A double-coated nanoemulsion of clove essential oil and its preparation method and use

Abstract

The present invention belongs to the technical field of biological preservatives, and specifically relates to a double-coated nanoemulsion of clove essential oil, which comprises clove essential oil, ε-polylysine, whey protein, lecithin, and corn starch. The present invention also provides a preparation method of the nanoemulsion and its use as a donkey meat preservative. The double-coated nanoemulsion of clove essential oil of the present invention not only retains the excellent properties of the original nanoemulsion, but also slows down the increase of pH value, the proliferation of microorganisms, and the oxidation of lipids and proteins to a certain extent, and greatly prolongs the shelf life.

Description

Technical Field

[0001] The invention belongs to the technical field of biological preservatives, and particularly relates to a double-coated nanoemulsion of clove essential oil, a preparation method thereof and use thereof as a donkey meat preservative. Background Art

[0002] Meat consumption accounts for an increasing proportion in people's diet structure, and the dosage and safety of meat additives are increasingly valued. Meat food is rich in nutrients and has a high water content, which is very easy to cause microbial growth and oxidation deterioration. Microorganisms degrade protein, fat and other components to produce volatile amines, skatole and other substances, which cause food safety problems in meat products. Microorganisms that cause meat corruption or foodborne infection mainly include Enterobacter, Salmonella, Clostridium perfringens, anaerobic Clostridium botulinum, Listeria, Staphylococcus aureus and Bacillus colitidis, which can decompose and utilize protein. Additives are needed to inhibit or kill these microorganisms during the processing, transportation and storage of meat food to prevent meat from spoiling or causing foodborne infection.

[0003] In recent years, donkey meat has become increasingly popular in daily meat consumption. Similarly, the high myoglobin content in donkey meat makes it more susceptible to color and oxidation quality deterioration. Donkey meat is richer in unsaturated fatty acids than other meats, and its high linoleic acid and linolenic acid content makes it more susceptible to fat oxidation than other animal muscles. At the same time, donkey meat has fine muscle fibers, which makes it more susceptible to muscle fiber rupture and causes microbial growth.

[0004] At present, the preservatives commonly used in meat production are mainly chemical synthetic substances such as benzoates, nitrites, and hydroxybenzoates. However, these widely used chemical preservatives are mostly carcinogenic and teratogenic to newborns. Biological preservatives have many advantages such as wide sources, small dosage, low energy consumption, low toxicity, and good effects; they also have many advantages such as bactericidal, antibacterial, antioxidant, deodorizing, giving food special flavor and physiological regulation functions. Biological preservatives are prepared by preparing one or several natural substances with antibacterial or antioxidant activity into a solution of appropriate concentration, and applying them to fresh food by soaking, spraying or coating, so as to achieve the effect of preservation. Biological preservatives are mostly produced by natural extraction or fermentation, which are efficient and safe, and have become a research hotspot in the field of meat preservation technology. Summary of the invention

[0005] In previous studies, the inventors found that the nanoemulsion of a mixture of polylysine coated with clove essential oil and carboxymethyl chitosan achieved good results in the cold storage of donkey meat, and could slow down the increase in pH, the proliferation of microorganisms, and the oxidation of lipids and proteins to a certain extent. However, the durability of the biological preservative was insufficient, mainly because the clove essential oil still had the problem of volatilization. The inventors continued to study and found that firstly, the clove essential oil was made into a Pickering emulsion, then made into a nanoemulsion by a high-pressure homogenization method, and then embedded with carboxymethyl chitosan, which could greatly extend the shelf life of the preservative, thereby completing the present invention.

[0006] Therefore, in one aspect, the present invention provides a double-encapsulated nanoemulsion of clove essential oil, the nanoemulsion comprising clove essential oil, carboxymethyl chitosan, ε-polylysine, whey protein, lecithin, and corn starch.

[0007] Furthermore, the nanoemulsion comprises, by weight, 10-20 parts of clove essential oil, 5-10 parts of ε-polylysine, 50-70 parts of carboxymethyl chitosan, 30-50 parts of whey protein, 1-2 parts of lecithin, and 2-3 parts of corn starch.

[0008] Furthermore, the nanoemulsion comprises, by weight, 15 parts of clove essential oil, 8 parts of ε-polylysine, 60 parts of carboxymethyl chitosan, 45 parts of whey protein, 1.5 parts of lecithin, and 2.5 parts of corn starch.

[0009] Furthermore, the PDI value of the nanoemulsion is 0.18-0.21.

[0010] Furthermore, after storage for 30 days, the PDI value of the nanoemulsion is 0.19-0.23.

[0011] In another aspect, the present invention provides a method for preparing the above-mentioned double-coated clove essential oil nanoemulsion, comprising:

[0012] Step 1, dissolving corn starch in deionized water to prepare a starch emulsion with a mass fraction of 5-8%, adding an equal amount of clove essential oil thereto, stirring for 5 minutes, and homogenizing at high speed to obtain a Pickering emulsion;

[0013] Step 2, preparing an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding ε-polylysine thereto, and then adding the Pickering emulsion of step 1 mixed with deionized water thereto, heating to 45-60° C., and high-speed homogenizing to obtain a nanoemulsion;

[0014] Step 3: dissolving carboxymethyl chitosan in pure water, stirring to form a transparent solution, and then adding the nanoemulsion of step 2 therein, and mixing to form a double-coated nanoemulsion of clove essential oil.

[0015] Furthermore, in step 1, the high-speed homogenization is performed using a high-speed homogenizer at a rotation speed of 19000-22000 rpm for 10 minutes.

[0016] Furthermore, in step 2, the high-speed homogenization is performed using a high-pressure homogenizer at a rotation speed of 21000-23000 rpm for 10 minutes.

[0017] In yet another aspect, the present invention provides use of the double-coated clove essential oil nanoemulsion in preserving donkey meat.

[0018] The invention is improved on the basis of a polylysine mixture nanoemulsion coated with clove essential oil and carboxymethyl chitosan. The clove essential oil is firstly made into a Pickering emulsion, then made into a nanoemulsion by a high-pressure homogenization method, and then mixed with a carboxymethyl chitosan solution to achieve double coating. The prepared donkey meat biological preservative not only retains the excellent properties of the original nanoemulsion, so as to slow down the increase of pH value, the proliferation of microorganisms and the oxidation of lipids and proteins to a certain extent, but also greatly prolongs the preservation time. DETAILED DESCRIPTION

[0019] ε-Polylysine is a homopolymer composed of 25-30 lysines obtained by fermentation of Streptomyces parvum, which can effectively inhibit Gram-negative and Gram-positive bacteria. ε-Polylysine inhibits bacterial proliferation by destroying cell walls, cell membranes and binding to DNA. ε-Polylysine and its acid salts are relatively mature in the research and industrial production in the United States, Japan and Europe. For example, CHISSO is the world's main supplier of ε-Polylysine. ε-Polylysine, as a mature preservative with good water solubility and a broad antibacterial spectrum, has long been used in meat preservation. Many scholars have found that its combination with natamycin chitosan has a more prominent antibacterial and preservation effect on meat products. Through joint research and development by universities and biological enterprises, my country has mastered the core technology of production and preparation, and in 2014, it was approved to add ε-Polylysine as a preservative to food.

[0020] Clove is an evergreen tree of the Myrtaceae family, and clove buds are one of China's famous traditional imported "southern medicines" (Zhao et al., 2006). Many studies have shown that clove essential oil (CEO) can effectively inhibit foodborne spoilage and pathogenic bacteria such as Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus (Anacarso et al., 2019; Cui et al., 2018; Zhang et al., 2017). Studies have found that CEO can effectively protect the texture of fish fillets and delay lipid oxidation in catfish fillets during cold storage (Binsi et al., 2017). Although CEO has good antibacterial and antioxidant activities, its volatility, low solubility, and aromatic properties limit its application in food preservation.

[0021] Pickering emulsion is an emulsion that uses solid nanoparticles as stabilizers, which are almost irreversibly adsorbed on the oil-water interface to form a strong interfacial film to prevent the merging of emulsion droplets. Compared with traditional emulsions, in Pickering emulsion, solid nanoparticles replace surfactants. Since the desorption energy of solid nanoparticles at the oil-water interface is very large, the emulsion formed is more stable and sufficient to protect the encapsulated essential oils. In addition, since the use of surfactants is reduced, it has lower toxicity and irritation. The nanoparticles used in Pickering emulsion are mainly divided into inorganic nanoparticles (silicon dioxide, metal particles, montmorillonite and bentonite, etc.) and organic nanoparticles (nanocellulose, starch, protein and polymer particles, etc.). The preparation technologies of Pickering emulsion include high-pressure homogenization, ultrasound, and membrane emulsification.

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Preparation Example 1

[0024] A method for preparing a double-coated nanoemulsion of clove essential oil, comprising:

[0025] Step 1, dissolving corn starch in deionized water to prepare 30 mL of 5% starch emulsion by mass, adding 30 mL of clove essential oil thereto, stirring for 5 min, and homogenizing for 10 min at a speed of 19000 rpm using a high-speed homogenizer to obtain a Pickering emulsion;

[0026] Step 2, preparing 100 mL of an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding 0.125 g of ε-polylysine thereto, and then mixing 1 mL of the Pickering emulsion of step 1 with 300 mL of deionized water and adding the mixture thereto, heating the mixture to 45-60° C., and homogenizing the mixture at a speed of 21000 rpm for 10 min to obtain a nanoemulsion;

[0027] Step 3: dissolving carboxymethyl chitosan in pure water, stirring to form a 3% transparent solution, and then adding the nanoemulsion of step 2 in a volume ratio of 1:3, and mixing to form a double-coated nanoemulsion of clove essential oil.

[0028] The above steps were repeated three times, and the particle size and polydispersity index (PDI) of the prepared double-coated nanoemulsion of clove essential oil were measured respectively, and the minimum particle sizes were 130.52±0.37 mm, 128.53±0.46 and 131.42±0.62, respectively. The above results show that the preparation method of the present invention can stably prepare the double-coated nanoemulsion of clove essential oil, and the batch-to-batch difference is small. And the PDI of the newly prepared nanoemulsion is 0.18, 0.18 and 0.21. After being stored at room temperature for 30, the PDI was measured again, which were 0.19, 0.20 and 0.23, respectively. It can be seen that the double-coated nanoemulsion of clove essential oil of the present invention can remain stable for a long time.

[0029] Comparative Example 1

[0030] Nanoemulsion made directly from clove essential oil and ε-polylysine, including:

[0031] Step 1, preparing 100 mL of an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding 0.125 g of ε-polylysine thereto, then mixing 0.5 g of clove essential oil with 300 mL of deionized water and adding the mixture thereto, heating the mixture to 45-60° C., and homogenizing the mixture at a speed of 21000 rpm for 10 min to obtain a nanoemulsion;

[0032] Step 2: dissolving carboxymethyl chitosan in pure water, stirring to form a 3% transparent solution, and then adding the nanoemulsion of step 1 in a volume ratio of 1:3, and mixing to form a nanoemulsion of coated clove essential oil.

[0033] The particle size and polydispersity index (PDI) of the prepared coated clove essential oil nanoemulsion were measured, and the minimum particle size was 112.52±0.44 mm, indicating that the particle size was smaller than that of the double-coated clove essential oil nanoemulsion of the present invention in Preparation Example 1.

[0034] The PDI of the newly prepared nanoemulsion was 0.18. After being stored at room temperature for 10 days, the PDI was measured again, and the PDI was 0.22; after being stored at room temperature for 30 days, the PDI was measured again, and the PDI was 0.34, indicating that the emulsion stability has deteriorated (it is generally believed that PDI < 0.25 is a stable emulsion).

[0035] Comparative Example 2

[0036] A double-coated nanoemulsion of clove essential oil was prepared by a method similar to that of Preparation Example 1, wherein nano-silicon dioxide was used as an emulsifier to prepare an intermediate Pickering emulsion, specifically comprising:

[0037] Step 1, 1.5 g of nano-silicon dioxide was dispersed in 30 mL of deionized water, 30 mL of clove essential oil and 0.2 g of polyvinyl pyrrolidone (stabilizer) were added thereto, stirred for 5 min, and homogenized for 10 min at a speed of 19000 rpm using a high-speed homogenizer to obtain a Pickering emulsion;

[0038] Step 2, preparing 100 mL of an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding 0.125 g of ε-polylysine thereto, and then mixing 1 mL of the Pickering emulsion of step 1 with 300 mL of deionized water and adding the mixture thereto, heating the mixture to 45-60° C., and homogenizing the mixture at a speed of 21000 rpm for 10 min to obtain a nanoemulsion;

[0039] Step 3: dissolving carboxymethyl chitosan in pure water, stirring to form a 3% transparent solution, and then adding the nanoemulsion of step 2 in a volume ratio of 1:3, and mixing to form a double-coated nanoemulsion of clove essential oil.

[0040] The particle size and polydispersity index (PDI) of the prepared double-coated clove essential oil nanoemulsion were measured, and the minimum particle size was 132.19±0.28 mm, indicating that the particle size was larger than that of the double-coated clove essential oil nanoemulsion of the present invention in Preparation Example 1.

[0041] The PDI of the newly prepared nanoemulsion was 0.21. After being stored at room temperature for 10 days, the PDI was measured again, and the PDI was 0.24; after being stored at room temperature for 30 days, the PDI was measured again, and the PDI was 0.28, and the emulsion stability became worse, but it was better than the nanoemulsion of the coated clove essential oil in Comparative Example 2 (PDI < 0.25 is generally considered to be a stable emulsion).

[0042] Comparative Example 3

[0043] A double-coated clove essential oil emulsion was prepared by a method similar to that of Preparation Example 1, wherein step 3 was no longer subjected to high-speed homogenization, and specifically comprised the following steps:

[0044] Step 1, dissolving corn starch in deionized water to prepare 30 mL of 5% starch emulsion by mass, adding 30 mL of clove essential oil thereto, stirring for 5 min, and homogenizing for 10 min at a speed of 19000 rpm using a high-speed homogenizer to obtain a Pickering emulsion;

[0045] Step 2, preparing 100 mL of an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding 0.125 g of ε-polylysine thereto, and then mixing 1 mL of the Pickering emulsion of step 1 with 300 mL of deionized water and adding the mixture thereto, heating the mixture to 45-60° C., and homogenizing the mixture at a speed of 2000 rpm for 20 min to obtain an emulsion;

[0046] Step 3: dissolve carboxymethyl chitosan in pure water, stir to form a 3% transparent solution, then add the emulsion of step 2 in a volume ratio of 1:3, and mix to form a double-coated clove essential oil emulsion.

[0047] The particle size and polydispersity index (PDI) of the prepared double-coated clove essential oil nanoemulsion were measured, and its minimum particle size was 202.28±0.39 mm, indicating that its particle size was much larger than that of the double-coated clove essential oil nanoemulsion of the present invention in Preparation Example 1.

[0048] The PDI of the newly prepared nanoemulsion was 0.23. After being stored at room temperature for 10 days, the PDI was measured again, and the PDI was 0.26; after being stored at room temperature for 30 days, the PDI was measured again, and the PDI was 0.39, indicating that the emulsion stability has deteriorated (it is generally believed that PDI < 0.25 is a stable emulsion).

[0049] Experimental Example 1

[0050] Experimental materials: Donkey hind leg meat was provided by Tianlong Food Co., Ltd., and other chemical reagents were obtained from the market.

[0051] Experimental group: emulsions prepared by the above-mentioned Preparation Example 1 and Comparative Examples 1-3;

[0052] Sample processing: Frozen sour donkey meat was transported to the laboratory through the cold chain. After removing the fascia and fat, the donkey meat was divided into rectangular blocks of 5×5×4cm (length×width×height). At the same time, each group (four experimental groups and one blank control group) selected 3 samples as indicators for day 0. Then, 15 portions of meat were aseptically sealed and stored at 4°C. The following indicators were evaluated on days 0, 5, 10, 20, and 30, and the average values ​​were taken. The results are shown in Table 1 below.

[0053] Table 1

[0054]

[0055]

[0056] Conclusion: Texture characteristics are an important intuitive indicator for evaluating meat quality (Paula & Conti-Silva, 2014). Compared with the traditional shearing method, its texture characteristics can describe the sample more comprehensively and objectively.

[0057] The hardness of food reflects the strength it needs to resist in order to maintain its shape when subjected to external forces (Caine et al., 2003). In all treatment groups, the hardness of donkey meat decreased significantly in the late stage of cold storage, which was caused by the degradation of muscle fibers mediated by microbial proteases; the preservative samples obtained in each embodiment and comparative example can slow down the reduction of donkey meat hardness to a certain extent, but the preservative sample of Preparation Example 1 of the present invention has a more obvious advantage in a longer preservation period.

[0058] The change in elasticity reflects the ability to resist external forces. At the beginning of storage, all groups of donkey meat showed high elasticity. As the storage period prolonged, the elasticity of donkey meat continued to decrease. At the end of storage, there was a significant difference between the treatment group and the blank control group (p<0.05), indicating that the use of clove essential oil can effectively inhibit protein degradation in donkey meat and protect muscle protein structure. In addition, the nanoemulsion double-coated with clove essential oil has a slow release function and has a better and longer protective effect on muscle protein.

[0059] Cohesion represents the strength of the internal bonding of meat (Rahman & AlFarsi, 2005). As the storage time increases, the adhesion of donkeys in each treatment group gradually decreases due to the gradual decrease in the adhesion of adjacent muscle fibers (Petracci & Cavani, 2012); the results support the important role of plant essential oils in inhibiting muscle fiber hydrolysis and oxidation (Zheng et al., 2019).

[0060] Chewiness refers to the sample's sustained resistance to chewing. As storage time increases, the chewiness of the donkey gradually decreases.

[0061] In summary, each treatment group has an effect on the various properties of donkey meat preservation, and there is a significant difference with the blank control group as the storage time increases; the nanoemulsion of double-coated clove essential oil prepared in Example 1 of the present invention has a better preservation effect on donkey meat for a longer period of time than other control treatment groups due to its slow release effect; specifically, the biological preservative double-coated by Pickering emulsion intermediate is more advantageous for extending the shelf life, and the preferred organic emulsifier (corn starch) of the present invention is better than the inorganic emulsifier (nano-silica), and high-speed homogenization is also an important step in preparing a long-acting biological preservative.

Claims

1. A method for preparing a double-coated nanoemulsion of clove essential oil, comprising: Step 1, dissolving corn starch in deionized water to prepare a starch emulsion with a mass fraction of 5-8%, adding an equal amount of clove essential oil thereto, stirring for 5 minutes, and homogenizing at high speed to obtain a Pickering emulsion; Step 2, preparing an emulsifier aqueous solution containing 1.5% whey protein and 0.05% lecithin, adding ε-polylysine thereto, and then adding the Pickering emulsion of step 1 mixed with deionized water thereto, heating to 45-60° C., and high-speed homogenizing to obtain a nanoemulsion; Step 3: dissolving carboxymethyl chitosan in pure water, stirring to form a transparent solution, and then adding the nanoemulsion of step 2 therein, and mixing to form a double-coated nanoemulsion of clove essential oil.

2. The preparation method according to claim 1, wherein In step 1, the high-speed homogenization is carried out using a high-speed homogenizer at a rotation speed of 19000-22000 rpm for 10 minutes.

3. The preparation method according to claim 1, wherein In step 2, the high-speed homogenization is carried out using a high-pressure homogenizer at a rotation speed of 21000-23000 rpm for 10 min.

4. The preparation method according to claim 1, wherein By weight, the nanoemulsion comprises 10-20 parts of clove essential oil, 5-10 parts of ε-polylysine, 50-70 parts of carboxymethyl chitosan, 30-50 parts of whey protein, 1-2 parts of lecithin and 2-3 parts of corn starch.

5. The preparation method according to claim 1, wherein By weight, the nanoemulsion comprises 15 parts of clove essential oil, 8 parts of ε-polylysine, 60 parts of carboxymethyl chitosan, 45 parts of whey protein, 1.5 parts of lecithin and 2.5 parts of corn starch.

6. A double-coated nanoemulsion of clove essential oil obtained by the preparation method according to any one of claims 1 to 5.

7. The nanoemulsion according to claim 6, wherein The PDI value of the nanoemulsion is 0.18-0.

21.

8. The nanoemulsion according to claim 6, wherein After the nanoemulsion is stored for 30 days, the PDI value is 0.19-0.

23.

9. Use of the double-coated clove essential oil nanoemulsion according to any one of claims 6 to 8 in preserving donkey meat.

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