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CN110074179B - Application of antibacterial peptide Jelleine-I in preparation of medicine for preventing and treating postharvest green mold of citrus fruits - Google Patents

Application of antibacterial peptide Jelleine-I in preparation of medicine for preventing and treating postharvest green mold of citrus fruits Download PDF

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CN110074179B
CN110074179B CN201910403367.0A CN201910403367A CN110074179B CN 110074179 B CN110074179 B CN 110074179B CN 201910403367 A CN201910403367 A CN 201910403367A CN 110074179 B CN110074179 B CN 110074179B
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jelleine
antibacterial peptide
citrus fruits
antibacterial
green mold
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CN110074179A (en
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曾凯芳
李心丹
邓丽莉
姚世响
阮长晴
易兰花
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Southwest University
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Southwest University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

Abstract

The invention discloses application of an antibacterial peptide Jelleine-I in preparation of a medicine for preventing and treating postharvest green mold of citrus fruits. Research results show that the antibacterial peptide Jelleine-I can effectively prevent and treat postharvest green mold of citrus fruits, has strong bactericidal capacity, instant bactericidal capacity superior to that of antibacterial peptides PAF56 and BP21 which are reported in the prior literature and can effectively control postharvest green mold of citrus fruits, long-acting bactericidal capacity superior to that of antibacterial peptide PAF56, good bacteriostatic stability, and no temperature, pH or Na influence+、K+Etc. The invention widens the application range of the antibacterial peptide Jelleine-I in the field of fruit and vegetable preservation, and also provides a new solution for preventing and treating postharvest green mold of citrus fruits.

Description

Application of antibacterial peptide Jelleine-I in preparation of drugs for preventing and treating postharvest green mold of citrus fruits
Technical Field
The invention relates to application of antibacterial peptide Jelleine-I in the field of fruit and vegetable preservation.
Background
Citrus fruits are susceptible to Penicillium digitatum (B) during storage and transportation after harvestPenicillium digitatum) The infection causes green mold disease, resulting in a great economic loss. In the existing control method for postharvest diseases of oranges, the chemical bactericide is most commonly used, but has high toxicity, potential threats to human health and environment, and resistance of pathogenic microorganisms to the chemical bactericide is easy to generate, so that the use of the chemical bactericide is gradually limited. Is resistant toAntimicrobial peptides (AMPs) are a general name of small molecular polypeptides with antibacterial activity, are widely existed in various organisms, and are difficult to generate resistance to pathogenic microorganisms and free of environmental pollution due to the action mechanism different from that of chemical bactericides and antibiotics, so that the peptides become ideal resources for replacing the chemical bactericides and the antibiotics. In recent years, antibacterial peptides have attracted more and more attention in the industries such as medicine, agriculture, food, and livestock, but there are few studies on disease control after fruit and vegetable harvest.
Disclosure of Invention
The invention aims to screen the antibacterial peptide which can effectively prevent and control the postharvest green mold of the citrus fruits and has strong bactericidal capability and good stability, so as to widen the application range of the antibacterial peptide in the field of fruit and vegetable preservation and provide a new solution for preventing and controlling the postharvest green mold of the citrus fruits.
Through research, the invention provides the following technical scheme:
the application of the antibacterial peptide Jelleine-I in preparing a medicine for preventing and treating postharvest green mold of citrus fruits; the amino acid sequence of the antibacterial peptide Jelleine-I is shown in SEQ ID No.5, and the C end of the antibacterial peptide is subjected to amidation modification.
The invention has the beneficial effects that: the invention discloses application of antibacterial peptide Jelleine-I in preparation of a medicine for preventing and treating postharvest green mold of citrus fruits. Research results show that the antibacterial peptide Jelleine-I can effectively prevent and treat postharvest green mold of citrus fruits, has strong bactericidal capacity, has instant bactericidal capacity superior to antibacterial peptides PAF56 and BP21 which are reported in the prior literature and can effectively control postharvest green mold of citrus fruits, has long-acting bactericidal capacity superior to antibacterial peptide PAF56, has good bacteriostatic stability and is not influenced by temperature, pH and Na+、K+Etc. The invention widens the application range of the antibacterial peptide Jelleine-I in the field of fruit and vegetable preservation, and also provides a new solution for preventing and treating the postharvest green mold of citrus fruits.
Drawings
FIG. 1 shows the control effect of seven antibacterial peptides on postharvest green mold of citrus fruits, wherein A is the incidence and B is the lesion diameter.
FIG. 2 shows inoculation P.digitatum6 days later, the control group and seven kinds of antibacterial agentsComparison of citrus fruits in peptide-treated groups.
FIG. 3 is a comparison of the control effect of five antimicrobial peptides on postharvest green mold of citrus fruits, wherein A and B are the antimicrobial peptides mixed with pathogenic bacteria and then immediately inoculated, A is the morbidity and B is the lesion diameter; c and D are inoculated after the antibacterial peptide and the pathogenic bacteria are mixed for 16 hours, wherein C is the morbidity and D is the diameter of a lesion.
FIG. 4 shows inoculation P.digitatumAfter 6 days, the control group was compared with citrus fruits of five antimicrobial peptide treatment groups of 0 h (the antimicrobial peptide was mixed with the pathogenic bacteria and immediately inoculated) and 16 h (the antimicrobial peptide was mixed with the pathogenic bacteria and inoculated) respectively.
FIG. 5 is a graph of temperature vs. antimicrobial peptide O3TR（A）、C12O3TR (B) and Jelleine-I (C) bacteriostatic stability.
FIG. 6 is pH vs. antimicrobial peptide O3TR（A）、C12O3TR (B) and Jelleine-I (C) bacteriostatic stability.
FIG. 7 shows the metal ion pair antibacterial peptide O3TR（A1，A2）、C12O3Effects of TR (B1, B2) and Jelleine-I (C1, C2) bacteriostatic stability.
In fig. 1 and 3, there is no significant difference (p > 0.05) between groups labeled with the same lower case english letters (a, b, c); there was a significant difference (p < 0.05) between the groups labeled with different lower case english letters (a, b, c).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The invention firstly screens seven cationic short-chain antibacterial peptides with broad-spectrum and high-efficiency bacteriostatic activity reported in literature from a large number of known antibacterial peptides: o is3TR (artificial sequence), C12O3TR (artificial sequence), dhvar5 (derived from human lactoferrin), hLF (1-11) (derived from human lactoferrin), LDP-NLS (artificial sequence), Jelleine-I (derived from Apis mellifera (L.))Apis mellifera) And Osmin (from Chouioia wasp: (A. Merrill.))Osmia rufa) And then synthesizing the seven antibacterial peptides by using a solid-phase synthesis method, determining the control effect of the seven antibacterial peptides on the postharvest green mold of the citrus fruit through a fruit test, screening the antibacterial peptides which can really and effectively control the postharvest green mold of the citrus fruit, comparing the instantaneous bactericidal ability with the long-acting bactericidal ability of the antibacterial peptides PAF56 and BP21 which can effectively control the postharvest green mold of the citrus fruit reported in the literature, screening the antibacterial peptides with stronger bactericidal ability, testing the bacteriostatic stability of the antibacterial peptides, and finally screening the antibacterial peptides with strong bactericidal ability and good bacteriostatic stability.
First, experimental material
1. Antibacterial peptide
Entrusted Nanjing Kinseri company to synthesize the following antibacterial peptides with a purity of >90% using a solid phase synthesis method:
O3TR: the amino acid sequence is shown as SEQ ID No.1, and the C-terminal of the amino acid sequence is amidated and modified (OOWW-NH)2O represents ornithine);
C12O3TR: the amino acid sequence is shown as SEQ ID No.1, and N terminal dodecanoylation modification and C terminal amidation modification ((C)12H23O)-OOWW-NH2）；
dhvar 5: the amino acid sequence is shown as SEQ ID No.2 (LLLFLLKKRKKRKY);
hLF (1-11): the amino acid sequence is shown as SEQ ID No.3 (GRRRRSVQWCA);
LDP-NLS: the amino acid sequence is shown as SEQ ID No.4 (KWRRKLKKLRPKKKRKV);
Jelleine-I: the amino acid sequence is shown as SEQ ID No.5, and the C-terminal amidation modification (PFKLSLHL-NH)2）；
Osmin: the amino acid sequence is shown as SEQ ID No.6 (GFLSALKKYLPIVLKHV);
PAF 56: the amino acid sequence is shown as SEQ ID No.7 (GHRKKWFW);
BP 21: the amino acid sequence is shown in SEQ ID No.8, and the N end of the amino acid sequence is modified by acetylation and the C end of the amino acid sequence is modified by amidation (Ac-FKLFKKILKVL-NH)2）。
The synthesized antibacterial peptide is prepared into 1 mmol/L mother liquor by respectively using sterile ultrapure water and stored at the temperature of minus 40 ℃.
2. Pathogenic bacteria
Penicillium digitatum (P. digitatum) Isolated from diseased citrus fruits and identified by molecular biological methods.
The pathogens were cultured on Potato Dextrose Agar (PDA) medium at 25 ℃ and after 7 days spores were collected and brought to the desired concentration with sterile water.
Second, Experimental methods
1. Control effect of antibacterial peptide on postharvest green mold of citrus fruits
Randomly grouping citrus fruits: a sterile water control group and an antimicrobial peptide treatment group, each group containing 15 fruits; soaking each group of fruits in 2% sodium hypochlorite solution for 2 min, cleaning to remove residual dirt on the surface, wiping and disinfecting the equator position of the fruit surface with 75% alcohol, and punching two holes (3 mm deep and 4 mm in diameter) at equal intervals on the equator position of the fruit by using a sterilization puncher; the sterile water control group was inoculated with 10. mu.L of sterile water per well; antimicrobial peptide-treated groups will be freshP. digitatumThe spore suspension was mixed with the antimicrobial peptide solution and inoculated immediately or 16 h later with 10. mu.L of the antimicrobial peptide solution into each well,P. digitatumthe final concentrations of spore and antibacterial peptide were 1X 104CFU/mL and 100. mu. mol/L; after the liquid in the holes is completely absorbed, packing the single fruit by a polyethylene film bag (170 mm multiplied by 140 mm), and storing in an environment with the temperature of 25 ℃ and the relative humidity of 90-95%; the incidence was counted daily and lesion diameter was measured. The experiment was repeated 3 times.
2. Influence of different temperatures on antibacterial activity of antibacterial peptide
Standing antibacterial peptide solutions with different concentrations at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 5 min, rapidly cooling to room temperature (25 deg.C), and standing unheated antibacterial peptide solution at room temperature (25 deg.C) as control; 180 μ L of liquid medium containing 20-fold dilution of potato dextrose (5% PDB)P. digitatumThe spore suspension and 20 mul of the antimicrobial peptide solution were mixed and immediately added to a sterile 96-well microplate,P. digitatumthe final concentration of spores was 1X 104CFU/mL, the final concentration of the antibacterial peptide is 0, 3.12, 6.25, 12.5, 25, 50 and 100 mu mol/L respectively; then placing 96-well plate at 25 deg.C for constant-temperature cultureCulturing in incubator, measuring OD 48 h with enzyme labeling instrument600The Minimum Inhibitory Concentration (MIC) was the lowest concentration that still completely inhibited the growth of pathogenic bacteria at 48 h. The experiment was repeated 3 times.
3. Effect of different pH on antibacterial peptide bacteriostatic Activity
Dissolving the antibacterial peptide in sterile water with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively, taking the sterile water without pH adjustment as a control, standing at room temperature for 1 h, adjusting the pH value to 7.0, diluting the antibacterial peptide solution with the sterile water, and performing a minimum inhibitory concentration determination test.
4. Influence of metal ions on antibacterial activity of antibacterial peptide
Respectively dissolving antibacterial peptide with KCl solution (5, 20, 35, 50 mmol/L) or NaCl solution (50, 100, 150, 200 mmol/L) with different concentrations sterilized at 121 deg.C for 20 min, and performing minimum inhibitory concentration determination test with sterile water as control.
Third, experimental results
1. Control effect of antibacterial peptide on postharvest green mold of citrus fruits
Firstly, seven antibacterial peptides (O) are investigated by adopting a mode of mixing the antibacterial peptides with pathogenic bacteria and then immediately inoculating3TR、C12O3TR, dhvar5, hLF (1-11), LDP-NLS and Jeleine-I, Osmin) have control effect on postharvest green mold of citrus fruits. As shown in FIG. 1 and FIG. 2, seven kinds of antibacterial peptides have great difference in control effect on postharvest green mold of citrus fruit, wherein the antibacterial peptide O3TR、C12O3TR and Jelleine-I can effectively control the occurrence of the postharvest green mold of the citrus fruit, and the antibacterial peptides dhvar5, hLF (1-11), LDP-NLS and Osmin have no obvious control effect on the postharvest green mold of the citrus fruit.
Secondly, the two inoculation modes of mixing the antibacterial peptide with the pathogenic bacteria and then immediately inoculating and mixing for 16 hours are compared with the antibacterial peptide O3TR、C12O3TR, Jelleine-I and the antibacterial peptides PAF56 and BP21 which are reported in the literature and can effectively control the postharvest green mold of citrus fruits have the capacity of instantaneous sterilization and long-acting sterilization. Comparative results of inoculation immediately after mixingAs shown in FIGS. 3A, 3B and 4, antimicrobial peptide O3TR、C12O3The disease control effect of TR and Jelleine-I is obviously better than that of antibacterial peptides PAF56 and BP21, wherein the peptide C12O3After TR treatment, the citrus fruit did not develop disease, indicating antimicrobial peptide O3TR、C12O3TR and Jelleine-I have superior transient bactericidal activity to the antimicrobial peptides PAF56 and BP 21. The results of comparison of inoculation after 16 h mixing are shown in FIGS. 3C, 3D and 4, where the disease control effect of the antimicrobial peptide PAF56 is the worst, and the antimicrobial peptides BP21 and O3TR、C12O3No significant difference in the control effects of TR and Jelleine-I: (P <0.05), indicating that the antimicrobial peptide O3TR、C12O3TR and Jelleine-I have long-term bactericidal activity superior to that of the antibacterial peptide PAF 56.
2. Influence of different temperatures on the bacteriostatic activity of the antimicrobial peptide
The results are shown in FIG. 5, antimicrobial peptide O3TR、C12O3TR and Jelleine-I pairP. digitatumThe growth inhibition effect of the three antibacterial peptides is not changed by the temperature change of the antibacterial peptides, which shows that the antibacterial activities of the three antibacterial peptides have thermal stability. Antibacterial peptide O3TR、C12O3The MICs of TR and Jelleine-I were 12.5, 6.25 and 6.25. mu. mol/L, respectively.
3. Effect of different pH on the bacteriostatic Activity of antimicrobial peptides
The results are shown in FIG. 6, antimicrobial peptide O3TR、C12O3TR and Jelleine-I can inhibit under different pH conditionsP. digitatumThe MIC value is unchanged, which indicates that the bacteriostatic activity of the three antibacterial peptides has acid-base stability.
4. Influence of metal ions on antibacterial activity of antibacterial peptide
The results are shown in FIG. 7, where the antimicrobial peptide C was present in NaCl solutions of different concentrations12O3TR and Jelleine-I pairP. digitatumThe growth inhibition ability of (A) is not changed, and the MIC value is not changed; antibacterial peptide O3TR is reduced in the bacteriostatic ability in high-concentration NaCl solution (150, 200 mmol/L), and MIC values are all 25 mu mol/L; in KCl solutions of different concentrations, antimicrobial peptide O3TR、C12O3TR and Jelleine-I can be kept in pairP. digitatumThe MIC value does not change; description of the antibacterial peptide C12O3The bacteriostatic ability of TR and Jelleine-I is not influenced by Na+、K+The influence of (c).
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest
Application of antibacterial peptide Jelleine-I in preparation of drugs for preventing and treating postharvest green mold of citrus fruits
<160> 8
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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<400> 1
Xaa Xaa Trp Trp
1
<210> 2
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 2
Leu Leu Leu Phe Leu Leu Lys Lys Arg Lys Lys Arg Lys Tyr
1 5 10
<210> 3
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 3
Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala
1 5 10
<210> 4
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Lys Trp Arg Arg Lys Leu Lys Lys Leu Arg Pro Lys Lys Lys Arg Lys
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<213> Italian bee (Apis mellifera)
<400> 5
Pro Phe Lys Leu Ser Leu His Leu
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<210> 6
<211> 17
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<213> Osmia rufa
<400> 6
Gly Phe Leu Ser Ala Leu Lys Lys Tyr Leu Pro Ile Val Leu Lys His
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Val
<210> 7
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<213> Artificial Sequence (Artificial Sequence)
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Gly His Arg Lys Lys Trp Phe Trp
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<213> Artificial Sequence (Artificial Sequence)
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Phe Lys Leu Phe Lys Lys Ile Leu Lys Val Leu
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Claims (1)

1. The application of the antibacterial peptide Jelleine-I in preparing a medicine for preventing and treating postharvest green mold of citrus fruits; the amino acid sequence of the antibacterial peptide Jelleine-I is shown in SEQ ID No.5, and the C end of the antibacterial peptide is subjected to amidation modification.
CN201910403367.0A 2019-05-15 2019-05-15 Application of antibacterial peptide Jelleine-I in preparation of medicine for preventing and treating postharvest green mold of citrus fruits Active CN110074179B (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN107455455A (en) * 2017-09-07 2017-12-12 西南大学 A kind of method for adopting rear infectious disease with antibacterial peptide PAF56 control citrusfruits

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN107455455A (en) * 2017-09-07 2017-12-12 西南大学 A kind of method for adopting rear infectious disease with antibacterial peptide PAF56 control citrusfruits

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Title
《The in vitro, in vivo antifungal activity and the action mode of Jelleine‑I against Candida species》;Fengjing Jia，等;《Amino Acids》;20171103(第50期);第229,230页 *
Identification and Characterization of a Hexapeptide with Activity Against Phytopathogenic Fungi That Cause Postharvest Decay in Fruits;Belén López-García，等;《Molecular Plant-Microbe Interactions》;20001231;第13卷(第8期);第837页 *
抗菌肽及其在果蔬病害控制中的应用;王文军等;《食品与机械》;20170228(第02期);全文 *
抗菌肽的研究进展;李孝东等;《山东医药工业》;20030310(第01期);全文 *

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