A methanolic extract of the fruit of phyllanthus emblica and uses thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a methanol phase extract of fruit of Phyllanthus emblica and application thereof. The methanol phase extract PeME is extracted from the fruit of Phyllanthus emblica by a methanol-chloroform extraction method, and a purified component PeME-F3 is obtained by further separation and purification of the methanol phase extract. The methanol phase extract PeME and the purified component PeME-F3 can inhibit Vibrio cholerae and Staphylococcus aureus, and can be used as bacteriostatic agents. The methanol phase extract of the fruit of Phyllanthus emblica can inhibit contamination of Vibrio cholerae and Staphylococcus aureus in crucian carp and Penaeus vannamei stored at low temperature, and can be applied to aquatic products; the methanol phase extract can also be used for preparation of food or medicine, and the prepared food or medicine also has the bacteriostatic effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, and in particular to a methanol-phase extract from the fruit of Phyllanthus emblica and its application. BACKGROUND

[0002] Infectious diseases caused by pathogens seriously threaten public health and human health, and have become the second largest cause of death in the world. According to the report of the World Health Organization (WHO), about 600 million people worldwide suffer from diseases every year due to the consumption of food contaminated by foodborne pathogens, resulting in about 420,000 deaths. Among them, Vibrio cholerae is the pathogen that causes the infectious disease of "cholera" type A. The bacterium spreads from environmental hosts to human hosts through contaminated water or food, causing the "cholera" disease that endangers human life. The main clinical symptoms include gastric ulcer, acute diarrhea, severe vomiting, severe dehydration, and even death. Staphylococcus aureus is one of the pathogens that cause high morbidity and mortality, and can cause skin infections, as well as fatal pneumonia and sepsis through contact with contaminants and human-to-human transmission.

[0003] Antibiotics play a crucial role in the treatment of infectious diseases. However, the inappropriate use of antibiotics leads to the development of antibiotic resistance in pathogenic bacteria, resulting in about 700,000 deaths worldwide each year. Long-term overuse of antibiotics also causes food safety risks and environmental pollution problems. Studies have shown that most current epidemic strains of Vibrio cholerae have multiple drug resistance, while clinical Staphylococcus aureus isolates are mostly methicillin- and vancomycin-resistant (MRSA and VRSA), further leading to higher mortality, more secondary infections, and higher medical costs. Therefore, it is urgent and important to screen and prepare new antibacterial agents.

[0004] Medicinal plants have become a green and natural compound resource library for antibacterial agent screening due to their outstanding advantages of low toxicity, safety, and low cost. Among them, the fruit of Phyllanthus emblica Linnaeus is often used as a traditional Chinese medicinal material for treating diseases such as diarrhea, jaundice, fever, and cough. In recent years, pharmacological studies have shown that various compounds extracted from the fruit of Phyllanthus emblica have the effects of lowering blood sugar, lowering blood lipids, protecting the liver, and treating sepsis, tuberculosis, heart and urinary system diseases, etc. However, there is no report on the bacteriostatic effect and application of the methanol-phase extract from the fruit of Phyllanthus emblica (PeME) at home and abroad. SUMMARY

[0005] The present application aims to provide a methanol phase extract of the fruit of Phyllanthus emblica, which can inhibit Vibrio cholerae and Staphylococcus aureus.

[0006] To achieve the above-mentioned purpose, the present application adopts the following specific technical solutions:

[0007] In the first aspect, the present application provides a methanol phase extract PeME of the fruit of Phyllanthus emblica, which is extracted from the fruit of Phyllanthus emblica by a methanol-chloroform solvent extraction method, and the specific steps are as follows:

[0008] S1. Fresh Phyllanthus emblica fruits are washed clean, dried, cut into pieces, pre-frozen at -80℃ for 2-4h, then freeze-dried at -80℃ for 48h, the freeze-dried sample is crushed and sieved with a 300-mesh sieve;

[0009] S2. Methanol and chloroform (1:2, v / v) are added to the sieved sample powder at a solid-liquid ratio of 1:10 (m / v) and mixed evenly, then sterile water is added and mixed evenly, and the ratio of sample powder to sterile water is controlled at 1:6 (m / v); the mixture is ultrasonically treated, then filtered with a 20-25μm microporous filter membrane, and the filtrate is collected; after the methanol phase and the chloroform phase are separated, evaporation and concentration are performed using a rotary evaporator to obtain PeME; the filtrate is extracted a second time using the above-mentioned methanol and chloroform solvent, and the obtained PeME is combined.

[0010] In the second aspect, the present application provides a purified component PeME-F3 of the methanol phase extract of the fruit of Phyllanthus emblica, which is obtained by separation and purification of the above-mentioned methanol phase extract PeME, and the separation and purification method is as follows:

[0011] The PeME is diluted with ultrapure water, and the supernatant is collected after centrifugation, then filtered through a sterile 0.22μm filter membrane, and the filtrate is collected; the filtrate sample is separated by a Waters 2707 connected to a UPLC Sunfire C 18 chromatographic column; a photodiode array detector is used to detect and collect single peaks in the wavelength range of 200-600nm, and three obvious separated peaks are detected at a wavelength of 280nm, and the substances corresponding to the separated peaks eluted within 8.6-10.2min are PeME-F3.

[0012] In the third aspect, the present application provides the use of the above-mentioned methanol phase extract PeME of the fruit of Phyllanthus emblica or the purified component PeME-F3 thereof as an antibacterial agent.

[0013] Further, the antibacterial object of the antibacterial agent is Vibrio cholerae and Staphylococcus aureus.

[0014] Further, the antibacterial agent exerts an antibacterial effect in the following aspects:

[0015] (a) inhibiting the growth and proliferation of Vibrio cholerae and Staphylococcus aureus;

[0016] (b) reducing the cell surface hydrophobicity, increasing the cell membrane fluidity and intracellular membrane permeability of Vibrio cholerae and Staphylococcus aureus;

[0017] (c) causing the cell structure damage of Vibrio cholerae and Staphylococcus aureus, and the leakage of intracellular nucleic acids and proteins;

[0018] (d) inhibiting the DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion metabolic pathways of Vibrio cholerae;

[0019] (e) inhibiting the cell wall, cell membrane and biofilm synthesis, protein synthesis, energy metabolism pathways of Staphylococcus aureus.

[0020] Further, the bacteriostatic agent can be used for the prevention and control of bacterial contamination of food, medicine or aquatic products, and can be used as an internal additive to prepare food, medicine or aquatic products, or as an external storage agent of food, medicine or aquatic products.

[0021] Further, the food is instant, fresh, refrigerated or frozen food.

[0022] Further, the dosage form of the medicine is liquid, powder, tablet, capsule, etc.

[0023] Further, the aquatic products include crucian carp and white shrimp, and the bacteriostatic agent has the effect of inhibiting the contamination of Vibrio cholerae and Staphylococcus aureus in the meat of crucian carp and white shrimp stored at low temperature (4℃).

[0024] The present application has the following beneficial effects:

[0025] 1. The present application provides a methanol phase extract of the fruit of Phyllanthus emblica, PeME, and a purified component PeME-F3 thereof, which have the effect of inhibiting Vibrio cholerae and Staphylococcus aureus.

[0026] 2. The present application also studies the bacteriostatic mechanism of the methanol phase extract of the fruit of Phyllanthus emblica, which is confirmed to be able to inhibit the growth and proliferation of Vibrio cholerae and Staphylococcus aureus; reduce the cell surface hydrophobicity, increase the cell membrane fluidity and intracellular membrane permeability, cause the leakage of intracellular nucleic acids and proteins, and cell structure damage; inhibit the DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion pathways of Vibrio cholerae; and inhibit the cell wall, cell membrane and biofilm synthesis, protein synthesis, energy metabolism pathways of Staphylococcus aureus; thereby causing cell death.

[0027] 3、The methanol phase extractive of the Phyllanthus emblica fruit in the application can be used for the preparation of food or medicine, and the prepared food or medicine also has the bacteriostatic effect.

[0028] 4、The methanol phase extractive of the Phyllanthus emblica fruit in the application can inhibit Vibrio cholerae and Staphylococcus aureus pollution in low-temperature stored crucian carp and South American white shrimp meat, and embodies the bacteriostatic effect in aquatic products. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is the bacteriostatic activity experimental results of PeME and PeME-F3 in the application. A: the experimental results of agar disc diffusion method; B: Prep-HPLC separation and purification of PeME; C: the experimental results of growth curve; D: the experimental results of time killing curve. C1 and D1, C2 and D2 are Vibrio cholerae GIM 1.449, Staphylococcus aureus ATCC 25923 respectively. *: p<0.05; **: p<0.01; ***: p<0.001.

[0030] Figure 2 It is the influence of PeME-F3 in the application on the exudation amount of CSH (A), CMF (B), ICMP (E), nucleic acid (C) and protein (D) of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923; wherein E includes E-1 and E-2, E-1 is the ICMP result graph of Vibrio cholerae GIM 1.449, and E-2 is the ICMP result graph of Staphylococcus aureus ATCC 25923. *: p<0.05; **: p<0.01; ***: p<0.001.

[0031] Figure 3 It is the cell surface structure change of Vibrio cholerae GIM 1.449 (A) and Staphylococcus aureus ATCC 259239 (B) before and after PeME-F3 treatment in the application.

[0032] Figure 4 It is the main metabolic pathway of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 caused by PeME-F3 in the application. It is the volcano graph of DGEs in Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 (A and B), and the main metabolic pathways enriched (C and D).

[0033] Figure 5 It is the experimental results of PeME-F3 in the application for inhibiting Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 pollution in crucian carp (A and B) and South American white shrimp (C and D) meat. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further illustrated below by examples. Obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0035] Note: The methods used in the examples are conventional methods, and the reagents not specifically mentioned are conventional reagents.

[0036] Example 1 Preparation of methanol phase extract of Phyllanthus emblica fruit and antibacterial test

[0037] (I) Preparation of methanol phase extract of Phyllanthus emblica fruit

[0038] Methanol-chloroform extraction method: Fresh Phyllanthus emblica fruit samples were washed with tap water, dried, cut into small pieces, and pre-frozen in a -80°C refrigerator for 2-4 h. Then, the samples were freeze-dried in a freeze dryer at -80°C for 48 h. The freeze-dried samples were pulverized using a multifunctional pulverizer, sieved using a 300-mesh sieve, and reserved for use.

[0039] 10 g of the above sample powder was weighed, 33 mL of methanol (analytical grade) and 66 mL of chloroform (analytical grade) (1:2, v / v) were added at a solid-liquid ratio of 1:10 (m / v), and vortex mixed using a magnetic stirrer for 5 h. Then, 60 mL of sterile water (analytical grade) was added, and vortex mixed for 2 h. The above mixture was ultrasonically treated using an ultrasonic cell disruptor for 20 min (300 W, ultrasonic probe diameter: 6 mm), filtered using a 20-25 μm microporous filter, and the filtrate was collected. After the methanol phase and the chloroform phase were separated, the PeME was obtained by evaporation and concentration using a rotary evaporator. The filtrate was extracted a second time using the above methanol-chloroform solvent, and the obtained PeME was combined and stored at 4°C in the dark for use.

[0040] After freeze-drying at -80°C for 48 h, the water loss rate of Phyllanthus emblica fruit was 87.9%, and the extraction rate of PeME was 41.0%.

[0041] (II) Antibacterial activity test of methanol phase extract of Phyllanthus emblica fruit

[0042] 1. Agar disc diffusion method

[0043] Test strains and culture conditions: Vibrio cholerae GIM1.449 strain (Table 1) was inoculated into sterilized Tryptic Soy Broth (TSB, pH 8.4-8.5, 3.0% NaCl) liquid medium at 1% volume fraction, and incubated at 37°C, 180 rpm for 16-18 h. After twice subculture activation, streaked on TSB agar plates and incubated at 37°C for 16-18 h. Single colonies were picked and inoculated into TSB liquid medium and incubated to mid-logarithmic growth phase (mid-LGP, OD 600 = 0.6-0.8) for determination of bacterial concentration by routine plate counting method.

[0044] Similarly, Staphylococcus aureus ATCC 29213 strain was inoculated into TSB medium (pH 7.0-7.2, 0.5% NaCl); Escherichia coli ATCC 25922 quality control strain was inoculated into Luria-Bertani (LB) medium (pH 7.0-7.2) and incubated at 37°C to mid-LGP (Table 1).

[0045] Table 1 Test strains and their culture media

[0046]

[0047] ATCC: American Type Culture Collection, USA; GCCC: Guangdong Culture Collection Center, China

[0048] The culture liquid (1×10 8 CFU / mL) of each test strain was uniformly coated on sterilized Mueller-Hinton Agar (M-HA) agar plates (100 μL / plate), and after the bacterial liquid was absorbed, sterile filter paper pieces (10 μL / piece, diameter: 6 mm) impregnated with PeME (100 mg / mL) were uniformly placed on the plates, and incubated at 37°C for 12 h. The diameter of inhibition zone (DIZ) was measured. Sterile water was used as negative control group (NC); gentamicin (Gentamicin, CN, 10 μg) test paper was used as positive control group (CN); Escherichia coli ATCC 25922 was used as quality control strain. Each group of tests was repeated three times. The results are shown in Table 2.

[0049] 2. Minimum inhibitory concentration method

[0050] In a sterilized 96-well bacterial culture plate, using an eight-channel pipetting gun, add sterilized M-H Broth (M-HB) liquid medium (100 μL / well). In the first column of wells, add PeME (100 μL / well, final concentration of 16.38 mg / mL), mix with the M-HB medium therein, then take 100 μL / well and add to the second column of wells, and so on, to the eleventh column of wells. Add sterile water (100 μL / well) to the twelfth column of wells as a negative control. Using an eight-channel pipetting gun, take the culture solution of the above-mentioned test strains (10 μL / well) and add to each well of the 96-well bacterial culture plate, and incubate at 37°C for 48 h. The minimum drug concentration contained in the culture well when the bacterial growth is completely inhibited is the minimum inhibitory concentration (MIC). The growth control well must have acceptable growth (≥2 mm of bottom coverage or clear turbidity). Each test is repeated three times. The results are shown in Table 2.

[0051] As can be seen from Table 2 and Figure 1 (A), PeME can inhibit the growth of Vibrio cholerae GIM1.449 and Staphylococcus aureus ATCC29213, and the observed DIZ values are 16.47±0.29 mm and 21.20±1.21 mm, respectively. In addition, the negative control has no inhibition zone; the DIZ values of the positive control are 18.0±1.41 mm and 18.25±1.06, respectively, indicating that PeME has similar antibacterial effect to the positive control antibiotic CN.

[0052] As can be seen from Table 2, the MIC values of PeME for inhibiting Vibrio cholerae GIM1.449 and Staphylococcus aureus are both 0.512 mg / mL.

[0053] Table 2 Experimental results of the antibacterial activity of the methanol phase extract of the Phyllanthus emblica fruit

[0054]

[0055] Example Two Separation and purification of the methanol phase extract of the Phyllanthus emblica fruit and antibacterial experiment of PeME-F3

[0056] Dilute PeME with ultrapure water (analytical grade) to a concentration of 10 mg / mL, centrifuge at 8000 x g for 20 min, collect the supernatant; then, filter through a sterile 0.22 μm filter membrane, collect the filtrate. Use a Waters 2707 (Waters, Milford, MA, USA) connected to a UPLC Sunfire C 18The filtrate samples were separated by a chromatographic column (5 μm, 10 x 250 mm, Waters, USA). The column temperature was 40 °C, and the injection volume was 100 μL. The mobile phase A was set as ultrapure water (analytical grade), and the mobile phase B was methanol (analytical grade) at a flow rate of 4 mL / min (isocratic elution: 0-15 min, 20% A, 80% B). The single peak was detected and collected by a photoelectric diode array detector at a wavelength range of 200-600 nm.

[0057] As shown in Figure 1 (B), three obvious separated peaks were detected at 280 nm wavelength within 14 min, namely Fraction 1-Fraction 3 (F1-F3), and Fraction 1-3 were collected respectively. The antibacterial activity of each fraction was analyzed by the above method.

[0058] As shown in Table 1 and Figure 1 (A), the DIZ values of PeME-F3 against Vibrio cholera GIM 1.449 and Staphylococcus aureus ATCC 25923 were 16.5 ± 0.41 mm and 15.80 ± 0.16, respectively, and the MIC values were 256 μg / mL and 512 μg / mL, respectively. Fraction 1 and Fraction 2 showed weak or no antibacterial activity.

[0059] Based on the above experimental results, the following examples used PeME-F3 to inhibit the growth of Vibrio cholera GIM 1.449 and Staphylococcus aureus ATCC 25923 at 1 x MIC values of 256 μg / mL and 512 μg / mL, respectively.

[0060] Example Three Growth and proliferation test of PeME-F3 against Vibrio cholera GIM 1.449 and Staphylococcus aureus ATCC 25923

[0061] (I) Growth curve analysis

[0062] Vibrio cholera GIM 1.449 and Staphylococcus aureus ATCC 25923 strains cultured to mid-LGP were inoculated into TSB medium added with PeME-F3 (1 x MIC or 1 / 2 x MIC) at a volume fraction of 1% respectively, and incubated at 37 °C, 180 rpm for 24 h. The growth curve was determined by using an automatic growth curve analyzer (Synergy, USA). TSB medium without PeME-F3 was used as a control, and the following analysis was similar.

[0063] As shown in Figure 1 (C-1), the maximum biomass (OD 600Compared to 1.09, under PeME-F3 treatments at concentrations of 1×MIC (256 μg / mL) and 1 / 2×MIC (128 μg / mL), the maximum biomass (OD) of Vibrio cholerae GIM was 1.449. 600 =0.652, OD 600 =0.788) decreased by 0.67 times and 0.77 times respectively (p<0.05).

[0064] Similarly, under the treatment conditions of 1×MIC (512 μg / mL) and 1 / 2×MIC (256 μg / mL) PeME-F3, the maximum biomass (OD) of Staphylococcus aureus ATCC 25923 was [not specified]. 600 =0.912, OD 600 =1.164) compared to the control group (OD 600 =1.521) decreased by 0.63 times and 0.77 times respectively (p<0.05) Figure 1 (C-2).

[0065] (II) Time-kill curve test

[0066] Vibrio cholerae GIM1.449 and Staphylococcus aureus ATCC25923 strains cultured to mid-LGP were inoculated at 1% (v / v) into TSB medium supplemented with PeME-F3 (1×MIC or 1 / 2×MIC) and cultured at 37°C and 180 rpm for 3 h, 6 h, 12 h, and 24 h, respectively. Using the standard plate counting method, the culture solutions at different time points were serially diluted, evenly spread on agar plates, and the colony counts (CFU / mL) were recorded.

[0067] like Figure 1 As shown in (D-1), compared with the control group with a PeME-F3 concentration of 0%, the viable count of Vibrio cholerae GIM 1.449 decreased by 1.84–4.27 log CFU / mL and 2.10–5.26 log CFU / mL, respectively, after treatment with PeME-F3 at concentrations of 1 / 2×MIC and 1×MIC for 3–24 h (p<0.05).

[0068] Similarly, after treatment with PeME-F3 at concentrations of 1 / 2×MIC and 1×MIC for 3–24 h, the viable count of Staphylococcus aureus ATCC25923 decreased by 1.82–4.27 log CFU / mL and 1.99–5.07 log CFU / mL, respectively (p<0.05). Figure 1 (D-2).

[0069] These experimental results show that Vibrio cholerae GIM 1.449 is more sensitive to PeME-F3 treatment, with a lower MIC value of inhibition (256 μg / mL); after 24 h of treatment at 1 x MIC, the maximum number of Vibrio cholerae was killed, with a decrease in the number of viable bacteria of 5.49 log CFU / mL (p < 0.05).

[0070] Example Four PeME-F3 alters key cell biophysical parameters of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0071] Cell Surface Hydrophobicity (CSH), Cell Membrane Fluidity (CMF) and Inner Cell Membrane Permeability (ICMP) are key biophysical parameters that maintain their cell structure and function, and are the main targets of antibacterial drugs.

[0072] CSH test: 1 mL of the bacterial suspension treated with PeME-F3 at a concentration of 1 x MIC for 2 h, 4 h and 6 h, respectively, was taken and mixed with 1 mL of hexadecane, then vortexed for 5 min, left to stand at room temperature for 30 min, and the supernatant was discarded. The OD 415 absorbance value was measured using a multifunctional microplate reader. Each test was repeated three times.

[0073] As can be seen from Figure 2 (A), compared with the control group with a PeME-F3 concentration of 0%, the CSH of Vibrio cholerae GIM 1.449 treated with PeME-F3 for 2 h, 4 h and 6 h decreased by 14.2%, 50.6% and 69.8%, respectively (p < 0.05), and showed a time-dependent decrease. Similarly, the CSH of Staphylococcus aureus ATCC 25923 decreased by 50.7%, 67.5% and 79.1%, respectively (p < 0.01).

[0074] CMF assay: The bacterial suspension treated with PeME-F3 at 1x MIC for 2h, 4h, 6h was added into sterilized 96-well bacterial culture plate (200 μL / well) respectively, and then 10 mM 1,6-Diphenyl-1,3,5-hexatrine (DPH) solution (2.0 μL / well) was added. The horizontal polarization (Ivh) and vertical polarization (Ivv) fluorescence of each well was detected by multifunctional microplate reader with 360 / 40 excitation filter, 460 / 40 detection filter and 400 nm dichroic mirror. The rDPH was calculated according to the following formula: rDPH=(Ivv-GxIvh) / (Ivv+2xGxIvh), where G=0.85. Each group of experiments was repeated three times.

[0075] As shown in Figure 2 (B), compared with the control group with 0% PeME-F3, the CMF of V. cholera GIM 1.449 and S. aureus ATCC 25923 treated with 1x MIC PeME-F3 for 2h increased significantly (1.73-fold, 1.56-fold) (p<0.05), and showed a time-dependent increase. After 6h treatment with 1x MIC PeME-F3, the CMF of V. cholera GIM 1.449 increased most significantly (3.17-fold) (p<0.05).

[0076] ICMP assay: The bacterial suspension treated with PeME-F3 at 1x MIC was added into sterilized 96-well bacterial culture plate (200 μL / well) respectively, and then 10 mM o-nitrophenyl-β-D-galactopyranoside (ONPG, 2.5 μL / well) was added. The OD 415 absorbance value was measured by multifunctional microplate reader every 30 min at 37°C. Each group of experiments was repeated three times.

[0077] As shown in Figure 2 (E), compared with the control group with 0% PeME-F3, the ICMP of V. cholera GIM 1.449 and S. aureus ATCC 25923 treated with 1x MIC PeME-F3 for 2-6h increased by 1.03-1.15-fold (Figure E-1) and 1.03-1.08-fold (Figure E-2) respectively, and showed a time-dependent increase trend.

[0078] These experimental results showed that PeME-F3 (1x MIC) could reduce the CSH of V. cholera GIM 1.449 and S. aureus ATCC 25923; at the same time, it could increase the ICMP and CMF of V. cholera GIM 1.449 and S. aureus ATCC 25923.

[0079] Example Five PeME-F3 causes leakage of intracellular nucleic acids and proteins of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0080] From the culture bacterial suspensions treated with PeME-F3 at 1 x MIC concentration for 2h, 4h, 6h, 1.5mL of each was taken and centrifuged at 3500rpm for 5min at 4°C. The supernatant was collected and the OD was measured using a multifunctional microplate reader. 260 Absorbance values. After 24h of treatment, the concentration of extracellular proteins in the supernatant samples was determined using the Bradford method protein concentration determination kit, according to the kit instructions. Each test was repeated three times.

[0081] As shown in Figure 2 (C), compared with the control group with PeME-F3 concentration of 0%, after 2h, 4h, 6h of treatment with PeME-F3, the extracellular nucleic acid leakage of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 increased by 1.65-2.38 times and 1.20-1.54 times (p<0.01), respectively.

[0082] As shown in Figure 2 (D), compared with the control group, after 24h of treatment with PeME-F3, the extracellular protein content of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 increased by 1.40 times and 2.16 times (p<0.001), respectively.

[0083] Example Six Scanning Electron Microscopy Observation of PeME-F3 Damaging the Cell Structure of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0084] The culture bacterial suspensions treated with PeME-F3 at 1 x MIC concentration for 2h, 4h, 6h were centrifuged at 4000rpm for 10min at 4°C, the supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was washed twice with sterilized 1 x PBS buffer (pH 7.2-7.4). Then, the cells were fixed with 2.5% glutaraldehyde at 4°C for 12h, and then dehydrated with gradient ethanol (30%, 50%, 80%, 90%, 100%) for 15min. After that, 20μL of the sample was dropped onto a cover glass and dried at room temperature. The cell morphology of the test strains was observed using a scanning electron microscope (SEM, 5.0kV, 30,000x). Each test was repeated three times.

[0085] As shown in Figure 3(A) As can be seen, in the control group with 0% PeME-F3 concentration, the cells of V. cholerae GIM 1.449 presented rod-shaped arc shape, full shape, flat surface and complete structure. In contrast, after 4h treatment with 1xMIC of PeME-F3, the cell surface shrunk, with obvious cracks and depressions; after 6h treatment, the cell structure was severely damaged, with intracellular material leakage and cell rupture.

[0086] Similarly, in the control group with 0% PeME-F3 concentration, the cells of S. aureus ATCC 25923 presented spherical shape, full shape, flat surface and complete structure. In contrast, after 4h treatment with 1xMIC of PeME-F3, the cell surface significantly shrunk and invaginated, with partial cell deformation and mutual adhesion; after 6h treatment, the cells ruptured, with massive content leakage and large-area cell adhesion Figure 3 , B).

[0087] Example Seven PeME-F3 alters multiple metabolic pathways of V. cholerae GIM 1.449 and S. aureus ATCC 25923

[0088] Illumina RNA sequencing test: In the culture bacterial suspension treated with 1xMIC of PeME-F3 for 4h as described above, RNAase inhibitor was added, the bacterial cells were collected by centrifugation, and total RNA of the bacterial cell samples was extracted using an RNA extraction kit according to the kit instructions. The purification, analysis and Illumina RNA sequencing of the RNA samples were completed by Shanghai Meiji Biomedicine Technology Co., Ltd. (China, Shanghai), using Illumina HiSeq2500 platform (Illumina, USA). Each test was repeated three times. Compared with the control group, the genes with Fold change (FC) ≥ 2.0 or ≤ 0.5, and Benjamini / Hochberg (BH corrected FDR) p<0.05 were defined as Differentially Expressed Genes (DEGs). Gene Set Rnrichment Analysis (GSEA) was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / KEGG / ).

[0089] (I) Changes in metabolic pathways of V. cholerae GIM 1.449

[0090] Comparative transcriptome analysis showed that, compared with the control group, the identified DEGs accounted for 61.8% (n=2320 / 3753) of the total number of Vibrio cholerae GIM 1.449 genes in the PeME-F3 treatment group. Among them, 2195 DEGs were significantly down-regulated at the transcription level (FC≤0.5), and 125 DEGs were significantly up-regulated (FC≥2.0). These DEGs were mainly enriched in 12 metabolic pathways, including Mismatch Repair (MMR), Base Excision Repair, Homologous Recombination, DNA Replication, Peptidoglycan Biosynthesis, Aminoacyl-tRNA Biosynthesis, Protein Transport, Bacterial Secretion System, Glycerophospholipid Metabolism, etc. Figure 4

[0091] For example, in MMR, about 19 DEGs were significantly inhibited at the transcription level (0.182-0.475-fold) (p<0.05). For example, single-stranded DNA binding protein (SSBP, GTH07_11935) was significantly down-regulated (0.227-fold, p<0.05). Single-stranded DNA exists transiently during DNA replication and gene transcription, and SSBP can protect it from damage to maintain the stability of the genome.

[0092] In the Base Excision Repair, Homologous Recombination, and Folate Biosynthesis pathways, about 55 DEGs were significantly down-regulated in Vibrio cholerae GIM 1.449 (0.101-0.493-fold, p<0.05). For example, RecA (GTH07_11315), a core enzyme for bacterial homologous recombination, was significantly inhibited at the transcription level (0.297-fold, p<0.05). RecA plays an important role in DNA repair, transformation, and SOS response activation. Uracil-DNA glycosylase (GTH07_02650) was also significantly down-regulated (0.192-fold, p<0.05). This enzyme catalyzes the first step of the Base Excision Repair pathway and plays an important role in repairing DNA damage and maintaining genome integrity.

[0093] In the Peptidoglycan Biosynthesis pathway, about 18 DEGs were significantly inhibited (0.147-0.439-fold, p<0.05). For example, undecaprenyl pyrophosphate synthase (UppS, GTH07_03105) was significantly down-regulated (0.283-fold, p<0.05). UppS is a key enzyme in bacterial cell wall biosynthesis and an important target for antibacterial agents.

[0094] ​In glycerophospholipid metabolism, about 18 DEGs were significantly inhibited (0.063-0.423 fold, p<0.05) in V. cholera GIM 1.449. For example, the expression of glycerol-3-phosphate dehydrogenase (GlpD, GTH07_16520) was strongly inhibited at the transcriptional level (0.063 fold, p<0.05). GlpD is a virulence factor in many pathogenic bacteria and is considered a potential drug target.

[0095] In aminoacyl-tRNA biosynthesis, about 24 DEGs were significantly inhibited (0.139-0.466 fold, p<0.05). For example, aminoacyl-tRNA synthetases (AARS, PQQ 26_08455, PQQ26_08220, PQQ26_09780, PQQ26_02235, etc.) were significantly inhibited (0.209-0.343 fold, p<0.05). AARS provides ribosomes with amino acids required for protein biosynthesis.

[0096] In bacterial secretion systems, about 32 DEGs were significantly down-regulated in V. cholera GIM 1.449 (0.027-0.481 fold, p<0.05). For example, Type II Secretion System (T2SS) protein GspC (GTH07_00830) was significantly down-regulated (0.301 fold, p<0.05). Gram-negative bacteria secrete enzymes and toxins through T2SS. Type VI Secretion System (T6SS) protein Hcp (GTH07_07215) was strongly inhibited (0.027 fold, p<0.05). Hcp is a core structure and effector protein of T6SS and has been shown to be a key factor in bacterial competition, invasion, adhesion, and virulence.

[0097] In summary, PeME-F3 can inhibit the DNA replication, mismatch repair, and homologous recombination pathways of Gram-negative V. cholera GIM 1.449, disrupting the stability of its genome; inhibit the peptidoglycan and glycerol ester synthesis pathways, and suppress the synthesis of cell walls, cell membranes, and biofilms; inhibit protein synthesis, secretion, and export pathways, reduce the pumping out of harmful substances, bacterial pathogenicity, and drug resistance; and further lead to cell disintegration and death.

[0098] (B) Changes in metabolic pathways of Staphylococcus aureus ATCC 25923

[0099] Comparative transcriptome analysis results showed that 63.9% (1696 / 2656) of the total number of S. aureus ATCC 25923 genes were identified as DEGs in the PeME-F3 treatment group compared to the control group. Among them, 1590 DEGs were significantly down-regulated at the transcriptional level (FC≤0.5), and 106 DEGs were significantly up-regulated (FC≥2.0). These DEGs were enriched in 11 metabolic pathways of S. aureus ATCC 25923, such as peptidoglycan biosynthesis, fatty acid biosynthesis, TCA, pentose phosphate pathway, glycolysis, aminoacyl-tRNA biosynthesis, pyrimidine metabolism, valine, leucine and isoleucine biosynthesis, etc. (p<0.05) Figure 4

[0100] For example, in the peptidoglycan biosynthesis, about 19 DEGs were significantly down-regulated in S. aureus ATCC 25923 (0.060-0.388-fold, p<0.05). For example, the cap8 DEFGOP gene cluster (PQQ26_00500, PQQ26_00505, PQQ26_00510, PQQ26_00515, PQQ26_00555, PQQ26_00560) encoding the capsular polysaccharide Cap8 was significantly inhibited (0.117-0.282-fold, p<0.05). Cap8 is essential for bacterial peptidoglycan synthesis, polymerization, and capsular polysaccharide transport, and plays an important role in bacterial virulence, immune evasion, and pathogen-host interaction. In addition, UDP-N-acetylglucosamine enolpyruvate phosphate transferase MurA (PQQ26_10550, 0.129-fold), glutamine-fructose-6-phosphate transaminase GlmM (PQ26_10845, 0.186-fold), UTP-glucose-1-phosphate uridylyltransferase GalU (PQQ26_12610, 0.217-fold) and other enzymes involved in cell wall and capsular polysaccharide biosynthesis were also significantly inhibited (p<0.05).

[0101] In the fatty acid biosynthesis pathway, about 14 DEGs were significantly down-regulated (0.164-0.464-fold, p<0.05), for example, acetyl-CoA carboxylase (ACC) (accABCD, PQQ26_08540, PQQ26_07710, PQQ26_07705, PQQ26_08545). ACC is the rate-limiting enzyme for the first step of fatty acid synthesis, which is essential for the synthesis and maintenance of the cell membrane.

[0102] ​In the biosynthesis of phenylalanine, tyrosine and tryptophan, about 18 DEGs were significantly inhibited at the transcriptional level (0.063-0.358 fold, p<0.05). For example, chorismate synthase AroC (PQQ26_07035) was significantly inhibited (0.317 fold, p<0.05). AroC is a key enzyme in the biosynthesis of tryptophan, involved in the formation of bacterial biofilm and virulence. The expression of 3-phosphoshikimate 1-carboxyvinyltransferase AroA (PQQ26_07025) and type I 3-dehydroquinate dehydratase AroD (PQQ26_03930) was also inhibited (0.23 fold and 0.195 fold, p<0.05). It was reported that aroA and aroD gene deletion affects the production of capsular polysaccharide and pili, inhibits the synthesis of Salmonella enterica biofilm. In addition, the expression of prephenate dehydratase PheA2 (PQQ26_09865) was strongly inhibited (0.063 fold). PheA2 is a key enzyme for the synthesis of L-phenylalanine through the shikimate pathway, involved in the biosynthesis of cell wall / membrane / coating.

[0103] In TCA, about 11 DEGs were significantly inhibited in Staphylococcus aureus ATCC 25923 (0.138-0.491 fold, p<0.05), for example, pyruvate dehydrogenase PdhA (PQQ26_05035), dihydroxyacyl dehydrogenase LpdA (PQQ26_05050), pyruvate carboxylase Pyc (PQQ26_05040) and L-lactate dehydrogenase Lqo (PQQ26_13150), etc. Among them, PdhA catalyzes the conversion of pyruvate to acetyl-CoA, which is a key reaction for entering TCA.

[0104] In summary, PeME-F3 can significantly inhibit the synthesis of peptidoglycan, fatty acid, amino acid and energy metabolism pathway of Gram-positive Staphylococcus aureus ATCC 25923, thereby blocking the formation of its cell wall, cell membrane and biofilm, destroying its cell structure, inhibiting its growth and proliferation, leading to cell death.

[0105] Example Eight Test of PeME-F3 inhibiting Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 contamination in the meat of crucian carp and South American white shrimp

[0106] Collection and analysis of samples: Fresh crucian carp and white prawn samples were collected from a seafood market in Shanghai, China, and transported to the laboratory in ice boxes. The fish and prawn samples were washed with running tap water. Under sterile conditions, the fish skin and prawn shell were removed, and the fish and prawn meat was cut into small pieces (about 1 x 1 x 1 cm) using a sterilized scalpel. The pieces were then washed three times with sterile water and sterilized under ultraviolet light for 20 min. In each 1 g of fish or prawn meat sample, 9 mL of sterilized 1x PBS buffer was added, and homogenized for 3 min. 100 μL of the homogenate was spread on agar plates, which were incubated at 37°C overnight, and the number of colonies was counted. Each test was repeated three times. If no colony growth was observed, the sample could be used for subsequent tests.

[0107] Preparation of artificially contaminated samples and grouping tests: As described above, Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 were inoculated into TSB medium and incubated at 37°C until mid-LGP. Then, they were diluted to 1 x 10 6 CFU / mL with 1x PBS and added to the homogenized fish and prawn meat samples described above. The negative control group was inoculated with the test strains without the addition of PeME-F3. The treatment group was inoculated with the test strains and added with 1x MIC concentration of PeME-F3 (512 μg / mL). The positive control group was inoculated with the test strains and added with deoxytetracycline (10 μg / mL). The three groups of test samples were stored at 4°C for 0 h, 6 h, 12 h, and 24 h, respectively. Then, each group of test samples was transferred to a sterile homogenization bag, 9 mL of 1x PBS was added, and homogenized for 3 min. The homogenized samples were serially diluted, and 100 μL of each dilution was spread on agar plates, which were incubated at 37°C for 12 h, and the number of colonies was counted. Each test was repeated three times. The inhibition rate (%) = (Ba - Bt) / Ba x 100%. Wherein, Ba is the number of viable bacteria (CFU / mL) of the test strains in the negative control group; Bt is the number of viable bacteria (CFU / mL) of the test strains in the treatment group.

[0108] As shown in Figs. Figure 5 (A and B), compared with the control group without the addition of PeME-F3, the number of viable Vibrio cholerae GIM 1.449 in the crucian carp samples was reduced by 47.57%, 73.89%, and 80.28% (p < 0.01) after 6 h, 12 h, and 24 h of treatment with 1x MIC concentration of PeME-F3, respectively. Similarly, the number of viable Staphylococcus aureus ATCC 25923 in the crucian carp samples was reduced by 63.95%, 77.93%, and 93.0% (p < 0.01), respectively.

[0109] As shown in Figs. Figure 5(C and D) showed that compared with the control group without adding PeME-F3, the viable cell count of Vibrio cholera GIM 1.449 in the Penaeus vannamei sample was reduced by 79.16%, 86.19% and 88.41% (p < 0.01) after 6h, 12h and 24h treatment with 1x MIC concentration of PeME-F3. Similarly, the viable cell count of Staphylococcus aureus ATCC 25923 in the Penaeus vannamei sample was reduced by 74.76%, 86.78% and 91.58% (p < 0.01), respectively.

[0110] These experimental results show that under low temperature (4℃) storage conditions, PeME-F3 can effectively inhibit the contamination of Vibrio cholera GIM 1.449 and Staphylococcus aureus ATCC 25923 in the meat of Carassius auratus and Penaeus vannamei, and the antibacterial effect is comparable to that of the positive control antibiotic terramycin.

[0111] In summary, the methanol phase extract of the fruit of Phyllanthus emblica provided by the present application can inhibit the growth and proliferation of Vibrio cholera and Staphylococcus aureus; reduce the cell surface hydrophobicity, increase the cell membrane fluidity and intracellular membrane permeability, leading to cell structure damage and leakage of intracellular nucleic acids and proteins; suppress the DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion pathways of Vibrio cholera; suppress the cell wall, cell membrane and biofilm synthesis, protein synthesis, energy metabolism, etc. of Staphylococcus aureus; and also has the effect of inhibiting the contamination of Vibrio cholera and Staphylococcus aureus in the meat of Carassius auratus and Penaeus vannamei stored at low temperature (4℃). The methanol phase extract of the fruit of Phyllanthus emblica can also be used for the preparation of food or medicine, and the prepared food or medicine also has antibacterial effect.

[0112] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A methanol-phase extract (PeME) of Phyllanthus emblica fruit, characterized in that, It was extracted from Phyllanthus emblica fruit by methanol-chloroform solvent extraction, and the specific steps are as follows: S1. Rinse the fresh amla fruits, air dry them, cut them into pieces, and pre-freeze them at -80°C for 2–4 h; then freeze-dry them at -80°C for 48 h; pulverize the freeze-dried samples and sieve them through a 300-mesh sieve; S2. Add methanol and chloroform to the sieved sample powder at a material-to-liquid ratio of 1:10 and mix thoroughly, wherein the volume ratio of methanol to chloroform is 1:2; then add sterile water and mix thoroughly, wherein the mass-to-volume ratio of sample powder to sterile water is 1:6; sonicate the mixture, then filter it using a 20–25 μm microporous membrane and collect the filtrate; after separating the methanol phase and the chloroform phase, evaporate and concentrate them using a rotary evaporator to obtain PeME; use the above methanol and chloroform solvents to perform a second extraction on the filtrate, and combine the results to obtain PeME.

2. A purified fraction PeME-F3, a methanol-phase extract of Phyllanthus emblica fruit, characterized in that, The PeME was obtained by separation and purification according to claim 1, and the separation and purification method is as follows: PeME was diluted with ultrapure water, centrifuged, and the supernatant was collected. The supernatant was then filtered through a sterile 0.22 μm filter membrane, and the filtrate was collected. A Waters 2707 was used to connect the UPLC Sunfire C100. 18 The filtrate sample was separated by chromatographic column; a photodiode array detector was used to detect and collect single peaks in the wavelength range of 200–600 nm. Three distinct separation peaks were detected at a wavelength of 280 nm. The substance corresponding to the separation peak within 8.6–10.2 min of elution was PeME-F3.

3. The application of the methanol phase extract PeME of Phyllanthus emblica fruit according to claim 1 or the purified component PeME-F3 of the methanol phase extract of Phyllanthus emblica fruit according to claim 2 in the preparation of an antibacterial agent, characterized in that, The antibacterial agent targets Vibrio cholerae and Staphylococcus aureus.

4. The application according to claim 3, characterized in that, The antibacterial agent is used for the prevention and control of bacterial contamination in food, medicine, or aquatic products.

5. The application according to claim 4, characterized in that, The food mentioned is ready-to-eat, fresh, refrigerated, or frozen.

6. The application according to claim 4, characterized in that, The dosage form of the medicine is liquid, powder, tablet or capsule.

7. The application according to claim 4, characterized in that, The aquatic products include crucian carp and whiteleg shrimp, and the antibacterial agent has the effect of inhibiting the contamination of Vibrio cholerae and Staphylococcus aureus in crucian carp and whiteleg shrimp meat stored at low temperature.

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