Enterobacter ludwigii YS3 and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, particularly relates to enterobacter ludwigii YS3, and further relates to application of the enterobacter ludwigii YS3 in fermentation of natural essence. According to the invention, tobacco rhizosphere soil is taken as a research object, and a dominant strain YS3 is obtained through separation. Through basic characteristics, 16S rRNA sequencing and phylogenetic analysis, the enterobacter ludwigii is identified to be enterobacter ludwigii, and the preservation number is GDMCC No: 66083. Volatile components in fermentation products of the strain are systematically analyzed through a GC-MS (Gas Chromatography-Mass Spectrometer) technology, the flavor metabolism characteristics of the strain are disclosed by combining sensory evaluation and enzyme activity determination, and a new way is provided for enriching tobacco flavor.

Description

Technical Field The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Enterobacter ludwigii YS3, and also relates to application of the Enterobacter ludwigii in fermenting natural flavors. Background Art In recent years, with increasing consumer demand for healthy and natural flavors, the use of natural flavors in the food industry has gradually become mainstream. Compared to traditional synthetic flavors, natural flavors not only offer superior flavor performance but also align with current consumer trends toward green and environmentally friendly practices. Microbial fermentation is a primary source of natural flavors, a process that not only provides natural flavor compounds but also boasts high production efficiency and sustainability.

[0001] Many studies have shown that the fermentation process of microbial metabolites produces rich flavor compounds, such as esters, alcohols, aldehydes and aromatic hydrocarbons. Among them, traditional fermentation microorganisms such as lactic acid bacteria and yeast have been widely used to improve the flavor of foods such as wine, dairy products and fruit juice.

[0002] However, despite the important role these traditional microorganisms play in flavor production, relatively little research has been conducted on the application of soil-derived microorganisms in natural flavor production, and the potential of soil microorganisms in natural flavor production has not yet been fully explored. The soil harbors a large number of microorganisms with unique metabolic capabilities. They can synthesize a variety of flavor substances by metabolizing organic matter. The application of these substances in food flavors has not received sufficient attention. Studies have shown that soil microorganisms can utilize different carbon sources to produce a variety of chemicals with unique flavors, thus providing new ideas and approaches for the production of natural flavors. Therefore, exploring the potential of soil microorganisms in natural flavor production will not only enrich the application areas of microbial metabolites, but also help improve the diversity and quality of flavor production.

[0003] Enterobacter ludwigii ( Enterobacter ludwigii Enterobacter ludwigii, a Gram-negative bacterium widely found in soil, water, and the rhizosphere, exhibits metabolic diversity, producing a wide range of compounds through glycolysis, amino acid conversion, and secondary metabolic pathways. For example, Enterobacter ludwigii exhibits strong acid production in food fermentation, and the organic acids (such as lactic acid and acetic acid) and esters it produces can significantly improve product flavor. Furthermore, Enterobacter ludwigii has been reported to exhibit metabolic diversity in bioremediation, plant growth promotion, and food fermentation. However, its systematic application in flavor and fragrance production has not yet been reported.

[0004] Hebei Province, as my country's primary sweet corn producer (accounting for 23% of national annual output), chose sweet corn as a fermentation base to avoid quality deterioration caused by long-distance transportation while aligning with local policies promoting high-value agricultural product utilization. Its large-scale cultivation system ensures a stable and low-cost supply of raw materials, achieving a deep integration of technological adaptability and regional industrial characteristics. Furthermore, sweet corn's sugar content (particularly sucrose, glucose, and fructose) is significantly higher than that of regular corn (approximately 10%-15% of dry weight), providing an ample carbon source for fermentation by microorganisms such as yeast and bacteria. Sweet corn's inherent sweet and neutral flavor reduces the impact of aromatic compounds produced during fermentation. Summary of the Invention This application isolated a dominant strain, YS3, from tobacco rhizosphere soil. It was identified as Enterobacter ludwigii through basic characterization, 16S rRNA sequencing, and phylogenetic analysis. GC-MS analysis of the volatile components of its fermentation products revealed the flavor metabolism characteristics of this strain, combined with sensory evaluation and enzyme activity assays, and provided a new approach to enrich tobacco flavor.

[0005] The purpose of the present invention can be achieved through the following technical solutions: One of the technical solutions provided by the present invention is a strain of Enterobacter Ludwigii, specifically Enterobacter Ludwigii ( Enterobacter ludwigii )YS3, the strain was deposited in Guangdong Provincial Microbial Culture Collection on April 1, 2025, with the deposit address at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, zip code 510075, and the deposit number is GDMCC No: 66083.

[0006] The present invention provides Enterobacter Ludwigii YS3, which is a dominant strain isolated and cultured from a soil sample in a tobacco-growing area in Shaoguan, Guangdong Province. It is identified as Enterobacter Ludwigii ( Enterobacter ludwigii ).

[0007] The strain Enterobacter ludwigii YS3 has the following characteristics: (1) Colony morphology: The colonies are usually round, with neat edges and smooth surfaces. They are milky white to off-white, moist, and slightly sticky. The diameter is about 1-3 mm. They grow rapidly and can form visible colonies within 24 hours at 37°C.

[0008] (2) Bacterial morphology: Gram staining of this strain shows red, and it is a Gram-negative short rod, arranged singly or in pairs.

[0009] (3) Physical and chemical properties: Oxygen requirement: facultative anaerobe; Spore formation: does not form spores; Temperature adaptability range: 15-42°C, optimum temperature is 30-37°C; pH adaptability range: can grow in the pH range of 5.5-8.5; (4) Amylase activity: It has obvious amylase activity (diameter of hydrolysis zone 1.0 cm).

[0010] The second technical solution provided by the present invention is the Enterobacter Ludwigii ( Enterobacter ludwigii ) Application of YS3, especially in the fermentation production of flavors and fragrances; Furthermore, it is used in the fermentation production of flavors and spices using sweet corn as raw material; Furthermore, the method for producing flavors and fragrances by fermentation using sweet corn as raw material is as follows: Fresh sweet corn kernels are crushed and sterilized, then water is added and inoculated with the YS3 strain. Fermentation is carried out at 30±2°C, pH 5.0-6.0, and microaerobicity of 3%-10%. Static fermentation is carried out for 7 days, and flavors and fragrances are extracted from the fermentation broth. In the early stage (1-3 days), the sensory properties of the fermented product are mainly fruity and sweet. In the middle stage (4-6 days), the strongest ester aroma is accompanied by mild fruity and sweet aromas. In the late stage (7 days), the aroma changes to lees. The fermentation product in the middle stage is preferably used for flavor and fragrance extraction. Furthermore, after the fermentation is completed, the fermentation liquid is taken and the flavor substances are extracted by steam distillation, continuous phase change extraction, or liquid-liquid extraction; preferably, liquid-liquid extraction is performed with 10% ethanol, and then filtered to obtain flavors and fragrances; Solid-phase microextraction combined with gas chromatography-mass spectrometry was used to detect the volatile components in the fermentation products of sweet corn produced by the YS3 strain. YS3 produced 78 volatile compounds, among which ethanol (2.10±0.75 μg / mL), isopentanol (0.08±0.03 μg / mL), glacial acetic acid (1.68±0.64 μg / mL), 3-hydroxy-2-butanone (1.01±0.31 μg / mL) and gamma-nonalactone (0.74±0.11 μg / mL) were the key flavor substances, presenting a complex aroma of ester and fruit.

[0011] The third technical solution provided by the present invention is the application of the flavors and spices produced in the second technical solution, especially in improving the flavor of tobacco leaves; Furthermore, the flavor of tobacco leaves can be improved by injecting the flavors and fragrances produced by the second technical solution into the filter of finished cigarettes or spraying them onto tobacco leaves.

[0012] Beneficial effects: The present invention isolates a strain of Enterobacter Ludwigii ( Enterobacter ludwigii) YS3. The aroma of its fermentation product initially exhibited fruity, estery, and sweet aromas, reaching a peak in the middle phase and shifting to a lees-like aroma in the later stages. Seventy volatile compounds were detected in the YS3 fermentation product, with key flavor compounds including ethanol (2.10±0.75 μg / mL), isopentanol (0.08±0.03 μg / mL), glacial acetic acid (1.68±0.64 μg / mL), 3-hydroxy-2-butanone (1.01±0.31 μg / mL), and gamma-nonalactone (0.74±0.11 μg / mL). Furthermore, 30 of these volatile compounds were detected only in the YS3 fermentation group, including 2,3-butanediol, glacial acetic acid, hexyl acetate, ethyl 3-hydroxybutyrate, ethyl DL-2-hydroxy-4-methylvalerate, and dimethyl phthalate. This suggests that the YS3 microorganism plays a key role in the formation of fermentation flavor compounds. YS3 forms a complex flavor characteristic of ester, fruity and sweet aroma through the synergistic action of specific metabolites. Its flavor intensity and complexity are obvious, showing its application potential as a fermentation strain for improving tobacco flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Strain screening process.

[0014] Figure 2 This is the Gram staining result.

[0015] Figure 3 Phylogenetic tree for YS3 strain identification.

[0016] Figure 4 Growth curve and spore formation test of strain YS3 at different temperatures.

[0017] Figure 5 Growth curves of strain YS3 at different pH levels.

[0018] Figure 6 Starch hydrolysis plate. Specific implementation plan The present invention will be further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and appropriate ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications made to the material composition and dosage in these embodiments also fall within the scope of protection of the present invention.

[0019] Example 1: Acquisition of YS3 strains Soil sampling was conducted in tobacco-growing areas in Shaoguan, Guangdong Province. Soil samples were collected using a five-point sampling method, with surface debris, such as fallen branches and leaves, removed before sampling. Surface soil was sampled from a depth of 0-20 cm, centered around the plant, ensuring uniformity between sampling points. Finally, soil samples from the five sampling points were mixed to create a final composite sample.

[0020] The soil sample was mixed with physiological saline, chopped and filtered to obtain a filtrate. The filtrate was inoculated into a conical flask containing 100 mL of YPD liquid medium and incubated at 30°C for 24 hours. The culture medium was found to be significantly turbid after incubation. After bacterial growth, the inoculation liquid was streaked onto YPD solid medium using an inoculation loop and incubated at 30°C until a single colony appeared. The specific process is as follows: Figure 1 As shown. A single colony was selected and inoculated into liquid culture medium. When the bacterial liquid reached an OD600 value of 0.5, it was used as seed liquid for subsequent fermentation experiments (using sweet corn fermentation medium). After fermentation, sensory (olfactory) evaluation was performed for initial screening, followed by GC-MS analysis of specific substance types and contents. Strains with high ester content were selected. This screening yielded a target strain, designated YS3.

[0021] At the same time, the bacterial liquid of a single colony was taken for Gram staining. Using an optical microscope, it was found that the Gram staining of the strain showed red, which was a Gram-negative short rod, arranged singly or in pairs ( Figure 2 ).

[0022] Example 2: 16S rRNA sequence determination A standardized process was used to extract DNA, and PCR amplification was performed to ensure the accuracy of the experimental data. As the most commonly used "molecular clock" in bacterial systematic taxonomy studies, 16S rRNA sequences contain rich evolutionary information and can provide a reliable basis for the classification and identification of bacterial species. During the comparison process, the results showed that strains such as YS3 have unique taxonomic positions. These strains are located in a specific branch that contains several strains of the genus Enterobacter, and these strains have strong bootstrap support rates. Bootstrap support is an important indicator for evaluating the credibility of branches in phylogenetic analysis. A higher bootstrap support rate means that the branch is more stable and reliable on the evolutionary tree. Further analysis showed that strains such as YS3 belong to Enterobacter Ludwigii ( Enterobacter ludwigii ) This particular strain. Through detailed comparison of strains such as YS3 with the corresponding strains, it was found that the bootstrap support rate between them was greater than 70 (such as Figure 3 shown).

[0023] Therefore, YS3 was identified as Enterobacter ludwigii ( Enterobacter ludwigii ), named Enterobacter Ludwigii ( Enterobacter ludwigii) YS3, and was deposited in Guangdong Provincial Microbiological Culture Collection on April 1, 2025, with the deposit number GDMCC No: 66083.

[0024] Example 3 Optimum Growth Temperature and pH (1) Experimental methods Spore resistance test: When strain YS3 reaches the logarithmic growth phase, heat at 80°C for 10 min. Take 0.5 mL of the bacterial solution and inoculate it into 19.5 mL of YPD medium. Cultivate at 30°C under microaerophilic conditions. Measure the absorbance of the bacterial solution at 600 nm every 12 h using a microplate reader.

[0025] Optimum growth temperature experiment: When strain YS3 reached the logarithmic growth phase, 0.5 mL of the bacterial solution was inoculated into 19.5 mL of YPD medium. The culture was carried out under microaerobic conditions and different temperature conditions (15°C, 30°C, 37°C, and 42°C). The absorbance of the bacterial solution at 600 nm was measured every 12 h using a microplate reader.

[0026] Optimal growth pH experiment: When strain YS3 reached the logarithmic growth phase, 0.5 mL of the bacterial culture was inoculated into 19.5 mL of YPD medium at different pH values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0). The cultures were incubated at 30°C under microaerophilic conditions. The absorbance at 600 nm was measured every 12 h using a microplate reader.

[0027] (2) Experimental results It is difficult to inactivate spores at high temperatures. The presence of spores can be determined by observing whether microorganisms grow after heating the bacterial solution at high temperatures. Figure 4 As shown, no obvious microbial growth was found in strain YS3 after being heated at 80°C for 10 min, indicating that strain YS3 failed to produce spores.

[0028] At the same time, by culturing the strains at different temperatures, it was found that strain YS3 can grow at 15-42°C, and the optimal growth temperature is 30-37°C.

[0029] like Figure 5 As shown, by culturing the strains in culture media with different pH values, it was found that strain YS3 could grow in the pH range of 5.5-8.5.

[0030] Example 4: Amylase activity YS3 was cultured in YPD medium. When the strain reached the logarithmic growth phase, the medium was used to dilute the bacterial suspension to an OD600 of 0.1. Subsequently, 10 μL of the bacterial suspension was dripped onto the surface of a starch hydrolysis medium plate. Under sterile conditions, the plate was placed in a 30°C incubator for 72 hours after the droplet dried naturally.

[0031] Determination of amylase activity: Evenly add 1 mL of 5% Lubricant's iodine solution to the surface of the plate for staining. After staining for 5 minutes, rinse the plate with saline and accurately measure the diameter of the clearing zone using the cross-counter method.

[0032] The results are as follows Figure 6 As shown, YS3 exhibited starch hydrolysis ability (hydrolysis zone diameter 1.0 cm), indicating that YS3 possessed an amylase system capable of degrading starch.

[0033] Example 5: Optimization of sweet corn fermentation conditions and sensory evaluation In the sweet corn fermentation experiment, fresh sweet corn kernels were crushed and weighed, with 20 g of each sample weighed. The mixture was bottled, placed in a sterilizer, and steamed at 100°C for 30 minutes. After steaming and cooling, 5 mL of sterile water and 0.4 mL of seed solution (prepared by inoculating frozen glycerol-stocked bacterial suspension into YPD liquid medium and incubating at 30°C for 24 hours) were added to each sample. Three blank control groups were also set up: 20 g of sterilized sweet corn was added with 5 mL of sterile water and 0.4 mL of YPD liquid medium, respectively.

[0034] All samples were fermented at a constant temperature of 30°C, and the sensory evaluation of odor was performed every 24 hours. Five experienced assessors were selected for the sensory evaluation process to ensure the reliability and consistency of the results. To avoid external interference, the samples were placed in transparent, colorless containers to eliminate the potential impact of the container on the odor. The assessors opened the sample container and shook it gently to promote even evaporation of the odor. Each evaluation lasted 2-3 minutes to ensure accuracy and avoid olfactory fatigue. The odor intensity was scored using a 9-point scoring method: weak: 1-3 points; medium: 4-6 points; strong: 7-9 points, and the corresponding score was assigned according to the odor concentration.

[0035] Through a 7-day cultivation experiment, combined with a sensory analysis system, the gas production characteristics and dynamic changes of flavor substances of isolated tobacco soil microorganisms were analyzed.

[0036] The results are shown in Table 1. The test samples did not show obvious gas production during the entire cultivation period. The aroma composition showed a phased change characteristic, among which: Initial stage (day 1): Fruity aroma (6 points), ester aroma (3 points), and sweet aroma (6 points) are the main aromas, which synergistically form a medium-intensity corn complex aroma. Mid-cultivation period (day 4): The ester aroma intensity significantly increased to a peak value (6 points), while the sweet aroma showed its first significant attenuation (4 points); In the late stage of cultivation (7th day), the fruity, ester and sweet aromas all decreased to weaker levels. At the same time, low-intensity wine lees smell (2 points) and off-odor (2 points) were detected, indicating that the direction of microbial metabolism may be transformed into secondary metabolites.

[0037] As shown in Table 1, tobacco soil microorganism YS3 exhibits different aromas at different stages of the fermentation process. In the early stages of fermentation, the fruit aroma score is high due to the high production of fruity substances in the substrate. As fermentation continues, the type and content of the substrate change, and the strain ferments starch to produce large amounts of acids and alcohols. Entering the mid-stage, these newly generated acids and alcohols provide ample substrate for the synthesis of esters. Simultaneously, the number of microorganisms with ester synthase activity increases, and their activity strengthens, further promoting the synthesis of esters. In the late stages of the culture, the intensity of the fruity, ester, and sweet aromas all decreases to lower levels, while low-intensity lees and off-flavors are detected. Based on these performance characteristics, the mid-stage (days 4-6) is clearly the optimal time to end fermentation, and the fermented material should be transferred to the extraction step as soon as possible.

[0038] Table 1 Sensory evaluation of aroma production characteristics of tobacco soil microorganisms at different fermentation time points

[0039] Example 6: GC-MS aroma component analysis To analyze the volatile aroma components during sweet corn fermentation, 6 g of the fermentation sample from day 4 in Example 5 was weighed, 0.5 g of sodium chloride and 1 μL of an internal standard (0.001 mg / mL in 2-octanol) were added, and the mixture was thoroughly mixed. The treated sample was equilibrated in an 80°C incubator for 10 minutes. Headspace extraction was then performed using a conditioned SPME fiber tip for 50 minutes. After extraction, the SPME fiber tip was directly inserted into the GC-MS inlet, and desorption was performed at 250°C for 5 minutes. Chromatographic analysis was performed using an RTX-POLARWAX capillary column (60 m × 250 μm × 0.25 μm), with high-purity helium as the carrier gas at a flow rate of 1 mL / min⁻¹ and a pressure of 112 kPa in splitless mode. Chromatographic conditions: Initial temperature 40°C, hold for 5 min, then increase to 120°C at 3°C ​​min⁻¹, hold for 1 min, then increase to 180°C at 5°C min⁻¹, hold for 1 min, and finally increase to 230°C at 10°C min⁻¹, hold for 5 min. Mass spectrometry conditions: electron impact ionization (EI) source, electron energy 70 eV; ion source temperature 230°C, interface temperature 210°C, quadrupole temperature 150°C; scanning mass range 33–500 m / z.

[0040] The results are shown in Table 2.

[0041] Table 2 GC-MS analysis of characteristic flavors of tobacco soil microorganism YS3 fermentation

[0042] According to the GC-MS analysis data in Table 2, significant differences in the types and contents of volatile components were observed between the fermentation products of tobacco soil microorganism YS3 and the blank control. The YS3 fermentation produced significantly different levels of flavor compounds compared to the blank control, with the total content in the YS3 fermentation group reaching 25.35 μg / mL, compared to only 4.50 μg / mL in the blank control. Of the 124 substances detected, most were found at higher levels in the YS3 fermentation group. Thirty substances were detected only in the YS3 fermentation group, including 2,3-butanediol, glacial acetic acid, hexyl acetate, ethyl 3-hydroxybutyrate, ethyl DL-2-hydroxy-4-methylvalerate, and dimethyl phthalate. This suggests that the YS3 microorganism plays a key role in the formation of fermentation flavor compounds.

[0043] Among the total volatile components, the content of key flavor substances such as alcohols, acids, ketones, and esters in YS3 was significantly higher than that in the blank group, forming a unique fermentation characteristic. The specific flavor differences are as follows: Among alcohol compounds, the types and contents of alcohols in the YS3 fermentation group were significantly higher than those in the blank group. Ethanol, the most abundant alcohol, reached 2.10±0.75 μg / mL, far exceeding the blank group's 0.28±0.03 μg / mL and 7.5 times that of the blank group. This constitutes the fermentation base aroma, a typical product of the microbial glycolysis pathway, indicating that the YS3 microorganism is relatively active in sugar metabolism. 2,3-Butanediol, at a high level of 3.65±1.14 μg / mL, contributes to fruity and sweet aromas. n-Hexanol (0.38±0.05 μg / mL) and 1-octen-3-ol (0.21±0.02 μg / mL) were significantly higher than those in the blank group, imparting a grassy and earthy aroma.

[0044] In terms of phenolic compounds, the contents of 2-methoxy-4-vinylphenol (0.65±0.00 μg / mL) and 2,4-di-tert-butylphenol (0.92±0.17 μg / mL) in the YS3 group were 3.8 times and 4.2 times that of the blank group, respectively, enhancing the eugenol-like and woody smoky flavors.

[0045] Among aldehydes, the contents of trans-2-nonanal (0.29±0.02 μg / mL) and trans-2-decenal (0.33±0.04 μg / mL) in the YS3 group were significantly higher than those in the blank group, enhancing the layering of fat and grass aroma, while benzaldehyde (0.18±0.01 μg / mL) contributed to the almond aroma.

[0046] Among the acidic compounds, glacial acetic acid (1.68±0.64 μg / mL) and isovaleric acid (0.53±0.10 μg / mL) were unique components in the YS3 group, dominating the acidic characteristics and contributing to the cheese aroma. Caprylic acid (0.81±0.22 μg / mL), which was 3.7 times higher than in the blank group, enhanced the fat aroma.

[0047] Among ketones, 3-hydroxy-2-butanone (1.01±0.31 μg / mL) was only present in the YS3 group, contributing to the creamy aroma, while the presence of 2-ethylcyclohexanone (0.16±0.02 μg / mL) enhanced the woody aroma characteristics.

[0048] The most prominent ester flavor was found in GC-MS analysis of the characteristic flavor of tobacco soil microorganism YS3 fermentation. The ester compounds after YS3 fermentation were diverse, with some components showing prominent concentrations, making a crucial contribution to the formation of fermentation aroma. Nine ester compounds were detected, of which hexyl acetate, ethyl 3-hydroxybutyrate, methyl 4-hydroxycinnamate, ethyl DL-2-hydroxy-4-methylvalerate, and dimethyl phthalate were found only in the YS3 fermentation group and were not detected in the blank group. Hexyl acetate has a fruity aroma, adding a refreshing flavor to tobacco. Its content in the YS3 fermentation group was 0.03±0.02 μg / mL. 3-Hydroxybutyrate has a sweet and fruity aroma, with a content of 0.29±0.11 μg / mL. Gamma-nonalactone exudes aromas such as coconut and peach, and its content in the YS3 fermentation group was relatively high at 0.74±0.11 μg / mL, significantly higher than the 0.17±0.04 μg / mL in the blank group. Ethyl palmitate has a faint fruity aroma, with a content of 0.66±0.12 μg / mL in the YS3 fermentation group, far exceeding the 0.12±0.03 μg / mL in the blank group.

[0049] Overall, YS3 formed a complex flavor characteristic of fruity and ester aroma through the synergistic effect of specific metabolites. Its flavor intensity and complexity far exceeded that of the blank group, showing its application potential as a fermentation strain for improving tobacco flavor.

[0050] The YS3 microbial fermentation produces a rich variety of flavor compounds, reflecting its complex and unique metabolic pathways. Multiple pathways, including glycolysis, polyol metabolism, fatty acid metabolism, and aromatic compound metabolism, work synergistically in the YS3 microorganism, resulting in its unique flavor synthesis pattern. Compared to the blank control group, the YS3 microorganism significantly altered the composition and content of flavor compounds, initiating or enhancing specific metabolic pathways to synthesize a large number of volatile compounds, playing a leading role in the formation of fermentation flavor. The YS3 strain is expected to enhance tobacco quality in terms of both aroma and taste. Alcohols and esters impart pleasant fruity and winey aromas to tobacco; aldehydes and ketones add grassy and sweet notes; and acids regulate tobacco acidity and improve taste. These substances work synergistically to contribute to tobacco's superior quality.

[0051] In summary, the present invention isolated a strain of Enterobacter Ludwigii ( Enterobacter ludwigii ) YS3, which has starch hydrolysis ability (amylase activity: hydrolysis zone diameter 1.0 cm). The aroma of its fermentation product initially exhibited fruity (6 points), ester aroma (3 points), and sweet aroma (6 points). The ester aroma peaked in the middle stage (6 points) and later shifted to a lees aroma (4 points). A total of 78 volatile compounds were detected in the YS3 group, with key flavor compounds including ethanol (2.10±0.75 μg / mL), isopentanol (0.08±0.03 μg / mL), glacial acetic acid (1.68±0.64 μg / mL), 3-hydroxy-2-butanone (1.01±0.31 μg / mL), and gamma-nonalactone (0.74±0.11 μg / mL). Its flavor complexity significantly surpassed that of previously reported strains.

[0052] Example 7: Sensory Evaluation of Cigarette Fragrance Extraction was performed using 20% ​​ethanol solution as the extractant, using the fermentation product from day 4 in Example 5 at weight ratios of 1:1, 1:5, and 1:10. The extraction time was 10 minutes, and the sample was filtered through a 0.45μm membrane. A 20μL sample was injected into the filter of a finished cigarette using a syringe, sealed, and allowed to stand for 10 minutes. A control group received the same volume of extractant and subsequent treatment. Seven smokers were selected for smoke evaluation (evaluation criteria see Table 3). Specific results are shown in Table 4.

[0053] Table 3 Evaluation criteria

[0054] Table 4 Sensory evaluation results

[0055] From the smoking evaluation results in Table 4, it can be seen that after adding the tobacco flavoring prepared by the present invention, the aroma characteristics of the cigarette are obvious. Sample 1 can significantly increase the aroma quality and aroma quantity, while increasing the sense of strength, dryness, comfort and aftertaste. The sensory organs such as irritation and miscellaneous smell are not significantly changed. Comprehensive comparison shows that Sample 1 can give the cigarette a special flavor characteristic.

[0056] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A strain of Enterobacter ludwigii, characterized in that Specifically, Enterobacter Ludwigii ( Enterobacter ludwigii )YS3, the deposit number is GDMCC No: 66083.

2. The Enterobacter Ludwigii of claim 1 ( Enterobacter ludwigii ) The application of YS3 is characterized by, It is used in the fermentation production of flavors and fragrances.

3. The use according to claim 2, characterized in that It is used in the production of flavors and spices by fermentation using sweet corn as raw material.

4. The use according to claim 3, characterized in that The method for producing flavors and spices by fermentation using sweet corn as raw material is as follows: Fresh sweet corn kernels were crushed and sterilized, then added with water and inoculated with YS3 strain. The fermentation conditions were 30±2℃, pH 5.0-6.0, microaerobicity 3%-10%, and static fermentation. The fermentation products from different fermentation cycles were selected for the extraction of flavors and fragrances.

5. The use according to claim 4, characterized in that The fermentation product after 4-6 days of fermentation is selected for extraction of flavors and fragrances.

6. The use according to claim 4, characterized in that After the fermentation is completed, the fermentation liquid is taken and the flavor substances are extracted by steam distillation, continuous phase change extraction, or liquid-liquid extraction.

7. The use according to claim 6, characterized in that After the fermentation is completed, the fermentation liquid is taken, liquid-liquid extraction is performed with 10% ethanol, and then filtration is performed to obtain flavors and fragrances.

8. Flavors and spices prepared according to any one of claims 3 to 7.

9. Use of the flavor and fragrance according to claim 8 in improving the flavor of tobacco leaves.

10. The use according to claim 9, characterized in that The flavor of tobacco leaves is improved by injecting the flavorings into the filter of finished cigarettes or spraying them onto tobacco leaves.

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