Application of essential oil sustained-release beads in the preparation of mildew-proof products for cigar leaves or cigar

Abstract

The application belongs to the tobacco mildew prevention technical field, and particularly relates to application of essential oil slow-release beads in preparation of cigar leaf and cigarette mildew-proof products. The application provides application of essential oil slow-release beads in preparation of cigar leaf and cigarette mildew-proof products, wherein the essential oil slow-release beads comprise a bacteriostatic active substance and a slow-release carrier, the bacteriostatic component is continuously and slowly released through the coating effect of the slow-release carrier, thereby achieving a convenient mildew-proof method which does not need to be sprayed and can be directly placed with finished cigar cigarettes, the method does not affect the quality of cigars, can prolong the duration of the mildew-proof effect, and avoids the problem of short-term effect caused by one-time release; meanwhile, the mildew-proof slow-release preparation has a broad-spectrum bacteriostatic effect, can effectively inhibit the growth of various molds such as Aspergillus corntus, Aureobasidium pullulans and Scopulariopsis, and is suitable for long-term mildew-proof protection of the fermentation process of cigar leaves and the aging process of finished cigars.

Description

Technical Field

[0001] This application belongs to the field of tobacco anti-mold technology, specifically relating to the application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves or cigarettes. Background Technology

[0002] During the fermentation process and the aging process of finished cigars, mold growth can occur in both the tobacco leaves and the cigar itself, severely impacting the quality and preservation of the cigars. Current techniques for combating mold growth in tobacco leaves primarily involve spraying anti-mold agents before fermentation, such as natamycin, rhamnolipids, organic acids, sodium dehydroacetate, linalool, and Bacillus amyloliquefaciens. These anti-mold agents can inhibit mold growth to a certain extent, protecting the quality of the tobacco leaves.

[0003] The significant differences in microbial communities between cigars and regular tobacco stem from their different processing methods. Regular tobacco undergoes high-temperature treatment during the re-drying process, inactivating most microorganisms on the leaf surface. The remaining microbial community is relatively simple, primarily consisting of heat-resistant bacteria such as Bacillus. The core objective during storage is to inhibit the growth of residual molds (such as Penicillium and Aspergillus) to prevent spoilage. Cigar tobacco leaves (especially the filler and binder) typically do not undergo high-temperature re-drying, preserving the complex microbial community naturally attached to the tobacco leaf, along with a prolonged period of suitable temperature and humidity for mold growth. The dominant microbial community consists of bacteria such as Staphylococcus, Corynebacterium, and Pseudomonas, accompanied by fungi such as Aspergillus, Penicillium, Alternaria, and Basidiomycetes. These microorganisms play a double-edged sword role in the fermentation of cigar tobacco leaves and the aging process of cigar cigarettes: beneficial bacteria can promote enzymatic reactions and mild fermentation, developing the unique mellow aroma and smooth taste of cigars; while harmful molds on the surface of tobacco leaves (such as Aspergillus niger and Aspergillus flavus) may also multiply rapidly during storage and fermentation, leading to mold growth.

[0004] Based on the aforementioned differences, traditional anti-mold agents are mainly suitable for spraying before tobacco leaf fermentation, and cannot be effectively applied to finished cigars. Furthermore, traditional anti-mold agents have a relatively narrow inhibitory range, typically effective only against specific types of mold, lacking broad-spectrum protection and failing to comprehensively address the various mold contamination problems that may occur during cigar storage and aging.

[0005] In addition, traditional anti-mold agents can only be sprayed before tobacco leaf fermentation and cannot be used to prevent mold growth on finished cigars, which severely restricts the long-term preservation and quality stability of cigar products. Therefore, there is an urgent need to develop an anti-mold technology that does not require spraying and can be placed directly with finished cigars. Summary of the Invention

[0006] Based on this, one embodiment of this application provides the application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves and cigarettes.

[0007] This application provides, on the one hand, the use of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves or cigarettes, wherein the essential oil slow-release beads contain essential oils with antibacterial activity and a slow-release carrier.

[0008] In some embodiments, the essential oil with antibacterial activity includes plant essential oils.

[0009] In some embodiments, the plant essential oil includes one or more of peppermint oil, cinnamaldehyde oil, and cinnamon oil.

[0010] In some embodiments, the concentration of the essential oil in the antibacterial essential oil is 5 w / w%.

[0011] In some embodiments, the essential oil slow-release beads have a particle size of 1.5mm-2.5mm.

[0012] In some embodiments, the sustained-release carrier comprises sodium alginate;

[0013] In some embodiments, the concentration of sodium alginate in the sustained-release carrier is 2.5 w / w%-3.5 w / w.

[0014] Another aspect of this application provides a method for preparing mold-resistant cigar tobacco leaves or cigarettes, comprising:

[0015] Place the essential oil slow-release beads in the same environment as tobacco leaves or cigarettes; so that the tobacco leaves or cigarettes are in the antibacterial environment constructed by the essential oil slow-release beads.

[0016] In some embodiments, the process includes: preparing an essential oil sustained-release bead intermediate, dissolving the essential oil sustained-release bead intermediate in a solution containing a sustained-release carrier, and then drying it.

[0017] In some embodiments, the solution containing the sustained-release carrier comprises a sodium alginate solution of 2.5 w / w% to 3.5 w / w%.

[0018] In some embodiments, the method for preparing the essential oil sustained-release beads includes:

[0019] The essential oil in the inner phase and the solution containing the slow-release carrier in the outer phase are simultaneously pumped into the solution through a microfluidic device at an inner phase flow rate of 0.3 mL / min - 0.5 mL / min and an outer phase flow rate of 0.5 mL / min - 0.7 mL / min, and then solidified in the coagulation liquid.

[0020] In some embodiments, the coagulating liquid comprises a calcium chloride solution of 4 w / w% to 6 w / w%.

[0021] In some embodiments, the drying conditions include a temperature of 38°C-42°C and a time of 1.5h-2.5h.

[0022] This application provides the application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves and cigarettes. The essential oil slow-release beads contain substances with antibacterial activity and a slow-release carrier. Through the encapsulation effect of the slow-release carrier, the antibacterial components are continuously and slowly released, thereby achieving a convenient anti-mold method that does not require spraying and can be placed directly with the finished cigars. This not only does not affect the quality of the cigars, but also prolongs the duration of the anti-mold effect, avoiding the problem of short-lasting anti-mold effect caused by one-time release. At the same time, the anti-mold slow-release preparation has a broad-spectrum antibacterial effect and can effectively inhibit the growth of various molds such as Aspergillus cristatus, Cladosporium brevicorum, and Cladosporium glomeratum. It is suitable for long-term anti-mold protection during the fermentation process of cigar tobacco leaves and the aging process of finished cigars. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 For essential oils and corresponding sustained-release beads to C3-2-1 (10 5 The antibacterial effect of CFU on plates (A: essential oil dropped on the plate surface; B: essential oil slow-release beads placed on the plate surface; C: control; ① is cinnamon essential oil / slow-release beads; ② is cinnamon essential oil / slow-release beads diluted 20 times; ③ is peppermint essential oil / slow-release beads diluted 20 times; ④ is cinnamaldehyde essential oil / slow-release beads diluted 20 times).

[0025] Figure 2 For essential oils and corresponding sustained-release beads to C3-2-1 (10 6 The antibacterial effect of CFU on plates (A: essential oil dropped on the plate surface; B: essential oil slow-release beads placed on the plate surface; C: control; ① is cinnamon essential oil / slow-release beads; ② is cinnamon essential oil / slow-release beads diluted 20 times; ③ is peppermint essential oil / slow-release beads diluted 20 times; ④ is cinnamaldehyde essential oil / slow-release beads diluted 20 times).

[0026] Figure 3 The antibacterial effect of peppermint oil sustained-release beads on untreated tobacco leaves (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of peppermint oil slow-release beads.

[0027] Figure 4The antibacterial effect of peppermint oil sustained-release beads on tobacco leaves inoculated with Y4-2 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of peppermint oil slow-release beads.

[0028] Figure 5 The antibacterial effect of peppermint oil sustained-release beads on tobacco leaves inoculated with Y6-3 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of peppermint oil slow-release beads.

[0029] Figure 6 The antibacterial effect of cinnamon essential oil sustained-release beads on tobacco leaves inoculated with Y4-2 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamon essential oil slow-release beads.

[0030] Figure 7 The antibacterial effect of cinnamon essential oil sustained-release beads on tobacco leaves inoculated with Y6-3 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamon essential oil slow-release beads.

[0031] Figure 8 The antibacterial effect of cinnamaldehyde essential oil sustained-release beads on untreated tobacco leaves (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamaldehyde essential oil slow-release beads.

[0032] Figure 9 The antibacterial effect of cinnamaldehyde essential oil sustained-release beads on tobacco leaves inoculated with Y4-2 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamaldehyde essential oil slow-release beads.

[0033] Figure 10 The antibacterial effect of cinnamaldehyde essential oil sustained-release beads on tobacco leaves inoculated with Y6-3 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamaldehyde essential oil slow-release beads.

[0034] Figure 11 The antibacterial effect of cinnamaldehyde essential oil sustained-release beads on tobacco leaves inoculated with C3-2-1 (A is a photograph of tobacco leaves; B is the result of elution microbial count of tobacco leaves (10)). -5 C represents the qPCR detection results of microorganisms eluted from tobacco leaves, where green indicates no slow-release beads were placed and red indicates the presence of cinnamaldehyde essential oil slow-release beads. Detailed Implementation

[0035] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0038] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0039] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0040] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0041] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0042] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0043] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0044] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0045] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0046] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0047] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0048] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0049] The term "sodium alginate" refers to a natural polysaccharide extracted from brown algae, which possesses gelling, film-forming, and biocompatible properties in the food, pharmaceutical, and antifungal technologies. Sodium alginate molecules contain a large number of carboxyl groups, enabling them to undergo ionic cross-linking reactions with divalent cations (such as calcium ions) to form a three-dimensional network gel structure. Specific types include low-viscosity sodium alginate, medium-viscosity sodium alginate, high-viscosity sodium alginate, and sodium alginate with different G / M ratios.

[0050] The term "microfluidic device" refers to a miniaturized device capable of precisely controlling the flow of tiny fluids. In the field of microcapsule fabrication technology, it enables precise control of droplet size, shape, and generation frequency. Microfluidic devices achieve precise segmentation and mixing of fluids through microchannel structures, thereby producing microcapsules with uniform particle size. Specific subcategories include: T-type microfluidic devices, Y-type microfluidic devices, coaxial microfluidic devices, and flow-focusing microfluidic devices, etc.

[0051] The term "Aspergillus cristatus" refers to a common mold that causes mildew on tobacco leaves, imparting a musty odor and reducing product quality during tobacco product storage. Aspergillus cristatus belongs to the genus Aspergillus and can rapidly grow and reproduce under suitable temperature and humidity conditions, producing numerous spores that cause green or black mold spots on the surface of tobacco leaves. Specific subspecies include different strains of Aspergillus cristatus, such as strain C3-2-1.

[0052] The term "Aureobasidium melanogenum" refers to a common mold that causes mildew, off-flavors, and compromises product quality in tobacco storage. Belonging to the genus Aureobasidium, it thrives in high humidity environments, producing black or dark brown colonies. Subspecies include various strains of Aureobasidium melanogenum, such as strain Y4-2.

[0053] The term "Scopulariopsis koningii" refers to a common mold that causes mildew on tobacco leaves, imparts a musty odor, and reduces the product's value during tobacco storage. Scopuliopsis koningii belongs to the genus Scopuliopsis and can grow on various substrates, producing gray or green colonies. Specific subspecies include different strains of Scopuliopsis, such as strain Y6-3.

[0054] An inhibition zone is an indicator for evaluating the activity of antimicrobial substances, providing a direct visual reflection of the strength of antimicrobial effects in the field of microbiology. An inhibition zone is a transparent area around an antimicrobial substance on a culture medium containing microorganisms, where no microorganisms grow. The larger the diameter of the inhibition zone, the stronger the antimicrobial effect. Specific types include circular inhibition zones, elliptical inhibition zones, and inhibition zones of varying clarity.

[0055] To address the problem that traditional anti-mold agents cannot be used to prevent mold growth in finished cigars and can only be used for spraying treatment before tobacco fermentation, which severely restricts the long-term preservation and quality stability of cigar products, this application provides the application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves and cigarettes.

[0056] This application provides the application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves and cigarettes. The essential oil slow-release beads contain an antibacterial substance and a slow-release carrier. Through the encapsulation effect of the slow-release carrier, the essential oil slow-release beads allow the antibacterial substance to be released continuously and slowly, thereby achieving long-term anti-mold protection for cigar tobacco leaves and cigarettes. Compared with traditional spray-on anti-mold agents, the essential oil slow-release beads of this application do not require spraying and can be placed directly with finished cigars, making them convenient to use and not affecting the quality of the cigars. The slow-release carrier can control the release rate of the antibacterial substance, prolonging the duration of the anti-mold effect and avoiding the problem of short-lasting anti-mold effect caused by one-time release. At the same time, the slow-release characteristics of the essential oil slow-release beads can reduce the volatilization loss of the antibacterial substance, improve anti-mold efficiency, and reduce usage costs. These essential oil slow-release beads are suitable for mold prevention and protection during the fermentation process of cigar tobacco leaves and the aging process of finished cigars. They can effectively inhibit the growth of various molds such as Aspergillus cristatus, Aspergillus brevicus, and Aspergillus spp., and have a broad-spectrum antibacterial effect.

[0057] In some embodiments, the antibacterial substance is a plant essential oil. Plant essential oils are natural volatile substances extracted from plants, possessing good antibacterial activity and safety, and can effectively inhibit the growth and reproduction of mold. Plant essential oils have advantages such as wide availability, low price, and environmental friendliness, making them suitable for mold prevention and protection of cigar tobacco leaves and cigarettes.

[0058] In some embodiments, the plant essential oil includes one or more of peppermint oil, cinnamaldehyde oil, and cinnamon oil. Peppermint oil has a refreshing aroma and good antibacterial effects, effectively inhibiting the growth of various molds; cinnamaldehyde oil is the main active ingredient in cinnamon, possessing strong antibacterial activity and antioxidant properties; cinnamon oil is an essential oil extracted from cinnamon bark and has broad-spectrum antibacterial effects. These three essential oils can be used alone or in combination; when used in combination, they produce a synergistic antibacterial effect and enhance antifungal properties.

[0059] In some embodiments, the sustained-release carrier is sodium alginate. Sodium alginate is a natural polysaccharide extracted from brown algae, possessing excellent biocompatibility, film-forming properties, and gelling characteristics. The sodium alginate molecule contains a large number of carboxyl groups, enabling it to undergo ionic cross-linking reactions with divalent cations (such as calcium ions) to form a three-dimensional network gel, thereby achieving the encapsulation and sustained release of substances with antibacterial activity. Sodium alginate has advantages such as wide availability, low price, and high safety, making it suitable for the preparation of essential oil sustained-release beads.

[0060] In some embodiments, the concentration of sodium alginate is 2.5 w / w%-3.5 w / w%. The concentration of sodium alginate has a significant impact on the molding and sustained-release performance of the controlled-release beads. When the sodium alginate concentration is too low, the mechanical strength of the controlled-release beads is insufficient, making them prone to breakage and leading to a rapid release of substances with antibacterial activity. When the sodium alginate concentration is too high, the density of the controlled-release beads is too high, resulting in a slow release rate of substances with antibacterial activity, which affects the antifungal effect. Specifically, the concentration of sodium alginate can be selected from any value among 2.5 w / w%, 2.8 w / w%, 3.0 w / w%, 3.2 w / w%, and 3.5 w / w%.

[0061] This application also provides a method for preventing mold growth in cigar tobacco leaves and cigarettes using essential oil slow-release beads, comprising the following steps:

[0062] Preparation of essential oil sustained-release beads: (1) Transfer the prepared internal phase solution (essential oil / essential oil diluted with caprylic / capric glycerol) and external phase solution (sodium alginate aqueous solution with a concentration of 3.0 w / w%) into two 30 mL syringes respectively, ensuring that there are no air bubbles remaining. Then, correctly install the syringes on the dual-channel injection pump and connect them to the corresponding inlet of the microfluidic device.

[0063] (2) Set the flow rate parameters of the syringe pump as follows: internal phase flow rate 0.4 mL / min, external phase flow rate 0.6 mL / min (internal and external phase flow rate ratio is 2:3). Start the syringe pump to allow the two liquid phases to meet stably in the microfluidic chip.

[0064] (3) Under the combined action of interfacial tension and shear force, the inner phase oil is enveloped by the outer phase water, forming a uniform initial droplet at the chip outlet. The droplet is directly dropped into a coagulation bath containing a 5 w / w% calcium chloride solution. Sodium alginate undergoes a transient ionic cross-linking reaction with calcium ions, and the droplet solidifies into a spherical slow-release bead.

[0065] (4) Collect the microcapsules formed in the coagulation bath and wash them repeatedly with sufficient deionized water 3-5 times to remove residual calcium ions and salts on the surface.

[0066] (5) Transfer the washed microcapsules to a drying tray and place them in a forced-air drying oven to dry at 40°C for 2 hours.

[0067] (6) After drying, place the microcapsules in a sealed container and store them in a dark, low-temperature environment for subsequent analysis and use. The essential oil may be selected from one or more of peppermint oil, cinnamaldehyde oil, and cinnamon oil; the sustained-release carrier is sodium alginate.

[0068] Post-processing: The slow-release beads do not produce residual pollution after use, do not need to be removed, and can be disposed of together with the tobacco leaves or cigarettes.

[0069] This application also provides a method for preparing essential oil sustained-release beads, comprising: preparing essential oil sustained-release beads by dissolving the essential oil sustained-release beads in a solution containing a sustained-release carrier, and then drying them. This preparation method utilizes microfluidic technology to prepare essential oil sustained-release beads with uniform particle size, enabling precise control of the size and structure of the beads, thereby achieving precise regulation of the release rate of substances with antibacterial activity. This method is simple to operate, easy to scale up for production, and the prepared essential oil sustained-release beads exhibit good stability and sustained-release performance.

[0070] In some embodiments, the solution containing the sustained-release carrier comprises a sodium alginate solution of 2.5 w / w% to 3.5 w / w%. The sodium alginate solution, as the external phase solution, can form stable droplets with the antibacterial substance in the internal phase within the microfluidic device, thereby preparing sustained-release beads with uniform particle size. Specifically, the concentration of the sodium alginate solution can be selected from any value among 2.5 w / w%, 2.8 w / w%, 3.0 w / w%, 3.2 w / w%, and 3.5 w / w%.

[0071] In some embodiments, the preparation method of the essential oil sustained-release beads includes: simultaneously pumping an internal phase containing an antibacterial substance and an external phase containing a sustained-release carrier into the solution via a microfluidic device at an internal phase flow rate of 0.3 mL / min - 0.5 mL / min and an external phase flow rate of 0.5 mL / min - 0.7 mL / min, forming the beads in a coagulating solution. Microfluidic technology can precisely control the flow rates of the internal and external phases, thereby preparing sustained-release beads with uniform particle size and stable structure. The ratio of the internal phase flow rate to the external phase flow rate has a significant impact on the size and structure of the sustained-release beads; when the internal phase flow rate is too high, the particle size of the sustained-release beads increases; when the external phase flow rate is too high, the particle size of the sustained-release beads decreases. Specifically, the internal phase flow rate can be selected from any value among 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, and 0.5 mL / min, and the external phase flow rate can be selected from any value among 0.5 mL / min, 0.55 mL / min, 0.6 mL / min, 0.65 mL / min, and 0.7 mL / min.

[0072] In some embodiments, the coagulation solution comprises a calcium chloride solution of 4 w / w% to 6 w / w%. The calcium chloride solution, as a coagulation solution, can undergo an ionic cross-linking reaction with sodium alginate, causing the sodium alginate to gel and thus form stable slow-release beads. The concentration of calcium chloride has a significant impact on the molding speed and mechanical strength of the slow-release beads. Specifically, the concentration of the calcium chloride solution can be selected from any value among 4 w / w%, 4.5 w / w%, 5.0 w / w%, 5.5 w / w%, and 6.0 w / w%.

[0073] In some embodiments, the post-treatment drying conditions include a temperature of 38℃-42℃ and a time of 1.5h-2.5h. Drying removes moisture from the slow-release beads, improving their stability and storage performance. The drying temperature and time significantly affect the quality of the slow-release beads. Excessive drying temperature or time may lead to the loss of antibacterial active substances through volatilization; conversely, insufficient drying temperature or time may result in incomplete moisture removal, affecting storage stability. Specifically, the drying temperature can be selected from any value among 38℃, 39℃, 40℃, 41℃, and 42℃, and the drying time can be selected from any value among 1.5h, 1.8h, 2.0h, 2.2h, and 2.5h.

[0074] The essential oil slow-release beads provided in this application are small in size, do not require spraying, and can be placed together with finished cigars to inhibit mold growth; moreover, the inhibitory effect of this application is broad-spectrum: peppermint and cinnamon essential oil slow-release beads have antibacterial effects against Aureobasidium melanogenum Y4-2 and Scopuliopsis koningii Y6-3; cinnamaldehyde essential oil slow-release beads have antibacterial effects against Aspergillus cristatus C3-2-1, Aureobasidium melanogenum Y4-2, and Scopuliopsis koningii Y6-3.

[0075] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0076] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0077] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0078] Example 1

[0079] This embodiment provides an essential oil slow-release bead for preventing mold growth in cigar tobacco leaves and cigarettes, and its preparation method.

[0080] 1. Materials required for the experiment

[0081] The tobacco leaves were provided by the Great Wall Cigar Factory of Sichuan Tobacco Industry Co., Ltd.; peppermint oil, cinnamaldehyde oil, and cinnamon oil were purchased from Wuyuan Biotechnology Co., Ltd.; potato dextrose agar (PDA) was purchased from Qingdao Haibo Biotechnology Co., Ltd.; sterile saline was purchased from Huankai Biotechnology; calcium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium alginate was purchased from Sinopharm Chemical Reagent Co., Ltd.; and the fungal DNA extraction kit was purchased from Feijing Biotechnology Co., Ltd.

[0082] 2. Instruments required for the experiment

[0083] Pipettes (Eppendorf), biochemical incubator (Shanghai Senxin Experimental Instrument Co., Ltd.), constant temperature and humidity chamber (BINDER), vernier calipers (Hong Kong Dinghao Measuring Tools Co., Ltd.), colony counter (Interscience Scan 4000).

[0084] 3. Experimental Procedure

[0085] 3.1 Plate Antibacterial Test

[0086] 3.1.1 Collection of mold spores

[0087] The activated mold, after two generations of inoculation, was inoculated onto PDA plates and incubated in a 28 ℃ biochemical incubator for 7 days. After incubation, 2 mL of sterile physiological saline was added to the plates in a clean bench, and the spores on the surface of the plates were scraped off with a spreader. The spores were then collected in sterile centrifuge tubes for later use.

[0088] 3.1.2 Preparation of essential oil sustained-release beads

[0089] (1) Solution preparation: Prepare an external phase sodium alginate solution at 3.0 w / w% and a coagulation solution (calcium chloride solution) at 5 w / w%.

[0090] (2) Preparation of sustained-release beads: The prepared internal and external phase solutions were drawn into 30 mL syringes respectively. The flow rates of the internal and external phases were set as follows: internal phase (essential oil) flow rate 0.4 mL / min, external phase flow rate 0.6 mL / min. The syringe pump was turned on, and the internal and external phase solutions were pumped in simultaneously. The solution was encapsulated at the inlet of the microfluidic device and dripped into the coagulation liquid to form microcapsules. The preparation of microcapsules was completed. The particle size of the prepared essential oil sustained-release beads was 2 mm, and the essential oil concentration was 5 w / w.

[0091] (3) Post-treatment and drying: The prepared slow-release beads were washed with deionized water and then transferred to an oven. The temperature was set at 40 °C and the time was 2 h. After drying, the beads were sealed and stored.

[0092] 3.1.3 Plate Antibacterial Test

[0093] In a clean bench, 200 μL of spore suspension was added to a PDA plate and spread evenly using a disposable sterile spreader. Each sample was tested in triplicate, with two replicates. After the plate surface had slightly dried, 2 μL of essential oil was added to one plate, and an essential oil slow-release bead was placed on the other plate. After completion, the plates were placed in a 28 ℃ biochemical incubator for 12 days, during which time photos were taken and recorded using a plate counter.

[0094] 3.2 Antibacterial Experiment of Tobacco Leaves

[0095] 3.2.1 Collection of mold spores

[0096] Same as 3.1.1 for culturing and collecting spores of Aspergillus cristatus (C3-2-1), Aureobasidium melanogenum (Y4-2), and Scopus koningii (Y6-3).

[0097] 3.2.2 Preparation of essential oil sustained-release beads

[0098] Same as 3.1.2.

[0099] 3.3.3 Inoculation of tobacco leaves with mold

[0100] After cutting the tobacco leaves into uniformly sized small pieces, each portion was placed in a plastic box, 20 g in total. Four portions of tobacco leaves were placed on a tray, and 1 mL of spore suspension was added to 3 mL of sterile physiological saline. The mixture was thoroughly mixed and then sprayed onto the tobacco leaves in the tray. After spraying, the tobacco leaves were thoroughly mixed again and then divided into four portions. One portion was used as a control, and four slow-release beads of three different essential oils were added to each of the remaining three portions. The plastic boxes were then covered and incubated in a constant temperature and humidity incubator at 28 ℃ and 80% relative humidity for 10 days.

[0101] 3.3.4. Microbial elution and DNA extraction from tobacco leaves

[0102] After incubation, 2 g of tobacco leaf sample was placed in a 50 mL sterile centrifuge tube, along with 4-5 glass beads (4-5 mm in diameter) and 20 mL of sterile physiological saline. The mixture was shaken on a high-speed shaker for 20 min. Subsequently, it was centrifuged at 4 ℃ and 1000 r / min for 10 min, and the supernatant was collected. This process of elution with physiological saline, shaking, and low-speed centrifugation was repeated twice, using 15 mL of physiological saline each time. All supernatants were combined and centrifuged at 4 ℃ and 10000 r / min for 10 min. The supernatant was discarded, and the resulting precipitate was the eluted microorganisms from the tobacco leaf surface. Finally, total DNA was extracted from the microbial precipitate using a fungal DNA extraction kit.

[0103] 3.3.5 Real-time quantitative PCR detection

[0104] To determine the number of molds inoculated into the microorganisms eluted from the surface of tobacco leaves, real-time quantitative PCR (qPCR) was used for detection. The primers used were:

[0105] 251F 5'-GARTCTTTGAACGCACATTGCG-3' (SEQ ID NO. 1).

[0106] 251R 5'-CTACCTGATCCGAGGTCAACCT-3' (SEQ ID NO. 2).

[0107] The probe used was 5'-FAM-CCGTAGGTCCAGCTCGCAGCTAGCC-BHQ1-3' (SEQ ID NO.3).

[0108] The qPCR addition system is shown in Table 1. The qPCR program used was: 95 ℃, 5 min; 95 ℃, 10 s; 60 ℃, 20 s; 72 ℃, 20 s. The last three steps were repeated 40 times. After the program was completed, the melting curve was detected. Each sample was tested in triplicate.

[0109] Table 1 qPCR addition system

[0110]

[0111] 4. Experimental Results

[0112] 4.1 Plate Antibacterial Test

[0113] This experiment selected three essential oils—peppermint, cinnamon, and cinnamaldehyde—and prepared corresponding sustained-release beads to evaluate their antibacterial effects on Aspergillus cristatus (C3-2-1) on agar plates. Figures 1-2 The results showed that at an inoculation concentration of 10... 5 In CFU plates, the three essential oils still exhibited similar antibacterial effects on day 6 as on day 3; however, the 20-fold diluted cinnamon essential oil showed a significantly weaker antibacterial effect than the undiluted oil. By day 12, the inhibition zones of all essential oils against *A. cristatus* C3-2-1 disappeared, indicating no antibacterial effect. However, at the same time point, inhibition zones remained around both the cinnamon essential oil slow-release beads and the 20-fold diluted cinnamaldehyde essential oil slow-release beads, indicating that both slow-release beads still had antibacterial effects, with the cinnamon essential oil slow-release beads showing the most significant effect. At an inoculation concentration of 10... 6 In the CFU plates, the antibacterial zones of the three essential oils had already shrunk by day 6, with mycelia spreading in the original antibacterial areas. By day 12, the antibacterial zones had also disappeared. In contrast, the antibacterial effect of the essential oil slow-release beads was still comparable to that on day 3, with the cinnamon essential oil slow-release beads exhibiting the largest diameter antibacterial zone, which remained clearly visible around them on day 12.

[0114] The above results indicate that cinnamon, peppermint, and cinnamaldehyde essential oils all have inhibitory effects on *A. cristatus* C3-2-1. However, preparing them into sustained-release beads not only did not weaken their antibacterial ability but also prolonged the duration of inhibition. Furthermore, sustained-release beads prepared from undiluted essential oils exhibited superior antibacterial performance.

[0115] 4.2 Effect of tobacco leaf mold control

[0116] Chuanxue No. 2 was selected as the experimental tobacco leaf sample, and its surface was sprayed with A. cristatus C3-2-1, Aureobasidium melanogenum Y4-2 and Scopus koningii Y6-3, with untreated tobacco leaves as the control.

[0117] 4.2.1 Antibacterial effect of peppermint essential oil slow-release beads

[0118] As shown in Figure 3, after 10 days of storage under constant temperature and humidity conditions (28℃, 80% relative humidity), untreated tobacco leaves showed obvious signs of mold, while tobacco leaves with peppermint oil slow-release beads showed no mold. Microorganisms eluted from the surface of the tobacco leaves were counted (diluted to 10). -5 The results showed that the total bacterial count in untreated tobacco leaves was significantly higher than that in tobacco leaves treated with slow-release beads. Further qPCR analysis revealed that the Cq value of the untreated tobacco leaf samples was lower than that of the slow-release bead-treated group, indicating a higher content of mold DNA in the eluent, i.e., a greater number of molds. These results collectively demonstrate that peppermint oil slow-release beads can effectively inhibit mold growth in tobacco leaves. Similarly, A. melanogenum Y4-2 (… Figure 4 ) and S. koningii Y6-3 ( Figure 5 After the treatment, the tobacco leaves without the slow-release beads showed obvious mold growth, while the treatment group with the added peppermint oil slow-release beads showed significantly reduced mold growth or even no mold growth at all, further confirming the inhibitory effect of peppermint oil slow-release beads on tobacco leaf mold growth.

[0119] 4.2.2 Antibacterial effect of cinnamon essential oil sustained-release beads

[0120] Similarly (e.g.) Figures 6-7 As shown in the figure, cinnamon essential oil slow-release beads can effectively inhibit the mold growth of tobacco leaves inoculated with A. melanogenum Y4-2 and S. koningii Y6-3.

[0121] 4.2.3 Antibacterial effect of cinnamaldehyde essential oil sustained-release beads

[0122] Similarly (e.g.) Figures 8-11 As shown in the figure, cinnamaldehyde essential oil slow-release beads can effectively inhibit mold growth in untreated tobacco leaves and tobacco leaves inoculated with A. melanogenum Y4-2, S. koningii Y6-3 and A. cristatus C3-2-1.

[0123] Result verification:

[0124] Table 2. Test results of the diameter of the inhibition zone in the examples (mm)

[0125]

[0126] Note: + indicates 0 < inhibition zone diameter < 10 mm, ++ indicates 10 ≤ inhibition zone diameter < 20 mm, +++ indicates 20 ≤ inhibition zone diameter < 30 mm, and ++++ indicates 30 ≤ inhibition zone diameter.

[0127] Table 3 Antibacterial effect of tobacco leaves in the examples (mold count, 10) 5 CFU / g)

[0128]

[0129] Note: Plate counts are performed by diluting the eluted microorganisms by 10⁻⁶. 5 Afterwards, a plate count was performed, with "-" indicating that no microorganisms grew at that dilution.

[0130] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. The application of essential oil slow-release beads in the preparation of anti-mold products for cigar tobacco leaves or cigarettes, characterized in that, The essential oil sustained-release beads contain essential oils with antibacterial activity and a sustained-release carrier.

2. The application according to claim 1, characterized in that, The essential oils with antibacterial activity include plant essential oils; Optionally, the plant essential oil includes one or more of peppermint oil, cinnamaldehyde oil, and cinnamon oil.

3. The application according to claim 1, characterized in that, The concentration of the essential oil with antibacterial activity is 5 w / w%. Optionally, the particle size of the essential oil slow-release beads is 1.5mm-2.5mm.

4. The application according to any one of claims 1 to 3, characterized in that, The sustained-release carrier includes sodium alginate; Optionally, the concentration of sodium alginate in the sustained-release carrier is 2.5 w / w%-3.5 w / w.

5. A method for preparing anti-mold cigar tobacco leaves or cigarettes, characterized in that, include: The essential oil slow-release beads as defined in any one of claims 1 to 4 are placed in the same environment as tobacco leaves or cigarettes; the tobacco leaves or cigarettes are placed in the antibacterial environment constructed by the essential oil slow-release beads.

6. A method for preparing essential oil sustained-release beads as defined in any one of claims 1 to 4, characterized in that, include: To prepare an essential oil sustained-release bead intermediate, the essential oil sustained-release bead intermediate was dissolved in a solution containing a sustained-release carrier and then dried.

7. The method for preparing essential oil sustained-release beads according to claim 6, characterized in that, The solution containing the sustained-release carrier includes a sodium alginate solution of 2.5 w / w% to 3.5 w / w%.

8. The method for preparing essential oil sustained-release beads according to claim 6, characterized in that, The preparation method of the essential oil sustained-release beads includes: The essential oil in the inner phase and the solution containing the slow-release carrier in the outer phase are simultaneously pumped into the coagulation liquid through a microfluidic device at an inner phase flow rate of 0.3 mL / min-0.5 mL / min and an outer phase flow rate of 0.5 mL / min-0.7 mL / min.

9. The method for preparing essential oil sustained-release beads according to claim 8, characterized in that, The coagulation solution comprises a calcium chloride solution of 4 w / v% to 6 w / v%.

10. The preparation method according to any one of claims 6 to 9, characterized in that, The drying conditions include a temperature of 38℃-42℃ and a time of 1.5h-2.5h.

Images