Evaluation method of effect of microbiological treatment of cigarette tobacco shreds

Abstract

The invention belongs to the technical field of tobacco processing, and relates to a method for evaluating the tobacco shred microbiological treatment effect of cigarettes. An effect evaluation system for microbiological treatment of cigarettes is established according to the change conditions of eight main chemical substances in the tobacco shreds of the cigarettes before and after microbiological treatment, and the fermentation treatment effect of the microorganisms is visually judged; comprising the following steps: step 1, sample pretreatment: selecting cigarette cut tobacco samples before and after microbiological treatment, and obtaining different components by using Soxhlet extraction and other pretreatment modes; 2, comprehensively judging the effect of treating the cigarette tobacco shreds by the microorganisms by comparing the change conditions of different components; according to the change rule combination of substances, the treatment effect of microorganisms is comprehensively analyzed; therefore, through comprehensive analysis, a complete microbiological treatment effect evaluation system can be formed.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco processing and relates to a method for evaluating the effect of microbial treatment of cigarette shredded tobacco. Background Art

[0002] Microbial treatment technology is widely used in cigarette tobacco processing. Microbial treatment technology can increase the total amount of neutral aroma substances in cigarette tobacco, thereby improving the aroma of tobacco, reducing tar release, and improving the quality of stems. Microbial treatment can reduce the cost of cigarette production, increase the utilization rate of low-grade tobacco leaves, and is in line with the trend of harm reduction. The microbial treatment effect evaluation system includes chemical index evaluation, sensory evaluation, and process adaptability evaluation. The evaluation of the existing microbial fermentation effect of cigarettes mainly focuses on three methods: ① Observe the appearance of the bacterial solution to see if it is contaminated with foreign bacteria and preliminarily judge the growth of the strain; ② Observe the colony morphology under a microscope to determine the number of live bacteria; ③ Determine the concentration of the culture medium, OD600 nm value to determine the activity of the strain.

[0003] However, existing evaluation methods only assess the effectiveness of microbial culture, and the relationship between colony growth, bacterial activity, and microbial efficacy is not completely linear. Consequently, there are cases where a bacterial culture is well established and bacterial activity is high, but the sensory evaluation of cigarette tobacco treated with these microorganisms may not be ideal.

[0004] Therefore, a comprehensive analysis and complete evaluation system or method is needed to solve the above technical problems. Summary of the Invention

[0005] The technical solution adopted by the present invention to solve the technical problem is: a method for evaluating the effect of microbial treatment of cigarette tobacco, which establishes an evaluation system for the effect of microbial treatment of cigarette tobacco by analyzing the changes in eight major chemical substances in cigarette tobacco before and after microbial treatment, and intuitively judges the effect of microbial fermentation treatment;

[0006] The evaluation method includes the following steps:

[0007] Step 1: Sample pretreatment: Select cigarette tobacco samples A and B before and after microbial treatment, and use Soxhlet extraction or other pretreatment methods to obtain different components; the extraction pretreatment methods include: simultaneous distillation extraction and accelerated solvent extraction;

[0008] Step 2: By comparing the changes in different components, comprehensively determine the effect of microbial treatment on cigarette tobacco.

[0009] Preferably, in step 1, the simultaneous distillation extraction comprises: separating and extracting alkaline, acidic, and neutral components in the cut tobacco sample by simultaneous distillation extraction; using dichloromethane and heating the water bath at 55-65° C.; and performing simultaneous distillation extraction for 1.5-2.5 hours, followed by washing to simultaneously extract the alkaline, acidic, and neutral components.

[0010] Preferably, in step 1, the accelerated solvent extraction comprises: extracting ester compounds in the tobacco sample by accelerated solvent extraction, using ethanol as the extraction solution, pressurized extraction time 8 to 12 minutes, 2 to 5 cycles, and then concentrating and injecting.

[0011] Preferably, in step 2, the changes in different components include: changes in conventional chemical indicators of tobacco, changes in nitrogenous compounds, changes in carbohydrates, changes in alkalis and organic acids, changes in pigments, changes in phenolic compounds, changes in ester compounds, and changes in anions and cations.

[0012] More preferably, the comparison of changes in conventional chemical indicators of tobacco includes: measuring the content of conventional chemical indicators of cut tobacco using a continuous flow analyzer, and comparing the change patterns of the total amount, wherein the conventional chemical indicators of cut tobacco include: total sugar, reducing sugar, nicotine, chlorine, potassium, and total nitrogen;

[0013] The comparison of changes in nitrogenous compounds includes: using an amino acid analyzer to measure free amino acids in the sample, comparing the changing trends of proteins and amino acids before and after microbial treatment, and analyzing possible protein change patterns.

[0014] More preferably, the comparison of carbohydrate changes includes: detecting the content of major monosaccharides and sugar derivatives in the sample using high performance liquid chromatography, detecting the content of lignin, cellulose and hemicellulose using a cellulometry instrument, and analyzing the change pattern of carbohydrates before and after microbial treatment;

[0015] The comparison of changes in bases and organic acids includes: using simultaneous distillation extraction to separate the alkaline, acidic and neutral components in the sample, washing and drying them separately, adding phenylethyl acetate as an internal standard, performing GC-MS analysis, and combining the changes in the content of pigment components to analyze the changing trends of organic acids.

[0016] More preferably, the comparison of pigment changes includes: detecting the pectin and plastid pigment content in the sample using ion chromatography and high performance liquid chromatography, and analyzing the degradation of macromolecular aroma precursors by microorganisms;

[0017] The comparison of changes in phenolic compounds includes: using high performance liquid chromatography to detect the content of phenolic compounds in samples, comparing the change patterns, and determining the effects of microorganisms on phenolic precursors.

[0018] More preferably, the comparison of the changes in ester compounds includes: pre-treating the sample using an accelerated solvent extraction method, analyzing and detecting the aroma-causing components sterols and higher fatty acids in the sample using GC-MS to determine the degradation of ester aroma precursors by microorganisms;

[0019] The comparison of changes in anions and cations includes: using ion chromatography to detect the anions and cations of potassium, calcium, sodium, magnesium, and citric acid in the sample, comparing the change patterns of anions and cations, analyzing the effects of microorganisms on anions and cations in tobacco, and then determining the impact on the sensory experience of cigarettes.

[0020] Preferably, in step 2, the effects of microorganisms are investigated from the perspectives of microbiome, genomics, and metabolomics.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention adopts simultaneous distillation extraction and accelerated solvent extraction to pre-treat the sample, which can achieve the simultaneous extraction of acidic, alkaline and neutral substances in tobacco. The operation is simple, the target object is extracted more thoroughly, the extraction time is shorter, and it is beneficial to improve work efficiency and work quality.

[0023] 2. The present invention not only simply detects and analyzes changes in the content of various substances, but also combines and comprehensively analyzes the treatment effects of microorganisms based on the changes in the substances. For example, protein content is combined with amino acid content to analyze the changing trends of protein substances; starch content is combined with total sugar and reducing sugar content to analyze the changing trends of carbohydrate substances; and aroma precursor substances such as carotenoids are combined with the content of neutral aroma components to analyze the formation patterns of aroma substances. Therefore, through comprehensive analysis, the present invention can form a complete microbial treatment effect evaluation system. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the relevant technologies in the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0025] This embodiment provides a method for evaluating the effect of microbial treatment of cigarette tobacco. By analyzing the changes in eight major chemical substances in cigarette tobacco before and after microbial treatment, an evaluation system for the effect of microbial treatment of cigarettes is established to intuitively judge the effect of microbial fermentation treatment.

[0026] The specific method is as follows:

[0027] 1.1 Sample processing

[0028] Cigarette tobacco samples A and B before and after microbial treatment were selected. Different components were obtained using pre-treatment methods such as Soxhlet extraction and simultaneous distillation extraction. The effect of microbial treatment on cigarette tobacco was comprehensively determined based on the changes in different components.

[0029] 1.2 Detection method

[0030] (1) Research on conventional chemical indicators of tobacco

[0031] The contents of conventional chemical indicators (total sugar, reducing sugar, nicotine, chlorine, potassium, and total nitrogen) in cut tobacco were measured using a continuous flow analyzer, and the changes in the total amount were compared.

[0032] (2) Research on nitrogen-containing compounds

[0033] The 20 free amino acids in the samples were determined using an amino acid analyzer. Combined with the changes in protein content detected in (1), the trends of protein and amino acid changes before and after microbial treatment were compared to analyze the possible patterns of protein changes.

[0034] (3) Research on carbohydrates

[0035] The contents of main monosaccharides and sugar derivatives in the samples were detected by high performance liquid chromatography, and the contents of lignin, cellulose and hemicellulose were detected by cellulometry. Combined with the contents of total sugar and reducing sugar detected in (1), the changes in carbohydrates before and after microbial treatment were analyzed.

[0036] (4) Research on bases and organic acids

[0037] The alkaline, acidic and neutral components in the sample were separated by simultaneous distillation and extraction, washed and dried separately, and phenylethyl acetate was added as an internal standard for GC-MS analysis. Combined with the changes in the content of pigment components, the changing trend of organic acids was analyzed.

[0038] (5) Research on pigments

[0039] Ion chromatography and high performance liquid chromatography were used to detect the pectin and plastid pigment contents in the samples, and to analyze the degradation of macromolecular aroma precursors by microorganisms.

[0040] (6) Research on phenolic compounds

[0041] By using high performance liquid chromatography to detect the content of phenolic compounds in samples and comparing their changing patterns, the effect of microorganisms on phenolic precursors can be determined.

[0042] (7) Research on ester compounds

[0043] The samples were pretreated using ASE (accelerated solvent extraction), and the aroma-causing components sterols and higher fatty acids in the samples were analyzed and detected using GC-MS to determine the degradation of ester aroma precursors by microorganisms.

[0044] (8) Research on anions and cations

[0045] Ion chromatography is used to detect common anions and cations such as potassium, calcium, sodium, magnesium, and citric acid in the samples, compare the changes in anions and cations, analyze the effects of microorganisms on anions and cations in tobacco, and then determine the impact on the sensory experience of cigarettes.

[0046] Example

[0047] Tobacco sample pretreatment

[0048] ① This method utilizes simultaneous distillation extraction to separate and extract alkaline, acidic, and neutral components from cut tobacco samples. Dichloromethane was used in a water bath heated to 60°C. After 2 hours of simultaneous distillation and extraction, washing was performed to simultaneously extract the alkaline, acidic, and neutral components.

[0049] Conventional practice: solvent extraction-silanization derivatization method to extract acidic substances in tobacco (about 1.2 hours) + solvent extraction method to extract alkaline substances in tobacco (about 1.5 hours) + distillation extraction method to extract neutral substances in tobacco (about 14.5 hours, extraction for 2.5 hours, adding anhydrous sodium sulfate overnight for 12 hours). It takes a total of 17.2 hours to extract the alkaline, acidic and neutral components in tobacco through these three methods.

[0050] This processing technology saves 15.2 hours per sample compared to traditional methods, increasing efficiency by 8.6 times. It also reduces sample and reagent consumption.

[0051] ② This method uses ASE (accelerated solvent extraction) to extract ester compounds in tobacco samples, using ethanol as the extraction solution, pressurized extraction time of 10 minutes, 3 cycles, and then concentrated and injected. The sample processing time is about 1 hour.

[0052] Standard procedure: Weigh 2g of tobacco dust sample into a 50mL stoppered centrifuge tube. Add 10mL of phosphate buffer adjusted to pH 3, vortex to completely soak the sample, and let it sit for 5 minutes. Then, add 10mL of acetonitrile and 80μL of 30.0mg / L acetophenone-d8 internal standard working solution. Vortex at 2500rpm for 10 minutes, then freeze at -18°C for 10 minutes. Add 4g of anhydrous magnesium sulfate and 1g of sodium chloride, shake rapidly, vortex at 2500rpm for 2 minutes, and centrifuge at 7000rpm for 3 minutes. Remove 1.5mL of the supernatant, add 0.2g of anhydrous magnesium sulfate, immediately vortex at 2500rpm for 2 minutes, and centrifuge at 7000rpm for 3 minutes. The supernatant is then filtered through a 0.22μm organic phase filter and analyzed by GC-MS / MS. Sample processing takes approximately 1 hour.

[0053] Traditional methods require vortexing, freezing, and multiple centrifugation steps, which are complex and prone to sample and target loss. The pretreatment method used in this application takes the same amount of time as the traditional method, but is simpler to operate and allows for more complete extraction of the target.

[0054] Data analysis results

[0055] According to the above scheme, the analytical data after treating tobacco shreds for a specific microorganism are shown in Tables 1 to 12.

[0056] ① Comprehensive analysis of the total nitrogen and protein test results (Table 5) and amino acid test results (Table 6): After microbial treatment, the total protein content in cigarette tobacco decreased, while the amino acid content increased. This indicates that the microorganisms have a protein-degrading effect, breaking down irritating large-molecule proteins into small-molecule amino acids, which is beneficial to the formation of Maillard reaction products during the subsequent combustion process and increasing the sensory aroma.

[0057] ② Comprehensive analysis of the total nitrogen and protein test results (Table 5) and the N-heterocyclic compound test results (Table 1): After microbial treatment, the total amount of protein and N-heterocyclic compounds in cigarette tobacco showed a downward trend, indicating that microorganisms further promoted the decomposition of nitrogen-containing compounds.

[0058] ③ Comprehensive analysis of the cell wall component detection results (Table 8) and phenolic substance detection results (Table 12): After microbial treatment, the pectin substance in the cell wall is reduced and converted into phenolic compounds. Phenolic compounds are important flavoring substances in cigarettes. Under different fermentation conditions, they can combine with amino acids and proteins to form pigments of different colors.

[0059] ④ Comprehensive analysis of the total nitrogen and protein test results (Table 5), amino acid test results (Table 6), phenolic compound test results (Table 12), and pigment compound test results (Table 9) showed that after microbial treatment, both protein and phenolic compounds decreased, while the carotenoid content of pigment compounds increased significantly, indicating that the microorganisms can promote the reaction between phenolic compounds and proteins to form carotenoids, the precursors of aroma components. This conclusion is also corroborated by the color of cigarette leaves.

[0060] ⑤ Analysis of the comprehensive detection results of pigment substances (Table 9) and neutral aroma components (Table 11): After microbial treatment, the total amount of pigment substances increased and the overall neutral aroma components increased. It is considered that some pigment substances may be degraded into aroma components such as neophytadiene, solanone, and geranylacetone. However, due to the supplementation of phenolic compounds and protein reaction products, the total amount of pigment substances increased.

[0061] ⑥ Comprehensive analysis of the volatile and semi-volatile acid test results (Table 2), non-volatile acid test results (Table 3) and higher fatty acid test results (Table 4): After microbial treatment, the total amount of volatile and semi-volatile acids decreased, the total amount of non-volatile acids remained basically unchanged, and the higher fatty acid test results increased, indicating that the microorganisms effectively reduced the irritating small molecular acid substances such as acetic acid, propionic acid, and 2-methyl-butyric acid, while promoting the chain formation of higher fatty acids. Higher fatty acids have a certain contribution to the aroma of the smoke.

[0062] ⑦ Analysis of the test results of starch, total sugar, reducing sugar and monosaccharides (Table 7): After microbial treatment, the starch content decreased, while the contents of total sugar, reducing sugar and monosaccharides such as fructose and glucose increased. This shows that the microorganisms accelerated the hydrolysis of starch, promoting its decomposition into reducing sugars and further decomposition into monosaccharides. The increase in sugar substances helps to enhance the sweetness of cigarette smoke.

[0063] By comparing different precursor substances and the total amount of decomposed or synthesized substances, it can be effectively determined that the microorganism has a degrading effect on proteins, starch, cell wall substances, volatile and semi-volatile acids; it has a promoting effect on the synthesis of non-enzymatic browning products and pigment substances; and has no obvious effect on semi-volatile acids.

[0064] Through this kind of detection and comparative analysis, a scientific theoretical basis can be provided for adjusting the temperature and time of the microbial fermentation process and determining the fermentation treatment effect.

[0065] The results of N-heterocyclic ring detection are shown in Table 1:

[0066] Table 1

[0067]

[0068] The results of volatile and semi-volatile acid tests are shown in Table 2:

[0069] Table 2

[0070]

[0071] The results of non-volatile acid test are shown in Table 3:

[0072] Table 3

[0073]

[0074] The results of higher fatty acid detection are shown in Table 4:

[0075] Table 4

[0076]

[0077] The test results of total nitrogen and protein are shown in Table 5:

[0078] Table 5

[0079]

[0080] The amino acid test results are shown in Table 6:

[0081] Table 6

[0082]

[0083] The results of sugar substance detection are shown in Table 7:

[0084] Table 7

[0085]

[0086] The results of cell wall component detection are shown in Table 8:

[0087] Table 8

[0088]

[0089] The results of pigment substance detection are shown in Table 9:

[0090] Table 9

[0091]

[0092] The results of volatile aldehydes and ketones detection are shown in Table 10:

[0093] Table 10

[0094]

[0095] The results of the neutral fragrance component test are shown in Table 11:

[0096] Table 11

[0097]

[0098] The results of phenolic substance detection are shown in Table 12:

[0099] Table 12

[0100]

[0101] This embodiment can also examine the effects of microorganisms from the perspectives of microbiome, genomics, and metabolomics.

[0102] This implementation characterizes the effectiveness of microbial treatment by analyzing the changes in chemical substances in tobacco after microbial treatment. It utilizes standard laboratory equipment, such as GC-MS (gas chromatography-mass spectrometry) and HPLC (liquid chromatography), requiring only basic chemistry knowledge and instrument operation skills. Furthermore, the solution features rapid peak elution times for the test indicators, enabling simultaneous elution of multiple substances using the same solvent, resulting in high efficiency.

[0103] In summary, the present invention not only simply detects and analyzes the changes in the content of each substance, but also combines and comprehensively analyzes the treatment effects of microorganisms based on the change patterns of the substances; therefore, the present invention can form a complete microbial treatment effect evaluation system through comprehensive analysis.

[0104] It should be emphasized that the above are only preferred embodiments of the present invention and do not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for evaluating the effect of microbial treatment of cigarette tobacco, characterized in that: By analyzing the changes in eight major chemical substances in cut tobacco before and after microbial treatment, an evaluation system for the effect of microbial treatment of cut tobacco was established to intuitively judge the effect of microbial fermentation treatment. The evaluation method comprises the following steps: Step 1: Sample pretreatment: Select cigarette tobacco samples before and after microbial treatment, and use Soxhlet extraction to obtain different components; the extraction pretreatment method includes: simultaneous distillation extraction and accelerated solvent extraction; Step 2: By comparing the changes in different components, comprehensively determine the effect of microbial treatment on cigarette tobacco.

2. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 1, characterized in that: In step 1, the simultaneous distillation extraction includes: separating and extracting the alkaline, acidic and neutral components in the tobacco sample by simultaneous distillation extraction; using dichloromethane and heating the water bath at 55-65° C.; and performing simultaneous distillation extraction for 1.5-2.5 hours and then washing to simultaneously complete the extraction of the alkaline, acidic and neutral components.

3. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 1, characterized in that: In step 1, the accelerated solvent extraction includes: extracting ester compounds in the tobacco sample using accelerated solvent extraction, using ethanol as the extraction solution, pressurized extraction time 8 to 12 minutes, 2 to 5 cycles, and then concentrating and injecting.

4. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 1, characterized in that: In step 2, the changes in the different components include: changes in conventional chemical indicators of tobacco, changes in nitrogen-containing compounds, changes in carbohydrates, changes in alkalis and organic acids, changes in pigments, changes in phenolic compounds, changes in ester compounds, and changes in anions and cations.

5. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 4, characterized in that: The comparison of changes in conventional chemical indicators of tobacco includes: measuring the content of conventional chemical indicators of tobacco using a continuous flow analyzer and comparing the change patterns of the total amount, wherein the conventional chemical indicators of tobacco include: total sugar, reducing sugar, nicotine, chlorine, potassium, and total nitrogen; The comparison of the changes in nitrogenous compounds includes: measuring free amino acids in the sample using an amino acid analyzer, comparing the changing trends of proteins and amino acids before and after microbial treatment, and analyzing possible protein change rules.

6. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 4, characterized in that: The comparison of carbohydrate changes includes: using high performance liquid chromatography to detect the content of major monosaccharides and sugar derivatives in the samples, using a cellulometry to detect the content of lignin, cellulose and hemicellulose, and analyzing the change pattern of carbohydrates before and after microbial treatment; The comparison of changes in bases and organic acids includes: using a simultaneous distillation extraction method to separate the alkaline, acidic and neutral components in the sample, washing and drying them separately, adding phenylethyl acetate as an internal standard, performing GC-MS analysis, and analyzing the change trend of organic acids in combination with the changes in the content of pigment components.

7. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 4, characterized in that: The comparison of pigment changes includes: using ion chromatography and high performance liquid chromatography to detect the content of pectin and plastid pigments in the samples, and analyzing the degradation of macromolecular aroma precursors by microorganisms; The comparison of the changes in the phenolic compounds includes: using high performance liquid chromatography to detect the content of the phenolic compounds in the sample, comparing the change patterns, and determining the effect of the microorganisms on the phenolic precursors.

8. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 4, characterized in that: The comparison of the changes in the ester compounds includes: pre-treating the samples using an accelerated solvent extraction method, analyzing and detecting the aroma components sterols and higher fatty acids in the samples using GC-MS, and determining the degradation of ester aroma precursors by microorganisms; The comparison of the changes in anions and cations includes: using an ion chromatograph to detect potassium, calcium, sodium, magnesium, and citric acid anions and cations in the sample, comparing the change patterns of anions and cations, analyzing the effects of microorganisms on anions and cations in the tobacco, and then determining the impact on the sensory perception of the cigarette.

9. The method for evaluating the effect of microbial treatment of cut tobacco according to claim 1, characterized in that: In step 2, the effects of microorganisms are investigated from the perspectives of microbiome, genomics, and metabolomics.

Images