Wickerhamomyces anomalus for preparing biological red date spice and application of Wickerhamomyces anomalus

Abstract

The invention belongs to the technical field of tobacco flavor prepared by microorganisms, and particularly relates to Wickerhamomyces anomalus for preparing biological red date flavor and application of the Wickerhamomyces anomalus. The Wickerhamomyces anomalus is classified and named as Wickerhamomyces anomalus, the strain name is HS-3, the Wickerhamomyces anomalus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30675. The Wickerhamomyces anomalus has the advantages that the Wickerhamomyces anomalus can be used for preparing the Wickerhamomyces anomalus; the strain can be used as a fermentation strain to play a role in fermentation of biological red date spices. When the Wickerhamomyces anomalus is used for fermenting the red date spice, the contents of aromatic substances such as isoamyl alcohol, octadecyl acetate and lauric acid in the spice can be increased, new aromatic substances such as isoamyl acetate, 3-hydroxy-2-butanone and phenethyl decanoate can be generated, the roasted sweet aroma, fruity aroma and sweet aroma in the red date spice can be obviously improved, the smoke taste is improved, and the flavor of the red date spice is improved. The fineness and comfort are improved, and the stimulation is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of preparing tobacco flavors with microorganisms, and specifically relates to an abnormal Wickham yeast for preparing biological red date flavors and an application thereof. 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] In the prior art, there is no abnormal Wickham yeast fermented red date tobacco flavor, and there is no report of adding it to cigarettes. Using the unique metabolic characteristics of abnormal Wickham yeast to produce a unique biological red date tobacco flavor is conducive to meeting the market demand for high-quality, safe and environmentally friendly flavors. Summary of the invention

[0004] The object of the present invention is to provide an abnormal Wickham yeast for preparing biological red date flavor and an application thereof. The abnormal Wickham yeast can be used as a fermentation strain in the preparation process of biological red date flavor, improve the roasted sweet aroma, fruity aroma and sweet aroma of the red date flavor, improve the mouthfeel of the smoke, increase the delicateness and comfort, and reduce irritation.

[0005] To achieve the above objectives, this application is implemented through the following technical solutions:

[0006] The invention discloses an abnormal Wickham yeast for preparing biological red date flavor. The abnormal Wickham yeast is classified and named as Wickerhamomyces anomalus, the strain is named HS-3, and is deposited in the General Biological Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No.30675.

[0007] Furthermore, the abnormal Wickham yeast HS-3 appears as a milky white circle with a raised center, neat edges, and a smooth, moist, and sticky surface on a YPD solid culture medium plate.

[0008] Furthermore, the culture method of the abnormal Wickham yeast is: inoculating the abnormal Wickham yeast liquid into YPD liquid culture medium at an inoculation rate of 1% and culturing for 12 hours.

[0009] Furthermore, the culture conditions of the abnormal Wickham yeast are a temperature of 30° C. and a rotation speed of 150 rpm.

[0010] Furthermore, the formula of the YPD liquid culture medium is: 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast powder; the formula of the YPD solid culture medium is: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast powder, and 20 g / L agar.

[0011] An application, using any of the above abnormal Wickham yeasts in the preparation of biological red date flavors.

[0012] Furthermore, the preparation method of biological red date flavor comprises the following steps:

[0013] (1) Preparation of abnormal Wickham yeast activation solution:

[0014] The abnormal Wickham yeast was activated and cultured at 30℃ and 150 r / min, and the abnormal Wickham yeast activation liquid was obtained after shaking culture for 12 hours.

[0015] (2) Single colony culture:

[0016] The abnormal Wickham yeast activation solution obtained in step (1) is diluted ten-fold, applied to YPD solid medium, and cultured in a 30°C constant temperature incubator for 48-72 h until a clear abnormal Wickham yeast single colony is observed;

[0017] (3) Seed solution preparation:

[0018] Pick a single colony of abnormal Wickham yeast obtained in step (2), inoculate it into YPD 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;

[0019] (4) Preparation of microbial preparations:

[0020] 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;

[0021] (5) Red date spice fermentation:

[0022] Take the microbial preparation from step (4) and treat with a bacterial amount of 1.85×10 8 - 2.65×10 8 cfu / g standard, the suspension was inoculated into sterilized red date juice, and then fermented in a shaker at 30°C and 150 rpm for 36 h;

[0023] (6) Red date spice extraction:

[0024] The red date juice fermented in step (5) is added with 75% ethanol, heated and refluxed for extraction for 2 h, and then vacuum filtered to separate the filtrate and the filter residue. 75% ethanol is further added to the filter 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 red date flavor.

[0025] Furthermore, the biological red date flavor is used in cigarettes.

[0026] The beneficial effects of the present invention are:

[0027] The present application utilizes abnormal Wickham yeast to ferment red date fruit flavors, and improves the aroma of the red date 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 roasted sweet aroma and fruity aroma increased; after GC-MS analysis of the flavor components, it was found that the content of flavor substances such as isopentanol, octadecyl acetate, and lauric acid increased, and new flavor substances such as isopentanol acetate, 3-hydroxy-2-butanone, and phenethyl decanoate were generated.

[0028] The abnormal Wickham yeast can be used as a fermentation strain in the fermentation of red date spices, improve the roasted sweet aroma, fruity aroma and sweet aroma of the red date spices, improve the taste of the smoke, increase the delicateness and comfort, and reduce irritation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a plate map of the abnormal Wickham yeast of this application.

[0030] Figure 2 This is an electron micrograph of the abnormal Wickham yeast of the present application.

[0031] Figure 3 This is a phylogenetic tree analysis diagram of the abnormal Wickham yeast of 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 abnormal Wickham yeast (Bacillus subtilis) HS-3 of the present application is currently deposited in the General Biological Center (CGMCC) of the China Microorganism Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the postal code is 100101, the preservation date is May 17, 2024, and the preservation number is CGMCC No.30675.

[0034] The formula of YPD liquid culture medium is: peptone 20g / L, glucose 20g / L, yeast powder 10g / L, and the formula of YPD solid culture medium is: peptone 20g / L, glucose 20g / L, yeast powder 10g / L, agar 20g / L.

[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 30°C and 150 r / min for 4 h, taken out, and then inoculated into 100 mL of YPD 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 The three dilution concentrations were evenly spread on solid YPD 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.

[0038] The purified strain was enriched and activated in YPD medium and then inoculated into red date juice. It was cultured at 30℃ and 150 r / min for 36 h. After the fermentation, a preliminary sensory evaluation was carried out. The strain with obvious aroma change was marked and saved as HS-3.

[0039] The screened HS-3 strain (screened from the soil of the jujube forest in September 2023) was spread on YPD solid medium and cultured in a constant temperature incubator at 30°C for 24 h. The colony morphology was observed. The results were as follows: Figure 1 .like Figure 1 As shown, the HS-3 strain appeared as a milky white circle with a raised center on the YPD solid culture medium plate, with neat edges and a smooth, moist and sticky surface. 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-3 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-3 strain had the highest similarity with KX839416.1Wickerhamomyces sp.strainYSY06, so the strain was named Wickerhamomyces anomalus.

[0043] Example 2

[0044] (1) Preparation of microbial preparations:

[0045] The abnormal Wickham yeast was activated and cultured at 30°C and 150 r / min. After shaking and culturing for 12 hours, the abnormal Wickham yeast activation solution was diluted tenfold and applied to YPD solid culture medium, and cultured in a 30°C constant temperature incubator for 48-72 hours until a clear abnormal Wickham yeast single colony was observed. The abnormal Wickham yeast single colony was inoculated into YPD liquid culture medium, and cultured at 30°C and 150 r / min until the OD 600nm value of the culture liquid reached 0.8-1.0 to obtain seed liquid; the seed liquid was centrifuged at 4°C and 10000 rpm / min for 8 minutes, the supernatant was discarded, and an equal amount of sterile water was re-dissolved, and the above centrifugation operation was repeated and the same amount of sterile water was used to re-dissolve the microbial preparation.

[0046] (2) Fermentation and extraction of red date spices:

[0047] The microbial preparation was adjusted to 1.85×10 8 - 2.65×10 8cfu / g standard, inoculated into sterilized red date juice, and fermented on a shaking table at 30℃ and 150 rpm for 36 h; added 75% ethanol to the fermented red date juice, heated and refluxed for 2 h, and 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 the biological red date flavor.

[0048] (3) Application of biological red date flavor in cigarettes:

[0049] Take biological red dates flavoring with a mass of 2.5‰ of cigarette tobacco, dilute it 2 times with ethanol, 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.

[0050] (4) Sensory evaluation:

[0051] 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 and fruity aroma of the test cigarettes were significantly better than those of the blank cigarettes, the taste of the smoke was improved, the delicacy and comfort were increased, and the stimulation was reduced.

[0052] Table 1

[0053]

[0054] (5) Analysis of neutral aroma components:

[0055] 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.

[0056] Chromatographic conditions:

[0057] Chromatographic column: DB-5MS column (60m x 250μm x 0.25μm);

[0058] Inlet temperature: 280°C;

[0059] Split ratio: 3:1;

[0060] Split flow rate: 3.0 mL / min;

[0061] Carrier gas: He, flow rate 1.0 mL / min:

[0062] 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).

[0063] Mass spectrometry conditions: electron impact (EI) ionization mode, electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; solvent delay 7 min; full scan detection, scanning mass range 35-500 amu.

[0064] The results are shown in Table 2, which is a comparison of the aroma substances in red date juice before and after treatment with the abnormal Wickham yeast.

[0065] Table 2

[0066]

[0067] Table 2

[0068]

[0069] Example 3

[0070] Abnormal Wickham yeast was spread on YPD solid culture medium and cultured in a constant temperature incubator at 30℃ 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.

[0071] 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.

[0072] The abnormal Wickham yeast slant was preserved and sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation.

[0073] 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. An abnormal Wickham yeast for preparing biological red date flavor, characterized in that: The abnormal Wickham yeast was classified and named as Wickerhamomyces anomalus, the strain was named HS-3, and it was deposited in the General Biological Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No.30675.

2. The abnormal Wickham yeast for preparing biological red date flavor according to claim 1, characterized in that: The abnormal Wickham yeast HS-3 appears as a milky white circle with a raised center, neat edges, and a smooth, moist, and sticky surface on a YPD solid culture medium plate.

3. The abnormal Wickham yeast for preparing biological red date flavor according to claim 1, characterized in that: The culture method of the abnormal Wickham yeast is as follows: the abnormal Wickham yeast liquid is inoculated into a YPD liquid culture medium at an inoculation rate of 1%, and cultured for 12 hours.

4. The abnormal Wickham yeast for preparing biological red date flavor according to claim 3, characterized in that: The culture conditions of the abnormal Wickham yeast are a temperature of 30° C. and a rotation speed of 150 rpm.

5. The abnormal Wickham yeast for preparing biological red date flavor according to claim 2, characterized in that: The formula of the YPD liquid culture medium is: 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast powder; the formula of the YPD solid culture medium is: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast powder, and 20 g / L agar.

6. An application, characterized in that: Use of the abnormal Wickham yeast according to any one of claims 1 to 5 in the preparation of biological red date flavors.

7. The use according to claim 6, characterized in that: The preparation method of biological red date flavor comprises the following steps: (1) Preparation of abnormal Wickham yeast activation solution: The abnormal Wickham yeast was activated and cultured at 30℃ and 150 r / min, and the abnormal Wickham yeast activation liquid was obtained after shaking culture for 12 hours. (2) Single colony culture: The abnormal Wickham yeast activation solution obtained in step (1) is diluted ten-fold, applied to YPD solid medium, and cultured in a 30°C constant temperature incubator for 48-72 h until a clear abnormal Wickham yeast single colony is observed; (3) Seed solution preparation: Pick a single colony of the abnormal Wickham yeast obtained in step (2), inoculate it into a YPD 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; (5) Red date spice fermentation: Take the microbial preparation from step (4) and treat with a bacterial amount of 1.85×10 8 - 2.65×10 8 cfu / g standard, the suspension was inoculated into sterilized red date juice, and then fermented in a shaker at 30°C and 150 rpm for 36 h; (6) Red date spice extraction: The red date juice fermented in step (5) is added with 75% ethanol, heated and refluxed for extraction for 2 h, and then vacuum filtered to separate the filtrate and the filter residue. 75% ethanol is further added to the filter 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 red date flavor.

8. The use according to claim 7, characterized in that: Application of biological red date flavor in cigarettes.

Images