A strain of Cossackie DS101 that improves the color of eggplant peel fermentation and its application

Abstract

This invention belongs to the field of industrial microbiology technology, specifically disclosing a strain of *Cossacchariformis* DS101 that improves the color of eggplant wrapper fermentation and its application. The strain's preservation number is CCTCC NO: M20241671. Adding this strain during eggplant wrapper fermentation can increase the total color difference value of the wrapper, deepen its color, and promote color changes during fermentation, thereby improving the quality of the wrapper. Therefore, *Cossacchariformis* DS101 can be used as an industrial microorganism in the fermentation of cigar tobacco leaves to produce dark brown wrappers, showing promising application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of industrial microorganisms, and relates to a strain of Klebsiella oxytoca DS101 for improving the fermentation color of tobacco jacket and application thereof. BACKGROUND

[0002] Cigar is a special tobacco product, which is made by hand after the cigar tobacco leaf is cured and fermented, and is composed of tobacco jacket, tobacco cover and tobacco core. The innermost part is the tobacco core, the tobacco cover is wrapped outside the tobacco core, and the tobacco jacket is wrapped outside. The tobacco jacket is the essence of cigar tobacco, although it accounts for the smallest proportion of cigar, but has the highest unit price, and plays a role of wrapping, protection and beautification for the whole cigar. The color of tobacco jacket tobacco leaf is required to be different due to the high and low of product grade, and the common color of tobacco jacket tobacco leaf can be divided into seven types from deep to shallow, i.e. black-brown, brown-brown, dark brown, medium brown, brown, light brown and green-brown. Different colors of tobacco jacket products have different positioning, and the demand of industrial manufacturers for different colors of tobacco jacket is also different. Generally speaking, the deeper the color of tobacco jacket, the more full-bodied flavor and the more mellow aroma it has, and it brings a more smooth and smooth taste experience, and the dark brown tobacco jacket is also more popular among cigar lovers.

[0003] Tobacco fermentation is the main processing method used by the tobacco industry to improve the quality of tobacco leaves. After fermentation, the appearance quality of the tobacco leaves becomes better, the color of the tobacco leaves deepens and becomes uniform, and the aroma becomes more mellow. The surface and inside of the tobacco leaves are rich in microorganisms, which play an important role in the fermentation process of tobacco. They can synthesize and secrete various extracellular degrading enzymes during growth and development, which can accelerate the degradation of macromolecular substances in tobacco leaves, thereby converting them into small molecules that are beneficial to improving the quality and safety of tobacco, and ultimately changing the quality of tobacco. However, the current understanding of cigar tobacco jacket fermentation microorganisms is limited, and their role in changing the color of tobacco jacket is rarely reported.

[0004] At present, the domestic cigar tobacco jacket has problems such as insufficient yield, unstable color and poor quality, which leads to excessive dependence on imported tobacco jacket. Therefore, it is crucial to improve the yield of cigar tobacco jacket, stabilize the color of cigar tobacco jacket and improve the grade of tobacco leaves. The temperature and humidity conditions of cigar tobacco fermentation create unique conditions for the application of biological technologies such as microorganisms and enzymes. Using microorganisms to improve the color of tobacco jacket during fermentation is an economical and effective way, and also a green production method. By mixing microorganisms with water evenly before tobacco fermentation and spraying them on the surface of tobacco jacket tobacco leaves, the microorganisms can be inoculated and placed in a tobacco pile for fermentation, which can increase the role of microorganisms in the fermentation process and promote the color change of tobacco jacket during fermentation. This is a safe, reliable and low-cost way.

[0005] The strain DS101 provided by the application belongs to the genus Kosakonia of the family Enterobacteriaceae. The first model strain of the genus was reported by Brady et al. in 2013. The genus Kosakonia has the functions of producing amylase, cellulase, tannase, phytase and the like, and is currently mainly applied to brewing of baijiu (Chinese liquor).

[0006] Currently, no strain of the genus Kosakonia of the family Enterobacteriaceae has been reported to have the function of the application. SUMMARY

[0007] The purpose of the application is to provide a strain of Kosakonia sp. DS101, and the strain has a preservation number of CCTCC NO: M20241671.

[0008] Another purpose of the application is to provide the application of the strain Kosakonia sp. DS101 in improving the color of eggplant clothes. In order to achieve the above purpose, the following technical measures are adopted in the application:

[0009] The applicant isolated and screened a strain DS101 with the ability of significantly changing the color of eggplant clothes in the fermentation process of cigar tobacco leaves from cigar tobacco leaves of different periods provided by the Hubei Province Enshi Tobacco Company, China. The applicant named the strain as Kosakonia sp. DS101. The strain was sent to the China Center for Type Culture Collection (CCTCC) on July 22, 2024, and has a preservation number of CCTCC NO: M20241671, a classification name of Kosakonia sp. DS101, and an address of China Wuhan, Wuhan University.

[0010] The culture and morphological characteristics of the strain are as follows:

[0011] The cell morphology of the strain DS101 is rod-shaped, 1-2 μm long and 0.5-1 μm wide. After being cultured on a YPDA agar plate medium at 28℃ for 48 h, the strain forms an opaque yellow colony, which is round, slightly convex, and has a complete and smooth edge.

[0012] The protection scope of the application includes:

[0013] A complex preparation containing the Kosakonia sp. DS101.

[0014] The application of the Kosakonia sp. DS101 or the complex preparation in improving the color of eggplant clothes.

[0015] In the above-mentioned application, preferably, the improvement is deepening the color of eggplant clothes.

[0016] The preferred method of the above-described application is to apply a 1×10⁻⁶ ppm solution to the eggplant wrapper according to its weight. 6 Up to 1×10 8 The inoculation amount was CFU / g of tobacco leaves, and Coxsackie bacterium DS101 strain was inoculated for composting to deepen the color of the eggplant wrapper.

[0017] Of the applications described above, the preferred method of inoculation is spraying.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The strain DS101 obtained in this invention is a strain that has the ability to improve the color of cigar wrapper, which was isolated and screened from cigar tobacco leaves at different times and promotes the color change during the fermentation process of cigar wrapper. It is an important supplement to the limited resources of cigar wrapper microbial strains.

[0020] (2) This invention provides a new way to promote color change in the fermentation process of tobacco leaves using beneficial microorganisms, which is green, economical and environmentally friendly. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the growth curve of strain DS101 in YPDA medium.

[0022] Figure 2 These are the measurement sites for tobacco leaf color parameters.

[0023] Figure 3 The effect of strain DS101 on improving the color of eggplant wrapper during tobacco fermentation is shown in the figure.

[0024] Where: Control represents the effect of spraying only water on the surface of the eggplant wrapper as a blank control.

[0025] Figure 4 A schematic diagram illustrating how adding strain DS101 during fermentation alters the color parameters of the eggplant skin. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. The embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, but the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0027] Example 1:

[0028] Isolation and identification of strain DS101

[0029] (1) Sample acquisition: The applicant collected cigar tobacco leaves during the drying and fermentation stages from Enshi Tobacco Company in Hubei Province from May 2023 to August 2023.

[0030] (2) Bacterial cell isolation and purification: 10g of tobacco leaves at different stages were weighed into Erlenmeyer flasks containing 90mL of sterile physiological saline and shaken in a shaker at 28℃ for 24h. Then, 1mL of each flask was added to 9mL of sterile physiological saline and gradually diluted to 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 , for 10 -5 10 -6 Two gradients of 0.1 mL were sequentially spread onto R2A, LB, 1 / 5LB, NB, TSA, CDM, YPDA, YPD, BG, and PCA solid media, with three plates for each gradient. The plates were incubated at 28°C for 48 hours, and single colonies were picked and streaked for purification. After purification, the culture was stored in screw-cap tubes at a 1:1 volume ratio of 50% glycerol to bacterial culture and kept at -80°C.

[0031] (3) Screening of bacteria that alter the color of eggplant husks during tobacco fermentation: The strains were cultured in liquid form, and the cultures were centrifuged and washed to obtain inoculum. Tobacco leaves with similar colors were selected and sprayed with a mixture of inoculum and water. The inoculum amount was 1×10⁻⁶. 7 CFU / g, the control group was sprayed with an equal amount of water, and then both were placed in a tobacco pile for fermentation. The color parameters of the fermented tobacco leaves were measured using a precision colorimeter. The color parameter measurement indicators were: brightness value L*, redness value a*, and yellowness value b*. Based on these, the total color difference value ΔE was calculated using the formula: ΔE=(ΔL*) / (b ... 2 +Δa* 2 +Δb* 2 ) 1 / 2 The larger the total color difference value ΔE, the darker the color. A difference greater than 2 indicates a significant color change that can be distinguished by the naked eye. If the difference between the total color difference value ΔE of the experimental group and the control group is greater than 2, the tobacco leaf color is significantly darker, indicating that this bacterium has the ability to improve the color of the eggplant wrapper during the tobacco fermentation process.

[0032] (4) Classification and identification of strain DS101: The obtained strain was subjected to 16S rRNA gene sequencing and its phylogenetic tree was constructed. Based on its physiological and biochemical characteristics, the bacterium was identified as Kosakonia. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 22, 2024, with accession number CCTCC NO: M20241671, classification name: Kosakonia sp. DS101, address: Wuhan University, Wuhan, China.

[0033] The culture and morphological characteristics of this strain are as follows:

[0034] The cells of strain DS101 are rod-shaped, 1–2 μm long and 0.5–1 μm wide. When cultured on YPDA agar plates at 28°C for 48 h, they form opaque yellow colonies that are round, slightly raised, and have intact, smooth edges.

[0035] (5) Growth curve: The bacterial strain was added to 50 mL of YPDA liquid medium at a 1% inoculum rate, with each treatment replicated three times. The culture was carried out at 28℃ and 150 rpm on a shaker. Every 12 hours, 2 mL of the bacterial cell growth concentration (OD600) was measured using a spectrophotometer. Growth curves were plotted, and the results are as follows: Figure 1 As shown, strain DS101 grows well in YPDA medium and reaches a stable growth phase within 48 hours.

[0036] Example 2:

[0037] Application of strain DS101 in improving eggplant wrapper color during tobacco fermentation:

[0038] The specific steps are as follows:

[0039] (1) Prepare sterilized YPDA liquid culture medium and conduct liquid amplification culture of strain DS101. The culture is centrifuged and washed to obtain inoculum. Prepare 3 tobacco leaves with similar color and divide them in half along the midrib. Spray the surface of one half of the tobacco leaf with a mixture of DS101 strain and water. The inoculum amount is 1×10⁻⁶ according to the weight of the tobacco leaf. 7 CFU / g, the other half of the tobacco leaves are sprayed with an equal amount of water, both of which are necessary for tobacco fermentation, and then put together into a tobacco pile for fermentation.

[0040] (2) Record the temperature of the tobacco pile daily. When the pile temperature reaches 50℃-55℃, turn the pile over according to the standard of "turning the top down and the inside out". Then re-stack the pile and repeat the operation 3 to 5 times. When the temperature of the tobacco pile remains basically unchanged, the tobacco fermentation is considered to be complete.

[0041] (3) The color parameters of the fermented tobacco leaves were measured using a precision colorimeter. The measurement sites were as follows:Figure 2 As shown, the tobacco leaf color parameter measurement area was divided into three regions: leaf base (2nd-3rd veins), leaf middle (5th-6th veins), and leaf tip (8th-9th veins). Detection points 1-3 and 4-6 were the color parameter measurement points for the left and right parts of the same leaf, respectively. Each measurement was repeated three times, and the average value of each point was taken as the color parameter of the tobacco leaf sample. The color parameter measurement indicators were: brightness value L*, redness value a*, and yellowness value b*, and the total color difference value ΔE was calculated based on these. L0 = 94.59, a0 = 0.01, and b0 = 1.04 were the color parameters of the white board. The calculation formula was: ΔE = (ΔL*) / (ΔB*). 2 +Δa* 2 +Δb* 2 ) 1 / 2 (ΔL* = L* - L0, Δa* = a* - a0, Δb* = b* - b0). The brightness value L* represents luminance, ranging from 0 to 100. A smaller value indicates a color closer to black, with the maximum value representing white. The redness value a* represents the red-green value; a larger value indicates a color closer to red. The yellowness value b* represents the yellow-blue value; a larger value indicates a color closer to yellow. The total color difference value ΔE represents a deeper color. A difference between 0 and 1 indicates almost no color change; between 1 and 2 indicates a moderate color change, discernible to the naked eye; and a difference greater than 2 indicates a significant color change, with larger differences indicating more pronounced color changes. If the difference between the experimental group's total color difference value ΔE and the control group is greater than 2, it indicates a significant deepening of the tobacco leaf color.

[0042] (4) Take photos of the fermented tobacco leaves and record the color changes. Figure 3 ).

[0043] (5) A schematic diagram was constructed with the eggplant peel color parameter as the x-axis and its value as the y-axis to illustrate how strain DS101 changed the eggplant peel color parameter during tobacco fermentation. Figure 4 ).

[0044] (6) Figure 4 As shown, the color parameters of the eggplant wrapper inoculated with strain DS101 during tobacco fermentation were compared with those of the control group that was only sprayed with water: the average brightness value L* decreased, the redness value a* and yellowness value b* remained basically the same, and the total color difference value 72.73 and 70.69 differed by 2.04. Since the difference was greater than 2, the eggplant wrapper color was significantly darker. Strain DS101 has a good ability to improve the color of eggplant wrapper during tobacco fermentation.

[0045]

Claims

1. An isolated strain of Cossackie ( Kosakonia sp.)DS101, the preservation number of the strain is CCTCCNO: M20241671.

2. A complex preparation comprising the Kosakonia DS101 of claim 1.

3. Use of the Kosakonia DS101 of claim 1 or the complex preparation of claim 2 for improving the color of eggplant skin, wherein the improvement is darkening the color of eggplant skin.

4. Use according to claim 3, wherein the improvement is in the range of 1 x 10 6 to 1 x 10 8 CFU / g of leaf, of the inoculum of K. oxytoca strain DS101, for the heap fermentation to deepen the color of the pericarp.

1. A method for improving the color of the pericarp of a tobacco plant, comprising inoculating the pericarp of the tobacco plant with a strain of K. oxytoca, and allowing the inoculated pericarp to ferment, wherein the strain of K. oxytoca is selected from the group consisting of K. oxytoca strains DS101, DS102, DS103, DS104, DS105, DS106, DS107, DS108, 5. The use of claim 4, wherein the inoculation is by spraying.

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