A method for preventing mildew of cigar tobacco during fermentation

Abstract

The present application relates to the technical field of cigar tobacco processing, and particularly relates to a method for preventing mildew of fermented cigar tobacco. The method comprises the following steps: first, spraying a rhamnolipid solution to the surface of the cigar tobacco, and then placing the cigar tobacco in a constant temperature and humidity environment for fermentation. In the present application, the rhamnolipid is mainly used to achieve the mildew prevention effect, and the mechanism is mainly to damage the cell wall and cell membrane. Both fungi and bacteria have cell walls and cell membranes, so compared with natamycin in the prior art which can only act on fungi, the present application has higher mildew prevention effect.

Description

Technical Field

[0001] This invention relates to the field of cigar tobacco processing technology, and in particular to a method for preventing mold growth during cigar tobacco fermentation. Background Technology

[0002] Fermentation is an important means of reducing the off-flavors and irritation of cigar tobacco leaves and improving their sensory quality. It is also a key step in the agricultural and industrial production of cigar tobacco leaves. Under normal circumstances, fermentation of cigar tobacco leaves under suitable temperature and humidity conditions can, to a certain extent, improve the maturity of the tobacco leaves, remove unpleasant odors such as raw grass, earthy smell, protein odor, and local off-flavors, improve the aroma and taste, and enhance the smoking quality.

[0003] For cigar tobacco leaves to achieve optimal fermentation results, a relatively high humidity (70%-80%) and a specific temperature (20-40℃) are often required. Mold thrives in humid and mild environments, with an optimal growth temperature of 22-35℃ and a relative humidity above 65%. Therefore, improper management during cigar tobacco fermentation can easily lead to mold growth. Moldy tobacco leaves exhibit significantly reduced appearance and sensory quality, decreased industrial usability, and severe economic losses. Furthermore, most molds are harmful to human health. If moldy tobacco leaves are not promptly or thoroughly cleaned, they can be used to make cigarettes and enter the market, posing a serious threat to consumer health. Therefore, implementing effective mold prevention measures during cigar tobacco fermentation is crucial.

[0004] Patent document CN101797071A discloses an additive for cigar anti-mold treatment. The additive's components and weight ratios are: 50-100 parts natamycin; 50-150 parts solubilizer; 0.1-2 parts antioxidant; and 0.1-3 parts surfactant. After mixing these components thoroughly, they are dissolved in 1000 parts water, the pH is adjusted to 3-7, and the mixture is stirred for 30 minutes. The additive obtained by this method, diluted with water, is applied to the surface of cigar tobacco leaves at a ratio of 100-2000 mg / kg natamycin to tobacco leaves by soaking or spraying. This effectively inhibits the growth of most mold-causing fungi without affecting the formation of the cigar's quality and style.

[0005] In this application, natamycin is primarily used to achieve its antifungal effect. Natamycin relies on its lactone ring structure to interact with sterol compounds on the fungal cell membrane, forming antibiotic-sterol compounds, thereby disrupting the structure of the fungal cell membrane. The hydrophilic portion (polyol portion) of the macrolide forms pores on the membrane, damaging cell membrane permeability, which in turn causes the leakage of amino acids, electrolytes, and other substances from the fungus, leading to cell death. When sterol compounds are absent from the cell membranes of certain microorganisms, natamycin has no effect on them. Therefore, natamycin only inhibits fungi and does not produce antibacterial activity against bacteria, thus its antifungal effect remains insufficient. Summary of the Invention

[0006] The present invention aims to solve the above problems by providing a method for preventing mold growth during the fermentation of cigar tobacco leaves.

[0007] The technical solution to the problem of this invention is to provide a method for fermenting cigar tobacco leaves to prevent mold growth, which includes the following steps: first, spraying a rhamnolipin solution onto the surface of the cigar tobacco leaves, and then placing the cigar tobacco leaves in a constant temperature and humidity environment for fermentation.

[0008] Rhamnolipids are anionic glycolipid surfactants produced by microorganisms and possessing biodegradable properties. They exhibit excellent chemical and biological characteristics, are non-toxic, and biodegradable, making them widely used in industry, agriculture, food processing, cosmetics, and medicine. Reports indicate that rhamnolipids possess strong antibacterial properties, effectively inhibiting the growth of most molds and bacteria. They can disrupt the cell walls of mold hyphae, altering cell membrane permeability and thus inhibiting mold spore germination and hyphal growth.

[0009] In this application, rhamnolipids are mainly used to achieve the antifungal effect. Its mechanism of action is mainly to destroy the cell wall and cell membrane. Since both fungi and bacteria have cell walls, it has a higher antifungal effect than natamycin, which can only act on fungi in the prior art.

[0010] The rhamnolipin solution may be simply an aqueous solution of rhamnolipin to facilitate its spraying onto tobacco leaves. Preferably, the rhamnolipin solution comprises rhamnolipin and water. The concentration of rhamnolipin should be limited; too low a concentration will not achieve the anti-mold effect, while too high a concentration may harm beneficial fermenting bacteria and result in poor fermentation of cigar tobacco leaves. Preferably, the mass ratio of rhamnolipin to water is 0.1%-1%. More preferably, the mass ratio of rhamnolipin to water is 0.5%-1%.

[0011] The mechanism by which rhamnolipids disrupt fungal cell walls is considered to be as follows: the cell wall is mainly composed of cellulose and glucan. Cellulose is a polysaccharide composed of D-glucose linked by β-1,4-glycosidic bonds. Similarly, the rhamnosyl ring in rhamnolipids is also linked by β-1,4-glycosidic bonds to form 2 or 3 sugars. This structural similarity may allow rhamnolipids to competitively bind to fungal cell wall polysaccharides, disrupting the synthesis or linkage of fungal cellulose. Therefore, enhancing the activity of the rhamnosyl ring in rhamnolipids can help improve the effectiveness of rhamnolipids in inhibiting fungi. Therefore, as a preferred embodiment of the present invention, the rhamnolipid solution further includes perfluoroiodobutane, with a mass ratio of perfluoroiodobutane to rhamnolipid of 10%-20%. The rhamnosyl ring forms a halogenated glycosyl complex with perfluoroiodobutane, which activates the rhamnosyl ring.

[0012] Further optimization involves fermenting the cigar tobacco leaves under visible light. Under visible light, the halogenated glycosyl complex can generate iodinated glycosyl intermediates via electron transfer, thereby further activating the cigar.

[0013] To further enhance the competitiveness of rhamnolipids, as a preferred embodiment of the present invention, the rhamnolipid solution further includes a nucleophilic reagent. This nucleophilic reagent attacks the iodinated glycoside intermediate to facilitate the formation of a glycosidic bond. The choice of nucleophilic reagent is not limited; as a preferred embodiment of the present invention, the nucleophilic reagent includes phosphonium trioxide and diammonium hydrogen phosphate. More preferably, the mass ratio of the nucleophilic reagent to rhamnolipid is 0.1%-0.5%.

[0014] To further improve the anti-mold effect, as a preferred embodiment of the present invention, the rhamnolipin solution also includes calcium bicarbonate, with a concentration of 1%-2%. On the one hand, calcium ions capture bacteria, which makes it easier for rhamnolipin to come into contact with the cell walls of bacteria to complete the antibacterial effect; on the other hand, the carbon dioxide produced expands the cells of cigar tobacco leaves, which also facilitates the penetration of rhamnolipin.

[0015] In this entire process, rhamnolipids are the primary active ingredient. However, rhamnolipids are a series of homologues composed of single or double rhamnosyl groups and fatty acids of varying carbon chain lengths. Dirhamnolipids have a high rhamnosyl content, which is beneficial for antibacterial activity, but their emulsifying properties are poor. Monorhamnolipids have better surface activity, facilitating the coating and penetration of the rhamnolipid solution onto cigar tobacco leaves. Therefore, the ratio of dirhamnolipids to monorhamnolipids significantly affects the final antibacterial and antifungal properties. Preferably, the rhamnolipids in this invention comprise monorhamnolipids and dirhamnolipids in a mass ratio of (2-3):(7-8).

[0016] The amount of rhamnolipin solution applied to cigar tobacco leaves should be limited. Too little will not achieve the anti-mold effect, while too much will result in excessive moisture content in the cigar tobacco leaves, which is also a factor in tobacco leaf mold growth. Preferably, the mass ratio of the rhamnolipin solution to the cigar tobacco leaves is 5%-15%. More preferably, the mass ratio is 5%-10%.

[0017] Besides the influence of rhamnolipin solution on the anti-mold effect, the amount of rhamnolipin solution absorbed by cigar tobacco leaves and the moisture content of the cigar tobacco leaves themselves also affect the anti-mold effect. To improve the antibacterial and anti-mold effect of rhamnolipin on cigar tobacco leaves, this invention involves heating the cigar tobacco leaves to 50-60°C before spraying; after spraying, the cigar tobacco leaves undergo vacuum freeze-drying; and then fermentation is carried out in a constant temperature and humidity environment. Heating first causes the pores of the cigar tobacco leaves to expand along with the leaves themselves, making it easier for the rhamnolipin solution to penetrate the pores after spraying. Finally, vacuum freeze-drying closes the pores of the tobacco leaves to retain the rhamnolipin and removes moisture from the cigar tobacco leaves, preventing mold growth during fermentation due to high moisture content. The vacuum freeze-drying parameters are not limited. As a preferred embodiment of the present invention, the freeze-drying process is first performed at -45°C and 0 Pa vacuum for 8 hours; then at -20°C and 20 Pa vacuum for 2 hours; and finally at 0°C and 20 Pa vacuum for 2 hours.

[0018] After spraying with rhamnolipin, the cigar tobacco leaves can be fermented using conventional fermentation parameters. However, to allow the rhamnolipin to function better, as a preferred method of this invention, fermentation is carried out in a constant temperature and humidity environment of 20-30°C and 70%-80% relative humidity for 10-20 days. More preferably, fermentation is carried out in a constant temperature and humidity environment of 20-25°C and 70%-75% relative humidity for 10-15 days.

[0019] The beneficial effects of this invention are:

[0020] 1. In this application, rhamnolipids are mainly used to achieve the anti-mold effect. The mechanism of action is mainly the destruction of cell walls and cell membranes. Fungi and bacteria both have cell walls and cell membranes, so they have a higher anti-mold effect.

[0021] 2. In this application, the cigar tobacco leaves are first heated and then freeze-dried under vacuum, which improves the absorption rate of rhamnolipids while reducing the moisture content of the cigar tobacco leaves, thereby further improving the anti-mold effect. Detailed Implementation

[0022] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described, but the present invention is not limited to these embodiments.

[0023] Example 1

[0024] A method for preventing mold growth in cigar tobacco leaves through fermentation includes the following steps:

[0025] We purchased pure monorhamnolipin (≥98%), pure dirhamnolipin (≥98%), perfluoroiodobutane (98%), phosphorus trioxide (98%), diammonium hydrogen phosphate (99%), and calcium bicarbonate (99%) from the market; and selected Grade 1 cigar tobacco leaves from the middle section of Yunxue No. 1.

[0026] According to the mass fractions, 2.5 parts of monorhamnolipid, 7.5 parts of dirhamnolipid, 30 parts of calcium bicarbonate, 0.01 parts of triphenylphosphine, and 0.02 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 1.5 parts of perfluoroiodobutane were added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipid solution.

[0027] One hundred portions of cigar tobacco leaves were heated to 55°C in a water bath. Heating was then stopped, and immediately 10 portions of rhamnolipin solution were evenly sprayed onto the surface of the 100 portions of cigar tobacco leaves. The cigar tobacco leaves were then placed in a vacuum freeze-drying apparatus. First, they were pre-frozen at -45°C and 0 Pa for 8 hours; then sublimated at -20°C and 20 Pa for 2 hours; finally, they were sublimated at 0°C and 20 Pa for 2 hours. The dried cigar tobacco leaves were then removed and transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity, equipped with visible light LEDs, for 15 days of constant temperature and humidity fermentation.

[0028] Example 2

[0029] This embodiment is basically the same as Embodiment 1, except that it is not fermented under visible light.

[0030] The cigar tobacco leaves were transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity. The chamber was placed in a dark, opaque room and fermented for 15 days.

[0031] Example 3

[0032] This embodiment is basically the same as Embodiment 1, except that: no nucleophilic reagent is used.

[0033] 2.5 parts of monorhamnolipid, 7.5 parts of dirhamnolipid, and 30 parts of calcium bicarbonate were mixed and added to 2000 parts of deionized water. Then, 1.5 parts of perfluoroiodobutane were added, and the mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipid solution.

[0034] Example 4

[0035] This embodiment is basically the same as Embodiment 1, except that calcium bicarbonate is not used.

[0036] According to the mass fractions, 2.5 parts of monorhamnolipid, 7.5 parts of dirhamnolipid, 0.01 parts of tribenzoxyphos and 0.02 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 1.5 parts of perfluoroiodobutane were added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipid solution.

[0037] Example 5

[0038] This embodiment is basically the same as Embodiment 1, except that only monorhamnolipid is used.

[0039] According to the mass fractions, 10 parts of monorhamnolipin, 30 parts of calcium bicarbonate, 0.01 parts of triphenylphosphine, and 0.02 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 1.5 parts of perfluoroiodobutane were added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipin solution.

[0040] Example 6

[0041] This embodiment is basically the same as Embodiment 1, except that only dirhamnolipid is used.

[0042] According to the mass fractions, 10 parts of dirhamnolipin, 30 parts of calcium bicarbonate, 0.01 parts of triphenylphosphine, and 0.02 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 1.5 parts of perfluoroiodobutane were added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipin solution.

[0043] Example 7

[0044] This embodiment is basically the same as embodiment 1, except that it does not perform the preheating followed by vacuum freeze-drying process.

[0045] At room temperature (25°C), 10 parts of rhamnolipin solution were evenly sprayed onto the surface of 100 parts of cigar tobacco leaves. The cigar tobacco leaves were then transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity, equipped with visible light LEDs, for 15 days of constant temperature and humidity fermentation.

[0046] Example 8

[0047] A method for preventing mold growth in cigar tobacco leaves through fermentation includes the following steps:

[0048] According to the mass fractions, 2 parts of monorhamnolipin, 8 parts of dirhamnolipin, 20 parts of calcium bicarbonate, 0.005 parts of triphenylphosphine, and 0.005 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 1 part of perfluoroiodobutane was added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipin solution.

[0049] Grade 1 cigar tobacco leaves from the upper part of Yunxue No. 6 were selected. 100 portions of the cigar tobacco leaves were heated in a water bath to 50°C, then heating was stopped. Immediately afterward, 5 portions of rhamnolipin solution were evenly sprayed onto the surface of the 100 portions of cigar tobacco leaves. The cigar tobacco leaves were then placed in a vacuum freeze-drying apparatus. First, they were pre-frozen at -45°C and 0 Pa for 8 hours; then sublimated at -20°C and 20 Pa for 2 hours; finally, they were sublimated at 0°C and 20 Pa for 2 hours. The dried cigar tobacco leaves were then removed and transferred to a constant temperature and humidity chamber at 20°C and 70% relative humidity, equipped with visible light LEDs, for 20 days of constant temperature and humidity fermentation.

[0050] Example 9

[0051] A method for preventing mold growth in cigar tobacco leaves through fermentation includes the following steps:

[0052] According to the mass fractions, 3 parts of monorhamnolipin, 7 parts of dirhamnolipin, 40 parts of calcium bicarbonate, 0.02 parts of triphenylphosphine, and 0.03 parts of diammonium hydrogen phosphate were mixed and added to 2000 parts of deionized water, and then 2 parts of perfluoroiodobutane were added. The mixture was homogenized at 10000 r / min for 3 min to obtain a rhamnolipin solution.

[0053] Selected Grade 1 cigar tobacco leaves from the middle section of Chuxue 80 variety were used. 100 portions of the tobacco leaves were heated in a water bath to 60°C, then heating was stopped. Immediately afterward, 15 portions of rhamnolipin solution were evenly sprayed onto the surface of the 100 portions of tobacco leaves. The tobacco leaves were then placed in a vacuum freeze-drying apparatus. First, they were pre-frozen at -45°C and 0 Pa for 8 hours; then sublimated at -20°C and 20 Pa for 2 hours; finally, they were sublimated at 0°C and 20 Pa for 2 hours. The dried tobacco leaves were then removed and transferred to a constant temperature and humidity chamber at 30°C and 80% relative humidity, equipped with visible light LEDs, for 10 days of constant temperature and humidity fermentation.

[0054] Example 10

[0055] A method for preventing mold growth in cigar tobacco leaves through fermentation includes the following steps:

[0056] According to the mass fractions, 2.5 parts of monorhamnolipin and 7.5 parts of dirhamnolipin were mixed and added to 2000 parts of deionized water to obtain a 0.5% rhamnolipin solution.

[0057] At room temperature (25°C), 10 parts of rhamnolipin solution were evenly sprayed onto the surface of 100 parts of cigar tobacco leaves. The cigar tobacco leaves were then transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity, equipped with visible light LEDs, for 15 days of constant temperature and humidity fermentation.

[0058] Examples 11-16

[0059] Examples 11-16 are basically the same as Example 10, except that the concentration of rhamnolipin is different. The concentration of rhamnolipin in each example is shown in Table 1 below.

[0060] Table 1.

[0061]

[0062] Examples 17-22

[0063] Examples 17-22 are basically the same as Example 10, except that the amount of rhamnolipin used on the cigar tobacco leaves is different. The amount of rhamnolipin used in each example is shown in Table 2 below.

[0064] Table 2.

[0065]

[0066] Example 23

[0067] This embodiment is basically the same as embodiment 10, except that:

[0068] According to the mass fractions, 2.5 parts of monorhamnolipin, 7.5 parts of dirhamnolipin, and 30 parts of calcium bicarbonate were mixed and added to 2000 parts of deionized water to obtain a rhamnolipin solution.

[0069] Comparative Example 1

[0070] At room temperature (25°C), 10 parts of deionized water were evenly sprayed onto the surface of 100 parts of cigar tobacco leaves. The cigar tobacco leaves were then transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity, equipped with visible light LEDs, for 15 days of constant temperature and humidity fermentation.

[0071] Comparative Example 2

[0072] According to the specified mass ratio, 10 parts natamycin and 0.1 parts surfactant were mixed and added to 2000 parts deionized water to obtain a solution. At room temperature (25°C), 10 parts of the solution were evenly sprayed onto the surface of 100 parts cigar tobacco leaves. The cigar tobacco leaves were then transferred to a constant temperature and humidity chamber at 25°C and 75% relative humidity, equipped with visible light LEDs, for 15 days of constant temperature and humidity fermentation.

[0073] [Anti-mold test]

[0074] Fermented cigar tobacco leaves from the examples and comparative examples were taken out, and the mold growth on the surface of the cigar tobacco leaves was observed. The mold growth rate of the tobacco leaves (mold growth rate = weight of moldy tobacco leaves / total weight of tobacco leaves) was statistically analyzed. The statistical results are shown in Table 3.

[0075] Table 3.

[0076] experimental group Is it moldy? mold rate experimental group Is it moldy? mold rate Example 1 no 0％ Example 14 yes 2.81％ Example 2 yes 1.56％ Example 15 yes 11.24％ Example 3 yes 1.41％ Example 16 yes 9.45％ Example 4 yes 0.55％ Example 17 yes 2.53％ Example 5 yes 1.01％ Example 18 yes 1.95％ Example 6 yes 0.46％ Example 19 yes 3.21％ Example 7 yes 0.98％ Example 20 yes 3.55％ Example 8 no 0％ Example 21 yes 7.04％ Example 9 no 0％ Example 22 yes 8.56％ Example 10 yes 2.50％ Example 23 yes 1.81％ Example 11 yes 4.25％ Comparative Example 1 yes 59.30％ Example 12 yes 3.26％ Comparative Example 2 yes 6.76％ Example 13 yes 2.02％

[0077] As shown in Table 3, a comparison of Example 10 with Comparative Examples 1 and 2 reveals that the rhamnolipin used in this application exhibits better anti-mold effects on cigar tobacco leaves compared to the water control group and natamycin. Examples 10-16 demonstrate that the concentration of rhamnolipin should be limited to the range of 0.1%-1%, as excessively high or low concentrations will negatively impact the anti-mold effect. Examples 10 and 17-22 show that the amount of rhamnolipin used on cigar tobacco leaves should be limited to 5%-15%, as excessively high or low concentrations will also affect the anti-mold effect. A comparison of Examples 1 and 10 shows that perfluoroiodobutane, visible light fermentation, nucleophilic reagents, the structural ratio of rhamnolipin, and the pre-heating followed by vacuum freeze-drying of cigar tobacco leaves all have a positive impact on the anti-mold effect of cigar tobacco leaves.

[0078] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for preventing mold growth during the fermentation of cigar tobacco leaves, characterized in that: Includes the following steps: First, the rhamnolipin solution is sprayed onto the surface of the cigar tobacco leaves, and then the cigar tobacco leaves are placed in a constant temperature and humidity environment for fermentation. The rhamnolipin solution comprises rhamnolipin and water, wherein the mass ratio of rhamnolipin to water is 0.1%-1%. The rhamnolipids comprise monorhamnolipids and dirhamnolipids in a mass ratio of (2-3):(7-8); Before spraying, heat the cigar tobacco leaves to 50-60℃; after spraying, vacuum freeze-dry the cigar tobacco leaves; and then place them in a constant temperature and humidity environment for fermentation.

2. The method for preventing mold growth during cigar tobacco fermentation according to claim 1, characterized in that: The rhamnolipid solution also includes perfluoroiodobutane, and the mass ratio of perfluoroiodobutane to rhamnolipid is 10%-20%.

3. The method for preventing mold growth during cigar tobacco fermentation according to claim 2, characterized in that: The cigar tobacco leaves are fermented under visible light.

4. The method for preventing mold growth during cigar tobacco fermentation according to claim 3, characterized in that: The rhamnolipid solution also includes a nucleophilic reagent.

5. A method for preventing mold growth during cigar tobacco fermentation according to claim 1 or 2, characterized in that: The mass ratio of the rhamnolipin solution to cigar tobacco leaves is 5%-15%.

6. A method for preventing mold growth during cigar tobacco fermentation according to claim 1 or 2, characterized in that: The rhamnolipid solution also includes calcium bicarbonate, with a concentration of 1%-2%.

7. The method for preventing mold growth during cigar tobacco fermentation according to claim 1, characterized in that: Cigar tobacco leaves are fermented in a constant temperature and humidity environment of 20-30℃ and 70%-80% relative humidity for 10-20 days.

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