Cyclic dipeptide precursor aroma component as well as preparation method and application thereof

Abstract

The invention discloses a cyclic dipeptide precursor aroma component as well as a preparation method and application thereof, the high-content cyclic dipeptide precursor aroma component, especially cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide and cyclic (proline-leucine) dipeptide, is produced through bacillus velezensis, and the cyclic (proline-valine) dipeptide, the cyclic (proline-isoleucine) dipeptide and the cyclic (proline-leucine) dipeptide are used as the precursor aroma component of the cyclic dipeptide. The cyclic dipeptide precursor aroma components and the fermentation liquor biological perfume can significantly improve the milk aroma and baking aroma of cigarettes, and enhance the corresponding aroma characteristics of the cigar tobacco leaves, so that the overall quality of the cigar tobacco leaves is improved. In addition, the cyclic dipeptide precursor aroma component generated by the bacillus velezensis and the fermentation liquor biological perfume provide a new way for improving the quality of tobacco products; the application of the components not only can enrich the sensory experience of tobacco products, but also opens up a new direction for product innovation and quality improvement in the tobacco industry.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco processing, in particular to a cyclic dipeptide latent aroma component, a preparation method and an application thereof. Background Art

[0002] In order to effectively safeguard the interests of consumers, reducing tar and reducing harm from cigarettes has been placed in a more prominent position. How to reduce tar without reducing aroma has become an important issue in the tobacco industry. Developing new natural tobacco flavors, especially those latent aroma components that release aroma during tobacco burning or heating, is crucial to solving this challenge.

[0003] Latent aroma components, also known as flavor precursors, are a class of compounds with low volatility and stable chemical properties at room temperature. They do not directly contribute to the aroma of tobacco products, but can be converted into substances with significant aroma during the burning or heating of tobacco, thereby enhancing the sensory quality of tobacco. At present, the flavors used in the tobacco industry are often volatile and have poor stability, and latent aroma components have become a hot topic in the research of flavor raw materials in the tobacco industry due to their low volatility and high stability at room temperature.

[0004] Cyclic dipeptides are small molecule peptides formed by condensation reaction of two amino acid molecules. This type of compound is widely present in nature, usually as a secondary metabolite of certain microorganisms [Chemical Research and Application, 2014, 26 (9): 1478-1482; Journal of Natural Products, 2003, 66 (10): 1299-1301]. Although cyclic dipeptide compounds have been reported to be used in skin whitening (CN118304208A), improving cognitive dysfunction (CN116685343A), treating mental illness (CN112439051A), and promoting wound healing (CN109646439B), there has been no research exploring their application as latent aroma components in tobacco products. Therefore, the use of biotechnology to screen microorganisms that can produce high value-added aroma enhancement effects and develop new cyclic dipeptide-type latent aroma components is of great significance for improving the overall quality of tobacco products, which is also a problem that tobacco industry technicians urgently need to solve. Summary of the invention

[0005] The purpose of the present invention is to provide a cyclic dipeptide latent aroma component, a preparation method and an application thereof in order to solve the above problems. The latent aroma component of the present invention is used for flavoring tobacco products, has a wide range of applicable raw materials, and is suitable for large-scale fermentation and production. The cyclic dipeptide latent aroma component and fermentation liquid biological flavor produced by Bacillus can improve the quality of tobacco products and also lay the foundation for the next step of industrial development.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A type of cyclic dipeptide latent fragrance ingredient, its structural formula is:

[0008]

[0009] On the other hand, the present invention also provides the use of the above-mentioned cyclic dipeptide latent aroma component in tobacco products, which can significantly enhance the milky aroma and roasted aroma of cigarette and cigar tobacco leaves and improve the quality of tobacco products.

[0010] Another aspect of the present invention also provides a method for preparing the above-mentioned cyclic dipeptide latent fragrance component, comprising the following steps:

[0011] (1) activating Bacillus velezensis and culturing it in LB liquid medium for 24 hours to obtain seed liquid;

[0012] (2) inoculating the bacterial seed liquid in step (1) into LB liquid culture medium at a fixed inoculum amount, and culturing at a certain temperature and rotation speed for a fixed time to obtain bacterial fermentation liquid;

[0013] (3) centrifuging the fermentation liquid of the strain in step (2) and harvesting the supernatant to obtain the fermentation liquid bio-flavor;

[0014] (4) extracting the fermentation broth biological flavor in step (3) with ethyl acetate for 3-5 times, and concentrating the organic phase by rotary evaporation at 48° C. until there is no ethyl acetate taste, to obtain a fermentation broth extract;

[0015] (5) The fermentation broth extract in step (4) is separated by column chromatography and monitored by GC-MS, the column chromatography eluates are combined to obtain the target segment, and the target segment is separated and purified by semi-preparative liquid phase to obtain the cyclic dipeptide latent aroma component.

[0016] Furthermore, the Bacillus velezensis is deposited in the BCCM Collection Center of Belgium with the deposit number of Bacillus velezensis LMG 22478.

[0017] Furthermore, the LB liquid culture medium is prepared by: 2-20 g of peptone, 2-20 g of yeast powder, 2-20 g of NaCl, and 500-2000 mL of water, and then sterilized under high pressure at 100-135° C. for 10-50 min.

[0018] Preferably, the LB liquid culture medium is prepared as follows: 5 g of peptone, 5 g of yeast powder, 10 g of NaCl, and 1000 mL of water, and then sterilized by high pressure at 121° C. for 20 min after dissolution.

[0019] Furthermore, in step (2), the amount of seed solution added is 1% to 40%, the culture temperature is 25 to 36° C., the culture time is 6 to 96 hours, and the shaking speed is 60 to 260 rpm.

[0020] Preferably, in step (2), the amount of seed solution added is 10%, the culture temperature is 32° C., the culture time is 24 h, and the shaking speed is 140 rpm.

[0021] Furthermore, in step (5), the column chromatography is preferably Sephadex LH-20 gel column chromatography, the elution solvent is CH2Cl2:MeOH=1:1, and the target segment is the 9th to 11th segments.

[0022] Furthermore, in step (5), the semi-preparative liquid phase elution solvent is MeOH:H2O, the solvent ratio is 15:85 to 55:45, preferably 35:65, and the detection wavelength is 210 nm.

[0023] In another aspect, the present invention provides a flavor for tobacco products, comprising one or more of the above-mentioned bacterial fermentation broth, fermentation broth biological flavor, fermentation broth extract or cyclic dipeptide latent aroma component.

[0024] Furthermore, the amount of the cyclic dipeptide latent aroma component applied to the tobacco product is 0.01% to 1%, and the amount of the fermentation liquid biological flavor applied to the tobacco product is 0.1% to 20%.

[0025] Preferably, the amount of the cyclodipeptide latent aroma component applied to tobacco products is 0.5%, the amount of the fermentation liquid biological flavor applied to cigarette products is 1.0%, and the amount applied to tobacco leaves is 2.0%.

[0026] The last aspect of the present invention also provides the use of the above tobacco product flavor in tobacco products;

[0027] The tobacco product is any one or more of cigarette products, flue-cured tobacco leaves, reconstituted tobacco leaves, cigar tobacco leaves and electronic cigarette liquid;

[0028] The applications include application as a flavor in a finished product or application as a fermentation medium in a raw material fermentation process.

[0029] The beneficial effects of the present invention are:

[0030] The present invention proposes for the first time that Bacillus Velezii can produce specific cyclic dipeptide latent aroma components, including cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide, cyclic (proline-leucine) dipeptide, etc. These components have important value in the flavor enhancement application of tobacco products.

[0031] The tobacco flavor provided in the present invention can effectively enhance the milky aroma and baking aroma of cigarettes, and can increase the milky aroma and baking aroma of cigar tobacco leaves. These characteristics make the cyclic dipeptide latent aroma components particularly suitable for improving the aroma of tobacco and tobacco products.

[0032] The process of using Bacillus Velezii to carry out microbial fermentation to produce latent aroma components and biological flavors has significant advantages: simple operation, easy control of conditions, and no need for complicated pretreatment or special equipment. It not only improves production efficiency but also reduces production costs, providing a cost-effective solution for aroma improvement in the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The figure is a sensory evaluation aroma profile diagram of the Bacillus Velez fermentation broth of the present invention.

[0035] Figure 2 The figure is the GC-MS total ion current diagram of the Bacillus Velez fermentation broth of the present invention.

[0036] Figure 3 The diagram is a structural diagram of three cyclic dipeptide latent fragrance components of the present invention.

[0037] Figure 4 This is the ESI-MS spectrum of compound 1 of the present invention.

[0038] Figure 5 The compound 1 of the present invention 1 H NMR spectrum (500 MHz, CD3OD).

[0039] Figure 6 The compound 1 of the present invention 13 C NMR spectrum (125 MHz, CD3OD).

[0040] Figure 7 This is the ESI-MS spectrum of compound 2 of the present invention.

[0041] Figure 8 The compound 2 of the present invention 1 H NMR spectrum (500 MHz, CD3OD).

[0042] Fig. 9 The compound 2 of the present invention 13C NMR spectrum (125 MHz, CD3OD).

[0043] Fig.10 This is the ESI-MS spectrum of compound 3 of the present invention.

[0044] Fig.11 is the compound 3 of the present invention 1 H NMR spectrum (500 MHz, CD3OD).

[0045] Fig.12 is the compound 3 of the present invention 13 C NMR spectrum (125 MHz, CD3OD).

[0046] Fig.13 This is the sensory evaluation fragrance profile diagram of the three cyclic dipeptide latent fragrance components of the present invention. DETAILED DESCRIPTION

[0047] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0048] Example 1 Preparation of biological flavors from bacterial fermentation broth

[0049] Bacillus velezensis was activated, and the strain was deposited in the Belgian BCCM Collection Center with the deposit number of Bacillus velezensis LMG 22478. After activation, the strain was inoculated in 2L LB liquid fermentation medium (5g peptone, 5g yeast powder, 10g NaCl, 1000mL water, and sterilized at 121°C for 20min after dissolution), and the liquid culture was used as seed liquid for 24h. 10L LB liquid fermentation medium was prepared, and the above seed liquid was inoculated with 10% inoculation amount, and fermented at 32°C and 140rpm for 24h. After the fermentation liquid was centrifuged at 4000 rpm, the supernatant was harvested to obtain the strain fermentation liquid biological flavor.

[0050] Example 2 Sensory evaluation of biological flavors of bacterial fermentation broth

[0051] The fermentation liquid was evaluated for aroma. The fermentation liquid had a distinct milky aroma, and the aroma of freshness and fermented yogurt was also strong. 6 μL of the fermentation liquid was injected into Sichuan Zhongyan 0# blank cigarettes, and sensory evaluation was performed after balancing. The results showed that compared with blank cigarettes without added flavorings, the milky aroma and baking aroma of cigarettes added with Bacillus Velez fermentation liquid were significantly improved ( Figure 1), the fruity and floral aromas are also enhanced, the aftertaste is obvious, and the irritation of the smoke is reduced.

[0052] Example 3 GC-MS analysis of aroma components in fermentation broth of strains

[0053] Take 20 mL of fermentation broth, add 10 mL of dichloromethane to extract twice, combine the dichloromethane layers and concentrate to 0.9 mL, add 0.1 mL of 0.8211 mg / mL phenylethyl acetate as an internal standard, and perform GC-MS analysis.

[0054] Gas chromatography (GC) conditions: Agilent HP-5MS column (model: 30m×250μm×0.25μm). Inlet temperature: 280°C. Carrier gas: high-purity helium (99.999%), flow rate: 1.0mL / min. Program temperature: initial temperature: 50°C, maintained for 4min, then raised to 240°C at a rate of 2°C / min, without retention. Injection volume: 1.0μL.

[0055] Mass spectrometry (MS) conditions: transfer line temperature 280°C; ion source temperature 230°C; quadrupole temperature 150°C; ionization mode electron impact (EI), electron energy 70 eV; solvent delay time 7 min. Full scan mass range (m / z) 35-550u, GC-MS adopts full scan mode.

[0056] The results are as follows Figure 2 The results show that the fermentation broth of Bacillus velez contains a large number of high content of cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide, cyclic (proline-leucine) dipeptide, cyclic (L-proline-L-alanine) dipeptide, cyclic (L-proline-glycine) dipeptide, cyclic (phenylalanine-proline) dipeptide and other aroma precursor components ( Figure 3 ), cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide, and cyclic (proline-leucine) dipeptide have high contents, reaching 7.15, 6.36, and 27.69 μg / mL, respectively. In addition, the contents of styroyl acetate and phenylethyl butyrate reached 1.61 μg / mL and 1.3 μg / mL, respectively. These two components have a certain sweet and floral fragrance. Terpene components 1-pentadecene, 1-eicosene, and (Z)-3-heptene can enhance the richness of the aroma.

[0057] Table 1 GC-MS analysis of main aroma components of Bacillus velez fermentation broth

[0058]

[0059]

[0060] Example 4 Separation and Identification of Latent Aroma Components of Milky Fragrance

[0061] Take 10L of fermentation broth bio-flavor after centrifugation, add 1 / 3 volume of ethyl acetate to extract three times, and concentrate the organic phase at 48°C to dryness to obtain the strain fermentation broth extract (262.8mg). Dissolve the extract with methanol, filter the membrane, and separate it on Sephadex LH-20 gel column chromatography, with the mobile phase being CH2Cl2:MeOH=1:1. After each bottle of gel column chromatography eluent evaporates, it is combined into 15 sections (M1~M15) and analyzed and detected by GC-MS. The target components are concentrated in the M9-M11 section. Combine the M9-M11 sections (103.2mg) and separate and purify them with semi-preparative liquid phase separation, with the mobile phase being MeOH:H2O=35:65 and the flow rate being 4.0mL / min. The peaks at 24.9 min, 35.3 min, and 36.2 min were collected and then dried to obtain cyclo(proline-valine) dipeptide (1, white solid, 5.3 mg), cyclo(proline-isoleucine) dipeptide (2, white solid, 1.5 mg), cyclo(proline-leucine) dipeptide (3, white solid, 6.1 mg) ( Figure 3 ). The three compounds were dried overnight, dissolved in deuterated methanol and then subjected to NMR data testing.

[0062] Low resolution mass spectrometry ESIMS positive ion mode of compound 1 ( Figure 4 ) shows ESI-MS: m / z 197.06[M+H] + Based on this, the molecular weight of compound 1 is judged to be 196. Combining its hydrogen spectrum and carbon spectrum data, it is inferred that the molecular formula of compound 1 is C 10 H 16 N2O2. Through calculation, it can be known that compound 1 has 4 unsaturations ([10*2+2+2-16] / 2=4). 1 H NMR spectrum ( Figure 5 ) indicates that compound 1 has two N-terminal methyl hydrogen signals (δ H 4.20, t, 7.3, H-6; 4.03, s, H-9), 1 group of N-methylene hydrogen signals (δ H 3.56,3.50 1H,m,H-3). The spectrum also shows the presence of two groups of methylene hydrogen signals (δ H 2.48, 2.32 each 1H, m, H-5; 2.02, 1.95 each 1H, m, H-4), 1 methine hydrogen signal (δ H 1.95,m,H-10), 2 double peak methyl hydrogen signals (δ H 1.09, d, 7.3, H3-11; 0.94, d, 6.9, H3-12). 13 The spectrum of C ( Figure 6) shows that the compound has 2 amide carbonyl signals (δc 173.0, C-7; 167.7, C-1), 2 methine carbon signals connected to N atoms (δc61.7, C-6; 60.2, C-9), 1 methylene carbon signal connected to N atoms (δc 46.3, C-3), 2 methylene carbon signals (δc 30.1, C-5; 23.4, C-4), 1 methine carbon signal (δc 29.4, C-10), and 2 methyl carbon signals (δc 19.0, C-11; 16.8, C-12). Through calculation, it can be known that compound 1 has a total of 4 unsaturations, two C=O occupy two unsaturations, and two rings occupy two unsaturations. The analysis conclusion is consistent with the actual situation. The carbon and hydrogen spectra of compound 1 (Table 2) are basically consistent with those reported in the literature [Chemical Research and Application, 2014, 26(9):1478-1482]. Therefore, compound 1 is determined to be a cyclic (proline-valine) dipeptide with a planar structure as shown in Figure 3 shown.

[0063] Table 2 Compound 1 1 H and 13 C NMR data

[0064]

[0065] a measured in CD3OD, 1 H(500MHz), 13 C(125MHz).

[0066] Compound 2 low resolution mass spectrometry ESIMS positive ion mode ( Figure 7 ) shows ESI-MS: m / z 211.09 [M+H] + Based on this, the molecular weight of compound 2 is judged to be 210. Combining its hydrogen spectrum and carbon spectrum data, it is inferred that the molecular formula of compound 2 is C 11 H 18 N2O2. Through calculation, it can be known that compound 2 has 4 unsaturations ([11*2+2+2-18] / 2=4). 1 H NMR spectrum ( Figure 8 ) indicates that compound 2 has two N-terminal methyl hydrogen signals (δ H 4.20,m,H-6; 4.03,s,H-9), 1 group of N-methylene hydrogen signals (δ H 3.55,3.50 1H,m,H-3). The spectrum also shows the presence of three groups of methylene hydrogen signals (δ H2.32, 1.96 each 1H, m, H-5; 2.02, 1.95 each 1H, m, H-4; 1.43, 1.32 each 1H, m, H-11), 1 methine hydrogen signal (δ H 2.16,m,H-10), 1 double peak methyl hydrogen signal (δ H 1.07, d, 7.1, H3-13), 1 triplet methyl hydrogen signal (0.93, t, 7.4, H3-12). 13 The spectrum of C ( Fig. 9 ) shows that the compound has 2 amide carbonyl signals (δc 172.6, C-7; 167.7, C-1), 2 methine carbon signals connected to N atoms (δc 61.5, C-6; 60.1, C-9), 1 methylene carbon signal connected to N atoms (δc 46.3, C-3), 3 methylene carbon signals (δc 29.7, C-5; 23.4, C-4; 23.3, C-11), 1 methine carbon signal (δc 37.2, C-10), and 2 methyl carbon signals (δc 15.7, C-13; 12.7, C-12). Through calculation, it can be known that compound 2 has a total of 4 unsaturations, two C=O occupy two unsaturations, and two rings occupy two unsaturations. The analysis conclusion is consistent with the actual situation. The carbon and hydrogen spectra of compound 2 (Table 3) are basically consistent with those reported in the literature [Chemical Research and Application, 2014, 26(9):1478-1482]. Therefore, compound 2 is determined to be a cyclic (proline-isoleucine) dipeptide with a planar structure as shown in Figure 3 shown.

[0067] Table 3 Compound 2 1 H and 13 C NMR data

[0068]

[0069] a measured in CD3OD, 1 H(500MHz), 13 C(125MHz).

[0070] Compound 3 is a white solid. Low-resolution mass spectrometry ESIMS positive ion mode ( Fig.10 ) shows ESI-MS: m / z 211.11 [M+H] + Based on this, the molecular weight of compound 3 is judged to be 210. Combining its hydrogen spectrum and carbon spectrum data, it is inferred that the molecular formula of compound 3 is C 11 H 18 N2O2. Through calculation, it can be known that compound 3 has 4 unsaturations ([11*2+2+2-18] / 2=4).1 H NMR spectrum ( Fig.11 ) indicates that compound 3 has two N-terminal methyl hydrogen signals (δ H 4.26, t, 7.3, H-6; 4.13, brs, H-9), 1 group of N-methylene hydrogen signals (δ H 3.51,2H,m,H-3). The spectrum also shows the presence of three groups of methylene hydrogen signals (δ H 2.30, 1.96 each 1H, m, H-5; 2.02, 2H, m, H-4; 1.90, 2H, m, H-10), 1 methine hydrogen signal (δ H 1.51, m, H-11), 2 double peak methyl hydrogen signals (δ H 0.97,d,6.0,H3-12;δ H 0.95,d,6.0,H3-13). 13 The spectrum of C ( Fig.12 ) shows that the compound has 2 amide carbonyl signals (δc 172.9, C-7; 169.1, C-1), 2 methine carbon signals connected to N atoms (δc 60.4, C-6; 54.8, C-9), 1 methylene carbon signal connected to N atoms (δc 46.6, C-3), 3 methylene carbon signals (δc 29.2, C-5; 23.8, C-4; 39.6, C-10), 1 methine carbon signal (δc 25.9, C-11), and 2 methyl carbon signals (δc 23.4, C-12; 22.4, C-13). Through calculation, it can be known that compound 3 has a total of 4 unsaturations, two C=O occupy 2 unsaturations, and two rings occupy 2 unsaturations. The analysis conclusion is consistent with the actual situation. The carbon and hydrogen spectra of compound 3 (Table 4) are basically consistent with those reported in the literature [Chemical Research and Application, 2014, 26(9):1478-1482]. Therefore, compound 3 is determined to be a cyclic (proline-leucine) dipeptide with a planar structure as shown in Figure 3 shown.

[0071] Table 4 Compound 3 1 H and 13 C NMR data

[0072]

[0073] a measured in CD3OD, 1 H(500MHz), 13 C(125MHz).

[0074] Example 5 Sensory evaluation of milky latent aroma components

[0075] The three cyclodipeptides were dissolved in anhydrous ethanol to prepare a 1 mg / mL solution. Using the center injection method, 3 μL of the solution was evenly injected into each blank cigarette. After equilibration at 22°C and 60% relative humidity, sensory evaluation was performed. The average sensory score was taken. The results showed (Table 5 and Fig.13 ), the milky and baking aromas of cigarettes with the addition of cyclic (proline-leucine) dipeptide are significantly improved, the aroma is strong, the aroma volume is large, and the floral and fruity aromas are improved to a certain extent. The milky and baking aromas of cigarettes with the addition of cyclic (proline-isoleucine) dipeptide are also significantly improved, the aroma is strong, the aroma volume is large, and the floral and fruity aromas are significantly improved compared with cyclic (proline-leucine) dipeptide. The milky and baking aromas of cigarettes with the addition of cyclic (proline-valine) dipeptide are not as obvious as those of cyclic (proline-leucine) dipeptide, but the sweet aroma is more obvious, and the floral and fruity aromas are more obvious than those of cyclic (proline-leucine) dipeptide.

[0076] Table 5 Sensory evaluation results of three cyclic dipeptide cigarette flavoring

[0077]

[0078] Example 6 Thermal cracking of milky aroma latent components

[0079] 3 mg of each of the high-content cyclic (proline-valine) dipeptide (1) and cyclic (proline-leucine) dipeptide (3) were taken for Py-GC / MS analysis. The results are shown in Tables 6 and 7. The Py-GC / MS conditions were: pyrolysis atmosphere 10% oxygen and 90% nitrogen; pyrolysis accessory temperature 250°C; transfer line temperature 250°C; adsorption trap conditions: adsorption temperature 50°C, desorption temperature 280°C; heating program: initial temperature 50°C, heating rate 20°C / ms; set temperature 600°C, maintained for 10s. Chromatographic column: HP-5MS chromatographic column (30m×250μm×0.25μm); injection port temperature: 200℃; heating program: initial temperature 50℃, hold for 2min, increase to 150℃ at 3℃ / min, hold for 5min, then increase to 280℃ at 8℃ / min, hold for 0min; carrier gas: He (99.999%); flow rate: 1mL / min; split ratio: 20:1. Ion source: EI; electron energy: 70eV; ion source temperature: 230℃; quadrupole temperature: 150℃; scanning mode: full scan; scanning range: 30~1000amu.

[0080] The results show that there are 22 main cleavage products of cyclo(proline-valine) dipeptide at 600 degrees Celsius. In addition to the original sample, 4-(pyrrolidin-1-yl)-1,5-dihydro-2H-pyrrole-2-one, 5,6-dihydro-6-pentyl-2H-pyran-2-one, and L-hydroxyproline are the main cleavage products of cyclo(L-pro-L-valine) dipeptide, and the peak area accounts for a high proportion. Among them, 5,6-dihydro-6-pentyl-2H-pyran-2-one is a typical lactone milk aroma component with the aroma of coconut cream fat, isobutyric acid has the aroma of yogurt and fruit, and n-hexanol and tetradecanoic acid are also milk aroma-related components that can enrich the milk aroma. 4-(Pyrrolidin-1-yl)-1,5-dihydro-2H-pyrrol-2-one, 2-pyrrolidone, 1-piperidine-1-butanone, and 2,5-dimethyl-3-isopropylpyrazine have significant roasted and nutty aromas, which can enrich the aroma of cigarettes and coordinate with the aroma of tobacco.

[0081] Table 6 Main thermal decomposition products of compound 1 at 600℃

[0082]

[0083]

[0084] There are 28 main cleavage products of cyclo(proline-leucine) dipeptide at 600°C, which are richer than the cleavage products of 1. In addition to the original sample, 4-(pyrrolidin-1-yl)-1,5-dihydro-2H-pyrrole-2-one, 2-ethyl-3-methylpyrazine, and 2-methylpyrrole are the main cleavage products of cyclo(proline-leucine) dipeptide, and the peak area accounts for a high proportion. Among them, there are a certain amount of isovaleric acid, tetradecanoic acid and methylcyclopentenolone in the cleavage products of cyclo(proline-leucine) dipeptide. Tetradecanoic acid has the aroma of coconut and cream, isovaleric acid has the aroma of fruit and yogurt, and methylcyclopentenolone has caramel and milk aroma. These three are the main milk aroma-related aroma components. Styrene has a resinous and floral aroma, and 4-methyl-2-oxopentanoic acid and methyl 4-methyl-2-oxopentanoate have a fruity aroma, which can enhance the floral and fruity aroma of the smoke. 4-(Pyrrolidin-1-yl)-1,5-dihydro-2H-pyrrol-2-one, 2-ethyl-3-methylpyrazine, 2-methylpyrrole, 3-methylpyrrole, pyrrole, 2,4-dimethylpyrrole, 2-pyrrolidone, 1-acetyl-2-pyrrolidone, etc. are typical roasted aroma components with nutty, roasted and roasted sweet aromas, which can enrich the aroma of cigarettes and coordinate with the aroma of tobacco.

[0085] Table 7 Main thermal decomposition products of compound 3 at 600℃

[0086]

[0087]

[0088] Example 7 Optimization of the amount of cyclic dipeptide latent aroma component applied to cigarettes

[0089] The cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide, and cyclic (proline-leucine) dipeptide were dissolved in anhydrous ethanol to prepare a 1 mg / mL solution. Using the central injection method, 1.5 μL, 3 μL, and 6 μL of the solution were evenly injected into each blank cigarette. Based on the weight of 0.6 g of tobacco per cigarette, the application amount was 0.25%, 0.5%, and 1.0%, respectively. The test cigarettes were equilibrated at 22°C and 60% relative humidity for sensory evaluation. The results showed that when the application amount of cyclic (proline-leucine) was 0.5%, the milky and baking aroma of the cigarettes was significantly improved, the aroma was strong, the aroma was large, and the floral and fruity aromas were improved to a certain extent. When the application amount of cyclic (proline-isoleucine) dipeptide was 0.5%, the milky and baking aroma of the cigarettes was also obvious, the aroma was relatively strong, and the floral and fruity aromas were improved to a certain extent. When the cyclic (proline-valine) dipeptide is applied at 0.5%, the milky and roasted aroma of the cigarette is also improved, and the sweet, floral and fruity aromas are significantly improved. When the three cyclic dipeptides are applied at 0.25%, the aroma is not significantly improved, and the aroma volume is slightly insufficient. When the three cyclic dipeptides are applied at 1.0%, there is a slight smell of impurities, and the aftertaste is slightly uncomfortable. Therefore, the application amount of cyclic dipeptide ingredients in cigarette products is 0.5%, which is excellent.

[0090] Example 8 Study on the Stability of Cyclic Dipeptide Latent Fragrance Components in Cigarettes

[0091] The cyclic (proline-leucine) dipeptide, cyclic (proline-valine) dipeptide, and cyclic (proline-isoleucine) dipeptide were dissolved in anhydrous ethanol to prepare a 1 mg / mL solution. Using the central injection method, 3 μL of the solution was evenly injected into each blank cigarette, and the application amount was 0.5%. The control group was applied with a mixed ethanol solution of 1.0 mg / mL proline and 1.0 mg / mL leucine, a mixed ethanol solution of 1.0 mg / mL proline and 1.0 mg / mL valine, and a mixed ethanol solution of 1.0 mg / mL proline and 1.0 mg / mL isoleucine, each injected with 3 μL. The test cigarettes were stored at 22°C and 60% relative humidity for 1 month, 2 months, 4 months, 6 months, and 10 months for sensory evaluation. The results showed that the milky and baking aromas of the test cigarettes applied with the three cyclic dipeptides were significantly improved at 1 month and 2 months, and the floral and fruity aromas were improved to a certain extent, and the aroma volume was large. After 4 months, the aroma volume decreased slightly, but the overall milky and baking aromas were still significantly improved. The sensory effects of the 6-month and 10-month test cigarettes were the same as those of the 4-month samples, and the aroma volume of the cigarettes was in a stable state. The control group test cigarettes with the amino acid mixed solution had rich milky and baking aromas at 1 month and 2 months, with a certain improvement in floral and fruity aromas, and obvious sweetness. However, at 4 months, the aroma volume decreased significantly, and the milky and baking aromas were not significantly improved. The aroma volume of the 6-month and 10-month test cigarettes further decreased, and the milky and baking aromas were almost not improved. During the storage of cigarettes, the applied amino acid mixed solution will be consumed during the metabolism of microorganisms in the tobacco. At 4 months, the flavoring effect has been significantly reduced. Cyclic (proline-leucine) dipeptide, cyclic (proline-valine) dipeptide, and cyclic (proline-isoleucine) dipeptide are all peptide condensation polymers, and microorganisms cannot directly use this component. They still have a strong flavoring effect at 4 months, and it is also very obvious at 10 months.

[0092] Example 9 Optimization of the amount of fermentation liquid bio-flavor cigarettes applied

[0093] The fermentation broth biological flavor prepared in Example 1 was injected into Sichuan Zhongyan 0# blank cigarettes at 3 μL, 6 μL, and 12 μL, respectively. The application amount was 0.5%, 1.0%, and 2.0%, respectively, based on the mass of 0.6 g of tobacco per cigarette, and sensory evaluation was performed after balance. The results show that compared with blank cigarettes without adding flavors, the milky and baking aromas of cigarettes with an application amount of 1.0% are significantly improved, the fruity and floral aromas are also improved, the sweet aftertaste is obvious, and the smoke irritation is reduced. The milky and baking aromas of cigarettes with an application amount of 0.5% are slightly improved, and the irritation is not significantly improved. For cigarettes with an application amount of 2.0%, although the milky and baking aromas are significantly improved, the miscellaneous gases are somewhat prominent, and the aftertaste is slightly uncomfortable. The application amount of fermentation broth biological flavors in cigarette products is preferably 1.0%.

[0094] Example 10 Stability test of fermented liquid bio-flavor cigarettes

[0095] 6 μL of the fermentation broth bio-flavor prepared in Example 1 was injected into Sichuan Zhongyan 0# blank cigarettes, and the application amount was 1.0%. The test cigarettes were stored at 22°C and 60% relative humidity for 1 month, 2 months, 4 months, 6 months, and 10 months for sensory evaluation. The results showed that when the fermentation broth bio-flavor was applied for 1 month and 2 months, the milk aroma and baking aroma of the cigarettes were significantly improved, the fruity aroma and floral aroma were also improved, the sweet aftertaste was obvious, and the smoke irritation was reduced. After 4 months, the aroma volume decreased slightly, but the overall milk aroma and baking aroma were still significantly improved. The sensory effects of the 6-month and 10-month test cigarettes were the same as those of the 4-month samples, and the aroma volume of the cigarettes was in a stable state. This shows that the flavoring effect of the biological flavor is relatively stable, and the effect is still obvious after 10 months.

[0096] Example 11 Optimization of the application amount of cyclic dipeptide latent aroma components in flue-cured tobacco leaves

[0097] Cyclic (proline-leucine) dipeptide, cyclic (proline-valine) dipeptide, and cyclic (proline-isoleucine) dipeptide were dissolved in anhydrous ethanol to prepare a 1 mg / mL solution. The cyclic dipeptide latent aroma components were sprayed on the surface of Chongqing flue-cured tobacco leaves (variety Honghua Dajinyuan, grade C3F, 2015) according to 0.25%, 0.5%, 1.0%, and 2.0% of the weight of tobacco leaf tissue. The tobacco shreds were balanced at 22°C and 60% relative humidity for 48 hours. The tobacco shreds were rolled into test cigarettes for sensory evaluation. The original aroma of Chongqing C3F flue-cured tobacco leaves was acceptable, with a certain amount of impurities and irritation. When the amount of cyclic (proline-leucine) dipeptide applied was 0.5%, the milky and baking aroma of the cigarettes was significantly improved, the aroma was rich, the floral and fruity aromas were also improved, and the irritation was reduced. When the amount of cyclic (proline-isoleucine) dipeptide applied is 0.5%, the milky and baking aroma of the cigarette is significantly improved, the floral and fruity aromas are also significantly improved, the aroma quality is improved, and the impurities are reduced. When the amount of cyclic (proline-valine) dipeptide applied is 0.5%, the milky and baking aroma of the cigarette is significantly improved, the sweet aroma is more significant, and the floral and fruity aromas are also improved to a certain extent. When the three cyclic dipeptides are applied at 0.25%, the aroma improvement is not obvious enough, and the aroma volume is slightly insufficient. When the three cyclic dipeptides are applied at 1.0% and 2.0%, there is a slight impurity and the aftertaste is slightly uncomfortable. Therefore, the application amount of cyclic dipeptide ingredients to flue-cured tobacco leaves is 0.5%, which is excellent.

[0098] Example 12 Optimization of the amount of fermentation liquid bio-flavor applied to cigar tobacco leaves

[0099] The fermentation liquid biological flavor prepared in Example 1 was sprayed on the surface of Chuxue No. 14 cigar core tobacco leaves at 0.5%, 1.0%, 2.0%, and 4.0% of the tobacco leaf mass. The control group was sprayed with an equal amount of sterile water. The cigar tobacco leaves were fermented at 32°C and 70% relative humidity for 96 hours. The control group and the fermentation liquid application group tobacco leaves were rolled and sensory evaluated. The results showed that the original aroma of the Chuxue No. 14 control group cigar core tobacco leaves was acceptable, the irritation was strong, and the aftertaste was uncomfortable. After fermentation treatment, the baking aroma and milk aroma of the 2.0% fermentation liquid group were significantly improved, the sweetness was enhanced, the irritation was significantly reduced, the quality was significantly improved, and the usability was increased. The 0.05% and 1.0% fermentation liquid application groups were not as obvious as the 2.0% group, and there was a slight irritation. The aroma of the 4.0% fermentation liquid application group was acceptable, with a certain amount of protein fermentation miscellaneous gas, and the aftertaste was slightly uncomfortable. Therefore, the optimal amount of fermentation liquid bio-flavor applied to cigar tobacco leaves is 2.0%.

[0100] In summary, Bacillus Velez has a remarkable ability in producing high-content cyclic dipeptide latent aroma components, especially cyclic (proline-valine) dipeptide, cyclic (proline-isoleucine) dipeptide and cyclic (proline-leucine) dipeptide. These cyclic dipeptide latent aroma components and fermentation liquid biological flavors can significantly enhance the milky and roasted aroma of cigarettes, while enhancing the corresponding aroma characteristics of cigar leaves, thereby improving the overall quality of cigar leaves.

[0101] In addition, the cyclic dipeptide aroma components and fermentation liquid bio-flavors produced by Bacillus Velezii provide a new way to improve the quality of tobacco products; the application of these ingredients can not only enrich the sensory experience of tobacco products, but also open up new directions for product innovation and quality improvement in the tobacco industry.

[0102] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations. In addition, the various different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A class of cyclic dipeptide latent aroma components, characterized in that: Its structural formula is:

2. Use of the cyclic dipeptide latent aroma component as claimed in claim 1 in tobacco products.

3. The method for preparing the cyclic dipeptide latent fragrance component according to claim 1, characterized in that: The following steps are involved: (1) activating Bacillus velezensis and culturing it in LB liquid medium for 24 hours to obtain seed liquid; (2) inoculating the bacterial seed liquid in step (1) into LB liquid culture medium at a fixed inoculum amount, and culturing at a certain temperature and rotation speed for a fixed time to obtain bacterial fermentation liquid; (3) centrifuging the fermentation liquid of the strain in step (2) and harvesting the supernatant to obtain the fermentation liquid bio-flavor; (4) extracting the fermentation broth biological flavor in step (3) with ethyl acetate for 3-5 times, and concentrating the organic phase by rotary evaporation at 48° C. until there is no ethyl acetate taste, to obtain a fermentation broth extract; (5) The fermentation broth extract in step (4) is separated by column chromatography and monitored by GC-MS, the column chromatography eluates are combined to obtain the target segment, and the target segment is separated and purified by semi-preparative liquid phase to obtain the cyclic dipeptide latent aroma component.

4. The method for preparing the cyclic dipeptide latent aroma component according to claim 3, characterized in that: The Bacillus velezensis is deposited in the Belgian BCCM Collection Center with the deposit number of Bacillus velezensisLMG 22478.

5. The method for preparing the cyclic dipeptide latent aroma component according to claim 3, characterized in that: The preparation method of the LB liquid culture medium is as follows: 2-20 g of peptone, 2-20 g of yeast powder, 2-20 g of NaCl, and 500-2000 mL of water, and the mixture is sterilized at 100-135° C. for 10-50 min after dissolution; or the preparation method of the LB liquid culture medium is as follows: 5 g of peptone, 5 g of yeast powder, 10 g of NaCl, and 1000 mL of water, and the mixture is sterilized at 121° C. for 20 min after dissolution.

6. The method for preparing the cyclic dipeptide latent aroma component according to claim 3, characterized in that: In the step (2), the amount of seed liquid added is 1% to 40%, the culture temperature is 25 to 36° C., the culture time is 6 to 96 hours, and the shaking speed is 60 to 260 rpm; or the amount of seed liquid added is 10%, the culture temperature is 32° C., the culture time is 24 hours, and the shaking speed is 140 rpm.

7. The method for preparing the cyclic dipeptide latent aroma component according to claim 3, characterized in that: In the step (5), the column chromatography is Sephadex LH-20 gel column chromatography, the elution solvent is CH2Cl2:MeOH=1:1, and the target segment is the 9th to 11th segments; In the step (5), the semi-preparative liquid phase elution solvent is MeOH:H2O, the solvent ratio is 15:85 to 55:45, and the detection wavelength is 210 nm; or the solvent ratio is 35:

65.

8. A flavor for tobacco products, comprising one or more of the bacterial fermentation broth, fermentation broth biological flavor, fermentation broth extract or cyclic dipeptide latent aroma component as claimed in claim 3.

9. The tobacco product flavor according to claim 8, characterized in that The amount of the cyclic dipeptide latent aroma component applied in tobacco products is 0.01% to 1%, and the amount of the fermentation liquid biological flavor applied in tobacco products is 0.1% to 20%; or the amount of the cyclic dipeptide latent aroma component applied in tobacco products is 0.5%, the amount of the fermentation liquid biological flavor applied in cigarette products is 1.0%, and the amount applied in tobacco leaves is 2.0%.

10. Use of the tobacco product flavoring agent according to claim 8 or 9 in tobacco products; The tobacco product is any one or more of cigarette products, flue-cured tobacco leaves, reconstituted tobacco leaves, cigar tobacco leaves and electronic cigarette liquid; The applications include application as a flavor in a finished product or application as a fermentation medium in a raw material fermentation process.

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