Preparation method of recombinant antibacterial peptide Hc-CATH and application of recombinant antibacterial peptide Hc-CATH in blueberry preservation

Abstract

The invention discloses a preparation method of a recombinant antibacterial peptide Hc-CATH and application of the recombinant antibacterial peptide Hc-CATH in blueberry preservation, and the preparation method comprises the following steps: synthesizing an Hc-CATH target gene as shown in SEQ ID NO: 2, and connecting to a pPICZ alpha A carrier to form a recombinant plasmid pPICZ alpha A-Hc-CATH; the method comprises the following steps: carrying out linear treatment on a recombinant plasmid pPICZ alpha A-Hc-CATH by using Sac I restriction enzyme, and transferring the treated plasmid into pichia pastoris to obtain a recombinant strain with a target gene; performing inducible expression on the screened positive transformant by using methanol, and collecting the fermented supernate to obtain the high-concentration antibacterial peptide; wherein the original amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. The recombinant Hc-CATH antibacterial peptide reduces the number of blueberry bacterial colonies, weightlessness and anthocyanin loss, and weakens the respiration intensity of blueberries.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of genetic engineering and biological application technology, and particularly relates to a preparation method of a recombinant antibacterial peptide Hc-CATH and application of the antibacterial peptide Hc-CATH in blueberry preservation. BACKGROUND

[0002] Hc-CATH is an antibacterial peptide isolated and characterized from Hydrophis cyanocinctus, and has good inhibitory effect on bacteria and fungi, and has anticancer activity and safety, so that the Hc-CATH can be used as a promising antibacterial drug and can be used in the fields of biological medicine, food, agriculture, animal husbandry and the like.

[0003] However, the yield of the antibacterial peptide extracted from nature is low, the chemical synthesis of the antibacterial peptide is complex and high in cost, and the use of genetic cloning technology can improve the antibacterial property and productivity of the polypeptide.

[0004] At present, no scholar has used genetic engineering technology to heterologously express Hc-CATH, and meanwhile, although Hc-CATH has certain antibacterial effect, the application of Hc-CATH in blueberry preservation to reduce the weight loss and anthocyanin loss of blueberries has not been reported. SUMMARY

[0005] This section aims to summarize some aspects of the embodiments of the application and briefly introduce some preferred embodiments.

[0006] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0007] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a preparation method of a recombinant antibacterial peptide Hc-CATH.

[0008] To solve the above technical problems, the present application provides the following technical scheme: a preparation method of a recombinant antibacterial peptide Hc-CATH, comprising,

[0009] Recombinant expression vector: the Hc-CATH target gene shown in SEQ ID NO: 2 is synthesized and connected to the pPICZ alpha A vector to form a recombinant plasmid pPICZ alpha A-Hc-CATH;

[0010] Recombinant strain construction: the recombinant plasmid pPICZ alpha A-Hc-CATH is linearized by Sac I restriction endonuclease, and the treated plasmid is transformed into Pichia pastoris to obtain a recombinant strain with the target gene;

[0011] Expression of antibacterial peptide: the positive transformants selected are induced to express by methanol, and the supernatant after fermentation is collected to obtain high-concentration antibacterial peptide;

[0012] The original amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1.

[0013] As a preferred solution of the preparation method, the preparation method of the recombinant plasmid pPICZ alpha A-Hc-CATH comprises the following steps:

[0014] An EcoRI enzyme cutting site is introduced at the 5' end of the Hc-CATH target gene sequence, and a start codon ATG and a 6His-tag are added, a termination codon TAA is added at the 3' end of the sequence, and an Xba I enzyme cutting site is introduced, to obtain the recombinant plasmid pPICZ alpha A-Hc-CATH.

[0015] As a preferred solution of the preparation method, the expression is induced by using methanol, and the expression is induced by 1% methanol, and the induction expression time is 5 days.

[0016] Still another object of the present application is to provide a recombinant antibacterial peptide Hc-CATH, which overcomes the defects in the prior art, and the minimum inhibitory concentration of the recombinant antibacterial peptide Hc-CATH to Staphylococcus aureus is 2.5 μg / mL, the minimum inhibitory concentration to Listeria monocytogenes and Escherichia coli O157 is 20 μg / mL, the minimum inhibitory concentration to Bacillus subtilis and Escherichia coli is 10 μg / mL, and the minimum inhibitory concentration to Salmonella is 5 μg / mL.

[0017] Still another object of the present application is to provide an application of the recombinant antibacterial peptide Hc-CATH in the preservation of blueberries, which overcomes the defects in the prior art.

[0018] As a preferred solution of the application, the blueberries are sprayed with the Hc-CATH solution, and the water is naturally drained in a sterile environment.

[0019] As a preferred solution of the application, the concentration of the Hc-CATH solution is 10 μg / mL.

[0020] As a preferred solution of the application, the spraying volume is 2 ml.

[0021] As a preferred solution of the application, the Hc-CATH antibacterial peptide can reduce the respiration intensity of blueberries, reduce the weight loss of blueberry fruits, reduce the loss of anthocyanins, and reduce the increase of the total number of colonies.

[0022] The present application has the following beneficial effects:

[0023] (1) The present application successfully and efficiently expresses the antibacterial peptide Hc-CATH through the Pichia pastoris expression system, and the concentration of the target protein in the expression product reaches 22 mu g / mL, providing a material basis for large-scale production of antibacterial peptides.

[0024] (2) The Pichia pastoris expression system used in the present application includes a methanol inducible promoter (AOX1), a resistance gene, a multiple cloning site, an alpha signal peptide, a gene copy number, and a ribosome binding site; wherein the low cost of methanol leads to the wide application of AOX1 inducible promoter in laboratory and industrial scale; the resistance gene is zeocin, and the screening marker is 6His, which can greatly improve the accuracy and efficiency of screening; the multiple cloning site is used for the insertion of exogenous genes, and its design allows the convenient introduction of the required gene sequence; the alpha signal peptide plays a crucial role in the construction of the vector, which can promote the successful secretion of exogenous proteins, ensure that the target protein can be correctly folded and secreted outside the cell, which is crucial for subsequent protein purification and functional research.

[0025] (3) The present application modifies the Hc-CATH antibacterial peptide, inserts it into the pPICZ alpha A vector after codon optimization of Pichia pastoris, and finally obtains high expression of antibacterial peptides by methanol induction expression. The research on its antibacterial effect, stability and hemolytic activity shows that the obtained antibacterial peptide shows antibacterial effect on several common pathogenic bacteria, at the same time, has low hemolytic activity, is less affected by temperature, pH and enzyme, has good stability, and has good application prospect in food preservation.

[0026] (4) The present application studies the preservation application of the obtained recombinant antibacterial peptide Hc-CATH, and compares it with common preservatives such as potassium sorbate, cecropin and water, and finds that it can obviously inhibit the reproduction of surface spoilage bacteria of blueberries, slow down the loss of anthocyanins, weaken the respiration intensity, weaken the weight loss, prolong the preservation time, and the related information of the present application is rarely reported at home and abroad, and there is no public use at home and abroad. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creating any creative labor.

[0028] Among them:

[0029] Figure 1The figure is a schematic diagram of PCR gel running of the antibacterial peptide gene Hc-CATH in the embodiment of the present application; wherein, the negative control is the PCR product of the pPICZ alpha A empty vector plasmid, the positive control adopts the PCR product of the recombinant plasmid, and the Hc-CATH is the PCR product of the transformed single clone yeast strain.

[0030] Figure 2 The figure is a schematic diagram of the inhibitory effect of the product expressed at different times on Staphylococcus aureus in the embodiment of the present application.

[0031] Figure 3 The figure is a schematic diagram of the antibacterial activity of Hc-CATH on 6 test strains in the embodiment of the present application.

[0032] Figure 4 The figure is a schematic diagram of the stability detection of Hc-CATH in the embodiment of the present application; wherein, (a) thermal stability detection, (b) pH stability, and (c) protease stability.

[0033] Figure 5 The figure is a schematic diagram of the hemolytic activity analysis of Hc-CATH on human red blood cells in the embodiment of the present application.

[0034] Figure 6 The figure is a schematic diagram of the influence of Hc-CATH on blueberry spoilage in the embodiment of the present application.

[0035] Figure 7 The figure is a schematic diagram of the influence of Hc-CATH, potassium sorbate, cecropin and water on the total number of colonies of blueberries in the embodiment of the present application.

[0036] Figure 8 The figure is a schematic diagram of the influence of Hc-CATH, potassium sorbate, cecropin and water on the respiration intensity of blueberries in the embodiment of the present application.

[0037] Figure 9 The figure is a schematic diagram of the influence of Hc-CATH, potassium sorbate, cecropin and water on the weight loss of blueberries in the embodiment of the present application.

[0038] Figure 10 The figure is a schematic diagram of the influence of Hc-CATH, potassium sorbate, cecropin and water on the anthocyanin of blueberries in the embodiment of the present application. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below in combination with the embodiments of the specification. The raw materials in the embodiments of the present application are all ordinary implementation products.

[0040] Embodiment 1

[0041] Construction of the recombinant antibacterial peptide Hc-CATH positive yeast strain:

[0042] The Hc-CATH gene sequence was optimized according to the codon bias of Pichia pastoris.

[0043] An EcoR I enzyme cutting site was introduced at the 5' end of the sequence, and a start codon ATG and a 6His-tag were added, at the 3' end of the sequence, a stop codon TAA and an Xba I enzyme cutting site were added, and the sequence was connected to the Pichia pastoris expression vector pPICZαA (Pint Bioengineering Co., Ltd.) through the two enzyme cutting sites.

[0044] The original amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1:

[0045] KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL

[0046] The optimized base sequence is shown as SEQ ID NO: 2:

[0047] AAG TTT TTTAAG AGA TTG TTG AAG TCT GTTAGA AGA GCT GTT AAG AAG TTTAGAAAG AAG CCAAGA TTG ATT GGT TTG TCTACT TTG TTG.

[0048] The recombinant plasmid obtained above was linearized by Sac I enzyme and then transformed into Pichia pastoris X33 (Pint Bioengineering Co., Ltd.), and was plated on YPD plates containing Zeocin (final concentration 25 μg / mL) to grow single colonies, and then the single colonies were incubated on YPD containing Zeocin (final concentration 100 μg / mL) for two days, and the strains were screened and verified by PCR, as shown in Figure 1 , verification was successful, and a positive yeast strain was successfully obtained.

[0049] The PCR system is shown in Table 1.

[0050] Table 1 PCR system

[0051]

[0052] Example 2

[0053] Expression and verification of recombinant antibacterial peptide Hc-CATH:

[0054] (1) Pick the positive clones which are verified to be correct, and place them in 5 ml BMGY medium (70 mL YP70, 10 mL 10% glycerol, 10 mL PBS, 10 mL 13.4% YNB, 200 μL biotin) at 30°C, 220 rpm, constant temperature culture for 24 h, then transfer the initial bacterial liquid to 25 ml BMGY medium for expansion culture at a 5% inoculation amount, when the OD600 of the bacterial liquid reaches 8 to 10, replace it into BMMY medium for continuous culture to induce the expression of the target protein, take a sample every 24 h during the period, and add 1% methanol, continuously culture for 144 h, and detect the bacteriostatic effect by the agar hole diffusion method to verify the expression of the target protein.

[0055] As shown in Figure 2 , with the change of time, the bacteriostatic circle gradually increases, indicating that the expression amount of the antibacterial peptide gradually increases, and it can be seen from the figure that the 5th day is the best induction expression time.

[0056] (2) ELISA determination of protein concentration.

[0057] Dilute the standard protein GFP (6His-tag) with the coating solution, coat 200 μL of the standard protein and the fermentation supernatant on a 96-well plate, and incubate at 4°C overnight;

[0058] Wash the plate with TBST for 3 times, block with 5% skimmed milk at room temperature for 1 h;

[0059] Wash the plate with TBST for 3 times, add His-tag mouse first antibody and incubate for 1.5 h;

[0060] Wash the plate with TBST for 3 times, add HRP-goat-anti-mouse second antibody and incubate for 1.5 h;

[0061] Wash the plate with TBST for 3 times, add 150 μL TMB color developing liquid to develop color for 30 min in the dark, and then add 50 ml 0.5M H2SO4 to terminate the reaction;

[0062] Measure the optical density at 450 nm to draw a standard curve.

[0063] The linear regression equation of the obtained protein content is y = 0.001x + 1.926, wherein R 2 = 0.9938, and the correlation is good. According to the standard curve, the protein concentration of the fermentation supernatant is 22 μg / mL.

[0064] Example 3

[0065] Performance determination of recombinant antibacterial peptide Hc-CATH:

[0066] (1) Bacteriostatic spectrum determination

[0067] The standard agar well diffusion method was used to determine the antibacterial spectrum. 15 μL of bacterial suspension in the logarithmic growth phase was mixed with 30 ml of LB solid medium to prepare the plate. After solidification, the holes were punched with a sterile gun head, 50 μL of antibacterial peptide was added, and the culture was incubated at 37°C overnight. Gentamicin was used as a positive control, and PBS was used as a negative control in this experiment.

[0068] As shown in Figure 3 , the Hc-CATH expression supernatant showed inhibitory effect on several common pathogenic bacteria, and the inhibitory effect of Hc-CATH on Staphylococcus aureus, Listeria monocytogenes and Salmonella was more obvious.

[0069] (II) Minimum inhibitory concentration (MIC) determination

[0070] The bacterial suspension of the logarithmic growth phase of the test strain was diluted to 10 6 CFU / mL.

[0071] Take 100 μL of bacterial solution and add it to a 96-well plate, then add 20 μL of Hc-CATH fermentation supernatant of different concentrations.

[0072] 100 μL of LB liquid medium was used as a blank control, and 100 μL of diluted bacterial suspension was used as a positive control.

[0073] Incubate at 37°C for 12 to 16 hours, and measure the OD600 value of each well using a microplate reader. The minimum inhibitory concentration of Hc-CATH on different strains is shown in Table 2.

[0074] Table 2

[0075]

[0076] (III) Stability analysis

[0077] (1) Thermal stability

[0078] The antibacterial peptide supernatant (final concentration 10 μg / ml) was treated at 4°C, 25°C, 37°C, 65°C and 90°C for 1 h, and Tris-Hcl (50 mM, pH 7.4) treated for 1 h under the same conditions was used as a negative control for the antibacterial experiment.

[0079] (2) pH stability

[0080] The antibacterial peptide was dissolved in buffers with pH values of 2, 4, 6, 8 and 10 (final concentration 10 μg / ml), and treated at 37°C for 4 h. Buffers with different pH values were used as negative controls for the antibacterial experiment.

[0081] (3) Protease stability

[0082] Take 50 μL of antibacterial peptide to add 0.24 U of pepsin, papain, protease K and trypsin, 37℃ for 2h, with different protease liquid as negative control, carry out antibacterial experiment.

[0083] As shown in Figure 4 , Hc-CATH has good antibacterial activity against Staphylococcus aureus at 4-100℃. But after 1h treatment at 100℃, there is no significant difference in the antibacterial activity of Hc-CATH, indicating that Hc-CATH is not affected by temperature and has good thermal stability.

[0084] In the pH range of 2 to 8, the antibacterial activity of Hc-CATH is less affected, and when the pH value is 10, the antibacterial activity of Hc-CATH is slightly reduced, indicating that acid and alkali have little effect on antibacterial peptides.

[0085] In addition, the antibacterial activity of antibacterial peptides in papain, protease K, trypsin and other three proteases has no obvious change, but it has a slight decrease under the action of pepsin.

[0086] (Four) hemolytic activity

[0087] Human red blood cells were used to measure the hemolytic activity of Hc-CATH. The blood cells were washed with PBS and diluted to 4% (v / v), 10 μL of different concentrations (20, 10, 5, 2.5, 1.25 μg / mL) of antibacterial peptides and 90 μL of blood cells were mixed, incubated at 37℃ for 1h, centrifuged to take the supernatant, measured the absorbance at 576nm, and calculated the hemolysis rate.

[0088] Its PBS is negative control; 0.1% Triton X-100 is positive control.

[0089] As shown in Figure 5 , within the range of 1.25-20 μg / mL, the hemolysis rate of Hc-CATH on human red blood cells is much lower than that of 0.1% Tritom X-100, showing low hemolytic activity.

[0090] Example 4

[0091] Application research of recombinant antibacterial peptide Hc-CATH in blueberry preservation:

[0092] Blueberry treatment:

[0093] Select several blueberries of uniform size, picked from the same time and from the same place.

[0094] Divide the blueberries into two groups: the experimental group is sprayed with 10 μg / mL of rHc-CATH 2ml, and the control group is sprayed with 2ml of distilled water. After natural draining of water in a sterile environment, cover with PP film, store at 30℃, and evaluate the indicators every 48h.

[0095] (1) Mold rate and total bacterial count determination

[0096] Five blueberries were selected respectively and placed in the same temperature, humidity and other environmental conditions for 15 days. The mold coverage or fruit decay of blueberry fruits was observed. As shown in Figure 6 , after five days, the control group appeared complete mold colonies under the same environmental conditions.

[0097] As shown in Figure 7 , the total number of colonies of the rHc-CATH experimental group was lower than that of the control group of potassium sorbate, cecropin and water. Therefore, the rHc-CATH antibacterial peptide can inhibit the reproduction of colonies when the blueberry is static at room temperature 25℃, thereby prolonging the shelf life.

[0098] (2) Respiratory intensity determination

[0099] The same size and number of blueberries were placed in beakers containing phenolphthalein indicator alkaline solution and sealed with sterile preservative film. After 10 days of standing, the control group decayed the fastest, produced the most CO2, consumed the most NaOH, and required the most oxalic acid for titration.

[0100] As shown in Figure 8 , on the second day, the respiratory intensity of the control group of potassium sorbate, cecropin and water was 185.39 mg / (kg*h), 180.74 mg / (kg*h), and 187.73 mg / (kg*h), respectively, and the respiratory intensity of the rHc-CATH experimental group was 174.39 mg / (kg*h);

[0101] The respiration intensity of the control group of potassium sorbate, cecropin and water was 190.86 mg / (kg*h), 183.56 mg / (kg*h) and 202.51 mg / (kg*h) respectively, and the respiration intensity of the rHc-CATH experimental group was 117.4 mg / (kg*h) on the 4th day; the respiration intensity of the control group of potassium sorbate, cecropin and water was 168.31 mg / (kg*h), 169.54 mg / (kg*h) and 199.14 mg / (kg*h) respectively, and the respiration intensity of the rHc-CATH experimental group was 158.89 mg / (kg*h) on the 6th day; the respiration intensity of the control group of potassium sorbate, cecropin and water was 185.23 mg / (kg*h), 156.33 mg / (kg*h) and 175.30 mg / (kg*h) respectively, and the respiration intensity of the rHc-CATH experimental group was 130.95 mg / (kg*h) on the 8th day; the respiration intensity of the control group of potassium sorbate, cecropin and water was 162.83 mg / (kg*h), 160.89 mg / (kg*h) and 166.29 mg / (kg*h) respectively, and the respiration intensity of the rHc-CATH experimental group was 106.84 mg / (kg*h) on the 10th day. Overall, the respiration intensity of the rHc-CATH experimental group was lower than that of the control group, indicating that the rHc-CATH antibacterial peptide had the effect of reducing the respiration intensity.

[0102] (3) Weight loss determination

[0103] The same number of blueberries, 15 g in total, were taken, and a multi-hole box was used to keep the environment the same.

[0104] As shown in Figure 9 , compared with the treatment results of the control group of potassium sorbate, cecropin and water, the rHc-CATH treatment slowed down the weight loss effect and prolonged the shelf life by about 2d.

[0105] (4) Anthocyanin determination

[0106] Accurately weigh 25.00 g of cut blueberries, add 200 mL of pre-cooled (4°C) extraction solution (methanol: water: formic acid = 60:38:2, V:V:V), homogenize for 45 s, stand in the dark for 5 min, centrifuge at 8000 r / min for 20 min, remove the supernatant, and store at -20°C. Then dilute the sample extract with 0.025 mol / L potassium chloride buffer (pH 1.0) and 0.4 mol / L sodium acetate buffer (pH 4.5) respectively, mix well, stand in the dark for 20 min, and then measure the absorbance at 530 nm and 700 nm respectively. The anthocyanin content in the sample is calculated according to the formula:

[0107]

[0108] ​A = A 530nm,pH1.0 -A 700nm,pH1.0 -(A 530nm,pH4.5 -A 700nm,pH4.5 )

[0109] Anthocyanin content (mg·100g -1 ) = (A x MW x 1000 x k) / (ε x L)

[0110] In the formula: A is the absorbance; MW is the molecular weight of cyanidin-3-O-glucoside (449.2 g / mol); k is the dilution factor; ε is the molar absorption coefficient of cyanidin-3-O-glucoside (26900 L·mol-1·cm-1); L is the optical path length (1 cm).

[0111] As Figure 10 shown, the anthocyanin content of the rHc-CATH experimental group and the control group of sorbic acid, cecropin and water all showed a significant downward trend over time. On the 2nd day, the rHc-CATH experimental group lost 52.8% of the original anthocyanin, while the control group of sorbic acid, cecropin and water lost 55.3%, 53.3% and 28.7%, respectively. On the 6th day, the loss rate of the rHc-CATH experimental group was 77.2%, while the loss rate of the control group of sorbic acid, cecropin and water after treatment was all above 95%. The results showed that the anthocyanin content value of the control group decreased significantly faster than that of the experimental group, which means that the loss process of blueberry anthocyanin in the control group was more rapid. In contrast, the rHc-CATH experimental group showed better preservation effect and successfully slowed down the loss rate of anthocyanin.

[0112] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, which should be covered in the scope of the present application.

Claims

1. A method for preparing recombinant antimicrobial peptide Hc-CATH, characterized in that: include, Recombination of expression vector: The Hc-CATH target gene shown in SEQ ID NO:2 was synthesized and ligated into the pPICZαA vector to form the recombinant plasmid pPICZαA-Hc-CATH; Construction of recombinant strains: The recombinant plasmid pPICZαA-Hc-CATH was linearized using Sac I restriction endonuclease, and the treated plasmid was then transformed into Pichia pastoris to obtain recombinant strains carrying the target gene. Expression of antimicrobial peptides: The screened positive transformants were induced to express with methanol, and the supernatant after fermentation was collected to obtain high concentrations of antimicrobial peptides; The original amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The preparation method according to claim 1, characterized in that: The method for preparing the recombinant plasmid pPICZαA-Hc-CATH includes, An EcoRI restriction site was introduced at the 5' end of the Hc-CATH target gene sequence, and start codons ATG and 6His-tag were added. A stop codon TAA was added at the 3' end of the sequence, and an XbaI restriction site was introduced to obtain the recombinant plasmid pPICZαA-Hc-CATH.

3. The preparation method according to claim 1 or 2, characterized in that: The expression was induced using methanol, specifically by inducing expression with 1% methanol for 6 days.

4. The recombinant antimicrobial peptide Hc-CATH prepared by any one of the preparation methods described in claims 1 to 3.

5. The recombinant antimicrobial peptide Hc-CATH as described in claim 4, characterized in that: The recombinant antimicrobial peptide Hc-CATH has a minimum inhibitory concentration (MIC) of 2.5 μg / mL against Staphylococcus aureus, 20 μg / mL against Bacillus subtilis and Escherichia coli, 10 μg / mL against Listeria monocytogenes and Escherichia coli O157, and 5 μg / mL against Salmonella spp.

6. The application of the recombinant antimicrobial peptide Hc-CATH according to claim 4 in the preservation of blueberries.

7. The application as described in claim 6, characterized in that: Spray blueberries with Hc-CATH solution and let them drain naturally in a sterile environment.

8. The application as described in claim 7, characterized in that: The concentration of the Hc-CATH solution is 10 μg / mL.

9. The application as described in any one of claims 6 to 8, characterized in that: The Hc-CATH antimicrobial peptide reduces weight loss and anthocyanin loss in blueberries.

10. The application as described in any one of claims 6 to 8, characterized in that: The Hc-CATH antimicrobial peptide reduces the respiration rate of blueberries.

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