Alginate oligosaccharide molecule for improving sweetness of tobacco leaves by activating transcription factors

Abstract

The invention belongs to the field of tobaccos, and provides an alginate oligosaccharide molecule for improving the sweetness of tobacco leaves by activating transcription factors. By screening alginate oligosaccharide fragment molecules and activating a signal channel, carbon and nitrogen metabolism of the tobacco leaves is promoted, degradation of macromolecular substances such as starch is promoted, a metabolic pathway of aroma substances such as flavone is activated, and the sweetness of the tobacco leaves is improved.

Description

Technical Field

[0001] The invention belongs to the field of tobacco, and particularly relates to a brown algae oligosaccharide molecule capable of activating transcription factors to improve the sweetness of tobacco leaves. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The sweetness of tobacco leaves is one of the taste evaluation contents in tobacco leaf sensory identification. The smoke substances after tobacco leaves are burned can feel sweet taste at the tip of the tongue, tongue surface and nasal cavity. The sweetness in the smoke is caused by a variety of substances and it is difficult to determine it by sweetness value. At present, when selecting raw materials, the tobacco industry uses the sweetness of tobacco leaves as one of the main indicators to measure the availability of tobacco leaves. The sweetness of tobacco leaves is extremely significantly positively correlated with the sugar-alkali ratio.

[0004] Light intensity has an important influence on the photosynthetic capacity, identity and sweetness of flue-cured tobacco leaves. It can enhance the photosynthesis of tobacco leaves and the time within a certain range, thereby increasing the accumulation of carbon and nitrogen products in flue-cured tobacco leaves. After curing, the sugar content of flue-cured tobacco leaves is high, the nicotine content is moderate, the internal substances are well coordinated, and the sweetness of tobacco leaves is obvious. Under poor light conditions, the nicotine, total nitrogen, potassium content and neutral aroma components of flue-cured tobacco leaves increase, the starch content in tobacco leaves increases, the total sugar and reducing sugar content decreases, and the sweetness of tobacco leaves decreases.

[0005] In addition, temperature also has a great impact on the sweetness of flue-cured tobacco. When the growth environment of the tobacco plants is below 30°C, substance synthesis is restricted and respiratory consumption increases, leading to metabolic disorders in the tobacco leaves and accelerated substance consumption rates, reducing the tobacco plants' ability to absorb and utilize light energy. After curing, the coordination of substances contained in the tobacco leaves becomes worse, and the sweetness of the tobacco leaves decreases.

[0006] In Shandong's mountain tobacco-growing areas, there are cloudy and rainy days and low temperatures during the tobacco leaf ripening period, which affect the carbon and nitrogen metabolism of the tobacco leaves and the sweetness of the tobacco leaves after curing. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a brown algae oligosaccharide molecule that activates transcription factors to increase the sweetness of tobacco leaves. The present invention screens brown algae oligosaccharide fragment molecules to activate signal pathways, thereby promoting carbon and nitrogen metabolism in tobacco leaves, promoting the degradation of macromolecular substances such as starch, activating the metabolic pathways of aroma substances such as flavonoids, and increasing the sweetness of tobacco leaves.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides the use of brown algae oligosaccharides in improving the sweetness of tobacco leaves.

[0010] The invention increases the photosynthesis of tobacco leaves and promotes the degradation of macromolecular substances such as tobacco leaf starch during the tobacco leaf maturation period by means of biotechnology, which is beneficial to enhancing the sweetness of tobacco leaves.

[0011] In some embodiments, at least one of the following aspects of the tobacco leaf is also improved:

[0012] a) Appearance quality;

[0013] b) smoke rate after baking;

[0014] c) tobacco leaf production;

[0015] b) stem content;

[0016] e) Chemical composition content determination and coordination;

[0017] f) Sensory quality.

[0018] The second aspect of the present invention provides a method for improving the sweetness of tobacco leaves by activating transcription factors, comprising:

[0019] For tobacco leaves in the mature stage, apply brown algae oligosaccharides to the leaves at least once on cloudy days with little sunlight or when the temperature drops.

[0020] In some embodiments, the tobacco leaves in the mature stage are 10-15 days after the tobacco leaves are toppled.

[0021] In some embodiments, the amount of the brown algae oligosaccharide applied is at least 100 g per mu each time.

[0022] In some embodiments, the application is by spraying.

[0023] In some embodiments, the number of times of applying brown algal oligosaccharide is 1 time or 2 times.

[0024] In some embodiments, the interval between two consecutive administrations of algal oligosaccharides is 7-8 days.

[0025] The third aspect of the present invention provides the prepared tobacco leaves mentioned above.

[0026] A fourth aspect of the present invention provides a tobacco product comprising the above-mentioned tobacco leaves.

[0027] Beneficial effects of the present invention

[0028] (1) The present invention screens brown algae oligosaccharide fragment molecules to activate signal pathways, thereby promoting carbon and nitrogen metabolism in tobacco leaves, promoting the degradation of macromolecular substances such as starch, activating the metabolic pathways of aroma substances such as flavonoids, and increasing the sweetness of tobacco leaves.

[0029] (2) The method of the present invention is simple, highly practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 : Observation of the ultrastructure of tobacco leaves after spraying brown algae oligosaccharides; (AB, electron microscope pictures of the ultrastructure of tobacco leaves in the control group, B is the enlargement of the red frame in Figure A, and the arrow indicates the chloroplast; CD, electron microscope pictures of the ultrastructure of tobacco leaves in the experimental group sprayed with brown algae oligosaccharides, D is the enlargement of the red frame in Figure C);

[0032] Figure 2 : Heat map of changes in the contents of some differentially expressed genes and metabolites (A is differentially expressed genes, B is differentially expressed metabolites). DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0035] The tests in the following embodiments were conducted in 2023 in Kunming (Yiling), Baoshan (Bingma, Xiyi, Zhujie, Lujie), Qujing, Yunnan Province, Xuchang, Pingdingshan, Henan Province, Yan'an (Fuxian, Huanglong), Shaanxi Province, Enshi, Hubei Province, Linyi (Yishu), Weifang (Changle), Shandong Province, and other 13 tobacco producing areas, and the tested flue-cured tobacco variety was K326. The local conventional seedling raising method was adopted for seedling raising, and after the tobacco seedlings were toppled, 18 leaves were left per plant, and other field management measures were carried out in accordance with the local high-quality flue-cured tobacco production technical regulations.

[0036] Example 1 Experimental method

[0037] Ten days after the tobacco leaves were toppled, brown algae oligosaccharides were sprayed continuously twice at low temperature (below 20°C), with an interval of 1 week, 100g / mu each time, and the leaves were sprayed evenly. The control group was sprayed with clean water. On the day of flue-cured tobacco topping, 200 tobacco plants with consistent growth were selected and marked as experimental sampling tobacco plants. The sampling time was the time when tobacco leaves in each tobacco area matured and harvested, specifically from September to October. Each treatment tobacco leaf was uniformly baked in a dense baking room, and 3.0kg of representative middle and upper post-baked tobacco was evenly selected for the physical properties, chemical composition and sensory quality evaluation of post-baked tobacco leaves. The samples for transcriptome, metabolome and electron microscopy observation were normal mature fresh tobacco leaves. The sampling time was consistent with the time when the tobacco leaves matured and were harvested. The leaf position was the third leaf from the top of the tobacco plant. When sampling, 7 tobacco leaf samples were mixed as one biological replicate. Three replicates were taken each time. The main vein and thicker branches were removed, the samples were fully mixed and wrapped in tin foil, immediately frozen in liquid nitrogen, embedded in dry ice for transportation, and stored in a -80°C refrigerator.

[0038] Example 2 Determination of physical indicators

[0039] The physical indices of tobacco leaves were measured according to YC / T 142-1998, where the leaf thickness was measured using a manual thickness meter, and the stem content was measured by selecting tobacco leaves with an equilibrium moisture content of 16.5±0.5% for stem extraction. The weight of the tobacco leaves and stems was then weighed using a 100%-per-day balance, and the stem content was calculated using the formula: stem content (%) = (stem weight / leaf weight) × 100.

[0040] Example 3 Determination of chemical composition

[0041] The flue-cured tobacco leaves were crushed at high speed and passed through a 60-mesh sieve. The water-soluble sugar was determined by the continuous flow method (YC / T159-2002); the total nitrogen in tobacco and tobacco products was determined by the continuous flow method (YCT 161-2002); the total alkaloids in tobacco and tobacco products were determined by the continuous flow method (potassium thiocyanate) (YC / T468-2013); the potassium in tobacco and tobacco products was determined by the flame photometry (YC / T173-2003); the chlorine in tobacco and tobacco products was determined by the continuous flow method (YC / T162-2011); the starch in tobacco and tobacco products was determined by the continuous flow method (YC / T216-2013), and the ratio of reducing sugar content to total sugar content was calculated.

[0042] Example 4 Sensory Evaluation and Identification

[0043] Sensory evaluation is to rate the sensory indicators such as aroma quality, aroma quantity, impurities, concentration, strength, irritation, aftertaste, etc., and the scoring standards are formulated with reference to YC / T 138-1998 Sensory Evaluation Method for Tobacco Products.

[0044] Example 5 Electron Microscope Observation

[0045] Cut several small pieces of about 2mm×2mm from the lower right side of the midrib of fresh tobacco leaves, put them into 2.5% glutaraldehyde phosphate buffer (pH 7.2), and evacuate at room temperature until the material sinks, and place them in a 4℃ refrigerator for more than 72 hours. The samples were cleaned, dehydrated, dried, mounted, and observed under a Hitachi S-3000 scanning electron microscope and photographed. Data were measured through 10 random fields of view and the average value was measured.

[0046] Example 6 Transcriptome and metabolome analysis

[0047] Transcriptome analysis used the number of fragments per kilobase of transcript million fragments (FPKM) to calculate the expression of transcripts. Differential analysis was performed using DESeq2 software, and the screening criteria for differentially expressed genes (DEGs) were FC>2 and false discovery rate (FDR)<0.01. Among them, the screening conditions for differentially expressed metabolites (DEMs) and differentially expressed genes (DEGs) were |log2(Fold Change)|≥1 and p<0.05.

[0048] Metabolome analysis performed qualitative and quantitative analysis on all mass spectrometry data, and the differential expressed metabolites (DEMs) between groups were screened by combining orthogonal partial least squares discriminant analysis (OPLS-DA) and difference fold value (Fold_Change). The screening criteria were (variable importance inprojection, VIP) ≥ 1, Fold_Change ≥ 2 or Fold_Change ≤ 0.5 to obtain differential metabolites.

[0049] Based on the screening results of differential genes and differential metabolites, the transcriptome and metabolome analysis results are mapped on the KEGG map, pathway annotation is performed, common synthesis pathway information is obtained, and the association between genes and metabolites is further analyzed. The present invention calculates the Pearson correlation coefficient (PCC) for the DEGs and DAMs screened from the transcriptome and metabolome. With the PCC threshold ≥ 0.6 and P < 0.05 as the standard, the metabolome and transcriptome data are jointly analyzed. PCC ≥ 0.6 indicates that there is a significant correlation between DEGs and DAMs.

[0050] From the test results of Example 2 to Example 6, it can be seen that:

[0051] Spraying brown algae oligosaccharides can effectively improve the appearance quality of flue-cured tobacco leaves in various production areas. The color, structure, identity and chroma of the middle and upper tobacco leaves have increased, and the orange rate of flue-cured tobacco is significantly higher, which significantly improves the grade of tobacco leaves. Spraying brown algae oligosaccharides can significantly increase the yield of tobacco leaves, reduce the stem content, and improve the effective utilization rate of tobacco leaves.

[0052] The results of the determination of the chemical components of flue-cured tobacco leaves and the comparative analysis of their coordination showed that the quality grade of flue-cured tobacco leaves was improved, the contents of nicotine, chlorine and starch in tobacco leaves were reduced, the contents of total sugar, reducing sugar, total nitrogen and potassium were increased, the sugar-alkali ratio, nitrogen-alkali ratio and two-sugar ratio were increased, and the Schmuck value was reduced. By spraying brown algae oligosaccharides, the content and ratio of chemical components in tobacco leaves were adjusted, which tended to the evaluation standards of high-quality flue-cured tobacco. Among the chemical components, the content of starch, chlorine and reducing sugar changed the most, with starch decreasing by 32.23% on average, chlorine decreasing by 28.00% on average, and reducing sugar increasing by 25.78% on average. The content of total nitrogen and protein changed the least, all below 10%. From the perspective of chemical composition coordination, the potassium-chlorine ratio, sugar-alkali ratio, and nitrogen-alkali ratio increased by more than 40%, among which the potassium-chlorine ratio increased the most, reaching 75.23%. The reducing sugar content of tobacco leaves increased by more than 20%, and the starch content in 9 tobacco-growing areas also decreased by more than 20%. Among them, the change in Enshi, Hubei was the most obvious, with an increase of 89.19% in reducing sugar and a decrease of 61.60% in starch. After using brown algae oligosaccharides, it reached the standard of high-quality flue-cured tobacco. All 13 tobacco-growing areas showed an increase in potassium content and a decrease in chlorine content after spraying brown algae oligosaccharides, and the ratio of potassium-chlorine ratio also increased. Except for Yiliang and Enshi, the total nitrogen content meets the standards of high-quality flue-cured tobacco, and the ratios of sugar-alkali ratio and nitrogen-alkali ratio in 13 tobacco areas treated with brown algae oligosaccharides are more reasonable. Although the protein content has increased or decreased, the difference is not obvious. It can be seen that spraying brown algae oligosaccharides can significantly change the content of chemical components in flue-cured tobacco leaves, especially the content of potassium and chlorine, optimize the composition of chemical components of tobacco leaves in different tobacco areas, make them more coordinated, and improve the quality grade of tobacco leaves.

[0053] From the perspective of sensory quality evaluation indicators, the sensory evaluation scores and quality grades of tobacco leaves were improved after spraying brown algae oligosaccharides in each tobacco area, with scores above 75.00 points and quality grades above 3.5. Among them, the tobacco leaves in Hubei Enshi tobacco area had the largest improvement in tobacco leaf smoking quality after spraying brown algae oligosaccharides, with a score increase of 5.5 points, a quality grade increase of 0.7, and significant improvements in aroma quality, aroma volume, aftertaste, miscellaneous smell, and irritation; followed by tobacco leaves in Linyi Yishui tobacco area, with a sensory evaluation score increase of 4.0 points and a quality grade increase of 0.4. From the perspective of various evaluation indicators, spraying brown algae oligosaccharides has the greatest impact on aroma volume, with the average score of aroma volume in 13 tobacco areas increased by 0.88 points; followed by aftertaste, with an average score increase of 0.62 points. There was no significant difference in the scores of combustibility and gray. Overall, spraying brown algae oligosaccharides can improve the aroma quality and aroma volume of tobacco leaves, reduce aftertaste, miscellaneous smell and irritation, improve the quality grade of tobacco leaves, and achieve better sensory effects.

[0054] pass Figure 1 Ultrastructural observation of tobacco leaves showed that the cell membrane and cell wall structure of the control group's leaves were intact and clear, and the organelles were squeezed to the edge of the cell by the vacuole and arranged in order. The thylakoid structure of the chloroplast was clearly visible, and there were a small number of starch grains. It can be judged that the synthesis of starch in the leaves of the control group was vigorous and the leaves were still in the development stage. The electron microscopy results of the group treated with brown algae oligosaccharides showed that organelles such as chloroplasts were gradually decomposed, the plastid membrane was ruptured, and starch grains accumulated in large quantities and began to degrade. It can be judged that the leaves of the experimental group are already in the mature stage, which significantly increases the sweetness of the tobacco leaves.

[0055] In carbohydrate metabolism, the contents of various carbohydrates such as mannose (D-Mannose), galactose (D-galactose), isomaltulose (Isomaltulose), L-glucose (L-Glucose), D-threonic acid (D-Threonic Acid), 2,4-di(2-methyl)butanoyl-3-(2-methylcrotonyl)sucrose (2,4-Di(2-Methylbutanoyl)-3-(2-Methylcrotonyl)Sucrose) increased. Some key synthases related to carbohydrate metabolism, such as mannosidase gene α-Man (gene_49519), galactosidase gene α-Gal (gene_26470), and furanosidase gene (gene_34644), were also upregulated. Combined with the result that the detection data of total sugar and reducing sugar in tobacco leaves increased in different situations, the spraying of brown algae oligosaccharides accelerated the accumulation of sugars in tobacco, laying the foundation for improving the quality of tobacco leaves.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of brown algae oligosaccharides in improving the sweetness of tobacco leaves.

2. The use according to claim 1, characterized in that At least one of the following aspects of the tobacco leaf is also improved: a) Appearance quality; b) smoke rate after baking; c) tobacco leaf production; b) stem content; e) Chemical composition content determination and coordination; f) Sensory quality.

3. A method for improving the sweetness of tobacco leaves by activating transcription factors, characterized in that: include: For tobacco leaves in the mature stage, apply brown algae oligosaccharides to the leaves at least once on cloudy days with little sunlight or when the temperature drops.

4. The method for improving the sweetness of tobacco leaves by activating transcription factors according to claim 3, characterized in that: The tobacco leaves in the mature stage are 10-15 days after the tobacco leaves are toppled.

5. The method for improving the sweetness of tobacco leaves by activating transcription factors according to claim 3, characterized in that: The amount of brown algae oligosaccharide applied is at least 100 g per mu each time.

6. The method for improving the sweetness of tobacco leaves by activating transcription factors according to claim 3, characterized in that: The application method is spraying.

7. The method for improving the sweetness of tobacco leaves by activating transcription factors according to claim 3, characterized in that: The number of times of applying brown algal oligosaccharide was once or twice.

8. The method for improving the sweetness of tobacco leaves by activating transcription factors according to claim 3, characterized in that: The interval between two consecutive applications of brown algae oligosaccharides is 7-8 days.

9. The prepared tobacco leaf according to any one of claims 3 to 8.

10. A tobacco product, characterized in that: Comprising the tobacco leaf according to claim 9.

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