Bacillus inoculant for improving fermentation quality of cigar tobacco leaves as well as preparation method and application of bacillus inoculant

Abstract

The invention relates to the technical field of tobacco processing, in particular to a bacillus inoculant for improving the fermentation quality of cigar tobacco leaves and a preparation method and application thereof, and the bacillus inoculant is composed of bacillus cereus, bacillus pumilus and bacillus thermophilus. The preparation method comprises the following steps: 1) respectively inoculating bacillus cereus, bacillus pumilus and bacillus thermophilus to a TSA culture medium under a sterile condition, sealing, inversely placing a culture dish into an incubator, and culturing at 37 DEG C for 48 hours; (2) respectively picking the purified single colonies, inoculating the single colonies into an LB liquid culture medium, and culturing for 48 hours under the conditions of 37 DEG C and 180r / min until OD600 is equal to 0.5; (3) diluting the three bacterial suspensions with sterile water, and respectively preparing bacillus fermentation broth with required concentration; and 4) mixing the bacillus cereus fermentation broth, the bacillus pumilus fermentation broth and the bacillus thermophilus fermentation broth to obtain the bacillus inoculant.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco processing, and specifically relates to a bacillus agent for improving the fermentation quality of cigar tobacco leaves, a preparation method and an application thereof. Background Art

[0002] Fermentation is an indispensable part of the cigar production process and plays a decisive role in the quality of cigar tobacco leaves and finished products. Studies have shown that the role of microorganisms is always present during the fermentation of tobacco leaves. Microorganisms on the tobacco leaf surface decompose macromolecules in the tobacco leaves through their own metabolism, resulting in a series of physical and chemical changes, which are crucial to improving the quality of tobacco leaves.

[0003] Bacillus is a type of aerobic or facultative anaerobic bacteria that can survive under extreme conditions and produce stress-resistant spores. Its species include many special functional strains that can produce a large amount of amylase and protease. It has important research and application value in scientific fields such as industry, agriculture, and medicine. In the process of tobacco leaf fermentation, beneficial Bacillus can be appropriately added to use organic macromolecules such as protein, sugar and fat in tobacco leaves as nutrients for the growth and reproduction of microorganisms, so that it can use its own growth metabolism to degrade macromolecules and produce biological enzymes to promote the internal biochemical metabolism of tobacco leaves, thereby improving the quality of tobacco leaves.

[0004] At present, domestic cigars have many problems such as insufficient tobacco aroma, heavy impurities, poor sensory quality evaluation, and low industrial availability. Therefore, it is necessary to further improve the fermentation quality of cigar core tobacco. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus inoculum for improving the fermentation quality of cigar tobacco leaves, a preparation method and an application thereof, and to improve the fermentation quality of cigar core tobacco leaves by using different Bacillus species that are resistant to high temperatures and strong acids and alkalis.

[0006] To achieve the above object, the present invention provides the following technical solutions: A bacillus agent for improving the fermentation quality of cigar tobacco leaves consists of bacillus cereus, bacillus brevis and thermophilic bacillus.

[0007] Wherein, the mass fraction of the Bacillus cereus is 10%.

[0008] Wherein, the mass fraction of the Bacillus pumilus is 6%.

[0009] Wherein, the mass fraction of the thermophilic Bacillus is 14%.

[0010] The preparation method of the bacillus agent of the present invention comprises the following steps: (1) Inoculate Bacillus cereus, Bacillus pumilus and Bacillus thermophilus onto TSA medium under sterile conditions, seal the culture dish, place it upside down in an incubator, and culture it at 37°C for 48 hours; (2) Pick out the purified single colonies and inoculate them into LB liquid culture medium, then culture them at 37°C and 180 rpm for 48 h until OD600 = 0.5; (3) Diluting the three bacterial suspensions with sterile water to prepare Bacillus fermentation broths of the required concentrations; (4) The fermentation broths of Bacillus cereus, Bacillus pumilus and Bacillus thermophilus are mixed to obtain a Bacillus inoculum.

[0011] The TSA culture medium is prepared by heating and boiling to dissolve, packaging, and sterilizing at 121° C. for 20 minutes with 15 g of casein trypsin digest, 5 g of sodium chloride, 5 g of soy flour papain digest, 15 g of agar powder, and 1 L of distilled water.

[0012] The LB liquid culture medium was prepared by sterilizing at 121° C. for 20 min using 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

[0013] The bacillus inoculant of the present invention can be used for cigar tobacco leaf fermentation. Before fermentation, the tobacco leaves are moistened, and then the prepared bacillus inoculant is evenly sprayed on the surface of the tobacco leaves with a small sprayer, and the moisture content of the tobacco leaves is controlled to be 26%±1%. The sprayed tobacco leaves are placed in a constant temperature and humidity chamber for fermentation. The fermentation conditions are a temperature of 45°C, a relative humidity of 80%, and a fermentation time of 10 days.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The bacillus inoculant of the present invention adopts three different bacillus, namely, bacillus cereus, bacillus pumilus and thermophilic bacillus. Moreover, through experimental research, it is found that high concentrations of bacillus cereus and bacillus pumilus have the most significant effect on reducing the total nitrogen content of tobacco leaves after fermentation and increasing the total sugar reducing sugar of tobacco leaves; low concentrations of thermophilic bacillus have the most significant effect on degrading protein, and high concentrations of bacillus cereus and bacillus pumilus inoculants can also effectively reduce the protein in tobacco leaves. In addition, bacillus pumilus and thermophilic bacillus can also reduce the nicotine content of tobacco leaves.

[0015] Moreover, the treatment with the bacillus agent of the present invention also has a significant effect on degrading starch in tobacco leaves, among which the treatment with bacillus cereus has the best effect. In addition, the bacillus pumilus and thermophilic bacillus agents can also effectively degrade the cellulose content in tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 and Figure 2 The total sugar and reducing sugar contents of tobacco leaves after fermentation with different treatments; Figure 3-Figure 5 The total nitrogen, protein and nicotine contents of filler tobacco leaves after fermentation under different treatments; Figure 6-Figure 8 Changes in NP, β-amylase and CL activities of filler tobacco leaves after fermentation with different treatments. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] 1. Experimental Materials and Methods

[0019] Test tobacco leaves: CX-81 upper grade cigar core tobacco leaves blended in Laifeng, Hubei in 2022.

[0020] Test strains: Bacillus pumilus (Beijing Solebold Technology Co., Ltd.), Bacillus cereus (Beijing Solebold Technology Co., Ltd.), and Bacillus thermophilus (Shanghai Luwei Technology Co., Ltd.).

[0021] TSA culture medium: 15 g of casein trypsin digest, 5 g of sodium chloride, 5 g of soy flour papain digest, 15 g of agar powder, 1 L of distilled water, heat and boil to dissolve, divide into portions, and sterilize at 121°C under high pressure for 20 min.

[0022] LB liquid medium: 10 g peptone, 5 g yeast powder, 10 g sodium chloride, 1 L distilled water, sterilized at 121°C for 20 min.

[0023] Culture of Bacillus: Different Bacillus were inoculated on TSA medium under sterile conditions and sealed, and the culture dish was placed upside down in an incubator and cultured at 37°C for 48 hours. A purified single colony was picked and inoculated into LB liquid culture medium, and then cultured at 37°C and 180 r / min for 48 hours until OD600 = 0.5.

[0024] Preparation of fermentation liquid: dilute the three types of Bacillus mentioned above according to the weight and moisture content of the treated tobacco leaves (tobacco leaves have their own initial moisture content before fermentation, and it is necessary to ensure that the tobacco leaves reach a suitable fermentation moisture content after spraying the bacterial agent. Therefore, the weight and initial moisture content of the tobacco leaves before fermentation should be considered, and the total amount of the bacterial agent used should be determined according to the weight and initial moisture content of the treated tobacco leaves, so as to ensure that the requirements for the fermentation moisture content of the tobacco leaves are met after spraying the bacterial agent), and prepare Bacillus fermentation liquids with concentrations of 2%, 6%, 10%, and 14%, respectively.

[0025] Fermentation of tobacco leaves: The half-leaf method was used to remove the main vein of the test tobacco leaves. The same number was marked on the same tobacco leaf, and half of the tobacco leaves were rehumidified with sterile water to a moisture content of 25% as a control. Three exogenous Bacillus agents (Bacillus cereus, Bacillus pumilus, and Bacillus thermophilus) were added, and each agent was set at 4 concentrations (2%, 6%, 10%, and 14%). Sterile water was used as the control, for a total of 13 treatments. The experimental design is shown in Table 1. The tobacco leaves were rehumidified before fermentation, and then the prepared enzyme preparation was evenly sprayed on the surface of the tobacco leaves with a small sprayer. The control was an equal amount of sterile water. The moisture content of the tobacco leaves was controlled at 26% ± 1%, and the sprayed tobacco leaves were placed in a constant temperature and humidity chamber for fermentation. Fermentation conditions: temperature 45 ° C, relative humidity 80%, and fermentation time 10 d. Each treatment was repeated 3 times.

[0026] Table 1 Experimental design table

[0027] Orthogonal test to optimize the ratio of compound microbial agent: Using the same variety of tobacco leaves as the test material, under the same conditions, on the basis of single factor test, taking the total score of tobacco leaf sensory quality evaluation as the index, the effects of different concentrations of Bacillus cereus, Bacillus pumilus and Bacillus thermophilus on tobacco leaf sensory quality were investigated, and a three-factor three-level orthogonal test was designed to optimize the ratio of compound microbial agent. The orthogonal test factor level table is shown in Table 2. The three-factor three-level L9 (33) orthogonal table was used to design the experiment as shown in Table 3.

[0028] Table 2 Orthogonal test factor level table

[0029] Table 3 Orthogonal experimental design table

[0030] 2. Experimental process

[0031] (1) Equal amount of sterile water treatment group The surface of the CX-81 upper first-level cigar core tobacco leaves produced in Laifeng, Hubei in 2022 was evenly sprayed with sterile water for moisture rehydration. The moistened tobacco leaves were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80% for 24 hours to balance the moisture content of the tobacco leaves to 25%±1%.

[0032] The tobacco leaves after moisture balance were fermented in a constant temperature and humidity chamber at 45°C and 80% humidity for 10 days. The fermented samples were sampled and marked as QS, and the conventional chemical composition, cellulase, β-amylase and neutral protease activities and sensory quality were tested and evaluated.

[0033] (2) Bacillus cereus treatment group The surface of the CX-81 upper first-level cigar core tobacco leaves produced in Laifeng, Hubei in 2022 was evenly sprayed with 2%, 6%, 10%, and 14% Bacillus cereus fermentation liquid for moisture adjustment. The moistened tobacco leaves were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80% for 24 hours to balance the moisture content of the tobacco leaves to 25%±1%.

[0034] The tobacco leaves after moisture balance were fermented in a constant temperature and humidity chamber at 45°C and 80% humidity for 10 days. The fermented samples were sampled and marked as L1, L2, L3, and L4, and the conventional chemical composition, cellulase, β-amylase and neutral protease activities, and sensory quality were tested and evaluated.

[0035] (3) Bacillus pumilus treatment group The surface of the CX-81 upper first-level cigar core tobacco leaves produced in Laifeng, Hubei in 2022 was evenly sprayed with 2%, 6%, 10%, and 14% Bacillus pumilus fermentation liquid for moisture adjustment. The moistened tobacco leaves were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80% for 24 hours to balance the moisture content of the tobacco leaves to 25%±1%.

[0036] The tobacco leaves after moisture balance were fermented in a constant temperature and humidity chamber at 45°C and 80% humidity for 10 days. The fermented samples were sampled and marked as D1, D2, D3, and D4, and the conventional chemical composition, cellulase, β-amylase and neutral protease activities, and sensory quality were tested and evaluated.

[0037] (4) Thermophilic Bacillus treatment group The surface of the CX-81 upper first-level cigar core tobacco leaves produced in Laifeng, Hubei in 2022 was evenly sprayed with 2%, 6%, 10%, and 14% of Thermophilic Bacillus fermentation liquid for moisture adjustment. The moistened tobacco leaves were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80% for 24 hours to balance the moisture content of the tobacco leaves to 25%±1%.

[0038] The tobacco leaves after moisture balance were fermented in a constant temperature and humidity chamber at 45°C and 80% humidity for 10 days. The fermented samples were sampled and marked as S1, S2, S3, and S4, and the conventional chemical composition, cellulase, β-amylase and neutral protease activities, and sensory quality were tested and evaluated.

[0039] (5) Composite microbial agent treatment group The surface of the CX-81 upper first-level cigar core tobacco leaves of Hubei Laifeng after blending in 2022 was evenly sprayed with the fermentation liquid of the composite bacterial agent according to the proportion of the orthogonal experimental design in Table 3 for rehydration. The rehydrated tobacco leaves were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80% for 24 hours to balance the moisture content of the tobacco leaves to 25%±1%.

[0040] The tobacco leaves after moisture balance were fermented in a constant temperature and humidity chamber at 45°C and 80% humidity, and the entire fermentation time was 10 days. The fermented samples were sampled and marked according to the treatment number in Table 3, and the conventional chemical composition, cellulase, β-amylase and neutral protease activities and sensory quality were tested and evaluated.

[0041] 3. Evaluation Method

[0042] Conventional chemical composition determination: refer to the standard methods of YC / T 159-2002, YC / T 161-2002, YC / T 261-2002 and YC / T 468-2013 to determine the total sugar, reducing sugar, total nitrogen, protein and nicotine content (mass fraction) of tobacco leaves.

[0043] Enzyme activity determination: The activity of cellulase, β-amylase and neutral protease was determined by ultraviolet spectrophotometry using a cellulase, β-amylase and neutral protease activity assay kit (Suzhou Keming Biotechnology Co., Ltd.).

[0044] Sensory quality evaluation: After fermentation, the sensory quality of tobacco leaves of each treatment sample was evaluated for eight indicators, including aroma quality, aroma volume, aftertaste, miscellaneous smell, sweetness, irritation, burningness, and grayness, with a full score of 100. The total score was calculated according to the following formula, and the result was rounded to one decimal place. The higher the total score, the better the sensory quality of tobacco leaves.

[0045]

[0046] Where: T is the sensory quality evaluation score; Pi is the weight of a single indicator (aroma quality 18%, aroma volume 16%, aftertaste 16%, impurities 10%, sweetness 10%, irritation 12%, flammability 10% and gray 8%). is the average score of a single indicator. The evaluation criteria are shown in Table 4.

[0047] Table 4 Evaluation criteria for sensory quality of cigar tobacco leaves

[0048] 4. Experimental Results and Analysis Contents of total sugar and reducing sugar in tobacco leaves after fermentation with different treatments Figure 1 , Figure 2 The total nitrogen, protein and nicotine contents of tobacco leaves after fermentation with different treatments are shown in Figure 3-Figure 5 The changes of NP, β-amylase and CL activities in tobacco leaves after fermentation with different treatments are shown in Figure 6-Figure 8 shown.

[0049] The sensory quality evaluation results of cigar filler tobacco leaves after fermentation treated with different bacterial agents are shown in Table 5. The orthogonal test results of the Bacillus bacterial agent (composite bacterial agent) prepared by the present invention are shown in Table 6.

[0050] Table 4 Sensory quality evaluation of cigar filler tobacco leaves after fermentation treated with different bacterial agents

[0051] Table 5 Orthogonal test results of Bacillus inoculants prepared by the present invention

[0052] The above experimental results show that there are significant differences in the chemical composition, surface enzyme activity and sensory quality of tobacco leaves after fermentation treated with different Bacillus agents. The total sugar and reducing sugar content of tobacco leaves increased and the total nitrogen content decreased after fermentation treated with different Bacillus agents, and the effects of each on the total sugar and reducing sugar content of tobacco leaves were similar. And high concentrations of Bacillus cereus and Bacillus pumilus treatments have the most significant effect on reducing the total nitrogen content of tobacco leaves after fermentation and increasing the total sugar and reducing sugar content of tobacco leaves. The three different Bacillus treatments can reduce the protein content of tobacco leaves after fermentation, among which low concentrations of thermophilic Bacillus cereus have the most significant effect on protein degradation, while high concentrations of Bacillus cereus and Bacillus pumilus can also effectively reduce the protein in tobacco leaves. In addition, Bacillus pumilus and thermophilic Bacillus can also reduce the nicotine content of tobacco leaves, among which 10% Bacillus pumilus treatment has the best effect on nicotine degradation, and 2% and 14% thermophilic Bacillus can also significantly reduce the nicotine content of tobacco leaves. The treatment with Bacillus inoculants also had a significant effect on degrading starch in tobacco leaves. The starch content of tobacco leaves decreased after fermentation with different Bacillus inoculants, among which the treatment with Bacillus cereus had the best effect. In addition, 10% of Bacillus brevis and Bacillus thermophilus inoculants could also effectively degrade the cellulose content in tobacco leaves.

[0053] Treatment with different Bacillus agents also affects the enzymatic activity of different enzymes on the tobacco leaf surface. The ZTE protease activity on the tobacco leaf surface treated with 2% Bacillus cereus and 6% Bacillus pumilus was the highest, and the enzyme activity began to decrease with the increase of concentration. Treatment with 2% Bacillus pumilus can significantly increase the β-amylase activity on the tobacco leaf surface, while the β-amylase activity on the tobacco leaf surface first increased and then decreased with the increase of the concentration of Thermophilic Bacillus. Treatment with Thermophilic Bacillus agents can significantly increase the activity of cellulase on the tobacco leaf surface. In addition, Bacillus pumilus and 14% Bacillus cereus agents also have a certain effect on increasing the activity of cellulase on the tobacco leaf surface.

[0054] In terms of sensory quality: the total scores of sensory quality evaluation of tobacco leaves fermented by different Bacillus agents increased. The aroma of tobacco leaves was improved, the smell was reduced, the aftertaste was more comfortable, and the sweetness was improved. Among them, the tobacco leaves treated with 10% Bacillus pumilus had the highest total score, the most abundant aroma, and the smell and irritation were smaller than those of other treatments. In the treatments with Bacillus cereus and Bacillus thermophilus, the treatments with concentrations of 10% and 14% had the highest total sensory quality scores in each group. Afterwards, based on the single factor experiment, the total score of tobacco leaf sensory quality evaluation was used as an indicator, and the concentrations of different Bacillus in composite agent 3 were optimized through orthogonal experiments, among which the concentrations of Bacillus cereus, Bacillus pumilus and Bacillus thermophilus were added at 10%, 6% and 14% respectively.

[0055] Through the above analysis, it is finally determined that the Bacillus inoculant for improving the fermentation quality of cigar tobacco leaves of the present invention is composed of Bacillus cereus, Bacillus pumilus and Bacillus thermophilus, wherein the concentration of Bacillus cereus is 10%, the concentration of Bacillus pumilus is 6%, and the concentration of Bacillus thermophilus is 14%.

[0056] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Bacillus inoculant for improving the fermentation quality of cigar tobacco leaves, characterized in that: It is composed of Bacillus cereus, Bacillus brevis and Bacillus thermophilus.

2. The Bacillus inoculant for improving the fermentation quality of cigar tobacco leaves according to claim 1, characterized in that: The mass fraction of the Bacillus cereus is 10%.

3. The Bacillus inoculant for improving the fermentation quality of cigar tobacco leaves according to claim 1, characterized in that: The mass fraction of the Bacillus pumilus is 6%.

4. The Bacillus inoculant for improving the fermentation quality of cigar tobacco leaves according to claim 1, characterized in that: The mass fraction of the thermophilic bacillus is 14%.

5. The method for preparing the bacillus agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Bacillus cereus, Bacillus pumilus and Bacillus thermophilus were inoculated onto TSA medium under sterile conditions, sealed, and the culture dish was inverted and placed in an incubator for 48 h at 37°C; (2) Pick out the purified single colonies and inoculate them into LB liquid culture medium, and then culture them at 37°C and 180 rpm for 48 h until OD600 = 0.5; (3) diluting the three bacterial suspensions with sterile water to prepare Bacillus fermentation broths of required concentrations; (4) Mixing the fermented broths of Bacillus cereus, Bacillus pumilus and thermophilic Bacillus to obtain a Bacillus inoculum.

6. The method for preparing the bacillus agent according to claim 5, characterized in that: The TSA culture medium is prepared by heating and boiling 15 g of casein trypsin digest, 5 g of sodium chloride, 5 g of soy flour papain digest, 15 g of agar powder and 1 L of distilled water to dissolve, packaging, and sterilizing at 121° C. for 20 min.

7. The method for preparing the bacillus agent according to claim 5, characterized in that: The LB liquid culture medium is prepared by sterilizing at 121° C. for 20 minutes using 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

8. Use of the Bacillus inoculant according to any one of claims 1 to 4 in cigar tobacco fermentation.

9. The use of the Bacillus inoculant in the fermentation of cigar tobacco leaves according to claim 8, characterized in that: Before fermentation, the tobacco leaves are moistened, and then the prepared Bacillus agent is evenly sprayed on the surface of the tobacco leaves with a small sprayer. The moisture content of the tobacco leaves is controlled at 26%±1%, and the sprayed tobacco leaves are placed in a constant temperature and humidity box for fermentation.

10. The use of the Bacillus inoculant in the fermentation of cigar tobacco leaves according to claim 9, characterized in that: The fermentation conditions were as follows: temperature 45°C, relative humidity 80%, and fermentation time 10 days.

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