A broad-spectrum antibacterial natural active ingredient nanoemulsion, its preparation method and application

Abstract

This invention provides a broad-spectrum antibacterial natural active ingredient nanoemulsion, its preparation method, and its application, relating to the field of food preservation technology. The preparation method of the broad-spectrum antibacterial natural active ingredient nanoemulsion provided by this invention includes the following steps: mixing soy protein isolate and citrate buffer to obtain an aqueous phase solution; mixing thymol, eugenol, ginger essential oil, spearmint oil, and soybean oil to obtain an oil phase solution; mixing the aqueous and oil phase solutions and performing shearing treatment to obtain a crude emulsion; and subjecting the crude emulsion to ultrasonic disruption to obtain the broad-spectrum antibacterial natural active ingredient nanoemulsion. This invention can improve the stability and water solubility of active ingredients such as eugenol, thymol, carvone, limonene, and α-soybeanene, improve their antibacterial properties, and effectively extend the shelf life of refrigerated prepared meat products.

Description

Technical Field

[0001] This invention relates to the field of food preservation technology, specifically to a broad-spectrum antibacterial natural active ingredient nanoemulsion, its preparation method, and its application. Background Technology

[0002] my country is a major consumer of meat products, and due to their convenience and excellent flavor, consumer demand for prepared meat products is increasing daily. Based on processing conditions and storage performance, prepared meat products can be divided into refrigerated and frozen types. Refrigerated prepared meat products should be stored and transported at temperatures between 0 and 4°C, but they are susceptible to microbial contamination and lipid and protein oxidation, leading to quality degradation and a shorter shelf life. In recent years, nanoemulsion encapsulation methods have gradually attracted attention. Nanoemulsions typically have a particle size of less than 200 nm, possessing high surface activity, good adsorption properties, and thermodynamic stability, and have been widely used in the food, chemical, and pharmaceutical industries. In the food sector, nanoemulsions are frequently used to encapsulate pigments, preservatives, probiotics, and other substances, not only improving their stability during storage but also providing sustained-release and controlled-release properties.

[0003] Plant essential oils and phenolic antioxidants are secondary metabolites of plants extracted twice, possessing strong antibacterial and antioxidant properties. Among them, eugenol, the main antibacterial component in clove essential oil, has been proven to have excellent bactericidal and antioxidant properties due to the phenolic hydroxyl groups in its chemical structure. Thymol, extracted from plants such as thyme and oregano, has monotert-phenol as its main active ingredient, exhibiting significant antibacterial, antioxidant, and antimutagenic properties. The main active ingredients in spearmint essential oil, carvone and limonene, as well as the main active ingredients in ginger essential oil, α-shogaene, β-sesquicaprene, and camphene, have all been proven to have strong antibacterial properties. However, the sulfur and nitrogen compounds in these essential oils produce a pungent odor, severely affecting the sensory appeal of the products. Furthermore, plant phenols and essential oils are easily affected by environmental factors such as light, heat, or extreme pH, resulting in a loss of biological activity and reduced bioavailability, thus hindering the widespread application of natural plant-based antibacterial agents in food. Summary of the Invention

[0004] The purpose of this invention is to provide a broad-spectrum antibacterial natural active ingredient nanoemulsion, its preparation method and application. This invention can improve the stability and water solubility of active ingredients such as eugenol, thymol, carvone, limonene, and α-gingerene, improve their antibacterial properties, and effectively extend the shelf life of refrigerated prepared meat products.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing a nanoemulsion of a broad-spectrum antibacterial natural active ingredient, comprising the following steps:

[0007] Soy protein isolate and citrate buffer were mixed to obtain an aqueous solution;

[0008] Thymol, eugenol, ginger essential oil, spearmint oil and soybean oil are mixed to obtain an oil phase solution;

[0009] The aqueous and oil phase solutions are mixed and subjected to shearing to obtain a crude emulsion.

[0010] The crude emulsion was subjected to ultrasonic disruption to obtain a nanoemulsion containing natural active ingredients with broad-spectrum antibacterial properties.

[0011] Preferably, the citric acid concentration in the citric acid buffer is 1-2% by mass; and the pH of the citric acid buffer is 3.5-4.0.

[0012] Preferably, the mass concentration of soy protein isolate in the aqueous solution is 0.5% to 2.5%.

[0013] Preferably, the oil phase solution contains 0.3-0.8% thymol, 0.05-0.1% eugenol, 0.05-0.1% ginger essential oil, and 2-6% spearmint oil.

[0014] Preferably, the volume ratio of the oil phase solution to the aqueous phase solution is 3-15:85-97.

[0015] Preferably, the high-speed shearing machine used in the shearing process has a rotation speed of 8000-12000 rpm, and the shearing process takes 2-5 minutes.

[0016] Preferably, the power of the ultrasonic fragmentation treatment is 20 kHz; the ultrasonic fragmentation treatment program is 3 seconds of operation followed by 3 seconds of interval; and the total ultrasonic duration of the ultrasonic fragmentation treatment is 8 to 16 minutes.

[0017] This invention provides a broad-spectrum antibacterial natural active ingredient nanoemulsion prepared by the preparation method described in the above technical solution.

[0018] This invention provides the application of the broad-spectrum antibacterial natural active ingredient nanoemulsion described in the above technical solution in preservatives.

[0019] Preferably, the broad-spectrum antibacterial natural active ingredient nanoemulsion is used as a preservative for the antibacterial and preservation of refrigerated prepared meat products.

[0020] This invention provides a method for preparing a nanoemulsion of natural active ingredients with broad-spectrum antibacterial properties. In this invention, the adsorption and stability properties of the nanoemulsion not only encapsulate the pungent odor of essential oils, reducing their impact on the product's sensory experience, but also improve the stability of essential oils and phenolic preservatives. This invention uses eugenol, thymol, ginger essential oil, and spearmint oil, which have inhibitory effects on the growth of meat spoilage microorganisms, coliform bacteria, Escherichia coli, and Pseudomonas, as natural active ingredients. These are encapsulated in the prepared nanoemulsion and applied to refrigerated prepared meat products to achieve broad-spectrum antibacterial, antioxidant, and shelf-life-extending effects.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) The nanoemulsion containing thymol, eugenol, ginger essential oil and spearmint oil prepared by the present invention has high stability, small particle size and good dispersion performance, and its particle size can reach 153.57nm.

[0023] (2) The nanoemulsion containing thymol, eugenol, ginger essential oil and spearmint oil prepared by the present invention has been proven to have broad-spectrum antibacterial properties through experiments. In the inhibition zone test, it has strong antibacterial properties against total bacterial count, coliform bacteria, Escherichia coli and Pseudomonas fluorescens.

[0024] (3) This invention successfully encapsulates thymol, eugenol, ginger essential oil and spearmint oil, which improves the stability of these bioactive components and prevents the volatilization of irritating odors. This is of great significance for the application of bioactive components in the food industry.

[0025] (4) In the application of this invention in chilled prepared steak, the modified atmosphere packaging chilled prepared steak coated with natural active ingredient nanoemulsion showed significantly lower values ​​for thiobarbituric acid reactants, volatile basic nitrogen, and total bacterial count compared to chilled prepared steak without nanoemulsion coating (p < 0.05) under storage conditions of 4°C. Furthermore, the shelf life was extended from 9 days to 14 days, with the total bacterial count decreasing from 6.10 log CFU / g to 5.57 log CFU / g on day 9. Attached Figure Description

[0026] Figure 1 Particle size and PDI value of natural active ingredient nanoemulsions at different soybean protein concentrations (0.5%, 1%, 1.5%, 2%, 2.5%); lowercase letters indicate significant differences between groups, p < 0.05;

[0027] Figure 2 Particle size and PDI value of natural active ingredient nanoemulsions with different water-oil phase ratios; lowercase letters represent significant differences between groups, p < 0.05;

[0028] Figure 3 The results of the inhibition zone experiments on (a) total bacterial count, (b) Escherichia coli CICC 10305 and (c) Pseudomonas fluorescens CICC 21620 were obtained from the natural active ingredient nanoemulsion and the blank control group.

[0029] Figure 4 The inhibition zone diameters of the natural active ingredient nanoemulsion and the blank control group were compared to the total bacterial count, Escherichia coli CICC10305, and Pseudomonas fluorescens CICC 21620. Lowercase letters indicate significant differences between groups, p < 0.05.

[0030] Figure 5 The total bacterial count of chilled prepared steak coated with natural active ingredient nanoemulsion and the control group at 4℃ on days 0, 4, 9 and 14; lowercase letters indicate significant differences between groups, p < 0.05;

[0031] Figure 6 The volatile basic nitrogen values ​​of chilled prepared steak coated with natural active ingredient nanoemulsion and the control group were measured at 4°C on days 0, 4, 9, and 14. Lowercase letters indicate significant differences between groups, p < 0.05.

[0032] Figure 7 The values ​​of thiobarbituric acid reactants in chilled prepared steak coated with a nanoemulsion of natural active ingredients and in the control group at 4°C on days 0, 4, 9, and 14 are given; lowercase letters indicate significant differences between groups, p < 0.05. Detailed Implementation

[0033] This invention provides a method for preparing a nanoemulsion of a broad-spectrum antibacterial natural active ingredient, comprising the following steps:

[0034] Soy protein isolate and citrate buffer were mixed to obtain an aqueous solution;

[0035] Thymol, eugenol, ginger essential oil, spearmint oil and soybean oil are mixed to obtain an oil phase solution;

[0036] The aqueous and oil phase solutions are mixed and subjected to shearing to obtain a crude emulsion.

[0037] The crude emulsion was subjected to ultrasonic disruption to obtain a nanoemulsion containing natural active ingredients with broad-spectrum antibacterial properties.

[0038] This invention involves mixing soy protein isolate and citrate buffer to obtain an aqueous solution. In this invention, the preferred mass concentration of citric acid in the citrate buffer is 1-2%; the preferred pH of the citrate buffer is 3.5-4.0, more preferably 3.6-3.7. The preferred method for preparing the citrate buffer in this invention includes: adding citric acid to deionized water and magnetically stirring at room temperature for 0.5 h to obtain the citrate buffer.

[0039] In this invention, the mixing of soy protein isolate and citrate buffer is preferably carried out under stirring conditions, more preferably under magnetic stirring. In this invention, the mixing time is preferably 3 hours; the mixing temperature is preferably room temperature.

[0040] In this invention, the mass concentration of soy protein isolate in the aqueous solution is preferably 0.5-2.5%, more preferably 1.0-2.0%, and even more preferably 1.5%.

[0041] This invention involves mixing thymol, eugenol, ginger essential oil, spearmint oil, and soybean oil to obtain an oil phase solution. In this invention, the preferred mass concentration of thymol in the oil phase solution is 0.3–0.8%, more preferably 0.5–0.6%; the preferred mass concentration of eugenol is 0.05–0.1%, more preferably 0.07–0.08%; the preferred mass concentration of ginger essential oil is 0.05–0.15%, more preferably 0.08–0.12%; and the preferred mass concentration of spearmint oil is 2–6%, more preferably 3–4%.

[0042] In this invention, the mixing of thymol, eugenol, ginger essential oil, spearmint oil, and soybean oil is preferably carried out under stirring conditions, more preferably by magnetic stirring. In this invention, the mixing time is preferably 0.5 hours; the mixing temperature is preferably room temperature.

[0043] After obtaining the aqueous and oil phase solutions, the present invention mixes the aqueous and oil phase solutions and performs shearing treatment to obtain a crude emulsion. In the present invention, the volume ratio of the oil phase solution to the aqueous phase solution is preferably 3-15:85-97, more preferably 6:94.

[0044] In this invention, the rotation speed of the high-speed shearing machine used for the shearing process is preferably 8000-12000 rpm, more preferably 9000-10000 rpm; the shearing process time is preferably 2-5 min, more preferably 3-4 min.

[0045] After obtaining the crude emulsion, the present invention subjectes the crude emulsion to ultrasonic disruption to obtain a nanoemulsion of natural active ingredients with broad-spectrum antibacterial properties. In the present invention, the power of the ultrasonic disruption treatment is preferably 20 kHz; the ultrasonic disruption treatment program is preferably 3 seconds of operation followed by 3 seconds of pause; the total ultrasonic disruption treatment duration is preferably 8–16 min, more preferably 10–13 min.

[0046] This invention provides a broad-spectrum antibacterial natural active ingredient nanoemulsion prepared by the preparation method described in the above technical solution. In this invention, the natural active ingredients in the broad-spectrum antibacterial natural active ingredient nanoemulsion include thymol, eugenol, ginger essential oil, and spearmint oil.

[0047] This invention provides the application of the broad-spectrum antibacterial natural active ingredient nanoemulsion described in the above-mentioned technical solution in preservatives, preferably as a preservative for the antibacterial and preservation of refrigerated prepared meat products. In this invention, the preferred method of application is to apply the natural active ingredient nanoemulsion to the surface of the refrigerated prepared meat product. In this invention, the application amount of the natural active ingredient nanoemulsion is preferably 5-10%, more preferably 6-8%. In this invention, the application amount refers to the percentage of the mass of the natural active ingredient nanoemulsion to the mass of the refrigerated prepared meat product.

[0048] The natural active ingredient nanoemulsion prepared by this invention can significantly inhibit the growth of meat-derived spoilage microorganisms, coliform bacteria, Escherichia coli, and Pseudomonas, exhibiting broad-spectrum antibacterial properties. Simultaneously, it solves the problems of strong odor and poor stability associated with plant-derived natural active ingredients. When applied to refrigerated prepared meat products, this natural active ingredient nanoemulsion can effectively extend their shelf life, showing broad application prospects in the food preservation field.

[0049] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] In the embodiments of the present invention, the eugenol reagent has a eugenol content of ≥98.5%, the thymol reagent is of analytical grade, and the ginger essential oil and spearmint oil have a purity of 100%.

[0051] Example 1

[0052] Optimization of soy protein isolate concentration in nanoemulsions based on particle size and PDI (particulate density).

[0053] (1) Add citric acid to deionized water and adjust the pH to 3.6 to obtain citric acid buffer.

[0054] (2) Add 0.15g, 0.3g, 0.45g, 0.6g and 0.75g of soy protein isolate to 30mL of citrate buffer, respectively, and stir magnetically for 3h at room temperature to obtain aqueous solutions with concentrations of 0.5%, 1%, 1.5%, 2% and 2.5%, respectively.

[0055] (3) Add 30.86 mg of thymol, 3.73 mg of eugenol, 0.005 mL of ginger essential oil and 0.2 mL of spearmint oil to 5 mL of soybean oil, and stir magnetically for 0.5 h at room temperature to obtain an oil phase solution.

[0056] (4) Mix 28.2 mL of aqueous solution with 1.8 mL of oil solution and shear at 10000 r / min for 3 min using a high-speed shearing machine to obtain crude emulsion.

[0057] (5) The crude emulsion was ultrasonically broken up at 20 kHz for 13 min with a cycle of 3 s running and 3 s intermittent to obtain a natural active ingredient nanoemulsion.

[0058] The natural active ingredient nanoemulsion prepared above was diluted with citrate buffer and the average particle size and PDI value were determined using a Malvern particle size analyzer. The refractive index was 1.33 and the emission wavelength was 633 nm.

[0059] The results are as follows Figure 1 As shown, the particle size and PDI value of the natural active ingredient nanoemulsions prepared at different soy protein isolate concentrations showed significant differences (p < 0.05). It is known that the smaller the average particle size of the emulsion, the more stable the system. When the soy protein isolate concentration was 1.5%, the emulsion had the smallest particle size, reaching 153.8 nm, at which point the PDI value was 0.10. Therefore, the optimal soy protein isolate concentration was 1.5%, and this was used as the optimal concentration for subsequent studies.

[0060] Example 2

[0061] Optimization of the ratio of aqueous and oil phase solutions for natural active ingredient nanoemulsions based on particle size and PDI.

[0062] (1) Add citric acid to deionized water and adjust the pH to 3.6 to obtain citric acid buffer.

[0063] (2) Add 0.45g of soy protein isolate to 30mL of citrate buffer and stir magnetically for 3h at room temperature to obtain an aqueous solution with a concentration of 1.5%.

[0064] (3) Add 30.86 mg of thymol, 3.73 mg of eugenol, 0.005 mL of ginger essential oil and 0.2 mL of spearmint oil to 5 mL of soybean oil, and stir magnetically for 0.5 h at room temperature to obtain an oil phase solution.

[0065] (4) The aqueous solution and the oil solution were mixed at volume ratios of 85:15, 88:12, 91:9, 94:6 and 97:3 respectively, and the mixture was sheared for 3 minutes at a speed of 10000 r / min using a high-speed shearing machine to obtain a crude emulsion.

[0066] (5) The crude emulsion was broken up by ultrasonication at 20 kHz for 13 min, with a cycle of 3 s running and 3 s intermittent to obtain a natural active ingredient nanoemulsion.

[0067] The natural active ingredient nanoemulsion prepared above was diluted with citrate buffer and the average particle size and PDI value were determined using a Malvern particle size analyzer. The refractive index was 1.33 and the emission wavelength was 633 nm.

[0068] The results are as follows Figure 2 As shown, the particle size and PDI value of the natural active ingredient nanoemulsions prepared under different water-oil phase ratios exhibit significant differences (p < 0.05). The emulsion with the smallest particle size (163.7 nm) and PDI value (0.09) is achieved when the water-oil phase ratio is 94:6. Therefore, the optimal water-oil phase ratio is 94:6, and this ratio will be used for subsequent studies.

[0069] Example 3

[0070] Study on the antibacterial properties of natural active ingredient nanoemulsions against total bacterial count, Escherichia coli, and Pseudomonas fluorescens.

[0071] (1) Add citric acid to deionized water and adjust the pH to 3.6 to obtain citric acid buffer.

[0072] (2) Add 0.45g of soy protein isolate to 30mL of citrate buffer and stir magnetically for 3h at room temperature to obtain an aqueous solution with a concentration of 1.5%.

[0073] (3) Add 30.86 mg of thymol, 3.73 mg of eugenol, 0.005 mL of ginger essential oil and 0.2 mL of spearmint oil to 5 mL of soybean oil, and stir magnetically for 0.5 h at room temperature to obtain an oil phase solution.

[0074] (4) Mix the aqueous solution and the oil solution at a volume ratio of 94:6, and shear them for 3 minutes at a speed of 10000 r / min using a high-speed shearing machine to obtain a crude emulsion.

[0075] (5) The crude emulsion was broken up by ultrasonication at 20 kHz for 13 min, with a cycle of 3 s running and 3 s intermittent to obtain a natural active ingredient nanoemulsion.

[0076] Test Example 1

[0077] A total bacterial count of 1.0 × 10⁻⁶ was selected. 6 CFU / g beef samples were homogenized and pounded, and the bacterial culture was collected for later use. Additionally, *Escherichia coli* (CICC 10305) and *Pseudomonas fluorescens* (CICC 21620) were activated by second-generation culture and cultured to a concentration of 1.0 × 10⁻⁶. 6 CFU / mL. Take 100 μL of the above bacterial suspensions (total bacterial count suspension, Escherichia coli suspension, and Pseudomonas fluorescens suspension) and spread them onto different nutrient agar plates.

[0078] Take 10 μL of the natural active ingredient nanoemulsion prepared in Example 3 onto a 6 mm filter paper and place it on a plate containing bacterial solution. Use the nanoemulsion without the addition of natural active ingredients (eugenol, thymol, ginger essential oil, spearmint oil) as a blank control.

[0079] The plates with total bacterial count and CICC 10305 were incubated at 37℃ for 48 h and 24 h, respectively, while the plates with CICC 21620 were incubated at 28℃ for 48 h. After incubation, the diameter of the inhibition zone was measured.

[0080] Figure 3 (a) shows the inhibition zone plate results of the natural active ingredient nanoemulsion (experimental group) and blank control group on total bacterial count. In (a), the four regions, clockwise from the upper right corner, are blank control, experimental group, experimental group, and experimental group, respectively. (b) shows the inhibition zone plate results of the natural active ingredient nanoemulsion and blank control group on Escherichia coli CICC 10305. In (b), the four regions, clockwise from the upper right corner, are blank control, experimental group, experimental group, and experimental group, respectively. (c) shows the inhibition zone plate results of the natural active ingredient nanoemulsion and blank control group on Pseudomonas fluorescens CICC 21620. In (c), the four regions, clockwise from the upper right corner, are blank control, experimental group, experimental group, and experimental group, respectively. Figure 3 The results showed that the natural active ingredient nanoemulsion prepared in this invention had a good inhibitory effect on total bacterial count, Escherichia coli, and Pseudomonas fluorescens. Escherichia coli usually reflects the processing environment and the quality of raw meat, while Pseudomonas fluorescens is a major spoilage bacterium in meat under refrigeration conditions; therefore, inhibiting this type of bacteria can effectively extend the shelf life of meat products. Figure 4As shown, compared with the blank control group, the diameter of the inhibition zone in the experimental group was significantly higher than that in the blank control group. The diameters of the inhibition zones for total colony count, CICC 10305 and CICC 21620 were 1.36 cm, 1.27 cm and 1.11 cm, respectively, indicating that the natural active ingredient nanoemulsion has good broad-spectrum antibacterial properties.

[0081] Application examples

[0082] Application of natural active ingredient nanoemulsions in chilled prepared steaks

[0083] (1) After slicing the beef sirloin into 2cm pieces, add salt, white sugar, monosodium glutamate, sodium carbonate, sodium tripolyphosphate and water and vacuum tumble and marinate for 2 hours.

[0084] (2) A natural active ingredient nanoemulsion was prepared according to the preparation method in Example 3;

[0085] The natural active ingredient nanoemulsion was then applied to the surface of the vacuum-tumbled steak (experimental group), with an application amount of 8% of the steak's mass. The prepared steak, which was not coated with the natural active ingredient nanoemulsion, served as the control group.

[0086] (3) The steak was packaged in high oxygen modified packaging and refrigerated at 4°C. Samples were taken on days 0, 4, 9 and 14 to test the total bacterial count, volatile basic nitrogen and thiobarbituric acid reactant values.

[0087] (4) At the sampling time mentioned in step (3), take 25g of meat sample and add 225mL of physiological saline with a mass fraction of 0.85% for homogenization and tapping. Take 1mL of diluted bacterial solution and add it to a plate. Pour it into plate counting agar using the pouring method. Incubate at 37℃ for 48h and then count.

[0088] (5) At the sampling time mentioned in step (3), take 20g of meat sample, chop it, pour it into 100mL of deionized water, let it stand for 30min, and then filter it for later use. Add 1mL of boric acid and 1 drop of mixed indicator to the center of a Conway dish, add 1mL of saturated K2CO3 solution to the outer chamber of the dish, seal the dish, and place it in a constant temperature incubator at 37℃ for 2h. Titrate with hydrochloric acid standard solution. Record the volume of hydrochloric acid titrant consumed V1 (mL) and the concentration C (mol / L). The volume of hydrochloric acid consumed by the blank sample (deionized water) is V2 (mL). The result is calculated as follows: Volatile basic nitrogen content (mg / 100g) = (V1-V2)×C×140000÷m 肉样 (g)

[0089] (6) At the sampling time mentioned in step (3), take 10g of meat sample and add 50mL of 7.5% trichloroacetic acid solution, shake for 30min, filter, add 5mL of 0.02mol / L thiobarbituric acid solution, incubate at 90℃ for 40min, centrifuge, take the supernatant, add 5mL of chloroform, let stand, and take the supernatant for colorimetric analysis at 532nm and 600nm, recording the absorbance A532 and A600. The result is calculated as: TBA (mg / kg) = (A 532 -A 600 )×4684÷m 肉样 (g)

[0090] Total bacterial count results as follows Figure 5 As shown, the total bacterial count in both the experimental and control groups increased with prolonged storage. On day 4, the total bacterial count was low in both groups, so there was no significant difference between them. On day 9, the total bacterial count in the prepared steak with the added natural active ingredient nanoemulsion was significantly lower than that in the control group (p < 0.05), at only 5.57 log CFU / g, while the total bacterial count in the control group was 6.10 log CFU / g, indicating spoilage. On day 14, the total bacterial count in the experimental group steak reached 6.11 log CFU / g, also indicating spoilage. These results demonstrate that the active ingredient nanoemulsion effectively inhibits microbial growth, extending the shelf life of prepared steak from 9 days to 14 days at 4°C.

[0091] In the examples, the results for volatile basic nitrogen are as follows: Figure 6 As shown. Besides total bacterial count, volatile basic nitrogen (VBI) is also an indicator used to evaluate the freshness of meat and meat products. Generally, the higher the VBI value, the higher the degree of spoilage; a value exceeding 15 mg / 100g indicates the sample is near-fresh, and a value exceeding 20 mg / 100g indicates complete spoilage. Figure 6 It was found that on days 4, 9, and 14, the volatile basic nitrogen (VNI) values ​​of the control group were significantly higher than those of the experimental group (p < 0.05). The VNI value of the control group reached 15.65 mg / 100g on day 9 and 20.44 mg / 100g on day 14. The VNI value of the experimental group reached 14.98 mg / 100g on day 14. The experimental results indicate that the natural active ingredient nanoemulsion in the preservative has a good antibacterial and preservative effect on processed steaks and can effectively delay product spoilage.

[0092] In the examples, the reactant values ​​of thiobarbituric acid are as follows: Figure 7As shown, thiobarbituric acid can react with aldehyde oxidation products to form colored compounds, and is therefore commonly used to reflect the degree of fat oxidation. With increasing storage days, the amount of secondary fat oxidation products increases, and an increase in the thiobarbituric acid reactant value indicates a deeper degree of fat oxidation. On day 4, fat oxidation was relatively slow in both the experimental and control groups, with no significant difference between them. On day 14, the degree of fat oxidation in the controlled group's prepared steak was significantly deeper than that in the experimental group. At this point, the thiobarbituric acid reactant value in the control group reached 0.75 mg / kg, while that in the experimental group was 0.61 mg / kg. These experimental results demonstrate that the natural active ingredient nanoemulsion has a significant inhibitory effect on fat oxidation in refrigerated prepared steak, and has an antioxidant and preservative effect on the product.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a nanoemulsion of a broad-spectrum antibacterial natural active ingredient, comprising the following steps: Soy protein isolate and citrate buffer were mixed to obtain an aqueous solution; the mass concentration of soy protein isolate in the aqueous solution was 1.5-2%. Thymol, eugenol, ginger essential oil, spearmint oil, and soybean oil are mixed to obtain an oil phase solution; the oil phase solution contains 0.3-0.8% thymol, 0.05-0.1% eugenol, 0.05-0.1% ginger essential oil, and 2-6% spearmint oil. The aqueous solution and the oil solution are mixed and sheared to obtain a crude emulsion; the volume ratio of the oil solution to the aqueous solution is 3-6:94-97. The crude emulsion was subjected to ultrasonic disruption to obtain a nanoemulsion containing broad-spectrum antibacterial natural active ingredients.

2. The preparation method according to claim 1, characterized in that, The citrate buffer solution has a citric acid concentration of 1-2% by mass and a pH of 3.5-4.

0.

3. The preparation method according to claim 1, characterized in that, The high-speed shearing machine used in the shearing process has a rotation speed of 8000-12000 rpm, and the shearing process takes 2-5 minutes.

4. The preparation method according to claim 1, characterized in that, The ultrasonic fragmentation process has a power of 20 kHz; the ultrasonic fragmentation process consists of a 3-second run followed by a 3-second pause; and the total ultrasonic duration of the ultrasonic fragmentation process is 8–16 minutes.

5. The broad-spectrum antibacterial natural active ingredient nanoemulsion prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the broad-spectrum antibacterial natural active ingredient nanoemulsion as described in claim 5 in preservatives.

7. The application according to claim 6, characterized in that, The broad-spectrum antibacterial natural active ingredient nanoemulsion is used as a preservative for the antibacterial and preservation of refrigerated prepared meat products.

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