A compound microbial inoculant, a preparation method and application thereof

Abstract

This invention discloses a compound microbial agent, its preparation method, and its application. The preparation method involves culturing *Pseudomonas paraxanthum* ZY-03, *Klebsiella variegata* HNYJ-3, *Enterobacter* HNYJ-4, and *Micrococcus* ZY02 in specific culture media to form seed cultures. These seed cultures are then inoculated into tobacco extract for fermentation. The fermented broth is then compounded in a specific ratio, centrifuged, and resuspended in propylene glycol aqueous solution to form the compound microbial agent. This compound microbial agent degrades lignin and macromolecular substances, while also degrading carotenoids, flavonoids, and other aroma precursors in tobacco stems and reconstituted tobacco leaves, synergistically improving the quality of tobacco stems and reconstituted tobacco leaves. Sensory evaluation results show significant improvements in aroma quality, aroma quantity, off-flavors, concentration, fineness, irritation, and aftertaste, thus enhancing the overall flavor and smoking experience of cigarettes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent, its preparation method, and its application. Background Technology

[0002] Tobacco stems, consisting of the coarse, hard veins of tobacco leaves, make up about 25% of tobacco leaves and are the main raw material for producing the stem strands that provide support in cigarettes. In early cigarette manufacturing, tobacco stems were generally considered to contain more impurities and have poor smokeability after combustion, thus they were only used in the production of low-grade cigarettes. However, with advancements in cigarette manufacturing technology and health awareness, it is now generally believed that the application of an appropriate amount of stem strands not only improves the filling properties of the tobacco, enhances the combustibility of cigarettes, and reduces the consumption of tobacco raw materials, but also effectively reduces the content of harmful substances such as tar, nicotine, and CO in cigarette formulations. The proportion of stem strands used in tobacco blends has been gradually increasing. Currently, their use in cigarette products has generally increased, and they have even become an important component of cigarette formulations.

[0003] Reconstituted tobacco, also known as tobacco sheet, is an important form of tobacco waste recycling. The raw materials come from the following sources: (1) the lower leaves, axillary buds, and stems other than fresh tobacco leaves during the tobacco harvesting process; (2) low-grade tobacco leaves that cannot be processed into cigarettes during tobacco production, curing, re-curing, and cigarette production, as well as a large amount of upper tobacco leaves, tobacco dust, shredded tobacco, and tobacco stalks; (3) fragments and tobacco dust generated during tobacco processing, and tobacco stems that account for 23-25% of the tobacco leaf weight; (4) fiber materials from wood pulp added to the reconstituted tobacco raw materials to increase their fiber content; and (5) product recycling. Finished products collected from distribution channels are then recycled back to the factory for reprocessing.

[0004] Both tobacco stems and reconstituted tobacco leaves contain a large amount of lignin, cellulose, and various macromolecules, which have a negative effect on the quality of smoking. At present, the improvement of the quality of tobacco stems and reconstituted tobacco leaves mainly focuses on degrading lignin, cellulose, and macromolecules through physical, chemical, and biological means, and then adding flavorings to enhance the aroma. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a compound microbial agent, its preparation method and application. The compound microbial agent can degrade lignin and macromolecular substances, and at the same time, it can degrade carotenoids, flavonoids and other aroma precursors in tobacco stems and reconstituted tobacco leaves, and work synergistically to further improve the quality of tobacco stems and reconstituted tobacco leaves.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] A compound microbial agent, the preparation method of which includes the following steps:

[0008] (1) Inoculate Pseudomonas paraxanthizobium ZY-03 into a liquid culture medium, the liquid culture medium comprising 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose, and culture at 25-37℃ and 120-200 r / min for 24-48 hours with shaking to form a seed culture;

[0009] (2) Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4, and Micrococcus ZY02 were inoculated into liquid culture medium, which included 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract. The culture was shaken at 35-40℃ and 120-200 r / min for 24-48 hours to form a seed culture.

[0010] (3) Mix tobacco powder and water at a mass ratio of 1:15, extract and filter to obtain tobacco extract, sterilize, and add 1% anhydrous ethanol to the tobacco extract according to the volume ratio to form a mixture;

[0011] (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03 and the seed culture of Micrococcus ZY02 into the tobacco extract at a ratio of 1% and ferment at 30°C for 36 hours. Inoculate the seed culture of Klebsiella variegata HNYJ-3 and the seed culture of Enterobacter HNYJ-4 into the tobacco extract at a ratio of 1.5% and ferment at 37°C for 36 hours.

[0012] (5) Deionized water and propylene glycol are mixed at a mass ratio of 10:(0.5-1), sterilized, and then an aqueous solution of propylene glycol is obtained.

[0013] (6) The fermentation broths of Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4, Pseudomonas paraxanthizobium ZY-03, and Micrococcus ZY02 were mixed in a certain mass ratio. The mixed solution was centrifuged, the supernatant was discarded, and the precipitate was resuspended in propylene glycol aqueous solution with a volume of 50% of the volume of the mixed solution to become a compound bacterial agent.

[0014] Preferably, in the above technical solution, the *Pseudomonas parafulva* ZY-03 was deposited at the China General Biotechnology Center (CGMCC) on February 23, 2022, with accession number CGMCC No. 24422; the *Klebsiella variicola* HNYJ-3 was deposited at the CGMCC on January 18, 2024, with accession number CGMCC No. 29654; the *Enterobacter p.* HNYJ-4 was deposited at the CGMCC on January 18, 2024, with accession number CGMCC No. 29655; and the *Micrococcus*... Pribram ZY02 was deposited on December 7, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 21313.

[0015] Preferably, in the above technical solution, the culture conditions in step (1) are: temperature 30℃, 180r / min, shaking culture for 24 hours, OD 600 The value is 1.5-2.0.

[0016] Preferably, in the above technical solution, in step (2), the culture conditions are: 37℃, 180r / min, shake culture for 24h, OD 600 The value is 1.5-2.0.

[0017] Preferably, in the above technical solution, in step (3), the mass ratio of tobacco powder to water is 1:15, the extraction is carried out at 80℃ for 3 hours, filtered to obtain tobacco extract, and sterilized at 121℃ for 15 minutes.

[0018] Preferably, in the above technical solution, in step (5), the mass ratio of deionized water to propylene glycol is 10:0.5, and sterilization is performed at 121°C for 15 minutes.

[0019] Preferably, in the above technical solution, in step (6), the mass ratio of Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4, Pseudomonas paraxanthizobium ZY-03 and Micrococcus ZY02 is 8:5:5:2.

[0020] Application of a compound microbial agent in tobacco stems and reconstituted tobacco leaves.

[0021] Preferably, in the above technical solution, the application involves uniformly spraying 8%-12% of the microbial agent onto tobacco stems or reconstituted tobacco leaves according to the mass percentage, fermenting for 48-72 hours at 30-40℃ and 60-80% humidity, shredding and sterilizing the shredded leaves, and adjusting the moisture content to 10-15%.

[0022] Preferably, in the above technical solution, the application is to roll tobacco shreds with adjusted moisture into cigarettes.

[0023] The above-described technical solution of the present invention has the following beneficial effects:

[0024] This application provides a compound microbial agent that significantly improves the quality of tobacco stems and reconstituted tobacco leaves through a specific combination of bacteria and its fermentation process. Specifically:

[0025] (1) The combined use of Pseudomonas paraxanthizobium ZY-03, Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4 and Micrococcus ZY02 can more comprehensively degrade macromolecules such as lignin, cellulose, carotenoids and flavonoids, and work synergistically to further improve the smoking quality of tobacco stems and reconstituted tobacco leaves.

[0026] (2) The fermentation process was optimized. By precisely controlling the fermentation conditions, the efficient degradation effect of the microbial agent was ensured, while avoiding damage to the original aroma of the tobacco leaves.

[0027] (3) The tobacco stems treated with compound microbial agents showed significant improvements in aroma quality, aroma quantity, off-flavors, concentration, fineness, irritation and aftertaste, thus improving the overall flavor and smoking experience of cigarettes. Detailed Implementation

[0028] 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.

[0029] Information on the strains used in this application is as follows:

[0030] (1) Pseudomonas parafulva ZY-03 was deposited on February 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The accession number is CGMCC No. 24422.

[0031] (2) Klebsiella variicola HNYJ-3 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Accession number: CGMCC No. 29654.

[0032] (3) Enterobacter sp. HNYJ-4 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Accession number: CGMCC No. 29655.

[0033] (4) Micrococcus prbram ZY02 was deposited on December 7, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The accession number is CGMCC NO.21313.

[0034] The tobacco dust and tobacco stems used in the following embodiments of this application are all from Henan China Tobacco Industry Co., Ltd.

[0035] Example 1

[0036] Preparation of compound microbial agents:

[0037] (1) Select *Pseudomonas paraxanthizobium* ZY-03 and inoculate it into a sterile liquid culture medium composed of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose. Incubate at 30℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0038] (2) Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4, and Micrococcus ZY02 were inoculated into a sterile liquid culture medium composed of NaCl 10 g / L, peptone 10 g / L, and yeast extract 5 g / L, respectively. The medium was cultured at 37℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.7, forming seed liquid.

[0039] (3) Mix tobacco powder with water at a ratio of 1:15 (mass ratio), extract at 80℃ for 3 hours, filter to obtain tobacco extract, sterilize at 121℃ for 15 minutes, add 1% anhydrous ethanol to the tobacco extract according to the volume ratio to form a mixture.

[0040] (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03 and the seed culture of Micrococcus ZY02 into the mixed solution at a ratio of 1%, and ferment at 30°C for 36 hours. Inoculate the seed culture of Klebsiella variegata HNYJ-3 and the seed culture of Enterobacter HNYJ-4 into the mixed solution at a ratio of 1.5%, and ferment at 37°C for 36 hours.

[0041] (5) Mix deionized water and propylene glycol at a mass ratio of 10:0.5 and sterilize at 121°C for 15 min to obtain an aqueous solution of propylene glycol.

[0042] (6) Mix 40 parts of fermentation broth of Klebsiella variegata HNYJ-3, 25 parts of fermentation broth of Enterobacter HNYJ-4, 25 parts of fermentation broth of Pseudomonas paraxanthizobium ZY-03, and 10 parts of fermentation broth of Micrococcus ZY02 (i.e., mass ratio 8:5:5:2). Centrifuge the mixed solution at low temperature, discard the supernatant, and resuspend the precipitate in propylene glycol aqueous solution at a volume of 50% of the volume of the mixed solution to obtain the compound bacterial agent.

[0043] Application Example 1

[0044] (1) Spray 8% of the microbial agent evenly onto the tobacco stems at 35°C and 70% humidity according to the mass percentage, ferment for 72 hours, cut into shreds, and then sterilize by drying at high temperature. Then adjust the moisture content to about 10%.

[0045] (2) The prepared stems are used for cigarette processing according to existing technology and rolled into cigarettes, denoted as T.

[0046] Comparative Example 1

[0047] (1) Select *Pseudomonas paraxanthizobium* ZY-03 and inoculate it into a sterile liquid culture medium composed of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose. Incubate at 30℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0048] (2) Mix tobacco powder and water at a mass ratio of 1:15, extract at 80℃ for 3 hours, filter to obtain tobacco extract, sterilize at 121℃ for 15 minutes, add 1% anhydrous ethanol to tobacco extract by volume to form a mixture.

[0049] (3) Inoculate the seed liquid of Pseudomonas paraxanthizobium ZY-03 into the mixed liquid at a ratio of 1%, and ferment at 30°C for 36 hours.

[0050] (4) Mix deionized water and propylene glycol at a mass ratio of 10:0.5 and sterilize at 121°C for 15 min to obtain an aqueous solution of propylene glycol.

[0051] (5) Centrifuge the fermentation broth of Pseudomonas paraxanthizobium ZY-03 at low temperature, discard the supernatant, and resuspend the precipitate in propylene glycol aqueous solution with a volume of 50% of the volume of the compound solution to become the inoculum.

[0052] (6) Spray 8% of the microbial agent evenly onto the tobacco stems at 35°C and 70% humidity according to the mass percentage, ferment for 72 hours, cut into shreds, dry and sterilize under high temperature, and then adjust the moisture content to about 10%.

[0053] (8) The prepared stems are used to make cigarettes according to existing technology, CK1.

[0054] Comparative Example 2

[0055] (1) Select *Pseudomonas paraxanthizobium* ZY-03 and inoculate it into a sterile liquid culture medium composed of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose. Incubate at 30℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0056] (2) Micrococcus ZY02 was inoculated into a sterile liquid culture medium composed of NaCl 10 g / L, peptone 10 g / L, and yeast extract 5 g / L. The medium was cultured at 37℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.7, forming seed liquid.

[0057] (3) Mix tobacco powder and water at a mass ratio of 1:15, extract at 80℃ for 3 hours, filter to obtain tobacco extract, sterilize at 121℃ for 15 minutes, add 1% anhydrous ethanol to tobacco extract by volume to form a mixture.

[0058] (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03 and the seed culture of Micrococcus ZY02 into the mixed liquid at a ratio of 1% respectively, and ferment at 30°C for 36 hours.

[0059] (5) Mix deionized water and propylene glycol at a mass ratio of 10:0.5 and sterilize at 121°C for 15 min to obtain an aqueous solution of propylene glycol.

[0060] (6) Mix the fermentation broth of Pseudomonas paraxanthizobium ZY-03 and the fermentation broth of Micrococcus ZY02 at a mass ratio of 5:2. Centrifuge the mixed solution at low temperature, discard the supernatant, and resuspend the precipitate in propylene glycol aqueous solution at a volume of 50% of the volume of the mixed solution to form a compound bacterial agent.

[0061] (7) Spray 8% of the microbial agent evenly onto the tobacco stems at 35°C and 70% humidity according to the mass percentage, ferment for 72 hours, cut into shreds, dry and sterilize under high temperature, and then adjust the moisture content to about 10%.

[0062] (8) The prepared stems are used to make cigarettes according to existing technology, and are denoted as CK2.

[0063] Comparative Example 3

[0064] (1) Select *Pseudomonas paraxanthizobium* ZY-03 and inoculate it into a sterile liquid culture medium composed of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose. Incubate at 30℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.8, forming seed liquid.

[0065] (2) Enterobacter HNYJ-4 and Micrococcus ZY02 were inoculated into a sterile liquid culture medium composed of NaCl 10 g / L, peptone 10 g / L, and yeast extract 5 g / L, respectively, and cultured at 37℃ and 180 r / min for 24 h with shaking. OD 600 The value is approximately 1.7, forming seed liquid.

[0066] (3) Mix tobacco powder and water at a mass ratio of 1:15, extract at 80℃ for 3 hours, filter to obtain tobacco extract, sterilize at 121℃ for 15 minutes, add 1% anhydrous ethanol to tobacco extract by volume to form a mixture.

[0067] (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03 and the seed culture of Micrococcus ZY02 into the mixed solution at a ratio of 1%, and ferment at 30°C for 36 hours. Inoculate the seed culture of Enterobacter HNYJ-4 into the mixed solution at a ratio of 1.5%, and ferment at 37°C for 36 hours.

[0068] (5) Mix deionized water and propylene glycol at a mass ratio of 10:0.5 and sterilize at 121°C for 15 min to obtain an aqueous solution of propylene glycol.

[0069] (6) The fermentation broth of Enterobacter HNYJ-4, the fermentation broth of Pseudomonas paraxanthizobium ZY-03, and the fermentation broth of Micrococcus ZY02 were mixed in a mass ratio of 5:5:2. The mixed solution was centrifuged at low temperature, the supernatant was discarded, and the precipitate was resuspended in propylene glycol aqueous solution with a volume of 50% of the volume of the mixed solution to become a compound bacterial agent.

[0070] (7) Spray 8% of the microbial agent evenly onto the tobacco stems at 35°C and 70% humidity according to the mass percentage, ferment for 72 hours, cut into shreds, dry and sterilize under high temperature, and then adjust the moisture content to about 10%.

[0071] (8) The prepared stems are used for cigarette processing according to existing technology and rolled into cigarettes, which are denoted as CK3.

[0072] Comparative Example 4

[0073] Unprocessed stems, when used in cigarette manufacturing according to existing technology and rolled into cigarettes, are denoted as CK.

[0074] result:

[0075] The processed stems are added to the blended tobacco. After being balanced in a constant temperature and humidity chamber, professional judges evaluate the aroma quality, aroma quantity, concentration, fineness, aftertaste, off-flavors, and irritation. Each indicator is scored on a 9-point scale, and the total sensory evaluation score is the sum of the scores of each sub-item.

[0076] The sensory evaluation results are shown in Table 1. Compared with the control group (CK), single-strain fermentation, two-strain fermentation, and three-strain fermentation can improve the sensory quality of cigarettes, but the improvement effect is smaller compared with the compound microbial agent of this application. The compound microbial agent of this application significantly improves the aroma quality and aroma quantity, reduces irritation and off-flavors, and further improves the quality of tobacco stems.

[0077] Table 1. Sensory evaluation results

[0078]

[0079] In summary, the tobacco stems treated with the compound microbial agent of this application showed significant improvements in aroma quality, aroma quantity, off-flavors, concentration, smoothness, irritation, and aftertaste. Compared with the untreated control sample CK, the sample T treated with the compound microbial agent scored higher in all indicators, achieving a total score of 42.67, significantly higher than other treatment groups. This indicates that the application of the compound microbial agent significantly improves the sensory quality of tobacco stems or reconstituted tobacco leaves, enhancing the overall flavor and smoking experience of cigarettes.

[0080] 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 compound microbial agent, characterized in that, The preparation method of the compound microbial agent includes the following steps: (1) Inoculate Pseudomonas paraxanthizobium ZY-03 into a liquid culture medium, wherein the liquid culture medium comprises 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose, and culture at 25-37℃ and 120-200 r / min for 24-48 hours with shaking to form a seed culture; (2) Klebsiella variegata HNYJ-3, Enterobacter HNYJ-4, and Micrococcus ZY02 were inoculated into liquid culture medium, which included 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract. The culture was shaken at 35-40℃ and 120-200 r / min for 24-48 hours to form a seed culture. (3) Mix tobacco powder and water at a mass ratio of 1:15, extract at 80℃ for 3 hours, filter to obtain tobacco extract, sterilize at 121℃ for 15 minutes, add 1% anhydrous ethanol to tobacco extract according to volume ratio to form a mixture; (4) Inoculate the seed culture of Pseudomonas paraxanthizobium ZY-03 and the seed culture of Micrococcus ZY02 into the tobacco extract at a ratio of 1% and ferment at 30°C for 36 hours. Inoculate the seed culture of Klebsiella variegata HNYJ-3 and the seed culture of Enterobacter HNYJ-4 into the tobacco extract at a ratio of 1.5% and ferment at 37°C for 36 hours. (5) Deionized water and propylene glycol are mixed at a mass ratio of 10:0.5 and sterilized at 121°C for 15 min to obtain an aqueous solution of propylene glycol; (6) The fermentation broth of Klebsiella variegata HNYJ-3, the fermentation broth of Enterobacter HNYJ-4, the fermentation broth of Pseudomonas paraxanthizobium ZY-03, and the fermentation broth of Micrococcus ZY02 were mixed in a mass ratio of 8:5:5:

2. The mixed solution was centrifuged, the supernatant was discarded, and the precipitate was resuspended in propylene glycol aqueous solution with a volume of 50% of the volume of the mixed solution to become a compound bacterial agent. The *Pseudomonas parafulva* ZY-03 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 23, 2022, with accession number CGMCC No. 24422; the *Klebsiella variicola* HNYJ-3 was deposited at the CGMCC on January 18, 2024, with accession number CGMCC No. 29654; the *Enterobacter sp.* HNYJ-4 was deposited at the CGMCC on January 18, 2024, with accession number CGMCC No. 29655; and the *Micrococcaceae Pribram* ZY02 was deposited at the CGMCC on December 7, 2020, with accession number CGMCC. NO.21313.

2. The compound microbial agent according to claim 1, characterized in that, In step (1), the culture conditions were: temperature 30℃, 180 r / min, shaking culture for 24 hours, OD 600 The value is 1.5-2.

0.

3. The compound microbial agent according to claim 1, characterized in that, In step (2), the culture conditions are: 37℃, 180r / min, and shaking culture for 24 h, OD 600 The value is 1.5-2.

0.

4. The application of the compound microbial agent according to any one of claims 1-3 in tobacco stems and reconstituted tobacco leaves.

5. The application according to claim 4, characterized in that, The application involves uniformly spraying 8%-12% of the microbial agent onto tobacco stems or reconstituted tobacco leaves according to the mass percentage, fermenting for 48-72 hours at 30-40℃ and 60-80% humidity, then shredding and sterilizing the shredded leaves, and adjusting the moisture content to 10-15%.

6. The application according to claim 5, characterized in that, The application involves rolling tobacco shreds after adjusting their moisture content into rolls.

Images