Biological red date spice strain, preparation method and application thereof
The preparation of biological red date spices through fermentation of Bacillus subtilis has solved the safety doubts of the existing spice production methods and the waste of environmental resources, achieved the production of high-quality red date spices and applied in cigarettes, and improved the softness of aroma and smoke.
Patent Information
- Application Number
- CN202411802629.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-02-28
AI Technical Summary
The existing spice production methods have problems with safety and waste of environmental resources, and lack the application of spices for fermenting red date tobacco using Bacillus subtilis.
Bio-red date fragrances were prepared by fermentation of Bacillus subtilis hs-1, and bacterial culture was cultured and activated using LB liquid and solid culture medium. They were then fermented in red date juice, and bio-red date fragrances were obtained by ethanol extraction and concentration under reduced pressure, and added to cigarettes.
The aroma and sensory evaluation of the red date spice was improved, and the toasted sweet, fruity and sweet fragrance in the cigarette was increased, and the smoke became softer and more delicate. GC-MS analysis showed that the fragrance substance content increased and new fragrance substances were generated.
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Figure CN119530087A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial tobacco flavors, and specifically relates to a strain of biological red date flavor, a preparation method and application thereof. Background Art
[0002] At present, the production of flavors and fragrances in the world mainly relies on two sources: artificial synthesis and natural plant and animal extraction. However, both methods have their own limitations. On the one hand, artificial synthetic fragrances are made by chemical methods. The methods are efficient, but the safety of the products is often questioned by the public. The harmful raw materials and by-products that may be used in the chemical synthesis process, as well as the potential risks to human health caused by long-term contact or ingestion of these chemical fragrances, are all reasons for the public's concerns about their safety. On the other hand, natural fragrances are derived from animals and plants, and the extraction process is relatively complex and inefficient. Due to the limitations of the extraction process, it is often difficult to obtain fragrances that meet the application standards. This not only limits the scope of use of natural fragrances, but may also lead to waste of environmental resources.
[0003] The use of microbial fermentation technology to prepare flavors and fragrances, on the one hand, utilizes the metabolism of microorganisms, the process is relatively green and environmentally friendly, and can effectively reduce potential risks to human health. On the other hand, biological fermentation can achieve efficient production by optimizing strains and fermentation conditions, increase the yield of flavors, and produce flavors with specific aromas and characteristics to meet different market needs. In the prior art, there is no report of red date tobacco flavors fermented by Bacillus subtilis, and there is no report of adding it to cigarettes for application. Summary of the invention
[0004] The purpose of the present invention is to provide a strain of biological red date spices, a preparation method and application thereof, so as to meet the current high quality demand for red date spices.
[0005] To achieve the above objectives, this application is implemented through the following technical solutions:
[0006] The invention discloses a strain of biological red date flavor, which is a bacillus subtilis. The bacillus subtilis is classified and named as Bacillus subtilis, the strain is named as hs-1, and is preserved in the General Biological Center of China Microbiological Culture Collection Administration, with the preservation number of CGMCC No.30676.
[0007] Furthermore, the Bacillus subtilis hs-1 exhibits an opaque dirty white colony on the LB solid culture medium plate, with a rough and wrinkled surface, expanded edges, and an irregular circular colony shape.
[0008] Furthermore, the culture method of Bacillus subtilis is as follows: inoculating Bacillus subtilis liquid into LB liquid culture medium at an inoculation rate of 1%, and the culture conditions are as follows: temperature 30° C., rotation speed 150 rpm, and culture time 12 h.
[0009] Furthermore, the LB liquid culture medium formula is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, and the LB solid culture medium formula is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, and agar 20 g / L.
[0010] Furthermore, a method for preparing a microbial agent using any of the above strains comprises the following steps:
[0011] (1) Preparation of Bacillus subtilis activation solution:
[0012] Bacillus subtilis was activated and cultured at 30℃ and 150 r / min, and the activated solution of Bacillus subtilis was obtained after shaking culture for 12 h.
[0013] (2) Single colony culture:
[0014] Dilute the activated Bacillus subtilis solution obtained in step (1) ten-fold, apply it to LB solid culture medium, and culture it in a constant temperature incubator at 30°C for 48-72 hours until a clear single colony of Bacillus subtilis is observed;
[0015] (3) Seed solution preparation:
[0016] Pick a single colony of Bacillus subtilis obtained in step (2), inoculate it into LB liquid culture medium, and culture it under shaking conditions of 30°C and 150 r / min until the OD 600nm value of the culture liquid reaches 0.8-1.0, thereby obtaining a seed liquid;
[0017] (4) Preparation of microbial preparations:
[0018] The seed solution obtained in step (3) is centrifuged at high speed under the conditions of 4°C, 10,000 rpm / min, 8 min, the supernatant is discarded, and an equal amount of sterile water is used to reconstitute the solution. The above centrifugation operation is repeated and the solution is reconstituted again with an equal amount of sterile water to finally obtain a microbial preparation.
[0019] A method for preparing biological red date flavor, using the above-mentioned microbial agent, comprises the following steps:
[0020] S1, take the microbial preparation, according to the treatment amount of 1.85×10 8 - 2.83×10 8cfu / g standard, the suspension was inoculated into sterilized dry red date juice, and then fermented in a shaker at 30°C and 150 rpm for 36 h;
[0021] S2. Add 75% ethanol to the red date juice fermented in step S1, heat and reflux for extraction for 2 hours, then vacuum filter to separate the filtrate and the residue, continue to add 75% ethanol to the residue, repeat the above operation twice, combine the filtrates obtained three times, let it stand for 24 hours, take the upper clear liquid and concentrate it under reduced pressure to obtain the biological red date flavor extract.
[0022] An application is the application of the biological jujube flavor prepared above in cigarettes.
[0023] Furthermore, the biological jujube flavor is added to the tobacco at a mass percentage of 0.05-0.1% of the cigarette tobacco.
[0024] The beneficial effects of the present invention are:
[0025] The present application utilizes Bacillus subtilis fermentation to prepare biological red date flavor, and the aroma of the red date flavor is improved through microbial fermentation. The sensory evaluation and GC-MS analysis results are both good. After sensory evaluation of cigarette flavoring, it is found that the roasted sweet aroma, fruity aroma, and sweet aroma are increased, and the smoke is softer and more delicate; after GC-MS analysis of the aroma components, it is found that the content of aroma substances such as isopentanol and octadecyl acetate is increased, and new aroma substances such as phenylethyl alcohol, isopentanyl acetate, 3-hydroxy-2-butanone, and phenylethyl acetate are generated. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a plate map of Bacillus subtilis of this application.
[0027] Figure 2 This is an electron microscope image of Bacillus subtilis of the present application.
[0028] Figure 3 This is a phylogenetic tree analysis diagram of Bacillus subtilis of this application. DETAILED DESCRIPTION
[0029] The technical solution of the present invention is described in detail below through examples. The following examples are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.
[0030] The Bacillus subtilis hs-1 of the present application is currently deposited in the General Biological Center of the China Microorganism Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 17, 2024, and the deposit number is CGMCC No. 30676.
[0031] The formula of LB liquid culture medium is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L; the formula of LB solid culture medium is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, agar 20 g / L.
[0032] Example 1
[0033] Under sterile conditions, 10 g of tea garden soil and 90 mL of sterile water were added to a conical flask, cultured at 30°C and 150 r / min for 4 h, taken out, and then inoculated into 100 mL of LB liquid culture medium at a volume fraction of 10% for enrichment culture for 48 h.
[0034] The enriched solution was diluted in series, and 10 -4 , 10 -6 , 10 -8 The three dilution concentrations were evenly spread on solid LB medium and cultured at 30°C for 24-48 h until clear single colonies were formed. Single colonies were picked and purified by streak method. The purified colonies were numbered and preserved.
[0035] The preserved strains were enriched and activated in LB medium and then inoculated into red date juice. They were cultured at 30°C and 150 r / min for 36 h. After the fermentation, a preliminary sensory evaluation was performed and the strains with obvious aroma changes were marked and preserved as hs-1.
[0036] The hs-1 strain obtained by screening (screened in tea garden soil in September 2023) was spread on LB solid medium and cultured in a constant temperature incubator at 30°C for 24 h. The colony morphology was observed. The results are as follows: Figure 1 .like Figure 1 As shown, the hs-1 strain appeared as an opaque dirty white colony on the LB solid culture medium plate, with a rough and wrinkled surface, expanded edges, and an irregular circular colony shape; the strain morphology was observed using a cryo-type high-resolution field emission scanning electron microscope, and the results were as follows Figure 2 shown.
[0037] Use a DNA extraction kit to extract the genomic DNA of the hs-1 strain, and the PCR amplification system is 25 μL: 1 μL of template DNA (20-50 ng / μL), 0.5 μL of each primer (10 μmol / L), 1 μL of dNTP mixed solution (2.5 mmol / L), 0.2 μL of Taq enzyme, 2.5 μL of 10×Buffer, and 19.8 μL of ultrapure water.
[0038] PCR amplification program: 94°C for 4 min; 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 30 min.
[0039] The PCR product was sent to Shanghai Bioengineering Co., Ltd. for 16S rDNA sequencing. The sequencing results were compared in NCBI GeneBank using BLAST software, and the target strain was identified based on homologous sequence similarity. MEGA 11 software was used to construct a phylogenetic tree based on the 16S rDNA sequencing results using the Neighbor-joining algorithm. Figure 3 As shown, the hs-1 strain had the highest similarity with Bacillus cereus, so the strain was named Bacillus subtilis.
[0040] Example 2
[0041] (1) Preparation of bacterial preparations:
[0042] Bacillus subtilis is activated and cultured to obtain an activation solution, which is diluted according to a certain gradient and then applied to LB solid culture medium, cultured in a constant temperature incubator at 30°C for 48-72 h, a single colony on the plate is picked up, inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 30°C and 150 r / min for 12 h to obtain a seed solution, which is inoculated into LB liquid culture medium at a 1% inoculum amount, and cultured in a constant temperature shaker at 30°C and 150 r / min until the OD600nm value is 0.8-1.0, the bacterial solution is centrifuged at 4°C and 10000 r / min for 10 min, the supernatant is discarded, and the solution is re-dissolved with an equal amount of sterile water, the centrifugation is repeated, and the solution is re-dissolved with an equal amount of sterile water to obtain a bacterial preparation.
[0043] (2) Red date spice fermentation:
[0044] According to the treatment amount of 1.85×10 8 -2.83×10 8 The bacterial preparation was inoculated into the sterilized red date juice at a 1% inoculation rate, and the fermentation was carried out at a fermentation temperature of 30°C and a rotation speed of 150 rpm for 36 h. An equal amount of red date juice was treated with an equal amount of sterile water according to the above method to prepare a control red date juice sample.
[0045] (3) Application of biological red date flavor in cigarettes:
[0046] Take 1.0‰ of the biological red dates flavoring in the amount of cigarette tobacco, dissolve it in ethanol twice the amount of the biological red dates flavoring, spray it on the cigarette tobacco, put it in a constant temperature and humidity chamber, balance it for 48 hours under the conditions of humidity 60%±2 and temperature 22℃±2, and roll it into cigarette sticks, which are the test cigarettes containing biological red dates flavoring.
[0047] (4) Sensory evaluation:
[0048] The test cigarettes and blank cigarettes were given to professional smokers to evaluate the aroma style of the cigarettes. The olfactory aroma, aroma characteristics, sensory quality and other aspects were evaluated and compared. The results are shown in Table 1. The roasted sweet aroma, fruity aroma and sweet aroma of the test cigarettes were significantly better than those of the blank cigarettes. The smoke was richer, the sweeter, and the smoke was softer and more delicate.
[0049] Table 1
[0050]
[0051] (5) Analysis of neutral aroma components:
[0052] The vortex cold extraction method was used to extract the aroma components of red jujube juice before and after fermentation. After processing according to the optimal fermentation conditions, 15 mL of the fermentation broth sample was added with an equal amount of dichloromethane solution, and the dichloromethane extract was collected after vortex oscillation extraction for 10 minutes. After repeating three times, the dichloromethane extracts were combined and dried overnight with anhydrous sodium sulfate, and 50 μL of 2,6-dichlorotoluene (0.6426 mg / mL) was added as an internal standard. After being concentrated to 1 mL at normal pressure, the chemical composition was analyzed by GC-MS.
[0053] Chromatographic conditions:
[0054] Chromatographic column: DB-5MS column (60m x 250μm x 0.25μm);
[0055] Inlet temperature: 280°C;
[0056] Split ratio: 3:1;
[0057] Split flow rate: 3.0 mL / min;
[0058] Carrier gas: He, flow rate 1.0 mL / min:
[0059] The heating program was as follows: initial temperature 50 °C (held for 3 min), then increased to 130 °C at a rate of 2 °C / min (held for 3 min), then increased to 180 °C at a rate of 5 °C / min (held for 2 min), and finally increased to 280 °C at a rate of 3 °C / min (held for 5 min).
[0060] Mass spectrometry conditions: ionization mode is electron impact (EI), electron energy is 70 eV; ion source temperature is 230℃; quadrupole temperature is 150℃; solvent delay is 7 min; full scan detection, scanning mass range is 35~500 amu, the results are shown in Table 2. Table 2 is the comparison results of aroma substances in red date juice before and after treatment with Bacillus subtilis.
[0061] Table 2
[0062]
[0063] Table 2
[0064]
[0065] Example 3
[0066] Bacillus subtilis was spread on LB solid culture medium and cultured in a constant temperature incubator at 30°C for 24 h, and its colony morphology was observed; the strain morphology was observed using a cryo-type high-resolution field emission scanning electron microscope.
[0067] The target strain genomic DNA was extracted using a DNA extraction kit, and the primers used in the PCR amplification reaction were synthesized by Shanghai Bioengineering Co., Ltd. The target strain was sequenced by 16S rDNA, and the sequencing was completed by Shanghai Bioengineering Co., Ltd. The obtained gene sequence was compared in NCBI GeneBank using BLAST software, and the target strain was identified based on the similarity of homologous sequences. MEGA 11 software was used to construct a phylogenetic tree based on the 16S rDNA sequencing results using the Neighbor-joining algorithm.
[0068] The Bacillus subtilis slant was preserved and sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation.
[0069] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.
Claims
1. A strain of biological red date flavor, characterized in that: The invention is a Bacillus subtilis, the classification name of the Bacillus subtilis is Bacillus subtilis, the strain name is hs-1, and it is preserved in the General Biological Center of China Microorganism Culture Collection Administration, and the preservation number is CGMCC No.30676.
2. The strain of biological red date flavor according to claim 1, characterized in that: The Bacillus subtilis colonies on the LB solid culture medium plate are opaque and dirty white, with a rough and wrinkled surface, expanded edges, and irregular circular shapes.
3. The strain of biological red date flavor according to claim 1, characterized in that: The culture method of Bacillus subtilis is as follows: inoculating Bacillus subtilis liquid into LB liquid culture medium at an inoculation rate of 1%, and the culture conditions are as follows: temperature 30° C., rotation speed 150 rpm, and culture time 12 h.
4. The strain of biological red date flavor according to claim 3, characterized in that: The LB liquid culture medium formula is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, and the LB solid culture medium formula is: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, and agar 20 g / L.
5. The strain of biological red date flavor according to claim 1, characterized in that: A method for preparing a microbial agent using the strain of any one of claims 1 to 4, comprising the following steps: (1) Preparation of Bacillus subtilis activation solution: Bacillus subtilis was activated and cultured at 30℃ and 150 r / min, and the activated solution of Bacillus subtilis was obtained after shaking culture for 12 h. (2) Single colony culture: Dilute the activated Bacillus subtilis solution obtained in step (1) ten-fold, apply it to LB solid culture medium, and culture it in a constant temperature incubator at 30°C for 48-72 hours until a clear single colony of Bacillus subtilis is observed; (3) Seed solution preparation: Pick a single colony of Bacillus subtilis obtained in step (2), inoculate it into LB liquid culture medium, and culture it under shaking conditions of 30°C and 150 r / min until the OD 600nm value of the culture liquid reaches 0.8-1.0, thereby obtaining a seed liquid; (4) Preparation of microbial preparations: The seed solution obtained in step (3) is centrifuged at high speed under the conditions of 4°C, 10,000 rpm / min, 8 min, the supernatant is discarded, and an equal amount of sterile water is used to reconstitute the solution. The above centrifugation operation is repeated and the solution is reconstituted again with an equal amount of sterile water to finally obtain a microbial preparation.
6. A method for preparing biological red date flavor, using the microbial agent according to claim 5, characterized in that: The following steps are involved: S1, take the microbial preparation, according to the treatment amount of 1.85×10 8 - 2.83×10 8 cfu / g standard, the suspension was inoculated into sterilized dry red date juice, and then fermented in a shaker at 30°C and 150 rpm for 36 h; S2. Add 75% ethanol to the red date juice fermented in step S1, heat and reflux for extraction for 2 hours, then vacuum filter to separate the filtrate and the residue, continue to add 75% ethanol to the residue, repeat the above operation twice, combine the filtrates obtained three times, let it stand for 24 hours, take the upper clear liquid and concentrate it under reduced pressure to obtain the biological red date flavor extract.
7. An application, characterized in that: The use of the biological jujube flavor prepared according to claim 6 in cigarettes.
8. The use according to claim 7, characterized in that: The biological jujube flavor is added to the tobacco shreds at 0.05-0.1% by mass of the cigarette tobacco shreds.