Enterobacter hallii C4 and its application

Abstract

The present application relates to the field of microbial technology, and specifically to Enterobacter hormaechei C4 and its applications. Enterobacter hormaechei C4 is deposited at the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen Road West, Chaoyang District, Beijing, on January 13, 2023, with the deposit number CGMCC No. 26480. The Enterobacter hormaechei C4 provided in the present application can utilize cellulose, starch, and other components in tobacco during the fermentation process to produce more flavor substances, thereby improving the aroma quality, aroma volume, concentration, stimulation, aftertaste, and sweetness of the tobacco, giving the tobacco better sensory quality.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to Enterobacter hallii C4 and its application. Background Art

[0002] Common methods for improving tobacco quality include soil improvement, breeding high-quality varieties, improving cultivation techniques, and optimizing processing technology. In recent years, the use of microorganisms to enhance tobacco quality has become a new and important research direction in the tobacco industry. Currently, microorganisms such as Bacillus subtilis, Bacillus amyloliquefaciens, and Saccharomyces cerevisiae are being used to improve tobacco quality through fermentation.

[0003] Enterobacter hallii is a bacterium that has the function of catalyzing the degradation of flavor precursors. It can degrade substances such as carotene, carotenoids and polysaccharides, thereby enhancing flavor and improving quality. However, there are currently few reports on the application of Enterobacter hallii to improve tobacco quality. The prior art CN117126767B discloses that Enterobacter hallii HDC12 can increase the biomass of tobacco, but has not studied whether it can improve the sensory quality of tobacco; the prior art CN111254092B discloses that Enterobacter hallii Y399, Bacillus velezensis YBN3 and Bacillus saltitudinis Y120 are prepared into a bacterial agent in proportion, which can improve the sensory quality of tobacco through fermentation. However, this technology uses a mixture of multiple bacteria to achieve the effect of improving the sensory quality of tobacco, and it is also clearly recorded in paragraph

[0037] that "when Enterobacter hallii Y399 acts alone on tobacco raw materials, its main function is classified as significantly improving oral comfort." It can be seen from this that Enterobacter hallii Y399 cannot improve the comprehensive sensory quality of tobacco; Wang Wenting et al. reported that Enterobacter hallii F8-1 can improve the sensory quality of tobacco through fermentation, but the inoculation amount is relatively high.

[0004] Therefore, it is necessary to strengthen the selection and breeding of Enterobacter hallii strains to solve the current problem of scarcity of Enterobacter hallii strains that can improve the sensory quality of tobacco through fermentation. Summary of the Invention

[0005] The problem that the present application aims to solve is to provide Enterobacter hallii C4, which can utilize cellulose, starch and other components in tobacco during the fermentation process to produce more flavor substances, thereby improving the aroma quality, aroma volume, concentration, stimulation, aftertaste and sweetness of tobacco, making the tobacco have better sensory quality.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] On the one hand, the present application provides Enterobacter hormaechei C4, characterized in that the Enterobacter hormaechei C4 is deposited in the General Microbiology Center of the China Culture Collection Administration Committee, the deposit address is No. 3, No. 1, Beichen Road West, Chaoyang District, Beijing, the deposit date is January 13, 2023, and the deposit number is CGMCC No. 26480.

[0008] The source of the Enterobacter hallii C4 is from the surface of tobacco leaves of Yunyan 87 tobacco, and is isolated and screened by plate streak culture method. The sampling site is the tobacco plantation in Huidong County, Liangshan Prefecture, Sichuan Province.

[0009] This application identifies Enterobacter hallii C4 from the perspective of morphology and molecular biology:

[0010] Morphological identification: The colonies of Enterobacter hallii C4 are milky white, translucent, with a smooth and moist surface, and Gram staining is red, indicating that they are Gram-negative bacteria.

[0011] Molecular biological identification: The nucleotide sequence of the 16s rDNA fragment of Enterobacter hormaechei C4 is shown in SEQ ID NO. 1, which is about 1500 bp in size and has a homology of 99.71% with that of Enterobacter hormaechei subsp. xiangfangensis.

[0012] In one aspect, the present application provides the use of the above-mentioned Enterobacter hallii C4 in producing at least one of cellulase or amylase.

[0013] In one aspect, the present application provides a bacterial agent containing the above-mentioned Enterobacter hallii C4, wherein the bacterial agent includes a freeze-dried powder.

[0014] In one aspect, the present application provides the use of the above-mentioned Enterobacter hallii C4 or the above-mentioned bacterial agent in improving tobacco quality, comprising the following steps:

[0015] The tobacco is dried to obtain dried tobacco; the Enterobacter hallii C4 or the bacterial agent is diluted to obtain a diluted bacterial solution; and the diluted bacterial solution is inoculated into the dried tobacco leaves for fermentation to obtain improved tobacco.

[0016] In some embodiments, the tobacco comprises cloud smoke 87 .

[0017] In some embodiments, the moisture content of the oven-dried tobacco leaves is 8% to 12%.

[0018] In some embodiments, the bacterial content of the diluted bacterial solution is 1.25×1010 ~1.75×10 10 CFU / mL.

[0019] In some embodiments, the weight ratio of the diluted bacterial solution to the dried tobacco leaves is 1:(50-150).

[0020] In some embodiments, the tobacco qualities include aroma quality, aroma quantity, off-flavor, strength, stimulation, aftertaste, and sweetness.

[0021] This application has the following beneficial effects:

[0022] (1) The Enterobacter hallii C4 provided in the present application has the function of producing cellulase and amylase, and can effectively utilize the cellulose and starch of tobacco during the fermentation process to grow and reproduce, and produce some flavor substances.

[0023] (2) The Enterobacter hallii C4 provided in this application can effectively improve the sensory quality of tobacco during the fermentation process, thereby obtaining better quality cigarettes.

[0024] (3) The fermentation performance of the Enterobacter hallii C4 provided in this application is excellent, and only a small amount of use is required to improve the quality of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present disclosure, the following briefly introduces the drawings required for use in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure.

[0026] Figure 1 The morphological characteristics of Enterobacter hallii C4 are shown in Figure 2 (a. Partial magnification of colony plate growth; b. Gram staining);

[0027] Figure 2 This is the electrophoresis detection result of the PCR amplification product of Enterobacter hallii C4 (lane M is 2000bp Maker);

[0028] Figure 3 It is the closest strain sequence to Enterobacter hallii C4;

[0029] Figure 4 This is the glucose standard curve of Enterobacter hallii C4. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in some embodiments of the present disclosure. Obviously, the embodiments described are only some of the embodiments of the present disclosure, not all of them. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present disclosure.

[0031] Example 1: Screening of C4 strain

[0032] S1. Weigh 10 g of Yunyan 87 tobacco leaves from the tobacco plantation in Huidong County, Liangshan Prefecture, Sichuan Province, cut them into pieces, mellowed them, and added them to 100 mL of sterile water. Oscillate them at 150 rpm for 30 min at 30°C and let them stand for 10 min. Take the supernatant and dilute it to 10 by 10-fold dilution. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 5 dilution gradients, 10 mL each;

[0033] S2. Each of the dilution gradient solutions was evenly coated on a beef extract peptone solid plate, with 1 mL of each dilution gradient solution coated on 3 plates;

[0034] S3. Place each of the plates upside down in a constant temperature incubator and incubate at 37°C for 24 hours, then collect single colonies with different morphologies and colors;

[0035] S4. Inoculate single colonies of different morphologies and colors onto solid plates of new beef extract peptone medium and repeat this step three times to obtain different single pure strains;

[0036] S5. Each of the different individual pure strains was inoculated into a flask containing 200 mL of beef extract peptone liquid culture medium, and cultured at 37°C with shaking at 200 rpm for 24 h to obtain several purified bacterial solutions;

[0037] S6. Conduct preliminary tobacco fermentation experiments on the purified bacterial liquids to screen a strain that can improve the overall sensory quality of tobacco, and name it C4.

[0038] Example 2 Identification of strain C4

[0039] (1) Morphological identification: The colonies of strain C4 are milky white, translucent, with a smooth and moist surface, and Gram staining is red, belonging to Gram-negative bacteria ( Figure 1 ).

[0040] (2) Molecular biological identification:

[0041] The 16S rDNA fragment of strain C4 was amplified, and the amplified product was subjected to gene sequencing and agarose gel electrophoresis. The gene sequencing result is shown in SEQ ID NO.1. The gel electrophoresis result showed that the size of the 16S rDNA fragment of strain C4 was about 1500 bp ( Figure 2 ).

[0042] The sequencing results of the 16S rDNA fragment amplification products of strain C4 were compared with the known sequences in GenBank using BLAST, and the sequences of the strains with the closest genetic relationship were downloaded ( Figure 3 ), in the homology comparison results of NCBI, strain C4 is most closely related to E. hormaechei subsp. xiangfangensis, with a percentage homology of 99.71%.

[0043] According to the results of molecular biological identification, strain C4 was determined to be Enterobacter hormaechei.

[0044] Example 3: Preparation of Enterobacter hallii C4 bacterial solution and sterile fermentation broth

[0045] S21. Enterobacter hallii C4 was inoculated into beef extract peptone broth and incubated at 37°C on a shaker at 200 rpm for 24 h.

[0046] S22. 2 mL of the activated bacterial solution was inoculated into a new beef extract peptone liquid medium and cultured on a 37°C constant temperature shaker at 200 rpm for 24 h to obtain the bacterial solution;

[0047] S23. Place part of the bacterial solution in a low-temperature high-speed centrifuge and centrifuge at 3500 r / min for 10 min. The supernatant is aspirated and filtered using a 0.22 μm aqueous filter to obtain the sterile fermentation solution. The bacterial count in the sterile fermentation solution is 5×10 10 ~7×10 10 .

[0048] Example 4: Preparation of Enterobacter hallii C4 bacterial agent

[0049] S31. Enterobacter hallii C4 was inoculated into activation medium under sterile conditions and incubated on a 37°C shaker at 160 rpm for 24 h. Colony growth was observed to ensure successful activation.

[0050] S32. The strain was expanded using a 50 L fermentor, with the strain added at 5%; the medium composition consisted of 10 g / L NaCl, 0.4 g / L MgSO4, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L glucose. After adding the strain, the fermentor temperature was set at 37 ° C, the speed was 160 rpm, the ventilation volume was 5 L / min, and the pH was 6.0. After 48 h of incubation, the culture was centrifuged at 14,000 rpm using a tubular centrifuge, and the separated cells were collected;

[0051] S33. 40% trehalose was added to the bacterial cells as a freeze-drying protective agent, mixed evenly, and then dried for 48 h using a vacuum freeze-drying method with a vacuum degree of 15 Pa, a freezing temperature of -65°C, and a heating temperature of 40°C. The viable bacterial count of the obtained bacterial powder was 6.2 × 10 11 CFU / g of the bacterial agent;

[0052] S34. Package the bacterial agent using sterile packaging materials, and indicate the production date, shelf life and other information. Store the packaged bacterial agent in a cool, dry place away from light.

[0053] Example 5: Determination of cellulase activity produced by Enterobacter hallii C4

[0054] (1) Draw a glucose standard curve

[0055] Take 7 25mL graduated test tubes, add 0mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL, and 2.5mL of 1.0mg / mL glucose standard solution, respectively, then add distilled water to make up the solution to 3mL, then add 4.5mL DNS reagent, mix well and heat in a boiling water bath for 5min, take out the test tubes after heating and immediately cool them with cold water, then add distilled water to make up the volume to 25mL and shake well, then measure the absorbance A at 540nm with a 10mm cuvette, draw a glucose standard curve (y=0.3077x-0.0119, R2=0.9993) with absorbance as ordinate and glucose content as abscissa ( Figure 4 ).

[0056] (2) Sample determination

[0057] Take four 25 mL graduated test tubes, add 50 ± 0.5 mg of filter paper (1 cm × 6 cm) to each, select three test tubes, number them in sequence and add 0.5 mL of the sterile fermentation broth prepared in Example 3 to each of them as the experimental group, add 0.5 mL of distilled water to the remaining test tube as the control group, then add 1 mL of citric acid-sodium citrate buffer to each of the four test tubes, keep the temperature at 50 ° C for 60 min, add 3 mL of DNS reagent, then add 0.5 mL of the sterile fermentation broth prepared in Example 3 to each of the experimental group test tubes, add 0.5 mL of distilled water to each of the control group test tubes, mix all the test tubes and heat them in a boiling water bath for 5 min. After heating, take out the test tubes and immediately cool them with cold water, then add distilled water to make the volume 25 mL, and then measure the absorbance with a 10 mm cuvette at a wavelength of 540 nm.

[0058] (3) Calculation of enzyme activity

[0059] Definition of enzyme activity unit: Using filter paper as substrate, under certain reaction conditions (pH 4.8, 50℃, constant temperature for 1h), 1mL of cellulase solution in the hydrolysis reaction catalyzes cellulose to produce 1μg of glucose for 1min, which is one enzyme activity unit, expressed in U / mL.

[0060] Calculation of cellulase activity:

[0061]

[0062] X: enzyme activity, unit is U / mL;

[0063] m: The amount of glucose obtained from the glucose standard curve, in mg;

[0064] n: total dilution multiple of enzyme solution;

[0065] t: reaction time, in min;

[0066] V: sample volume, in mL.

[0067] After calculation, the cellulase production activity of Enterobacter hallii C4 was obtained as shown in Table 1.

[0068] Table 1 Cellulase activity of Enterobacter hallii

[0069]

[0070] Example 6: Amylase activity assay

[0071] (1) Determination of sample enzyme concentration (GB 1886.174-2016)

[0072] The sterile fermentation liquid obtained by embodiment 3 is diluted and placed in 70 ℃ of water baths with constant temperature and preheated, then draw 20mL soluble starch and be dissolved in 4 test tubes, add 5mL phosphate buffer and shake up, be placed in 60 ℃ of water baths with constant temperature and preheat 8min after, add the sterile fermentation liquid of 1mL preheated, start timing immediately, shake up and accurately react 5min.After reaction is completed, draw 1mL reaction solution with liquid-transfer gun immediately, add 3 test tubes that are filled with 0.5mL hydrochloric acid solution and 5mL dilute iodine solution in advance and carry out numbering as experimental group, add 1mL distilled water as control group in 1 test tube that is filled with 0.5mL hydrochloric acid solution and 5mL dilute iodine solution simultaneously.By experimental group and control group under 660nm wavelength, rapidly measure its habit of loneliness (A) with ultraviolet spectrophotometer.According to absorbance, look up table appendix B, obtain test enzyme liquid concentration.

[0073] (2) Calculation of enzyme activity

[0074] Enzyme activity is expressed as 1g solid enzyme powder or 1mL liquid enzyme, at 60℃ (70℃ for thermostable enzyme), pH 6.0, liquefies 1g soluble starch in 1h, which is 1 unit of enzyme activity, expressed as U / g (U / mL).

[0075] Calculation of amylase activity:

[0076] X=c×n

[0077] X: enzyme activity, unit: U / mL;

[0078] c: test enzyme solution concentration, u / mL;

[0079] n: sample dilution factor.

[0080] After calculation, the amylase production activity of Enterobacter hallii C4 was obtained as shown in Table 2.

[0081] Table 2 Amylase activity of Enterobacter hallii

[0082]

[0083] Example 7: Tobacco quality improvement effect test of Enterobacter hallii C4

[0084] S41. Select Yunyan 87 tobacco YQ2 grade tobacco leaves for drying until the moisture content is 8% to 12%;

[0085] S42. The bacterial solution prepared in Example 3 was diluted 4 times with distilled water to obtain a bacterial content of 1.25×10 10 ~1.75×10 10 CFU / mL of diluted bacterial solution;

[0086] S43. Different weights of the diluted bacterial solution were added to a portion of the dried tobacco leaves. The mixture was then mixed to form the experimental groups. The weight ratio of the diluted bacterial solution to tobacco leaves in experimental group 1 was 1:50, the weight ratio of the diluted bacterial solution to tobacco leaves in experimental group 2 was 1:100, and the weight ratio of the diluted bacterial solution to tobacco leaves in experimental group 3 was 1:150. Another portion of the dried tobacco leaves was not added with the diluted bacterial solution and served as the control group. The experimental and control groups were fermented simultaneously at a temperature of 25°C and a humidity of 65% for 3 months.

[0087] S44. The main chemical components of the fermented tobacco leaves were tested, with the test results shown in Table 3. The fermented tobacco leaves were also made into cigarettes, and five professional smokers conducted a sensory evaluation using the "Sensory Evaluation Method for Chinese Cigarette Styles" (YC / T 497-2014). The average evaluation results were calculated, and the evaluation results are shown in Table 4.

[0088] Table 3 Main chemical composition test results

[0089]

[0090] Table 4 Sensory evaluation table

[0091] Group Fragrance Aroma volume Miscellaneous gases concentration Stimulate Aftertaste sweetness Score control group 5.00 5.00 5.00 5.50 5.00 5.00 4.85 35.35 Experimental Group 1 5.50 5.50 5.50 6.00 5.50 5.50 5.00 38.50 Experimental Group 2 5.75 5.58 5.58 5.75 5.54 5.45 5.67 39.32 Experimental Group 3 5.50 5.50 5.20 6.20 5.10 5.10 5.00 37.60

[0092] The results showed that the experimental group had lower levels of total alkaloids, total sugars, total nitrogen, and starch than the control group, while higher levels of reducing sugars and neophytodienes. This indicates that during the fermentation process, the Enterobacter C4 bacterial solution was able to fully utilize the main chemical components in tobacco, thereby producing more flavor substances. All sensory scores in the experimental group were higher than those in the control group, indicating that the Enterobacter C4 bacterial solution can improve the aroma quality, aroma volume, concentration, stimulation, aftertaste, and sweetness of tobacco through fermentation, and can also bring a unique aroma to tobacco.

[0093] Example 8: Tobacco Quality Improvement Effect Test of Enterobacter hallii C4 Inoculum

[0094] S51 select Yunyan 87 tobacco YQ2 grade tobacco leaves are dried until the moisture content is 8% to 12%;

[0095] S52. The bacterial agent prepared in Example 4 was dissolved in a ratio of bacterial agent mass: water mass = 1:1000 to obtain a dissolved bacterial solution;

[0096] S53. The dissolved bacterial solution was diluted 4-fold with distilled water to obtain a diluted bacterial solution;

[0097] S54. Add 4% of the diluted bacterial solution by weight of the tobacco leaves to a portion of the dried tobacco leaves, mix well, and serve as the experimental group. The other portion of the dried tobacco leaves was not added with the diluted bacterial solution and served as the control group. Both the experimental and control groups were fermented at a temperature of 25°C and a humidity of 65% for 3 months.

[0098] S55. The main chemical components of the fermented tobacco leaves were tested. The test results are shown in Table 5. The fermented tobacco leaves were also made into cigarettes. Five professional smokers conducted a sensory evaluation using the "Sensory Evaluation Method for Chinese Cigarette Style" (YC / T 497-2014). The average evaluation results were calculated and shown in Table 6.

[0099] Table 5 Main chemical composition test results

[0100]

[0101] Table 6 Sensory evaluation table

[0102] Group Fragrance Aroma volume Miscellaneous gases concentration Stimulate Aftertaste sweetness Score control group 5.25 5.50 5.00 5.85 5.00 5.50 5.00 37.10 Experimental group 6.00 5.50 5.50 5.65 5.00 5.75 5.80 39.20

[0103] The results showed that the experimental group had lower levels of total alkaloids, total sugars, total nitrogen, starch, and neophytadiene than the control group, while higher levels of reducing sugars. This indicates that during the fermentation process, the inoculum of Enterobacter hallii C4 was able to fully utilize the main chemical components in tobacco, thereby producing more flavor substances. The experimental group also achieved higher sensory scores than the control group, indicating that the inoculum of Enterobacter hallii C4 can improve the aroma quality, concentration, pungency, aftertaste, and sweetness of tobacco through fermentation without affecting the aroma volume, and can also bring a unique aroma to tobacco.

[0104] In summary, the Enterobacter hallii C4 provided in this application has the ability to produce cellulase and amylase. It can utilize cellulose, starch, and other components in tobacco during fermentation to produce more flavor substances, thereby improving the sensory qualities of tobacco. Using the method of this application to improve tobacco quality can produce higher-quality tobacco products in a more environmentally friendly manner, providing the tobacco industry with a biological solution that can effectively improve tobacco quality. Through in-depth research and application development in the future, Enterobacter hallii C4 is expected to become a quality-enhancing microorganism for a variety of cash crops, promoting the development of the agricultural economy.

[0105] The foregoing description is merely a preferred embodiment of the present disclosure. It should be understood that the present disclosure is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present disclosure is applicable to various other combinations, modifications, and environments and can be modified within the scope of the concepts described herein through the above teachings or techniques or knowledge in the relevant fields. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present disclosure are intended to be protected by the claims appended hereto.

Claims

1. Enterobacter hormaechei C4, characterized in that The Enterobacter hallii C4 is deposited in the General Microbiology Center of the China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen Road West, Chaoyang District, Beijing, the deposit date is January 13, 2023, and the deposit number is CGMCC No. 26480.

2. Use of the Enterobacter hallii C4 according to claim 1 in producing at least one of cellulase or amylase.

3. The bacterial agent containing the Enterobacter hallii C4 according to claim 1, characterized in that The bacterial agent includes freeze-dried powder.

4. Use of the Enterobacter hallii C4 according to claim 1 or the bacterial agent according to claim 3 in improving tobacco quality, characterized in that: The following steps are involved: drying the tobacco to obtain dried tobacco; Diluting the Enterobacter hallii C4 or the bacterial agent to obtain a diluted bacterial solution; as well as The diluted bacterial solution is inoculated into the dried tobacco leaves for fermentation to obtain improved tobacco.

5. The use according to claim 4, characterized in that The tobacco includes Yunyan 87.

6. The use according to claim 4, characterized in that The moisture content of the dried tobacco leaves is 8% to 12%.

7. The use according to claim 4, characterized in that The bacterial content of the diluted bacterial solution was 1.25×10 10 ~1.75×10 10 CFU / mL.

8. The use according to claim 4, characterized in that The weight ratio of the diluted bacterial solution to the dried tobacco leaves is 1:(50-150).

9. The use according to claim 4, characterized in that The tobacco quality includes aroma quality, aroma quantity, off-flavor, concentration, stimulation, aftertaste and sweetness.

Images