Extraction method and application of pepper polyphenol with pseudomonas sp. Inhibitory activity

Abstract

The invention provides a method for extracting pepper polyphenol with pseudomonas sp. Inhibitory activity. The method comprises the following steps: preparing a crude polyphenol aqueous solution, and further respectively and sequentially extracting free pepper polyphenol, esterified pepper polyphenol, glucoside pepper polyphenol and insoluble combined pepper polyphenol from the crude polyphenol. According to the extraction method provided by the invention, the crude polyphenol solution is further extracted to respectively obtain different forms of pepper polyphenols with different mycopseudomonas inhibition activities, so that a basis is provided for subsequent deeper research and application, and the pepper polyphenols with higher antibacterial activity can be obtained. Through analysis, the insoluble-binding form polyphenol extracted by taking fresh pepper fruits with medium maturity as a raw material has the highest pseudomonas sp. Inhibitory activity. The antibacterial ability of the refrigerated food preservative adopting the insoluble-combined pepper polyphenol is also improved.

Description

Technical Field

[0001] The present invention relates to the field of extraction and utilization of natural products, and particularly to an extraction method and application of piper polyphenols with Pseudomonas fragi inhibitory activity. Background Art

[0002] Piper nigrum L. is a plant of the genus Piper in the Piperaceae family, one of the oldest and most well-known spice crops, containing volatile oils, piperine and other substances, having various functional activities, and widely used in the food and pharmaceutical industries. At present, most of the produced pepper products are still primary products, and there are few deep-processed products in the forms of pepper essential oil, oleoresin, piper polyphenol extract, etc. on the market. The development research on deep-processed products currently remains at the laboratory level and has not reached the level of large-scale production. As an important secondary metabolite, the content of polyphenols is related to various factors such as plant species, growth environment, and plant maturity stage, and its functional activities have attracted people's research interest. Natural phenolic substances have antibacterial activity, and they can exert their antibacterial activity by disrupting the integrity of cell membranes, inhibiting the synthesis and metabolism of biological macromolecules, etc. Pseudomonas fragi, as a Gram-negative bacterium, is the main inducer of the spoilage of frozen beef, pork, chicken and the deterioration of milk under aerobic conditions. Pseudomonas fragi can grow and reproduce rapidly at low temperatures, and can decompose proteins and fats, actively participate in catabolism, and ultimately lead to a large amount of food waste. Polyphenols mainly exist in plants in conjugated and bound forms, and only a small part exists in free form, but most current studies focus on the functional activities of free-form polyphenols, and there is less research on the activities of conjugated and bound forms of polyphenols. Pseudomonas fragi, as the dominant spoilage flora of meat products, its rapid reproduction at low temperatures has led to a large amount of meat product waste, and due to the widespread use of antibiotics, many bacteria have developed drug resistance, and there is an urgent need to develop new natural antibacterial agents to inhibit the growth and reproduction of pathogenic bacteria. Summary of the Invention

[0003] The first object of the present invention is to provide a new natural antibacterial agent to inhibit the growth and reproduction of pathogenic bacteria, so as to solve the problem that due to the widespread use of antibiotics in the prior art, many bacteria have developed drug resistance.

[0004] The first aspect of the present invention provides an extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity, including the steps of: (1) Preparing a crude polyphenol aqueous solution, adjusting the pH value of the crude polyphenol aqueous solution to 1-3, adding an equal volume of ethyl acetate and shaking well, and separating and collecting the first organic phase and the first aqueous phase. The first organic phase is the free-form piper polyphenol; (2) Add an antioxidant to the first aqueous phase, perform a primary alkali hydrolysis under an inert atmosphere, then adjust the pH value to 1-3 with an acid, add an equal volume of ethyl acetate and shake well, and separate and collect the second organic phase and the second aqueous phase. The second organic phase is the esterified form of piper polyphenols. (3) Add an acid to the second aqueous phase to adjust the pH value to 1-3, and perform an acid hydrolysis at 75-90 °C. After cooling to room temperature, adjust the pH value to 1-3 with a base, add an equal volume of ethyl acetate and shake well, and separate and collect the third organic phase and the third aqueous phase. The third organic phase is the glycoside form of piper polyphenols. (4) Filter the third aqueous phase, add a base to the filter residue, and perform a secondary alkali hydrolysis under an inert atmosphere; then adjust the pH value to 1-3 with an acid, centrifuge and collect the supernatant, add an equal volume of ethyl acetate and shake well, and separate and collect the fourth organic phase, which is the insoluble-bound form of piper polyphenols.

[0005] The extraction method provided by the present invention further extracts and separates the crude polyphenol solution to obtain different forms of piper polyphenols with different inhibitory activities against Pseudomonas syringae, providing a basis for subsequent in-depth research and application, and facilitating the obtaining of products with higher inhibitory activities against Pseudomonas syringae.

[0006] In some embodiments, the crude polyphenol solution is prepared by the following method: using freeze-dried pepper powder as the raw material, adding a 70% vol methanol aqueous solution at an addition amount of 9-11 mL / g, performing ultrasonic extraction, filtering, and removing the solvent from the filtrate by rotary evaporation. Preferably, the temperature during ultrasonic extraction is 20-25 °C, and the ultrasonic time is 1.5-3 h.

[0007] In some embodiments, the freeze-dried pepper powder is obtained by crushing and freeze-drying fresh pepper fruits; preferably, the fresh pepper fruits are selected from those at 200-220 days after flowering, 230-250 days after flowering, and 260-270 days after flowering; more preferably, the fresh pepper fruits are picked at 200-220 days after flowering.

[0008] In some embodiments, the reaction temperature of the primary alkali hydrolysis and the secondary alkali hydrolysis is 20-25 °C, and the reaction time is 3-5 h.

[0009] In some embodiments, the reaction time of the acid hydrolysis is 20-40 min.

[0010] In some embodiments, the inert atmosphere is selected from at least one of a nitrogen atmosphere, a helium atmosphere, and an argon atmosphere. Performing alkali hydrolysis under an inert atmosphere effectively prevents the reactants from contacting oxygen or moisture in the air, avoids unnecessary side reactions, and ensures the purity and efficiency of the reaction.

[0011] In some embodiments, the antioxidant is a 3-5M alkaline solution containing 0.5-2% of component A, ascorbic acid, and 9-11 mM of component B, ethylenediaminetetraacetic acid; wherein, component A is selected from at least one of ascorbic acid, esterified vitamin C, and acerola cherry powder, component B is selected from at least one of ethylenediaminetetraacetic acid, tetrasodium glutamate diacetate, and methylglycine diacetate, and the alkaline solution is selected from at least one of sodium hydroxide solution and potassium hydroxide solution. When performing alkaline hydrolysis, adding an antioxidant can effectively avoid the oxidation of polyphenols, thereby ensuring the extraction amount of polyphenol substances.

[0012] In some embodiments, the acid is selected from 5-7 M hydrochloric acid; the base is selected from 3-5M sodium hydroxide.

[0013] In some embodiments, in order to maximize the extraction rate and extraction amount of polyphenols, steps (1)-(4) are repeated 4-6 times, and the first organic phase, the second organic phase, the third organic phase, and the fourth organic phase are respectively combined to obtain piper polyphenols with different Pseudomonas syringae inhibitory activities.

[0014] The present invention also provides an insoluble-bound form of piper polyphenols with Pseudomonas syringae inhibitory activity, obtained by using the extraction method.

[0015] The present invention also provides a free form of piper polyphenols with Pseudomonas syringae inhibitory activity, obtained by using the extraction method.

[0016] The present invention also provides an esterified form of piper polyphenols with Pseudomonas syringae inhibitory activity, obtained by using the extraction method.

[0017] The present invention also provides a glycoside form of piper polyphenols with Pseudomonas syringae inhibitory activity, obtained by using the extraction method.

[0018] The present invention also provides an analysis method for the Pseudomonas syringae inhibitory activity of piper polyphenols. Using the extraction method, different forms of piper polyphenols are extracted from fresh pepper fruits, and at least one of the determination of the minimum inhibitory concentration, the determination of the minimum bactericidal concentration, the determination of the particle size of the bacterial suspension, the determination of the nucleic acid leakage of the bacterial suspension, the determination of the protein leakage of Pseudomonas syringae, the determination of the intracellular reactive oxygen content of Pseudomonas syringae, and the observation of cell membrane integrity and cell morphology is performed on different forms of piper polyphenols.

[0019] Through the analysis method, different forms of piper polyphenols with stronger Pseudomonas syringae inhibitory activity at different maturity stages can be clearly obtained. It has been verified that the four forms of piper polyphenols have different degrees of inhibitory effects on Pseudomonas syringae. Among them, the insoluble-bound form of piper polyphenols has the best antibacterial effect.

[0020] Based on this, the present invention also provides the application of the four forms of pepper polyphenols obtained by the extraction method in inhibiting Pseudomonas syringae pv. tomato; in particular, the application in refrigerated food preservatives.

[0021] Among them, the refrigerated food is selected from at least one of cream, poultry eggs, and meat.

[0022] In some embodiments, the preservative includes more than 40% of the insoluble-bound form of pepper polyphenols.

[0023] The extraction method of pepper polyphenols with Pseudomonas syringae pv. tomato inhibitory activity provided by the present invention can efficiently and comprehensively extract different forms of pepper polyphenols with different Pseudomonas syringae pv. tomato inhibitory activities, providing a basis for subsequent research and application.

[0024] Using fresh pepper fruits at different maturity stages as raw materials, different forms of pepper polyphenols were extracted. Through the analysis of total phenol content, it was found that the total phenol content was the highest in fresh pepper fruits at the mid-maturity stage. Based on this, indicators such as MIC, MBC, particle size distribution, nucleic acid and protein leakage, generation of reactive oxygen species, laser confocal microscopy, and transmission electron microscopy were measured to observe the changes in cell activity and explore the antibacterial mechanism of different forms of polyphenols against Pseudomonas syringae pv. tomato. The results showed that the MIC and MBC of the insoluble-bound form of polyphenols were the smallest, with the best antibacterial ability, followed by the esterified form, and the free form had the worst antibacterial ability. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a schematic diagram of the total phenol content extracted from fresh pepper fruits at different maturity stages in Examples 1-3. Among them, MM represents fresh pepper fruits at the mid-maturity stage (MM), picked 210 days after flowering; LM represents fresh pepper fruits at the late maturity stage, photographed 240 days after flowering; FM represents fully mature fresh pepper fruits, picked 270 days after flowering.

[0027] Figure 2 It is the particle size distribution result of the bacterial suspension treated with the four forms of pepper polyphenols obtained by the extraction method of pepper polyphenols with Pseudomonas syringae pv. tomato inhibitory activity in Example 1 for 8 h.

[0028] Figure 3It is the result of nucleic acid leakage of Pseudomonas fragi after being treated with four forms of piper polyphenols obtained by the extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity in Example 1 for different times.

[0029] Figure 4 It is the result of protein leakage of Pseudomonas fragi after being treated with four forms of piper polyphenols obtained by the extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity in Example 1 for different times.

[0030] Figure 5 It is the intracellular reactive oxygen species content of Pseudomonas fragi after being treated with four forms of piper polyphenols obtained by the extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity in Example 1 for different times.

[0031] Figure 6 It is a diagram showing the damaged condition of the cell membrane of Pseudomonas fragi observed by a laser confocal microscope after being treated with four forms of piper polyphenols obtained by the extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity in Example 1 for 8 h.

[0032] Figure 7 It is a TEM image of Pseudomonas fragi cells after being treated with four forms of piper polyphenols obtained by the extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity in Example 1 for 8 h. Detailed implementation mode

[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without any creative effort fall within the scope of protection of the present invention.

[0034] In the prior art, since the content of polyphenols in pepper leaves is the highest, much higher than that in fresh pepper fruits, generally pepper leaves and the like are used as raw materials to extract crude polyphenol aqueous solutions, and the crude polyphenol solutions are directly used for their activity research. However, the components in piper polyphenols are relatively complex, and it is impossible to clarify the specific active components and their mechanisms in piper polyphenols. Based on this, the present invention provides an extraction method of piper polyphenols with Pseudomonas fragi inhibitory activity, including the steps: (1) Prepare a crude polyphenol aqueous solution, adjust the pH value of the crude polyphenol aqueous solution to 1-3, add an equal volume of ethyl acetate and shake well, and then separate and collect the first organic phase and the first aqueous phase. The first organic phase is the free form of piper polyphenols; (2) Add an antioxidant to the first aqueous phase, and under the condition of passing an inert gas, carry out primary alkali hydrolysis by reacting at 20 - 25 °C for 3 - 5 h. Then adjust the pH value to 1 - 3 with 5 - 7 M hydrochloric acid. Add an equal volume of ethyl acetate and shake well, then separate and collect the second organic phase and the second aqueous phase. The second organic phase is the esterified form of piper polyphenols. (3) Adjust the pH value of the second aqueous phase to 1 - 3 with 5 - 7 M hydrochloric acid, and carry out acid hydrolysis by reacting at 75 - 90 °C for 20 - 40 min. After cooling to room temperature, adjust the pH value to 1 - 3 with 3 - 5 M sodium hydroxide. Add an equal volume of ethyl acetate and shake well, then separate and collect the third organic phase and the third aqueous phase. The third organic phase is the glycoside form of piper polyphenols. (4) Filter the third aqueous phase, add an alkali solution with a concentration of 3 - 5 M sodium hydroxide solution to the filter residue, and under the condition of passing an inert gas, carry out secondary alkali hydrolysis by reacting at 20 - 25 °C for 3 - 5 h. Then adjust the pH value to 1 - 3, centrifuge and collect the supernatant. Add an equal volume of ethyl acetate and shake well, then separate and collect the fourth organic phase, which is the insoluble - bound form of piper polyphenols.

[0035] Steps (1) - (4) are repeated 4 - 6 times respectively, such as 4 times, 5 times, 6 times or more times, and the first organic phase, the second organic phase, the third organic phase and the fourth organic phase are respectively combined to improve the extraction rate of each form of piper polyphenols, and further improve the antibacterial property of the finally obtained piper polyphenols.

[0036] In steps (1), (2) and (4), 5 - 7 M hydrochloric acid is used as the pH regulator. In step (3), 3 - 5 M NaOH solution is used as the pH regulator. The pH value can be adjusted to 1, 2 or 3, preferably the pH value is 2; the concentration of hydrochloric acid can be 5 M, 6 M or 7 M, preferably 6 M; the concentration of the NaOH solution can be 3 M, 4 M, 5 M, etc., preferably 4 M.

[0037] In the examples, the crude polyphenol solution is prepared by the following method: Using freeze - dried pepper powder as the raw material, add 70% vol methanol aqueous solution at an addition amount of 9 - 11 mL / g, carry out ultrasonic extraction, filter and remove the solvent from the filtrate by rotary evaporation. The addition amount of the methanol solution can be 9 mL / (g pepper powder), 10 mL / (g pepper powder), 11 mL / (g pepper powder), for example, for 20 g of freeze - dried pepper powder, add 180 mL, 200 mL or 220 mL of methanol solution. The concentration of the methanol solution can be 65%, 70% or 75%.

[0038] In the examples, the ultrasonic extraction is repeated 4 - 6 times, and the first organic phase, the second organic phase, the third organic phase, and the fourth organic phase are respectively combined to obtain different forms of piper polyphenols with Pseudomonas syringae inhibitory activity. Repeating the ultrasonic extraction multiple times can improve the extraction rate of polyphenolic substances in fresh pepper fruits, thereby enhancing the antibacterial property of the obtained piper polyphenols.

[0039] In the examples, the freeze - dried pepper powder is obtained by freeze - drying and crushing fresh pepper fruits at three different maturities: medium - mature (mid - maturity), nearly - mature (late - maturity), and fully - mature. In some examples, the fresh pepper fruits are picked 200 - 220 days after flowering (such as 200 days, 210 days, 220 days, preferably 210 days). Fresh pepper fruits at mid - maturity 200 - 220 days after flowering have the highest total piper phenol content.

[0040] The total phenol content in pepper leaves is significantly higher than that in fresh pepper fruits, which is the reason why pepper leaves are generally used as raw materials for polyphenol extraction in the current existing technology. Since fresh pepper fruits have less polyphenol content, they are currently less used for polyphenol extraction and antibacterial applications, especially for the inhibition of Pseudomonas syringae. The present invention uses fresh pepper fruits with relatively less total phenol content as raw materials. Through the extraction method provided by the present invention, different forms of piper polyphenols are successfully separated, and the components are further analyzed. The results show that among the polyphenols, the content of free - form polyphenols is the highest, but its inhibitory effect on Pseudomonas syringae is poor. The insoluble - bound form of piper polyphenols with less content has the highest inhibitory activity against Pseudomonas syringae. The insoluble - bound form of piper polyphenols obtained by the extraction method provided by the present invention is used as a preservative for refrigerated foods, which can reduce the use of antibiotics, etc., and is more ecological and environmentally friendly.

[0041] The present invention also provides an insoluble - bound form of piper polyphenols with Pseudomonas syringae inhibitory activity, which is obtained by using the extraction method.

[0042] The present invention also provides a free - form of piper polyphenols with Pseudomonas syringae inhibitory activity, which is obtained by using the extraction method.

[0043] The present invention also provides an esterified form of piper polyphenols with Pseudomonas syringae inhibitory activity, which is obtained by using the extraction method.

[0044] The present invention also provides a glycoside form of piper polyphenols with Pseudomonas syringae inhibitory activity, which is obtained by using the extraction method.

[0045] The present invention also provides a method for analyzing the inhibitory activity of pepper polyphenols against Pseudomonas syringae. Using the extraction method, different forms of pepper polyphenols are extracted from fresh pepper fruits, and at least one of the following determinations is carried out on different forms of pepper polyphenols respectively: determination of minimum inhibitory concentration, determination of minimum bactericidal concentration, determination of the particle size of the bacterial suspension, determination of nucleic acid leakage of the bacterial suspension, determination of protein leakage of Pseudomonas syringae, determination of the intracellular reactive oxygen content of Pseudomonas syringae, and observation of cell membrane integrity and cell morphology.

[0046] The present invention also provides the application of four forms of pepper polyphenols obtained by the extraction method in inhibiting Pseudomonas syringae; especially in the application as a preservative for refrigerated foods.

[0047] Among them, the refrigerated foods are selected from at least one of cream, poultry eggs, and meat.

[0048] In some embodiments, the preservative includes more than 40% of the insoluble-bound form of pepper polyphenols.

[0049] Example 1 This example provides a method for extracting pepper polyphenols with the activity of inhibiting Pseudomonas syringae pv. syringae, using fresh pepper fruits at the mid-maturity stage (MM) as raw materials: The fresh pepper fruits are picked 210 days after flowering, immediately transported back to the laboratory after harvesting, pre-frozen at -80 °C, and then vacuum freeze-dried for 2 days to remove moisture. After that, they are crushed and passed through an 80-mesh sieve to obtain freeze-dried pepper powder. Accurately weigh 20 g of the freeze-dried pepper powder, add 200 mL of methanol (7:3 v / v, that is, the methanol concentration is 70%), and perform extraction by ultrasonic wave for 1 h at 25 °C. After filtering to obtain the filtrate, remove the solvent with a rotary evaporator to obtain a crude polyphenol aqueous solution. Adjust the pH of the crude polyphenol aqueous solution to 2 with 6 M hydrochloric acid, then add an equal volume of ethyl acetate, fully oscillate to obtain an organic layer and an aqueous layer, separate and collect the first organic phase and the first aqueous phase. The first organic phase is the free component. The first aqueous phase is treated with 4 M sodium hydroxide (10 mM ethylenediaminetetraacetic acid, 1% ascorbic acid) for 4 h under the conditions of room temperature (such as 20 °C) and nitrogen passing for one-time alkali hydrolysis. Then, adjust the pH of the aqueous solution to 2 with 6 M hydrochloric acid, add an equal volume of ethyl acetate, oscillate and layer, separate and collect the second organic phase and the second aqueous phase. The second organic phase is the esterified component. Treat the second aqueous phase with 6 M hydrochloric acid at 85 °C for 30 min for acid hydrolysis, cool it, adjust the pH to 2 with 4 M sodium hydroxide, add an equal volume of ethyl acetate, oscillate and layer, separate and collect the third organic phase and the third aqueous phase. The third organic phase is the glycoside component. Filter the third aqueous phase, and the filter residue is subjected to secondary alkali hydrolysis with 4 M sodium hydroxide for 4 h under the conditions of room temperature (such as 20 °C) and nitrogen passing. Then, adjust the pH value to 2 with 6 M hydrochloric acid, centrifuge at 8000 rpm for 15 min, collect the supernatant, and then add an equal volume of ethyl acetate, oscillate and layer, separate and collect the fourth organic phase. The fourth organic phase is the insoluble-bound component.

[0050] In this example, the above process is repeated 5 times. The organic phases are combined separately, the organic phases are rotary evaporated until viscous and with low fluidity, and finally, they are made up to 25 mL in a brown volumetric flask with absolute ethanol to obtain different forms of pepper polyphenols with the activity of inhibiting Pseudomonas syringae pv. syringae.

[0051] Example 2 This example provides a method for extracting pepper polyphenols with the activity of inhibiting Pseudomonas syringae pv. syringae, which is basically the same as Example 1, except that the fresh pepper fruits at the late-maturity stage (LM, 240 days after flowering) are used as raw materials.

[0052] Example 3 This example provides a method for extracting pepper polyphenols with the activity of inhibiting Pseudomonas syringae pv. syringae, which is basically the same as Example 1, except that the fresh pepper fruits at the fully mature stage (FM, 270 days after flowering) are used as raw materials.

[0053] Example 4 This example provides an analysis method for the inhibitory activity of pepper polyphenols against Pseudomonas syringae, including: (1) Using the extraction method provided in Example 1, insoluble-bound form pepper polyphenols, free form pepper polyphenols, esterified form pepper polyphenols, and glycoside form pepper polyphenols are extracted from fresh pepper fruits in the mid-maturity stage. (2) The minimum inhibitory concentration, minimum bactericidal concentration, determination of the particle size of the bacterial suspension, determination of nucleic acid leakage of the bacterial suspension, determination of protein leakage of Pseudomonas syringae, determination of the intracellular reactive oxygen content of Pseudomonas syringae, and observation of cell membrane integrity and cell morphology are carried out on the different forms of pepper polyphenols extracted in step (1) to analyze the inhibitory activity and mechanism of different forms of pepper polyphenols against Pseudomonas syringae.

[0054] Test Example 1 Total Polyphenol Content Test object: Pepper polyphenols extracted in Examples 1-3.

[0055] Test method: Pipette 1 mL of each fraction extract into a 25 mL brown volumetric flask, then add 1 mL of 1N Folin-Ciocalteu reagent. After shaking well, add 4 mL of 7% sodium carbonate solution, and shake well again to adjust the final volume to 25 mL. The reaction system reacts in the dark at room temperature for 2 h, and then the absorbance value is measured at 750 nm. The standard curve of gallic acid is prepared according to the above steps. The total polyphenol content of each fraction extract is expressed as milligrams of gallic acid equivalent per gram of freeze-dried pepper powder (mg GAE / gDW).

[0056] Test results: The total phenol content in different forms of polyphenols extracted from fresh pepper fruits at three maturity stages is as Figure 1 shown.

[0057] From Figure 1 it can be seen that the total phenol content decreases from 10.63 mg GAE / g DW (unit: milligrams of gallic acid equivalent per dry weight (mg GAE / g DW)) in the medium maturity (MM) stage to 8.39 mg GAE / g DW in the fully mature (FM) stage. The data shows that as the maturity of pepper fruits increases, the total phenol content shows a downward trend. The total phenol content changes significantly between the MM stage and the FM stage (p < 0.05), but there is only a slight change between the nearly mature (LM) stage and the FM stage. From Figure 1It can also be seen that for fresh pepper fruits at different maturity stages, among the four forms of polyphenols, the free form is the main form constituting the total phenols, and its content varies between 4.17 mg GAE / g DW and 3.20 mg GAE / g DW. Followed by the esterified form and the insoluble-bound form, and the fruits in the MM stage contain the richest insoluble-bound form of polyphenols, indicating that the waste from traditional pepper extraction is another good source of Pseudomonas syringae inhibitory substances, which makes full use of the traditional crude polyphenol extraction residue.

[0058] Experimental Example 2 Pseudomonas syringae Inhibitory Activity Test Object: Different forms of pepper polyphenols obtained in Example 1.

[0059] Test Method: (1) Activation of Bacteria and Preparation of Bacterial Suspension After the nutrient agar is sterilized and cooled to solidify into a slant, Pseudomonas syringae is streaked and cultured, and the test tube is cultured in a constant temperature incubator at 30 °C for standby.

[0060] Pseudomonas syringae is cultured to the logarithmic phase, inoculated in a liquid medium, and the medium is oscillated and cultured in a constant temperature shaker at 150 r / min and 30 °C for 12 h to prepare a bacterial suspension. The concentration of the bacterial suspension is adjusted to 106 - 107 CFU / mL by the McFarland turbidity method for standby.

[0061] (2) Determination of Minimum Inhibitory Concentration (MIC) Dissolve different forms of polyphenol extracts in 1% ethanol, then dilute them in gradients. Mix the extract (2 mL) and the nutrient agar medium (18 mL) so that the final concentration of the extract is 0.625, 1, 1.25, 2, 2.5, 4, 5, 8 mg / mL. In addition, add sterile water and 1% ethanol as the blank group and the positive control group. After the medium solidifies, evenly disperse 200 μL of the bacterial suspension (about 1×10 6 CFU / mL) on the cooled medium and culture it at 30 °C for 24 h. The lowest extract concentration that inhibits visible bacterial growth is used as the MIC.

[0062] (3) Determination of Minimum Bactericidal Concentration (MBC) The operation is the same as that described for the determination of the minimum inhibitory concentration (MIC). The medium is cultured at 30 °C for 48 h, and the lowest extract concentration without bacterial growth is used as the MBC.

[0063] (4) Determination of the Particle Size of the Bacterial Suspension The extract with a concentration of MIC and the bacterial suspension were cultured in a constant temperature shaker at 30 °C and 150 r / min for 8 h. Meanwhile, a blank group and a negative control group were set up. The cultured bacterial suspension was centrifuged at 8000 rpm and 4 °C for 6 min. The bacterial cells were washed 3 times with sterilized PBS buffer, and finally the bacterial cells were resuspended in 5 mL of phosphate buffer for testing on the machine. Each experiment was repeated three times.

[0064] (5) Determination of nucleic acid leakage from the bacterial suspension The cells cultured to the logarithmic phase were washed three times with sterile PBS buffer and resuspended in PBS, and the concentration was adjusted to 106 - 107 CFU / mL. The extract with a concentration of MIC, 1% ethanol, and sterile water were respectively added to the bacterial suspension, and cultured in a shaker at 30 °C and 150 r / min. The bacterial suspension cultured for 0, 2, 4, and 8 h was centrifuged, and the supernatant was collected. The absorbance value of the supernatant at 260 nm was measured and a curve was plotted.

[0065] (6) Determination of protein leakage from the bacterial suspension The operation steps were carried out according to the steps for the determination of nucleic acid leakage from the bacterial suspension. The absorbance value of the supernatant at 280 nm was measured and a curve was plotted.

[0066] (7) Determination of reactive oxygen species in the bacterial suspension The four extracts with a concentration of MIC, 1% ethanol, and sterile water were respectively added to the bacterial suspension and cultured in a shaker at 30 °C and 150 r / min. The bacterial suspension cultured for 0, 2, 4, and 8 h was centrifuged at low temperature, and the bacterial cells were washed 3 times with PBS and resuspended in 1 mL of phosphate buffer. Subsequently, 200 μL of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 mg / L) was added and incubated in the dark for 30 min. The bacterial cells were washed three times with PBS, and finally the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0067] (8) Observation by laser confocal microscope Propidium iodide (PI) and Calcein-AM were used to evaluate the integrity of the cell membrane. The four extracts with a concentration of MIC, 1% ethanol, and sterile water were respectively added to the bacterial suspension and cultured in a shaker at 30 °C and 150 r / min for 8 h. Then the bacterial cells were washed 3 times with PBS, and then operated according to the instructions on the kit. Finally, observation was carried out with a laser confocal microscope.

[0068] (9) Observation by transmission electron microscope The extract at a concentration of MIC, 1% ethanol, and sterile water were added to the bacterial suspension, and the mixture was treated at 30 °C and 150 r / min for 8 h. The bacterial suspension was dropped onto a copper grid for drying, and the samples were observed under a transmission electron microscope.

[0069] Test results: (1)Analysis of the minimum inhibitory concentration and minimum bactericidal concentration of the four forms of piper polyphenols obtained in Example 1 The MIC and MBC of the four forms of polyphenols inhibiting the growth of Pseudomonas fragi are shown in Table 1.

[0070] Table 1 Antibacterial activity against Pseudomonas fragi

[0071] Note: "+++" indicates a large number of colonies As can be seen from Table 1, all forms have antibacterial activity against Pseudomonas fragi, with the concentration varying between 1 mg / mL and 4 mg / mL. Among them, the insoluble-bound form has the best antibacterial activity against Pseudomonas fragi, with the MIC and MBC values both being 1 mg / mL. The least effective is the free form, with the MIC and MBC values being 2 mg / mL and 4 mg / mL respectively. A large number of colonies grew in the blank group (sterile water) and the positive control group (1% ethanol), indicating that they did not significantly inhibit the growth of Pseudomonas fragi. Previous studies have explored the antibacterial effects of myrtle extract (extracted with water-ethanol as a solvent) and 3-carene as antibacterial agents against Pseudomonas fragi, with their MICs being 25 mg / mL and 8.7 mg / mL respectively. This shows that compared with myrtle fruit extract and 3-carene, the antibacterial effect of pepper extract against Pseudomonas fragi is better.

[0072] (2)Effect on the particle size distribution of Pseudomonas fragi The particle size distribution of the suspension after treating the cells with the four forms of polyphenols for 8 h is as Figure 2 shown. In the blank group and the positive control group, the cell suspension mainly consisted of particles of 1.5 μm. After the cells were treated with the extract for 8 h, the particle size distribution of the bacterial suspension shifted to the left, indicating that all four forms may cause cell aggregation. A similar phenomenon was also found in the study of treating Pseudomonas aeruginosa with linalool. The size distribution peaks of the suspensions treated with the free form and the esterified form showed a significant leftward shift. In addition, the free form produced a bimodal phenomenon with large particle sizes, indicating that cell aggregation occurred.

[0073] (3)Effect on the nucleic acid leakage of Pseudomonas fragi The cell membrane plays a crucial role in the metabolism of bacteria. Damage to the cell membrane first leads to the leaching of small molecules, followed by the outflow of macromolecules such as DNA, RNA, and proteins. Therefore, the change in cell membrane permeability is characterized by the leakage amount of nucleic acids, and nucleic acids play an important role in the growth and development of cells. The leakage of nucleic acids will lead to cell death. Nucleic acids have the maximum absorbance at 260 nm. Therefore, the absorbance value of the bacterial suspension at 260 nm is used to reflect the leakage of nucleic acids. The results are as Figure 3 shown. The OD values of the cell suspensions treated in four forms all showed an upward trend over time. Compared with the treatment groups, the upward trends of the blank group and the positive control group were slow and the OD values were both less than those of the treatment groups. At 0 - 2 h, the OD values of all treatment groups were close to those of the blank group and the positive control group, indicating that the extract did not play a significant role within 2 h. Compared with other treatment groups, the OD value of the free form was significantly higher than that of other treatment groups at 4 h. All treatment groups showed the fastest growth rate within 4 - 8 h, indicating that the destructive effect of the extract was the most obvious within 4 - 8 h. And the extract could change the permeability of the cell membrane of Pseudomonas fragi, resulting in the leakage of nucleic acids and thus causing cell death.

[0074] (4)Effect on the protein leakage of Pseudomonas fragi The protein leakage conditions of the bacterial suspension at different time points are as Figure 4 shown. The OD values of all treatment groups were higher than those of the blank group and the positive control group, and showed a similar trend to the nucleic acid leakage situation. The massive leakage of proteins further indicated the severe damage of the cell membrane. This result showed that different forms of pepper polyphenol extract exerted their antibacterial function by destroying the integrity of the cell membrane of the bacteria. Similar results were also found in previous studies on the treatment of Escherichia coli and Staphylococcus aureus with cinnamon essential oil, and it was found that some phenolic substances, such as p - coumaric acid, p - hydroxybenzoic acid, protocatechuic acid, caffeic acid, vanillic acid, and syringic acid, etc., could diffuse through the plasma membrane into the cells and increase the permeability of their cell membranes, further leading to the leakage of intracellular ions and macromolecules. The above - mentioned phenolic acids were found in all four forms.

[0075] (5)Effect on the intracellular reactive oxygen species content of Pseudomonas fragi The excessive accumulation of reactive oxygen species (ROS) can cause the oxidation of basic components within cells, further exerting its mutagenic effect and leading to carcinogenesis. On the other hand, the excessive production of ROS can also trigger the oxidative stress response in cells, ultimately resulting in the premature death of organisms. Previous studies have shown that antibiotics kill bacteria by increasing the intracellular ROS content through the Fenton reaction. DCFH-DA, as a lipophilic fluorescent probe, is often used to detect the intracellular ROS content. DCFH-DA itself does not produce fluorescence and can freely enter the cell membrane. After entering the cell, it will be hydrolyzed by intracellular esterase into dichlorofluorescin (DCFH). DCFH cannot cross the cell membrane and is oxidized by intracellular ROS into fluorescent 2',7'-dichlorofluorescein (DCF). Within a certain range, the fluorescence intensity is positively correlated with the ROS content generated. Therefore, the relative fluorescence intensity is used to represent the ROS content. After the cells were treated with the MIC of the four components of the extract, the changes in the intracellular ROS content at different time points were as Figure 5 shown. There was no significant change in the ROS content of the blank group and the positive control group with the extension of time. In the treatment group, with the extension of the treatment time, the intracellular ROS level increased to varying degrees. The ROS content increased slowly within 2 h after the cells were treated with each component of the extract, increased rapidly at 4 - 8 h of treatment, and reached the peak at 8 h. There were significant differences in the ROS content among the treatment groups. After 8 h of treatment with the glycoside-form extract, a high content of ROS was generated in the cells, approximately 5 times that of the blank group. Followed by the esterified form, the insoluble-bound form, and the free form. Among them, the ROS content generated after treatment with the glycoside-form extract was significantly different statistically from that generated after treatment with the esterified form and the free form (p < 0.05). The trend of ROS content increase was similar to the change trend of DNA and protein leakage, indicating that the treatment of cells with the four forms of polyphenols could promote the accumulation of intracellular ROS. The ROS level of Hafnia alvei treated with linalool increased significantly, and similar results were also found in Escherichia coli treated with silver nanoparticles.

[0076] (6) Effect on cell membrane integrity To further observe the effects of different forms of polyphenols on the cell membrane integrity of Pseudomonas fragi, Calcein-AM and PI were used as fluorescent dyes in this study to observe the cell membrane damage under a laser confocal microscope. Calcein-AM has good hydrophobicity and can easily penetrate the cell membrane of living cells. Calcein-AM is hydrolyzed by esterase to Calcein, emitting strong green fluorescence and remaining inside the cell membrane. PI is a red dye that cannot penetrate the intact cell membrane. Only when the cell membrane is damaged can PI enter the cell and bind to DNA to emit fluorescence. The fluorescence color may emit red, orange or yellow according to the degree of cell membrane damage. The effects of the four components of the extract on cell membrane integrity are as Figure 6 shown. The results show that the blank group (a) and the positive control group (b) show a large amount of green fluorescence and a small amount of red fluorescence, indicating that most cells have intact cell membranes. After the cells were treated with the four components for 8 h, the intensity of red fluorescence increased significantly, and only a small amount of green fluorescence remained. Among them, the fluorescence density of the insoluble-bound component treatment group (f) was low, indicating that the number of cells in this treatment group was less than that of other treatment groups. The possible reason is that the insoluble-bound component has a strong effect and completely destroys the cell membrane, and then the cells are broken into fragments. Similar results were found in the studies of Houttuynia cordata Thunb. crude extract on Cronobacter sakazakii and carvacrol on Escherichia coli and Salmonella.

[0077] (7) Observation of cell morphology by transmission electron microscopy To further observe the cell damage, transmission electron microscopy was used for observation. Figure 7 shows the morphological changes of the cells after being treated with the four forms. The images show that the cell surfaces of the blank group and the positive control group are smooth, the cytoplasm inside the cells is uniform, and no cell damage is shown. After the cells were treated with the free-form extract (c), the rod-shaped structure of the cells was deformed, the internal substances flowed out, and the cells aggregated in large numbers; after the cells were treated with the MIC glycoside-form extract (d), the cell boundaries were blurred, the cell membrane was ruptured, and a large amount of intracellular components leaked; after the cells were treated with the MIC esterified-form extract (e), the cell membrane was significantly ruptured, the contents aggregated and partially flowed out; after the cells were treated with the insoluble-bound form extract (f), the cells showed adhesion, the cytoplasm flowed out, and the normal cell morphology could hardly be distinguished. These results indicate that the pepper polyphenol extract can reduce cell viability and further cause cell death by changing cell morphology, destroying the integrity of the cell membrane and releasing intracellular substances to varying degrees. The above experimental results show that different components of the pepper polyphenol extract have good antibacterial effects on Pseudomonas fragi.

[0078] In summary, the MIC and MBC of the insoluble-bound form of polyphenols are the lowest, indicating the best antibacterial ability, followed by the esterified form, and the free form has the worst antibacterial ability. The particle size distribution shows that treating the bacterial suspension with esterified and free forms of polyphenols leads to bacterial aggregation, which may be due to cell membrane rupture and cell adhesion, ultimately resulting in an increase in the particle size of the bacterial suspension. After treatment with the four forms of polyphenols at different time points, significant leakage of cellular nucleic acids and proteins occurs, and the intracellular ROS level increases significantly. By observing the cell morphology through live-dead cell staining and transmission electron microscopy, after treatment with the four polyphenols, a large number of cells die, and the cell morphology shows varying degrees of deformation, cell membrane rupture, and cytoplasmic outflow. All the index data increase significantly after 8 h of treatment. The above results indicate that the natural polyphenol extract exhibits a slow inhibitory effect and can induce apoptosis by disrupting the integrity of the cell membrane and generating ROS, suggesting that Piper polyphenols have the activity to inhibit Pseudomonas fragi.

[0079] The above examples use fresh Piper fruits as raw materials, and through the described extraction method, different forms of Piper polyphenols are extracted. After analysis, the insoluble-bound form of Piper polyphenols with the highest inhibitory activity against Pseudomonas fragi is obtained. The insoluble-bound form of Piper polyphenols can be further applied to the inhibition of Pseudomonas fragi, such as in the application as a preservative for refrigerated foods. For example, as a preservative for cream, eggs, or meat products, the addition amount of the insoluble-bound form of Piper polyphenols in the preservative can be more than 40%, such as 40%, 50%, 60%, 70%, 80%, 90%, etc. Necessary components can also be combined as needed in the preservative.

[0080] Finally, it should be noted that the above examples 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 foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features. However, such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting pepper polyphenols having Pseudomonas aeruginosa inhibitory activity, characterized in that: Includes steps: (1) preparing a crude polyphenol aqueous solution, and adjusting the pH value of the crude polyphenol aqueous solution to 1-3 with an acid, adding an equal volume of ethyl acetate and shaking sufficiently, and then separating and collecting a first organic phase and a first aqueous phase, wherein the first organic phase is free-form pepper polyphenol; (2) adding an antioxidant to the first aqueous phase, performing alkaline hydrolysis under an inert atmosphere, adjusting the pH value to 1-3 with an acid, adding an equal volume of ethyl acetate and shaking sufficiently, and then separating and collecting a second organic phase and a second aqueous phase, wherein the second organic phase is the esterified form of pepper polyphenols; (3) adding an acid to the second aqueous phase to adjust the pH value to 1-3, and performing acid hydrolysis at 75-90° C., cooling to room temperature, and adjusting the pH value to 1-3 with a base, adding an equal volume of ethyl acetate and shaking sufficiently, and then separating and collecting a third organic phase and a third aqueous phase, wherein the third organic phase is the glycoside form of pepper polyphenol; (4) filtering the third aqueous phase, adding alkali to the filter residue, and performing secondary alkaline hydrolysis under an inert atmosphere; adjusting the pH value to 1-3 with acid, centrifuging and collecting the supernatant, adding an equal volume of ethyl acetate and shaking sufficiently, and separating and collecting the fourth organic phase, which is the insoluble-bound form of pepper polyphenols.

2. The extraction method according to claim 1, characterized in that The crude polyphenol solution is prepared by the following method: freeze-dried pepper powder is used as a raw material, and a 70% vol methanol aqueous solution is added at an addition amount of 9-11 mL / g, ultrasonic extraction is performed, and the filtrate is filtered and the solvent is removed by rotary evaporation; Preferably, the temperature during the ultrasonic extraction is 20-25°C, and the ultrasonic time is 1.5-3 h; Preferably, the freeze-dried pepper powder is obtained by crushing fresh pepper fruits and freeze-drying; Preferably, the fresh pepper fruit is selected from 200-220 days after flowering, 230-250 days after flowering, and 260-270 days after flowering; Preferably, the fresh pepper fruit is picked 200-220 days after flowering.

3. The extraction method according to claim 1 or 2, characterized in that The reaction temperature of the primary alkaline hydrolysis and the secondary alkaline hydrolysis is 20-25°C, and the reaction time is 3-5h; Preferably, the reaction time of the acid hydrolysis is 20-40 min; Preferably, the inert atmosphere is selected from at least one of a nitrogen atmosphere, a helium atmosphere, and an argon atmosphere; Preferably, the antioxidant is a 3-5M alkaline solution of ethylenediaminetetraacetic acid containing 0.5-2% of component A ascorbic acid and 9-11 mM of component B; wherein component A is selected from at least one of ascorbic acid, esterified vitamin C, and acerola cherry powder, component B is selected from at least one of ethylenediaminetetraacetic acid, tetrasodium glutamate diacetate, and methylglycine diacetate, and the alkaline solution is selected from at least one of sodium hydroxide solution and potassium hydroxide solution; Preferably, the acid is selected from 5-7 M hydrochloric acid; Preferably, the base is selected from 3-5M sodium hydroxide.

4. The extraction method according to any one of claims 1 to 3, characterized in that Repeat steps (1) to (4) 4 to 6 times, respectively combine the first organic phase, the second organic phase, the third organic phase and the fourth organic phase to obtain different forms of pepper polyphenols with different Pseudomonas aeruginosa inhibitory activities.

5. An insoluble-bound pepper polyphenol having Pseudomonas aeruginosa inhibitory activity, characterized in that: The extract is obtained by the extraction method according to claims 1-4.

6. A free form pepper polyphenol having Pseudomonas aeruginosa inhibitory activity, characterized in that: The extract is obtained by the extraction method according to claims 1-4.

7. An esterified form of pepper polyphenol having Pseudomonas aeruginosa inhibitory activity, characterized in that: The extract is obtained by the extraction method according to claims 1-4.

8. A glycoside-form pepper polyphenol having Pseudomonas aeruginosa inhibitory activity, characterized in that: The extract is obtained by the extraction method according to claims 1-4.

9. A method for analyzing the inhibitory activity of pepper polyphenols against Pseudomonas aeruginosa, characterized in that: The method according to any one of claims 1 to 4 is used to extract different forms of pepper polyphenols from fresh pepper fruit, and the different forms of pepper polyphenols are subjected to at least one of the following conditions: minimum inhibitory concentration determination, minimum bactericidal concentration determination, bacterial suspension particle size determination, bacterial suspension nucleic acid leakage determination, Pseudomonas fragariae protein leakage determination, Pseudomonas fragariae intracellular active oxygen content determination, cell membrane integrity and cell morphology observation.

10. Use of the pepper polyphenols according to any one of claims 5 to 8 in inhibiting Pseudomonas aeruginosa; preferably in a refrigerated food preservative; Preferably, the refrigerated food is selected from at least one of cream, eggs and meat; Preferably, the preservative comprises more than 40% of the insoluble-bound pepper polyphenols according to claim 5.

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