Bacteriostatic polyphenol extracted based on probiotic fermentation sea-buckthorn and application thereof

Abstract

The invention discloses bacteriostatic polyphenol extracted based on probiotic fermented sea-buckthorn and application of the bacteriostatic polyphenol. The polyphenol is obtained by firstly inoculating lactobacillus plantarum Lp10211 into sea-buckthorn juice for fermentation, then freeze-drying the fermented sea-buckthorn juice and extracting freeze-dried powder. The sea-buckthorn is used as a main raw material, probiotic fermentation treatment is performed, polyphenol obtained after fermentation has an antibacterial effect and can be applied to fresh keeping of fresh milk, a feasible thought is provided for exploring green, healthy and efficient antibacterial components, and meanwhile the economic additional value of the sea-buckthorn is increased.

Description

Technical Field

[0001] The invention belongs to the field of food technology, and specifically relates to antibacterial polyphenols extracted from sea buckthorn through probiotic fermentation and application thereof. Background Art

[0002] In recent years, the widespread use of antibiotics has led to the emergence of drug-resistant bacteria, posing a serious threat to public health and safety. However, various types of antibiotics must be used in food storage, agricultural production, and animal husbandry. Therefore, finding natural and safe antibacterial ingredients without the use of synthetic preservatives has become a hot topic in food preservation.

[0003] At present, many studies have found that plant secondary metabolism, such as phenolic compounds, flavonoids and tannins, have a certain inhibitory effect on microorganisms and foodborne pathogens that cause food spoilage. For example, tea polyphenols show a significant inhibitory effect on the proliferation of Gram-positive bacteria; gallic acid extracted from pomegranate peel, red wine and various vegetables has been verified to be able to block the efflux pump of multidrug-resistant Staphylococcus aureus, thereby achieving antibacterial activity. The antibacterial activity of various plant polyphenols shows a wide range of diversity and complexity, but their inhibitory mechanism is still not fully understood.

[0004] Seabuckthorn (Hippophae rhamnoides Linn.) is a plant that is both a medicine and food. It contains a variety of bioactive substances such as flavonoids, phenolic acids and polysaccharides. It is a rich source of phenolic compounds, mainly epicatechin, gallic acid, rutin and ferulic acid. Related studies have shown that seabuckthorn berry extracts have strong antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans, and its n-hexane and chloroform extracts show significant antibacterial activity comparable to vancomycin. The differences in the chemical composition, extraction method and extraction solvent contained in seabuckthorn berries will greatly affect their antibacterial activity.

[0005] Fermentation technology is one of the main methods for extracting natural preservatives from plant-based raw materials. The decomposition of cell wall structure caused by fermentation can release and / or induce the synthesis of various bioactive compounds. Among them, polyphenol substrates in plant-based raw materials will be converted into small molecule phenolic compounds after probiotic fermentation, releasing phenolic acids and flavonoids, etc., promoting the release of bound and conjugated polyphenols and improving their various functional activities.

[0006] In general, fermented plant-based raw materials (mainly fruits) are a good source of high-concentration polyphenol compounds, and the fermentation process is accompanied by the generation and transformation of various bioactive compounds. Therefore, the present invention is designed to ferment sea buckthorn with probiotics to extract polyphenols, providing a theoretical basis for the development of efficient antibacterial active ingredients. Summary of the invention

[0007] One of the purposes of the present invention is to provide a polyphenol extracted from sea buckthorn by probiotic fermentation, the preparation method of which is as follows: firstly inoculating Lactobacillus plantarum Lp10211 into sea buckthorn juice for fermentation, then freeze-drying the fermented sea buckthorn juice, and extracting the freeze-dried powder to obtain polyphenol.

[0008] Furthermore, the sea buckthorn juice is obtained by squeezing sea buckthorn and water at a mass volume ratio of 1g:3-6mL.

[0009] Furthermore, Lactobacillus plantarum Lp10211 is inoculated into sea buckthorn juice at a mass percentage of 0.01-0.1% for fermentation, and the fermentation conditions are: fermentation temperature 35-40° C., initial fermentation pH 4-6, and fermentation time 28-36 hours.

[0010] Furthermore, the specific process of extracting the lyophilized powder to obtain the antibacterial polyphenol is as follows: taking the lyophilized powder, adding 80% ethanol for ultrasonic extraction, separating the supernatant and the residue for later use; rotary evaporating the supernatant to an alcohol-free state, adding water to dissolve it, adding n-hexane to defatted, taking the aqueous phase and adding ethyl acetate to extract, and the obtained organic phase is free phenol; adding 2M NaOH to the remaining aqueous phase for alkaline hydrolysis, then adding 6M HCl to acidify to pH 2.0, adding ethyl acetate to extract, and the obtained organic phase is conjugated phenol; after alkaline hydrolysis and acidification of the residue, adding n-hexane to defatted, taking the aqueous phase and adding ethyl acetate to extract, and the obtained organic phase is bound phenol.

[0011] Furthermore, the usage ratio of the lyophilized powder and 80% ethanol is 8 g:50 mL.

[0012] The second object of the present invention is to provide the use of the above polyphenols in the preparation of antibacterial agents.

[0013] The third object of the present invention is to provide the use of the above polyphenols in the preparation of food preservatives.

[0014] Furthermore, the food is fresh milk.

[0015] The present invention uses seabuckthorn as the main raw material, carries out probiotic fermentation treatment, studies the content change and antibacterial mechanism of three-state polyphenols (free state, bound state and conjugated state) before and after fermentation, provides a theoretical basis for developing high-efficiency antibacterial active ingredients, applies them to the preservation of fresh milk, further determines their antibacterial effects, provides a feasible idea for exploring green, healthy and efficient antibacterial ingredients, and at the same time improves the economic added value of seabuckthorn. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The changes of phenolic substances in sea buckthorn juice before and after fermentation.

[0017] Figure 2 Results of changes in the content of three-state polyphenols after extraction of sea buckthorn juice before and after fermentation (A, B).

[0018] Figure 3 These are the inhibition zone results of tri-state polyphenols against Escherichia coli and Staphylococcus aureus before and after fermentation.

[0019] Figure 4 The minimum inhibitory concentration of conjugated phenol before and after fermentation (A), time-growth curve (B, C), protein leakage (D) and nucleic acid leakage (E) results.

[0020] Figure 5 Effects of conjugated phenols on Escherichia coli biofilm formation (A), cell membrane surface hydrophobicity (B), cell membrane permeability (C), and cell outer membrane permeability (D) before and after fermentation.

[0021] Figure 6 The results show the quenching effect of conjugated phenol on the main fluorophore in the Escherichia coli cell membrane protein before and after fermentation.

[0022] Figure 7 The effect of conjugated phenol on the microstructure of Escherichia coli before and after fermentation.

[0023] Figure 8 Effects of post-fermentation conjugated phenols on the acidity (A), lactose (B) and microbial proliferation (C, D, E) of milk during storage. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources, among which Lactobacillus plantarum Lp10211 was obtained from Jiangsu Junyao Life Science and Technology Development Co., Ltd.

[0027] Example 1

[0028] 1. Extraction of tri-state polyphenols before and after fermentation

[0029] In this embodiment, Lactobacillus plantarum Lp10211 was used as a seabuckthorn fermentation agent to ferment freshly squeezed seabuckthorn juice.

[0030] Preparation of freshly squeezed seabuckthorn juice: weigh 20 g of fresh seabuckthorn fruit and 80 mL of sterile water in a ratio of 1:4 (W / V), squeeze the juice and set aside.

[0031] Preparation of fermented seabuckthorn juice: The freshly squeezed seabuckthorn juice was adjusted to pH 5.12 with anhydrous sodium carbonate solution and then pasteurized (65°C, 30 min). 0.07 g (0.07%, W / W) of Lactobacillus plantarum Lp10211 was inoculated under aseptic conditions and fermented at 37°C for 32.6 h.

[0032] The phenolic components in probiotic fermented seabuckthorn juice and freshly squeezed seabuckthorn juice were determined by high performance liquid chromatography. The samples were separated on a reverse phase C18 column (4.6 mm × 250 mm, 5 μm). Before analysis, the samples were centrifuged (8000 rpm) at 4 ° C for 8 minutes, and the supernatant was filtered through a 0.45 μm PES membrane. The mobile phase consisted of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B), and the gradient program was as follows: 0-10 min, 10% B; 10-15 min, 20% B; 15-23 min, 30% B; 23-40 min, 10% B. Phenolic substances were detected by ultraviolet detector, the detection wavelength was 280 nm, the flow rate was 0.6 mL / min, and the external standard method was used to quantitatively analyze the HPLC results according to the peak retention time.

[0033] Test results such as Figure 1 As shown in the results, compared with the fresh sea buckthorn juice group (SJ), the content of phenolic compounds in the fermented (FS) group increased by 37.06%, indicating that probiotic fermentation can promote the release of phenolic compounds, among which the contents of rutin, ferulic acid and epicatechin increased by 82.37%, 23.47% and 15.17%, respectively. Studies have shown that the content of phenolic components has a characterization significance for its antibacterial potential.

[0034] Afterwards, the two groups of sea buckthorn juice were freeze-dried, and free phenol (FP), bound phenol (BP) and conjugated phenol (CP) were extracted before and after fermentation to explore the differences in the antibacterial activity of the extracts.

[0035] Freshly squeezed seabuckthorn juice and fermented seabuckthorn juice are freeze-dried to powder for later use. 8g of pure seabuckthorn freeze-dried powder and probiotic fermented seabuckthorn freeze-dried powder are taken, and 50mL of 80% ethanol is added for ultrasonic extraction 3 times, and the supernatant and the residue are separated for later use; the supernatant is rotary evaporated to an alcohol-free state, and then 10mL of water is added for redissolution, 30mL of n-hexane is added for defatting 2 times, and the aqueous phase is added with 30mL of ethyl acetate for extraction for multiple times until the organic phase is colorless to obtain free phenol (organic phase); 40mL of 2M NaOH is added to the remaining aqueous phase for alkaline hydrolysis for 4h, and then 6M HCl is added for acidification to pH 2.0, and ethyl acetate is added for extraction for multiple times until the organic phase is colorless to obtain conjugated phenol (organic phase); the residue is subjected to alkaline hydrolysis and acidification, and then 30mL of n-hexane is added for defatting 2 times, and the aqueous phase is added with ethyl acetate for extraction for multiple times until the organic phase is colorless to obtain conjugated phenol (organic phase).

[0036] The Folin-Ciocalteu colorimetric method and aluminum nitrate complex method were used to determine the content of polyphenols (calculated as gallic acid) and total flavonoids (calculated as rutin) per gram of dry weight. Figure 2 As shown in A and B, the polyphenol concentration of the CP component increased significantly after fermentation, and the polyphenol content increased by 21%; the total flavonoid content of the BP and CP components changed significantly, and the flavonoid content increased by 27% and 117%, respectively.

[0037] 2. Determination of the antibacterial activity of tri-state polyphenols before and after fermentation

[0038] Common pathogenic bacteria Escherichia coli and Staphylococcus aureus were selected as indicator strains to evaluate the antibacterial activity of samples.

[0039] The agar diffusion method was used to determine the antibacterial activity of six samples against Escherichia coli and Staphylococcus aureus. First, pour 15mL of LB solid culture medium into the plate and punch a hole after solidification. Take the bacterial solution that has been cultured for 12 hours, dilute it with sterile water, draw 200μL of the bacterial solution and add it to the solid culture medium to spread it evenly, finally add 100μL of sample solution into the hole, place the plate in a 37℃ incubator and culture it for 12 hours to observe the inhibition zone and measure the diameter of the inhibition zone. The test components include 5mg / mL tri-state polyphenol solution (sample to be tested), 50% ethanol solution (solvent control), and sterile water (blank control), among which the tri-state polyphenol solution is prepared with 50% ethanol solution. Each group of experiments was repeated three times.

[0040] Test results such as Figure 3 As shown in the figure, the CP component after fermentation has the best inhibitory ability against Escherichia coli and Staphylococcus aureus, especially the inhibition zone size of Escherichia coli can reach 3.9 cm. Therefore, the antibacterial mechanism of the CP component before and after fermentation was studied for Escherichia coli.

[0041] 3. Study on the antibacterial mechanism of conjugated phenols (CPs) on Escherichia coli before and after fermentation

[0042] 1. Determination of minimum inhibitory concentration (MIC) and time-growth curve

[0043] The minimum inhibitory concentration was determined by microdilution method. The overnight culture solution was diluted with sterile water to OD 600 =0.4, take 1mL of diluted bacterial solution and add CPs solution, so that the final concentration of CPs in the bacterial solution is 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25mg / mL, take 200μL of each component to 96-well plate, and culture at 37℃ for 10h. Determine the absorbance of the bacterial solution at 600nm before and after treatment, and the minimum concentration that significantly inhibits bacterial growth is the MIC.

[0044] The growth curve of E. coli under CPs treatment was determined based on MIC. The overnight culture solution was diluted with LB liquid medium to OD 600 =0.1. Add CPs solution to bacterial solution to make the final concentration of 0, 1 / 2, 1, 2, 4 MIC, take 200 μL to 96-well plate, culture at 37 ° C for 7 hours, and measure the absorbance of 600nm of bacterial solution every 1 hour using full wavelength microplate reader. Draw the growth curve with incubation time as the horizontal axis and absorbance at 600nm as the vertical axis.

[0045] Test results such as Figure 4 As shown in A, the antibacterial effects of post-fermentation conjugated phenol (FCP) and pre-fermentation conjugated phenol (CCP) treatments changed significantly at concentrations of 0.39 and 0.79 mg / mL, i.e., the MIC of FCP was 0.39 mg / mL and the MIC of CCP was 0.79 mg / mL. Figure 4 As shown in B and C, at the same concentration, the inhibitory effect of FCP within 7 h was significantly better than that of CCP.

[0046] 2. Impact on leakage of contents (protein, nucleic acid)

[0047] Proteins and nucleic acids are key biomacromolecules present in bacterial cell membranes and cytoplasm, and play an important role in life activities. Cell membrane damage can lead to leakage of cell contents, so the integrity of the cell membrane can be indicated by the release of proteins and nucleic acids. Take 1mL of Escherichia coli in the logarithmic growth period, centrifuge at 3000g for 3min at room temperature to collect the bacteria, add PBS buffer to wash the bacteria three times and resuspend the bacteria, add CPs solution to the bacterial suspension to make the concentration 1MIC, culture for 6h, and take samples every 1h to centrifuge and determine the content of protein and nucleic acid in the supernatant. Use a full-wavelength microplate reader to measure the absorbance of the supernatant at 260nm to determine the release of DNA, and use the SN / T 3926-2014 Coomassie Brilliant Blue method to determine the protein content.

[0048] Test results such as Figure 4 As shown in D and E. After 2 h of CPs treatment, the content of nucleic acid and protein in the E. coli suspension increased significantly (P < 0.05), and the content of nucleic acid and protein in the E. coli suspension continued to increase slowly with the increase of treatment time.

[0049] 3. Effects on biofilm formation and cell membrane surface hydrophobicity

[0050] The nonspecific adhesion of bacteria can enable bacteria to adapt to changes in the environment and promote bacterial reproduction; the nonspecific adhesion of bacteria is closely related to the hydrophobicity of their surface. Take 1 mL of Escherichia coli in the logarithmic growth phase, wash the bacteria three times with PBS buffer, and dilute to OD 600=0.5. Take 1 mL of bacterial suspension and add CPs to make the final concentration 1 / 4, 1 / 2, 1, 2, 4 MIC, add 1 mL of toluene, mix well and let stand for 30 min. After the aqueous phase and organic phase of the sample are separated, aspirate the lower aqueous phase and measure the OD 600 The component without CPs was used as blank control (A0), and the cell hydrophobicity was calculated according to the following formula.

[0051] Hydrophobicity (%) = ((A0-A1) / A0) × 100

[0052] Biofilm is a complex microbial aggregate formed in the environment that can attach to solid surfaces. The formation of biofilm has an important impact on the physiological function of microorganisms and the stability of the ecosystem. The crystal violet staining method can be used to evaluate the number of bacteria on the biofilm attachment surface. Use LB liquid medium to gradient dilute the overnight cultured Escherichia coli, dilute 1000 times, and then add CPs solution to make the final concentration 1 / 4, 1 / 2, 1, 2, 4 MIC. Take 200μL and add it to a 96-well plate and culture it at 37℃ for 48h. After the culture is completed, the bacterial suspension is aspirated, the wells are washed with PBS buffer, and air-dried for 30min. Add 200μL of 2.5% glutaraldehyde fixative to each well for 1h, aspirate the fixative, add 200μL of PBS buffer to each well for washing, air-dry for 30min, and wash 3 times in total. Add 0.1% crystal violet solution and stain for 15min. After aspirating the crystal violet solution, add 200μL of PBS buffer to each well again for washing, air-dry for 30min, and wash 3 times in total to wash away excess crystal violet. Then, 200 μL of 33% acetic acid solution was added to each well to re-dissolve the crystal violet, and the absorbance was measured at 570 nm using a full-wavelength microplate reader.

[0053] Test results such as Figure 5 As shown in A. After 48 hours of CPs treatment, there was a significant difference between the Blank group and the 1 / 4MIC group, indicating that CPs can significantly reduce the number of bacteria attached to the biofilm attachment surface and inhibit biofilm formation, and it is also concentration-dependent. Figure 5 As shown in Figure B, with the increase of FCP concentration, the surface hydrophobicity of E. coli decreased significantly, and the hydrophobicity was only 37.25% at 4MIC concentration, indicating that CPs can effectively reduce the surface hydrophobicity of E. coli, resulting in reduced adhesion and thus inhibiting the formation of biofilm. This is consistent with the results of the biofilm formation experiment.

[0054] 4. Effects on cell membrane and extracellular membrane permeability

[0055] Using ONPG as a substrate, the ability of CPs to damage bacterial cell membranes was evaluated by measuring the activity of bacterial β-galactosidase released into the culture medium. Overnight cultured bacteria were washed and resuspended in sterile PBS buffer solution (10 8 CFU / mL), take 1mL of bacterial suspension and 1mL of CPs solution of different concentrations to react for 2h, then take 1mL of supernatant and add 1mL ONPG solution (2.5mM), and use a microplate reader to measure the absorbance of the treated bacterial suspension and ONPG solution at 405nm when incubated at 37℃.

[0056] Escherichia coli is a Gram-negative bacterium with two membranes, the outer membrane and the intracellular membrane. The outer membrane mainly serves as a permeability barrier to protect bacteria from harmful compounds in the environment, such as antibiotics and bile salts. NPN is a hydrophobic fluorescent probe that remains quenched in an aqueous environment but emits strong fluorescence in a hydrophobic environment. Therefore, the increase in NPN fluorescence intensity can be used as an indicator of increased permeability of the cell outer membrane. 8 CPs solution was added to the cell suspension with a final concentration of 1 / 4, 1 / 2, 1, 2, and 4 MIC. After adding 20 μL of NPN (1 mM) fluorescent agent, the mixture was cultured at 37°C for 2 h and the fluorescence intensity was measured by a fluorescence spectrophotometer with an excitation wavelength of 350 nm and an emission wavelength of 430 nm.

[0057] Test results such as Figure 5 C. Compared with the Blank group, as the concentration of CPs increased, the absorbance increased in a dose-dependent manner, that is, the amount of β-galactosidase leaked from the cells increased, indicating that CPs would increase the permeability of the E. coli cell membrane, which is consistent with the results of content leakage. Figure 5 As shown in D, the fluorescence intensity of untreated cells is low, indicating that the outer membrane of E. coli remains intact and only very little NPN is absorbed. As time increases, the fluorescence intensity gradually increases, indicating that CPs can cause increased permeability of the cell outer membrane. The fluorescence intensity of cells treated with high concentrations of FCP is significantly higher than that of cells treated with CCP, indicating that FCP significantly damages the cell outer membrane, causing NPN to be distributed to the hydrophobic environment and increasing its fluorescence intensity.

[0058] 5. Effect on the quenching of major fluorophores in cell membrane proteins

[0059] Membrane proteins are mainly responsible for material transport, signal transduction, energy conversion, and the process of maintaining thermodynamic non-equilibrium to keep cells alive and fully function. Tryptophan, tyrosine, and phenylalanine are the main fluorophores in membrane proteins. Potassium iodide is a fluorescence quencher that can quench the surface residues of membrane proteins. It can quench their emitted fluorescence by directly binding to the residues on the surface of membrane proteins.

[0060] To the cell suspension (1×10 8 CFU / mL), added different concentrations of CPs (sample) and potassium iodide (negative control), incubated at 37°C for 1 h, and measured the changes in fluorescence intensity at emission wavelengths of 300-400 nm and excitation wavelengths of 258 nm, 280 nm and 296 nm.

[0061] Test results such as Figure 6 shown. Figure 6 A is the effect of potassium iodide on the fluorescence emission spectra of tryptophan, tyrosine and phenylalanine residues on the surface of Escherichia coli membrane proteins at fixed excitation wavelengths of 258nm, 280nm and 296nm. As the concentration of potassium iodide increases, the fluorescence intensity of tryptophan residues gradually decreases, but the fluorescence intensity of tyrosine and phenylalanine residues does not change significantly, indicating that potassium iodide cannot effectively quench tyrosine and phenylalanine residues. Figure 6 B and C represent the quenching effect of post-fermentation conjugated phenol (FCP) and fermentation prefix conjugated phenol (CCP) on Escherichia coli membrane proteins, respectively. With the increase of the concentration of the two, the fluorescence intensity of each residue gradually decreased and red-shifted, indicating that CPs would change the conformation of membrane proteins, and the degree of red shift of FCP was higher than that of CCP. At the same time, at a concentration of 4 MIC, post-fermentation conjugated phenol reduced the fluorescence intensity of tryptophan, tyrosine and phenylalanine residues by 75.2%, 83.85% and 81.37%, respectively, which was significantly better than the 38.65%, 56.84% and 36.34% achieved before fermentation.

[0062] In summary, CPs can achieve the antibacterial effect by changing the conformation of cell membrane proteins, thereby causing cell membrane damage.

[0063] 6. Effects on the microstructure of E. coli

[0064] The changes in the cell membrane of Escherichia coli after treatment with different concentrations of CPs were observed by scanning electron microscopy. Take 1mL of overnight cultured Escherichia coli, add CPs solution to make the final concentration of 1MIC and 2MIC and culture for 12h, and use the water treatment group as a blank control. After the culture is completed, the bacteria are collected by centrifugation at 3000g for 3min at room temperature, and 1mL of PBS buffer is added to wash the bacteria 3 times. Remove PBS, add 2mL of 2.5% glutaraldehyde fixative, fix at 4℃ overnight, remove the glutaraldehyde fixative, and wash the bacteria again 3 times. Dehydrate with 20%, 50%, 80%, 90%, 95%, and 100% ethanol solutions for 15min in turn, freeze-dry and send the samples for scanning electron microscopy.

[0065] Test results such as Figure 7 shown. Figure 7 A is the water treatment group, Figure 7 B and C are FCP-treated groups. Figure 7D and E are CCP treatment groups. As can be seen from the figure, the bacteria in the water treatment group have a complete cell membrane structure, but the dense structure of the E. coli cell membrane in the sample treatment group is disintegrated, the surface of the bacteria is severely damaged, and the whole is sparsely dispersed. That is, CPs will have a certain degree of influence on the formation and structure of E. coli biofilm, which is consistent with the previous results of content leakage and biofilm formation.

[0066] 3. Application of fermentation conjugated phenol (FCP) in fresh milk preservation

[0067] Next, the prepared FCP was subjected to relevant tests on the freshness characterization indicators during the storage period of fresh milk.

[0068] The E. coli cultured overnight was collected by centrifugation (4°C, 4000 g, 10 min), washed with sterile saline, resuspended in sterile saline, and added to pasteurized fresh milk until the bacterial concentration was 10 2 CFU / mL. FCP solution was added to each group of samples to make the final concentrations of 0, 1 / 2, 1, and 2 MIC. The amount added was relatively small and would not affect the taste or color of the milk. Each group of samples was incubated at 4, 25, and 37°C, and the quality indicators of the milk were analyzed at different time intervals. The acidity of milk was determined according to the Chinese national standard GB 5009.239-2016. The lactose content in milk was determined by the Fehling titration method with reference to GB 5009.7-2016. The total number of bacteria in milk was determined according to the national standard method GB 4789.2-2016.

[0069] Test results such as Figure 8 The main sugar in milk is lactose, which is easily decomposed into lactic acid by microorganisms, thereby increasing the acidity of milk. Therefore, the acidity and lactose content of fresh milk are key indicators for evaluating the freshness of fresh milk. Figure 8 A is a graph showing the change of acidity over time in each group of fresh milk samples at different storage temperatures. It can be found that storage temperature has a great influence on the change of milk acidity. At 4°C, the acidity changes very slowly due to the slow reproduction of E. coli, but the degree of change increases significantly at 25 and 37°C. And FCP can effectively reduce the increase of fresh milk acidity in a dose-dependent manner. Figure 8 As shown in Figure B, the lactose content in milk stored at different temperatures for 8 days changes. As time goes by, lactose is consumed by E. coli and shows a downward trend. Due to the inhibitory effect of FCP on E. coli, the degree of lactose decrease in the presence of FCP is significantly lower than that in the control group, that is, FCP can inhibit the reproduction of E. coli in fresh milk.

[0070] In order to further determine the effect of FCP on the preservation of fresh milk, the effect of different concentrations of FCP on the total bacterial count of milk during storage was determined. Figure 8As shown in C, D, and E, at 25 and 37°C, microorganisms multiplied rapidly, and the addition of FCP significantly inhibited the proliferation of microorganisms. This trend is consistent with the results of acidity and lactose content in milk. In addition, at 4°C, 1MIC and 2MIC concentrations had a bactericidal effect on Escherichia coli.

[0071] In conclusion, low temperature (4°C) can effectively inhibit the growth of bacteria in milk. However, at 25 and 37°C, FCP significantly reduced the total colony count in fresh milk in a dose-dependent manner, thereby reducing lactose loss and acidity increase.

[0072] The above experimental results all show that the three-state polyphenols of seabuckthorn have a certain antibacterial effect. In general, the antibacterial performance of FCP after fermentation is significantly better than that of the other groups. It achieves antibacterial effect by destroying the integrity of bacterial cell walls and membranes and inhibiting the formation of bacterial biofilms. At the same time, the fresh milk preservation experiment proves that during the storage of milk, FCP significantly delays the increase of acidity and the loss of lactose in milk by inhibiting the proliferation of bacteria. Therefore, in addition to its biological activity that promotes health, FCP also has a positive impact on food preservation and storage due to its antibacterial activity.

Claims

1. A polyphenol extracted from seabuckthorn by probiotic fermentation, characterized in that: The preparation method of the polyphenols comprises the following steps: firstly inoculating Lactobacillus plantarum Lp10211 into seabuckthorn juice for fermentation, then freeze-drying the fermented seabuckthorn juice, and extracting the freeze-dried powder to obtain the polyphenols.

2. The polyphenol according to claim 1, characterized in that The seabuckthorn juice is obtained by squeezing seabuckthorn and water at a mass volume ratio of 1g:3-6mL.

3. The polyphenol according to claim 1, characterized in that: Lactobacillus plantarum Lp10211 is inoculated into sea buckthorn juice at a mass percentage of 0.01-0.1% for fermentation. The fermentation conditions are: fermentation temperature 35-40°C, initial fermentation pH 4-6, and fermentation time 28-36 h.

4. The polyphenol according to claim 1, characterized in that The specific process of extracting the lyophilized powder to obtain the antibacterial polyphenol is as follows: taking the lyophilized powder, adding 80% ethanol for ultrasonic extraction, separating the supernatant and the residue for later use; rotary evaporating the supernatant to an alcohol-free state, adding water to dissolve it again, adding n-hexane to defatted, taking the aqueous phase and adding ethyl acetate to extract, and the obtained organic phase is free phenol; adding 2M NaOH to the remaining aqueous phase for alkaline hydrolysis, then adding 6M HCl to acidify to pH 2.0, adding ethyl acetate to extract, and the obtained organic phase is conjugated phenol; after alkaline hydrolysis and acidification of the residue, adding n-hexane to defatted, taking the aqueous phase and adding ethyl acetate to extract, and the obtained organic phase is bound phenol.

5. The polyphenol according to claim 4, characterized in that The usage ratio of lyophilized powder and 80% ethanol is 8g:50mL.

6. Use of the polyphenols according to any one of claims 1 to 5 in the preparation of antibacterial agents.

7. Use of the polyphenols according to any one of claims 1 to 5 in the preparation of food preservatives.

8. The use according to claim 7, characterized in that: The food is fresh milk.

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