CN104780919A - Composition, containing cis-cyclo(l-leu-l-pro) or cis-cyclo(l-phe-l-pro), for use as an antimicrobial and antiviral agent, and preparation method therefor - Google Patents

Composition, containing cis-cyclo(l-leu-l-pro) or cis-cyclo(l-phe-l-pro), for use as an antimicrobial and antiviral agent, and preparation method therefor Download PDF

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CN104780919A
CN104780919A CN201380052609.7A CN201380052609A CN104780919A CN 104780919 A CN104780919 A CN 104780919A CN 201380052609 A CN201380052609 A CN 201380052609A CN 104780919 A CN104780919 A CN 104780919A
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pro
leuconostoc
cis
leu
composition
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CN104780919B (en
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姜思旭
郭珉逵
刘芮
权俊吾
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SNU R&DB Foundation
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
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    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • A61P31/12—Antivirals
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The present invention provides an antimicrobial composition containing cis-cyclo(L-Leu-L-Pro) as an active ingredient and having specific antimicrobial activity against the genus Streptococcus or bacteria of the genus Shigella, a pesticide composition, and a method for producing an antimicrobial agent. The present invention exhibits excellent antimicrobial activity against the genus Streptococcus or Shigella and thus can be applied to various industrial fields requiring the removal and prevention of the genus Streptococcus or Shigella bacteria. In particular, the present invention is economically advantageous in terms of the use thereof because even a small amount thereof exhibits antimicrobial activity. In addition, the present invention provides an antiviral composition containing cis-cyclo(L-Leu-L-Pro) or cis-cyclo(L-Phe-L-Pro).

Description

Comprise antibacterial and antiviral compositions and the manufacture method thereof of cis ring (L-Leu-L-PROLINE) or cis ring (L-Phe-L-PROLINE)
Technical field
The present invention relates to the novelty teabag of cis ring (L-Leu-L-PROLINE).Further illustrate namely, the present invention is by cis ring (L-Leu-L-PROLINE) streptococcus as effective ingredient or the technology of streptococcus compositions.
The present invention relates to the technology of cis ring (L-Leu-L-PROLINE) and cis ring (L-Phe-L-PROLINE) novelty teabag.Further illustrate namely, the present invention is by cis ring (L-Leu-L-PROLINE) or cis ring (L-Phe-L-PROLINE) technology as the antiviral composition of effective ingredient.
Background technology
Typically, lactate fermentation effect depends on organic acid accumulation and substrate oxidation. [1,2,3]the metabolites such as acetaldehyde, diacetylation compound, hydrogen peroxide and carbon dioxide, effectively can control the growth of antibacterial, pathogenic bacteria and putrefaction bacteria. [1,2,3]according to another research report, nonprotein substrate effectively can also contain gram positive bacteria, the growing of mouldy bacterium and gram-negative bacteria. [4]
At first, low-molecular-weight lactobacillus (Reutericyclin) antibiotic refines from the lactobacillus LTH2584 (Lactobacillus reuteri LTH2584) contained by fermentation bread.Lactobacillus is more responsive to microorganisms such as gram positive bacteria, gram-negative bacteria, yeast, mycetes. [5]for a long time, lactobacillus or lactobacillus (Lactic acid bacteria:LAB) are used as the food of the mankind and animal, as yeast, serve very important effect when food fermentation and storage. [1,2,3]
Typically, LAB can secrete extracellular agonist compounds, to resist many microorganisms, and changes extracellular environment by endogenous metabolites. [6]in the compound excreted by LAB, various antibacterial metabolite can be categorized into less than 1000 low-molecular-weight compounds and be assessed as the bacteriocin (bacteriocins) having less than 1000 molecular weight [7].Up to now, the research to bacteriocin is mainly concentrated on about the research topic of LAB antibacterial activity effect.The bacteriocin generated from LAB, in activity, model of action, molecular weight, hereditary source and biochemical characteristic, one of foreign peoples (heterogeneous) group belonging to the antibacterial peptides and proteins with SPECTRAL DIVERSITY, [8,9] and this type of peptides and proteins are divided into three types on the basis of mature peptide and leader peptide (prepeptide). [10,11,12,13]pediocin is the one of the bacteriocin being originated in Pediococcus pentosaceus (Pediococcus pentecostals), [14]all containing cation high molecular and little substrate in the compound that major part is identical therewith.[ 15]in addition, this compound is also used to kill gram-positive cocci and the pathogenic cocci such as bacillus cereus (Bacillus subtilis), Listerella (Listeria species), staphylococcus aureus strains (Staphylococcus aureus).[ 16,17,18]in addition, some bacteriocin can also kill coliform (Escherichia coli) the food pathogenic coccus the same with Salmonella.[ 19]up to now, mainly concentrated research and investigation have been carried out to two amino acids (Proline-containing 2,5-diketopiperazine) that contract containing 2,5-rings of proline Cyclic dipeptides. [6,20,22,23,24,25,26,27]all the time, this cyclic peptide or Cyclic dipeptides are considered to the effect playing substrate in biology, [22,23,24,25,26]especially such Cyclic dipeptides, in potential treatment and prevention effects, has the antibacterial activity effect to antibacterium, mycete, virus and cancerous cell. [6,23,26,27,28]in addition, this antibacterial activity Cyclic dipeptides culture yeasts, lichens plant and mould time confirmed. [20,29]according to another interrelated data, from streptomycete, isolate Cyclic dipeptides (1-leucyl-1-propyl) and Cyclic dipeptides (1-phenylalanyl-1-prolyl). [20,24,25,29]antibacterial activity effect peptide can be found Anywhere natural, and on the biological target contributing to human survival, there is extremely important impact. [22,23,25]
Therefore, having for various bacteria in the urgent need to developing, especially the novel substance that bacterium has remarkable antibacterial activity effect being belonged to for streptococcus (Streptococcus) genus or shigella (Shigella).
This description with reference to and refer to the content of many papers and patent documentation.In addition, the paper quoted in this description and patent documentation content, its object is to be described in more detail the technical field belonging to the present invention and content thereof.
Summary of the invention
Technical task of the present invention
The present invention is intended to develop various antibacterial, especially streptococcus (Streptococcus) genus or shigella (Shigella) accessory is had to the novel substance of remarkable antibacterial activity effect.According to result of study of the present invention, the cis ring (L-Leu-L-PROLINE) (cis-cyclo (L-Leu-L-Pro)) in dipeptides has special antibacterial activity effect to Streptococcus or Shigella.
The object of the invention is to, provide one by cis ring (L-Leu-L-PROLINE) as effective ingredient, and Streptococcus or Shigella are had to the compositions of antibacterial activity effect.
Another object of the present invention is to, provide one by cis ring (L-Leu-L-PROLINE) as effective ingredient, and Streptococcus or Shigella are had to the composition pesticide of antibacterial activity effect.
In addition, the present invention also aims to provide a kind of manufacture method streptococcus (Streptococcus) genus or shigella (Shigella) accessory being had to the antibacterial of antibacterial action.
By detailed description of the invention, claim and drawing, other object for the present invention and advantage will be further illustrated below.
The solution of technical task
The present invention will provide one by cis ring (L-Leu-L-PROLINE) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and Streptococcus or Shigella be had to the bactericidal composition of special antibacterial activity effect.
The present invention is intended to develop various antibacterial, especially streptococcus (Streptococcus) genus or shigella (Shigella) accessory is had to the novel substance of remarkable antibacterial activity effect.According to result of study of the present invention, the cis ring (L-Leu-L-PROLINE) (cis-cyclo (L-Leu-L-Pro)) in dipeptides has special antibacterial activity effect to Streptococcus or Shigella.
The present invention belongs to streptococcus (Streptococcus) or shigella (Shigella) accessory has remarkable antibacterial activity effect.
" antibacterial " the word representative containment growth of antibacterial adopted in this description, propagation and killing action, and be the same meaning with " antibacterium " in this explanation.
The cis ring (L-Leu-L-PROLINE) being used as effective ingredient in the present invention comprises the cis ring (L-Leu-L-PROLINE) of occurring in nature.In addition, about the chemical structural formula of cis ring (L-Leu-L-PROLINE) is as shown in accompanying drawing 21 of the present invention.
As shown in the embodiment of the invention, the cis ring (L-Leu-L-PROLINE) being used as effective ingredient is in the present invention the cis ring (L-Leu-L-PROLINE) separated from Leuconostoc (Leuconostoc) bacterium
In the Leuconostoc of the cis ring (L-Leu-L-PROLINE) being used as effective ingredient in the present invention when being separated from Leuconostoc, be included in more than one Leuconostoc selected in the flora be made up of leuconostoc mesenteroide (Leuconostoc mesenteroides), leuconostoc lactis (Leuconostoclactis), cold leukonid (Leuconostoc gelidum).In addition, when isolating from Leuconostoc cis ring (L-Leu-L-PROLINE) that be used as effective ingredient in the present invention, Leuconostoc is leuconostoc mesenteroide.
Cis ring (L-Leu-L-PROLINE) in the present invention has antibacterial activity effect to streptococcus
According to the embodiment of the present invention, the Streptococcus in the present invention is selected more than one streptococcus from the flora that streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus agalactiae (Streptococcus agalactiae), bargen's streptococcus (Streptococcus bovis), Streptococcus oralis (Streptococcusoralis), micrococcus scarlatinae (Streptococcus pyogenes), Streptococcus viridans (Streptococcus viridans) form.More correct is selected more than one Streptococcus from the flora of streptococcus pneumoniae, micrococcus scarlatinae, streptococcus agalactiae composition, and the most correct is bargen's streptococcus.
In addition, the present invention has special antibacterial activity effect to streptococcus
According to another the embodiment of the present invention, the Streptococcus in the present invention is selected more than one streptococcus from the flora that shigella sonnei (Shigella sonnei), shigella boydii (Shigella boydii), Shigella dysenteriae (Shigella dysenteriae), shigella flexneri (Shigella flexneri) form.More correct is selected more than one streptococcus from the flora of Shigella dysenteriae, shigella sonnei composition, and the most correct is Shigella dysenteriae.
According to another kind of pattern of the present invention, cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and is provided composition pesticide streptococcus (Streptococcus) or shigella (Shigella) to special antibacterial activity effect by the present invention.
In addition, compositions provided by the invention is a kind of composition pesticide containing above-mentioned cis ring (L-Leu-L-Pro) pesticide active ingredient.
Composition pesticide provided by the invention is the composition pesticide of selected more than one composition or additive in the group of solvent, carrier, emulsifying agent and the dispersant composition approved in Pesticide Science from pesticide active ingredient and (b) of cis ring (L-Leu-L-Pro).Term " pesticide active ingredient " in this explanation refers to the antibiotic effect of above-mentioned cis ring (L-Leu-L-Pro) streptococcus (Streptococcus) or shigella (Shigella) or reaches active sufficient quantity.
When the compositions in the present invention is made composition pesticide, in composition pesticide of the present invention, the solvent approved in Pesticide Science will be contained.In addition, in above-mentioned solvent containing water, aromatic solvent (such as, solvesso (Solvesso)) product, dimethylbenzene, paraffin (such as, mineral oil fractions material matter), ethanol (such as, methanol, ethanol, amylalcohol, toluene nine prefecture), ketone (such as, Ketohexamethylene, gamma-butyrolacton), ketopyrrolidine (NMP, NOP), acetate fiber (ethylene glycol acetate fiber), ethylene glycol, fatty acid dimethylamino, fatty acid and fatty acid ester (in principle, can also solvent mixture be utilized).
In addition, when the compositions in the present invention is made composition pesticide, containing the carrier approved in Pesticide Science in composition pesticide of the present invention.Such as, above-mentioned carrier comprises the natural mineral matter (such as, Kaolin, clay, Talcum, Chalk) of pulverizing and the synthetic mineral (such as, silicon dioxide, the silicate of high dispersive) of pulverizing.
In addition, when the compositions in the present invention is made composition pesticide, composition pesticide of the present invention is containing the emulsifying agent that Pesticide Science is approved.Such as, mentioned emulsifier comprises nonionic and anionic emulsifier (such as, Polyoxyethylene fatty ethanol ether, alkyl ore deposit hydrochlorate, aryl ore deposit hydrochlorate).
In addition, when the compositions in the present invention is made composition pesticide, containing the dispersant approved in Pesticide Science in composition pesticide of the present invention.Such as, above-mentioned dispersant comprises Lignin sulfite waste liquid, methylcellulose.
The present invention is the composition pesticide containing the cis ring be contained in antibacterial compositions (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) effective ingredient.In order to avoid too much repeat specification in the present note, thus eliminate the identical content about these two kinds of compositions.
According to another embodiment of the present invention, utilize the present composition, manufacture and have the method for the antibacterial of antibacterial action to be divided into following two stages to streptococcus (Streptococcus) genus or shigella (Shigella) accessory: namely, (a) manufactures the stage of lactic acid bacteria culture solution after cultivating lactobacillus; B cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) in above-mentioned culture fluid is condensed into the stage of applicable antimicrobial concentration by ().
In (a) of the present invention stage, when Leuconostoc is used as lactobacillus, upper Leuconostoc is at leuconostoc mesenteroide (Leuconostoc mesenteroides), leuconostoc lactis (Leuconostoc lactis), cold leukonid (Leuconostocgelidum), leuconostoc paramesenteroides (Leuconostoc paramesenteroides), Fructus Citri Limoniae leukonid (Leuconostoccitreum), leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides), selected more than one Leuconostoc lactobacillus in the flora that Leuconostoc holzapfelii forms.More correct, upper Leuconostoc is leuconostoc mesenteroide.
When (a) of the present invention stage manufactures lactic acid bacteria culture solution, the culture medium constituent and additive that can adopt in the industry can be utilized.
(b) of the present invention enriching stage, also comprises the stage of separation and refine cis ring (L-Leu-L-Pro) from culture fluid or the stage of the concentrated culture fluid containing cis ring (L-Leu-L-Pro).
Stage (b) of the present invention, when being separated and refining cis ring (L-Leu-L-Pro), putative various method can be adopted.
Such as, the above-mentioned ultrafiltration membrane allowing water pass through to have certain molecular weight CUT-OFF value, to obtain the method for separated component; The various refinement methods separated are carried out by various chromatography (size, electrolysis, hydrophobicity or affinity make).
The manifestation mode " concentrated by valid density " in the present invention refers to that (lactic acid bacteria culture solution of L-Leu-L-Pro or cis ring (L-Leu-L-Pro) is to the antibacterial activity concentration that streptococcus (Streptococcus) belongs to or shigella (Shigella) belongs to containing cis ring.
Such as, refer to that the cis ring (L-Leu-L-Pro) be contained in above-mentioned cis ring (L-Leu-L-Pro) or culture fluid is 2.65mg (37.86mM) to the minimum antibacterial activity concentration that streptococcus (Streptococcus) belongs to or shigella (Shigella) belongs to.
The invention relates to the antibacterial manufacture method containing the cis ring be contained in above-mentioned antibacterial compositions (L-Leu-L-Pro).Therefore, in order to avoid too much repeat specification in the present note, thus the identical content about these two kinds of compositions is eliminated.
In another embodiment of the invention, the present composition is by cis ring (L-Phe-L-PROLINE) (cis-cyclo (L-Phe-L-Pro)) or cis ring (L-Leu-L-Pro) (cis-cyclo (the L-Leu-L-Pro)) antiviral composition as effective ingredient.But being not limited to above-mentioned cis ring (L-Phe-L-PROLINE), also can be the antiviral composition separated from Leuconostoc (Leuconostoc).Upper Leuconostoc can from leuconostoc mesenteroide (Leuconostoc mesenteroides), leuconostoc lactis (Leuconostoc lactis), cold leukonid (Leuconostocgelidum), leuconostoc paramesenteroides (Leuconostoc paramesenteroides), Fructus Citri Limoniae leukonid (Leuconostoccitreum), selected a kind of Pseudomonas in the flora that leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides) and Leuconostoc holzapfelii form.But the antiviral composition be not limited to for above-mentioned virus also can be for influenza A virus (H 3n 2) antiviral compositions.
In addition, in another embodiment of the invention, the present composition is by cis ring (L-Phe-L-PROLINE) (cis-cyclo (L-Phe-L-Pro)) or cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and to influenza A virus (H 3n 2) there is the medical composition of special antibacterial activity effect.
In addition, in another embodiment of the invention, manufacture in the present invention influenza A virus (H 3n 2) the method with the antiviral agent of antibacterial action be divided into following two stages: namely, (a) manufactures the stage of lactic acid bacteria culture solution after cultivating lactobacillus; B cis ring (L-Phe-L-PROLINE) (cis-cyclo (L-Phe-L-Pro)) in above-mentioned culture fluid or cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) are condensed into the stage of applicable antimicrobial concentration by ().
Invention effect
Below by feature of the present invention for simple declaration and advantage:
(II) in addition, the invention provides and a kind of cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro) as effective ingredient, and to be belonged to streptococcus (Streptococcus) or shigella (Shigella) accessory has the manufacture method of the bactericidal composition of special antibacterial activity, composition pesticide and antibacterial.
(II) present composition has outstanding antibacterial activity effect to Streptococcus or shigella, is therefore applicable to the various industrial circles needing to remove or prevent Streptococcus, shigella.Because a small amount of present composition also can give play to outstanding antibacterial activity effect, so have high economic benefit.
(II) in addition, the present invention is the antiviral composition containing cis ring (L-Leu-L-Pro) or cis ring (L-Phe-L-PROLINE), in the middle of the treatment that therefore also can be used for multiple virus.
(iv) the present invention also for the pesticide industry and pharmaceuticals industry that utilize dipeptides provide a kind of pesticide about cis ring (L-Leu-L-Pro) or cis ring (L-Phe-L-PROLINE) and medical in basic data.
Accompanying drawing explanation
Fig. 1 is about the antibacterial activity comparison diagram in plant extract.When comparing experiment, we have prepared some disks containing culture fluid.Containing Si Shi lactobacillus (W.cibaria) LBP-B06 culture fluid Wei in No. 1 disk, containing Lactobacillus saki (L.sakei) LBP-S02 culture fluid in No. 2 disks, containing lactobacillus Pickles (L.kimchii) LBP-B02 culture fluid in No. 3 disks, containing Lactobacillus plantarum (Lb.plantarum) LBP-K10 culture fluid in No. 4 disks, lactobacillus is contained addicted to citric acid leukonid LBP-K11 (L.citreum LBP-K11) culture fluid in No. 5 disks, containing leuconostoc mesenteroide LBP-K06 (L.mesenteroides LBP-K06) culture fluid in No. 6 disks.As shown in the figure, Left-Hand Panel make use of the Bacteria Indicators of bacillus cereus (Bacillussubtilis), and right panel make use of the Bacteria Indicators of coliform (Escherichia coli).At this, all experiments have all independently carried out three times.
Fig. 2 is by 600nm absorbance and pH change, embodies the normal growth signal chart of Lactobacillus plantarum LBP-K10.After 28 hours, after having carried out the pH (closed hoop) and 600 (open ring-type) of two time-of-weeks, have and carried out pH process with the degree of pH about 3.8 (Left-Hand Panel).In addition, in Lactobacillus plantarum LBP-K10 normal growth process, disk diffusion method (disk diffusion assay) is adopted to carry out observing (right panel) to antibacterial activity change, and after cultivation one time, carry out pH (closed hoop) and antibacterial activity (open ring-type) process of week age.
Fig. 3 is the ability schematic diagram of containment pathogenicity bacteria growing.On a lbmc agar plate, the suppression ring of culture fluid to the Candida albicans Pseudomonas (C.albicans7) with gram positive bacteria (B.subtilis, L.monocytogens and S.aureus), gram-negative bacteria (S.typhimurium, S.sonnei and E.coli) and opportunistic fungi pathogenicity is utilized to observe.
Fig. 4 is in Lactobacillus plantarum LBP-K10 culture fluid, the thermal stability schematic diagram of the non-protein antibiotic substance of the extraction extracted.Observed by the diameter of the growth inhibitor of disk diffusion method and learn, the antibacterial activity of non-protein material to gram positive bacteria (B.subtilis) and gram-negative bacteria (E.coli) is similar to through heat treated culture fluid.
Fig. 5 is when processing protease, has the feature schematic diagram of the culture fluid of non-protein antibiotic substance effect.At this, after multiple protein catabolic enzyme is processed, its antibacterial activity is observed.As shown in following embodiment, in the middle of E.C. 3.4.21.64 and chymotrypsin protein ferment treatment to culture fluid, and with 1000 molecular weight for standard, test after culture fluid being divided into YM-1cutoff (YM1C) and YM-1 supernatant (YM1S), control group.
Fig. 6 utilizes CH 2cl 2, extract the schematic diagram of Lactobacillus plantarum LBP-K10 culture fluid.A in Fig. 6 utilizes CH 2cl 2(dichloromethane), extracts the antibacterial activity schematic diagram of the supernatant after Lactobacillus plantarum LBP-K10 culture fluid; B is not containing CH 2cl 2culture supernatant antibacterial activity schematic diagram; C utilizes CH 2cl 2the subnatant antibacterial activity schematic diagram that (dichloromethane) extracts.
Fig. 7 utilizes ZORBAX C18 octadecyl Si hydrophobic resin, Lactobacillus plantarum LBP-K10 is prepared to the result schematic diagram of chromatograph HPLC analysis.Now, collected fractional distillation material matter matter by the extraction method of liquid to liquid, 210,260 and 280nm ultraviolet wavelength condition under, have recorded above-mentioned analysis content.All fractional distillation material matter matter all can be divided into more than 10, and by shown in following embodiment, collects above-mentioned 10 fractional distillation material matter matter and concentrate.
Fig. 8 is the method utilizing least concentration culture medium, the test portion separated is carried out to the result schematic diagram of antibacterial activity test from Pediococcus pentosaceus (P.pentecostals).At this, utilize CH 2cl 2be extracted all culture fluid, and utilize dilution method, Bacteria Indicators is placed on 6 orifice plate plates of each test portion, then utilize the method for least concentration culture medium to carry out the result of testing, in gram positive bacteria and gram-negative bacteria, found antibacterial activity effect simultaneously.Now, by the method for implementing least concentration culture medium, and the CH of the culture fluid be not separated 2cl 2subnatant is used as control group.In addition, in the bacillus cereus (Bacillus subtils) of gram positive bacteria.Observe the antibacterial activity of the separation test portion separated from Pediococcus pentosaceus (Pediococcus pentosaceus).Show according to result 6a, 6b, 7a, 7b, 8,9,10 being separated to the antibacterial activities of test portions and carrying out observation analysis, the antibacterial activity be separated in test portion with 8 and 10 at 7b is the most obvious.Utilizing the method for least concentration culture medium to carry out testing result to 7b to be separated test portion least concentration with 8 and 10, is 1.45mg (20.71mM), 1.37mg (19.6mM), 2.35mg (33.57mM) respectively.
Fig. 9 is the method utilizing least concentration culture medium, the antibacterial activity of Lactobacillus plantarum LBP-K10 separation test portion is carried out to the result schematic diagram of observation analysis.Now, utilize gram positive bacteria bacillus cereus (Bacillus subtils), analyze the antibacterial activity that Lactobacillus plantarum LBP-K10 is separated test portion.In addition, isolate the separation test portion of 3.0x 106CFU (colonyforming unit) respectively in every 6 orifice plates after, detect for bacillus cereus (Bacillus subtils).The result of carrying out observation analysis according to the antibacterial activity being separated test portion to 2,3,4,5,7,8,9,10 shows, the antibacterial activities in 7b, 8 and 10 separation test portions are the most obvious.Utilizing the method for least concentration culture medium to carry out testing result to 7b to be separated test portion least concentration with 8 and 10, is 1.30mg (20.62mM), 1.25mg (17.86mM), 2.35mg (33.57mM) respectively.
Figure 10 utilizes the method for least concentration culture medium, the antibacterial activity of leuconostoc mesenteroide LBP-K06 separation test portion antibacterial activity is carried out to the result schematic diagram of observation analysis.Now, utilize the bacillus cereus (Bacillus subtils) of gram positive bacteria, analyze the antibacterial activity that leuconostoc mesenteroide LBP-K06 is separated test portion.According to 3,4,5,6a, 6b, 7a, 7b, 8,9,10 antibacterial activities being separated test portions result of carrying out observation analysis shows, the antibacterial activity be separated in test portion with 10 at 7b is the most obvious.Utilizing the method for least concentration culture medium to carry out testing result to 7b, 10 least concentrations being separated test portion, is 1.43mg (20.43mM), 5.50mg (78.6mM) respectively.
Figure 11 carries out the result chromatogram of observation analysis to the antibacterial activity of Pediococcus pentosaceus, Lactobacillus plantarum LBP-K10, leuconostoc mesenteroide LBP-K06 separation test portion antibacterial activity.At this, all bacterial strains all adopt CH 2cl 2extraction method has carried out separating treatment.
Figure 12 is the chromatogram of various lactobacillus fractional distillation material matter matter.That is, be about utilizing CH 2cl 2the chromatography figure of the various lactobacillus preparative hplc HPLC that extraction method is extracted.In this experiment, have employed Pediococcus pentosaceus MCPP (Pediococcuspentosaceus MCPP), leuconostoc mesenteroide LBP-K06 (Leuconostoc mesenteroides LBP-K06), Lactobacillus plantarum LBP-K10 (Lactobacillus plantarum LBP-K10), Si Shi lactobacillus LBP-K15 Wei (Weissella cibariaLBP-K15), Wei Si Salmonella LBP-K16 (Weissella confusa LBP-K16), Lactobacillus saki LBP-S01 (L.sakeiLBP-S01), Lactobacillus plantarum/Pan Tuosi LBP-S02 (Lactobacillus plantarum/pentos LBP-S02), lactococcus lactis LBP-S03 (Lactococcus lactis LBP-S03), lactococcus lactis LBP-S06 (Lactococcus lactis LBP-S06), the bacterial strains such as staphylococcus sciuri LBP-S07 (Staphylococcus sciuri LBP-S07).
Figure 13 is the result schematic diagram that the electron impact ionization method (electron ionization, EI) utilizing Pediococcus pentosaceus MCPP to be separated P8 carries out observation analysis.
Figure 14 is the result schematic diagram that the chemical ioni zation method (chemical ionization, CI) utilizing Pediococcus pentosaceus MCPP to be separated P8 carries out observation analysis.
Figure 15 is separated P8 to Pediococcus pentosaceus MCPP 1the observation analysis result schematic diagram of H-nuclear magnetic resonance method (500MHz, 100%DMSO).
Figure 16 is separated P8 to Pediococcus pentosaceus MCP 1h-nuclear magnetic resonance method (containing 500MHz, 10%D 2the DMSO of O) observation analysis result schematic diagram.
Figure 17 is separated P8 to Pediococcus pentosaceus MCPP 13the observation analysis result schematic diagram of C-nuclear magnetic resonance method (500MHz, 100%DMSO).
Figure 18 is separated P8 to Pediococcus pentosaceus MCPP 13c-nuclear magnetic resonance method is (containing 500MHz, 10%D 2the DMSO of O) observation analysis result schematic diagram.
Figure 19 utilizes X-line to discount method, from the expected structure formula of the P8 that Pediococcus pentosaceus MCPP separates.
Figure 20 utilizes X-line fold-line method, from the structural formula of the P8 that Pediococcus pentosaceus MCPP separates.
Figure 21 is the final structure schematic diagram of the P8 separated from Pediococcus pentosaceus MCPP.Analysis result shows according to observations, and the antibiotic substance separated has C 11h 18o 2n 2cis ring (L-Leu-L-Pro) (the cis ring (L-Leu-L-Pro) of molecular formula.
The antiviral effect figure of Figure 22 separating compound.At this, antiviral activity experiment have employed the epithelioid cell being derived from and can blocking dog kidney (Madin-Darby, canine kidney).In experimentation, infect the influenza A virus (H3N2) of three days by a definite date to epithelioid cell.The preparative hplc HPLC of Lactobacillus plantarum LBP-K10 is shunted goods and materials be mixed in the middle of the minimum key element culture medium (complete minimalessential medium, MEM agarose) of 0.7%, and put into by influenza A virus (H 3n 2) on the blocked dog epithelial cell (MDCK) that infects.Now, all antiviral activity experiment groups all have employed bacterial plaque (plaque) analytic process.(A) check plot; (B) virus control district; (C) the 8th fractional distillation material matter, 5.0mM cis ring (L-Leu-L-Pro); (D) 8 fractional distillation material matter, 10.0mM cis ring (L-Leu-L-Pro); (E) the 10th fractional distillation material matter, 5.0mM cis ring (L-Phe-L-PROLINE); (F) the 10th fractional distillation material matter, 10.0mM cis ring (L-Phe-L-PROLINE).
Figure 23 is the result schematic diagram that the electron impact ionization method (electronionization, EI) utilizing Lactobacillus plantarum LBP K-10 to be separated N10 carries out observation analysis.
Figure 24 is the result schematic diagram that the chemical ioning method (chemical ionization, CI) utilizing Lactobacillus plantarum LBP K-10 to be separated N10 carries out observation analysis.
Figure 25 is separated N10 to Lactobacillus plantarum LBP K-10 1the analysis result figure of H-nuclear magnetic resonance method (600MHz, 100%DMSO).
Figure 26 be to Lactobacillus plantarum LBP K-10 be separated N10 2D HSQC according to 1h/ 13the analysis result figure of C NMR (600MHz, in100%MeOD).
Figure 27 be to Lactobacillus plantarum LBP K-10 be separated N10 2D COSY according to 1h/ 13h NMR (600MHz, in100%DMSO (left side) and 10%D 2o comprises DMSO (right side)) spectrum schematic diagram.
Figure 28 utilizes X-line fold-line method, the structure of the N10 separated from Lactobacillus plantarum LBP K-10.
Figure 29 utilizes X-line fold-line method, from the structure of the N10 that Lactobacillus plantarum LBP K-10 separates.
Figure 30 is the final structure schematic diagram of the N10 separated from Lactobacillus plantarum LBP K-10.Analysis result shows according to observations, and the antibiotic substance separated has C 14h 16o 2n 2l-Leu-the L-Pro of molecular formula.
Detailed description of the invention
Below will the present invention will be described in more detail by embodiment.But state in advance, following embodiment is only used to illustrate in greater detail the technology of the present invention, according to present inventive concept, scope of the present invention is not limited to following embodiment.
< embodiment 1> is separated from plant, the LAB (lactic acid bacteria) of qualification.
In this manual, under the qualification that nothing illustrates separately, in order to the concentration embodying predetermined substance adopt " % ", represent solid/solid (w/w) %, solid/liquid (weight/volume) %, liquid/liquid (volume/volume) % respectively.
Material and method
The separation of experimental strain and lactobacillus
First, various lactobacillus has been isolated respectively from Caulis et Folium Brassicae junceae Pickles and Herba Sedi Pickles, cabbage kimchi, and utilize agitator to be stirred by off-the-shelf various material, and in Caulis et Folium Brassicae junceae Pickles and Herba Sedi Pickles, with the addition of the NaCl of 5% respectively, prepared again the Caulis et Folium Brassicae junceae Pickles and the Herba Sedi Pickles that do not add 5%NaCl, and respectively 4 DEG C, 22 DEG C, 30 DEG C temperature conditions under given storage.Then, within one-month period, every 24 hours, all plant experimental groups are detected, and after utilizing the concentration of carrying out the distilled water diluting of sterilization treatment each experiment group, spread upon on MRS (the de Mann Rogosa Sharpe) solid medium of lactobacillus isolation medium, and under the temperature conditions of 30 DEG C, cultivated 2-3 days time.After cultivation terminates completely, filter out separating effect some thalline preferably, carried out the process of 16srDNA base sequence, and for compare with medium information, and carried out again cultivating in liquid MRS culture medium.
The lactobacillus qualification be separated
In this experiment, in order to identify the lactobacillus separated from fermenting plant, and by 16S rDNA base sequence method, lactobacillus is identified.For carrying out PCR (polymerase chain reaction), and have employed 27F (5 '-AGA GTT TGA TCM TGG CTC AG-3 ' (serial number 1) and 1492R (5 '-GGYTAC CTT GTTACG ACT T-3 ' (serial number 2)) of general Eubacteria primer.In addition, for carrying out PCR reaction, and under the temperature conditions of 94 DEG C after denaturation (pre-denaturation) process of 5 minutes, degeneration (denaturation) process of 1 minutes has been carried out again under the temperature conditions of 94 DEG C, and under the temperature conditions of 55 DEG C, carry out annealing (annealing) process of 1 minutes, and through 1 minutes under the temperature conditions of 72 DEG C, expansion (extention) process of 30 times, final under the temperature conditions of 72 DEG C through additional expansion (extra-extention) process of 7 minutes, and stored under the temperature conditions of 4 DEG C.After polymerase chain reaction, confirm because of 0.7% agarose gel electrophoresis and the 16S rDNA of amplification, and after isolating DNA, carried out base sequence analysis.In addition, utilize the nucleotide BLAST chart of NCBI, the 6S rDNA base sequence homology of the bacterial strain filtered out is compared.(http://www.ncbi.nlm.nih.gov)。
Meet antibacterial activity test and the normal bacterial bacterial strain needed for condition of culture, multi-agent patience leather is blue-positive and remove from office orchid-negative bacterial strain
The present invention, in the middle of the lactobacilli strain be separated, mainly have employed Lactobacillus plantarum LBP-K10.At this, all fractional distillation lactobacilli strain containing Lactobacillus plantarum LBP-K10 in the present invention have employed MRS (mMRS) culture medium or 1.7% solid medium (Difco laboratory, Detroit, MI) of distortion.In the present invention, in order to find the antibiotic substance generated by LAB, and have employed the lactobacilli strain such as Pediococcus pentosaceus, Lactobacillus plantarum and Leuconostoc mesenteroides separated from Korean traditional fermented foodstuff.In addition, in order to detect antibacterial activity, the bacterium such as contrast such as gram negative bacteria such as gram-positive bacterium contrast bacterium and E.coli, S.typhimurium, S.sonnei etc. such as B.subtilis, S.areus, L.innocua, S.pneumoniae are used as antibacterial index bacterial strain by the present invention.In addition in the present invention, mankind 19A cerebrospinal fluid is derived from also by state-run for Korea S health care hospital, and have in penicillin, erythromycin, tetracycline, clindamycin for repellence Streptococcus pneumonia 14596, state-run health care hospital of Korea S the mankind B that is derived from defecate, and to have in ACSSuT, H1:1 and H2:1.2 repellence Salmonella typhimurium 12219, to have new green grass or young crops two (MEC A+) repellence methicillin-repellence staphylococcus aureus strains (methicillin-resistant staphylococcus aureus); MRSA) 11471 these three kinds of multi-agent patience antibacterials are used in the middle of the minimum concentration of ordinary dissolution (MIC) of antibacterium compound.Except Listerella (Listeria spp.), cultivate in major part pathogenic strains equal liquid medium within (Luria-Bertani:LB) broth (1%tryptone, 0.5%yeastextract, 1%NaCl), Listerella (Listeria spp.) then cultivates in brain heart infusion (brain heart infusion) culture fluid (Difco, Laboratories, Detroit, MI).In addition, all bacterial cells are all cultivated in MRS, and under the condition do not stirred or rock, the inoculation microbial inoculum (seeding inoculum) of 0.1 ~ 0.2% have been carried out to the cultivation of 3 day time in 30 DEG C of temperature.In addition, also by the mode of centrifugalize, under 8000rpm condition, through 15 minutes, twice cultivation has been carried out to the cell of growth.By culture supernatant (Culturesupernatant; CS) for Analysis of Antimicrobial Activity.
Antibacterial activity is assessed
In order to understand antibacterial activity, and carry out the experiment of media dilution method (broth microdilution test), disk diffusion method (diskdiffusion assay) and antagonism method (antagonism test)
Above-mentioned various experimental technique is as follows.
In the experiment of media dilution method, after serial dilution is carried out to concentrated culture supernatant, whether observe microbiological indicator bacterial strain with activity.In addition, in the experiment of antagonism method, in order to observe antibacterial activity, and the LAB cell cultivated through 24 hours is made to reach OD 600the optimum state of=1.0, and with unit point drop on MRS plate, then under the temperature conditions of 30 DEG C, carried out the cultivation of 24 hours.After this, the index bacterial strain cultivated in advance 3 ~ 5 hours is placed in drop to be had on the MRS plate of LAB cell, and under the temperature conditions of 30 DEG C, after cultivating the time, calculates the diameter (mm) of growth district.
In the experiment of disk diffusion method, culture supernatant (CS) or antiviral substance point are dropped on the paper disc (Toyo Roshikaisha, ltd) of 6mm, then will cultivate the agar plate being seeded in 1% through the index bacterial strain of 3 ~ 5 hours in advance.After agar plate solidifies, oriented disk paper (disk paper) is placed on agar plate, and under suitable temperature conditions, has carried out the cultivation of 24 hours, then the diameter (mm) of containment growth ring is calculated, to measure antibacterial activity.
According to pH and the antibacterial activity change of LAB growth
In units of 1.0%, the Lactobacillus plantarum LBP-K10 at cultivation one night is inoculated in distortion MRS culture medium, then under the temperature conditions of 30 DEG C, carries out quiescent culture.Until enter resting stage (stationary phase) after inoculation terminates, under 600mm condition, within every 4 hours, measure an absorbance, and obtain supernatant after centrifugation, and measure pH and antibacterial activity.After utilizing above-mentioned media dilution method (broth microdilution test), disk diffusion method (disk diffusion assay), antagonism method (antagonism test) to inoculate, within a week, within every 24 hours, measure an antibacterial activity.
To the effect analysis of proteolytic enzyme
Under the temperature conditions of 37 DEG C, to E.C. 3.4.21.64 (proteinase K, Sigma-Aldrich, Inc., USA), chymase (chymotrypsin, Boehringer MannheimGmbH., Germany), E.C. 3.4.21.64 (proteinaseK, Sigma-Aldrich, Inc., USA), trypsin trypsin, Sigma-Aldrich, Inc., USA) carry out the hatching process of hour after, except E.C. 3.4.21.64, under room temperature (25 DEG C) condition, the ultimate density of other protease is adjusted to after, observe protein substance except cell and non-protein substance whether to pathogenic microbes have activity and for CS (utilize cellulose acetate film YM-1 to filter, molecular weight below 1000 and more than 1000) sensitivity of proteolytic enzyme.Carry out, after isothermal reaction, carrying out degenerative treatments to mixture through appropriate time, and for carry out deactivation process to these protease, and under the temperature conditions of 95 DEG C, carry out the heat treatment of 5 minutes.As mentioned above, utilize disk diffusion method, determine antibacterial activity.
To the heat treatment of antibiotic substance
In order to confirm the impact that the activity of temperature on antibiotic substance causes, and in the water-bath (water bath) of 100 DEG C, through 60 minutes, 120 minutes, CS is heat-treated, again under 121 DEG C of temperature conditions, utilize high pressure steam sterilizer (autoclave), to heat treatment CS being carried out to 15 minutes.Before activity analysis is carried out to sample, under CS is placed in normal temperature condition.Now, CS is used as control group.In addition, after carrying out high-temperature process, remaining antibacterial activity is measured by disk diffusion method.
The separation of antibacterial substance prepares
For in the Lb.plantarum LBP-K10 be separated, utilize the MRS (mCS) of distortion, from CS, extract antibacterial compounds, and illustrate its characteristic and determine following element.MMRS comprises following element:
Often liter containing 10g peptone, 2g ammonium citrate, 5g sodium acetate fiber, 0.1g magnesium sulphuric acid, 0.05g manganese sulphuric acid, 2g potassium phosphate, 5g yeast extract and 20g glucose.
Before being mixed with other source of supply by D-Glucose, carry out sterilization treatment respectively.CS (being also like this in P.pentosaceus and Lc.Mesenteroides) is extracted from the Lb.plantarum LBPK-10 cultivating three days among mMR, and as mentioned above, 30 minutes centrifuging treatments have been carried out under 8000rpm condition, then by after concentrated 10 times of Lb.plantarumLBPK-10 bacterial strain, carry out lyophilization (freeze-drying) process, and according to Liquid-liquid extraction method, utilize separatory funnel (separating funnel), extract the dichloromethane (CH of a time 2cl 2), and under the temperature conditions of 55 DEG C, utilize vaporizer to carry out processed.Then, obtain and be separated organic facies (organic phase), and utilize vapour removal organic solvent.In addition, for more water base phase (aqueous phase) and organic facies and CS activity and be only extracted water base phase (aqueous phase).Make eluent (eluent) by after C18SPE print cartridge (WatersSep-pak C18plus cartridge, Millipore Corp., Marlborou, MA), allow the sample overflowed be placed on SPE print cartridge, and have employed the methanol solvate (isocratic flow) of 100%.Before being placed on SPE by sample, activation is carried out to print cartridge, and after utilizing the flowing water of MeOH and 15ml of 15ml to carry out the cleaning of 100%, sample is placed on SPE, and whom has carried out the cleaning of 100% with.By print cartridge and 100%MeOH, complete the final spilling of sample.In addition, for continuing the sample of collection spilling and removing remaining organic solvent and carried out evaporation process.In order to implement following HPLC process, and utilize the TDW suitably carrying out sterilization treatment, dissolution process has been carried out to each concentrated sample.
The antiviral substance analyzed by HPLC (high performance liquid chromatography) method (HPLC) is separated
Utilize appropriate distilled water to dissolve the material extracted, and utilize the cellulose acetate membrane of 0.22 μm to carry out filtration treatment.The sample filtered is by adopting ODS C-18 vertical axis (octadedosyl-C18hydrophobic semi-preparativecolumn) (9.4 × 250mm, Agilent, USA) HPLC (High Performance Liquid Chromatography (semi-prep HPLC system, Agilent1200series, USA)) system carried out analysis and separating treatment.In addition, after the temperature of vertical axis is fixed on 40 DEG C, in the mobile phase in 35 minutes, 67% be water, 3% be acetonitrile (ACN), 30% is ethanol, and flow velocity is now 1.5ml/ minute.In addition, utilize the multiple ripple of UV to open induction system (UV multi-wavelengthdetector system), observe be respectively the condition of 210nm, 260nm, 280nm at wavelength under.In order to obtain each powder sample, and by the mode of evaporating and freeze, collection and concentration are carried out to each separation (fraction).
Minimum inhibitory concentration method (MIC) is utilized to measure antibacterial activity
After utilizing HPLC to collect fractional distillation material matter matter (fractionation), as citing document [Mathur et al., 2005; Messens et al., 2002] described, by minimum inhibitory concentration method (minimum inhibitory concentration; MIC), the activity of each acquisition sample is detected.By identical method, in the sample be so separated, additional refinement has the separation (fraction) of the most highly active suitable peak value, then obtains single compound again.To this, also undertaken adding by multiple analytic process such as GC/MS, elementary analysis (elemental analysis), NMR spectrographic method and X-ray scattering technologies (X-ray crystallography) and analyze.
Cell culture and virus strains, culture medium and condition of culture
The complete constituent of DMEM (v/v) culture medium is as follows: namely, the DMEM (high glucose, HycloneLaboratories, USA) of 76.5%, the penicillin streptomycin (Gibco, USA) of 1.0%, 2.5% 1.0MHEPES buffer solution, the interior Ox blood serum sterilizing of phosphoric acid-buffered saline (7.5%DPBS) of Dulbecco, BioXtra, be applicable to cell culture 2.5% protein solution (Sigma, USA) and 10.0% N of clear (FBS) (NOVA, USA) of fetal blood.
In addition, VIM by 94.0% DMEM (high glucose) (Hyclone Laboratories, USA), penicillin/streptomycin (the Gibco of 1.0%, USA), 2.5%1.0M HEPES buffer (Gibco, USA), the BioXtra of Ox blood serum sterilization treatment is carried out in the phosphoric acid-buffered saline (7.5%DPBS) of Dulbecco, be applicable to the protein solution (Sigma of 2.5% of cell culture, and clear (the FBS) (NOVA of 10.0% N of fetal blood USA), USA), trypsin tosyl phenylalanyl chloromethyl ketone-treated trypsin (TPCK-treatedtrypsin) (2 μ g/ml) carrying out the acyl chlorides methyl ketone-process of toluene ore deposit of 0.1% forms.In order to carry out dental plaque analysis, and VIM agarose is attached in 2X VIM, and from the agarose SEG (TNT research, Korea) of 1.4%, is finally used as the VIM agarose of 0.7%.
Antiviral activity test-dental plaque analytic process
A.MDCK cell culture condition
As above-mentioned research, with dental plaque (plaque) for coefficient has carried out antiviral test.[Gaush et al., 1968; Huprikar et al., 1980; Chapel et al., 2006; Chapel et al., 2007; Furuta et al., 2009] in embodiments of the present invention, in order to observe antiviral activity, and by Madin-Darby dog kidney (Madin Darby canine kidney; MDCK) epithelial cell is used as antiviral index.In the present invention, for each wild type influenza virus cell of each dental plaque-refine, have employed the monolayer 5x10 of 5pfu 5intracellular mdck cell.[Gaush et al.1968; Huprikar etal.1980; Chapel et al.2006; Chapel et al.2007; Furuta et al.2009] in above-mentioned DMEM, cultivated mdck cell.At first, melted mdck cell slowly in the tank of 37 DEG C, and for removing dimethyl sulfoxine (DMSO) contained in cell, and carried out soft mixed processing with the complete DMEM of more than 10 times, and under 12,000rpm condition, carried out the centrifuging treatment of 3 minutes, then remove supernatant, swim to prevent mdck cell.At this, three said process are repeatedly carried out.After utilizing DMEM culture medium to clean mdck cell, allow mdck cell be suspended in the complete DMEM culture medium of 10ml, and by stirring gently, making mdck cell be scattered in cell culture, being then transferred on 100 Π cell culture.After this, at the 5%CO of 37 DEG C of temperature 2in couveuse, the cultivation of 72 hours is carried out to mdck cell.Utilize as the method, repeatedly cultivated mdck cell (90 ~ 95% of cell culture).After outwelling DMEM culture medium, the PCK-trypsin of 1.5ml is utilized to process the mdck cell (2x105) cultivated, and at the 5%CO of 37 DEG C of temperature 2in couveuse, mdck cell is carried out to the cultivation of 10-15 minutes.In addition, utilize the DMEM culture medium hybrid separation mdck cell out of 10 times, in order to prevent the reservation of auxocyte, and said mixture is transferred in the 50ml conical pipe of sterilization treatment, then carry out the centrifuging treatment of 3 minutes, and utilize phosphoric acid buffer normal saline solution (PBS) to clean three times.After utilizing PBS cleaning to terminate, add the complete DMEM culture medium of 10ml, and continue the complete DMEM culture medium that with the addition of 30ml.Then, the mdck cell of each 10ml is transferred in 10 Π cell culture, and at the 5%CO of 37 DEG C of temperature 2in couveuse, carry out cultivating for 48 hours.
B. the dental plaque analytic process of antiviral activity
(Gaush et al.1968 as described above; Huprikar et al.1980; Chapel et al.2006; Chapel etal.2007; Furuta et al.2009), the analysis having carried out dental plaque prepares.The mdck cell growing to cell culture 90 ~ 95% is transplanted in 6 orifice plate cell dish, then will the mdck cell of cell culture 80% have been grown to, and utilized PBS to clean twice, then will contain viral infection culture medium (virus infection media; VIM) multiple single compound is put in the middle of mdck cell.Carry out 90 minutes constant temperature culture under 34 DEG C of temperature conditions during, within every 15 minutes, shake gently and infect viral 6 orifice plate dishes.After this, remove the culture medium infecting virus, and 0.7%DMEM agarose (1:1) is placed in respective orifice plate, then at the 5%CO of 34 DEG C of temperature 2the cultivation of 72 hours is carried out, to observe the generation qualification of dental plaque in incubator.
By adopting the electron impact ionization method (electron ionization:EI) of GS-MS and the analysis of chemical ioni zation method (chemicalionization:CI)
In the present invention, adopt and possess 7679 serial automated fluid samplers, and the chromagram system be made up of the serial GC of Agilent 6890 (AgilentTechnologies, Waldron, Germany).High-resolution mass-synchrometer (JEOLJMS-700, Tokyo is make use of in mass analysis, Japan), to carry out electron ionization and compound ionsization research in Lb.plantarum, and gas chromatograph drawing system and high-resolution mass-synchrometer (JEOLJMS-700, Tokyo, Japan) is adopted to implement experiment.In addition, also by JMS-700M Station software [T, 2006; Ahi et al., 2008], gas chromatography system and high-resolution mass-synchrometer are adjusted.
1h, 13c, HSQC and 2D-COSY nuclear magnetic resonance spectroscopy
In Lb.plantarum, utilize NMR spectrographic method, at D 2the sample (1.0mg) extracted in O and DMSO processes, to carry out hydrion and carbon distribution, to combine and order-checking.Utilize under the condition of 300K, possess XWIN-NMR3.5 software [Reynolds et al.2002; Lu et al.2004; Baranovskii et al.2007] the BrukerAVANCE-500 spectrogrph of CryoProbe16, have recorded NMR spectrum.All 2-D NMR test the standard pulse sequence (standard pulse sequence) all utilizing Bruker to provide and have given enforcement.On PC (Windows Professional 2000, Microsoft), XWIN-NMR 3.5software packages (Karlsruhe, Germany) is utilized to process NMR data.On AVANCE 500 spectrogrph (Bruker-Biospin), utilize the frequency of 500.13 and 125.77MHz, right 1the NMR spectrum of H and 13C has carried out record, and have employed the anti-detector in broadband (the broadband inverse detector possessing Z axis dipmeter (Z gradient) in the middle of experiment; BBI).In addition, at D 2o and containing D 2in the DMSO solution of O, record is carried out to the spectrum of [Pateletal.2010] 293K sample temperature.
Be separated elementary analysis (the Elemental analysis of single compound; EA)
For determining the elemental composition of fractional distillation material matter matter in Lt, plantarum LBp-K10, and adopt elemental analyser, and utilize this elemental analyser, the element of C, H, N and O is analyzed.(CE Instruments EA1110, EA1112) [Yuet al.2008; Okada et al.2009]. when assessing explosive, generally employ oxygen balance (Oxygen balance; OB) pattern.In addition, the element ratio of C/H/O/N is also utilized to calculate OB.
The structure crystal of the fractional distillation material matter matter utilizing x-ray to reflect
In order to determine the structure of the single compound be separated from Lt.plantarum LBP-K10, and have employed X-ray analysis of crystal structure method.Ethanol 35% and 65% dichloromethane (CH 2cl 2) in dirty solution mixture, the crystal of single compound is cultivated, and utilizes Bruker Proteum 300CCD, obtain in Pohang Light Source (Kim et al.2005) light beam line (beamline) 6B (Angstrom) data set (data set).
Experimental result
Lactobacillus in plant identification
Various lactobacillus has been isolated from each plant material of Korea S and Caulis et Folium Brassicae junceae Pickles, Herba Sedi Pickles and cabbage kimchi, and the LAB of more than 400 is isolated from this three kind of plant source of supply, then by antagonism method of testing, 200 LAB becoming most of antibacterial activity root are filtered out.In addition, utilize 16s DNA sequencing method, and by the amplification of PCR and compare, the LAB filtered out is identified.Ultimately, Leuconostoc spp., Lactobacillusspp., Lactococcus spp. and Weisella spp (table 1) is identified in the present invention.In above-mentioned three kind of plant, mainly isolate Leuconostoc spp. and Lactobacillus spp.In addition, be also separated in Herba Sedi Pickles and identify Lactococcus lactis.In order to cultivate above-mentioned fermented material, the Preliminary fermentation stage (1 ~ 2 day) in incubation, regular observation has been carried out to Leuconostoc spp., and phase (2-3 days) after incubation, Leuconostoc spp. has then become main bacterial isolates.
Table 1
[table 1]
Be separated and identify that the antibacterial activity of lactobacillus compares
At the growing period of LAB, cell according to respective needs, can generate many 2 metabolites.Therefore, for carry out adding research to LAB and antibacterial action, and continue through antagonism test and dilution method experiment, to be separated and the antibacterial activity of the LAB identified has carried out observation and comparative analysis widely.The antibacterial activity of isolated strains is as shown in table 2.In addition, in the bacterial strain of above-mentioned lactic acid bacteria, by comparing with other LAB isolated, and utilize disk diffusion method, to the W.cibaria LBP-B06 with stronger antibacterial activity, L.sakei LBP-S01, L.kimchii LBP-B02, Lb.plantarumLBP-K10, L.citreum LBP-K11 and L.mesenteroides LBP-K06 observe.In the present invention, by B.subtilis (gram-positive) and escherichia coli (gram negative) as index bacterial strain (indicator).As shown in Figure 1, in the LAB through detecting, the antibacterial activity of Lb.plantarum LBP-K10 bacterial strain is far above other various bacteria bacterial strain.Accordingly, Lb.plantarum LBP-K10 bacterial strain is mainly used as the antibiotic substance of plant material by the present invention.
Table 2
[table 2]
A represents the diameter (tabulation bacterial strain: Caulis et Folium Oryzae bacillus cereus) of antibacterial activity scope.+ represent diameter <15mm, ++ represent diameter <22mm, +++ represent diameter >=22 ㎜.
B represents minimal inhibitory concentration (Minimum inhibitory concentration:MIC).
C represents the multiple of minimal inhibitory concentration.+ represent 1 times, ++ represent 0.5 times, +++ represent 0.25 times.(index bacterial strain: Caulis et Folium Oryzae bacillus cereus).
The Analysis of Antimicrobial Activity of < embodiment 3>Lb.plantarum LBP-K10
What compare according to the 16S rDNA sequence of Lb.plantarum LBP-K10 and NCBI nucleotide blast program found that, identical with Lb.plantarum bacterial strain IMAU10173100%.According to the explanation of embodiment, the Lb.plantarum LBP-K10 identified is cultivated, then the change (Fig. 2 A, left plate) that the corresponding pH profile between the absorbance, cultured cell of 600nm and the pH value during Lb.plantarum normal growth must be paid close attention to was observed through 28 hours.PH analysis result according to LAB Growth of Cells and corresponding CS shows, and absorbance and pH inversely, and from 8 hours, enter exponential phase (logarithmic phase), and entered lag phase after 28 hours.After 28 hours lag phasees, absorbance and pH are almost in changeless state.(Fig. 2 A, left plate), for understanding the change along with the time, on the impact that antibacterial active zone comes, and in units of 1 day, gathered culture fluid through 3 days and implements experiment.According to experimental result display, within the 1st day, start to generate active, within the 2nd day, be in the state continued to increase, and the antibacterial activity of the 3rd day is in most high state, and maintain about two time-of-weeks (Fig. 2 right plate).PH continues minimizing state, and before and after lag phase, pH value reaches about 4.1, and the time is afterwards in and maintains above-mentioned numeric state.In addition, antibacterial activity is in increase trend two days later in cultivation, and reaches peak at the 3rd day.Whether (Fig. 2 A, right plate) has antibacterial activity to pathogenic bacteria for understanding Lactobacillus plantarum LBP-K10, and according to the method described above, by disk diffusion method, has carried out antibacterial activity detection (Figure 28) to various bacterial strain.At this, utilize disk diffusion method, altogether 3 experiments are separately implemented to the culture fluid of Lactobacillus plantarum LBP-K10, and by calculating Developing restraint ring (mm), the antibacterial activity of each bacterial strain is measured.Analyze display according to observations, Lactobacillus plantarum LBP-K10 culture fluid is to Caulis et Folium Oryzae bacillus cereus (Bacillus subtilis), staphylococcus aureus (Staphylococcusaureus), listera innocua (Listeria innocua), gram positive bacteria and the salmonella typhimuriums (Salmonella typhimuruium) such as streptococcus pneumoniae (Streptococcus pneumonia), the gram negative bacterias such as shigella sonnei (Shigella sonnei), the opportunistic infection hospital Mycophytas such as Candida albicans (Candida albicans) also have antibacterial activity (Fig. 2 B).
In addition, whether to heat, there is stability for understanding antibiotic substance, and after under numerous conditions heat treated being carried out to it, antibacterial activity test (Fig. 2 C) has been carried out to culture fluid.According to experimental result display, the culture fluid after Overheating Treatment changes without any antibacterial activity.After the salmonella typhimurium of the Caulis et Folium Oryzae bacillus cereus of gram positive bacteria and gram negative bacteria is used as index bacterial strain, the antibacterial activity of culture fluid is carried out to the result of observation analysis, experiment group after Overheating Treatment is with when comparing without heat treated control group, and its state is almost similar to control group.In addition, when heating 15 minutes in the steriliser of 121 DEG C of temperature, the antibacterial activity of experiment group still maintains former state.Show according to above-mentioned experimental result, the material with antibacterial activity can not the degeneration because being heated, and has stability to heat.(Fig. 2 C)
For illustrating the substance characteristics suppressing CS, and specify effect of proteolytic enzyme (proteinase), and carried out testing (protein kinase K, chymase and trypsin) (Figure 20) to multiple proteins catabolic enzyme.In order to implement the present invention's experiment, and the cultivation of 72 hours has been carried out to CS, and for preventing the molecular weight of CS solution higher or lower than 1000, and utilize YM-1 Acetate Film (Amicon, USA), CS is divided into protein (proteinous) and nonprotein two parts.In addition, also prepare the CS control group of YM1S (the CS solution of molecular weight more than 1000) and YM1C (the CS solution of molecular weight below 1000), and the concentration of proteolytic enzyme is adjusted to 1mg/ml [Pangsomboon, 2009].After the concentration of adjustment proteolytic enzyme, in CS and YM1S two laboratory sample solution, all to some extent (Figure 20) is reduced to the bacillus subtilis of Developing restraint root and colibacillary antibacterial activity.In bacillus subtilis, trypsin is down to 10.5% in control group CS, and chymase is then down to 6.5%, but the antibacterial activity of protein kinase K does not almost change.Compared with bacillus subtilis, escherichia coli decrease 7.0% (Figure 20) in protein kinase K and trypsin.Owing to pressing the molecular weight of 1000, Acetate Film is divided into 3 parts, therefore antibacterial activity effect may depend on CS and YM1S protein.Compare in the molecular weight below 1000, fail to change the YM1C of antibacterial activity, the overall antibacterial characteristics of CS and YM1S reduces a little to some extent, but still remains most antibacterial activity.According to experimental judgment that is hot and breaks down proteins stability, the material antibacterial activity of nonprotein is stronger.Conclude accordingly, in Lb.plantarum LBP-K10, most of antiviral activity of culture supernatant is all derived from nonprotein class material (Fig. 2 C, 2D).
The separation of antibacterial or antiviral substance and characteristic
On the basis of above-mentioned kinds of experiments result, adopt the supernatant cultivated through 3 days, implement the experiment that Lactobacillus plantarum is separated antiviral substance.In separation process, by Liquid-liquid extraction method, utilize CH 2cl 2be separated antibiotic substance from culture fluid after, recycling preparative hplc (semi-prep) carries out fractional distillation, collection with HPLC, and observes fractional distillation result (Fig. 6 and Fig. 7) by HPLC chromatogram.In this experiment, for implementing above-mentioned experiment, and in separation antiviral substance process, by Liquid-liquid extraction method, to utilizing CH 2cl 2extract lower floor's culture fluid, utilize CH 2cl 2culture supernatants and do not utilize CH 2cl 2the antibacterial activity of culture fluid extracted compares, to remove the unnecessary acid impurities D-ALPHA-Hydroxypropionic acid of part or acetic acid, and in the middle of experiment after being applicable to.(Fig. 6)
By this experiment, from all antibiotic substance of culture fluid, extract CH 2cl 2after, utilize subnatant to implement experiment (Fig. 6) after this.That is, by said method, utilize in culture fluid and extract CH 2cl 2upper liquid and subnatant and do not extract CH 2cl 2concentrated culture fluids, implement antibacterial activity test result display, extract CH 2cl 2upper liquid and subnatant and do not extract CH 2cl 2the antibacterial activity of Concentrated culture fluids similar, and the antibacterial activity of subnatant is far above other two kinds experiments group (Fig. 6).Conclude accordingly, utilize and extract CH 2cl 2subnatant be separated antibiotic substance more effective (Fig. 6).
In the experiment group prepared by above-mentioned pretreatment process, after appropriate concentration is carried out to the fractional distillation material matter in preparative hplc HPLC chromatogram, implement antibacterial activity test.According to experimental result, in Lactobacillus plantarum chromatogram, find more than 10 fractional distillation material matter.At this, be separated, have collected the peak value of each time period, and antibacterial activity detection (Fig. 7 and table 3) has been carried out to each fractional distillation.Then, utilize minimal inhibitory concentration (MIC), carried out detecting (Fig. 8 ~ Figure 10) to the antibacterial activity of 2,7,8 and No. 10 fractional distillation material matter.Find according to above-mentioned experiment, the antibacterial activity the highest (Fig. 8 ~ Figure 10) of No. 8 fractional distillation material matter.
Table 3
[table 3]
A represents minimal inhibitory concentration (Minimum inhibitory concentration:MIC).+ represent 1 times, ++ represent 0.5 times, +++ represent 0.25 times.
The result display of experiment is implemented by minimal inhibitory concentration method, in all fractional distillation material matter of Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus brevis, the fractional distillation material matter P8 of identical chromatogram to each bacterial strain, the antibacterial activity the highest (Fig. 8, Fig. 9, Figure 10 and table 3) of N8, M8.Wherein, except except the acidic materials of the previous section fractional distillation of chromatogram, the pH of each fractional distillation material matter is nearly all close to neutral pH (table 4), and this result is in Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus brevis, also show similar tendency (Fig. 8, Fig. 9 and Figure 10).
Table 4
[table 4]
In addition, based on the result being implemented experiment by minimal inhibitory concentration method, the how various lactobacilluss identified are utilized in this experiment, and by HPLC, the result that analysis chromatogram compares is shown, in Pediococcus pentosaceus, Lactobacillus plantarum and leuconostoc pseudomesenteroides, the distribution of fractional distillation material matter almost similar (Figure 11).In addition, in the chromatogram of other lactobacilli strain, fractional distillation tendency (Figure 12) being similar to Pediococcus pentosaceus, Lactobacillus plantarum and leuconostoc pseudomesenteroides is also demonstrated.Conclude accordingly, lactobacillus, in growth course, can generate antibiotic substance, and the metabolite generated almost similar (Figure 11 and Figure 12).
Antiviral activity is analyzed
In antiviral activity experiment, have employed the epithelioid cell being derived from and can blocking dog kidney (Madin-Darby, caninekidney).In experimentation, infect the influenza A virus (H of three days by a definite date to epithelioid cell 3n 2).In the middle of the complete MEM (completeminimalessential medium, MEM agarose) the preparative hplc HPLC fractional distillation material matter of Lactobacillus plantarum LBP-K10 being mixed into 0.7%, and put into by influenza A virus (H 3n 2) on the blocked dog epithelial cell (MDCK) that infects.Now, all antiviral activity experiment groups all have employed bacterial plaque (plaque) analytic process.As in Figure 22, the meaning of each English symbol is as follows: namely, (A) check plot; (B) virus control district; (C) the 8th fractional distillation material matter, 5.0mM cis ring (L-Leu-L-Pro); (D) the 8th fractional distillation material matter, 10.0mM cis ring (L-Leu-L-Pro); (E) the 10th fractional distillation material matter, 5.0mM cis ring (L-Phe-L-PROLINE); (F) the 10th fractional distillation material matter, 10.0mM cis ring (L-Phe-L-PROLINE).As shown in figure 22, the 8th fractional distillation material, 10.0mM cis ring (L-Leu-L-Pro) and the 10th fractional distillation material, 10.0mM cis ring (L-Phe-L-PROLINE), show antiviral activity in plaque assay.Especially the 10th fractional distillation material, 10.0mM cis ring (L-Phe-L-PROLINE), show high antiviral activity.
The structural analysis of antibacterial and antiviral substance
In order to analyze the structure of antibacterial and antiviral substance, and utilize Freeze Dryers, the 8th and the 10th fractional distillation material of the Pediococcus pentosaceus of separating from HPLC, Lactobacillus plantarum, Lactobacillus brevis is made Powdered.As mentioned above, in experiment material and method, utilize direct insertion probe (DIP, Direct Insertion Probe; And chromatography mass-synchrometer (Agilent6890series GC, Agilent Technologies, Waldron, Germany DIP); High-resolutionmassspectrometer, JEOL JMS-700, Tokyo, Japan) (GC-MS), carry out analyzing (Figure 13, Figure 14, Figure 23 and Figure 24) to molecular weight.In addition, also to be analyzed by electron impact ionization method (electron ionization, EI) and chemical ioni zation method (chemical ionization, CI), and grasp and continuity between reference value.According to testing result display, each molecular weight of reference value of the 8th and the 10th fractional distillation molecular weight of material is respectively 210 and 244.According to another electron ionization method (electron ionization, EI) and the display of chemical ioning method (chemical ionization, CI) analysis result, 8th fractional distillation material of Pediococcus pentosaceus and Lactobacillus plantarum is 210, and the 10th fractional distillation material is then 244 (Figure 13 and Figure 14).
In order to grasp molecular structure more accurately, and carry out analysis and comparison by the leading experiment of electron impact ionization method (electronionization, EI) and chemical ioning method (chemical ionization, CI), and utilize 500MHz magnetic resonance, observation and analysis (Figure 15) has been carried out to the structure of the 8th fractional distillation material and the 10th fractional distillation material.In addition, also utilize and adopt 1H nuclear magnetic resonance method and 13C nuclear magnetic resonance method, to through the P8 of 100%DMSO process and 10% heavy water (D 20) the mix with DMSO the 8th and the 10th fractional distillation material is analyzed.(Figure 15 ~ Figure 18, Figure 25 ~ Figure 27).According to analysis result display, through the P8 of 100%DMSO process, in 8.0ppm, demonstrate typical nitrogen-hydrogen peak value (Figure 15), and containing 10%D 2the P8 of 0, be then replaced as heavy water in nitrogen-hydrogen peak value, and the peak value of 8.0ppm then disappears totally (Figure 16).Moved (Chemical shift) by the chemistry of 13C nuclear magnetic resonance method process by the 8th and the 10th fractional distillation material, in the DMSO test portion containing DMSO and 10% heavy water, 8th fractional distillation material and the 10th fractional distillation material are shown as the material (Figure 17 having 11 carbon elements and 14 carbon elements respectively, 18, Figure 25 to 27).
The crystal structure analysis result reflected according to another utilizing x-ray and being shown by the result of various structural analysis, the 8th fractional distillation material of Pediococcus pentosaceus, Lactobacillus brevis LBP-K06, Lactobacillus plantarum LBP-K10 is for having C 11h 18o 2n 2the cis ring (L-Leu-L-Pro) (L-Leu-L-Pro) (Figure 21) of structure, this final structure as shown in figure 21.
ESI-CID mass spectrum m/z 211 [MH] +base peak, 195 [MH-O] +, 154 [MH-C 4h 9] +
1h NMR (DMSO-d 6) δ 0.83 and 0.85 (6H at C-12 and C-13, d, J=6.49Hz and 6.45Hz), 1.34-1.36 (1H at C-5, m), 1.73-1.76 (1H at C-5, m), 1.801.87 (3H at C-4 and C-10, m), 2.10-2.13 (1H atC-10, m), 3.31-3.34 (2H at C-3, m), 3.98-4.02 (1H at C-6, m), 4.16-4.19 (1H at C-9, m), (8.02 1H atN-8, s).
13c NMR (DMSO-d 6) δ 170.8 (C-1), 167.0 (C-7), 58.8 (C-9), 52.9 (C-6), 45.2 (C-3), 37.9 (C-5), 27.7 (C-10), 24.4 (C-4), 23.0 (C-11), 22.7 and 22.2 (C-12 and C-13).
According to another analysis confirmation, the 10th fractional distillation material of Pediococcus pentosaceus, Lactobacillus brevis LBP-K06 and Lactobacillus plantarum LBP-K10 is for having C 14h 16o 2n 2the cis ring (L-Phe-L-Pro) (Figure 28 to Figure 30) of structure.
ESI-CID mass spectrum m/z 245 [MH] +base peak, 153 [MH-C 7h 8] +, 125 [MH-C 7h 8-CO] +
1H NMR(DMSO-d 6)1.41-1.45(1H at C-5,m)、1.69-1.74(2H at C-4,m)、1.98-2.03(1H atC-5,m)、3.01-3.08(2H at C-10,m)、3.24-3.42(2H at C-3,m)、4.05-4.08(1H at C-6,m)、4.33-4.35(1H at C-9,m)、7.17-7.27(5H at phenyl group,m)7.98(1Hat N-8,s)。
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Claims (20)

1. a bactericidal composition, it is characterized in that, described bactericidal composition is by cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and Streptococcus (Streptococcus) or Shigella (Shigella) is had to the bactericidal composition of special antibacterial activity.
2. bactericidal composition according to claim 1, it is characterized in that, described cis ring (L-Leu-L-Pro) (L-Leu-L-Pro) is the cis ring (L-Leu-L-Pro) separated from leukonid (Leuconostoc).
3. bactericidal composition according to claim 2, it is characterized in that, described leukonid is at leuconostoc mesenteroide (Leuconostoc mesenteroides), leuconostoc lactis (Leuconostoc lactis), cold leukonid (Leuconostocgelidum), leuconostoc paramesenteroides (Leuconostoc paramesenteroides), Fructus Citri Limoniae leukonid (Leuconostoccitreum), leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides), the leukonid of selected more than one strain composition in leuconostoc lactis (Leuconostocholzapfelii).
4. bactericidal composition according to claim 1, it is characterized in that, described streptococcus is the streptococcus of selected more than one strain composition in streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus agalactiae (Streptococcus agalactiae), bargen's streptococcus (Streptococcusbovis), Streptococcus viridans (Streptococcus viridans).
5. antibacterial compositions according to claim 1, it is characterized in that, described shigella is the shigella of selected more than one strain composition in shigella sonnei (Shigellasonnei), shigella boydii (Shigella boydii), Shigella dysenteriae (Shigella dysenteriae) and good fortune shigella flexneri (Shigella flexneri).
6. a composition pesticide, it is characterized in that, described composition pesticide is by cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and Streptococcus (Streptococcus) or Shigella (Shigella) is had to the composition pesticide of special antibacterial activity.
7. antibacterial composition pesticide according to claim 6, is characterized in that, described composition pesticide is the composition pesticide of selected more than one composition or additional additive in the group formed from solvent, carrier, emulsifying agent and dispersant.
8. Streptococcus (Streptococcus) or Shigella (Shigella) are had to a manufacture method for the antibacterial of special antibacterial activity, it is characterized in that described manufacture method is divided into following two stages: (a) manufactures the stage of lactic acid bacteria culture solution after cultivating lactobacillus; B cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) in above-mentioned culture fluid is condensed into the stage of applicable antimicrobial concentration by ().
9. an antiviral composition, is characterized in that, described antiviral composition is by the antiviral composition of cis ring (L-Phe-L-Pro) (cis-cyclo (L-Phe-L-Pro)) as effective ingredient.
10. antiviral composition according to claim 9, it is characterized in that, described cis ring (L-Phe-L-Pro) is the cis ring (L-Phe-L-Pro) separated from leukonid (Leuconostoc).
11. antiviral compositions according to claim 10, it is characterized in that, described leukonid is at leuconostoc mesenteroide (Leuconostoc mesenteroides), leuconostoc lactis (Leuconostoc lactis), cold leukonid (Leuconostocgelidum), leuconostoc paramesenteroides (Leuconostoc paramesenteroides), Fructus Citri Limoniae leukonid (Leuconostoccitreum), leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides), the leukonid of selected more than one strain composition in leuconostoc lactis (Leuconostocholzapfelii).
12. antiviral compositions according to claim 9, is characterized in that, described virus is influenza A virus (H3N2).
13. 1 kinds of medical compositions, it is characterized in that, described medical composition is by cis ring (L-Phe-L-Pro) (cis-cyclo (L-Phe-L-Pro)) as effective ingredient, and influenza A virus (H3N2) is had to the medical composition of special antibacterial activity.
14. 1 kinds have the manufacture method of the antiviral agent of antivirus action to influenza A virus (H3N2), it is characterized in that, described manufacture method is divided into following two stages: (a) cultivates lactic acid, to manufacture the stage of lactic acid bacteria culture solution; B cis ring (L-Phe-L-Pro) (cis-cyclo (L-Phe-L-Pro)), in above-mentioned culture fluid, is condensed into the stage of effective antiviral concentration by ().
15. 1 kinds of antiviral compositions, is characterized in that, described antiviral composition is by the antiviral composition of cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient.
16. antiviral compositions according to claim 15, is characterized in that, described cis ring (L-Leu-L-Pro) is the cis ring (L-Leu-L-Pro) separated from leukonid.
17. antiviral compositions according to claim 16, it is characterized in that, described leukonid is leuconostoc mesenteroide (Leuconostoc, mesenteroides), leuconostoc lactis (Leuconostoc lactis), cold leukonid (Leuconostocgelidum), leuconostoc paramesenteroides (Leuconostoc paramesenteroides), Fructus Citri Limoniae leukonid (Leuconostoccitreum), leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides), the leukonid of selected more than one strain composition in leuconostoc lactis (Leuconostocholzapfelii).
18. antiviral compositions according to claim 17, is characterized in that, described virus is influenza A virus (H3N2).
19. 1 kinds of medical compositions, it is characterized in that, described medical composition is by cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)) as effective ingredient, and influenza A virus (H3N2) is had to the medical composition of special antibacterial activity.
20. 1 kinds have the manufacture method of the antiviral agent of antivirus action to influenza A virus (H3N2), it is characterized in that, described manufacture method is divided into following two stages: (a) cultivates lactic acid, to manufacture the stage of lactic acid bacteria culture solution; B cis ring (L-Leu-L-Pro) (cis-cyclo (L-Leu-L-Pro)), in above-mentioned culture fluid, is condensed into the stage of effective antiviral concentration by ().
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