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CN107338206A - A kind of culture medium for being enriched with Bacillus acidi lactici and preparation method thereof - Google Patents

A kind of culture medium for being enriched with Bacillus acidi lactici and preparation method thereof Download PDF

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CN107338206A
CN107338206A CN201710605470.4A CN201710605470A CN107338206A CN 107338206 A CN107338206 A CN 107338206A CN 201710605470 A CN201710605470 A CN 201710605470A CN 107338206 A CN107338206 A CN 107338206A
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翟岱青
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Qingdao Biochemical Equipment Co Ltd Of Gold Allusion Quotation
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Abstract

The present invention relates to field of microbial culture technology, more particularly to a kind of culture medium for being enriched with Bacillus acidi lactici and preparation method thereof, the culture medium of the enrichment Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the additive includes following material:2~12 parts of peptone, 3~12 parts of sodium chloride, 20~50 parts of meat soup, 20~80 parts of cereal fermentation liquor, 5~12 parts of milk powder, 2~8 parts of glucose, 1~10 part of lactose, 1.2~5.6 parts of phosphorus source, 0.02~0.15 part of Pig cholate, 0.6~2.9 part of Sodium Pyruvate, 0.1~3.8 part of amino acid, 0.1~2 part of antibiotic, 0.05~0.5 part of metal salt, 0.01~0.05 part of surfactant, 500 parts of distilled water;Pass through the growth of cereal fermentation liquor other bacteriums always in the present invention first, realize the selective growth of lactic acid bacteria, by adding abundant nutriment in the medium, increase the growth vigor of lactic acid bacteria, promote the growth of lactic acid bacteria, so as to improve the yield of lactic acid bacteria, the enrichment of lactic acid bacteria is realized, is had broad application prospects in industrialized production and detection.

Description

A kind of culture medium for being enriched with Bacillus acidi lactici and preparation method thereof
Technical field
The present invention relates to field of microbial culture technology, more particularly to a kind of culture medium for being enriched with Bacillus acidi lactici and its preparation Method.
Background technology
Lactic acid bacteria refers to a kind of general name without gemma, gram-positive bacterium of the fermenting carbohydrate primary product for lactic acid. Every bacterium that lactic acid can be produced from the fermentation process of glucose or lactose is referred to as lactic acid bacteria.This is that a group is quite numerous and jumbled Bacterium, it can at least be divided into 18 category at present, share kind more than 200.In addition to only a few, wherein the overwhelming majority is all must can not in human body Flora few and with important physiological function, it is widely present in the enteron aisle of human body.At present by domestic and international biologist Confirmed, enteral lactic acid bacteria has detection of the very close direct relation currently for the pathogenic bacteria in food with good health and a long life Typically carried out according to relevant standard method, and the establishment of relevant criterion method depends on traditional separation identification side Method.It is directed to every kind of pathogenic bacteria and is required to its specific enriched medium and carries out the steps such as increasing bacterium, selective enrichment;Same sample Need to carry out the detection of various pathogens simultaneously, thus need while carry out samples on a variety of specific enriched medium Increase bacterium processing, cause detection work increasing early stage bacterium process cumbersome.
The method of traditional microculture is still the authoritative method for screening microorganism so far.National standard, which increases bacterium method, to be needed 3-4 time carried out to sample increase bacterium that the increasing bacterium process of bacterium could be completed, and it is compound increase that bacterium only needs once to increase bacterium process can be complete Into the breeding of bacterium, it is easy to operate to be compared to national standard increasing bacterium method.Existing enriched medium single increases bacterium and can only increased 1~2 kind of pathogenic bacteria of bacterium, the purpose of quick detection can not be realized, therefore, to realize that the quick increasing bacterium of pathogenic bacteria detects, it is necessary to It is improved for conventional medium and method.
The content of the invention
In view of the shortcomings of the prior art, the main object of the present invention be to provide it is a kind of be enriched with Bacillus acidi lactici culture medium and its Preparation method, the culture medium can cultivate salmonella, staphylococcus aureus, ETEC, Listeria monocytogenes simultaneously With the compound increasing bacterium of vibrio parahemolyticus, the purpose of quick detection is realized.
To solve the above problems, the invention provides it is a kind of be enriched with Bacillus acidi lactici culture medium, including basal medium and Additive, in terms of mass fraction, the additive includes following material:2~12 parts of peptone, 3~12 parts of sodium chloride, meat soup 20 ~50 parts, 20~80 parts of cereal fermentation liquor, 5~12 parts of milk powder, 2~8 parts of glucose, 1~10 part of lactose, phosphorus source 1.2~5.6 Part, 0.02~0.15 part of Pig cholate, 0.6~2.9 part of Sodium Pyruvate, 0.1~3.8 part of amino acid, 0.1~2 part of antibiotic, metal 0.05~0.5 part of salt, 0.01~0.05 part of surfactant, 500 parts of distilled water.
Preferably, the culture medium of described enrichment Bacillus acidi lactici, in terms of mass fraction, the additive includes following thing Matter:5~10 parts of peptone, 5~10 parts of sodium chloride, 25~42 parts of meat soup, 36~64 parts of cereal fermentation liquor, 8~10 parts of milk powder, Portugal 3~5 parts of grape sugar, 2~8 parts of lactose, 2.4~3.8 parts of phosphorus source, 0.05~0.12 part of Pig cholate, 1.2~2.4 parts of Sodium Pyruvate, ammonia 0.5~3.6 part of base acid, 1.2~1.8 parts of antibiotic, 0.1~0.4 part of metal salt, 0.01~0.03 part of surfactant, distillation 500 parts of water.
Preferably, the culture medium of described enrichment Bacillus acidi lactici, in terms of mass fraction, the additive includes following thing Matter:8 parts of peptone, 6 parts of sodium chloride, 35 parts of meat soup, 48 parts of cereal fermentation liquor, 8 parts of milk powder, 3 parts of glucose, 6 parts of lactose, phosphorus source 3 parts, 0.08 part of Pig cholate, 1.8 parts of Sodium Pyruvate, 2.6 parts of amino acid, 1.6 parts of antibiotic, 0.3 part of metal salt, surfactant 0.02 part, 500 parts of distilled water.
The present invention does not have special requirement to the species of basal medium, can often know for those skilled in the art, example Such as, the culture medium is selected from MEM culture mediums, DMEM culture mediums, DMEM-F12 culture mediums (1:1), HB culture mediums, BEM culture mediums In one kind.
In the present invention, peptone provides nitrogen source and carbon source for the growth of bacterium, is an important factor for maintaining cell growth, originally In invention, the peptone can be phytone or animal protein peptone, it is preferred that the peptone be soy peptone, At least one of blood peptone, tryptone, meat peptone.
Contain nitrogen-containing compound such as creatine, creatinine, purine base, amino acid in meat soup, while fat also can be partly in meat It is dissolved in soup, in addition, also has water-soluble B vitamin in soup, such as B1, B2 and nicotinic acid, there may also be some nothings in soup Machine salt, such as sylvite and trace elements iron and a small amount of protein, these nutriments are advantageous to the breeding and growth of bacterium, described Meat soup can be soup, plain boiled pork soup and the seafood soup of red meat, it is preferred that the meat soup is pork soup, steamed beef soup, mutton At least one of soup, chicken soup, duck soup, fish soup.
Containing substantial amounts of trace element in cereal fermentation liquor, and it is in faintly acid, the growth that can suppress most of bacteriums is numerous Grow, but be advantageous for the breeding of the growth of lactic acid bacteria, therefore play a part of selectivity culture bacterium, simultaneously as other thin Bacterium can not breed, it is possible to increase and lactic acid bacteria is to the utilization rate of nutriment in culture medium, so as to improve the growth vigor of lactic acid bacteria, Accelerate the breeding of lactic acid bacteria, improve the yield of lactic acid bacteria, contribute to the enrichment of lactic acid bacteria.Preferably, the cereal fermentation liquor is The zymotic fluid of at least one of rice, corn, sorghum, wheat, millet, soybean.
Amino acid is one kind of growth factor, the growth factor amount very little required for microorganism growth, and microorganism sheet Body can not synthesize or synthetic quantity deficiency, it is impossible to which required for meeting body growth, the growth of microorganism or the synthesis of enzyme need amino Acid, but microorganism lacks the ability of synthesizing amino acid in itself, it is necessary to amino acid is added in the medium or contains amino acid Polypeptide, the normal growth of bacterium could be maintained.Preferably, the amino acid is glycine, ALANINE, Pidolidone, L- paddy Glutamine, L-arginine, L-Histidine, L-Leu, ILE, Serine, L-threonine, TYR, L- rely In propylhomoserin, L-Methionine, L-phenylalanine, L-PROLINE, L-Trp, Valine, L-Aspartic acid and altheine At least one.
Phosphate can both provide enough P elements for microorganism, can also pH value in buffer culture medium, it is preferred that institute Phosphorus source is stated as at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
Surfactant can change the permeability of cell, and so as to be advantageous in cell building-up process, the release of enzyme is excellent Choosing, the surfactant is SDS and/or CTAB.
Antibiotic can suppress the breeding of other strains, prevent other strains from being polluted to caused by culture medium, it is preferred that institute Antibiotic is stated as at least one of beta-lactam antibiotic, aminoglycoside antibiotics, polypeptide antibiotics, more preferably , the beta-lactam antibiotic is benzyl penicillin, ospen, tardocillin, methicillin, naphthlazole, benzene azoles west At least one of woods, flucloxacillin, Cloxacillin, dicloxacillin;The aminoglycoside antibiotics is mycin, celebrating is big mould At least one in element, kanamycins, TOB, amikacin, neomycin, ribostamycin, micronomicin, A Si mycins Kind;The polypeptide antibiotics is at least one of vancomycin, Norvancomycin, teicoplanin.
Metal salt be also maintain cell growth another key factor, it is preferred that the metal salt be molysite, mantoquita, At least one of zinc salt, cobalt salt, manganese salt, chromic salts, selenium salt, nickel salt, molybdenum salt, vanadic salts, pink salt, strontium salt.
A kind of preparation method for the culture medium for being enriched with Bacillus acidi lactici, comprises the following steps:
The preparation of meat soup:Meat is put into 2~4 water, is then warming up to 85~100 with 3~5 DEG C/min speed DEG C, 30~180min is then incubated, after being cooled to room temperature, obtains meat soup;
The preparation of cereal fermentation liquor:Cereal is soaked into 5~48h in 5~6 times of water, then cereal is pulverized, obtained To cereal slurry, yeast is then added in cereal slurry, is fermented after 12~72h, is filtered to remove sediment, obtains fermented grain Liquid;
By peptone, sodium chloride, meat soup, cereal fermentation liquor, milk powder, glucose, lactose, phosphorus source, Pig cholate, pyruvic acid Sodium, amino acid, antibiotic, metal salt, surfactant are well mixed in water, are then adjusted pH value to 7.2-7.5, high temperature and are gone out After bacterium, lactic acid bacteria culturing medium is obtained.
Compared with prior art, the present invention has following technique effect:
The selective growth of lactic acid bacteria is realized by the growth of cereal fermentation liquor other bacteriums always first in the present invention, By adding abundant nutriment in the medium, increase the growth vigor of lactic acid bacteria, promote the growth of lactic acid bacteria, so as to carry The yield of high lactic acid bacteria, the enrichment of lactic acid bacteria is realized, had broad application prospects in industrialized production and detection.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, following examples, the present invention is carried out It is further described.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit The present invention.
Embodiment 1
A kind of culture medium for being enriched with Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the addition Agent includes following material:
8 parts of soy peptone, 6 parts of sodium chloride, 35 parts of mutton soup, 48 parts of rice fermentation liquid, 8 parts of milk powder, 3 parts of glucose, 6 parts of lactose, 3 parts of phosphorus source, 0.08 part of Pig cholate, 1.8 parts of Sodium Pyruvate, 2.6 parts of glycine, 1.6 parts of beta-lactam antibiotic, 0.3 part of molysite, 0.02 part of surfactant, 500 parts of distilled water;
The present embodiment additionally provides a kind of preparation method of the culture medium of the enrichment Bacillus acidi lactici, comprises the following steps:
The preparation of mutton soup:Mutton is put into 3 times of water, is then warming up to 100 DEG C with 4 DEG C/min speed, then 60min is incubated, after being cooled to room temperature, obtains mutton soup;
The preparation of rice fermentation liquid:Rice is soaked into 24h in 5 times of water, then grinds rice to obtain rice slurry, Then yeast is added in rice slurry, is fermented after 36h, is filtered to remove sediment, obtains rice fermentation liquid;
By soy peptone, sodium chloride, mutton soup, rice fermentation liquid, milk powder, glucose, lactose, phosphorus source, Pig cholate, third Ketone acid sodium, glycine, beta-lactam antibiotic, molysite, surfactant in water be well mixed, then adjust pH value to 7.4, after high-temperature sterilization, obtain lactic acid bacteria culturing medium.
Embodiment 2
A kind of culture medium for being enriched with Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the addition Agent includes following material:
5 parts of blood peptone, 5 parts of sodium chloride, 25 parts of mutton soup, 36 parts of corn fermentation liquid, 8 parts of milk powder, 3 parts of glucose, 2 parts of lactose, 2.4 parts of phosphorus source, 0.05 part of Pig cholate, 1.2 parts of Sodium Pyruvate, 0.5 part of ALANINE, aminoglycoside antibiotics 1.2 parts, 0.1 part of chromic salts, 0.01 part of surfactant, 500 parts of distilled water;
The present embodiment additionally provides a kind of preparation method of the culture medium of the enrichment Bacillus acidi lactici, comprises the following steps:
The preparation of mutton soup:Mutton is put into 2 times of water, is then warming up to 85 DEG C with 3 DEG C/min speed, Ran Houbao Warm 30min, after being cooled to room temperature, obtain mutton soup;
The preparation of corn fermentation liquid:Corn is soaked into 12h in 5 times of water, corn mill is then obtained into corn syrup, Then yeast is added in corn syrup, is fermented after 32h, is filtered to remove sediment, obtains corn fermentation liquid;
By blood peptone, sodium chloride, mutton soup, corn fermentation liquid, milk powder, glucose, lactose, phosphorus source, Pig cholate, third Ketone acid sodium, ALANINE, aminoglycoside antibiotics, chromic salts, surfactant in water be well mixed, then adjust pH value to 7.3, after high-temperature sterilization, obtain lactic acid bacteria culturing medium.
Embodiment 3
A kind of culture medium for being enriched with Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the addition Agent includes following material:
10 parts of tryptone, 10 parts of sodium chloride, 42 parts of chicken soup, 64 parts of sorghum zymotic fluid, 10 parts of milk powder, 5 parts of glucose, 8 parts of lactose, 3.8 parts of phosphorus source, 0.12 part of Pig cholate, 2.4 parts of Sodium Pyruvate, 0.5~3.6 part of Glu, polypeptide antibiosis 1.8 parts of element, 0.4 part of cobalt salt, 0.03 part of surfactant, 500 parts of distilled water;
The present embodiment additionally provides a kind of preparation method of the culture medium of the enrichment Bacillus acidi lactici, comprises the following steps:
The preparation of chicken soup:Chicken is put into 4 times of water, is then warming up to 85 DEG C with 3 DEG C/min speed, Ran Houbao Warm 30min, after being cooled to room temperature, obtain chicken soup;
The preparation of sorghum zymotic fluid:Sorghum is soaked into 5h in 5 times of water, then grinds sorghum to obtain sorghum slurry, Then yeast is added in sorghum slurry, is fermented after 12h, is filtered to remove sediment, obtains sorghum zymotic fluid;
By tryptone, sodium chloride, meat soup, sorghum zymotic fluid, milk powder, glucose, lactose, phosphorus source, Pig cholate, pyruvic acid Sodium, Glu, polypeptide antibiotics, cobalt salt, surfactant are well mixed in water, then adjust pH value to 7.4, high After temperature sterilizing, lactic acid bacteria culturing medium is obtained.
Embodiment 4
A kind of culture medium for being enriched with Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the addition Agent includes following material:
2 parts of meat peptone, 3 parts of sodium chloride, 20 parts of duck soup, 20 parts of wheat zymotic fluid, 5 parts of milk powder, 2 parts of glucose, breast 1 part of sugar, 1.2 parts of phosphorus source, 0.02 part of Pig cholate, 0.6 part of Sodium Pyruvate, 0.1 part of L-arginine, beta-lactam antibiotic 0.1 Part, 0.05 part of selenium salt, 0.01 part of surfactant, 500 parts of distilled water;
The present embodiment additionally provides a kind of preparation method of the culture medium of the enrichment Bacillus acidi lactici, comprises the following steps:
The preparation of duck soup:Duck is put into 3 times of water, is then warming up to 90 DEG C with 4 DEG C/min speed, Ran Houbao Warm 100min, after being cooled to room temperature, obtain duck soup;
The preparation of wheat zymotic fluid:Wheat is soaked into 12h in 5 times of water, wheat flour milling is then obtained into wheat slurry, Then yeast is added in wheat slurry, is fermented after 32h, is filtered to remove sediment, obtains wheat zymotic fluid;
By meat peptone, sodium chloride, duck soup, wheat zymotic fluid, milk powder, glucose, lactose, phosphorus source, Pig cholate, acetone Sour sodium, L-arginine, beta-lactam antibiotic, selenium salt, surfactant in water be well mixed, then adjust pH value to 7.4, after high-temperature sterilization, obtain lactic acid bacteria culturing medium.
Embodiment 5
A kind of culture medium for being enriched with Bacillus acidi lactici, including basal medium and additive, in terms of mass fraction, the addition Agent includes following material:
12 parts of soy peptone, 12 parts of sodium chloride, 50 parts of fish soup, 80 parts of soybean fermented liquid, 12 parts of milk powder, 8 parts of glucose, 10 parts of lactose, 5.6 parts of phosphorus source, 0.15 part of Pig cholate, 2.9 parts of Sodium Pyruvate, 3.8 parts of L-Histidine, 2 parts of polypeptide antibiotics, 0.5 part of vanadic salts, 0.05 part of surfactant, 500 parts of distilled water;
The present embodiment additionally provides a kind of preparation method of the culture medium of the enrichment Bacillus acidi lactici, comprises the following steps:
The preparation of fish soup:Fish is put into 4 times of water, is then warming up to 90 DEG C with 5 DEG C/min speed, is then incubated 150min, after being cooled to room temperature, obtain fish soup;
The preparation of soybean fermented liquid:Soybean is soaked into 48h in 6 times of water, then grinds soybean to obtain soybean slurry, Then yeast is added in soybean slurry, is fermented after 60h, is filtered to remove sediment, obtains soybean fermented liquid;
By soy peptone, sodium chloride, fish soup, soybean fermented liquid, milk powder, glucose, lactose, phosphorus source, Pig cholate, acetone Sour sodium, L-Histidine, polypeptide antibiotics, vanadic salts, surfactant are well mixed in water, then adjust pH value to 7.4, high After temperature sterilizing, lactic acid bacteria culturing medium is obtained.
Foregoing description is only the description to section Example of the present invention, not to any restriction of the scope of the invention, one's own profession The those of ordinary skill of industry can make improvement or modification according to the present invention to above-described embodiment, but belong to present invention protection model Enclose.

Claims (10)

1. a kind of culture medium for being enriched with Bacillus acidi lactici, it is characterised in that including basal medium and additive, with mass fraction Meter, the additive include following material:2~12 parts of peptone, 3~12 parts of sodium chloride, 20~50 parts of meat soup, cereal fermentation liquor 20~80 parts, 5~12 parts of milk powder, 2~8 parts of glucose, 1~10 part of lactose, 1.2~5.6 parts of phosphorus source, Pig cholate 0.02~0.15 Part, 0.6~2.9 part of Sodium Pyruvate, 0.1~3.8 part of amino acid, 0.1~2 part of antibiotic, 0.05~0.5 part of metal salt, surface 0.01~0.05 part of activating agent, 500 parts of distilled water.
2. the culture medium of enrichment Bacillus acidi lactici according to claim 1, it is characterised in that described to add in terms of mass fraction Agent is added to include following material:5~10 parts of peptone, 5~10 parts of sodium chloride, 25~42 parts of meat soup, 36~64 parts of cereal fermentation liquor, 8~10 parts of milk powder, 3~5 parts of glucose, 2~8 parts of lactose, 2.4~3.8 parts of phosphorus source, 0.05~0.12 part of Pig cholate, pyruvic acid 1.2~2.4 parts of sodium, 0.5~3.6 part of amino acid, 1.2~1.8 parts of antibiotic, 0.1~0.4 part of metal salt, surfactant 0.01~0.03 part, 500 parts of distilled water.
3. the culture medium of enrichment Bacillus acidi lactici according to claim 2, it is characterised in that described to add in terms of mass fraction Agent is added to include following material:8 parts of peptone, 6 parts of sodium chloride, 35 parts of meat soup, 48 parts of cereal fermentation liquor, 8 parts of milk powder, glucose 3 Part, 6 parts of lactose, 3 parts of phosphorus source, 0.08 part of Pig cholate, 1.8 parts of Sodium Pyruvate, 2.6 parts of amino acid, 1.6 parts of antibiotic, metal salt 0.3 part, 0.02 part of surfactant, 500 parts of distilled water.
4. the culture medium of the enrichment Bacillus acidi lactici according to claims 1 to 3 any one, it is characterised in that the albumen Peptone is at least one of soy peptone, blood peptone, tryptone, meat peptone.
5. the culture medium of enrichment Bacillus acidi lactici according to claim 4, it is characterised in that the meat soup is pork soup, ox At least one of meat soup, mutton soup, chicken soup, duck soup, fish soup.
6. the culture medium of enrichment Bacillus acidi lactici according to claim 4, it is characterised in that the cereal fermentation liquor is big The zymotic fluid of at least one of rice, corn, sorghum, wheat, millet, soybean.
7. it is according to claim 4 enrichment Bacillus acidi lactici culture medium, it is characterised in that the amino acid be glycine, ALANINE, Pidolidone, Glu, L-arginine, L-Histidine, L-Leu, ILE, Serine, L-threonine, TYR, 1B, L-Methionine, L-phenylalanine, L-PROLINE, L-Trp, Valine, L- At least one of aspartic acid and altheine.
8. the culture medium of enrichment Bacillus acidi lactici according to claim 1, it is characterised in that the antibiotic is beta-lactam At least one of class antibiotic, aminoglycoside antibiotics, polypeptide antibiotics.
9. enriched medium according to claim 1, it is characterised in that the metal salt is molysite, mantoquita, zinc salt, cobalt At least one of salt, manganese salt, chromic salts, selenium salt, nickel salt, molybdenum salt, vanadic salts, pink salt, strontium salt.
10. a kind of preparation method of the culture medium of enrichment Bacillus acidi lactici according to claim 1~9 any one, it is special Sign is, comprises the following steps:
The preparation of meat soup:Meat is put into 2~4 times of water, is then warming up to 85~100 DEG C with 3~5 DEG C/min speed, Then 30~180min is incubated, after being cooled to room temperature, obtains meat soup;
The preparation of cereal fermentation liquor:Cereal is soaked into 5~48h in 5~6 times of water, then grinds cereal to obtain cereal slurry Material, yeast is then added in cereal slurry, ferment after 12~72h, be filtered to remove sediment, obtain cereal fermentation liquor;
By peptone, sodium chloride, meat soup, cereal fermentation liquor, milk powder, glucose, lactose, phosphorus source, Pig cholate, Sodium Pyruvate, ammonia Base acid, antibiotic, metal salt, surfactant are well mixed in water, then regulation pH value to 7.2-7.5, after high-temperature sterilization, Obtain lactic acid bacteria culturing medium.
CN201710605470.4A 2017-07-24 2017-07-24 A kind of culture medium for being enriched with Bacillus acidi lactici and preparation method thereof Pending CN107338206A (en)

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CN109971690A (en) * 2019-05-08 2019-07-05 无锡中顺生物技术有限公司 A kind of pool mouth spreads cultivation the culture medium and propagation method of lactic acid bacteria
CN110628684A (en) * 2019-10-25 2019-12-31 华中农业大学 Lactobacillus enrichment agent and application thereof in high-density fermentation of lactobacillus
CN115305201A (en) * 2022-09-16 2022-11-08 中南大学 Microbial agent for efficiently treating oily sludge and preparation method thereof

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Application publication date: 20171110