Tripeptide substance capable of inhibiting staphylococcus aureus as well as preparation method and application of tripeptide substance

Abstract

The invention belongs to the technical field of biology, discloses a tripeptide substance capable of inhibiting staphylococcus aureus and a preparation method, and particularly discloses an active peptide, and the amino acid sequence of the active peptide is KSK. The antibacterial active peptide provided by the invention has the activity of inhibiting staphylococcus aureus and does not have the activity of inhibiting escherichia coli, the antibacterial activity of the active peptide is influenced by the environmental pH, and the antibacterial effect is the best when the pH is 6.0. The active peptide can be synthesized by a common liquid-phase or solid-phase synthesis method, and can also be obtained by fermenting allium macrostemon pasture and then carrying out water extraction on the allium macrostemon pasture. Aiming at the antibacterial performance of the active peptide, the allium macrostemon can be used for fermenting forage grass to optimize intestinal flora of poultry (organic acids, such as lactic acid, acetic acid and propionic acid, capable of reducing pH are contained in the fermented forage grass), so that the intestinal tract of the poultry is healthier, the digestion and absorption functions can be improved, and a feasible scheme for replacing antibiotics is provided for the livestock and poultry industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a tripeptide substance capable of inhibiting Staphylococcus aureus, a preparation method and an application thereof. Background Art

[0002] The use of antibiotic feed additives has caused many safety issues, such as the development of drug resistance in pathogens, endogenous infections in livestock and poultry, and the impact of antibiotic residues in livestock products on the immune effects of humans and animals. Therefore, the restriction and ban of antibiotics in feed has become an inevitable trend, and "antibiotic-free, green, and efficient" feed will also become a research hotspot and development direction of the feed industry.

[0003] Europe was the first to propose a ban on growth-promoting antibiotics, starting with Sweden in 1986. Denmark banned antibiotics from livestock and poultry feed in 2000, and the European Union fully banned their use in feed by 2006. South Korea also announced a ban on feed antibiotics in 2011. In July 2020, my country announced a complete halt to the production and sale of commercial feed containing growth-promoting drug feed additives (excluding herbal feed). With the ban on antibiotics, the development of alternatives to antibiotics will become a hot topic in the livestock and poultry industry.

[0004] Antimicrobial peptides are small peptide molecules found widely in nature. Most antimicrobial peptides contain 10 to 100 amino acids and exhibit broad-spectrum antimicrobial activity. Compared to antibiotics, antimicrobial peptides can effectively reduce bacterial resistance. They also offer advantages such as broad-spectrum antimicrobial activity, good thermal stability, and low dosage requirements, providing an effective approach to addressing the problem of resistance to traditional antibiotics.

[0005] Staphylococcus aureus is a common foodborne pathogen that is commonly found in the intestines and stomachs of humans or animals. Under suitable conditions, it can produce enterotoxins and cause host diseases. Desmodium intortum, also known as green-leafed mountain leech, green-leafed mountain leech, etc., is a creeping perennial herb of the genus Desmodium intortum of the Leguminosae family, native to Central and South America. Plants of the genus Desmodium are a very potential source of high-protein feed in tropical and subtropical regions. The prior art discloses the effect of fermentation of Desmodium intortum on the silage quality. However, there is currently no evidence that the fermentation products of Desmodium intortum have any active effect on pathogenic bacteria in the digestive tract of livestock and poultry. Summary of the Invention

[0006] The present invention utilizes the twisted mung bean grass forage to undergo fermentation treatment, and the organic acid produced reduces the pH of the contents of the digestive tract of livestock and poultry, while also producing an antimicrobial active peptide (KSK) that has the activity of inhibiting Staphylococcus aureus, a pathogenic bacterium in the digestive tract of livestock and poultry under acidic conditions, thereby providing a feasible alternative to antibiotics for the livestock and poultry breeding industry.

[0007] The first aspect of the present invention aims to provide an active peptide.

[0008] The second aspect of the present invention aims to provide a nucleic acid molecule encoding the active peptide of the first aspect of the present invention.

[0009] The third aspect of the present invention aims to provide an expression cassette, a recombinant vector or a transgenic cell comprising the nucleic acid molecule of the second aspect of the present invention.

[0010] The fourth aspect of the present invention aims to provide a method for preparing the active peptide of the first aspect of the present invention.

[0011] The purpose of the fifth aspect of the present invention is to provide the use of the active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention or the extract of fermented Mung Bean Grass in at least one of (b1) to (b).

[0012] The sixth aspect of the present invention aims to provide a product.

[0013] The seventh aspect of the present invention aims to provide a method for inhibiting bacteria.

[0014] In order to achieve the above object, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides an active peptide, the amino acid sequence of the active peptide is:

[0016] 1) KSK; or

[0017] 2) An amino acid sequence shown in 1) having the same or similar function after one or more amino acids are modified, substituted or deleted.

[0018] The second aspect of the present invention provides a nucleic acid molecule encoding the active peptide of the first aspect of the present invention.

[0019] The third aspect of the present invention provides an expression cassette, a recombinant vector or a transgenic cell comprising the nucleic acid molecule of the second aspect of the present invention.

[0020] In some embodiments of the invention, the transgenic cells do not contain reproductive material.

[0021] The fourth aspect of the present invention provides a method for preparing the active peptide of the first aspect of the present invention, which is any one of (a1) to (a3):

[0022] (a1) Using Mung Bean as raw material, through fermentation and extraction;

[0023] (a2) Synthesis by liquid phase or solid phase synthesis;

[0024] (a3) Cultivating the transgenic cell according to the third aspect of the present invention.

[0025] In some embodiments of the present invention, the preparation method (a1) specifically comprises the following steps: mixing the radix gypsophila with sucrose and lactic acid bacteria, fermenting, and drying to obtain fermented radix gypsophila; mixing the fermented radix gypsophila with water and extracting.

[0026] In some embodiments of the present invention, the fermentation time is 0.5 to 60 days, preferably 1 to 30 days.

[0027] In some embodiments of the present invention, the added amount of sucrose is 0.5% to 2% of the weight of the Vigna volvacea; preferably 1% to 2%.

[0028] In some embodiments of the present invention, the amount of lactic acid bacteria added is (0.5-9)×10 6 CFU / g weight of Mung Bean.

[0029] In some embodiments of the present invention, the extraction condition is extraction at room temperature for 2 to 6 hours, preferably 3 to 4 hours.

[0030] In some embodiments of the present invention, the fermented Mung Bean is crushed before extraction.

[0031] A fifth aspect of the present invention provides the use of the active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention, or the extract of fermented M. truncatum in (b1) and / or (b2):

[0032] (b1) antibacterial activity;

[0033] (b2) preparing products with antibacterial activity;

[0034] The extract of the fermented V. truncatula forage grass contains the active peptide of the first aspect of the present invention.

[0035] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Enterobacter aerogenes, and Bacillus subtilis; preferably, the bacteria is Staphylococcus aureus.

[0036] In some embodiments of the present invention, the extract of the fermented Mung Bean forage is prepared by the following preparation method: Mung Bean is mixed with sucrose and lactic acid bacteria, fermented, and dried to obtain fermented Mung Bean; the fermented Mung Bean is mixed with water and extracted to obtain the extract of the fermented Mung Bean forage.

[0037] In some embodiments of the present invention, the fermentation time is 0.5 to 60 days, preferably 1 to 30 days.

[0038] In some embodiments of the present invention, the added amount of sucrose is 0.5% to 2% of the weight of the Vigna volvacea; preferably 1% to 2%.

[0039] In some embodiments of the present invention, the amount of lactic acid bacteria added is (0.5-9)×10 6 CFU / g weight of Mung Bean.

[0040] In some embodiments of the present invention, the extraction condition is extraction at room temperature for 2 to 6 hours, preferably 3 to 4 hours.

[0041] In some embodiments of the present invention, the fermented Mung Bean is crushed before extraction.

[0042] In some embodiments of the present invention, the product is at least one of feed, feed additive and reagent.

[0043] The sixth aspect of the present invention provides a product comprising the active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention.

[0044] In some embodiments of the present invention, the product is at least one of feed, feed additive and reagent.

[0045] In some embodiments of the present invention, the product has the function of inhibiting bacteria (at least one of Staphylococcus aureus, Enterobacter aerogenes, and Bacillus subtilis).

[0046] The seventh aspect of the present invention provides a method for inhibiting bacteria, comprising the step of treating bacteria using the active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention or the product of the sixth aspect of the present invention.

[0047] In some embodiments of the present invention, the treatment method includes mixing the active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention or the product of the sixth aspect of the present invention with bacteria and culturing.

[0048] In some embodiments of the present invention, the pH value of the culture system is 5-8; preferably 5-6.5; more preferably 5.5-6.0.

[0049] In some embodiments of the present invention, the culture time is 20 to 30 hours.

[0050] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Enterobacter aerogenes, and Bacillus subtilis; preferably, the bacteria is Staphylococcus aureus.

[0051] The beneficial effects of the present invention are:

[0052] The antimicrobial active peptide (KSK) provided by the present invention has the activity of inhibiting Staphylococcus aureus, but has no inhibitory activity against Escherichia coli, and the antibacterial activity of the active peptide is affected by the environmental pH, and the antibacterial effect is best at pH 6.0. The active peptide can be synthesized by common liquid phase or solid phase synthesis methods, or can be obtained by water extraction after fermentation of Mung Bean forage. In view of the antibacterial properties of the active peptide, Mung Bean fermented forage can be used to optimize the intestinal flora of poultry (the fermented forage contains organic acids that lower the pH, such as lactic acid, acetic acid, propionic acid, etc.), thereby making the intestinal tract of poultry healthier, so as to improve the digestion and absorption functions, and provide a feasible alternative to antibiotics for the livestock and poultry farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 To study the effect of fermented Mung Bean Grass extract on the growth of Staphylococcus aureus (A), Enterobacter aerogenes (B), Escherichia coli (C) and Bacillus subtilis (D).

[0054] Figure 2 The effect of different pH on the antibacterial effect (A is Staphylococcus aureus, B is Enterobacter aerogenes, C is Escherichia coli, and D is Bacillus subtilis) of fermented Mung Bean Grass extract. In the figure, different lowercase letters indicate significant differences among different treatment groups (p<0.05).

[0055] Figure 3 This is the Sephadex G-25 chromatogram of the fermented M. rotundus herb extract.

[0056] Figure 4 This is the Sephadex G-25 elution peak of the fermented Mung Bean grass extract.

[0057] Figure 5 The effect of Sephadex G-25 eluate on the growth of Staphylococcus aureus is shown in each tube.

[0058] Figure 6 The figure shows the HPLC spectrum of the component having the activity of inhibiting the growth of Staphylococcus aureus.

[0059] Figure 7 This is the liquid phase spectrum of liquid chromatography-mass spectrometry.

[0060] Figure 8 This is the secondary mass spectrum of Lys-Ser-Lys.

[0061] Figure 9 This is the mass spectrum of artificially synthesized KSK.

[0062] Figure 10 Figure 3 shows the effect of KSK on the growth of Staphylococcus aureus under different pH conditions. In the figure, different lowercase letters indicate significant differences among different treatment groups (p<0.05).

[0063] Figure 11 This is the standard curve of artificially synthesized KSK. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below through specific examples.

[0065] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0066] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0067] The test strains in the examples are: Staphylococcus aureus (CMCC(B)26003), Escherichia coli (CMCC(B)44102), Enterobacter aerogenes (ATCC 13048) and Bacillus subtilis (CMCC(B)63501), all of which were provided by the Microbiology Teaching and Research Section of the School of Life Sciences, South China Normal University.

[0068] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0069] Example 1 Preparation of fermented Mung Bean grass extract

[0070] Preparation of fermented grass of Mung Bean: freshly harvested Mung Bean grass was chopped into 1-2 cm pieces, sucrose was added at 1% of the weight of fresh grass, and 1×10 6 CFU / g of lactic acid bacteria (disclosed in "Tian Jing, Tang Guojian, Peng Jianzong, Yang Chengwei, Zhang Jianguo. Nutritional components and silage fermentation quality of Mung Bean. Grassland Science, 2020, 37(2): 370-376"), then bagged, sealed and fermented at a temperature of 20-40°C.

[0071] The Mung Bean Grass fermented for 2 weeks was dried at 80°C and then crushed to prepare grass powder, that is, the fermented grass powder was obtained.

[0072] Preparation of fermented Mung Bean Forage Extract: Accurately weigh 1g of fermented forage powder, add 15mL of distilled water, extract at 20-35℃ for 4h, filter with qualitative filter paper, collect the filtrate, and filter the filtrate with a 0.22μm filter membrane to sterilize the obtained fermented Mung Bean Forage Extract (recorded as fermented forage extract), and store it in a -20℃ refrigerator for future use.

[0073] Example 2 Antibacterial experiment of fermented forage extract

[0074] (1) Culture medium

[0075] LB medium (for E. coli cultivation): 10 g peptone, 10 g sodium chloride, 5 g yeast extract, 1000 mL water. Adjust the pH of the medium to 7.0 and sterilize at 121°C for 20 min.

[0076] NB medium (for the cultivation of Staphylococcus aureus, Enterobacter aerogenes, and Bacillus subtilis): 10 g peptone, 3 g beef extract, 5 g sodium chloride, and 1000 mL water. Adjust the pH of the medium to 7.2 and sterilize at 121°C for 20 min.

[0077] (2) Method

[0078] 200 μL of fermented forage extract was added to 1 mL of culture medium, and then 1 μL of 1×10 9 CFU / mL bacterial solution. A control group replaced 200 μL of fermented forage extract with 200 μL of distilled water (filtered and sterilized), with three replicates for each. Based on the growth characteristics of different bacteria, the OD value of the bacterial solution was measured at 600 nm after a certain incubation period. The incubation time for Staphylococcus aureus, Escherichia coli, and Bacillus subtilis was 10 hours, and the incubation time for Enterobacter aerogenes was 24 hours. The effect of the extract on bacterial growth was determined based on the OD value.

[0079] (3) Results

[0080] This example measured the effects of the extracts of Mung Bean Herb before and after fermentation on the growth of four bacteria. The results are as follows: Figure 1 Compared with the control group, the fermented forage extract (i.e., the fermented forage extract prepared in Example 1) had a significant inhibitory effect on the growth of Staphylococcus aureus ( Figure 1 A) had no significant inhibitory effect on the growth of Enterobacter aerogenes, Escherichia coli and Bacillus subtilis ( Figure 1 The forage extract before fermentation (i.e., the extract obtained by crushing and drying freshly harvested V. truncatula forage grass and then subjecting it to water extraction, with the same extraction conditions as in Example 1) not only had no inhibitory effect on the growth of Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Bacillus subtilis, but instead had a certain promoting effect.

[0081] The effect of pH on the antibacterial activity of the fermented forage extract was further investigated as follows: 200 μL of the extract was added to each 1 mL of culture medium, and then 1 μL of a 1×10 9 CFU / mL bacterial solution. 200 μL of distilled water (filter sterilized) was used instead of 200 μL of the extract in the control group. The pH was then adjusted with 0.1N HCl to obtain culture systems at pH 5.5, pH 6.0, and pH 7.0, respectively. Based on the growth characteristics of different bacteria, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Bacillus subtilis were cultured for 9 hours, 20 hours, 8 hours, and 9 hours, respectively. The OD value of the bacterial solution was measured at 600 nm. The effect of the extract on bacterial growth was determined based on the OD value.

[0082] The effects of fermented forage extract on the growth of four bacteria in pH 5.5, pH 6.0 and pH 7.0 culture medium were measured. Figure 2 As shown in the results, in the culture systems of pH 5.5, pH 6.0 and pH 7.0, the fermented forage extract had inhibitory activity on the growth of Staphylococcus aureus, and the inhibitory activity was most obvious at pH 6.0. 600 The value of nm showed a decrease of 56.5% compared with the control group ( Figure 2 For Enterobacter aerogenes and Bacillus subtilis, the fermented forage extract only had inhibitory activity at pH 5.5, and showed growth promotion effects at pH 6.0 and pH 7.0 ( Figure 2 B and D); For E. coli, the fermented forage extract showed a promoting effect under the three pH conditions. The extracts under different pH conditions had no inhibitory effect on the growth of E. coli ( Figure 2 Middle C).

[0083] Example 3 Isolation and Identification of Active Ingredients in Fermented Forage Extract

[0084] In order to analyze which specific components in the fermented forage extract have antibacterial effects, this example used Sephadex G-25 dextran gel column chromatography to separate the antibacterial components in the fermented forage extract, used high-performance liquid chromatography to preliminarily separate the active components, and used LC-MS / MS to identify the structures of the active components, as follows:

[0085] 1.Sephadex G-25 dextran gel column chromatography to separate antibacterial components

[0086] Slowly pour 5-10 times the volume of deionized water into the Sephadex G-25 dry gel, allow it to fully swell, and then use the tilting method to remove small particles suspended on the surface before loading into the column. Gel column loading requires the gel to naturally settle within the column, achieving uniform density and the absence of voids and bubbles. The chromatography column is 1 cm x 100 cm in size and is equilibrated with ultrapure water. The eluent flow rate is adjusted to 0.25 mL / min. After freeze-drying and concentrating the fermented forage extract 75 times, a 1 mL sample is loaded. Deionized water is used as the eluent at a flow rate of 0.25 mL / min. A total of 61 tubes (approximately 2 mL per tube) are collected. The OD value of the eluent in each tube is measured using a spectrophotometer at four wavelengths: 215 nm, 220 nm, 245 nm, and 280 nm.

[0087] Take each tube of eluate and perform the antibacterial test on Staphylococcus aureus to determine the tube number where the antibacterial component is located.

[0088] The OD values ​​of the eluates collected from the gel chromatography fractions were measured at four wavelengths to obtain the corresponding elution curves ( Figure 3 ). According to OD 280 The absorbance values ​​of nm are divided into 5 components ( Figure 4 ).

[0089] After concentrating each tube of eluate, the effect of each tube of eluate on the growth of Staphylococcus aureus was measured. The results showed that ( Figure 5 ), the eluates from tubes 36 to 39 and 41 to 43 showed significant antibacterial activity, that is, the antibacterial components of the fermented forage extract were mainly concentrated in Figure 4 Therefore, the eluate from this part was collected, sterilized by filtration through a 0.22 μm filter membrane, and stored at -20°C for further separation, purification, and identification.

[0090] 2. Preliminary separation of active components by high performance liquid chromatography

[0091] Preparation of mobile phase:

[0092] Mobile phase A (ultrapure water): Measure 900 mL of ultrapure water solution into a beaker, vacuum filter through a 0.48 μm pore size aqueous filter membrane, and transfer to a blue-mouth bottle. Loosen the bottle cap before ultrasonic removal of bubbles. The ultrasonication time is 15 min.

[0093] Mobile phase B (acetonitrile solution, chromatography grade): Measure 900 mL of acetonitrile solution into a beaker, vacuum filter through a 0.48 μm pore size organic phase filter, and then transfer to a blue-mouth bottle. Loosen the bottle cap before ultrasonically removing bubbles. Ultrasonication lasts for 15 min.

[0094] method:

[0095] The test sample is a component separated by Sephadex G-25 chromatography that has the activity of inhibiting the growth of Staphylococcus aureus.

[0096] First, replace the mobile phases with the corresponding mobile phases A and B. Turn on the equipment step by step, starting from top to bottom and from left to right. Turn on the computer, set the initial mobile phase volume, and loosen the exhaust valve. Also, deaerate the mobile phase solution, setting the flow rate to 2 mL / min and the exhaust time to 5 minutes. After purging, tighten the exhaust valve and set the flow rate to 1 mL / min. Install the chromatographic column (Superlu C18, 250 × 4.6 mm). In reversed-phase HPLC, compounds with high polarity elute earlier, while compounds with low polarity elute later. This property can be exploited to find suitable separation conditions by adjusting the mobile phase ratio. Run the baseline for at least 30 minutes until it remains level. Then, load the sample, set the sequence table, and begin the run. After all measurements are complete, wash the column with acetonitrile for at least 20 minutes, copy the data, and shut down the equipment. Separation conditions are shown in Table 1.

[0097] Table 1 Reverse HPLC conditions

[0098]

[0099] The results showed that the liquid phase separation spectrum had three main peaks, the separation effect was good, and the peaks were well separated ( Figure 6 ). On this basis, the sample can be further subjected to LC-MS / MS structural analysis.

[0100] 3. LC-MS / MS identification of the structure of the active component

[0101] The above active component samples were sent to the Analytical Testing Center of South China Normal University for mass spectrometry analysis. The specifications of the ultra-high pressure liquid chromatography column used were: ZORBAX Eclipse Plus C18, 2.1×50mm. Based on the separation conditions provided by the above high performance liquid chromatography analysis, the mobile phase used for the test was 0.1% formic acid water and 10% acetonitrile. Mainly through ESI multi-charge analysis, on the basis of obtaining accurate MS and MS / MS, the amino acid sequence was determined by MS / MS retrieval of ion fragments, and qualitative analysis was performed using Database or Library software. Mass spectrometry detection was performed based on the good separation conditions of high performance liquid chromatography. First, a preliminary screening was carried out based on the molecular formula, matching degree and other relevant information of the target compound given by the primary mass spectrometry, and then the parent ion with a stronger signal was selected for secondary mass spectrometry, which was further dissociated to obtain the molecular structure of the target compound.

[0102] Liquid chromatography-mass spectrometry (LC-MS) Figure 7) showed that the peaks of the active components could be well separated at this time, and three compounds were identified, with the matching scores of the primary mass spectrometry being 71.34, 87.28 and 90.85, respectively. A preliminary screening was performed based on the molecular formula of the target compound, the matching scores and other related information (Table 2) given by the primary mass spectrometry. The component with a high matching score of the primary mass spectrometry and an m / z value of 362.2414 was selected for secondary mass spectrometry and further dissociated, and the molecular structure of the target compound was obtained to be lysyl-seryl-lysine (Lys-Ser-Lys, abbreviated as KSK) ( Figure 8 ).

[0103] Table 2 Primary mass spectrometry test results (partial)

[0104]

[0105]

[0106] Example 4 Artificial Synthesis of Lys-Ser-Lys

[0107] Shanghai Bioengineering Co., Ltd. was commissioned to use the Fmoc solid-phase synthesis method to synthesize KSK, and the structure of the synthetic product was determined by mass spectrometry.

[0108] The mass spectrometry results of the synthetic product were KSK( Figure 9 ).

[0109] Example 5 Minimum inhibitory concentration (MIC) test of artificially synthesized KSK

[0110] Take six sterile test tubes and add 1 mL of a 16 mg / mL KSK solution to the first tube. Dilute the solution to the fifth tube in a doubling ratio. At this point, the KSK concentrations in each tube are 16, 8, 4, 2, and 1 mg / mL, respectively. The sixth tube serves as a negative control without KSK. KSK samples (prepared in Example 4) of various concentrations are then added to a 96-well bacterial culture plate. Samples are added to wells 1 through 5, and ultrapure water is added to well 6, with 20 μL per well. An ampicillin MIC plate is prepared using the same method, with the first well serving as a positive drug control.

[0111] NB medium (pH 6.0) was used to culture activated Staphylococcus aureus (1.5×10 8 CFU / mL) was diluted 100-fold and 100 μL was added to each well. At this point, the KSK concentrations in each well were 2.667, 1.333, 0.667, 0.333, and 0.167 mg / mL, respectively. The cells were incubated at 37°C for 24 hours. The absorbance was measured using a microplate reader (595 nm). The lowest concentration with an inhibition rate greater than 95% was calculated as the MIC. The formula is as follows:

[0112]

[0113] Where: Y is the inhibition rate; X P As positive control, X N As negative control, X S For the sample to be tested.

[0114] The antibacterial effects of different concentrations of synthetic KSK are shown in Table 3. The results show that synthetic KSK also has the activity of inhibiting Staphylococcus aureus. Under the condition of pH 6.0, the minimum inhibitory concentration of KSK against Staphylococcus aureus was measured to be 1.333 mg / mL.

[0115] Table 3 MIC determination results

[0116]

[0117] The antibacterial effect of synthetic KSK under different pH conditions was further investigated as follows: the pH of the NB culture medium for culturing Staphylococcus aureus was adjusted to pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0, respectively. The amount of KSK added was 200 μg / mL, and the control group was replaced with the same volume of sterile water. The cells were cultured in a 37°C incubator for 24 h, and then the OD was measured. 600 nm.

[0118] The results showed that compared with the control group, the antibacterial effect of synthetic KSK was most significant at pH 6.0, and the inhibitory effect was not obvious at pH 6.5, 7.0, 7.5 and 8.0 ( Figure 10 ), such a result is similar to Figure 2 The antibacterial effect of the fermented forage extract in medium A is consistent.

[0119] Example 6 Determination of KSK content changes during the fermentation of Mung Bean Grass

[0120] The KSK artificially synthesized in Example 4 was used as a standard sample, and the content of KSK in the fermented forage was determined by high performance liquid chromatography.

[0121] Reagent preparation:

[0122] KSK standard solution: accurately prepare KSK stock solution (10 mg / mL) and dilute it to different amounts of concentrations of 1.0, 3.0, 4.0, 5.0, and 6.0 mg / mL. Filter through a 0.22 μm filter membrane and transfer to a chromatographic vial for use.

[0123] Mobile phase A (ultrapure water): Measure 900 mL of ultrapure water solution into a beaker, vacuum filter through a 0.48 μm pore size aqueous filter membrane, and transfer to a blue-mouth bottle. Loosen the bottle cap before ultrasonic removal of bubbles. The ultrasonication time is 15 min.

[0124] Mobile phase B (acetonitrile solution, chromatography grade): Measure 900 mL of acetonitrile solution (99.9%) into a beaker and vacuum filter through a 0.48 μm pore size organic phase filter. Then transfer to a blue-mouth bottle and loosen the bottle cap before ultrasonically removing bubbles. Ultrasonication lasts for 15 min.

[0125] Sample extraction and determination:

[0126] The forage grass before fermentation (0h) and 12h, 24h, 48h, 3d, 7d, 14d, 30d, and 60d after fermentation (fermentation conditions are the same as in Example 1) was dried and prepared into grass powder respectively. After extraction at room temperature for 4h, 0.5g of forage grass powder was added to 5mL of distilled water, and the extract was filtered with qualitative filter paper to collect the filtrate. The filtrate was filtered and sterilized through a 0.22μm filter membrane to obtain an extract, which was stored at -20°C for future use.

[0127] The HPLC conditions are shown in Table 4.

[0128] Table 4 Reverse HPLC determination conditions

[0129]

[0130] The standard curve constructed using KSK standards is as follows Figure 11 As shown in Table 5, no KSK peak was detected before fermentation, but KSK was detectable in the extract after 12 hours of fermentation. By day 30, the KSK content had increased to 2.621 g / kg DM, 3.35 times the level at day 12. However, by day 60, the KSK content was similar to that at day 30 (Table 5), indicating that KSK content stabilizes after 30 days of fermentation.

[0131] Table 5 Changes in KSK content in fermented forage extracts

[0132]

[0133]

[0134] Note: ND means not detected; different lowercase letters in the same column indicate significant differences among different fermentation times (p<0.05).

[0135] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. An active peptide, the amino acid sequence of the active peptide is: 1) KSK; or 2) An amino acid sequence shown in 1) having the same or similar function after one or more amino acids are modified, substituted or deleted.

2. A nucleic acid molecule encoding the active peptide according to claim 1.

3. An expression cassette, recombinant vector or transgenic cell comprising the nucleic acid molecule of claim 2.

4. The method for preparing the active peptide according to claim 1, which is any one of (a1) to (a3): (a1) Using Mung Bean as raw material, through fermentation and extraction; (a2) Synthesis by liquid phase or solid phase synthesis; (a3) obtained by culturing the transgenic cell according to claim 3.

5. Use of the active peptide according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant vector or transgenic cell according to claim 3 or the extract of fermented M. truncatum in (b1) and / or (b2): (b1) inhibiting bacterial activity; (b2) preparing products with antibacterial activity; The extract of the fermented M. convolvulus herb contains the active peptide according to claim 1.

6. The use according to claim 5, characterized in that The bacteria include at least one of Staphylococcus aureus, Enterobacter aerogenes, and Bacillus subtilis.

7. The use according to claim 5, characterized in that The fermented Mung Bean forage extract is prepared by the following preparation method: Mung Bean is mixed with sucrose and lactic acid bacteria, fermented, and dried to obtain fermented Mung Bean; the fermented Mung Bean is mixed with water and extracted to obtain the fermented Mung Bean forage extract; Preferably, the fermentation time is 0.5 to 60 days.

8. A product comprising the active peptide according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant vector or transgenic cell according to claim 3.

9. A method for inhibiting bacteria, comprising the step of treating bacteria with the active peptide according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant vector or transgenic cell according to claim 3 or the product according to claim 8.

10. The method according to claim 9, characterized in that The treatment method comprises mixing the active peptide according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette, recombinant vector or transgenic cell according to claim 3 or the product according to claim 8 with bacteria and culturing; Preferably, the pH value of the culture system is 5-8.

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