A starved microbial inoculant and its application in tobacco fermentation

Abstract

This invention discloses a starved microbial agent and its application in tobacco fermentation. The preparation method of the starved microbial agent is as follows: Klebsiella pneumoniae is inoculated into LB liquid medium to obtain a primary seed culture; the primary seed culture is mixed with LB liquid medium to obtain a secondary seed culture; the secondary seed culture is mixed with tertiary seed medium to obtain a tertiary seed culture; the tertiary seed culture is centrifuged to remove the supernatant, and the precipitate is added with deionized water to obtain a primary starved seed; the primary starved seed is mixed with tobacco aqueous extract to obtain a secondary starved seed; the secondary starved seed is centrifuged, and the precipitate is redissolved to obtain the starved microbial agent. After treatment with this starved microbial agent, the reducing sugar, total sugar, and sugar-alkali ratio of the tobacco leaves increase significantly, indicating an improvement in the intrinsic chemical quality of the tobacco leaves; after treatment with the starved microbial agent, the aroma quality and aroma quantity of the tobacco leaves are significantly improved, the off-flavors are reduced, the smoke is more delicate, the irritation is reduced, the aftertaste is improved, and the quality of the tobacco leaves is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a starved microbial agent and its application in tobacco fermentation. Background Technology

[0002] Domestically produced tobacco leaves generally suffer from a high content of macromolecules and a low content of aroma compounds, resulting in a strong off-flavor, high irritation, and insufficient aroma when inhaled. Current efforts to improve this situation involve optimizing processing techniques and adding flavorings, but the results are not particularly ideal. Processing optimization primarily focuses on improving the tobacco curing process, but it's difficult to efficiently reduce macromolecules, let alone increase aroma components. Adding flavorings mainly masks off-flavors and increases aroma compound content, but this is often not well-balanced and is costly. Microbial fermentation of tobacco leaves can effectively improve their internal chemical quality, increase aroma compound content, and is a lower-cost and simpler process.

[0003] However, due to the low moisture content of tobacco leaves, which falls far short of the optimal moisture requirements for microbial fermentation, and the presence of nicotine in tobacco leaves, some microorganisms struggle to colonize and ferment stably on the leaf surface. Therefore, increasing the inoculum size and extending the fermentation time are typically employed to ensure optimal fermentation results. Starved microorganisms possess strong environmental adaptability; selecting appropriate starvation methods can enable them to function better on the tobacco leaf surface, improving fermentation efficiency. The preparation of starved microbial agents requires tailoring process parameters based on the characteristics of the microorganisms themselves. Furthermore, starved microbial agents used in tobacco leaf treatment must also consider the tolerance of the tobacco leaf surface environment. This necessitates extensive experimentation and cannot be simply applied blindly. Inappropriate preparation processes can lead to low microbial activity and poor treatment outcomes. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a starved microbial agent and its application in tobacco fermentation, which overcomes the above-mentioned defects of the prior art.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] A starved microbial agent, said starved microbial agent being prepared by the following method:

[0007] (1) Seed liquid preparation:

[0008] A single colony of Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 24327, was inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ and 100-200 r / min for 6-36 h to obtain the primary seed culture.

[0009] Mix the primary seed culture with LB liquid medium at a volume ratio of 1:100 to 1:5 and incubate at 25-40℃ and 100-200r / min for 6-36h to obtain the secondary seed culture.

[0010] The secondary seed culture and the tertiary seed culture medium are mixed at a volume ratio of 1:100 to 1:5 and cultured in a shaker at 25-40℃ and 100-200r / min for 6-36h to obtain the tertiary seed culture.

[0011] (2) Preparation of starved microbial inoculants:

[0012] Take a volume of V1 of the third-stage seeds, centrifuge to remove the supernatant, add 80% of V1 volume of deionized water to the precipitate, and place it in a shaker at 25-40℃ and 10-100 r / min for 1-10 h to obtain the first-stage starved seeds.

[0013] Primary starved seeds were mixed with tobacco aqueous extract at a volume ratio of 10:1 to 100:1 and placed at 4-30℃ for 6-36 hours to obtain secondary starved seeds.

[0014] (3) Starvation microbial inoculants:

[0015] Centrifuge the secondary starved seeds (volume V2), remove the supernatant, reconstitute the precipitate with 80% (volume V2) of deionized water, and let it stand at 4°C for 6-36 hours to obtain the starved microbial inoculant.

[0016] Preferably, in the above technical solution, the culture conditions for the primary seed culture are: cultured for 12 hours at a temperature of 30°C and a rotation speed of 150 r / min in a shaker; in the preparation of the secondary seed culture, the volume ratio of the primary seed culture to LB liquid culture medium is 1:10, and the culture is carried out for 12 hours at a temperature of 30°C and a rotation speed of 150 r / min in a shaker.

[0017] Preferably, in the above technical solution, the tobacco aqueous extract is obtained by mixing tobacco leaf fragments and water at a solid-liquid ratio of 1:20 to 1:5, extracting at a constant temperature of 70°C for 2 hours, filtering and collecting the filtrate.

[0018] Preferably, in the above technical solution, the tertiary seed culture medium is prepared by the following method: tobacco aqueous extract and LB liquid culture medium are mixed at a volume ratio of 1:1 to 1:10 to obtain the tertiary seed culture medium.

[0019] Preferably, in the above technical solution, tobacco leaf fragments and water are mixed at a solid-liquid ratio of 1:10, tobacco water extract and LB liquid culture medium are mixed at a volume ratio of 1:3, secondary seed liquid and tertiary seed culture medium are mixed at a volume ratio of 1:10, and cultured for 12 hours in a shaker at a temperature of 30°C and a rotation speed of 150 r / min.

[0020] Preferably, in the preparation of the starved microbial agent in the above technical solution, the centrifugation conditions are as follows: centrifuge at 4000 rpm and 4℃ for 10 min, and then place in a shaker at 30℃ and 80 r / min for 2 h; the primary starved seeds and tobacco aqueous extract are placed at 10℃ and allowed to stand for 12 h at a volume ratio of 20:1.

[0021] Application of a starving microbial agent in tobacco fermentation.

[0022] Preferably, in the above technical solution, tobacco leaves are treated with starved microbial agents. The starved microbial agents are mixed with tobacco leaves or shredded tobacco at a mass ratio of 1:100 to 1:10. The agents are sprayed evenly onto the surface of the tobacco leaves using a mist sprayer. The leaves are then placed in an environment with a temperature of 25-40°C and a humidity of 30%-100% and left to stand for 6-36 hours until fermentation is complete.

[0023] Preferably, in the above technical solution, the mass ratio of the starving microbial agent to tobacco leaves or shredded tobacco is 1:50, and the cultivation conditions after spraying are: temperature 30℃, humidity 70%, and open standing for 24 hours.

[0024] Preferably, in the above technical solution, after treatment with starvation microbial agents, the reducing sugar, total sugar and sugar-alkali ratio of tobacco leaves are significantly increased; the aroma quality and aroma quantity of tobacco leaves are significantly improved, impurities are reduced, the smoke is more delicate, the irritation is reduced, the aftertaste is improved, and the quality of tobacco leaves is significantly improved.

[0025] The above-described technical solution of the present invention has the following beneficial effects:

[0026] The starved microbial agent prepared in this application significantly increases the reducing sugar, total sugar, and sugar-alkali ratio of tobacco leaves after treatment, indicating an improvement in the intrinsic chemical quality of the tobacco leaves. After treatment with the starved microbial agent, the aroma quality and aroma quantity of the tobacco leaves are significantly improved, the off-flavors are reduced, the smoke is more delicate, the irritation is reduced, the aftertaste is improved, and the total score increases from 64.10 to 66.32, showing a significant improvement in the quality of the tobacco leaves. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0028] This application utilizes Klebsiella sp. HNYJ-1 to prepare starvation microbial inoculum. Klebsiella sp. HNYJ-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327, deposited on January 17, 2022, and located in Beijing, China.

[0029] Example 1: Seed Liquid Preparation

[0030] (1) Preparation of primary seed culture: Klebsiella pneumoniae HNYJ-1 single colony is inoculated into LB liquid medium with an inoculation loop and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 12h) to obtain primary seed culture.

[0031] (2) Preparation of secondary seed culture: Mix the primary seed culture with LB liquid culture medium at a volume ratio of 1:100 to 1:5 (optimal 1:10) and culture in a shaker at 25-40℃ (optimal 30℃) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 12h) to obtain the secondary seed culture.

[0032] (3) Preparation of three-stage seed liquid:

[0033] Tobacco aqueous extract: Mix tobacco leaf fragments at a solid-liquid ratio of 1:20 to 1:5 (optimal 1:10), extract at a constant temperature of 70℃ for 2 hours, filter and collect the filtrate to obtain tobacco aqueous extract;

[0034] Tertiary seed culture medium: Tobacco aqueous extract and LB liquid culture medium are mixed at a volume ratio of 1:1 to 1:10 (optimal 1:3) to obtain tertiary seed culture medium;

[0035] The secondary seed culture and the tertiary seed culture medium are mixed at a volume ratio of 1:100 to 1:5 (optimal 1:10) and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200 r / min (optimal 150 r / min) for 6-36 h (optimal 12 h) to obtain the tertiary seed culture.

[0036] In the preparation of seed culture, the first two stages of seed culture can obtain a larger amount of inoculum, and the third stage of seed culture adds tobacco water extract, which can enable the seeds to adapt to the tobacco environment in advance.

[0037] Example 2: Preparation of starved microbial inoculants

[0038] (1) Preparation of primary starved seeds: Measure a volume of V1 of the tertiary seeds, centrifuge to remove the supernatant (4000 rpm, 4℃ for 10 min), add 80% of V1 volume of deionized water to the precipitate, and place it in a shaker at 25-40℃ (optimal 30℃) and 10-100 r / min (optimal 80 r / min) for 1-10 h (optimal 2 h) to obtain primary starved seeds.

[0039] (2) Preparation of secondary starved seeds: Primary starved seeds and tobacco aqueous extract are mixed in a volume ratio of 10:1 to 100:1 (optimal 20:1) and placed at 4-30℃ (optimal 10℃) for 6-36h (optimal 12h) to obtain secondary starved seeds.

[0040] Starvation seed preparation allows the strain to preferentially recognize the tobacco substrate under starvation conditions.

[0041] (3) Starvation microbial inoculant: Centrifuge the secondary starvation seeds with a volume of V2, remove the supernatant, reconstitute the precipitate with 80% V2 volume of deionized water, and place it in a 4℃ environment for 6-36h (optimal 24h) to obtain the starvation microbial inoculant.

[0042] The purpose of this step is to allow the microbial cells to consume as much of the remaining nutrients as possible in a low-temperature environment, until they reach a state of complete starvation.

[0043] The three-step method for preparing starved microbial cells in this application differs from general starved cell preparation methods. It adopts a gradual approach, which is gentle and includes a certain degree of starvation induction, resulting in higher activity and better treatment effects of the obtained microbial agents.

[0044] Example 4: Method for treating tobacco leaves with starved microbial agents

[0045] The starving microbial inoculant is mixed with tobacco leaves or shredded tobacco at a mass ratio of 1:100 to 1:10 (optimal 1:50). The inoculant is sprayed evenly onto the surface of the tobacco leaves using a mist sprayer. The mixture is then placed in an environment with a temperature of 25-40℃ (optimal 30℃) and a humidity of 30%-100% (optimal 70%) and left to stand for 6-36 hours (optimal 24 hours) until fermentation is complete.

[0046] This fermentation process is simple and easy to operate. It requires a small inoculum size and has a short fermentation time, greatly improving fermentation efficiency.

[0047] Application Example 1

[0048] The experimental tobacco leaves were selected from C3F tobacco leaves grown in Xiangxian County, Henan Province in 2020. Microbial inoculant was sprayed evenly onto the tobacco leaf surface using a 1:50 mass ratio with tobacco leaves, and left to stand for 24 hours at 70% humidity and 30℃; this group was designated T. Deionized water was sprayed evenly onto the tobacco leaf surface using a 1:50 mass ratio with tobacco leaves, and this group was designated CK. The tobacco leaves were then thoroughly turned and left to stand for 24 hours at 30℃ and 70% humidity. After fermentation, the leaves were cut into shreds and sterilized by high-temperature drying. Flow spectroscopy was used to analyze the chemical composition of the shredded tobacco. The remaining shredded tobacco was adjusted for moisture content, rolled into cigarettes, and subjected to sensory evaluation by professional tobacco tasters.

[0049] Results analysis:

[0050] (1) Routine chemical composition detection

[0051] The chemical composition analysis of the two types of tobacco leaves is shown in Table 1. The results show that after treatment with starvation microbial agents, the reducing sugar, total sugar, and sugar-alkali ratio of the tobacco leaves increased significantly, indicating that the intrinsic chemical quality of the tobacco leaves was improved.

[0052] Table 1. Analysis of conventional chemical components of tobacco leaves

[0053]

[0054] (2) Sensory evaluation of the two types of cigarettes

[0055] The sensory evaluation results of the two types of cigarettes are shown in Table 2. After treatment with starvation microbial agents, the aroma quality and quantity of the tobacco leaves were significantly improved, the off-flavors were reduced, the smoke was more delicate, the irritation was reduced, and the aftertaste was improved. The total score increased from 64.10 to 66.32, indicating a significant improvement in the quality of the tobacco leaves.

[0056] Table 2. Sensory evaluation results of the two types of cigarettes

[0057]

[0058] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A starved microbial inoculant characterized in that, The starvation microbial agent is prepared by the following method: (1) Seed liquid preparation: Klebsiella (Klebsiella sp.) HNYJ-1 preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 24327 Klebsiella sp. The single colony of Klebsiella (Klebsiella sp.) HNYJ-1 is inoculated into LB liquid medium by an inoculating ring, and then cultured in a shaker at a temperature of 25-40 ℃ and a speed of 100-200 r / min for 6-36 h to obtain a first-stage seed liquid. The first-stage seed liquid is mixed with LB liquid medium at a volume ratio of 1:100-1:5, and cultured at 25-40°C and 100-200 r / min in a shaker for 6-36 h to obtain a second-stage seed liquid; The second-stage seed liquid is mixed with a third-stage seed culture medium at a volume ratio of 1:100-1:5, and cultured at 25-40°C and 100-200 r / min in a shaker for 6-36 h to obtain a third-stage seed liquid; The third-stage seed culture medium is prepared by mixing tobacco water extract and LB liquid medium at a volume ratio of 1:1-1:10; (2) Preparation of the starvation microbial agent: A volume of V1 of the third-stage seed liquid is centrifuged to remove the supernatant, and the precipitate is added with 80% V1 of deionized water, and then placed in a shaker at 25-40°C and 10-100 r / min for 1-10 h to obtain a first-stage starvation seed; The first-stage starvation seed is mixed with tobacco water extract at a volume ratio of 10:1-100:1, and then placed at 4-30°C for 6-36 h to obtain a second-stage starvation seed; A volume of V2 of the second-stage starvation seed is centrifuged to remove the supernatant, and the precipitate is resuspended with 80% V2 of deionized water, and then placed at 4°C for 6-36 h to obtain the starvation microbial agent.

2. The starved microbial inoculant of claim 1, characterized by, The culture conditions of the first-stage seed liquid are as follows: temperature 30°C, rotation speed 150 r / min in a shaker for 12 h; in the preparation of the second-stage seed liquid, the volume ratio of the first-stage seed liquid to LB liquid medium is 1:10, the temperature is 30°C, and the rotation speed is 150 r / min in a shaker for 12 h.

3. The starved microbial inoculant of claim 2, characterized by, The tobacco water extract is prepared by mixing tobacco fragments and water at a solid-liquid ratio of 1:20-1:5, extracting at 70°C for 2 h, filtering to obtain the filtrate, and then obtaining the tobacco water extract.

4. The starved microbial inoculant of claim 1, wherein, The solid-liquid ratio of tobacco fragments to water is 1:10, the volume ratio of the tobacco water extract to LB liquid medium is 1:3, and the volume ratio of the second-stage seed liquid to the third-stage seed culture medium is 1:10, and the culture is carried out at 30°C and 150 r / min in a shaker for 12 h.

5. The starved microbial inoculant of claim 1, wherein, In the preparation of the starvation microbial agent, in the preparation process of the first-stage starvation seed of step (2), the centrifugation conditions are as follows: 4000 rpm, 4°C for 10 min, and then placed at 30°C and 80 r / min in a shaker for 2 h; the volume ratio of the first-stage starvation seed to the tobacco water extract is 20:1, and the mixture is placed at 10°C for 12 h.

6. The starvation microbial agent according to any one of claims 1-5 for use in tobacco leaf fermentation.

7. Use according to claim 6, characterized in that, The tobacco leaves are treated by using the starved microbial agent, the starved microbial agent is mixed with the tobacco leaves or tobacco shreds according to a mass ratio of 1:100-1:10, the agent is sprayed onto the surface of the tobacco leaves in a misty spray pot, and the mixture is placed in an environment with a temperature of 25-40 DEG C and a humidity of 30%-100% for open standing for 6-36 hours until fermentation is completed.

8. Use according to claim 7, characterized in that, The mass ratio of the starved microbial agent to the tobacco leaves or tobacco shreds is 1:50, and the culture condition after spraying is a temperature of 30 DEG C, a humidity of 70%, and open standing for 24 hours.

9. Use according to claim 8, characterized in that, After the treatment by the starved microbial agent, the reducing sugar, total sugar and sugar-base ratio of the tobacco leaves are obviously increased, the aroma quality and aroma amount of the tobacco leaves are obviously improved, the miscellaneous smell is reduced, the smoke is more delicate, the stimulation is reduced, the aftertaste is improved, and the quality of the tobacco leaves is obviously improved.

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