Application of carotenoid-producing grease yeast in flue-cured tobacco fermentation and flue-cured tobacco shred fermentation method

Abstract

According to the application of the carotenoid-producing grease yeast in flue-cured tobacco fermentation, tobacco shreds are subjected to fermentation treatment, so that the smoking quality of the tobacco shreds is obviously improved, the smoke is thickened, the aroma is rich, the tobacco shreds are slightly burnt, the permeability is enhanced, the fineness is improved, and the quality improvement and aroma enhancement of the tobacco shreds are realized. The application of the grease yeast in tobacco leaf fermentation has important significance on excavation of microbial strains capable of improving the quality and flavor of flue-cured tobacco products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco processing, in particular to application of carotenoid-producing oleaginous yeast in fermentation of flue-cured tobacco. BACKGROUND

[0002] There are various chemical substances in cured tobacco leaves, however, the imbalance of some substance contents will affect the sensory quality, such as high starch content leading to poor burnability and rough smoke of flue-cured tobacco; high cellulose content making flue-cured tobacco poor in burnability and strong in irritation, and harmful substances will be produced through high-temperature thermal degradation; high protein content of flue-cured tobacco not only affects the burnability and makes the smoke contain harmful ingredients, but also produces pungent and bitter taste. Unlike the above, carotenoids are one of the most important terpenes in tobacco, which not only directly affect the appearance quality such as color and luster of tobacco leaves, but also the related degradation products are closely related to the aroma quality and quantity of tobacco leaves; in addition to increasing the oil content of tobacco leaves, the degradation products of oil components on the surface of tobacco leaves are also beneficial to the improvement of tobacco aroma quality.

[0003] Flue-cured tobacco fermentation is an important link in the process of tobacco processing, which aims to degrade macromolecular substances such as sugars, proteins and fatty acids in tobacco leaves into volatile flavor components such as acids, esters, phenols, pyridines and terpenes through the action of microorganisms and enzymes under a certain temperature and relative humidity environment, so as to alleviate the defects of uncoordinated components, lack of aroma, strong irritation and heavy bitterness of tobacco leaves, and significantly improve the aroma and taste quality of tobacco leaves. In addition to the aging process of tobacco leaves, many current studies achieve macromolecular substance degradation by treating flue-cured tobacco with enzymes and microbial fermentation, so as to promote the formation of terpene flavor substances and Maillard reaction products. Among them, applying microbial resources to tobacco fermentation is an effective strategy to improve the quality of tobacco leaves and enhance the aroma of tobacco leaves. Various enzymes produced by microorganisms can degrade macromolecular substances to form small molecular products or flavor precursor products with aroma characteristics; in addition, secondary metabolites of microorganisms such as terpenes and flavonoids also have volatile flavor characteristics and antioxidant physiological activities, which can improve the quality of tobacco leaves and form characteristic flavor quality. Based on microbial fermentation treatment of tobacco leaves, the content of macromolecular substances can be significantly degraded, and the content ratio of substances in flue-cured tobacco can be improved.

[0004] Rhodotorula is a kind of oil industrial yeast that can naturally produce carotenoids, and has strong robustness and wide substrate utilization spectrum. It can be fermented at high density by using cheap carbon sources such as cellulose hydrolysate, and has superior fermentation performance. The yeast can utilize the nutrients in cured tobacco leaves for fermentation, and can also produce carotenoids and oils that have a positive effect on the quality of tobacco leaves. Many studies have shown that the degradation products of carotenoids such as beta-carotene and lycopene in cured tobacco, such as beta-ionone and dihydroactinidiolide, are important components of tobacco aroma; the oil in tobacco leaves also has a positive effect on the senses, and may improve the quality of tobacco and the experience of consumers. At the same time, the sensory quality of cured tobacco is determined by the mutual influence of different substances, and under certain conditions, the chemical substances in tobacco leaves are degraded to the appropriate range, which is a key technical problem for improving the quality of tobacco leaves. Therefore, how to ferment the tobacco leaves based on the yeast to improve the flavor quality of the tobacco leaves is a key technical problem that needs to be solved. SUMMARY

[0005] The purpose of the present application is to provide an application of carotenoid-producing Rhodotorula in the fermentation of cured tobacco, which can significantly improve the sensory quality of the tobacco by fermenting the tobacco with Rhodotorula, making the smoke denser, the aroma more abundant, the aroma more focused, the permeability stronger, and the delicacy improved, thereby realizing the quality improvement and aroma enhancement of the tobacco. The application of the red yeast in tobacco fermentation has important significance for mining microbial strains that can improve the quality and flavor of cured tobacco products.

[0006] To solve the above technical problems, the present application provides an application of carotenoid-producing oil yeast in the fermentation of cured tobacco, wherein the oil yeast is used as a yeast preparation during fermentation.

[0007] The viable cell count of the yeast preparation is 1x10 8 CFU / mL-3x10 8 CFU / mL.

[0008] The oil yeast is Rhodotorula.

[0009] The present application also provides a method for fermenting cured tobacco, characterized in that the method comprises spraying the bacterial slurry of the above-mentioned oil yeast on the cured tobacco and mixing evenly, so that the moisture content of the cured tobacco is maintained at 10%-30% for fermentation.

[0010] The bacterial slurry has a concentration of 1x10 8 CFU / mL-3x10 8 CFU / mL, and the spraying amount of the bacterial slurry is 2%-6% of the mass of the cured tobacco.

[0011] The fermentation temperature is 25-30℃, the relative humidity of the fermentation environment is 40%-60%, and the fermentation time is 2-4 days.

[0012] The fermented tobacco is dried at 70-100 DEG C for 10-30 min, so that the moisture content of the tobacco is 5-10%.

[0013] The application further provides the flue-cured tobacco prepared by the fermentation method.

[0014] Advantages of the application

[0015] The application provides application of carotenoid-producing round red-spored yeast in fermentation of flue-cured tobacco, so that the smoking quality of the tobacco is obviously improved, the smoke is thick, the aroma is rich, the smoke is slightly focused, the permeability is enhanced, and the delicacy is improved, and the quality and aroma of the tobacco are improved. The application of the round red-spored yeast in fermentation of tobacco leaves has important significance for mining microbial strains capable of improving the quality and flavor of flue-cured tobacco products. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Oil content accumulated by round red-spored yeast Y4 in different culture media;

[0017] Figure 2 Carotenoid yield synthesized by round red-spored yeast Y4 in different culture media;

[0018] Figure 3 Growth state of round red-spored yeast Y4 in different culture media. DETAILED DESCRIPTION

[0019] The application provides application of carotenoid-producing round red-spored yeast in fermentation of flue-cured tobacco, wherein the round red-spored yeast is used as a yeast preparation in the fermentation process.

[0020] The viable cell count of the yeast preparation is 1x10 8 CFU / mL-3x10 8 CFU / mL.

[0021] The yeast preparation is activated and cultured in a nitrogen-limited culture medium, and after centrifugal collection of the fermentation liquor, the fermentation liquor is washed twice with sterile water to obtain a bacterial slurry.

[0022] The carbon-nitrogen ratio in the nitrogen-limited culture medium formula is 50-300, the carbon source is 15-30 g / L, anhydrous magnesium sulfate is 0.1 g / L, potassium dihydrogen phosphate is 0.1 g / L, and sodium phosphate dibasic is 0.1 g / L.

[0023] Preferably, the carbon source includes but is not limited to glucose, xylose, mixed sugar, and cellulose hydrolysate.

[0024] Preferably, the nitrogen source includes but is not limited to ammonium sulfate, ammonium chloride, urea, and yeast extract powder.

[0025] Preferably, the fermentation culture temperature is 25-30℃, and the culture time is 48-96h.

[0026] Preferably, the centrifugal collection of the bacterial slurry is at room temperature, 7000-9000g for 2-5min.

[0027] The application also provides a method for fermenting flue-cured tobacco shreds, characterized in that the method comprises spraying the bacterial slurry of the above-mentioned Rhodosporidium toruloides into flue-cured tobacco shreds, mixing uniformly, and fermenting the flue-cured tobacco shreds with a water content of 10%-30%.

[0028] The bacterial slurry has a concentration of 1x10 8 CFU / mL-3x10 8 CFU / mL, and the spraying amount of the bacterial slurry is 2%-6% of the mass of the flue-cured tobacco shreds.

[0029] The fermentation temperature is 25-30℃, the relative humidity of the fermentation environment is 40%-60%, and the fermentation time is 2-4 days.

[0030] The fermented tobacco shreds are dried at 70-100℃ for 10-30min to make the water content of the tobacco shreds reach 5-10%.

[0031] The application also provides flue-cured tobacco shreds prepared by the above-mentioned fermentation method.

[0032] The following embodiments and drawings are used to illustrate the embodiments of the application in detail, so that the process of how the application applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented.

[0033] 1. The ability of nitrogen-limited Rhodosporidium toruloides to produce oil and carotenoids

[0034] 1.1 Materials and methods

[0035] YPD medium: 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose.

[0036] Nitrogen-limited medium NL10: 20 g / L glucose, 0.5 g / L yeast extract powder, 3 g / L NH4Cl, 1.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 1 g / L Na2HPO4, adjust pH = 6.

[0037] Nitrogen-limited medium NL60: 20 g / L glucose, 0.5 g / L yeast extract powder, 0.33 g / L NH4Cl, 1.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 1 g / L Na2HPO4, adjust pH = 6.

[0038] Nitrogen-limited medium NL120: 20 g / L glucose, 0.25 g / L yeast extract powder, 0.166 g / L NH4Cl, 1.5 g / LMgSO4·7H2O, 1 g / L KH2PO4, 1 g / L Na2HPO4, adjust pH to 6.

[0039] Rhodosporidium toruloides AS2.1389 was purchased from China General Microbiological Culture Collection Center. It was cultured in NL10 until OD 600 After 10 days, the strains were washed and transferred to 50 mL liquid culture medium of YPD, NL10, NL60, and NL120, respectively, and cultured for 72 h for the detection of oil and carotenoid content.

[0040] Oil detection: The acid-heat method is used to extract oil from oil-producing yeast.

[0041] Carotenoid detection: 500 μL of fermentation broth was centrifuged at 10,000 × g for 1 min, and the supernatant was discarded. The cells were washed with acetone and then centrifuged and the organic phase was discarded. 0.3 g of glass beads and 500 μL of acetone were added to the cells, vortexed for 2 min, and placed on ice for 1 min. This step was repeated three times. The vortexed system was placed in a non-contact cell disruptor and disrupted for 10 min, with a 10-second pause and a 5-second rest period, at 40% power and 20°C. After ultrasonic disruption, the cells were vortexed again for 2 min, and the acetone extraction step was repeated until the cells turned bleached. After bleaching, the cells were centrifuged at 10,000 × g for 1 min, and the organic phase was filtered through a 0.22 μm membrane for high-performance liquid chromatography (HPLC). Quantitative analysis was performed using a Shimadzu LC-2030PLUS HPLC system equipped with a Waters T-nature C18 column (4.6 × 250 mm, 5 μm) and a UV detector. The mobile phase was acetonitrile:methanol:isopropanol = 1:1:1, the flow rate was 1 mL / min, the injection volume was 10 μL, the column temperature was 40°C, and the detection wavelength was 470 nm.

[0042] 1.2 Results

[0043] Rhodosporidium toruloides Y4 is more conducive to the accumulation of oil and carotenoids under nitrogen-limited culture conditions, such as Figure 1 As shown, compared to cells fermented in YPD medium, oil accumulation initially increased and then decreased with increasing carbon-nitrogen ratios in nitrogen-limited medium. Oil accumulation was highest in NL60, reaching 3.394 g / L, representing 76% of the total cell volume. Research suggests that the presence of these oils improves the oil content of tobacco leaves, transforming them into pleasant flavor components in cut tobacco.

[0044] Similar to oil synthesis, Rhodosporidium toruloides Y4 also favors the accumulation of carotenoids under nitrogen-limited culture conditions. Figure 2 As shown, the strain fermented in NL60 increased lycopene, rhodotorula erythrophyllin, γ-carotene, and β-carotene production by 114.8%, 60.8%, 226.6%, and 88.8%, respectively, compared to NL10. Therefore, Y4 accumulated the highest amounts of lipids and carotenoids in NL60, making it more suitable for fermenting tobacco.

[0045] 2. Growth status of Rhodosporidium toruloides in flue-cured tobacco leaves

[0046] 2.1 Materials and Methods

[0047] Nitrogen-limited medium (NL): 20 g / L glucose, 0.5 g / L yeast extract powder, 0.33 g / L NH4Cl, 1.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 1 g / L Na2HPO4, adjust pH to 6.

[0048] Flue-cured tobacco culture medium: Take tobacco cuts from the upper leaves B2F of Yunyan 87 produced in Yunnan, Henan, and Shandong areas, place 5 g of tobacco cuts in a 250 mL conical flask, add 100 mL of ultrapure water, and sterilize with high-pressure steam.

[0049] Rhodosporidium toruloides AS2.1389 was purchased from China General Microbiological Culture Collection Center. After activation on NL plates for 48 h, single colonies were cultured in 5 mL of liquid NL medium for 24 h at a speed of 200 rpm and a temperature of 28 °C to a value of OD 600 The Y4 seed solution was inoculated into different culture media to make the initial OD 600 The OD value was 1.5, and the cells were cultured at 28°C. The OD value was measured every 24 hours. 600 .

[0050] 2.2 Experimental Results

[0051] Growth status of Rhodosporidium toruloides Y4 in different culture media Figure 3 As shown. In the ordinary nitrogen-limited medium, Y4 reached its maximum value at 96h. Strain Y4 can grow by utilizing the nutrients slowly released by tobacco. The medium made from Yunnan tobacco can reach its maximum value at 96h, and then the growth becomes slow. The medium made from Henan tobacco also maintains a stable growth after 72h, and the growth increases at 216h. The medium made from Shandong tobacco makes the OD of the strain 600The tobacco cut filler medium was smaller than that of the other two places, but it increased at 240 h. Round red spore yeast Y4 can utilize the nutritional components in tobacco cut filler for normal growth and reproduction, and the maximum growth of the strain has little difference with that of ordinary medium, and can continuously supply the growth of the strain.

[0052] 3. Changes in the contents of substances in flue-cured tobacco after fermentation with round red spore yeast

[0053] 3.1 Materials and methods

[0054] Tobacco raw material pretreatment: Place an appropriate amount of tobacco cut filler under the ultraviolet lamp of a clean bench for 1 h.

[0055] Experimental group: Take 50 g of tobacco cut filler of upper leaves B2F of Yunnan Yunyan 87, cultivate strain Y4 in NL medium to OD 600 20, centrifuge the bacterial cells, wash twice with sterile water to make bacterial slurry. The concentration of the bacterial slurry is 2×10 8 CFU / mL, and the spraying amount of the bacterial slurry is 5% of the mass of the flue-cured tobacco cut filler. Place the treated tobacco cut filler in a fermentation environment with a relative humidity of 60% at 28°C for 4 days. After fermentation, dry the tobacco cut filler at 75°C for 10 min to make the moisture content of the tobacco cut filler reach 5-10%. The control group is 50 g of tobacco cut filler of upper leaves B2F of Yunyan 87 sprayed with an equal amount of sterile water, which is treated according to the above method.

[0056] Chemical component determination: Determine the contents of total sugar, reducing sugar, starch, total plant alkaloid, and cellulose in tobacco according to industry standard methods YC / T159-2019, YC / T216-2013, YC / T468-2013, and YC / T347-2010. Extract carotenoids in tobacco leaves using ultrasonic method, use petroleum ether-acetone (1:1) as the extraction agent, ultrasonic power is 100 W, extraction temperature is 30°C, extraction time is 20 min, and material ratio is 1:25; calculate the content of carotenoids by ultraviolet spectrophotometer, and the detection wavelength is 450 nm.

[0057] 3.2 Experimental results

[0058] Table 1 Changes in chemical components of tobacco cut filler after fermentation

[0059] Sample Total sugar / % Reducing sugar / % Starch / % Nicotine / % Cellulose / % Carotenoid / % Control 35.94 27.61 6.31 2.03 12.52 0.003 Fermented tobacco 42.35 39.52 4.30 1.78 10.32 0.1

[0060] Microbial treatment significantly increased the total sugar and reducing sugar contents of cut tobacco, while reducing the starch, nicotine, and cellulose contents. Compared to unfermented cut tobacco, the total sugar and reducing sugar contents of fermented cut tobacco increased by 17.8% and 43.13%, respectively; starch, nicotine, and cellulose contents decreased by 46.7%, 14%, and 21.3%, respectively; and carotenoid content increased by 97%. These results demonstrate that microbial treatment effectively modulates the chemical composition of tobacco leaves, achieving a more balanced balance of various substances and enhancing the content of flavor precursors.

[0061] 4. Changes in flavor composition and content of fermented tobacco leaves

[0062] 4.1 Materials and methods

[0063] Place 1g each of the tobacco treated with strain Y4 and purified water in 2.1 into a 10mL centrifuge tube. Add 2mL of dichloromethane to each tube and extract in an ultrasonic extractor at room temperature for 1 hour. Remove the organic phase after extraction and dehydrate it with anhydrous sodium sulfate. Pass the dehydrated organic phase through a 0.22μm organic filter membrane before analyzing volatile flavor components using GC-MS.

[0064] GC / MS detection conditions: (1) Gas chromatography conditions. The chromatographic column was DB-1MSUI, with specifications of (60 m × 0.25 mm × 0.25 μm). Carrier gas: He, constant flow mode; column flow rate: 1.0 mL / min; split ratio: 10:1. Inlet temperature: 250°C; programmed temperature: initial temperature 40°C, hold for 2.0 min, then increase to 260°C at a rate of 5°C / min, hold for 10.0 min. (2) Mass spectrometry conditions. Transfer line temperature: 250°C; ionization mode: electron impact source (EI); ionization energy: 70 eV; ion source temperature: 200°C; solvent delay time: 3.0 min; detection mode: full scan monitoring mode, mass scan range: 30 amu to 500 amu.

[0065] 4.2 Results

[0066] Compared to unfermented tobacco, fermented tobacco contains an increased variety of volatile flavor components and a higher content (Table 2). Fermentation added new compounds such as 4-methylhexane, 3,7,11-trimethyldodecanol, and scopolamine. Compared to unfermented tobacco, the content of new phytadiene increased by 27%, 2,3-octanedione by 369%, megastrodienone by 26%, 4-hydroxy-β-dihydrodamascone by 14%, 4,7,9-megastrodien-3-one by 22%, 9-hydroxy-4,7-megastrodien-3-one by 108%, benzyl alcohol by 60%, phenylethanol by 8%, α-ceramic acid diterpenoid alcohol and β-ceramic acid diterpenoid alcohol by 27% and 79%, respectively, myosmine and 2,3'-bipyridine by 186% and 109%, respectively. The flavor components limonene and squalene increased by 67% and 10.96%, respectively. However, the content of some ingredients decreased, such as nicotine, 2-hydroxyquinoline, α-linolenic acid, and 1-heptanetriol. The results show that microbial treatment can significantly increase the flavor components of tobacco while reducing the harmful effects of nicotine.

[0067] Table 2 Changes in volatile flavor components of tobacco after fermentation

[0068]

[0069]

[0070] 5. Sensory evaluation of fermented tobacco leaves

[0071] 5.1 Materials and Methods

[0072] After fermentation with microbial Y4 slurry, the experimental group of cut tobacco leaves from Yunnan Province and the control group, treated with sterile water, were dried at 75°C for 20 minutes to a moisture content of 5-10%. The tobacco was then rolled into cigarettes for smoking. Five experts with cigarette evaluation qualifications from the Qujing branch of the Yunnan Tobacco Company conducted the evaluation. Sensory quality was calculated according to the Yunnan Tobacco Industry Standard QYNZY.J07.022-2015, the new cigarette sensory evaluation method. The detailed evaluation criteria included smoke volume (10 points), aroma and flavor (30 points), physiological intensity (10 points), harmony (10 points), pungency (15 points), and mouthfeel (25 points). Each score was the average of the five experts.

[0073] 5.2 Results

[0074] The sensory evaluation results, shown in Table 3, showed that tobacco treated with microbial Y4 sludge had higher overall sensory scores, with significantly higher smoke volume, aroma, physiological intensity, harmony, and mouthfeel than the control group. Pungency was also lower than that of unfermented tobacco. This suggests that treatment with microbial Y4 significantly improved the aroma quality and volume of tobacco, enhancing its quality.

[0075] Table 3 Sensory evaluation of fermented tobacco

[0076] Smoke amount Flavor aroma Physiological intensity Harmony Irritation Mouthfeel Total score CK 7.92±0.24 21.65±0.47 7.5±0.41 8.2±0.24 13.21±0.24 21.17±0.47 79.65±0.14 Y4 8±0.01 25.53±0.24 8.1±0.24 8.7±0.04 12.98±0.24 23.53±0.24 86.84±0.41

[0077] All of the above are intended to be primary implementations of this intellectual property and do not constitute limitations on other implementations of such new products and / or methods. Those skilled in the art will utilize this important information and modify the above to achieve similar implementations. However, all modifications or adaptations based on this invention to new products are reserved.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. An application of a carotenoid-producing oil yeast in flue-cured tobacco fermentation, characterized in that: The oil yeast is used as a yeast preparation during the fermentation process.

2. The use of the carotenoid-producing oil yeast in tobacco fermentation as claimed in claim 1, characterized in that: The viable count of the yeast preparation is 1×10 8 CFU / mL-3×10 8 CFU / mL.

3. The use of the carotenoid-producing oil yeast in tobacco fermentation as claimed in claim 1, characterized in that: The oil yeast is Rhodosporidium toruloides.

4. A method for fermenting flue-cured tobacco shreds, characterized in that: The method comprises spraying the sludge of the oil yeast according to claim 1 or 2 onto cut flue-cured tobacco and mixing the mixture evenly, so as to maintain the moisture content of the cut flue-cured tobacco between 10% and 30% for fermentation.

5. The method for fermenting flue-cured tobacco shreds according to claim 4, wherein: The concentration of the bacterial sludge is 1×10 8 CFU / mL—3×10 8 CFU / mL, and the amount of bacterial sludge sprayed is 2%-6% of the mass of flue-cured tobacco.

6. The method for fermenting flue-cured tobacco shreds according to claim 4, wherein: The fermentation temperature is 25-30° C., the relative humidity of the fermentation environment is 40%-60%, and the fermentation time is 2-4 days.

7. The method for fermenting flue-cured tobacco shreds according to claim 4, wherein: The fermented tobacco is dried at 70-100° C. for 10-30 minutes to reduce the moisture content of the tobacco to 5-10%.

8. Flue-cured tobacco shreds prepared by the fermentation method according to any one of claims 3 to 7.

Images