Enterobacter hormaechei C4 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Enterobacter hormaechei C4 and application thereof, the Enterobacter hormaechei C4 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, Yard 1, West Beichen Road, Chaoyang District, Beijing, the preservation date is January 13, 2023, and the preservation number is CGMCC No.26480. The enterobacter hormaechei C4 provided by the invention can generate more flavor substances by utilizing cellulose, starch and other components in the tobacco in the fermentation process, so that the aroma quality, aroma quantity, concentration, irritation, aftertaste and sweetness of the tobacco are improved, and the tobacco has more excellent 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] China is the world's largest tobacco producer and consumer, and the tobacco industry occupies an important position in the national economy. With the development of the economy, market competition in the tobacco industry has become increasingly fierce. Due to the improvement of consumers' health awareness and changes in taste preferences, the requirements for tobacco quality are also getting higher and higher, and high-quality tobacco products are often more competitive. In order to improve the quality of tobacco, the commonly used methods currently include improving soil, breeding high-quality varieties, improving planting techniques, and optimizing processing technology. In recent years, the use of microorganisms to improve tobacco quality has become a new and important research direction in the tobacco industry. At present, microorganisms such as Bacillus subtilis, Bacillus amyloliquefaciens and Saccharomyces cerevisiae can be 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 achieving the effect of enhancing flavor and improving quality. However, there are currently few reports on the application of Enterobacter hallii to improve tobacco quality. Prior art CN117126767B discloses that Enterobacter holmii HDC12 can increase the biomass of tobacco, but does not study whether it can improve the sensory quality of tobacco; Prior art CN111254092B discloses that Enterobacter holmii Y399, Bacillus velezensis YBN3 and Bacillus saltitudinis Y120 are prepared into a bacterial agent in proportion, and the sensory quality of tobacco can be improved by 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 holmii Y399 acts on tobacco raw materials alone, its main function classification is to significantly improve oral comfort", from which it can be seen that Enterobacter holmii Y399 cannot improve the comprehensive sensory quality of tobacco; Wang Wenting et al. reported that Enterobacter holmii F8-1 can improve the sensory quality of tobacco by fermentation, but the inoculation amount is relatively high.

[0004] Therefore, it is necessary to strengthen the selection and breeding of Enterobacter hollandii strains to solve the current problem of scarce Enterobacter hollandii 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, so that tobacco has 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, the deposit address is No. 3, Yard 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 holmesii C4 is from the surface of tobacco leaves of Yunyan 87 tobacco, which is separated and screened by plate streak separation and culture method. Sampling site: Tobacco planting area of ​​Huidong County, Liangshan Prefecture, Sichuan Province.

[0009] This application identified Enterobacter holmesii 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, which means they are Gram-negative bacteria.

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

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

[0013] On the one hand, the present application provides a bacterial agent containing the above-mentioned Enterobacter hallii C4, wherein the bacterial agent includes a lyophilized powder.

[0014] On the one hand, 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 dried tobacco leaves is 8% to 12%.

[0018] In some embodiments, the bacterial content of the diluted bacterial solution is 1.25×10 10 ~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 holmesii C4 provided in the present application has the function of producing cellulase and amylase, and can effectively utilize the cellulose and starch of tobacco to grow and reproduce during the fermentation process, and produce some flavor substances.

[0023] (2) The Enterobacter holmesii C4 provided in the present 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 holmesii C4 provided in the present 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 drawings required for use in some embodiments of the present disclosure will be briefly introduced below. 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 1 (a. Partial magnification of colony plate growth; b. Gram staining image);

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

[0028] Figure 3 It is the strain sequence that is most closely related to Enterobacter hallii C4;

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

[0030] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0031] Example 1: Screening of C4 strain

[0032] S1. Weigh 10g of Yunyan 87 tobacco leaves from the tobacco plantation in Huidong County, Liangshan Prefecture, Sichuan Province, cut them into pieces, mellowed them, added them into 100mL of sterile water, vibrated them at 150r / min at 30℃ for 30min, and then let them stand for 10min. Take the supernatant and dilute it to 10 by 10-fold dilution method. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 5 dilution gradient solutions, each dilution gradient solution is 10mL;

[0033] S2. Take 1 mL of each dilution gradient solution and evenly spread it on a beef extract peptone solid plate, and spread 3 plates for each dilution gradient solution;

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

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

[0036] S5. Inoculate each of the different individual pure strains into a conical flask containing 200 mL of beef extract peptone liquid culture medium, and culture at 37°C with shaking at 200 r / min for 24 h to obtain a number of purified bacterial liquids;

[0037] S6. The several purified bacterial liquids are respectively subjected to preliminary tobacco fermentation experiments to screen the strain that can improve the overall sensory quality of tobacco, and the strain is named C4.

[0038] Example 2 Identification of strain C4

[0039] (1) Morphological identification: The colonies of strain C4 are milky white, translucent, smooth and moist, and Gram-stained 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 product of strain C4 were compared with the known sequences in GenBank using BLAST, and the sequences of the strains with the closest 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 bacterial liquid and sterile fermentation liquid of Enterobacter hallii C4

[0045] S21. Enterobacter holmesii C4 was inoculated into beef extract peptone liquid culture medium, and the culture was activated and cultured on a constant temperature shaker at 37°C and 200 rpm for 24 h;

[0046] S22. 2 mL of the activated bacterial solution was inoculated into a new beef extract peptone liquid culture 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 3500r / min for 10min, aspirate the supernatant, and use a 0.22μm aqueous phase filter to filter the bacteria to obtain the sterile fermentation solution. The bacterial content in the sterile fermentation solution is 5×10 10 ~7×10 10 .

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

[0049] S31. After inoculating the activation medium with Enterobacter hallii C4 under sterile conditions, the activation culture was placed on a constant temperature shaker at 37°C and shaken at 160 rpm for 24 hours, and the growth of the colonies was observed to ensure successful colony activation;

[0050] S32. The strain was expanded and cultured in a 50L fermenter, with a strain addition amount of 5%, a medium composition 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 temperature of the fermenter was set to 37 ° C, the speed was 160 rpm, the ventilation volume was 5 L / min, and the pH was 6.0. After 48 h of culture, the centrifuge was centrifuged at 14000 rpm to collect the separated bacteria.

[0051] S33. 40% trehalose was added to the bacterial cells as a freeze-drying protective agent, and the mixture was evenly mixed and dried for 48 hours 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 to obtain a bacterial powder with a viable bacterial count of 6.2×10 11 CFU / g of the bacterial agent;

[0052] S34. The bacterial agent is packaged using sterile packaging materials, and the production date, shelf life and other information are noted, and the packaged bacterial agent is stored in a cool and dry place away from light.

[0053] Example 5: Determination of cellulase activity produced by Enterobacter holmesii 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, 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 with cold water, then add distilled water to make up to 25mL and shake well, then measure the absorbance A at 540nm with a 10mm colorimetric dish, and draw a glucose standard curve (y=0.3077x-0.0119, R2=0.9993) with absorbance as the ordinate and glucose content as the abscissa ( Figure 4 ).

[0056] (2) Sample determination

[0057] Take 4 25mL graduated test tubes, add 50±0.5mg filter paper (1cm×6cm) to each of them, select 3 test tubes, number them in sequence and add 0.5mL of the sterile fermentation broth prepared in Example 3 to each of them as the experimental group, add 0.5mL of distilled water to the remaining 1 test tube as the control group, then add 1mL of citric acid-sodium citrate buffer to each of the 4 test tubes, accurately keep the temperature at 50°C for 60min, add 3mL of DNS reagent, then add 0.5mL of the sterile fermentation broth prepared in Example 3 to each of the test tubes of the experimental group, add 0.5mL of distilled water to each of the test tubes of the control group, mix all the test tubes and heat them 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 the volume to 25mL, and then measure the absorbance with a 10mm cuvette at a wavelength of 540nm.

[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°C, 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 as 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 multiple of enzyme solution dilution;

[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 produced by Enterobacter hallii

[0069]

[0070] Example 6: Amylase activity assay

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

[0072] The sterile fermented liquid obtained by embodiment 3 is diluted and placed in 70 ℃ of thermostatic water baths for preheating, then 20mL soluble starch is drawn and dissolved in 4 test tubes, after adding 5mL phosphate buffer and shaking up, after being placed in 60 ℃ of thermostatic water baths and preheating for 8min, 1mL preheated sterile fermented liquid is added, and timing is started immediately, and shake up and accurately reacts for 5min. After the reaction is completed, 1mL reaction solution is drawn with a pipette gun immediately, 3 test tubes filled with 0.5mL hydrochloric acid solution and 5mL dilute iodine solution in advance and numbered are added as the experimental group, and 1mL distilled water is added to 1 test tube filled with 0.5mL hydrochloric acid solution and 5mL dilute iodine solution as the control group. The experimental group and the control group are rapidly measured by ultraviolet spectrophotometer at a wavelength of 660nm for their habit of loneliness (A). According to the absorbance table look-up Appendix B, the concentration of test enzyme solution is obtained.

[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) and pH 6.0, can liquefy 1g soluble starch in 1h, which is 1 enzyme activity unit, 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 activity of Enterobacter hallii C4 was obtained as shown in Table 2.

[0081] Table 2 Amylase activity produced by Enterobacter hallii

[0082]

[0083] Example 7: Test on tobacco quality improvement effect of Enterobacter holmesii 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. adding different weights of the diluted bacterial solution to a portion of the dried tobacco leaves, mixing them evenly to form an experimental group, wherein the weight ratio of the diluted bacterial solution to the tobacco leaves in experimental group 1 is 1:50, the weight ratio of the diluted bacterial solution to the tobacco leaves in experimental group 2 is 1:100, and the weight ratio of the diluted bacterial solution to the tobacco leaves in experimental group 3 is 1:150; another portion of the dried tobacco leaves is not added with the diluted bacterial solution to form a control group, and each experimental group and the control group are simultaneously placed in an environment of 25° C. and 65% humidity for fermentation for 3 months;

[0087] S44. The main chemical components of the tobacco leaves after fermentation were tested, and the test results are shown in Table 3. At the same time, the tobacco leaves after fermentation were made into cigarettes, and 5 professional cigarette tasters were asked to conduct sensory evaluation according to the "Chinese Cigarette Style Sensory Evaluation Method" YC / T 497-2014 and take the average value. 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 gas 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 total alkaloids, total sugars, total nitrogen, and starch contents of the experimental group were lower than those of the control group, and the contents of reducing sugars and neophytodienes were higher than those of the control group, indicating that during the fermentation process, the bacterial solution of Enterobacter holmesii C4 can make full use of the main chemical components in tobacco, thereby producing more flavor substances. The sensory scores of the experimental group were all higher than those of the control group, indicating that the bacterial solution of Enterobacter holmesii C4 can improve the aroma quality, aroma volume, concentration, stimulation, aftertaste and sweetness of tobacco through fermentation, and can bring a unique smell to tobacco.

[0093] Example 8: Test on the tobacco quality improvement effect of Enterobacter holmesii C4 bacterial agent

[0094] S51. Select Yunyan 87 tobacco YQ2 grade tobacco leaves for drying 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. Dilute the dissolved bacterial solution 4 times 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 an experimental group, and do not add the diluted bacterial solution to another portion of the dried tobacco leaves as a control group. The experimental group and the control group are placed in an environment at a temperature of 25° C. and a humidity of 65% for fermentation for 3 months;

[0098] S55. The main chemical components of the tobacco leaves after fermentation were tested, and the test results are shown in Table 5. At the same time, the tobacco leaves after fermentation were made into cigarettes, and 5 professional cigarette tasters were asked to conduct sensory evaluation according to the "Chinese Cigarette Style Sensory Evaluation Method" YC / T 497-2014 and take the average value. The evaluation results are 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 gas 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 total alkaloids, total sugars, total nitrogen, starch, and neophytadiene contents of the experimental group were lower than those of the control group, and the reducing sugar content was higher than that of the control group, indicating that during the fermentation process, the inoculum of Enterobacter holmesii C4 can fully utilize the main chemical components in tobacco to produce more flavor substances. The multiple sensory scores of the experimental group were higher than those of the control group, indicating that the inoculum of Enterobacter holmesii C4 can improve the aroma quality, concentration, stimulation, aftertaste and sweetness of tobacco by fermentation without affecting the aroma volume, and can bring a unique smell to tobacco.

[0104] In summary, the E. coli C4 provided by this application has the ability to produce cellulase and amylase, and can use cellulose, starch and other components in tobacco during fermentation to produce more flavor substances, thereby improving the various sensory qualities of tobacco. The method of this application is used to improve the quality of tobacco, and tobacco products with better quality can be obtained in a greener and more environmentally friendly way, providing the tobacco industry with a biological solution that can effectively improve the quality of tobacco. In the future, through in-depth research and application development, E. coli C4 is expected to become a quality-enhancing microorganism for a variety of cash crops, promoting the development of the agricultural economy.

[0105] The above is only 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 regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present disclosure, and should be within the scope of protection of the claims attached to the present disclosure.

Claims

1. Enterobacter hormaechei C4, characterized in that The Enterobacter hallii C4 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the storage address is No. 3, No. 1, Beichen Road West, Chaoyang District, Beijing, the storage date is January 13, 2023, and the storage 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. A 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 Enterobacterium hallii C4 according to claim 1 or the bacterial agent according to claim 4 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 cloud tobacco 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 is 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, offal, concentration, stimulation, aftertaste and sweetness.

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