Preparation method and application of biological apricot spice

Abstract

The invention belongs to the technical field of microbial spice preparation, and particularly relates to a preparation method and application of biological apricot spice. Saccharomyces cerevisiae HS-1 is used as a fermentation strain Saccharomyces cerevisiae and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30677. The biological apricot spice is prepared by fermentation, so that the content of aromatic substances such as 3-hydroxy-2-butanone and lauric acid in the spice can be remarkably increased, and the flavor of the biological apricot spice is improved. The biological apricot spice can generate new flavor substances such as isoamyl alcohol, isoamyl acetate, phenethyl alcohol, phenethyl acetate and the like, is applied to cigarettes, and can obviously improve the sweet aroma and fruity aroma of the cigarettes, increase the sweet and moist feeling of smoke and reduce irritation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial flavor preparation, and specifically relates to a preparation method of biological apricot flavor and application thereof. Background Art

[0002] In today's global flavor and fragrance industry, bio-fermentation technology utilizes the metabolic pathways in microorganisms to produce flavor molecules with exquisite structure and unique aroma through enzymatic reactions. It produces less waste liquid and waste residue, is more environmentally friendly, and the metabolic products have their own flavors. They are developed and researched on demand to accurately satisfy the taste buds of a diverse market.

[0003] In the tobacco flavoring segment, brewer's yeast fermented apricot tobacco flavoring is still in "no man's land". Cigarettes are a popular fast-moving consumer product in the world, and there is a strong demand for improvement in tobacco flavor quality, and high requirements for the safety and adaptability of flavors. It is necessary to have a rich sweet fragrance and a refreshing fruity aroma to "add points and color" to the smoke and improve the comfort of smoking, but it is also necessary to strictly adhere to the bottom line of safety and eliminate the residue of harmful chemicals. As a microorganism widely used in the food and beverage industry, brewer's yeast has good fermentation performance and metabolic characteristics, and can produce a variety of beneficial compounds during the fermentation process. The development of brewer's yeast fermented apricot flavor meets the rigid market demand for high-quality tobacco apricot flavors. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method of biological apricot flavor and application thereof, so as to meet the current high quality demand for apricot flavor.

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

[0006] A method for preparing biological apricot flavor comprises the following steps:

[0007] 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 apricot juice, and then fermented in a shaker at 30°C and 150 rpm for 36-48 h;

[0008] S2. Add 75% ethanol to the apricot 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 apricot flavor extract.

[0009] Furthermore, in step S1, the microorganism is Saccharomyces cerevisiae, which is classified and named Saccharomyces cerevisiae, with a strain name of HS-1, and is deposited in the General Biological Center of China Microorganism Culture Collection Administration with a deposit number of CGMCC No.30677.

[0010] Furthermore, the brewer's yeast HS-1 appears as milky white round colonies on the YPD solid culture medium plate, with a smooth and moist surface, a dense texture and easy to pick up.

[0011] Furthermore, the culturing method of the brewer's yeast is: inoculating the brewer's yeast liquid into the YPD liquid culture medium at an inoculation rate of 1%, and the culturing conditions are: temperature 30°C, rotation speed 150 rpm, and culturing time 12-18 h.

[0012] 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.

[0013] Furthermore, a method for preparing a microbial preparation, using any of the above-mentioned brewer's yeast, comprises the following steps:

[0014] S11. Preparation of Activated Solution of Saccharomyces cerevisiae:

[0015] The Saccharomyces cerevisiae was activated and cultured at 30℃ and 150 r / min, and the activated liquid of Saccharomyces cerevisiae was obtained after shaking culture for 12 h.

[0016] S12, single colony culture:

[0017] After diluting the activated Saccharomyces cerevisiae solution obtained in step S11 ten-fold, 100 μL was evenly applied to the YPD solid medium, and cultured in a constant temperature incubator at 30° C. for 48-72 h until a clear single colony of Saccharomyces cerevisiae was observed;

[0018] S13, seed solution preparation:

[0019] Pick a single colony of Saccharomyces cerevisiae obtained in step S12, 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;

[0020] S14. Preparation of microbial preparations:

[0021] The seed solution obtained in step S13 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.

[0022] An application, using the biological apricot flavor prepared by any one of the above items on cigarettes.

[0023] Furthermore, the amount of the biological apricot flavor applied to the cigarette is 0.25-0.35% of the mass of the tobacco.

[0024] The beneficial effects of the present invention are:

[0025] The present application utilizes brewer's yeast fermentation to prepare biological apricot flavor, and through the action of microbial fermentation, the aroma of the apricot flavor is improved, and the sensory evaluation and GC-MS analysis results both show good results. Through the sensory evaluation of cigarette flavoring, it is found that the fragrance is sweet and fruity, and the sweetness of smoke is increased and the irritation is reduced; after GC-MS analysis of the flavor components, it is found that the content of flavor substances such as 3-hydroxy-2-butanone and lauric acid is increased, and new flavor substances such as isopentanol, isopentanyl acetate, phenylethyl alcohol, and phenylethyl acetate are generated.

[0026] The biological apricot flavor is applied to cigarettes to significantly enhance the sweetness and fruity aroma of cigarettes, increase the sweetness of smoke and reduce irritation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a plate map of Saccharomyces cerevisiae used in this application.

[0028] Figure 2 This is an electron micrograph of the Saccharomyces cerevisiae of this application.

[0029] Figure 3 This is a phylogenetic tree analysis diagram of Saccharomyces cerevisiae used in this application. DETAILED DESCRIPTION

[0030] 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.

[0031] The brewer's yeast (Saccharomyces cerevisiae) HS-1 of the present application is currently deposited in the General Biological Center (CGMCC) of the China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 17, 2024, and the deposit number is CGMCC No. 30677.

[0032] The YPD liquid culture medium formula of the present application is: peptone 20g / L, glucose 20g / L, yeast powder 10g / L, and the YPD solid culture medium formula is: peptone 20g / L, glucose 20g / L, yeast powder 10g / L, agar 20g / L.

[0033] Example 1

[0034] Under sterile conditions, 5 g of tea garden soil and 50 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 volume fraction of 10% for enrichment culture for 48 h.

[0035] 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.

[0036] The preserved strains were enriched and activated in YPD medium and then inoculated into apricot juice. The culture was carried out at 30°C and 150 r / min for 36 h. After the fermentation, a preliminary sensory evaluation was carried out. The strains with obvious aroma changes were marked and preserved as HS-1.

[0037] The screened HS-1 strain (screened from tea garden soil in May 2023) was spread on YPD solid medium and cultured in a 30°C constant temperature incubator for 24 h to observe its colony morphology. The results are as follows: Figure 1 .like Figure 1 As shown, the HS-1 strain appeared as milky white round colonies on the YPD solid culture medium plate, with a smooth and moist surface, a dense texture and easy to pick up; 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.

[0038] 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.

[0039] 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.

[0040] 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 The 26S rDNA of HS-1 strain was most similar to that of AY048154.1 Saccharomyces cerevisiae NRRL Y-12632, so the strain was named Saccharomyces cerevisiae.

[0041] Example 2

[0042] (1) Preparation of bacterial preparations:

[0043] The cerevisiae yeast was activated and cultured to obtain an activation solution, which was diluted according to a certain gradient and then applied to a YPD solid culture medium, and cultured in a constant temperature incubator at 30°C for 48-72 h. A single colony on the plate was picked and inoculated into a YPD 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 was inoculated into a YPD liquid culture medium at a 1% inoculation rate, and cultured in a constant temperature shaker at 30°C and 150 r / min until the OD600nm value was 0.8-1.0, and the bacterial solution was centrifuged at 4°C and 10000 r / min for 10 min, the supernatant was discarded, and an equal amount of sterile water was re-dissolved, the centrifugation was repeated, and an equal amount of sterile water was re-dissolved to obtain a bacterial preparation.

[0044] (2) Apricot spice fermentation:

[0045] The amount of bacteria treated was 1.85×10 8 -2.83×10 8 The bacterial preparation was inoculated into the sterilized apricot juice at a 1% inoculation rate and fermented for 36 h at a fermentation temperature of 30 °C and a rotation speed of 150 rpm. An equal amount of apricot juice was treated with an equal amount of sterile water according to the above method to prepare a control apricot juice sample.

[0046] (3) Application of biological apricot flavor in cigarettes:

[0047] Take biological apricot flavor accounting for 3.0‰ of the mass of cigarette tobacco, dissolve it in ethanol twice the amount of the biological apricot flavor, 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 cigarettes, which are the test cigarettes containing biological apricot flavor.

[0048] (4) Sensory evaluation:

[0049] 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 sweetness and fruity aroma of the test cigarettes are significantly better than those of the blank cigarettes. The sweetness of the smoke is increased, it is softer and more delicate, and the irritation is reduced.

[0050] Table 1

[0051]

[0052] (5) Analysis of neutral aroma components:

[0053] The vortex cold extraction method was used to extract the aroma components of apricot 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.

[0054] Chromatographic conditions:

[0055] Chromatographic column: DB-5MS column (60m x 250μm x 0.25μm);

[0056] Inlet temperature: 280°C;

[0057] Split ratio: 3:1;

[0058] Split flow rate: 3.0 mL / min;

[0059] Carrier gas: He, flow rate 1.0 mL / min:

[0060] 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).

[0061] 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.

[0062] The results are shown in Table 2, which is a comparison of the aroma substances of apricot juice before and after the treatment with the brewer's yeast.

[0063] Table 2

[0064]

[0065] Example 3

[0066] Saccharomyces cerevisiae was spread on YPD 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 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.

[0068] The brewer's yeast 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 method for preparing biological apricot flavor, 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 apricot juice, and then fermented in a shaker at 30°C and 150 rpm for 36-48 h; S2. Add 75% ethanol to the apricot 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 apricot flavor extract.

2. The method for preparing biological apricot flavor according to claim 1, characterized in that: In step S1, the microorganism is Saccharomyces cerevisiae, which is classified and named Saccharomyces cerevisiae, and the strain name is HS-1, which is deposited in the General Biological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC No.30677.

3. The method for preparing biological apricot flavor according to claim 2, characterized in that: The brewer's yeast appears as milky white round colonies on the YPD solid culture medium plate, with a smooth and moist surface, a dense texture and easy to pick up.

4. The method for preparing biological apricot flavor according to claim 3, characterized in that: The culturing method of the brewer's yeast is as follows: the brewer's yeast liquid is inoculated into a YPD liquid culture medium at an inoculation rate of 1%, and the culturing conditions are as follows: a temperature of 30° C., a rotation speed of 150 rpm, and a culturing time of 12-18 h.

5. The method for preparing biological apricot flavor according to claim 4, 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. The method for preparing biological apricot flavor according to claim 1, characterized in that: The preparation method of the microbial preparation comprises the following steps: S11. Preparation of Activated Solution of Saccharomyces cerevisiae: The Saccharomyces cerevisiae was activated and cultured at 30℃ and 150 r / min, and the activated liquid of Saccharomyces cerevisiae was obtained after shaking culture for 12 h. S12, single colony culture: After diluting the activated Saccharomyces cerevisiae solution obtained in step S11 ten-fold, 100 μL was evenly applied to the YPD solid medium, and cultured in a constant temperature incubator at 30° C. for 48-72 h until a clear single colony of Saccharomyces cerevisiae was observed; S13, seed solution preparation: Pick a single colony of Saccharomyces cerevisiae obtained in step S12, 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; S14. Preparation of microbial preparations: The seed solution obtained in step S13 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. An application, characterized in that: Use of the biological apricot flavor prepared according to any one of claims 1 to 6 in cigarettes.

8. The use according to claim 7, characterized in that: The amount of the biological apricot flavor applied to the cigarette is 0.25-0.35% of the mass of the tobacco.

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