Cigar tobacco leaf fermentation by constructing stable microbial community based on adaptive evolution method

Abstract

The invention discloses a method for constructing a stable microbial community based on adaptive evolution to ferment cigar tobacco leaves. The method comprises the following steps: firstly, separating and enriching the microbial community from the cigar tobacco leaves; then, a culture medium prepared after target cigar tobacco leaves are sterilized is used for continuous subculture of microbial communities; on the basis of the difference of nutritional ingredients in different cigar tobacco leaves and the interaction among microorganisms, a microbial community is driven to generate adaptive evolution, and finally a stable microbial community is formed; finally, the cigar tobacco leaves are inoculated with the stable microbial community for solid-state fermentation, and the quality and fragrance of the cigar tobacco leaves can be obviously improved after fermentation is finished. The method has the characteristics of simplicity and convenience in operation, easiness in repetition, high efficiency and the like, and can effectively solve the problems of poor quality, unstable quality and the like of domestic cigar tobacco leaves at present. By applying the method disclosed by the invention, stable microbial community construction can be completed within 15 days, so that the construction of synthetic microbial communities is greatly accelerated, the time is shortened, and the cost is saved.

Description

Technical Field

[0001] The invention relates to the application field of microbial technology in the tobacco industry, and in particular to a method based on adaptive evolution to construct a stable microbial community for cigar tobacco fermentation. Background Art

[0002] Cigar tobacco fermentation is an important process to improve the appearance color, physical properties, aroma concentration and aroma quality of cigar tobacco, and is a key step in cigar tobacco production. The microbial community in tobacco plays an important role in the fermentation process. The microbial community of cigar tobacco comes from the uncontrolled external migration of microorganisms in nature. Since cigar tobacco fermentation is completely dependent on microorganisms that migrate randomly in the natural environment, this dependence may lead to unstable fermentation effects and quality differences between different batches. Drawing on the experience of traditional liquor fermentation technology, pre-inoculation of fermentation initiators before fermentation may solve this problem. Fermentation initiators are microbial preparations used to initiate or promote fermentation. Mixed microbial agents (fermentation agents) have high genetic diversity, which enables them to adapt to the environment faster. There is a competitive and cooperative relationship between microorganisms, and the interaction between microorganisms makes them easier to play a role.

[0003] The preparation of mixed microbial inoculants (fermentation agents) usually adopts two methods to synthesize microbial communities. The first is based on the study of microbial individuals and communities, and synthesizes microbial communities that can achieve ideal functions according to the metabolic characteristics of individual microorganisms and the interactions between microorganisms. However, due to the limited understanding of microbial communities, most can only construct simple communities composed of limited species. The second is to obtain a synthetic microbiome with ideal functions by controlling environmental conditions to domesticate microbial communities. The adaptive evolution of microbial communities refers to the process in which microbial communities gradually change their community structure and function under specific environmental conditions through mechanisms such as gene mutation, gene flow and natural selection to better adapt to environmental changes, and gradually form stable microbial communities. Summary of the invention

[0004] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method based on adaptive evolution to construct a stable microbial community for cigar tobacco fermentation. Based on the basic material composition of the cigar tobacco to be fermented and the interaction between microorganisms, the present invention establishes a method based on adaptive evolution to construct a stable microbial community for cigar tobacco fermentation, which has the characteristics of simple operation, easy repeatability, high efficiency, etc.

[0005] The present invention separates microbial communities from imported high-quality cigar leaves and continuously cultures them on a culture medium prepared by sterilizing cigar leaves to be fermented, drives the microbial communities to perform adaptive evolution on the target leaves based on the basic material composition of the target cigar leaves and the interaction between the microorganisms, analyzes the microbial community construction process through high-throughput sequencing, determines the formation of a stable microbial community, inoculates the constructed stable microbial community into the target cigar leaves for solid-state fermentation, and verifies the effect of the microbial community in the fermentation of cigar leaves.

[0006] The present invention constructs a cigar tobacco leaf starter based on the adaptive evolution of microflora. First, a microbial community is separated and enriched from high-quality cigar tobacco leaves. Then, the microbial community is continuously subcultured using a culture medium prepared after sterilizing the target cigar tobacco leaves. Due to the different nutritional components in different cigar tobacco leaves, a stable microbial community will eventually be formed. Finally, the stable microbial community is inoculated into cigar tobacco leaves for solid-state fermentation to verify the fermentation effect of the constructed microflora.

[0007] The specific steps include:

[0008] (1) Select a certain mass of imported high-quality cigar tobacco leaves and add sterile saline or neutral phosphate buffer, shake to separate the tobacco leaves and microorganisms, centrifuge to collect the microorganisms, and then add 5 mL of sterile saline or neutral phosphate buffer to resuspend the microorganisms.

[0009] (2) The collected microorganisms were added to 100 mL of liquid culture medium prepared from ordinary cigar tobacco leaves to be fermented, and cultured in a shaking incubator at 30-37° C. for 5-7 days.

[0010] (3) After the culture is completed, step (2) is repeated and the transfer is continued multiple times to obtain a stable microbial community. High-throughput sequencing is used to determine whether the constructed microbial community is stable.

[0011] (4) The stable microbial community is evenly sprayed onto cigar tobacco leaves to be fermented to perform tobacco fermentation. The fermentation effect of the cigar tobacco leaves is verified by sensory evaluation.

[0012] In step (1), 1-5 g of tobacco leaves are weighed, sterile saline or neutral phosphate buffer is added at a mass volume ratio of 1 g:40-50 mL, and the mixture is shaken at a speed of 200-250 rpm for 3-4 hours, and then the tobacco leaf fragments and microorganisms are separated and the microorganisms are collected.

[0013] In step (2), the liquid culture medium prepared with common cigar tobacco leaves to be fermented has the following composition: 100 mL of distilled water is added to 1 g of tobacco leaves to be fermented, and sterilized at 115° C. for 20 min.

[0014] In step (2), the culture conditions are: 30-37°C, 180-220 rpm for 5-7 days.

[0015] In step (2), the culture medium needs to be transferred to a new culture medium for 2-3 times to form a stable microbial community.

[0016] In step (4), the constructed stable microbial community is diluted 5-10 times with water, physiological saline or other buffer, and the bacterial liquid is added to cigar tobacco leaves with a moisture content of 20-30% at a rate of 1-5% by weight of the tobacco leaves for fermentation at a fermentation temperature of 30-50° C. and a fermentation time of 15-30 days.

[0017] The beneficial effects of the present invention are embodied in:

[0018] 1. The present invention has the characteristics of simple operation, easy repeatability, high efficiency, etc., and can effectively solve the problems of poor quality and unstable quality of current domestic cigar tobacco leaves.

[0019] 2. By applying the method of the present invention, the construction of a stable microbial community was completed within 15 days, which greatly accelerated the construction of a synthetic microbial community, shortened the time, and saved costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present embodiment. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 This is the test process of the present invention.

[0022] Figure 2 Bacterial flora analysis constructed in the present invention, wherein A, B, and C are different samples.

[0023] Figure 3 β-diversity analysis of the bacterial flora constructed for the present invention. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0025] Example 1: Microbial community construction

[0026] Isolation of microorganisms: Take three different brands of high-quality cigar tobacco leaves, weigh 5g of each tobacco leaf, add 100mL of sterilized saline or phosphate buffer (pH 7), shake and elute to separate the tobacco leaves and bacterial solution, and collect the microorganisms by centrifugation.

[0027] Adaptive evolution of microbial communities: The collected microorganisms were added to a tobacco leaf culture medium prepared by sterilizing ordinary cigar tobacco leaves, and cultured in a shaker at 30°C and 220 rpm for 5 days. Then 10 mL of the cultured microorganisms were added to a new tobacco leaf culture medium for culture, and the culture was transferred continuously for 3-4 times to form a stable microbial community.

[0028] Example 2: Analysis of constructed microbial communities

[0029] High-throughput sequencing was used to analyze the changes in the microbial community during continuous cultivation, and the community composition formed at the end of each cultivation of the three samples was compared to determine whether a stable microbial community could be formed after several rounds of transfer cultivation. Figure 2 and Figure 3 As shown, after 3 rounds of cultivation, the structure of the bacterial community has changed greatly compared with the original sample, but the relative abundance of the dominant genera in the second and third rounds of the three communities is basically the same, and the difference in the community structure is small, indicating that after 2-3 rounds of transfer, the bacterial community structure remains basically stable.

[0030] Example 3: Cigar Tobacco Fermentation

[0031] The stable microbial community constructed by adaptive evolution was diluted 5-10 with sterile water, and then evenly sprayed on cigar tobacco leaves at a 30% inoculation amount (v / g), placed in a biochemical incubator, and fermented for 15 days at a temperature of 30°C and a humidity of 75%. According to the evaluation of professionals, the quality of cigar tobacco leaves fermented with mixed flora has been significantly improved, the aroma of cigar tobacco leaves has been significantly improved, the aroma has become richer, the smoke has a sweet taste, the impurities have been reduced, and the aftertaste has become more comfortable.

[0032] Table 1 Cigar tobacco leaf sample fermentation evaluation results

[0033]

[0034] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations. In addition, the various different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method based on adaptive evolution to construct a stable microbial community for cigar tobacco fermentation, characterized in that: First, the microbial community is isolated and enriched from cigar leaves; then, the microbial community is continuously cultured using a culture medium prepared after sterilizing the target cigar leaves; due to the different nutritional components in different cigar leaves, a stable microbial community will eventually be formed; finally, the stable microbial community is inoculated into cigar leaves for solid-state fermentation.

2. The method of constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 1, characterized in that The steps include: (1) taking 1-5 g of cigar tobacco leaves, adding sterile saline or neutral phosphate buffer, shaking to separate the tobacco leaves and microorganisms, centrifuging to collect the microorganisms, and then adding sterile saline or neutral phosphate buffer to resuspend the microorganisms; (2) adding the collected microorganisms to 100 mL of liquid culture medium prepared from ordinary cigar tobacco leaves to be fermented for culturing; (3) Repeat step (2) and continue to transfer multiple times to obtain a stable microbial community; determine whether the constructed microbial community is stable by high-throughput sequencing; (4) The stable microbial community is evenly sprayed onto cigar tobacco leaves to be subjected to enhanced fermentation for tobacco leaf fermentation.

3. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 2, characterized in that: In step (1), 1-5 g of tobacco leaves are weighed, sterile saline or neutral phosphate buffer is added at a mass volume ratio of 1 g:40-50 mL, and the mixture is shaken at a speed of 200-250 rpm for 3-4 hours, and then the tobacco leaf fragments and microorganisms are separated and the microorganisms are collected.

4. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 2, characterized in that: In step (2), the liquid culture medium prepared with common cigar tobacco leaves to be fermented has the following composition: 100 mL of distilled water is added to 1 g of tobacco leaves to be fermented, and sterilized at 115° C. for 20 min.

5. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 2, characterized in that: In step (2), the culture conditions are: 30-37°C, 180-220 rpm for 5-7 days.

6. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 2, characterized in that: In step (3), the operation of step (2) is repeated, and the culture medium is continuously transferred to a new culture medium for 2-3 times to form a stable microbial community.

7. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 2, characterized in that: In step (4), the constructed stable microbial community is diluted 5-10 times with water, physiological saline or other buffer solution, and the bacterial liquid is added to cigar tobacco leaves with a moisture content of 20-30% at a rate of 1-5% by weight of the tobacco leaves for fermentation.

8. The method for constructing a stable microbial community for cigar tobacco fermentation based on adaptive evolution according to claim 7, characterized in that: The fermentation temperature is 30-50°C and the fermentation time is 15-30 days.

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