Methanol phase extract of fruits of phyllanthus emblica and application of methanol phase extract

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a methanol phase extract of fruits of phyllanthus emblica and application of the methanol phase extract. According to the invention, a methanol-chloroform extraction method is adopted to extract a methanol phase extract PeME from emblic leafflower fruit, and the methanol phase extract PeME is further separated and purified to obtain a purified component PeME-F3. The methanol phase extract PeME and the purified component PeME-F3 of the methanol phase extract PeME can inhibit vibrio cholerae and staphylococcus aureus, and can be used as bacteriostatic agents. The methanol phase extract of the emblic leafflower fruit can inhibit vibrio cholerae and staphylococcus aureus pollution in meat of crucian and penaeus vannamei which are stored at low temperature, and can be applied to aquatic products; the method can also be used for preparing food or medicines, and the prepared food or medicines also have an antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a methanol-phase extract from the fruit of Phyllanthus emblica and its application. Background Art

[0002] Infectious diseases caused by pathogens pose a serious threat to public health and human health and have become the second leading cause of death globally. According to a report by the World Health Organization (WHO), approximately 600 million people worldwide contract diseases each year from consuming food contaminated with foodborne pathogens, resulting in approximately 420,000 deaths. Among them, Vibrio cholerae is the pathogen that causes the infectious "cholera" disease of type A. The bacterium is transmitted from environmental hosts to human hosts through contaminated water or food, triggering the life-threatening "cholera" disease. Clinical symptoms mainly include gastric ulcers, acute diarrhea, severe vomiting, severe dehydration, and even death. Staphylococcus aureus is one of the pathogens that cause high morbidity and mortality. It is transmitted through contact with contaminants and person-to-person contact, and can cause skin infections, as well as fatal pneumonia and sepsis.

[0003] Antibiotics play a crucial role in the treatment of infectious diseases. However, the inappropriate use of antibiotics has led to the emergence of antibiotic resistance in pathogenic bacteria, resulting in approximately 700,000 deaths globally each year. The long-term overuse of antibiotics has also caused food safety risks and environmental pollution problems. Research shows that most current epidemic strains of Vibrio cholerae are multi-drug resistant, while clinical Staphylococcus aureus isolates are mostly resistant to methicillin and vancomycin (MRSA and VRSA), leading to higher case fatality rates, more secondary infections, and higher medical costs. Therefore, it is extremely important to screen and prepare new antibacterial agents.

[0004] Medicinal and edible plants have become a green 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 commonly used as a traditional Chinese medicine 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 also 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 at home and abroad on the antibacterial effect and application of the methanol-phase extract from the fruit of Phyllanthus emblica (PeME). Summary of the Invention

[0005] The object of the present invention is 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 object, the specific technical solutions adopted by the present invention are as follows:

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

[0008] S1. Rinse the fresh fruit of Phyllanthus emblica, cut it into pieces after drying, and pre-freeze it at -80°C for 2–4 h; then freeze-dry it at -80°C for 48 h; crush the freeze-dried sample and sieve it through a 300-mesh sieve;

[0009] S2. Add methanol and chloroform (1:2, v / v) to the sieved sample powder according to a solid-liquid ratio of 1:10 (m / v) and mix evenly; then add sterile water and mix evenly, controlling the ratio of the sample powder to sterile water at 1:6 (m / v); ultrasonically treat the mixture, and then filter it through a 20–25 μm microporous filter membrane to collect the filtrate; after separating the methanol phase and the chloroform phase, use a rotary evaporator for evaporation and concentration to obtain PeME; use the above methanol and chloroform solvents to extract the filtrate for the second time, and combine the obtained PeME.

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

[0011] Dilute PeME with ultrapure water, centrifuge and collect the supernatant, and then filter it through a sterile 0.22 μm filter membrane to collect the filtrate; connect Waters 2707 to UPLC Sunfire C 18 Separate the filtrate sample with a chromatographic column; use a photodiode array detector to detect and collect single peaks in the wavelength range of 200–600 nm. At a wavelength of 280 nm, three obvious separated peaks are detected, and the substance corresponding to the separated peak eluted within 8.6–10.2 min is PeME-F3.

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

[0013] Furthermore, the antibacterial objects of the antibacterial agent are Vibrio cholerae and Staphylococcus aureus.

[0014] Furthermore, the antibacterial agent exerts antibacterial effects in the following aspects:

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

[0016] (b) Reduce the cell surface hydrophobicity of Vibrio cholerae and Staphylococcus aureus, increase the cell membrane fluidity and the permeability of the inner cell membrane;

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

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

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

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

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

[0022] Furthermore, the dosage form of the drug is liquid, powder, tablet, capsule, etc.

[0023] Furthermore, 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°C).

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

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

[0026] 2. The present invention also conducts a study on the bacteriostatic mechanism of the methanol-phase extract of Phyllanthus emblica fruits, and confirms that it can inhibit the growth and proliferation of Vibrio cholerae and Staphylococcus aureus; reduce their cell surface hydrophobicity, increase the cell membrane fluidity and the permeability of the inner cell membrane, cause leakage of intracellular nucleic acids and proteins, and damage to the cell structure; inhibit the pathways of DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion, etc. of Vibrio cholerae; inhibit the pathways of cell wall, cell membrane and biofilm synthesis, protein synthesis, energy metabolism, etc. of Staphylococcus aureus; thereby causing cell death.

[0027] 3. The methanol-phase extract of Phyllanthus emblica fruits of the present invention can be used for the preparation of foods or drugs, and the prepared foods or drugs also have antibacterial effects.

[0028] 4. The methanol-phase extract of Phyllanthus emblica fruits of the present invention can inhibit the contamination of Vibrio cholerae and Staphylococcus aureus in crucian carp and white shrimp meat stored at low temperature, reflecting its antibacterial effect in aquatic products. Description of the Drawings

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

[0030] Figure 2 The effects of PeME-F3 in the present invention on the exudation amounts of CSH (A), CMF (B), ICMP (E), nucleic acid (C) and protein (D) of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923; where E includes E-1 and E-2, E-1 is the ICMP result diagram of Vibrio cholerae GIM 1.449, and E-2 is the ICMP result diagram of Staphylococcus aureus ATCC 25923. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0031] Figure 3 The changes in the cell surface structures of Vibrio cholerae GIM 1.449 (A) and Staphylococcus aureus ATCC 25923 (B) before and after treatment with PeME-F3 in the present invention.

[0032] Figure 4 The main metabolic pathways changed by PeME-F3 in the present invention in Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923. They are the volcano plots (A and B) of DGEs in Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923, and their enriched main metabolic pathways (C and D).

[0033] Figure 5 The experimental results of PeME-F3 in the present invention inhibiting the contamination of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 in crucian carp (A and B) and white shrimp (C and D) meat. Detailed Embodiments

[0034] The technical solution of the present invention will be further described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work fall within the scope protected by the present invention.

[0035] Note: The methods used in the embodiments are all conventional methods unless otherwise specified, and the reagents are all conventional reagents unless otherwise specified.

[0036] Example 1 Preparation and antibacterial test of methanol-phase extract PeME of Phyllanthus emblica fruits

[0037] (1) Preparation of methanol-phase extract of Phyllanthus emblica fruits

[0038] Methanol-chloroform extraction method: Wash the fresh Phyllanthus emblica fruit samples with tap water, cut them into small pieces after drying, and place them in a -80 °C refrigerator for pre-freezing for 2 - 4 h. Then, place them in a freeze dryer and freeze-dry at -80 °C for 48 h. Crush the freeze-dried samples with a multi-functional crusher and sieve them through a 300-mesh sieve for standby.

[0039] Weigh 10 g of the above sample powder, add 33 mL of methanol (analytical grade) and 66 mL of chloroform (analytical grade) (1:2, v / v) at a solid-liquid ratio of 1:10 (m / v), and vortex mix with a magnetic stirrer for 5 h. Then, add 60 mL of sterile water (analytical grade) and vortex mix for 2 h. Ultrasonically treat the above mixture with an ultrasonic cell disruptor for 20 min (300 W, ultrasonic probe diameter: 6 mm), filter it with a 20 - 25 μm microporous filter membrane, and collect the filtrate. After separating the methanol phase and the chloroform phase, use a rotary evaporator for evaporation and concentration to obtain PeME. Use the above methanol-chloroform solvent to extract the filtrate for the second time, combine the obtained PeME, and store it in the dark at 4 °C for standby.

[0040] Under the condition of -80 °C, after freeze-drying for 48 h, the water loss rate of Phyllanthus emblica fruits is 87.9%, and the extraction rate of PeME is 41.0%.

[0041] (2) Antibacterial activity test of methanol-phase extract of Phyllanthus emblica fruits

[0042] 1. Agar disc diffusion method

[0043] Test strains and culture conditions: Vibrio cholerae strain GIM1.449 (Table 1) was inoculated into sterilized Tryptic Soy Broth (TSB, pH 8.4–8.5, 3.0% NaCl) liquid medium at a volume fraction of 1% respectively, and cultured at 37 °C with 180 rpm for 16–18 h. After two passages of activation, it was streaked on a TSB agar plate and cultured at 37 °C for 16–18 h. A single colony was picked and inoculated into TSB liquid medium, and cultured until the mid-logarithmic growth phase (mid-LGP, OD 600 = 0.6–0.8), and the bacterial concentration was determined by the conventional plate counting method for standby use.

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

[0045] Table 1 Test strains and their media

[0046]

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

[0048] The culture solutions of the above test strains (1×10 8 CFU / mL) were respectively evenly spread on sterilized Mueller-Hinton Agar (M-HA) agar plates (100 μL / plate). After the bacterial solution was absorbed, sterile filter paper disks (10 μL / disk, diameter: 6 mm) impregnated with PeME (100 mg / mL) were evenly placed on them, and cultured at 37 °C for 12 h, and the diameter of the inhibition zone (DIZ) was measured. Sterile water was used as the negative control group (NC); a gentamicin (CN, 10 μg) test paper disk was used as the positive control group (CN); Escherichia coli ATCC 25922 was used as the quality control strain. Each group of experiments was repeated three times. The results are shown in Table 2 below.

[0049] 2. Minimum inhibitory concentration method

[0050] In a sterilized 96-well bacterial culture plate, using an eight-channel pipette, add sterilized M-H Broth (M-HB) liquid medium (100 μL / well). In the wells of the first column, add PeME (100 μL / well, final concentration 16.38 mg / mL). After mixing it with the M-HB medium therein, aspirate 100 μL / well and add it to the wells of the second column, and so on, adding up to the wells of the 11th column. Add sterile water (100 μL / well) to the wells of the 12th column as a negative control. Using an eight-channel pipette, aspirate the culture solution of the above test strains (10 μL / well) and add it to each well of the 96-well bacterial culture plate. Incubate at 37 °C for 48 h. The lowest drug concentration when bacterial growth is completely inhibited in the culture well is its minimum inhibitory concentration (MIC). The growth control wells must show acceptable growth (≥2 mm bottom covered or distinct turbidity). Each group of experiments is repeated three times. The results are shown in Table 2.

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

[0052] As shown in Table 2, the MIC values of PeME against Vibrio cholerae GIM1.449 and Staphylococcus aureus are both 0.512 mg / mL.

[0053] Table 2 Antibacterial activity experimental results of the methanol extract of Phyllanthus emblica fruits

[0054]

[0055] Example 2 Separation and purification of the methanol extract of Phyllanthus emblica fruits and antibacterial experiment of PeME-F3

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

[0057] As Figure 1 (B) shows that three distinct separated peaks were detected by scanning at 280 nm for 14 min, namely components 1–3 (Fraction 3–Fraction 3, F1–F3), and components 1–3 were collected separately. Using the above method, the antibacterial activities of each component were analyzed.

[0058] As shown in Table 1 and Figure 1 (A), the DIZ values of component 3 of PeME (PeME-F3) against Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 were 16.5±0.41 mm and 15.80±0.16, respectively; the MIC values were 256 μg / mL and 512 μg / mL, respectively. Components 1 and 2 showed weak antibacterial activity or no antibacterial activity.

[0059] Based on the above test results, the 1×MIC values of PeME-F3 against Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 used in the following examples were 256 μg / mL and 512 μg / mL, respectively.

[0060] Example 3 Growth and proliferation inhibition tests of PeME-F3 against Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0061] (I) Growth curve analysis

[0062] Vibrio cholerae GIM1.449 and Staphylococcus aureus ATCC25923 strains cultured to mid-LGP were inoculated into TSB medium supplemented with PeME-F3 (1×MIC or 1 / 2×MIC) at a volume fraction of 1% respectively, and cultured at 37 °C and 180 rpm for 24 h. An automatic growth curve analyzer (Synergy, USA) was used to measure their growth curves. TSB medium without added PeME-F3 was used as a control, and the following analysis was similar.

[0063] As Figure 1 (C-1) shows that compared with the maximum biomass (OD 600Compared with the control group (OD 600 =1.09), under the treatment conditions of PeME-F3 at concentrations of 1×MIC (256 μg / mL) and 1 / 2×MIC (128 μg / mL), the maximum biomass (OD 600 =0.652, OD 600 =0.788) of Vibrio cholerae GIM 1.449 decreased by 0.67-fold and 0.77-fold, respectively (p<0.05). 600 = 0.652, OD 600 = 0.788) decreased by 0.67-fold and 0.77-fold, respectively (p<0.05).

[0064] Similarly, under the treatment conditions of PeME-F3 at 1×MIC (512 μg / mL) and 1 / 2×MIC (256 μg / mL), the maximum biomass (OD 600 =0.912, OD 600 =1.164) of Staphylococcus aureus ATCC 25923 was 0.63-fold and 0.77-fold lower than that of the control group (OD 600 =1.521), respectively (p<0.05) ( 600 = 0.912, OD 600 = 1.164) was 0.63-fold and 0.77-fold lower than that of the control group (OD 600 =1.521), respectively (p<0.05) ( 600 = 1.521) decreased by 0.63-fold and 0.77-fold, respectively (p<0.05) ( Figure 1 , C-2).

[0065] (II) Time-kill curve assay

[0066] Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 strains cultured to mid-LGP were inoculated into TSB medium supplemented with PeME-F3 (1×MIC or 1 / 2×MIC) at a volume fraction of 1% and cultured at 37°C with 180 rpm for 3 h, 6 h, 12 h, and 24 h. Using the conventional plate counting method, the culture solutions at different time points were serially diluted and evenly spread on agar plates, and the colony counts (CFU / mL) were recorded.

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

[0068] Similarly, after treatment with PeME-F3 at 1 / 2×MIC and 1×MIC concentrations for 3–24 h, the viable counts of Staphylococcus aureus ATCC 25923 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 indicate that Vibrio cholerae GIM 1.449 is more sensitive to the treatment with PeME-F3, and its inhibited MIC value is lower (256 μg / mL); after treatment with 1×MIC for 24 h, the most Vibrio cholerae are killed, and the viable cell count decreases by 5.49 log CFU / mL (p<0.05).

[0070] Example 4 PeME-F3 Alters Key Cellular Biophysical Parameters of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0071] Bacterial cell surface hydrophobicity (CSH), cell membrane fluidity (CMF), and inner cell membrane permeability (ICMP) are key biophysical parameters for maintaining their cell structure and function, and are also the main targets of antibacterial drugs.

[0072] CSH test: In the cultured bacterial suspensions treated with PeME-F3 at the above 1×MIC concentration for 2 h, 4 h, and 6 h, 1 mL was taken respectively, 1 mL of hexadecane was added, vortexed and mixed for 5 min, left standing 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 Figure 2 (A) shows that compared with the control group with a PeME-F3 concentration of 0%, after treatment with PeME-F3 for 2 h, 4 h, and 6 h, the CSH of Vibrio cholerae GIM 1.449 decreased significantly by 14.2%, 50.6%, and 69.8% respectively (p<0.05), and showed a time-dependent decrease with the treatment time. 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 culture bacterial suspensions treated with PeME-F3 at the above-mentioned 1×MIC concentration for 2 h, 4 h, and 6 h were respectively added into sterilized 96-well bacterial culture plates (200 μL / well), and then 10 mM 1,6-diphenyl-1,3,5-hexatriene (DPH) solution (2.0 μL / well) was added. Using a multifunctional microplate reader, with an excitation filter of 360 / 40, a detection filter of 460 / 40, and a dichroic mirror of 400 nm, the horizontal polarization (Ivh) and vertical polarization (Ivv) fluorescence of each well were respectively detected. Calculate according to the following formula: rDPH = (Ivv - G×Ivh) / (Ivv + 2×G×Ivh), where G = 0.85. Each group of experiments was repeated three times.

[0075] As Figure 2 (B) shows that compared with the control group with a PeME-F3 concentration of 0%, after treatment with PeME-F3 at 1×MIC concentration for 2 h, the CMFs of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 were significantly increased (1.73-fold and 1.56-fold respectively) (p < 0.05), and showed a treatment time-dependent increase. After treatment with 1×MIC of PeME-F3 for 6 h, the CMF of Vibrio cholerae GIM 1.449 increased most significantly (3.17-fold) (p < 0.05).

[0076] ICMP assay: The culture bacterial suspensions treated with PeME-F3 at the above-mentioned 1×MIC concentration were respectively added into sterilized 96-well bacterial culture plates (200 μL / well), and then 10 mM o-nitrophenyl-β-D-galactopyranoside (ONPG, 2.5 μL / well) was added, incubated at 37°C, and every 30 min, the OD 415 absorbance value of each well was measured using a multifunctional microplate reader. Each group of experiments was repeated three times.

[0077] As Figure 2 (E) shows that compared with the control group with a PeME-F3 concentration of 0%, after treatment with PeME-F3 at 1×MIC concentration for 2–6 h, the ICMPs of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 increased by 1.03–1.15-fold (Figure E-1) and 1.03–1.08-fold (Figure E-2) respectively, and showed a treatment time-dependent increasing trend.

[0078] These experimental results indicate that PeME-F3 (1×MIC) can reduce the CSH of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923; meanwhile, increase their ICMP and CMF.

[0079] Example 5: PeME-F3 causes leakage of intracellular nucleic acids and proteins in Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0080] In the cultured bacterial suspensions treated with PeME-F3 at the above 1×MIC concentration for 2 h, 4 h, and 6 h, 1.5 mL was taken respectively, centrifuged at 3500 rpm for 5 min at 4°C, and the supernatant was collected. The OD was measured using a multifunctional microplate reader 260 Absorbance value. After 24 h of treatment, a Bradford method protein concentration assay kit was used to measure the concentration of extracellular proteins in the supernatant samples according to the kit instructions. Each experiment was repeated three times

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

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

[0083] Example 6: Scanning electron microscopy observation of the destruction of cell structures of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 by PeME-F3

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

[0085] As Figure 3(A) It can be seen that in the control group with a PeME-F3 concentration of 0%, the cells of Vibrio cholerae GIM 1.449 showed a rod-shaped arc, full shape, flat surface, and complete structure. In contrast, after being treated with 1×MIC PeME-F3 for 4 hours, the cell surface shrank, with obvious cracks and depressions; after 6 hours of treatment, the cell structure was severely damaged, intracellular substances leaked, and the cells ruptured.

[0086] Similarly, in the control group with a PeME-F3 concentration of 0%, the cells of Staphylococcus aureus ATCC 25923 were spherical, plump, flat, and structurally intact. In contrast, after 4 hours of treatment with 1×MIC PeME-F3, the cell surface was significantly shrunk and invaginated, and some cells were deformed and adhered to each other; after 6 hours of treatment, the cells ruptured, a large amount of contents leaked, and a large area of ​​cell adhesion appeared ( Figure 3 , B).

[0087] Example 7 PeME-F3 changes multiple metabolic pathways in Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0088] Illumina RNA sequencing test: RNAse inhibitors were added to the culture suspension treated with 1×MIC concentration of PeME-F3 for 4 h, and the cells were collected by centrifugation. The total RNA of the cell samples was extracted using an RNA extraction kit according to the instructions of the kit. The purification, analysis and Illumina RNA sequencing of RNA samples were completed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) using the Illumina HiSeq2500 platform (Illumina, USA). Each group of experiments was repeated three times. Compared with the control group, 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 Enrichment Analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / KEGG / ).

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

[0090] The results of comparative transcriptome analysis showed that, compared with the control group, in the PeME-F3 treatment group, the identified DEGs accounted for 61.8% of the total number of Vibrio cholerae GIM 1.449 genes (n = 2320 / 3753). Among them, 2195 DEGs were significantly down-regulated at the transcriptional level (FC ≤ 0.5), while 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 , A and C).

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

[0092] In the base excision repair, homologous recombination, and folate biosynthesis pathways, about 55 DEGs were significantly down-regulated in Vibrio cholerae GIM1.449 (0.101–0.493 fold, p < 0.05). For example, the recombinase RecA (GTH07_11315) was significantly inhibited at the transcriptional level (0.297 fold, p < 0.05). RecA is the core enzyme of bacterial homologous recombination and 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 reaction in the base excision repair pathway and plays an important role in repairing DNA damage and maintaining genomic 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, approximately 18 DEGs were significantly inhibited in Vibrio cholerae GIM 1.449 (0.063–0.423-fold) (p<0.05). 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, approximately 24 DEGs were significantly inhibited (0.139–0.466-fold, p<0.05). For example, aminoacyl-tRNA synthetases (AARSs, PQQ 26_08455, PQQ26_08220, PQQ26_09780, PQQ26_02235, etc.) were significantly inhibited (0.209–0.343-fold, p<0.05). AARSs supply ribosomes with the amino acids required for protein biosynthesis.

[0096] In the bacterial secretion system, approximately 32 DEGs were significantly downregulated in Vibrio cholerae GIM 1.449 (0.027–0.481-fold, p<0.05). For example, the type II secretion system (T2SS) protein GspC (GTH07_00830) was significantly downregulated (0.301-fold, p<0.05). Gram-negative bacteria secrete enzymes and toxins through T2SS. The type VI secretion system (T6SS) protein Hcp (GTH07_07215) was strongly inhibited (0.027-fold, p<0.05). Hcp is the 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 Vibrio cholerae GIM 1.449, disrupting the stability of its genome; inhibit the synthesis pathways of peptidoglycan and glycerolipids, blocking the synthesis of cell walls, cell membranes, and biofilms; inhibit the protein synthesis, secretion, and export pathways, reducing the pumping out of harmful substances, bacterial pathogenicity, and drug resistance; and thus leading to cell lysis and death.

[0098] (2) Metabolic pathway changes in Staphylococcus aureus ATCC 25923

[0099] Comparative transcriptome analysis results showed that compared with the control group, the DEGs identified in the PeME-F3 treatment group accounted for 63.9% (1696 / 2656) of the total number of genes in Staphylococcus aureus ATCC 25923. Among them, 1590 DEGs were significantly downregulated at the transcriptional level (FC ≤ 0.5), while 106 DEGs were significantly upregulated (FC ≥ 2.0). These DEGs were enriched in 11 metabolic pathways of Staphylococcus aureus ATCC25923, 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 , B, and D).

[0100] For example, in peptidoglycan biosynthesis, about 19 DEGs were significantly downregulated in Staphylococcus aureus ATCC 25923 (0.060–0.388-fold, p < 0.05). For example, the cap8DEFGOP gene cluster encoding capsular polysaccharide Cap8 (PQQ26_00500, PQQ26_00505, PQQ26_00510, PQQ26_00515, PQQ26_00555, PQQ26_00560) 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 escape, and pathogen-host interaction. In addition, UDP-N-acetylglucosamine enolpyruvate transferase MurA (PQQ26_10550, 0.129-fold), glutamine-fructose-6-phosphate aminotransferase GlmM (PQ26_10845, 0.186-fold), UTP-glucose-1-phosphate uridylyltransferase GalU (PQQ26_12610, 0.217-fold), etc., which are 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 downregulated (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 first rate-limiting enzyme in fatty acid synthesis and is crucial for the synthesis and maintenance of cell membranes.

[0102] In the biosynthesis of phenylalanine, tyrosine, and tryptophan, approximately 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 tryptophan biosynthesis and is involved in the formation of bacterial biofilms and virulence. The expressions of 3 - phosphoshikimate 1 - carboxyvinyltransferase AroA (PQQ26_07025) and type I 3 - dehydroquinate dehydratase AroD (PQQ26_03930) were also inhibited (0.23 fold and 0.195 fold, p < 0.05). According to the literature, the deletion of aroA and aroD genes affects the production of capsular polysaccharides and fimbriae and inhibits the synthesis of biofilms in Salmonella enterica. Additionally, 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 via the shikimate pathway and is involved in the biosynthesis of cell wall / membrane / envelope.

[0103] In the TCA cycle, approximately 11 DEGs were significantly inhibited in Staphylococcus aureus ATCC 25923 (0.138 - 0.491 fold, p < 0.05), such as pyruvate dehydrogenase PdhA (PQQ26_05035), dihydrolipoamide 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 entry into the TCA cycle.

[0104] In summary, PeME - F3 can significantly inhibit the peptidoglycan synthesis, fatty acid synthesis, amino acid synthesis, and energy metabolism pathways 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, and leading to cell death.

[0105] Example 8: Experiment on the inhibition of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 contamination in the flesh of crucian carp and Litopenaeus vannamei by PeME - F3

[0106] Collection and analysis of crucian carp and Litopenaeus vannamei samples: Fresh crucian carp and Litopenaeus vannamei samples were collected from a seafood market in Shanghai, China, and quickly transported to the laboratory in an ice box. The collected fish and shrimp samples were washed with running tap water. Under sterile conditions, the fish skin and shrimp shell were removed, and the fish and shrimp meat were cut into small pieces (about 1×1×1 cm) with a sterilized scalpel, then rinsed three times with sterile water and sterilized by ultraviolet light for 20 min. In each 1 g of fish and shrimp meat sample, 9 mL of sterilized 1×PBS buffer solution was added and homogenized for 3 min. 100 μL of the homogenate was taken and spread on an agar plate respectively, and cultured overnight at 37 °C, and the number of colonies was counted. Each experiment was repeated three times. If no colony growth was observed, the sample could be used for subsequent experiments.

[0107] Preparation of artificially contaminated samples and grouped experiments: As described above, Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 were inoculated into TSB medium and cultured at 37 °C until mid-LGP. Then, it was diluted to 1×10 6 CFU / mL with 1×PBS and added to the above-mentioned homogenized meat samples of crucian carp and Litopenaeus vannamei respectively. A negative control group was set up in the experiment, inoculating the test strain without adding PeME-F3; a treatment group, inoculating the test strain and adding 1×MIC concentration of PeME-F3 (512 μg / mL); a positive control group, inoculating the test strain and adding oxytetracycline (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, the test samples of each group were transferred to a sterile homogenization bag, 9 mL of 1×PBS was added, and homogenized for 3 min. The homogenized samples were serially diluted, 100 μL was taken and evenly spread on an agar plate respectively, and cultured at 37 °C for 12 h, and the number of colonies was counted. Each experiment was repeated three times. Inhibitory rate (%) = (Ba–Bt) / Ba×100%. Where Ba is the viable count (CFU / mL) of the test strain in the negative control group; Bt is the viable count (CFU / mL) of the test strain in the treatment group.

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

[0109] As Figure 5As shown in (C and D), compared with the control group without PeME-F3, after treatment with 1×MIC concentration of PeME-F3 for 6 h, 12 h, and 24 h, the viable counts of Vibrio cholerae GIM 1.449 in the white shrimp samples decreased by 79.16%, 86.19%, and 88.41% respectively (p<0.01). Similarly, the viable counts of Staphylococcus aureus ATCC 25923 in the white shrimp samples decreased by 74.76%, 86.78%, and 91.58% respectively (p<0.01).

[0110] These experimental results indicate that under low-temperature (4°C) storage conditions, PeME-F3 can effectively inhibit the contamination of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 in crucian carp and white shrimp meat, and its antibacterial effect is comparable to that of the positive control antibiotic oxytetracycline.

[0111] In summary, the methanol-phase extract of Phyllanthus emblica fruits provided by the present invention can inhibit the growth and proliferation of Vibrio cholerae and Staphylococcus aureus; reduce their cell surface hydrophobicity, increase cell membrane fluidity and intracellular membrane permeability, resulting in damaged cell structure and leakage of intracellular nucleic acids and proteins; inhibit the DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion of Vibrio cholerae; inhibit the cell wall, cell membrane and biofilm synthesis, protein synthesis, energy metabolism of Staphylococcus aureus; and also has the effect of inhibiting the contamination of Vibrio cholerae and Staphylococcus aureus in crucian carp and white shrimp meat stored at low temperature (4°C). The methanol-phase extract of Phyllanthus emblica fruits of the present invention can also be used for the preparation of foods or drugs, and the prepared foods or drugs also have antibacterial effects.

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

Claims

1. A methanol extract PeME of the fruit of Phyllanthus emblica, characterized in that It is extracted from the fruit of Phyllanthus emblica by methanol-chloroform solvent extraction method, and the specific steps are as follows: S1. Rinse the fresh emblica fruits, dry them, cut them into pieces, and pre-freeze them at -80℃ for 2–4h; then freeze-dry them at -80℃ for 48h; crush the freeze-dried samples and sieve them through a 300-mesh sieve; S2. Methanol and chloroform are added to the sieved sample powder at a solid-liquid ratio of 1:10 and mixed evenly, wherein the volume ratio of methanol to chloroform is 1:2; sterile water is then added and mixed evenly, wherein the mass-volume ratio of the sample powder to sterile water is 1:6; the mixed solution is ultrasonically treated, and then filtered using a 20-25 μm microporous filter membrane to collect the filtrate; after separating the methanol phase and the chloroform phase, they are evaporated and concentrated using a rotary evaporator to obtain PeME; the filtrate is extracted a second time using the above-mentioned methanol and chloroform solvents, and the obtained PeME is combined.

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

3. Use of the methanol extract PeME of the fruit of Emblica officinalis according to claim 1 or the purified fraction PeME-F3 of the methanol extract of the fruit of Emblica officinalis according to claim 2 as an antibacterial agent.

4. The use according to claim 3, characterized in that: The antibacterial agent inhibits Vibrio cholerae and Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The antibacterial agent exerts an antibacterial effect through the following aspects: (a) Inhibit the growth and proliferation of Vibrio cholerae and Staphylococcus aureus; (b) Reduce the cell surface hydrophobicity of Vibrio cholerae and Staphylococcus aureus and increase the cell membrane fluidity and intracellular membrane permeability; (c) It causes damage to the cell structure of Vibrio cholerae and Staphylococcus aureus, and leakage of intracellular nucleic acids and proteins; (d) inhibiting DNA replication, repair and recombination, cell wall, cell membrane and biofilm synthesis, protein synthesis and secretion metabolic pathways of Vibrio cholerae; (e) Inhibit the cell wall, cell membrane and biofilm synthesis, protein synthesis, and energy metabolism pathways of Staphylococcus aureus.

6. The use according to claim 4, characterized in that: The antibacterial agent can be used for the prevention and control of bacterial contamination in food, medicine or aquatic products.

7. The use according to claim 6, characterized in that: The food is ready-to-eat, fresh, refrigerated or frozen food.

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

9. The use according to claim 6, 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 the crucian carp and whiteleg shrimp meat stored at low temperature.

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