MICROBIAL FERMENTATION METHOD FOR IMPROVING TOBACCO QUALITY

Abstract

A microbial fermentation method for improving tobacco quality, comprising the following steps: (1) inoculating flue-cured tobacco as an inoculation source into a tobacco powder medium, and cultivating at 20-40° C. for 20-50 h; (2) inoculating microbial liquid in step (1) into a fresh tobacco powder medium, cultivating at 20-40° C. for 20-50 h, and circulating for 15-25 cycles to obtain stable flue-cured tobacco microbial flora; (3) inoculating the microbial flora obtained in step (2) into the fresh tobacco powder medium, and culturing at 20-40° C. for 20-50 h to obtain seed liquid of the microbial flora; (4) centrifuging the microbial liquid obtained in step (3) and collecting microbes; (5) washing the microbes collected in step (4); (6) resuspending the washed bacteria in step (5) to obtain a microbial suspension; and (7) spraying the microbial suspension in step (6) to the tobacco leaves for fermentation for 6-8 days.

Claims

1. A microbial fermentation method for improving tobacco quality, comprising the following steps: (1) inoculating a flue-cured tobacco as an inoculation source to a tobacco powder medium for cultivation at 20-40° C. for 20-50 h; (2) inoculating microbial liquid in step (1) into a fresh tobacco powder medium, cultivating at for 20-50 h, and circulating for 15-25 cycles to obtain stable flue-cured tobacco microbial flora; (3) inoculating the microbial flora obtained in step (2) into a fresh tobacco powder medium, and cultivating at 20-40° C. for 20-50 h to obtain seed liquid of the microbial flora; (4) centrifuging the microbial liquid obtained in step (3) and collecting microbes; (5) washing the microbes collected in step (4); (6) resuspending the washed microbes in step (5) to obtain a microbial suspension; and (7) spraying the microbial suspension of step (6) on the tobacco leaves for fermenting for 6-8 days.

2. The method according to claim 1, wherein in step (2), the cultivation is performed at the temperature of step (1) for the same time.

3. The method according to claim 1, characterized in that in step (3), the cultivation is performed at the temperature of step (1) for the same time.

4. The method according to claim 1, wherein after step (2) is completed, the structure of the microbial flora obtained in step (2) is analyzed to obtain the relative abundance information of the top 10 species at the genus level.

5. The method according to claim 1, wherein in step (1), the cultivation is performed at 37° C. for 24 h.

6. The method according to claim 1, wherein in step (1), the cultivation is performed at 25° C. for 48 h.

7. The method according to claim 1, wherein in step (2), circulation is performed for 20 cycles.

8. The method according to claim 1, wherein in step (7), the fermentation is performed for 7 days.

9. The method according to claim 1, wherein in step (1), the tobacco powder medium comprises 8-12 g/L of tobacco powder, 8-12 g/L of tryptone, 4-6 g/L of yeast powder and 4-6 g/L of sodium chloride.

10. The method according to claim 1, wherein in step (1), the tobacco powder is pulverized into powder at low temperature and passes through a 200-mesh sieve.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a graph showing the relative abundance of the top 10 species at the genus level of the microbial flora obtained by enrichment and domestication for different cultivation time in Example 1, and

[0027] FIG. 2 is a graph showing the relative abundance of the top 10 species at the genus level of the microbial flora obtained by enrichment and domestication for different cultivation time in Example 2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to better clarify the purpose, technical scheme and advantages of the examples of the present invention, the technical scheme in the examples of the present invention will be clearly and completely illustrated below with reference to the drawings in the examples of the present invention. Clearly, the described examples are part of those of the present invention instead of all. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

[0029] Tobacco leaves fermented by microbial flora of flue-cured tobacco at 37° C.: [0030] (1) Enrichment of microbial flora of flue-cured tobacco at 37° C.: the flue-cured tobacco was inoculated into a tobacco powder medium as an inoculum source, and cultured at 37° C. for 24 h; the tobacco powder medium comprises 10 g/L of tobacco powder, 10 g/L of tryptone, 5 g/L of yeast powder and 5 g/L of sodium chloride; [0031] (2) Domestication of microbial flora of flue-cured tobacco at 37° C.: the microbial liquid in step (1) was inoculated into a fresh tobacco powder medium, cultivated continuously at 37° C. for 24 h, and circulated for 20 cycles to obtain stable flue-cured tobacco microbial flora;

[0032] Structural analysis of the microbial flora: the structure of the microbial flora of flue-cured tobacco at 37° C. was analyzed by high-throughput sequencing technology, and the relative abundance information of the top 10 species at the genus level was obtained. As shown in FIG. 1, the three genera with the highest relative abundance are Cronobacter (Cronobacter spp.), Bacillus (Bacillus spp.) and Franconibacter (Frankia). [0033] (3) Preparation of seed liquid: the microbial flora obtained in step (2) was inoculated into a fresh tobacco powder medium, and cultivated at 37° C. for 24 h to obtain seed liquid of the microbial flora at 37° C.; [0034] (4) Centrifugation: the microbial liquid obtained in step (3) was centrifuged at rotating speed of 8000 g for 5 min to collect microbes; [0035] (5) Washing: deionized water was added to the microbes collected in step (4) for washing, and step (4) was repeated twice; [0036] (6) Resuspending: the washed microbes in step (5) was resuspended with deionized water to obtain a microbial suspension; [0037] (7) Tobacco leaf fermentation: 0.4 mL/g of bacterial suspension was sprayed to the tobacco leaves, and the tobacco leaves were placed in an environment of 37° C. for 7 days of fermentation.

[0038] Analysis of principal chemical components of tobacco leaves: the contents of total sugar, reducing sugar, starch, protein and nicotine in tobacco leaves before and after fermentation were determined by a continuous flow method. The results are shown in Table 1. After fermentation, the content of total sugar and reducing sugar in tobacco leaves increased by 29.83% and 52.21%, respectively, while starch, protein and nicotine decreased by 32.55%, 10.05% and 43.71%, respectively;

[0039] Analysis of aroma components of tobacco leaves: the content of aroma components in tobacco leaves before and after fermentation were determined by a GC/MS fingerprint method. The results are shown in Table 2. The content of aroma components in the fermented tobacco leaves increased, and aldehydes, alcohols, ketones, lipids, alkenes, alkanes and derivatives thereof increased by 2.46, 1.88, 0.64, 1.69, and 2.96 times, respectively;

[0040] Preparation and inhaling evaluation of heat-not-burn cigarettes: the fermented tobacco leaves were prepared into heat-not-burn cigarettes (Heat-not-burn cigarettes) by the slurry process for papermaking, and inhaling evaluation experts were invited to inhale the smoke and evaluate the smoking. The inhaling evaluation results are shown in Table 3. After inhaling evaluation, it was found that the impact of the fermented tobacco leaves significantly increased, the coke aroma was stronger, the aroma was rich, and characteristics were apparent. The smoke volume, aroma, impact and coordination increased by 0.5, 0.5, 1.0 and 0.5 scores, respectively.

TABLE-US-00001 TABLE 1 Changes of principal chemical components in tobacco leaves before and after fermentation at 37° C. Principal chemical components Total Reducing Sample sugar sugar Starch Protein Nicotine Raw 30.41 24.40 5.53 5.57 3.50 tobacco leaves Fermented 39.48 37.14 3.73 5.01 1.97 tobacco leaves

TABLE-US-00002 TABLE 2 Changes of aroma components in tobacco leaves before and after fermentation at 37° C. Content (μg/g) Raw Fermented tobacco tobacco Type Aroma components leaves leaves Aldehydes Phenylacetaldehyde 0.63 2.31 n-pentadecanal 0.23 0.85 4-pentenal 0.13 0.12 3-methoxybenzaldehyde 0.09 0.42 4-diethylaminobenzaldehyde 0.09 0.15 oxime 2,4-heptadienal 0.01 0.27 Total 6 kinds 1.19 4.12 Alcohols Cembrenediol 4 9.17 7.04 Cembrenediol 3 3.35 1.82 Phenylethanol 1.62 3.73 Phytol 1.62 4.86 Cembrenediol 2 1.42 2.51 Cembrenediol 1 0.60 0.86 Geranylgeraniol 0.60 0.76 Black pinitol 0.49 0.80 Bicyclo[2.2.1] 0.13 0.23 hept-2,5-dien-7-ol Linalool 0.09 0.38 Farnesol 0.07 0.17 Benzyl alcohol 0.03 3.98 Total 12 kinds 19.19 27.14 Ketones Solanone 6.09 14.43 β-damascenone 2.96 9.83 Megastigmatrienone B 1.66 5.05 Megastigmatrienone D 1.24 4.49 Geranylacetone 1.17 2.89 Megastigmatrienone C 0.76 1.12 Megastigmatrienone A 0.40 0.86 5,6-dimethyl-2- 0.33 3.51 benzimidazole 1-[4-(1-methyl-2- 0.31 0.42 propenyl)phenyl]ethanone 4-hydroxy-β-damascenone 0.28 1.25 β-damascenone 0.16 0.51 Damascenone 0.09 0.16 4-oxoisophorone 0.09 0.16 1-ethyl-6-methyl-2(1H)- 0.03 0.10 pyridone Total 14 kinds 15.56 44.78 Phenols 2,6-di-tert-butyl-p-cresol 4.74 2.25 2,5-diethylphenol 0.35 0.36 2,6-dimethylphenol 0.25 0.33 4-vinyl-2-methoxyphenol 0.12 2.51 Total 4 kinds 5.47 5.45 Lipids Dibutyl phthalate 2.31 1.61 Methyl palmitate 1.38 3.08 Dihydroactinidiolide 0.39 2.02 Total 3 kinds 4.09 6.71 Olefins Neophytadiene 142.93 414.32 Caryophyllene oxide 4.14 5.02 Aromadendrene 3.91 1.04 Artemisia triene 2.20 1.15 β-elemene 1.62 0.37 Alloaromadendrene 1.42 1.05 Longifolene 0.79 0.36 α-Selinene 0.67 0.44 (E)-(β)-Farnesene 0.51 2.87 γ-terpinene 0.46 0.40 3-tetradecene 0.37 0.25 1,4-octadiene 0.07 0.24 Longifolene 0.07 0.19 Pinene 0.04 0.10 Total 14 kinds 159.19 427.80 Alkanes m-cymene 1.05 4.47 and their n-tridecane 0.33 0.34 derivatives 1-allyl-3-methylene- 0.23 0.39 cyclohexane 7-isopropyl-1- 0.21 0.23 methylnaphthalene 2-bromohexane 0.10 0.11 6,6-dimethyl-3- 0.05 1.23 methylenebicyclo [3.1.1]heptane 1,2-dihydro-1,4,6- 0.05 1.23 trimethylnaphthalene Total 7 kinds 2.02 8.00 Nitrogenous Myosming 1.07 0.52 chemicals 1,5,8-trimethyltetraline 0.39 0.17 4-hexyloxyaniline 0.23 0.94 1,2,3,4-tetrahydroquinoxaline 0.18 0.24 Indole 0.15 0.27 Pyrrole 0.07 0.11 Total 6 kinds 2.09 2.25

TABLE-US-00003 TABLE 3 Sensory quality index scores of tobacco leaves before and after fermentation at 37° C. Sample Smoke Description of group volume Aroma Impact Harmony Pungency Taste Total sensory quality Raw 8.0 23.0 7.0 7.5 13.5 22.5 81.5 Thin aroma, low tobacco impact and poor leaves fragrance Fermented 8.5 23.5 8.0 8.0 13.0 22.5 83.5 Obviously tobacco increased impact, leaves heavier coke aroma, rich fragrance and apparent characteristics

Example 2

[0041] Tobacco leaves fermented by microbial flora of flue-cured tobacco at 25° C.: [0042] (1) enrichment of the microbial flora of flue-cured tobacco at 25° C.: inoculating the flue-cured tobacco as the inoculation source to a tobacco powder medium for cultivation at 25° C. for 48 h; [0043] (2) Domestication of microbial flora of flue-cured tobacco at 25° C.: the microbial liquid in step (1) was inoculated into a fresh tobacco powder medium, cultivated continuously at 25° C. for 48 h, and circulated for 20 cycles to obtain stable flue-cured tobacco microbial flora;

[0044] Structural analysis of the microbial flora: the structure of the microbial flora of flue-cured tobacco at 25° C. was analyzed by high-throughput sequencing technology, and the relative abundance information of the top 10 species at the genus level was obtained. As shown in FIG. 2, the three genera with the highest relative abundance are Klebsiella (Klebsiella spp.), Lactobacillus (Lactobacillus spp.) and Lelliottia, respectively. [0045] (3) Preparation of seed liquid: the microbial flora obtained in step (2) was inoculated into a fresh tobacco powder medium, and cultivated at 25° C. for 48 h to obtain the seed liquid of the microbial flora at 25° C.; [0046] (4) Centrifugation: the microbial liquid obtained in step (3) was centrifuged at rotating speed of 8000 g for 5 min to collect microbes; [0047] (5) Washing: deionized water was added to wash the microbes collected in step (4), and step (4) was repeated twice; [0048] (6) Resuspending: the washed microbes in step (5) was resuspended with deionized water to obtain a microbial suspension; [0049] (7) Tobacco leaf fermentation: 0.4 mL/g of bacterial suspension was sprayed into the tobacco leaves, and the tobacco leaves were placed in an environment of 25° C. for 7 days of fermentation.

[0050] Analysis of principal chemical components of tobacco leaves: the contents of total sugar, reducing sugar, starch, protein and nicotine in tobacco leaves before and after fermentation were determined by a continuous flow method. The results are shown in Table 4. After fermentation, the content of total sugar and reducing sugar in tobacco leaves increased by 20.45% and 42.92%, respectively, while starch, protein and nicotine decreased by 61.12%, 22.80% and 65.43%, respectively;

[0051] Analysis of aroma components of tobacco leaves: the content of aroma components in tobacco leaves before and after fermentation were determined by a GC/MS fingerprint method. The results are shown in Table 5. The content of aroma components in the fermented tobacco leaves increased substantially, and aldehydes, alcohols, ketones, lipids, alkenes, alkanes and derivatives and nitrogenous compounds thereof increased by 2.78, 0.57, 2.51, 1.03, 3.07, 7.27, 3.82, and 0.32 times, respectively;

[0052] Preparation and inhaling evaluation of heat-not-burn cigarettes: the fermented tobacco leaves were prepared into heat-not-burn cigarettes (Heat-not-burn cigarettes) by the slurry process for papermaking, and inhaling evaluation experts were invited to inhale the smoke and evaluate the smoking. The inhaling evaluation results are shown in Table 6. After inhaling evaluation, it was found that the smoke concentration of the fermented tobacco leaves increased, the impact was high, but the pungency rose, the aroma was single, and the smoke volume and the impact increased by 0.5 and 1.0 score, respectively.

TABLE-US-00004 TABLE 4 Changes of principal chemical components in tobacco leaves before and after fermentation at 25° C. Principal chemical components Total Reducing Sample sugar sugar Starch Protein Nicotine Raw 30.41 24.40 5.53 5.57 3.50 tobacco leaves Fermented 36.63 34.71 2.15 4.30 1.21 tobacco leaves

TABLE-US-00005 TABLE 5 Changes of aroma components in tobacco leaves before and after fermentation at 25° C. Content (μg/g) Raw Fermented tobacco tobacco Type Aroma components leaves leaves Aldehydes Phenylacetaldehyde 0.63 2.34 n-pentadecanal 0.23 1.32 4-pentenal 0.13 0.13 3-methoxybenzaldehyde 0.09 0.20 4-diethylamino- 0.09 0.23 benzaldehyde oxime 2,4-heptadienal 0.01 0.25 Total 6 kinds 1.19 4.46 Alcohols Cembrenediol 4 9.17 6.36 Cembrenediol 3 3.35 1.41 Phenylethanol 1.62 5.90 Phytol 1.62 0.11 Cembrenediol 2 1.42 2.40 Cembrenediol 1 0.60 2.63 Geranylgeraniol 0.60 0.87 Black pinitol 0.49 2.92 Bicyclo[2.2.1]hept- 0.13 0.08 2,5-dien-7-ol Linalool 0.09 0.30 Farnesol 0.07 0.01 Benzyl alcohol 0.03 7.06 Total 12 kinds 19.19 30.06 Ketones Solanone 6.09 9.29 β-damascenone 2.96 14.41 Megastigmatrienone B 1.66 9.27 Megastigmatrienone D 1.24 7.53 Geranylacetone 1.17 2.82 Megastigmatrienone C 0.76 1.62 Megastigmatrienone A 0.40 2.25 5,6-dimethyl-2- 0.33 3.70 benzimidazole 1-[4-(1-methyl-2- 0.31 0.44 propenyl)phenyl]ethanone 4-hydroxy-β-damascenone 0.28 2.06 β-damascenone 0.16 0.64 Damascenone 0.09 0.36 4-oxoisophorone 0.09 0.08 1-ethyl-6-methyl-2(1H)- 0.03 0.19 pyridone Total 14 kinds 15.56 54.66 Phenols 2,6-di-tert-butyl-p-cresol 4.74 7.44 2,5-diethylphenol 0.35 0.33 2,6-dimethylphenol 0.25 0.35 4-ethenyl-2-methoxyphenol 0.12 2.96 Total 4 kinds 5.47 11.08 Lipids Dibutyl phthalate 2.31 7.45 Methyl palmitate 1.38 6.67 Dihydroactinidiolide 0.39 2.52 Total 3 kinds 4.09 16.64 Olefins Neophytadiene 142.93 1255.98 Caryophyllene oxide 4.14 37.78 Aromadendrene 3.91 6.42 Artemisia triene 2.20 2.39 β-elemene 1.62 0.56 Alloaromadendrene 1.42 4.60 Longifolene 0.79 0.06 α-Selinene 0.67 0.77 (E)-(β)-Farnesene 0.51 4.73 γ-terpinene 0.46 0.46 3-tetradecene 0.37 2.23 1,4-octadiene 0.07 0.17 Longifolene 0.07 0.23 Pinene 0.04 0.12 Total 14 kinds 159.19 1316.50 Alkanes m-cymene 1.05 5.51 and their n-tridecane 0.33 0.58 derivatives 1-allyl-3-methylene- 0.23 0.22 cyclohexane 7-isopropyl-1- 0.21 0.33 methylnaphthalene 2-bromohexane 0.10 0.12 6,6-dimethyl-3- 0.05 1.49 methylenebicyclo [3.1.1]heptane 1,2-dihydro-1,4,6- 0.05 1.49 trimethylnaphthalene Total 7 kinds 2.02 9.74 Nitrogenous Myosming 1.07 0.59 chemicals 1,5,8-trimethyltetraline 0.39 0.19 4-hexyloxyaniline 0.23 1.17 1,2,3,4- 0.18 0.30 tetrahydroquinoxaline Indole 0.15 0.44 Pyrrole 0.07 0.05 Total 6 kinds 2.09 2.75

TABLE-US-00006 TABLE 6 Sensory quality index scores of tobacco leaves before and after fermentation at 25° C. Sample Smoke Description of group volume Aroma Impact Harmony Pungency Taste Total sensory quality Raw 8.0 23.0 7.0 7.5 13.5 22.5 81.5 Thin aroma, low tobacco impact and poor leaves fragrance Fermented 8.5 23.0 8.0 7.5 13.0 22.5 82.5 The smoke tobacco concentration leaves increased, the impact is high, the pungency rose, and the aroma was single.

[0053] In the aforementioned steps of the embodiment, the components of the tobacco powder culture medium described in step (1) are 10 g/L of tobacco powder, 10 g/L of tryptone, 5 g/L of yeast powder, and 5 g/L of sodium chloride.

[0054] The aforementioned are merely examples of the present invention. For those skilled in the art, this application can still modify the technical solutions described in the aforementioned embodiments, or equivalently replace some of the technical features. However, these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of various examples of the present invention.