Application of a microcalorimetric method in the fermentation process of reconstituted tobacco concentrate

Abstract

This invention discloses the application of microcalorimetry in the fermentation process of reconstituted tobacco concentrate. Microcalorimetry is used to monitor the growth status of microorganisms during the fermentation process, and the fermentation endpoints of different types of microorganisms are determined through heat curves. This application utilizes microcalorimetry to effectively predict the fermentation time of reconstituted tobacco by aerobic and facultative anaerobic microorganisms, determine the optimal macromolecular degradation time and the optimal aroma component accumulation time, thereby better guiding production and processing. Furthermore, this method is simple to operate, provides accurate results, and effectively solves the problem of the fermentation endpoint in reconstituted tobacco concentrate.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of a microcalorimetric method in the fermentation process of reconstituted tobacco concentrate. Background Technology

[0002] Reconstituted tobacco using the papermaking method is a tobacco product made from tobacco materials (stems, dust, and broken tobacco leaves) as the main raw materials through processes such as extraction, pulping, concentration, papermaking, coating, and drying. After extraction, the extract is concentrated into a concentrate, which is then pressed into a solid sheet. The concentrate is then mixed with a coating solution and applied to the sheet to form reconstituted tobacco.

[0003] However, most of the concentrates produced during the reconstituted tobacco processing currently suffer from two major problems: firstly, high viscosity due to the high content of macromolecules, and secondly, insufficient aroma due to the loss of aroma components. These problems can be effectively solved through microbial fermentation. However, because the concentrate is complex in composition, thick, dark, and opaque, it is difficult to monitor the fermentation level and microbial growth status of the reconstituted tobacco concentrate in real time using conventional methods. This poses significant challenges to optimizing the concentrate fermentation process and determining the fermentation endpoint.

[0004] Microorganisms release heat during their growth; the more numerous and active the microorganisms, the higher the heat they produce. Microcalorimetry, a novel diagnostic method, rapidly and accurately detects microbial activity by measuring the minute amounts of heat generated by microbial metabolism. This method converts the extremely small amounts of heat generated by microbial fermentation into a real-time microbial growth curve using appropriate calibration and specialized software (the Seebeck effect). It boasts high detection sensitivity, capable of detecting even microbial quantities as small as 100 CFU. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of microcalorimetry in the fermentation process of reconstituted tobacco concentrate. It uses microcalorimetry to monitor the growth status of microorganisms in real time during the fermentation process of concentrate, and determines the fermentation endpoint of different types of microorganisms through heat release curves, thus providing practical guidance for the fermentation of reconstituted tobacco concentrate.

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

[0007] An application of microcalorimetry in the fermentation process of reconstituted tobacco concentrate: microcalorimetry is used to monitor the growth status of microorganisms in real time during the fermentation process of reconstituted tobacco concentrate, and the fermentation endpoint of different types of microorganisms is determined by heat curves.

[0008] A method for monitoring the fermentation endpoint of reconstituted tobacco concentrate using microcalorimetry, the method comprising:

[0009] (1) Take two identical transparent ampoules and add 20%-80% of the volume of reconstituted tobacco concentrate to each. After equilibration, inoculate one ampoule with active bacterial solution as the test sample and add the same volume of high-temperature inactivated bacterial solution to the other ampoule as the control sample.

[0010] (2) After the control channel and the detection channel stabilized, real-time monitoring was started to monitor the micro-thermal changes of the reconstituted tobacco concentrate after the addition of active bacterial solution and to obtain the heat curve;

[0011] (3) The heat curve of the reconstituted tobacco concentrate with time as the abscissa is single-peaked after aerobic fermentation. For the concentrate fermentation with the main requirement of degrading macromolecules, the fermentation endpoint is determined at the peak time by 2-12 hours. For the concentrate fermentation with the main requirement of enhancing aroma, the fermentation endpoint is determined at the peak time by 1-12 hours. The heat curve of the reconstituted tobacco concentrate with time as the abscissa is double-peaked after aerobic fermentation. For the concentrate fermentation with the main requirement of degrading macromolecules, the fermentation endpoint is determined at the second peak time by 2-12 hours. For the concentrate fermentation with the main requirement of enhancing aroma, the fermentation endpoint is determined at the second peak.

[0012] Preferably, in step (1), the concentration of the reconstituted tobacco concentrate is 40%.

[0013] Preferably, in step (1), the control sample is placed in the control channel for equilibration for 4.5 h.

[0014] Preferably, for reconstituted tobacco concentrate with added aerobic bacteria, the fermentation endpoint for macromolecular degradation is set 6 hours after the peak time of the heat curve, and 2 hours before the peak time, according to the heat curve. For reconstituted tobacco concentrate with added facultative anaerobic bacteria, the fermentation endpoint for macromolecular degradation is set 6 hours after the second peak time, according to the heat curve, and the second peak time is set as the fermentation endpoint for aroma enhancement.

[0015] Preferably, the reconstituted tobacco concentrate is tobacco concentrate TS-006.

[0016] Preferably, the aerobic bacteria is Micrococcus sp. ZY-02, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21313.

[0017] Preferably, the facultative anaerobic bacterium is Klebsiella sp. HNYJ-1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327.

[0018] The above-described technical solution of the present invention has the following beneficial effects:

[0019] This application utilizes microcalorimetry to effectively predict the fermentation time of reconstituted tobacco leaves by aerobic and facultative anaerobic microorganisms, determine the optimal macromolecular degradation time and the optimal aroma component accumulation time, thereby better guiding production and processing.

[0020] This method is simple to operate, yields accurate results, and can effectively solve the problem of the fermentation endpoint of reconstituted tobacco concentrate. Attached Figure Description

[0021] Figure 1 A schematic diagram for selecting the fermentation time endpoint for aerobic bacteria.

[0022] Figure 2 A schematic diagram illustrating the selection of the fermentation endpoint for facultative anaerobic bacteria. Detailed Implementation

[0023] 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.

[0024] Example 1: Method for detecting microcaloric substances in concentrated liquid

[0025] (1) Preheat the microcalorimeter, fill a transparent ampoule with 20%-80% of its maximum volume of concentrated solution (40% is optimal) as a control, place it in the control channel for equilibration for 4.5 h, and perform the operation under aseptic conditions;

[0026] (2) Add the same volume of concentrated solution to the same ampoule as the control, inoculate with active bacterial solution, and place it into the detection channel as the test group. At the same time, inactivate the same volume of bacterial solution at high temperature and add it to the concentrated solution of the control group. Real-time detection begins 1 hour after the control channel and the detection channel have stabilized.

[0027] Example 2: Monitoring of the concentrated liquid fermentation process

[0028] (1) Aerobic fermentation: The heat curve of the concentrate with time as the abscissa shows a "single peak" shape after aerobic fermentation. For the concentrate fermentation with the main requirement of degrading macromolecules, the fermentation endpoint is determined by delaying the "peak" time by 2-12 h (optimal 6 h); for the concentrate fermentation with the main requirement of enhancing aroma, the fermentation endpoint is determined by advancing the "peak" time by 1-12 h (optimal 2 h).

[0029] Aerobic bacterial fermentation involves a latent phase, a logarithmic phase, a stationary phase, and a decline phase. Heat production is low during the latent phase, peaks during the logarithmic phase, gradually decreases during the stationary phase, and finally disappears during the decline phase. Therefore, the calorimetric curve exhibits a single peak. For concentrated liquor fermentation primarily aimed at degrading macromolecules, the most important aspect is the degradation of these macromolecules. This requires sufficient bacterial growth to produce more degradative enzymes, which needs to continue until the stationary or decline phase. Therefore, the peak is shifted later in the fermentation process. Conversely, for concentrated liquor fermentation aimed at enhancing flavor, fermentation cannot be too complete to prevent further decomposition of many flavor components. Therefore, the peak is shifted earlier in the fermentation process. The specific timing is based on actual experiments.

[0030] (2) Fermentation by facultative anaerobic bacteria: The heat curve with time as the abscissa after fermentation by facultative anaerobic bacteria shows a "double peak" shape. For the fermentation of the concentrate with the main requirement of degrading macromolecules, the fermentation endpoint is determined to be 2-12 h after the "second peak" (optimal 6 h); for the fermentation of the concentrate with the main requirement of enhancing aroma, the fermentation endpoint is determined to be at the "second peak".

[0031] Facultative anaerobic bacteria fermentation involves both aerobic and anaerobic processes. Aerobic fermentation primarily produces degrading enzymes, while anaerobic fermentation generates more aroma components. Therefore, for concentrated liquid fermentation where the primary goal is to enhance aroma, it is best to allow the bacteria to undergo the anaerobic fermentation process. However, for concentrated liquid fermentation where the primary goal is to degrade macromolecules, degrading enzymes will still be produced during the anaerobic stage, and allowing them to fully ferment is beneficial for the effective degradation of macromolecules.

[0032] Application examples

[0033] Aerobic bacterial fermentation and reprocessing of tobacco leaf concentrate:

[0034] (1) Materials: Tobacco leaf concentrate TS-006 (intermediate aroma type) was provided by Henan Tobacco Industry Tobacco Sheet Co., Ltd.;

[0035] The fermenting microorganism is Micrococcus sp. ZY-02, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 7, 2020, with accession number CGMCC No. 21313. It can effectively degrade macromolecular substances in the concentrate and has a certain aroma-enhancing effect.

[0036] (2) A microcalorimeter was used for detection. Untreated samples and samples with added Micrococcus ZY-02 were placed in the control channel and the detection channel, respectively. The calorimetric curves were obtained after 96 h. According to the calorimetric curves, the optimal time for macromolecular degradation was 6 h after the peak time and the optimal time for flavoring fermentation was 2 h before the peak time.

[0037] (3) Based on the optimal time obtained from the heat curve, two batches of the same reconstituted tobacco concentrate were subjected to large-scale fermentation treatment. Fermentation was terminated at the optimal time for macromolecular degradation, and the resulting concentrate was denoted as F1; fermentation was terminated at the optimal time for aroma enhancement, and the resulting concentrate was denoted as F2. The concentrate that was not fermented was denoted as CK.

[0038] Facultative anaerobic bacteria fermentation and regeneration of tobacco leaf concentrate:

[0039] (1) Materials: Tobacco leaf concentrate TS-006 (intermediate aroma type) was provided by Henan Tobacco Industry Tobacco Sheet Co., Ltd.;

[0040] The fermentation microorganism is Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on January 17, 2022, with accession number CGMCC No. 24327. It can effectively increase the aroma components in the concentrate and has a certain ability to degrade macromolecules.

[0041] (2) A microcalorimeter was used for detection. Untreated samples and samples with Klebsiella pneumoniae HNYJ-1 were placed in the control channel and the detection channel, respectively. The samples were detected for 120 h to obtain the calorimetric curves. According to the calorimetric curves, the optimal time for macromolecular degradation was determined by delaying the time after the "second peak" by 6 h. The time of the "second peak" was taken as the optimal time for flavoring fermentation.

[0042] (3) Based on the optimal time obtained from the heat curve, two batches of the same reconstituted tobacco concentrate were subjected to large-scale fermentation treatment. Fermentation was terminated at the optimal time for macromolecular degradation, and the resulting concentrate was recorded as F3; fermentation was terminated at the optimal time for aroma enhancement fermentation, and the resulting concentrate was recorded as F4.

[0043] Results and Analysis:

[0044] Flow spectroscopy was used to detect the conventional chemical components in the macromolecules of reconstituted tobacco leaves. The concentrate was simultaneously distilled and extracted using dichloromethane as the extractant, and the results were analyzed by GC-MS. The content of macromolecular substances and aroma compounds in the concentrate after fermentation was analyzed.

[0045] The content of macromolecular substances in the reconstituted tobacco concentrate is shown in Table 1, and the content of aroma components is shown in Table 2. As can be seen from the tables:

[0046] (1) The aerobic micrococcus ZY-02 has a more prominent effect on degrading macromolecules, but its flavoring effect is average; while the facultative anaerobic Klebsiella HNYJ-1 has a prominent flavoring effect, but its ability to degrade macromolecules is average.

[0047] (2) For the concentrate fermented by Micrococcus ZY-02, the content of macromolecular substances in F1 was more significantly lower than that in F2, and the content of aroma components in F2 was higher than that in F1; for the concentrate fermented by Klebsiella HNYJ-1, the content of macromolecular substances in F3 was more significantly lower than that in F4, and the content of aroma components in F4 was higher than that in F3.

[0048] Table 1. Changes in the content of macromolecular substances in reconstituted tobacco concentrate

[0049]

[0050] Table 2. Changes in the content of aroma components in reconstituted tobacco concentrate

[0051]

[0052] It is evident that the microcalorimetric method can effectively predict the fermentation time of reconstituted tobacco leaves by aerobic and facultative anaerobic microorganisms, determine the optimal macromolecular degradation time and the optimal aroma component accumulation time, thereby better guiding production and processing. This method is simple to operate, yields accurate results, and can effectively solve the problem of the fermentation endpoint of reconstituted tobacco leaf concentrate.

[0053] 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 method for monitoring the end of fermentation of a reconstituted tobacco leaf concentrate using microcalorimetry, characterized in that, The method comprises: (1) two identical transparent ampoules are taken, 20%-80% volume of reconstituted tobacco concentrate is added into each of the ampoules, after balancing, active bacteria liquid is introduced into one of the ampoules as a test sample, and the same volume of high-temperature inactivated bacteria liquid is added into the other ampoule as a control sample; a microcalorimeter is used for detection, the test sample is placed in a detection channel, and the control sample is placed in a control channel; (2) after the control channel and the detection channel are stabilized, real-time monitoring is started, the microcalorimetric change of the reconstituted tobacco concentrate after the active bacteria liquid is added is monitored, and a heat curve is obtained; (3) the reconstituted tobacco concentrate is fermented by aerobic bacteria, the heat curve with time as the horizontal coordinate is in a single-peak shape, for the concentrate fermentation mainly requiring degradation of macromolecules, the fermentation end point is determined to be 2-12 h after the peak top time, for the concentrate fermentation mainly requiring flavoring, the fermentation end point is determined to be 1-12 h before the peak top time; the reconstituted tobacco concentrate is fermented by facultative anaerobic bacteria, the heat curve with time as the horizontal coordinate is in a double-peak shape, for the concentrate fermentation mainly requiring degradation of macromolecules, the fermentation end point is determined to be 2-12 h after the second peak top, for the concentrate fermentation mainly requiring flavoring, the fermentation end point is determined to be the second peak top; The aerobic bacteria are Micrococcus sp. ZY-02, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 21313; The facultative anaerobic bacteria are Klebsiella sp. HNYJ-1, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 24327.

2. The method of monitoring the end point of fermentation of reconstituted tobacco concentrate using microcalorimetry according to claim 1, characterized in that, In step (1), the concentration of the reconstituted tobacco concentrate is 40%.

3. The method of monitoring the end point of fermentation of reconstituted tobacco concentrate using microcalorimetry according to claim 1, wherein, In step (1), the control sample is placed in the control channel and balanced for 4.5 h.

4. The method of monitoring the end point of fermentation of reconstituted tobacco concentrate using microcalorimetry according to claim 1, wherein, For the reconstituted tobacco concentrate added with aerobic bacteria, according to the heat curve, the fermentation end point of macromolecule degradation is 6 h after the peak top time of the heat curve, and the fermentation end point of flavoring fermentation is 2 h before the peak top time; for the reconstituted tobacco concentrate added with facultative anaerobic bacteria, according to the heat curve, the fermentation end point of macromolecule degradation is 6 h after the second peak top, and the fermentation end point of flavoring fermentation is the second peak top time.

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