrations of alcohol, has great development prospects.
Along with the continuous improvement of the living standard of people, the chance and the drinking capacity of people for drinking wine are more and more. Proper drinking is beneficial to body health, but long-term excessive drinking can cause great harm to human health and even cause adverse effects on society. Meanwhile, people are more and more conscious of healthy drinking, and anti-alcohol products are produced at the same time, at present, researches on anti-alcohol medicines are mainly focused on the aspects of compound and initial extract of traditional Chinese medicines, and researches on probiotics capable of effectively tolerating and improving health hazards of drunkenness and alcohol are very limited.
Therefore, the research on the probiotics with excellent alcohol stress resistance has important significance for the development of anti-alcohol health products and further human health.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a pediococcus acidilactici strain with excellent alcohol stress resistance.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the Pediococcus acidilactici has excellent alcohol stress resistance, and is named as follows: LTJ28, category name: pediococcus acidilactici, the preservation number is: CGMCC No.20087, preservation date: year 2020, 6 and 15 days, Xilu No. 1 Hospital No. 3, Kyoto, Chaoyang, the depository: china general microbiological culture Collection center.
Moreover, the strain is separated from fermented grains of white spirit and is the dominant strain in the fermented grains.
The bacterial colony and the thallus characteristics of the strain are as follows: culturing for 24h on MRS culture medium, wherein the colony is white, opaque, and has the advantages of raised, moist, smooth edge, luster, and 0.4-0.6 mm diameter, and the cell morphology is observed under optical microscope, and the gram stain is G+The thallus is in a spherical shape.
Moreover, the strains are respectively subjected to 4%, 6%, 8%, 10% and 12% (volume fraction) alcohol stress treatment, and the survival rates of the strains are 89.52%, 68.41%, 55.50%, 41.65% and 26.21% in sequence.
Moreover, the survival rate of 14.49% after 4h culture under the condition of pH 3 and 6.89% after 4h culture in MRS medium containing 0.3% bile salt have the adhesion rate of 28.18%.
The pediococcus acidilactici with excellent alcohol stress resistance is applied to the aspect of relieving alcoholism.
The pediococcus acidilactici with excellent alcohol stress resistance is applied to functional foods and medicines.
The invention has the advantages and positive effects that:
1. the Pediococcus acidilactici is derived from fermented grains in the traditional white spirit fermentation process of fermented food, and belongs to a well-known and safe strain which can be used for food. The pediococcus acidilactici has excellent alcohol stress resistance, is the dominant strain in the fermented grains of the white spirit, and has very strong alcohol stress resistance.
2. After the Pediococcus acidilactici is subjected to stress treatment by 4%, 6%, 8%, 10% and 12% (volume fraction) of alcohol, the survival rates of the Pediococcus acidilactici are 89.52%, 68.41%, 55.50%, 41.65% and 26.21% in sequence, and the Pediococcus acidilactici has strong alcohol stress resistance. Can be used in the fields of food, medicine, etc., such as development of related functional food and medicine (anti-hangover product).
Drawings
FIG. 1 is a colony morphology and cell morphology diagram of Pediococcus acidilactici CGMCC No.20087 in the invention;
FIG. 2 is a 16S rDNA phylogenetic tree diagram of Pediococcus acidilactici CGMCC No.20087 in the present invention;
FIG. 3 shows the survival rate of Pediococcus acidilactici CGMCC No.20087 in the treatment of alcohol with different concentrations.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
The Pediococcus acidilactici has excellent alcohol stress resistance, and is named as follows: LTJ28, category name: pediococcus acidilactici, the preservation number is: CGMCC No.20087, preservation date: year 2020, 6 and 15 days, Xilu No. 1 Hospital No. 3, Kyoto, Chaoyang, the depository: china general microbiological culture Collection center.
Preferably, the strain is separated from fermented grains of Chinese liquor, and is the dominant strain in the fermented grains.
Preferably, the bacterial colony and thallus characteristics of the strain are as follows: culturing for 24h on MRS culture medium, wherein the colony is white, opaque, and has the advantages of raised, moist, smooth edge, luster, and 0.4-0.6 mm diameter, and the cell morphology is observed under optical microscope, and the gram stain is G+The thallus is in a spherical shape.
Preferably, the strains are respectively subjected to 4%, 6%, 8%, 10% and 12% (volume fraction) alcohol stress treatment, the survival rates of the strains are 89.52%, 68.41%, 55.50%, 41.65% and 26.21%, and the strains have strong alcohol stress resistance.
Preferably, the survival rate of the cells cultured for 4h under the condition of pH 3 is 14.49%, the survival rate of the cells cultured for 4h in MRS culture medium containing 0.3% of bile salt is 6.89%, the adhesion rate is 28.18%, and the cells have good in-vivo survival ability.
The pediococcus acidilactici with excellent alcohol stress resistance is applied to the aspect of relieving alcoholism.
The pediococcus acidilactici with excellent alcohol stress resistance is applied to functional foods and medicines.
Specifically, the following examples illustrate the technical solutions of the present invention from the characteristics of isolation and identification of strains, tolerance to alcohol treatment at different concentrations, acid resistance, bile salt resistance, and adhesion, respectively, and the details are as follows.
Example 1 isolation and identification of lactic acid bacteria
The following experiments were all performed under sterile conditions.
1. Enrichment of strains
Weighing 25g of fermented grains in the process of brewing white spirit, adding 225mL of sterile normal saline, uniformly mixing, diluting the obtained bacterial liquid by 10%3～105After doubling, 100 mu L of the suspension was spread on an MRS plate, and the suspension was cultured in an inverted state at 37 ℃ for 24 hours to observe the growth of colonies.
The preparation method of the MRS solid culture medium comprises the following steps: 10g of peptone, 5g of beef extract powder, 801 mL of Tween, 2g of dipotassium phosphate, 4g of yeast extract powder, 0.2g of magnesium sulfate, 2g of triammonium citrate, 20g of glucose, 0.05g of manganese sulfate, 5g of sodium acetate, 20g of agar, 1L of distilled water, pH 6.2 +/-0.2, and sterilizing at 121 ℃ for 20min for later use.
2. Isolation of the Strain
And (3) selecting the bacterial colony obtained in the step (1), performing microscopic examination by using a gram staining method, observing morphological characteristics of the bacterial colony, and selecting gram-positive bacteria as target strains.
3. Purification of the strains
And (3) selecting the bacterial colonies obtained in the step (2), streaking on an MRS plate, repeatedly purifying until no other mixed bacteria exist, and recording the states of different bacterial colonies.
4. Identification of strains
The bacterial genome DNA rapid extraction kit is adopted to extract the lactobacillus strain genome, and the lactobacillus strain genome is used as a template for PCR amplification. And sequencing the PCR products which are successfully amplified, splicing to obtain a DNA sequence, and comparing and analyzing sequence homology by using a BLAST retrieval system of NCBI. The known strain with the highest similarity was selected and analyzed for similarity. Phylogenetic trees were created using the Neighbor-Joining method (NJ) in MEGA 5 software.
Physiological and biochemical experimental research is carried out on the pure bacteria obtained in the step 3, and the bacterial strain is found to have strong activityThe anti-alcohol stress capacity is realized by culturing the strain on an MRS culture medium for 24 hours, the bacterial colony is white and opaque, the bacterial colony is protruded, moist, smooth in edge and glossy, the diameter of the bacterial colony is 0.4-0.6 mm, the cell morphology is observed under an optical microscope, and the gram stain is G+The cells are spherical (as shown in FIG. 1).
The result of homology comparison by BLAST software is shown in figure 2, and the result is combined with the colony morphological characteristics, the cell morphological characteristics and the 16S rDNA gene sequence result to determine the new Pediococcus acidilactici (Pediococcus acidilactici) in the Pediococcus, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) 6, 15 days 2020, the preservation number is CGMCC No.20087, and the address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
Example 2 tolerance of Pediococcus acidilactici CGMCC No.20087 to treatment with alcohol of different concentrations
Strains at the end of logarithmic growth are selected, 2 percent of inoculum size is added into MRS liquid culture medium with different contents (4 percent, 6 percent, 8 percent, 10 percent and 12 percent) of alcohol to carry out alcohol stress for 12 hours in an incubator at 37 ℃, and meanwhile, a culture group of the alcohol-free MRS liquid culture medium is used as a negative control. Determination of OD after stress600nmThe growth conditions were compared.
The preparation method of the MRS liquid culture medium comprises the following steps: 10g of peptone, 5g of beef extract powder, 801 mL of Tween, 2g of dipotassium phosphate, 4g of yeast extract powder, 0.2g of magnesium sulfate, 2g of triammonium citrate, 20g of glucose, 0.05g of manganese sulfate, 5g of sodium acetate, 1L of distilled water, pH 6.2 +/-0.2, and sterilization at 121 ℃ for 20min for later use.
The pure bacteria obtained in example 1 are subjected to alcohol treatment with different concentrations, the tolerance of pediococcus acidilactici CGMCC No.20087 is in a descending trend along with the increase of the alcohol concentration, and under the alcohol stress treatment of 4%, 6%, 8%, 10% and 12% (volume fraction), the survival rates are 89.52%, 68.41%, 55.50%, 41.65% and 26.21% in sequence, and the pure bacteria belong to strains with high alcohol stress resistance (as shown in figure 3).
Example 3 determination of Properties of Pediococcus acidilactici CGMCC No.20087
1. Determination of acid resistance
Selecting a strain at the end of logarithmic growth, adding the strain into a liquid MRS culture medium with the pH value of 3.0 according to the inoculum size of 2 percent, culturing for 4 hours in an incubator at 37 ℃, and simultaneously taking a conventional pH value MRS culture medium culture group as a negative control. Determination of OD after stress600nmThe growth conditions were compared.
2. Determination of the bile salt resistance
Strains at the end of logarithmic growth are selected, the strains are added into a liquid MRS culture medium containing 0.3% bile salt according to the inoculum size of 2% and cultured for 4 hours in an incubator at 37 ℃, and meanwhile, a conventional MRS culture medium culture group is used as a negative control. Determination of OD after stress600nmThe growth conditions were compared.
3. Measurement of adhesive Capacity
Rat tail collagen was added to 96-well plates and shaken to uncover the wells overnight for air drying. mu.L of the bacterial suspension was added to each well, and incubated at 37 ℃ for 1 hour with no addition of the bacterial suspension as a control. The non-adherent bacteria were washed with PBS, followed by addition of 10. mu.L of MTT per well for 4 hours, and the culture was terminated. Add 100. mu.L of Dimethylsulfoxide (DMSO) per well and put on a low speed shaker for 15min to dissolve the crystals sufficiently. Then measuring the absorbance (A) values of the lactobacillus rinsed by PBS at the wavelength of 570nm, the lactobacillus not rinsed and the blank control holes without bacteria in an enzyme-linked immunosorbent assay instrument, and determining the adhesion capability of the lactobacillus and the blank control holes.
The pure bacteria obtained in example 1 were studied for acid resistance, bile salt resistance and adhesion, and found that the survival rate of the bacteria was 14.49% when cultured for 4 hours at pH 3 and 6.89% when cultured for 4 hours in MRS medium containing 0.3% bile salt, and the adhesion rate was 28.18%.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (7)
1. A pediococcus acidilactici with excellent alcohol stress resistance is characterized in that: the name of the Pediococcus acidilactici (Pediococcus acidilactici) is as follows: LTJ28, category name: pediococcus acidilactici, the preservation number is: CGMCC No.20087, preservation date: year 2020, 6 and 15 days, Xilu No. 1 Hospital No. 3, Kyoto, Chaoyang, the depository: china general microbiological culture Collection center.
2. The pediococcus acidilactici with excellent alcohol stress resistance according to claim 1, wherein: the strain is separated from fermented grains of Chinese liquor, and is the dominant strain in the fermented grains.
3. The pediococcus acidilactici with excellent alcohol stress resistance according to claim 1, wherein: the bacterial colony and thallus characteristics of the strain are as follows: culturing for 24h on MRS culture medium, wherein the colony is white, opaque, and has the advantages of raised, moist, smooth edge, luster, and 0.4-0.6 mm diameter, and the cell morphology is observed under optical microscope, and the gram stain is G+The thallus is in a spherical shape.
4. The pediococcus acidilactici with excellent alcohol stress resistance according to claim 1, wherein: the strain is respectively subjected to 4%, 6%, 8%, 10% and 12% (volume fraction) alcohol stress treatment, and the survival rate of the strain is 89.52%, 68.41%, 55.50%, 41.65% and 26.21% in sequence.
5. The pediococcus acidilactici with excellent alcohol stress resistance according to claim 1, wherein: the survival rate of 14.49% after being cultured for 4h under the condition of pH 3, and the survival rate of 6.89% after being cultured for 4h in MRS culture medium containing 0.3% bile salt, and the adhesion rate is 28.18%.
6. The use of Pediococcus acidilactici with excellent alcohol stress resistance as claimed in any one of claims 1 to 5 for alleviating hangover.
7. Use of the pediococcus acidilactici with excellent alcohol stress resistance according to any one of claims 1 to 5 in functional foods and medicines.
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| CN116114763B (en) * | 2023-01-12 | 2024-01-16 | 四川省食品发酵工业研究设计院有限公司 | Application of Pediococcus acidilactici brew R036 as anti-alcohol agent |
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