Preparation method and application of aspergillus flavus, microbial preparation and apricot spice
By using Aspergillus flavus HS-4, the problem of using harmful raw materials and low extraction efficiency in the existing fragrance production technology is solved, and the preparation of high-quality fragrances is achieved, the fruity and sweet fragrance of the fragrance is increased, and new fragrance substances are generated.
Patent Information
- Application Number
- CN202411802622.6
- 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 flavor and fragrance production technology has the problem that artificial synthesis may use harmful raw materials and produce by-products, and the natural extraction process is complex and inefficient, making it difficult to meet the market demand for high-quality fragrances.
Aspergillus flavus HS-4 was used for fermentation, and characteristic biological apricot flavors were prepared through microbial metabolism to enhance the fruity and sweet aromas in the flavors.
Through sensory evaluation and GC-MS analysis, it was found that fruity and sweet fragrance increased, and new fragrance substances were generated, which improved the aroma richness and delicateness of the fragrance and reduced irritation.
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial spices, and specifically relates to a preparation method and application of aflatoxin, a microbial preparation and apricot spices. Background Art
[0002] In the field of flavor and fragrance production, both traditional artificial synthesis and natural plant and animal extraction methods have certain limitations. Although artificial synthetic fragrances are efficient, the public has doubts about their safety because they may use harmful raw materials and produce by-products. Long-term contact or ingestion may pose potential risks to human health. The extraction process of natural fragrances is complex and inefficient. Due to the limitations of the extraction process, it is difficult to meet the application standards, which not only limits the scope of use but also may cause waste of environmental resources. The use of microbial fermentation technology to prepare flavors and fragrances is relatively green and environmentally friendly through the metabolism of microorganisms, which can effectively reduce potential risks to human health. At the same time, microbial strains will produce different metabolites during the fermentation process, giving the fragrances a unique aroma and a richer sense of hierarchy.
[0003] At present, there is no apricot flavor fermented by Aspergillus flavus for tobacco, and there is no report of adding it to cigarettes. Using the unique metabolic characteristics of Aspergillus flavus to produce a unique biological apricot flavor for tobacco will help meet the market demand for high-quality flavors. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing apricot flavor, a microbial preparation and an application thereof. The aflatoxin can be used as a fermentation strain in the preparation process of apricot flavor to improve the fruity and sweet aroma of the apricot flavor.
[0005] To achieve the above objectives, this application is implemented through the following technical solutions:
[0006] A type of Aspergillus flavus, classified and named Aspergillus flavus, with a strain name of HS-4, deposited in the General Biological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.41231.
[0007] Furthermore, the Aspergillus flavus HS-4 appears milky white on the PDA solid culture medium plate, the surface of the colony is fluffy, the center part is relatively dense, the edge is relatively diffuse, and the colony texture is dry.
[0008] Furthermore, the culture method of Aspergillus flavus is as follows: inoculate the Aspergillus flavus liquid into PDA liquid culture medium at an inoculation rate of 1% and culture for 24 hours.
[0009] Furthermore, the culture conditions of the Aspergillus flavus are a temperature of 28° C. and a rotation speed of 150 rpm.
[0010] Furthermore, the formula of the PDA liquid culture medium is: 30% potato extract powder, 2% glucose, and the formula of the PDA solid culture medium is: 30% potato extract powder, 2% glucose, and 2% agar.
[0011] A method for preparing a microbial preparation, comprising any of the above-mentioned Aspergillus flavus, comprising the following steps:
[0012] S1. Preparation of Aspergillus flavus activation solution:
[0013] The Aspergillus flavus was activated and cultured at 28°C and 150 r / min, and the Aspergillus flavus activation solution was obtained after shaking culture for 24-48 hours;
[0014] S2. Single colony culture:
[0015] The activated Aspergillus flavus solution obtained in step S1 was diluted ten-fold, applied to a PDA solid culture medium, and cultured in a constant temperature incubator at 28° C. for 48-72 h until a clear single Aspergillus flavus colony was observed;
[0016] S3. Seed solution preparation:
[0017] Pick a single Aspergillus flavus colony obtained in step S2, inoculate it into a PDA liquid culture medium, and culture it under shaking conditions of 28° C. and 150 r / min until the OD 600nm value of the culture liquid reaches 0.8-1.0, thereby obtaining a seed liquid;
[0018] S4. Preparation of microbial preparations:
[0019] The seed solution obtained in step S3 is centrifuged at high speed, the centrifugation conditions are: 4°C, 10000 rpm / min, 8 min, the supernatant is discarded, and an equal amount of sterile water is used to dissolve it. The above centrifugation operation is repeated and the solution is again dissolved with an equal amount of sterile water to finally obtain a microbial preparation.
[0020] A method for preparing apricot flavor, using the above-mentioned microbial preparation, comprises the following steps:
[0021] (1) Apricot spice fermentation:
[0022] Take the microbial preparation and treat the bacteria with a volume of 10 6 cfu / g standard, inoculated into sterilized apricot juice at a volume ratio of 5%, and then fermented in a shaker at 28°C and 150 rpm for 60-72 h;
[0023] (2) Apricot spice extract:
[0024] Add 75% ethanol to the apricot juice fermented in step (1), heat and reflux for extraction for 2 h, then vacuum filter to separate the filtrate and the residue, add 75% ethanol to the residue, repeat the above operation twice, combine the filtrates obtained three times, let stand for 24 h, take the supernatant and concentrate under reduced pressure to obtain biological apricot flavor.
[0025] An application is the application of the biological apricot flavor prepared above in cigarettes.
[0026] The beneficial effects of the present invention are:
[0027] The present application utilizes Aspergillus flavus to ferment apricot flavor, and improves the aroma of apricot flavor raw materials through microbial metabolism. The sensory evaluation and GC-MS analysis results are both good. After sensory evaluation, it was found that the fruity and sweet aromas were increased; after GC-MS analysis of the flavor components, it was found that the content of flavor substances such as octadecyl acetate and lauric acid was increased, and new flavor substances such as 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one, 5-methylfurfural, and dibutyl phthalate were generated.
[0028] The Aspergillus flavus can be used as a fermentation strain in the fermentation of apricot flavor for tobacco, thereby improving the fruity and sweet aroma of the apricot flavor, increasing the fineness of the smoke, and reducing irritation. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is a plate image of Aspergillus flavus of this application.
[0030] Figure 2 This is an electron microscope image of Aspergillus flavus of this application.
[0031] Figure 3 This is the phylogenetic tree analysis diagram of Aspergillus flavus in this application. DETAILED DESCRIPTION
[0032] 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.
[0033] The Aspergillus flavus HS-4 of this application is currently deposited in the General Biological Center of the China Microbiological Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101. The deposit date is May 17, 2024, and the deposit number is CGMCC No.41231.
[0034] In the present application, the formula of PDA liquid culture medium is: 30% potato extract powder, 2% glucose, and the formula of PDA solid culture medium is: 30% potato extract powder, 2% glucose, and 2% agar.
[0035] Example 1
[0036] Under sterile conditions, 10 g of jujube forest soil and 90 mL of sterile water were added to a conical flask, cultured at 28°C and 150 r / min for 4 h, taken out, and then inoculated into 100 mL of PDA liquid culture medium at a 10% inoculum (volume fraction) for enrichment culture for 48 h.
[0037] The enriched solution was diluted in series, and 10 -4 , 10 -6 , 10 -8 100 uL of the three dilution concentrations were evenly spread on solid PDA medium and cultured at 28°C for 48-72 h until clear single colonies were formed. Single colonies were picked and purified by streak method. The purified colonies were numbered and stored.
[0038] The purified strain was enriched and activated in PDA medium and then inoculated into apricot juice. It was cultured at 28°C and 150 r / min for 60 h. After the fermentation, a preliminary sensory evaluation was carried out. The strains with obvious aroma changes were marked and saved as HS-4.
[0039] The screened HS-4 strain (screened from the soil of the jujube forest in September 2023) was spread on PDA solid culture medium and cultured in a constant temperature incubator at 28°C for 24 h. The colony morphology was observed. The results were as follows: Figure 1 .like Figure 1 As shown, the HS-4 strain appeared as a milky white irregular shape on the PDA solid culture medium plate, with a fluffy surface, a dense center, a diffuse edge, and a dry colony texture. 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.
[0040] Use a DNA extraction kit to extract the genomic DNA of the HS-4 strain, and the PCR amplification system is 25 μL: template DNA (20-50 ng / μL) 1 μL, primer (10 μmol / L) 0.5 μL, dNTP mixed solution (2.5 mmol / L) 1 μL, Taq enzyme 0.2 μL, 10×Buffer 2.5 μL, and ultrapure water 19.8 μL.
[0041] 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.
[0042] The PCR product was sent to Shanghai Bioengineering Co., Ltd. for 26S 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 26S rDNA sequencing results using the Neighbor-joining algorithm. The results are shown in Figure 3 As shown, the 26S rDNA of HS-4 strain had the highest similarity with MT528910.1 Aspergillus flavus clone EF 261, so the strain was named Aspergillus flavus.
[0043] The Aspergillus flavus strain HS-4 obtained by screening was cultivated in a conventional manner for 3-4 days, and then the content of aflatoxin produced by the metabolism of the Aspergillus flavus strain HS-4 was detected according to the fourth method of "GB5009.22-2016". The results are shown in Table 1.
[0044] Table 1 Aflatoxin metabolic yield
[0045] According to the test results in Table 1, the Aspergillus flavus strain HS-4 of the present application does not produce aflatoxin during the metabolic process and is safe.
[0046] Example 2
[0047] (1) Preparation of microbial preparations
[0048] The Aspergillus flavus was activated and cultured at 28°C and 150 r / min. After shaking culture for 24 hours, the Aspergillus flavus activation solution was diluted tenfold and applied to the PDA solid culture medium. The culture was cultured in a constant temperature incubator at 28°C for 60 hours until a clear single colony of Aspergillus flavus was observed. The single colony of Aspergillus flavus was inoculated into the PDA liquid culture medium, and the culture was shaken at 28°C and 150 r / min until the OD 600nm value of the culture liquid reached 0.8-1.0 to obtain the seed liquid; the seed liquid was centrifuged at 4°C and 10000 rpm / min for 8 minutes, the supernatant was discarded, and the solution was re-dissolved with an equal amount of sterile water. The above centrifugation operation was repeated and the solution was re-dissolved with an equal amount of sterile water to obtain the microbial preparation.
[0049] (2) Apricot flavor fermentation and extraction
[0050] The microbial preparation was adjusted to 1.75×10 8 - 2.65×10 8cfu / g standard, inoculated into sterilized apricot juice, and fermented on a shaking table at 28°C and 150 rpm for 65 h; added 75% ethanol to the fermented apricot juice, heated to reflux and extracted for 2 h, then vacuum filtered to separate the filtrate and residue, continued to add 75% ethanol to the residue, repeated the above operation twice, combined the filtrates obtained three times, and after standing for 24 h, took the supernatant and concentrated it under reduced pressure to obtain biological apricot flavor.
[0051] (3) Application of biological apricot flavor in cigarettes
[0052] Take biological apricot flavor with a weight of 4.5‰ of cigarette tobacco, dilute it 2 times with ethanol, spray it on the cigarette tobacco, put it into 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 cigarettes, which are the test cigarettes containing biological apricot flavor.
[0053] (4) Sensory evaluation
[0054] 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 2. The fruity and sweet aroma effects of the test cigarettes were significantly better than those of the blank cigarettes, and the smoke was more delicate, soft and comfortable, and the stimulation was reduced.
[0055] Table 2
[0056] (5) Analysis of neutral aroma components:
[0057] Take 15 mL of fermentation broth sample and add 15 mL of dichloromethane solution. After vortexing and extracting for 10 min, collect the dichloromethane extract. Repeat three times and combine the dichloromethane extracts. Add anhydrous sodium sulfate and dry overnight. Add 50 μL of 2,6-dichlorotoluene (0.6426 mg / mL) as an internal standard. After concentrating to 1 mL at normal pressure, perform GC-MS chemical composition analysis.
[0058] Chromatographic conditions: chromatographic column DB-5MS column (60m x 250μm x 0.25μm); injection port temperature 280℃; split ratio 3:1; split flow rate 3.0 mL / min; 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 was electron impact (EI), electron energy was 70 eV; ion source temperature was 230°C; quadrupole temperature was 150°C; solvent delay was 7 min; full scan detection, scanning mass range was 35-500 amu.
[0061] The results are shown in Table 3, which is a comparison of the aroma substances in apricot juice before and after Aspergillus flavus fermentation.
[0062] Table 3
[0063] Example 3
[0064] Aspergillus flavus was spread on PDA solid culture medium and cultured in a constant temperature incubator at 28°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.
[0065] 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 26S 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 26S rDNA sequencing results using the Neighbor-joining algorithm.
[0066] The Aspergillus flavus slant was preserved and sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation.
[0067] 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 flavus, characterized in that: The strain was named Aspergillus flavus and HS-4, and was deposited in the General Biological Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.41231.
2. The Aspergillus flavus according to claim 1, characterized in that The Aspergillus flavus appears milky white on the PDA solid culture medium plate, the surface of the colony is fluffy, the center part is relatively dense, the edge is relatively diffuse, and the colony texture is dry.
3. The Aspergillus flavus according to claim 1, characterized in that The culture method of Aspergillus flavus is as follows: inoculate Aspergillus flavus liquid at a 1% inoculation rate into PDA liquid culture medium and culture for 24 hours.
4. The Aspergillus flavus according to claim 3, characterized in that The culture conditions of the Aspergillus flavus are a temperature of 28° C. and a rotation speed of 150 rpm.
5. The Aspergillus flavus according to claim 3, characterized in that The formula of the PDA liquid culture medium is: 30% potato extract powder and 2% glucose; the formula of the PDA solid culture medium is: 30% potato extract powder, 2% glucose and 2% agar.
6. A method for preparing a microbial preparation, comprising the Aspergillus flavus of any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Preparation of Aspergillus flavus activation solution: The Aspergillus flavus was activated and cultured at 28°C and 150 r / min, and the Aspergillus flavus activation solution was obtained after shaking culture for 24-48 hours; S2. Single colony culture: The activated Aspergillus flavus solution obtained in step S1 was diluted ten-fold, applied to a PDA solid culture medium, and cultured in a constant temperature incubator at 28° C. for 48-72 h until a clear single Aspergillus flavus colony was observed; S3. Seed solution preparation: Pick a single Aspergillus flavus colony obtained in step S2, inoculate it into a PDA liquid culture medium, and culture it under shaking conditions of 28° C. and 150 r / min until the OD 600nm value of the culture liquid reaches 0.8-1.0, thereby obtaining a seed liquid; S4. Preparation of microbial preparations: The seed solution obtained in step S3 is centrifuged at high speed, the centrifugation conditions are: 4°C, 10000 rpm / min, 8 min, the supernatant is discarded, and an equal amount of sterile water is used to dissolve it. The above centrifugation operation is repeated and the solution is again dissolved with an equal amount of sterile water to finally obtain a microbial preparation.
7. A method for preparing apricot flavor, using the microbial preparation of claim 6, characterized in that: The following steps are involved: (1) Apricot spice fermentation: Take the microbial preparation and treat the bacteria with a volume of 10 6 cfu / g standard, inoculated into sterilized apricot juice at a volume ratio of 5%, and then fermented in a shaker at 28°C and 150 rpm for 60-72 h; (2) Apricot spice extract: The apricot juice fermented in step (1) is added with 75% ethanol, heated under reflux for extraction for 2 h, and then vacuum filtered to separate the filtrate and the residue. 75% ethanol is further added to the residue, and the above operation is repeated twice. The filtrates obtained three times are combined, and after standing for 24 h, the supernatant is taken and concentrated under reduced pressure to obtain the biological apricot flavor.
8. An application, characterized in that, Use of the biological apricot flavor prepared according to claim 7 in cigarettes.