Green preservative based on natamycin nano system as well as preparation method and application of green preservative

Abstract

The invention belongs to the field of fresh-keeping processing of agricultural products, and particularly relates to a green fresh-keeping agent based on a natamycin nano system as well as a preparation method and application of the green fresh-keeping agent. According to the preservative provided by the invention, a natural product natamycin obtained by fungus fermentation is used as a bacteriostatic active substance, sorghum prolamine is combined in a solution system, and a stabilizer is added, so that the green preservative with a stable nano-dispersion system is prepared. The fruit and vegetable preservative is simple in production process, low in cost, green and safe, and can be applied to fruits and vegetables such as tomatoes, grapes, blueberries, strawberries and cucumbers as a novel fruit and vegetable preservative.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product preservation and processing, and more specifically, relates to a green preservative based on natamycin nanosystem, its preparation method and application. Background Technology

[0002] With increasing demand, tomatoes have gradually gained prominence in daily diets and have become an important part of the fruit and vegetable industry. Tomatoes are typical climacteric fruits, making them susceptible to mechanical damage and pathogen infection during harvesting, post-harvest transportation, and storage. Common fungal diseases affecting tomatoes mainly include: Botrytis cinerea (…). Botrytis cinerea Gray mold caused by Penicillium ( ) Penicillium expansum Penicillium fruit rot caused by Phytophthora ( Phytophthora infestans This includes diseases such as late blight in tomatoes caused by *Botrytis cinerea*. Gray mold, caused by *Botrytis cinerea*, is a major post-harvest disease of tomatoes. In addition, this pathogen can also infect cucumbers, strawberries, blueberries, and other crops, causing significant losses to agricultural production.

[0003] For a long time, the prevention and control of postharvest diseases in tomatoes has mainly relied on the spraying of chemical pesticides. However, long-term use of chemical pesticides can easily lead to fungal resistance, damage the ecological environment, and affect the quality and safety of agricultural products. Therefore, there is an urgent need to develop green, safe, and eco-friendly biological control measures to provide technical means and theoretical support for the prevention and control of postharvest diseases in tomatoes and other fruits and vegetables.

[0004] Sorghum is a widely cultivated grain crop, with a protein content of approximately 11%, of which sorghum prolysin accounts for about 80% of the total protein. Among existing cereal prolysins, sorghum prolysin has the strongest hydrophobicity and the worst digestibility. Due to the low digestibility of sorghum protein, sorghum is mainly used for brewing fermentation, and the discarded residue contains a large amount of prolysin, resulting in resource waste. Sorghum prolysin possesses unique strong hydrophobicity; changing the solution polarity alters its protein conformation, thus allowing it to self-assemble into nanoparticles via antisolvent methods. These nanoparticles can then co-precipitate with hydrophobic drugs, making them potential carrier materials for encapsulating hydrophobic active ingredients. However, using a single protein to encapsulate active ingredients often results in insufficient stability, so a stabilizing agent is usually introduced for compound use. Sodium carboxymethyl cellulose is an anionic polymer obtained by chemically modifying natural cellulose. It is readily soluble in water, forming a transparent, viscous colloidal solution with excellent thickening, emulsifying, suspending, and stabilizing properties. It can stabilize proteins through steric hindrance, preventing protein aggregation and sedimentation in solution. It also has the advantages of being non-toxic, biodegradable, and having good film-forming properties, making it an ideal structural stabilizing material.

[0005] Natamycin is a kind of "polyene macrolide" fungal inhibitor fermented by Streptomycesnatalensis, and is also the only internationally approved high-efficiency, broad-spectrum, safe and natural antifungal biological food preservative. It is reported that natamycin can act on fungal cells to reduce the production of ergosterol, or rely on its lactone ring structure to act on the sterol on the fungal cell membrane to form a sterol compound, thereby destroying the structure of the cytoplasmic membrane, increasing the permeability of the cell membrane, and then causing the leakage of amino acids, electrolytes and other substances in the bacteria, resulting in the death of the bacteria. Since the target of natamycin as a polyene antibiotic is the sterol on the fungal cell membrane, the surface of human cells lacks sterol, and it is not sensitive to polyene antibiotics, so natamycin is harmless to the human body. However, the solubility of natamycin in water is extremely low at room temperature, only 0.03-0.05 mg / mL, and due to the cyclic structure in the molecular structure, it is sensitive to ultraviolet light and oxidants, and is easily degraded under the action of light or oxidants, which limits its application in fruit and vegetable preservation. SUMMARY

[0006] The purpose of the present application is to provide a green preservative based on a natamycin nanosystem and a preparation method and application thereof.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides a green preservative based on a natamycin nanosystem, which is a dispersion system containing natamycin-sorghum prolamin nanomicrospheres, the natamycin-sorghum prolamin nanomicrospheres are formed by embedding natamycin in sorghum prolamin, and the dispersion system also contains a stabilizer, which is sodium carboxymethyl cellulose, and the mass ratio of sodium carboxymethyl cellulose, natamycin and sorghum prolamin is 109.44 mg:10.64 mg:24 mg.

[0008] The green preservative provided by the present application is a microsphere formed by embedding natamycin in sorghum prolamin as a substrate as an effective component, and the nanodispersion system of the preservative has good stability, the solubility and light stability of the green preservative of the present application are significantly improved compared with natamycin, and the green preservative has good fresh-keeping and mildew-inhibiting effect, and can be used for the fresh-keeping of tomatoes and other fruits and vegetables.

[0009] Further, the average particle size of the microspheres is 201.83±2.05 nm.

[0010] The present application also provides a preparation method of the green preservative, comprising the following steps: S1, mixing a sorghum prolamin solution with a concentration of 1.6 mg / mL and a natamycin solution with a concentration of 53.2 mg / mL according to a volume ratio of 15:0.2 to obtain a natamycin-sorghum prolamin solution; S2. A sodium carboxymethyl cellulose solution with a concentration of 1.2 mg / mL is added dropwise to the natamycin-sorghum alcohol-soluble protein solution, and the solution is stirred to disperse it evenly. Then, the ethanol is removed to obtain the green preservative. The volume ratio of natamycin-sorghum lysozyme solution to sodium carboxymethyl cellulose solution is 1:6.

[0011] Furthermore, the dripping rate is 0.2 mL / s.

[0012] Furthermore, the stirring rate is 400 rpm.

[0013] Furthermore, ethanol was removed by rotary evaporation at 140 rpm and 40 °C.

[0014] The present invention also provides the application of the green preservative in the preservation of fruits and vegetables.

[0015] Furthermore, the preservation is achieved by antagonizing postharvest gray mold of fruits and vegetables.

[0016] Furthermore, the pathogen causing the gray mold disease is *Botrytis cinerea*.

[0017] The present invention has the following beneficial effects: This invention uses a 1.6 mg / mL sorghum prolysin solution with stronger hydrophobicity as a matrix and sodium carboxymethyl cellulose as a stabilizer. A specific amount of sorghum prolysin self-assembles with a 53.2 mg / mL natamycin solution via an antisolvent method to form nanoparticles. This method is simple to operate, low in cost, and pollution-free during the reaction process. The entire preparation process and product align with current green and environmentally friendly concepts.

[0018] The sorghum prolysin used in this invention is derived from sorghum, which is generally used primarily as animal feed or for brewing. Its fermentation waste contains a significant amount of prolysin, and directly discarding it results in substantial resource waste. This invention extracts and utilizes the sorghum prolysin, increasing the added value of sorghum-related agricultural products and reducing resource waste.

[0019] The green preservative prepared in this invention combines sodium carboxymethyl cellulose, natamycin, and sorghum prolysin in a specific mass ratio of 109.44 mg: 10.64 mg: 24 mg, achieving an encapsulation rate of 80% for natamycin. Furthermore, the particles are uniform in size, with an average particle size of 201.83 ± 2.05 nm, which increases the solubility and photostability of natamycin, which has extremely poor water solubility, allowing it to be stably dispersed in the system. This increases the effective contact area between natamycin and pathogenic microorganisms, facilitating its antibacterial activity. This green preservative can significantly inhibit the occurrence of gray mold. Attached Figure Description

[0020] Figure 1 This is a scanning electron microscope image of Nata-NP.

[0021] Figure 2 The graph shows the effect of sorghum gliadin concentration on the average particle size, PDI, and zeta potential of Nata-NP.

[0022] Figure 3 The graph shows the effect of sorghum prolysin concentration on the encapsulation rate and loading rate of Nata-NP.

[0023] Figure 4 The graph shows the effect of ethanol concentration on the average particle size, PDI, and zeta potential of Nata-NP.

[0024] Figure 5 The graph shows the effect of ethanol concentration on the encapsulation efficiency and loading rate of Nata-NP.

[0025] Figure 6 The diagram shows the effect of the ratio of forward to reverse phase on the average particle size, PDI, and zeta potential of Nata-NP.

[0026] Figure 7 The diagram shows the effect of the positive and negative phase systems on the encapsulation rate and loading rate of Nata-NP.

[0027] Figure 8 The graph shows the effect of sodium carboxymethyl cellulose concentration on the average particle size, PDI, and zeta potential of Nata-NP.

[0028] Figure 9 The graph shows the effect of sodium carboxymethyl cellulose concentration on the encapsulation efficiency and loading rate of Nata-NP.

[0029] Figure 10 Absorbance graphs of Nata-Water and Nata-NP with different natamycin contents.

[0030] Figure 11 The graph shows the retention rate of Nata in Nata-Water and Nata-NP after different times of UV irradiation.

[0031] Figure 12 This is a photograph of the sepals of a bunch of tomatoes taken on day 7 of storage.

[0032] Figure 13 This is a graph showing the incidence rates of different treatment groups at different storage times. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0034] The English and Chinese abbreviations of the materials involved in the following embodiments are shown in Table 1.

[0035] Table 1: Comparison of Chinese and English Abbreviations

[0036] Example 1: A method for preparing a green preservative based on a natamycin nanosystem.

[0037] The preparation method of this embodiment includes the following steps: (1) Preparation of reversed-phase solution: A CMC-Na solution with a concentration of 1.2 mg / mL was prepared by mixing CMC-Na and pure water at a mass-volume ratio of 1.2 mg: 1 mL. The solution was magnetically stirred at 500 rpm for 2 hours to ensure complete dissolution, and then placed at 4°C overnight to allow for complete hydration, thus obtaining the reversed-phase solution.

[0038] (2) Preparation of normal phase solution: a. Preparation of Nata solution: Add 10.64 mg of Nata powder to 0.2 mL of 1,2-propanediol, wrap with tin foil to protect from light, vortex for 2 min, let stand in the dark for 2 h, and obtain Nata solution after Nata dissolves.

[0039] b. Preparation of Kaf solution: Mix Kaf and 80% ethanol aqueous solution at a mass-volume ratio of 1.6 mg: 1 mL, stir magnetically at 400 rpm for 30 min at room temperature, and then homogenize using a high-speed shear machine at 10000 rpm for 1 min to obtain a Kaf solution of 1.6 mg / mL.

[0040] c. Mixing: Add 0.2 mL of the Nata solution prepared above (a) to 15 mL of Kaf solution, wrap with tin foil to protect from light, and magnetically stir at 400 rpm for 1 h at room temperature to mix evenly, thus obtaining the Nata-Kaf solution.

[0041] (3) Preparation of Nata-NP by anti-solvent method: The reversed-phase solution was added dropwise to the normal-phase solution at a rate of 0.2 mL / s, so that the final volume ratio of the normal-phase solution to the reversed-phase solution was 1:6. During the titration, the system was continuously stirred magnetically at 400 rpm, and stirred for 1 h after the titration was completed to ensure uniform dispersion. Then, the system was rotary evaporated at 140 rpm and 40 °C using a vacuum rotary evaporator to remove the ethanol in the system, thus obtaining Nata-NP.

[0042] Verification Example 1: The influence of different factors on the physicochemical properties of green preservatives.

[0043] I. Experimental Methods.

[0044] To verify the effects of Kaf concentration, ethanol concentration, the ratio of forward to reverse phases, and CMC-Na concentration on the performance of the preservative, the following experimental treatments were conducted. The influencing factors included Kaf concentrations of 0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL, 2 mg / mL, 2.4 mg / mL, and 2.8 mg / mL; ethanol concentrations of 60%, 70%, 80%, 90%, and 100%; forward to reverse phase volume ratios of 1:1, 1:3, 1:6, 1:9, 1:12, and 1:15; and CMC-Na concentrations of 0 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL. The remaining steps were the same as in Example 1.

[0045] II. Sample Testing.

[0046] 1. Scanning electron microscopy (SEM) detection.

[0047] After the sample prepared in Example 1 was freeze-dried, it was attached to the sample nail with conductive tape and then sputtered with gold using an ion sputtering coating machine. Its microstructure was observed using a scanning electron microscope with an accelerating voltage of 5 kV and a magnification of 2000x.

[0048] Experimental results are as follows Figure 1 As shown, a dense porous structure is visible in the Nata-NP dispersion system, in which Nata is embedded in Kaf to form a large number of relatively uniform microspheres with a particle size of 200~500nm, which belongs to the category of nanoparticles.

[0049] 2. Average particle size, PDI, and zeta potential.

[0050] The prepared sample was diluted 100 times with pure water, vortexed and mixed, and then its average particle size, PDI and ζ potential were measured using a Malvern nanoparticle size analyzer. Each measurement was repeated three times.

[0051] Experimental results are as follows Figure 2 , Figure 4 , Figure 6 and Figure 8 As shown, Kaf concentration, ethanol concentration, the ratio of the forward to reverse phase, and CMC-Na concentration all have a significant impact on these three indicators. Among them, when the Kaf concentration is 1.6 mg / mL, the ethanol volume fraction is 80%, the forward to reverse phase volume ratio is 1:6, and the CMC-Na concentration is 1.2 mg / mL, Nata-NP has a smaller average particle size and PDI, and at the same time has a larger zeta potential. At this time, the average particle size is 201.83±2.05 nm.

[0052] 3. Encapsulation rate and load rate.

[0053] The prepared Nata-NP was centrifuged at 10000×g at 4℃ for 30 min to remove some insoluble matter and prevent clogging of the ultrafiltration tube. 0.4 mL of the supernatant was transferred to a 3kD ultrafiltration tube and centrifuged at 10000×g at 4℃ for 20 min. The filtrate from the bottom of the ultrafiltration tube was diluted 20 times with pure water, and the absorbance was measured at 305 nm. The Nata content in the filtrate was calculated using the Nata standard curve, which represents the content of unencapsulated Nata. The Nata encapsulation rate and loading rate were calculated using the following formula:

[0054] EE=(Nata total -Nata free ) / Nata total ×100%.

[0055] LE=(Nata total -Nata free ) / Kaf×100%.

[0056] In the formula, EE is the encapsulation ratio, in percentage. LE is the load factor, in percentage. total For the total mass of natamycin, Nata free The mass of natamycin in the filtrate is given by Kaf, which represents the total mass of sorghum prolysin.

[0057] Experimental results are as follows Figure 3 , Figure 5 , Figure 7 and Figure 9 As shown, with increasing Kaf concentration, the encapsulation efficiency gradually increases while the loading rate gradually decreases; for ethanol volume fraction, from 60% to 90%, the encapsulation efficiency and loading rate gradually increase, but when the ethanol volume fraction reaches 100%, the encapsulation efficiency and loading rate decrease again; with increasing ratio of normal to reverse phase, the encapsulation efficiency and loading rate also gradually increase; when the CMC-Na concentration increases from 0 to 1.2 mg / mL, the encapsulation efficiency and loading rate increase accordingly, but with further increase of CMC-Na concentration, the encapsulation efficiency and loading rate begin to decrease again.

[0058] 4. Solubilizing effect.

[0059] Nata-NP and Nata-Water solutions with different Nata concentrations were prepared. The Nata concentration gradients in the two systems were: 0 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL. After preparation, Nata-NP and Nata-Water were filtered through a 0.45 μm microporous membrane and diluted 20-fold. Both solutions were zeroed using their respective Nata concentration of 0, and the absorbance at 305 nm was measured.

[0060] Depend on Figure 10 It can be seen that when the Nata concentration in Nata-Water reaches 0.15 mg / mL or higher, its absorbance essentially remains unchanged. This is because the solution is essentially saturated with Nata at this point; adding more Nata does not increase the amount of dissolved Nata, and the undissolved Nata is filtered through the membrane, thus the absorbance of the solution does not increase significantly. However, for Nata-NP, when the Nata concentration reaches 0.3 mg / mL, its absorbance is still significantly increased compared to the lower concentration group. This result indicates that, compared to Nata-Water, Nata-NP can effectively improve the solubility of Nata in solution.

[0061] 5. Light stability.

[0062] Dissolve 5.0 mg of Nata in 50 mL of pure water and stir magnetically for 2 h to prepare a Nata aqueous solution with a concentration of 0.1 mg / mL. Take 30 mL of the Nata aqueous solution and 30 mL of the sample prepared in Example 1 into a petri dish, then place them open in a clean bench and turn on the UV lamp, ensuring that the irradiation distance and angle are consistent. Take a sample every 30 min, filter the sample through a 0.45 μm microporous membrane and dilute it 20 times with pure water. Measure the absorbance at 305 nm and calculate the retention rate of Nata using the Nata standard curve.

[0063] Depend on Figure 11 It was found that under ultraviolet (UV) irradiation, the degradation rate of Nata in Nata-Water was significantly faster than that in Nata-NP. The degradation rate of Nata in Nata-NP slowed down after 120 min, while Nata-Water continued to degrade rapidly. After 180 min of UV irradiation, the Nata retention rate in Nata-Water dropped below 25%, while the Nata retention rate in Nata-NP remained above 50%. This indicates that Nata-NP helps resist the degradation effect of UV light on Nata and maintains a higher Nata retention rate.

[0064] 6. Application in the preservation of bunched tomatoes.

[0065] Take a PDA plate contaminated with *Botrytis cinerea* after 7 days of culture and collect *Botrytis cinerea* spores under aseptic conditions. Add sterile water to the contaminated plate to submerge the mycelium. Gently scrape off the spores with a spreading stick, then pipette the spore-containing liquid through four layers of sterile gauze into a 10 mL centrifuge tube and shake to disperse the spores. Count the spores using a hemocytometer, and then adjust the spore suspension to a concentration of 5 × 10⁻⁶ with sterile water. 6 The samples were counted at 1 / mL and used for subsequent experiments.

[0066] Botrytis cinerea ( Botrytis cinerea B05.10 has been published in the article “He,C.,Zhang,ZQ,Li,BQ,Xu,Y.,Tian,SP,2019.Effect of natamycin on Botrytis cinerea and Penicillium expansum—Postharvest pathogens of grape berries and jujube fruit.Postharvest Biology and Technology,151,134-141.https: / / orcid.org / 10.1016 / j.postharvbio.2019.02.009.” The applicant has committed to distributing the biological material to the public within twenty years of the applicant’s application and has provided information on how to obtain the biological material.

[0067] Address: Ocean University of China, No. 1299 Sansha Road, Huangdao District, Qingdao.

[0068] After rinsing and drying the sepals of *Tomato* clusters with pure water, they were immersed in a 1.5% sodium hypochlorite solution for 2 minutes for disinfection and sterilization, followed by rinsing with pure water and drying. The sepals were then immersed in Nata-NP solutions with effective Nata concentrations of 0 mg / mL, 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL for 3 minutes, removed, slightly drained, and air-dried in a clean bench. Pure water immersion served as a blank control group, with three replicates per group and two clusters of sepals per replicate. After drying, the sepals were sprayed with a spore suspension at a concentration of 1×10⁻⁶. 5 Spray once per mL of the solution onto both sides of the sepals, then air dry and store in a sealed container. Place the container in a 25°C constant temperature and humidity incubator, maintaining a humidity of 85%. Observe the sepals daily for mold growth, freshness, wrinkling, and greenness, and take photos for record-keeping.

[0069] Figure 12The image shows the disease incidence on the sepals of tomatoes under different treatments on day 7 of storage. For easier comparison, mold spots on the sepals are circled in red. Clearly, the number of mold spots in the pure water soaking group and the Nata-NP soaking group with an effective Nata concentration of 0 was significantly higher than in the Nata-NP soaking groups with effective Nata concentrations of 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL. Figure 13 It was found that the disease incidence rate was significantly higher in the pure water soaking group and the Nata-NP soaking group with an effective Nata concentration of 0 than in the Nata-NP soaking group with effective Nata concentrations of 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL. Furthermore, the incidence rate in the latter group was lower than that in the former group throughout the entire storage period. On day 7 of storage, the incidence rate was 65.07% in the pure water soaking group, 76.40% in the Nata-NP soaking group with an effective Nata concentration of 0, while the incidence rates in the Nata-NP soaking groups with effective Nata concentrations of 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL were only 6.91%, 4.76%, and 4.25%, respectively. Clearly, the Nata-NP soaking groups with effective Nata concentrations of 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL effectively inhibited the growth of pathogens on the calyxes of Tomato calyxes, achieving a good preservation and anti-mold effect.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A green preservative based on a natamycin nanosystem, characterized in that, The green preservative is a dispersion system containing natamycin-sorghum prolysin nanospheres. The natamycin-sorghum prolysin nanospheres are formed by encapsulating natamycin in sorghum prolysin. The dispersion system also contains a stabilizer, which is sodium carboxymethyl cellulose. The mass ratio of sodium carboxymethyl cellulose, natamycin and sorghum prolysin is 109.44 mg: 10.64 mg: 24 mg.

2. The green preservative according to claim 1, characterized in that, The average particle size of the microspheres is 201.83 ± 2.05 nm.

3. The method for preparing the green preservative according to claim 1, characterized in that, Includes the following steps: S1. A natamycin-sorghum prolysin solution with a concentration of 1.6 mg / mL and a natamycin solution with a concentration of 53.2 mg / mL are mixed at a volume ratio of 15:0.2 to obtain a natamycin-sorghum prolysin solution. The solvent of the sorghum prolysin solution is an 80% (v / v) aqueous ethanol solution, and the solvent of the natamycin solution is 1,2-propanediol. S2. A sodium carboxymethyl cellulose solution with a concentration of 1.2 mg / mL is added dropwise to the natamycin-sorghum alcohol-soluble protein solution, and the solution is stirred to disperse it evenly. Then, the ethanol is removed to obtain the green preservative. The volume ratio of natamycin-sorghum lysozyme solution to sodium carboxymethyl cellulose solution is 1:

6.

4. The preparation method according to claim 3, characterized in that, The dripping rate is 0.2 mL / s.

5. The preparation method according to claim 3, characterized in that, The stirring speed is 400 rpm.

6. The preparation method according to claim 3, characterized in that, Ethanol was removed by rotary evaporation at 140 rpm and 40 °C.

7. The application of the green preservative according to claim 1 in the preservation of fruits and vegetables.

8. The application according to claim 7, characterized in that, The preservation and anti-corrosion effect is achieved by antagonizing postharvest gray mold in fruits and vegetables.

9. The application according to claim 8, characterized in that, The pathogen causing gray mold is *Botrytis cinerea*.

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