Pithecellobium clypearia polysaccharide nano-emulsion as well as preparation method and application thereof

Abstract

The invention discloses pithecellobium clypearia polysaccharide nano-emulsion as well as a preparation method and application thereof, and belongs to the technical field of nano-emulsion preparation. The oil-in-water pithecellobium clypearia polysaccharide nano-emulsion (PCBP-NM) is prepared by taking protein as an emulsifier, taking pithecellobium clypearia polysaccharide as an emulsifier and a stabilizer and taking ocimum gratissimum essential oil as an oil phase on the basis of an ultrasonic-assisted high-energy method. A detection result shows that the PCBP-NM provided by the invention has good fluidity and stability, and shows remarkable tolerance and stability in an adverse environment. A free radical scavenging experiment result shows that the PCBP-NM has outstanding antioxidant activity. Bacteriostatic and bactericidal experiment results show that PCBP-NM has remarkable inhibiting and killing effects on various bacteria, and has broad-spectrum antibacterial activity. In a word, the pithecellobium clypearia polysaccharide nano-emulsion prepared by the method has extremely high application value.

Description

Technical Field

[0001] The invention relates to the technical field of nanoemulsion preparation, in particular to a monkey earring polysaccharide nanoemulsion and a preparation method and application thereof. Background Art

[0002] Nanoemulsion is a stable two-phase system formed by the action of emulsifiers, in which one phase is tightly dispersed in the other phase in the form of tiny droplets, mainly including water-in-oil and oil-in-water types. Its formulation is highly flexible, including both the free combination of multiple components such as surfactants, liquid lipids and even drug conjugates, and the flexible combination of preparation processes. This feature provides convenience for the design of innovative nanoemulsions with high application value. However, nanoemulsions are thermodynamically unstable systems, and their applications are limited by physical and chemical instabilities. Stabilizers need to be added to improve the stability of the emulsion, thereby improving the bioavailability or bioactivity of the nanoemulsion. The choice of emulsifier in nanoemulsion plays a vital role in the stability of its system and its release and absorption in the digestive system. Due to its amphiphilicity, biocompatibility and biosafety, food-grade proteins have been widely used in food processing and the preparation of nanoemulsions, including sodium caseinate (SC), whey glycoprotein (WGP), soy protein isolate (SPI), etc. Protein molecules form a continuous film between oil droplets and water droplets through their own electrostatic effects and amphiphilicity, preventing the accumulation of oil droplets, thus forming a stable nanoemulsion system. Polysaccharides have the characteristics of strong intermolecular interactions, strong stability, superior biological activity, strong hydrophilicity and viscosity, which can change the rheological properties of emulsions and improve the stability of emulsions. They have been widely used in the preparation of emulsions at all levels. More and more studies have shown that plant polysaccharides such as regenerated cellulose, gum arabic, soybeans, and tea polysaccharides can form covalent and non-covalent complexes with protein emulsifiers to improve the physical and chemical stability of nanoemulsions under various conditions.

[0003] Pithecellobium clypearia Benth is a plant of the genus Pithecellobium in the Fabaceae family, widely distributed in tropical Asia. Related studies have shown that Pithecellobium clypearia Benth polysaccharides (PCBPs) have the characteristics of low molecular weight, strong antioxidant, strong hypoglycemic, strong hypolipidemic and high potential value, which can not only improve the physical and chemical stability of nanoemulsions, but also bring them excellent biological activity. Summary of the invention

[0004] The purpose of the present invention is to provide a monkey earring polysaccharide nanoemulsion and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The monkey earring polysaccharide nanoemulsion prepared by the present invention has good fluidity and stability, strong environmental tolerance, and outstanding antioxidant activity and antibacterial activity.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a method for preparing a monkey earring polysaccharide nanoemulsion, comprising the following steps: mixing protein powder with water, stirring evenly, refrigerating overnight, adding monkey earring polysaccharide, stirring evenly, and obtaining a polysaccharide-protein solution;

[0007] The polysaccharide protein solution is mixed with clove basil essential oil, stirred evenly, and sheared to obtain a crude emulsion;

[0008] The coarse emulsion is ultrasonically crushed to obtain the monkey earring polysaccharide nanoemulsion.

[0009] Preferably, the mass volume ratio of the protein powder to water is 1 g:49 mL; the protein powder is any one of sodium caseinate, whey glycoprotein, soy protein isolate and collagen; the concentration of monkey earring polysaccharide in the polysaccharide protein solution is 1.0-4.0 mg / mL; the ultrasonic crushing is to ultrasonically treat the crude emulsion at 25kHz, 315W in an ice bath; and the time of the ultrasonic crushing is 4-15 min.

[0010] Preferably, the protein powder is whey glycoprotein; the concentration of monkey earring polysaccharide in the polysaccharide protein solution is 2.0 mg / mL; and the ultrasonic crushing time is 15 min.

[0011] Preferably, the mass volume ratio of the clove basil essential oil to the polysaccharide protein solution is 1 g:15 mL; and the shearing is performed at 14000 rpm for 7 min.

[0012] The invention also provides a monkey earring polysaccharide nanoemulsion obtained according to the preparation method.

[0013] The present invention also provides an application of the monkey earring polysaccharide nanoemulsion in preparing health food and / or anti-oxidation medicine that are helpful for anti-oxidation.

[0014] The invention also provides a health food that helps to resist oxidation, comprising the monkey earring polysaccharide nanoemulsion.

[0015] The invention also provides an antioxidant medicine, comprising the monkey earring polysaccharide nanoemulsion.

[0016] The invention also provides an application of the monkey earring polysaccharide nanoemulsion in preparing an antibacterial and bactericidal agent.

[0017] The invention also provides a bacteriostatic and sterilizing product, comprising the monkey earring polysaccharide nanoemulsion.

[0018] The present invention discloses the following technical effects:

[0019] The invention prepares a monkey earring polysaccharide nanoemulsion (PCBP-NM). The test results show that PCBP-NM has good fluidity and stability, exhibits significant tolerance and stability in adverse environments such as temperature (30-80°C), salinity (50-300mmol / L), storage time (4°C and 25°C, 30 days), and has a wide pH tolerance range. The free radical scavenging experiment results show that PCBP-NM has good scavenging effects on DPPH and ABTS· + The free radicals all have strong scavenging ability, and their scavenging rate shows a positive correlation trend with the concentration, indicating that PCBP-NM has outstanding antioxidant activity. The MIC and MBC experimental results show that PCBP-NM has significant inhibitory and bactericidal effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella paratyphi B and Bacillus subtilis, and has broad-spectrum antibacterial activity. In short, the monkey earring polysaccharide nanoemulsion prepared by the present invention has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a statistical diagram of the effect of ultrasound time on the particle size and PDI of monkey earring polysaccharide nanoemulsion performance;

[0022] Figure 2 This is a statistical diagram of the effect of monkey earring polysaccharide on the performance of nanoemulsion; where A: particle size, B: PDI, C: Zeta potential;

[0023] Figure 3 Statistical diagram of the viscosity properties of monkey earring polysaccharide nanoemulsion; wherein, A: PCBP-0NM, B: PCBP-1NM, C: PCBP-2NM, D: PCBP-4NM;

[0024] Figure 4 It is a statistical diagram of the viscosity and elastic modulus of the monkey earring polysaccharide nanoemulsion;

[0025] Figure 5 It is a statistical diagram of the effect of temperature on the performance of monkey earring polysaccharide nanoemulsion; wherein, A: PCBP-0NM, B: PCBP-1NM, C: PCBP-2NM, D: PCBP-4NM;

[0026] Figure 6 It is a statistical diagram of the effect of salinity on the performance of monkey earring polysaccharide nanoemulsion; wherein, A: PCBP-0NM, B: PCBP-1NM, C: PCBP-2NM, D: PCBP-4NM;

[0027] Figure 7 It is a statistical diagram of the effect of pH on the performance of monkey earring polysaccharide nanoemulsion; wherein, A: PCBP-0NM, B: PCBP-1NM, C: PCBP-2NM, D: PCBP-4NM;

[0028] Figure 8 Statistical diagram of the effect of storage time at different temperatures on PCBP-NM particle size; A: 4°C, B: 25°C;

[0029] Fig. 9 The scavenging rate curves of PCBP-NM and Vc on DPPH free radical (A) and ABTS free radical (B). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The reagents and strains used in the present invention are shown in Table 1:

[0036] Table 1 Main reagents and strain information

[0037]

[0038] The preparation method of monkey earring polysaccharide used in the present invention is as follows:

[0039] 1. Take fresh monkey earring branches and leaves produced in Yunfu City, Guangdong Province, dry them naturally, crush them, and pass them through a 40-mesh sieve to obtain monkey earring powder.

[0040] 2. Take 100 g of monkey earring powder, add deionized water at a liquid-to-solid ratio of 30 mL / g, and extract monkey earring polysaccharide in an ultrasonic cleaner (frequency of 40 kHz) at an ultrasonic power of 300 W and an ultrasonic time of 30 min.

[0041] 3. The monkey earring polysaccharide extract was filtered through a Buchner funnel, centrifuged (5500rpm, 10min), and rotary evaporated to obtain a monkey earring polysaccharide extract with a higher concentration.

[0042] 4. Add 1 / 4 volume of Sevag reagent (the volume ratio of chloroform and n-butanol is 4:1) of monkey earring polysaccharide extract, shake vigorously, centrifuge (5500rpm, 10min) to remove the middle white protein layer, repeat 2-3 times until no white flocs appear, and collect the supernatant.

[0043] 5. Add 4 times the volume of anhydrous ethanol to the supernatant while stirring. Polysaccharide precipitation can be observed. Let it stand in a 4°C refrigerator overnight.

[0044] 6. After centrifugation (4500rpm, 5min), the monkey earring polysaccharide was collected, reconstituted with a small amount of distilled water, transferred to a dialysis bag (Mw 3500Da) and dialyzed for 48h, and finally vacuum freeze-dried to obtain the monkey earring polysaccharide.

[0045] Example 1

[0046] In this example, an oil-in-water nanoemulsion (PCBP-NM) was prepared by ultrasound-assisted high-energy method, using monkey earring polysaccharides (PCBPs) as emulsifiers and stabilizers, different types of proteins as emulsifiers, and clove basil essential oil as the encapsulated oil phase. The effects of protein type, ultrasound time and PCBPs concentration on the performance of PCBP-NM were investigated, and the fluid properties of PCBP-NM were analyzed by rheometer.

[0047] 1. Experimental Methods

[0048] 1.1 Preparation of monkey earring polysaccharide nanoemulsions with different concentrations

[0049] Accurately weigh 0.2g of protein powder, add 9.8mL of deionized water, and stir at 1000rpm in a constant temperature magnetic stirrer for 4h. Put it in a 4℃ refrigerator overnight to ensure that the protein is fully hydrated. Then add monkey earring polysaccharide to make 0, 1.0, 2.0, 4.0mg / mL polysaccharide protein solution, and stir at 1000rpm in a constant temperature magnetic stirrer for 1h. Add clove basil essential oil (1:15, w / v) and stir at 1000rpm in a constant temperature magnetic stirrer for 1h to mix it thoroughly. A crude emulsion was obtained by shearing at 14000 rpm for 7 min using a high-speed shearing machine. Finally, the crude emulsion was prepared into monkey earring polysaccharide nanoemulsions (PCBP-NM) of different concentrations (0, 1.0, 2.0, 4.0 mg / mL) using an ultrasonic cell disruptor (25 kHz, 315 W) in an ice bath. According to the concentration of monkey earring polysaccharide, they were named PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM.

[0050] 1.2 Single Factor Experiment

[0051] 1.2.1 Screening of protein emulsifier types

[0052] The ultrasonic time was set to 10 min, and the concentration of monkey earring polysaccharide was set to 0 and 2.0 mg / mL, and the effect of emulsifier types (sodium caseinate, whey glycoprotein, soy protein isolate and collagen) on the performance of PCBP-NM was investigated.

[0053] 1.2.2 Screening of ultrasound time

[0054] Using whey glycoprotein (WGP) as an emulsifier and monkey earring polysaccharide concentration of 2.0 mg / mL, the effect of ultrasonic time (0, 2, 4, 6, 8, 10, 15 min) on the performance of PCBP-NM was investigated.

[0055] 1.2.3 Screening of monkey earring polysaccharide concentration

[0056] Using whey glycoprotein (WGP) as an emulsifier and the ultrasonic time set to 15 min, the effect of monkey earring polysaccharide concentration (0, 1.0, 2.0, 4.0 mg / mL) on the performance of PCBP-NM was investigated.

[0057] 1.3 Potential analysis of nanoemulsion particle size

[0058] To avoid multiple scattering effects, PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM were diluted 150 times and then their average particle size, dispersion coefficient and Zeta potential were measured.

[0059] 1.4 Rheological properties of nanoemulsions

[0060] The fluid properties of PCBP-NM were measured using a DHR-3 rheometer (TA USA). -1 The apparent viscosity of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM was measured in the steady-state flow mode at 25°C with an impact shear pressure of 1Pa and an angular frequency of 0.1-10 rad s -1 The linear viscoelastic viscosity of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM is determined within the range and the measurement process is in dynamic frequency sweep mode.

[0061] 2. Experimental results

[0062] 2.1 Single factor screening results

[0063] 2.1.1 Screening of emulsifier types

[0064] As shown in Table 2, the emulsifying ability of collagen (CG) is poor, the average particle size of PCBP-0NM (CG) is 51723.3nm, and after adding monkey earring polysaccharide, the average particle size of PCBP-2NM (CG) is 577.1nm, which shows that monkey earring polysaccharide has an emulsifying effect and can emulsify and stabilize nanoemulsions to a certain extent. Compared with CG, the three protein emulsifiers, soybean protein isolate (SPI), whey glycoprotein (WGP) and sodium caseinate (SC), all have good emulsifying effects. The PDI values ​​of nanoemulsions prepared by SPI, WGP and SC are not much different, indicating that the three nanoemulsions have good dispersibility. By comparing the particle size, it can be seen that the PCBP-NM particle size obtained by SC as an emulsifier is larger, exceeding 300nm, exceeding the general particle size range of nanoemulsions; the PCBP-NM particle size obtained by SPI is close to 300nm, which also exceeds the general particle size range of nanoemulsions; the PCBP-NM particle size obtained by WGP as an emulsifier is the smallest, which meets the particle size requirements of nano solutions. By comparing the Zeta potential value, it can be seen that: except CG, the absolute potential values ​​of the nanoemulsions obtained by other protein emulsifiers are all above 18.0mV, and the potential values ​​of PCBP-2NM are all higher than PCBP-0NM, which shows that after adding monkey earring polysaccharides, the stability of the nanoemulsion is better. Comprehensive PDI value, particle size and Zeta potential value, PCBP-NM obtained by WGP as an emulsifier has better performance, smaller particle size, higher bioavailability, and better nanoemulsion stability. In summary, the present invention selects WGP as a protein emulsifier for subsequent experiments.

[0065] Table 2 Various properties of PCBP-NM stabilized by different proteins

[0066]

[0067] 2.2.2 Screening of ultrasound time

[0068] Depend on Figure 1 It can be seen that when the ultrasonic time is 0min, the average particle size of PCBP-NM is close to 400nm, and when the ultrasonic time is 2min, the average particle size of PCBP-NM is close to 235nm. Thereafter, with the increase of ultrasonic time, the average particle size of PCBP-NM decreases to 220nm and remains stable; the PDI of PCBP-NM also decreases with the increase of ultrasonic time and eventually tends to be stable. This shows that PCBP-NM with smaller particle size and better dispersion performance can be successfully prepared by ultrasonic-assisted method, and the continuous increase of ultrasonic time will not cause PCBP-NM to degrade, and its particle size and PDI remain stable. In summary, the ultrasonic time of 15min was selected for subsequent experiments.

[0069] 2.2.3 Effect of monkey earring polysaccharide concentration on the properties of nanoemulsions

[0070] Depend on Figure 2 As shown in Figures A and B, compared with PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM, the particle size of PCBP-NM increases slightly with the increase of polysaccharide concentration, and its PDI value decreases first and then increases. This may be because, at low concentrations, the synergistic emulsification of polysaccharides and proteins promotes the formation of more stable polysaccharide nanoemulsions with better dispersion properties. At high concentrations, the excess polysaccharide molecules may increase the particle size and PDI due to self-aggregation and complexation with proteins.

[0071] The absolute value of the Zeta potential represents the stability of the nanoemulsion. Figure 2 As shown in Figure C, the potential values ​​of PCBP-1NM, PCBP-2NM and PCBP-4NM are all higher than that of PCBP-0NM, which indicates that the stability of the nanoemulsion is improved after the addition of monkey earring polysaccharide. The potential value of PCBP-4NM is lower than that of PCBP-2NM, which may be due to the high concentration of polysaccharide, which causes electrostatic complexation with whey glycoprotein, resulting in a decrease in the Zeta potential value. In summary, when the concentration of monkey earring polysaccharide is 2.0 mg / mL, the stability of the prepared PCBP-2NM is the best.

[0072] 2.3 Rheological properties of PCBP-NM

[0073] Depend on Figure 3 It can be seen that the greater the shear rate of the four PCBP-NMs, the lower their apparent viscosity. -1 The boundary point is the apparent viscosity decreasing trend before this point. This may be because with the increase of shear rate, the flocs formed by the droplets in the velocity field gradually deform or even decompose, resulting in a decrease in apparent viscosity. After this point, the apparent viscosity is infinitely close to 0. This may be because the flocs are completely decomposed or the rates of their formation and decomposition are equal, maintaining relative stability. The stability of the emulsion system can be reflected by rheological properties. The principle is that the movement speed of oil droplets in the water phase decreases with the increase of the viscosity of the nanoemulsion, which hinders the further interaction between the emulsifier and the oil droplets, thereby affecting the stability of the nanoemulsion system. At the same time, at the same shear rate, with the increase of the concentration of monkey ear ring polysaccharide, the viscosity of the nanoemulsion continues to decrease, which shows that the addition of monkey ear ring polysaccharide can improve the viscosity of the emulsion, improve the stability of the oil phase molecules, promote the interaction between the oil phase molecules, proteins, and monkey ear ring polysaccharides in the system, and improve the overall bioavailability and biological activity of the nanoemulsion.

[0074] Figure 4 The ordinate in represents tanδ (viscous modulus / elastic modulus). Figure 4It can be seen that in the angular frequency range of 0-10rad / s, the tanδ value of PCBP-0NM is always less than 1, which indicates that the elastic modulus of PCBP-0NM is greater than its viscous modulus, and its main property is elasticity. For PCBP-1NM, as the angular frequency increases, the overall trend of its tanδ value is continuously decreasing. When the angular frequency is <1.39rad / s, its tanδ>1, viscous modulus>elastic modulus, at this time PCBP-1NM mainly exhibits viscosity, and when the angular frequency>1.39rad / s, PCBP-1NM mainly exhibits elasticity. For PCBP-2NM, its tanδ value fluctuates around 1, indicating that it exhibits both elasticity and viscosity. For PCBP-4NM, its main property is viscosity.

[0075] In summary, after adding monkey earring polysaccharide, the viscosity of the nanoemulsion was significantly reduced, the ratio of the viscous modulus to the elastic modulus was significantly increased, and the overall bioavailability of the nanoemulsion was also improved accordingly.

[0076] Example 2

[0077] This example tests the tolerance and stability of PCBP-NM under different environments.

[0078] 1. Experimental Methods

[0079] 1.1 Effect of temperature on PCBP-NM performance

[0080] 2 mL of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM were placed in 10 mL glass vials and then treated at different temperatures (30, 40, 50, 60, 70, 80 °C) for 1.0 h. After the nanoemulsions were cooled to room temperature, their average particle size and dispersion coefficient were measured.

[0081] 1.2 Effect of pH on the properties of PCBP-NM

[0082] Take 0.5 mL of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM, add 2.0 mL of 0.01 mM PBS buffer solution with different pH values ​​(2, 3, 4, 5, 6, 7, 9, 11), stir at 100 rpm for 5 h, and measure the average particle size and dispersion coefficient.

[0083] 1.3 Effect of salinity on PCBP-NM performance

[0084] 3 mL of NaCl solution with different concentrations (50, 100, 150, 200, 250, 300 mM) was added to an equal volume of PCBP-NM and stirred at 100 rpm for 2 h, and the average particle size and dispersion coefficient were measured.

[0085] 1.4 Effect of storage time on PCBP-NM performance

[0086] PCBP-NM was stored at 4°C and 25°C for 30 days, and samples were taken at the same time every 10 days to measure the average particle size.

[0087] 2. Experimental results

[0088] 2.1 Effect of temperature on the properties of nanoemulsion

[0089] Depend on Figure 5 It can be seen from the AD that in the temperature range of 30-80℃, the particle size fluctuation ranges of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM are 217.3-205.1nm, 218.9-209.9nm, 226.7-209.3nm and 234.6-223.7nm, respectively, indicating that the temperature has little effect on the average particle size of PCBP-NM. When the temperature is high, the particle size of the nanoemulsion is slightly reduced. This may be because the thermodynamic motion inside the nanoemulsion is more intense at high temperature, which affects the non-covalent interaction between the oil phase molecules, protein molecules and polysaccharide molecules. From the PDI analysis, the PDI values ​​of PCBP-1NM, PCBP-2NM and PCBP-4NM are generally lower than those of PCBP-0NM, indicating that in the high temperature system, the addition of monkey earring polysaccharide can effectively maintain the stability of the nanoemulsion. In summary, PCBP-NM can maintain good performance at higher temperatures.

[0090] 2.2 Effect of salinity on the properties of nanoemulsions

[0091] Depend on Figure 6 It can be seen that the four PCBP-NMs can maintain a relatively stable state in different salinities, and their average particle size and PDT do not change much. As the salinity increases, the ionic charge in the solution becomes higher and higher, which reduces the electrostatic repulsion at the oil-water interface, but the particle size and PDI values ​​of the four PCBP-NMs remain stable, which indicates that the interaction between the emulsifier and the oil phase and the water phase in the four PCBP-NMs is stronger than the charge force brought by the salt ions, and has a strong ability to resist changes in salt ion concentration.

[0092] 2.3 Effect of pH on the properties of nanoemulsion

[0093] Depend on Figure 7As shown in A, B, C and D, the particle size and PDI of the four PCBP-NMs remained stable after adding buffer solutions of pH 4, 5, 6, 7, 9 and 11. When a buffer solution of pH 3 was added, the properties of the nanoemulsion mutated. The average particle size of PCBP-0NM jumped from 219.1nm to 622.0nm, and the PDI was as high as 0.33±0.04; the average particle size of PCBP-1NM increased from 225.3nm to 375.1nm, and the PDI was 0.23±0.02; the average particle size of PCBP-2NM increased from 218.5nm to 270.5nm, and the PDI was 0.23±0.01; the average particle size of PCBP-4NM increased from 226.1nm to 316.4nm, and the PDI was 0.14±0.01. When a buffer solution with a pH of 2 is added, the PDI of PCBP-0NM rises to 0.23 ± 0.06, and the PDI of PCBP-1NM, PCBP-2NM and PCBP-4NM remains stable. It can be concluded that the ability of the nanoemulsion to resist changes in environmental pH value is enhanced by adding monkey ear ring polysaccharide, and the effect is more significant as the concentration of polysaccharide increases. When a buffer solution with a pH of 3 is added, the pH value in the solution system is close to the isoelectric point of whey glycoprotein, resulting in the denaturation of the protein emulsifier and the enlargement of the particle size of the nanoemulsion. The added monkey ear ring polysaccharide itself has an emulsifying effect, and the negative charge it carries can be complexed with the H+ part in an acidic environment, maintaining the stability of the nanoemulsion, thereby making the emulsion system have a wider pH tolerance range and improving the application value of the nanoemulsion.

[0094] 2.4 Effect of storage days on the properties of nanoemulsions

[0095] Generally speaking, as the storage time increases, the microbial activity in the emulsion system and the thermal motion between molecules will lead to the denaturation of the nanoemulsion. Figure 8 It can be seen that when the storage temperature is 4 or 25°C, the particle sizes of the four PCBP-NMs remain stable, which indicates that PCBP-NM has good storage properties.

[0096] Example 3

[0097] In this example, the scavenging ability of DPPH and ABTS free radicals was used as an indicator to detect and compare the antioxidant activities of PCBP-0NM, PCBP-1NM, PCBP-2NM, and PCBP-4NM, and to explore the effect of monkey earring polysaccharide on the antioxidant activity of nanoemulsion.

[0098] 1. Experimental Methods

[0099] 1.1DPPH free radical scavenging experiment

[0100] 2mL of sample solution with different concentrations (40-200μg / mL) was mixed with an equal volume of DPPH-ethanol solution (0.1mmol / L). After the mixed solution was protected from light for 30 minutes, the absorbance of each group was measured at 517nm, and ascorbic acid (Vc) was used as a positive control. The sample group consisted of 2mL DPPH and an equal volume of sample solution, the control group consisted of 2mL anhydrous ethanol and an equal volume of sample solution, and the blank group consisted of 2mL DPPH and an equal volume of anhydrous ethanol. The DPPH free radical scavenging rate was calculated as follows:

[0101]

[0102] 1.2 ABTS free radical scavenging experiment

[0103] ABTS solution (7.0 mM) and potassium persulfate (2.45 mM) were mixed in equal volumes, and the mixture was reacted at 4°C in the dark for 12-16 h to obtain ABTS radical cation (ABTS· + ). Then dilute ABTS with PBS buffer (pH 7.2). + The solution was prepared so that the absorbance at 734 nm was 0.70 ± 0.02. 1.0 mL of samples of different concentrations were mixed with 3.0 mL of ABTS· + The solutions were mixed and reacted in the dark at room temperature for 6 min. The absorbance was measured at 734 nm. Ascorbic acid (Vc) was used as a positive control. + and 1 mL sample solution, the control group consisted of 3 mL PBS + and 1 mL sample solution, and the blank group consisted of 3 mL ABTS· + and 1mL PBS solution. The ABTS free radical scavenging rate is calculated as follows:

[0104]

[0105] 2. Experimental results

[0106] 2.1DPPH free radical scavenging experiment

[0107] Depend on Fig. 9 As shown in Figure A, within the range of 40-200 μg / mL, PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM all have strong scavenging capacity for DPPH, and the scavenging capacity increases with the concentration. By comparison, it can be concluded that under the same concentration conditions, the scavenging capacity of the nanoemulsion is: PCBP-4NM>PCBP-2NM>PCBP-1NM>PCBP-0NM. This indicates that the addition of monkey earring polysaccharide can improve the DPPH free radical scavenging activity of the nanoemulsion.

[0108] 2.2 ABTS free radical scavenging experiment

[0109] Depend on Fig. 9 As shown in Figure B, within the range of 20-100 μg / mL, PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM all had an effect on ABTS· + It has a strong scavenging ability, and increases with the increase of concentration. At the same time, under the same concentration conditions, the scavenging rate of the nanoemulsion is: PCBP-4NM>PCBP-2NM>PCBP-1NM>PCBP-0NM. This shows that the addition of monkey earring polysaccharide can improve the ABTS free radical scavenging activity of the nanoemulsion.

[0110] Example 4

[0111] In this example, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella paratyphi B and Bacillus subtilis were determined by the two-fold dilution method to evaluate the in vitro antibacterial activity of PCBP-NM.

[0112] 1. Experimental Methods

[0113] 1.1 Preparation of culture medium and resuscitation and activation of bacterial strains

[0114] Nutrient broth (NB) and Mueller-Hinton agar (MHA) media contain the basic nutrients required for the growth and reproduction of most bacteria, and are used for bacterial recovery, activation and preservation. Subsequent antibacterial experiments use Mueller-Hinton broth (MHB) media. Stir the agar and hydrolyzed casein peptone (MH) broth media powder in the conical flask under heating conditions until they are completely dissolved, autoclave at 121°C for 20 minutes, and pour into a sterile culture dish after the temperature drops to 50°C for subsequent experiments.

[0115] On the clean bench, four single colonies stored in glycerol cryopreservation tubes were picked up by inoculation loops and placed in NB liquid culture medium. Then, the four liquid culture mediums were cultured with an air-powered shaker and cultured at a speed of 120 r / min for 8-10 hours. The temperature was set to 37°C. After the culture medium became turbid, it was placed in a refrigerator at 4°C for later use. Before the experiment, the liquid culture medium was suspended in sterile saline and diluted with sterile saline to prepare a bacterial suspension with a turbidity of 0.5 McFarland, which is approximately 10 8 CFU / mL, and then dilute the bacterial suspension to 10 5 -10 6 CFU / mL for subsequent experiments.

[0116] 1.2 Determination of MIC and MBC

[0117] The MIC and MBC tests of PCBP-NM were based on relevant literature with appropriate modifications. Levofloxacin hydrochloride was diluted to 8.0 μg / mL with sterile water in a sterile clean bench. 0.5 g / mL PCBP-NM was prepared with sterile water. Using the two-fold dilution method, 0.5 g / mL was diluted to 0.9765 mg / mL in wells 1-10 of a 96-well plate, and then 50 μL of bacterial suspension (approximately 1×10 5 CFU / mL), 150 μL of culture medium was added to well 11, and 100 μL of culture medium and 50 μL of bacterial solution (about 1×10 5 CFU / mL). Place the 96-well plate in a 37°C incubator for 6 h. The sample concentration corresponding to the first well that begins to change color is the MIC.

[0118] Pipette 50 μL of the culture medium from all the wells without color change in the 96-well plate and spread it evenly on the solid culture medium. Then place the plate in a 37°C incubator and culture it for 24 hours. Observe whether there is bacterial growth in the culture dish. The lowest sample concentration corresponding to the culture dish with no bacterial growth is MBC.

[0119] 2. Experimental results

[0120] 2.1 MIC of monkey earring polysaccharide nanoemulsion

[0121] As shown in Table 3, the minimum inhibitory concentration of PCBP-NM against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella paratyphi B, and Bacillus subtilis is 31.25 mg / mL.

[0122] Table 3 MIC statistics of PCBP-NM

[0123]

[0124]

[0125] 2.2 MBC of monkey earring polysaccharide nanoemulsion

[0126] As shown in Table 5, the minimum bactericidal concentration of PCBP-NM for Escherichia coli, Staphylococcus aureus and Bacillus subtilis is 31.25 mg / mL. The minimum bactericidal concentration of PCBP-NM for Salmonella paratyphi type B and Pseudomonas aeruginosa is 62.50 mg / mL. Staphylococcus aureus and Bacillus subtilis are Gram-positive bacteria, Salmonella paratyphi type B and Pseudomonas aeruginosa are Gram-negative bacteria, and Escherichia coli is a Gram-negative bacterium whose structure is easily destroyed. Therefore, the experimental results show that PCBP-NM has a broad-spectrum antibacterial effect, and its antibacterial effect on Gram-positive bacteria is more significant.

[0127] Table 4 MBC statistics of PCBP-NM

[0128]

[0129] The experimental results show that PCBP-0NM, PCBP-1NM, PCBP-2NM and PCBP-4NM have significant inhibitory and bactericidal effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella paratyphi B and Bacillus subtilis, have broad-spectrum antibacterial effects, and their antibacterial effects on Gram-positive bacteria are more significant.

[0130] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a monkey earring polysaccharide nanoemulsion, characterized in that: The following steps are involved: Mix the protein powder with water, stir evenly, refrigerate overnight, add monkey earring polysaccharide, stir evenly, and obtain a polysaccharide-protein solution; The polysaccharide protein solution is mixed with clove basil essential oil, stirred evenly, and sheared to obtain a crude emulsion; The coarse emulsion is ultrasonically crushed to obtain the monkey earring polysaccharide nanoemulsion.

2. The preparation method according to claim 1, characterized in that The mass volume ratio of the protein powder to water is 1 g:49 mL; the protein powder is any one of sodium caseinate, whey glycoprotein, soy protein isolate and collagen; The concentration of monkey earring polysaccharide in the polysaccharide protein solution is 1.0-4.0 mg / mL; The ultrasonic crushing is to ultrasonically treat the crude emulsion in an ice bath; the time of the ultrasonic crushing is 4-15 min; the frequency of the ultrasonic treatment is 25 kHz; the power of the ultrasonic treatment is 315 W.

3. The preparation method according to claim 2, characterized in that: The protein powder is whey glycoprotein; The concentration of monkey earring polysaccharide in the polysaccharide protein solution is 2.0 mg / mL; The ultrasonic disruption time is 15 min.

4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the clove basil essential oil to the polysaccharide protein solution is 1g:15mL; The shearing is performed at 14000 rpm for 7 minutes.

5. A monkey earring polysaccharide nanoemulsion obtained according to the preparation method according to any one of claims 1 to 4.

6. Use of the monkey earring polysaccharide nanoemulsion according to claim 5 in the preparation of antioxidant health foods and / or antioxidant medicines.

7. A health food that helps with anti-oxidation, characterized in that: The invention comprises the monkey earring polysaccharide nanoemulsion according to claim 5.

8. An antioxidant drug, characterized in that: The invention comprises the monkey earring polysaccharide nanoemulsion according to claim 5.

9. Use of the monkey earring polysaccharide nanoemulsion as claimed in claim 5 in the preparation of an antibacterial and bactericidal agent.

10. A bacteriostatic and sterilizing product, characterized in that: The invention comprises the monkey earring polysaccharide nanoemulsion according to claim 5.

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