Method for preparing 2-phenethyl alcohol from waste tobacco

Abstract

The invention discloses a method for preparing 2-phenethyl alcohol from waste tobacco. The method comprises the following steps: (1) preparing gluconacetobacter ZT-01 seed liquid; (2) preparing a tobacco extract: mixing waste tobacco powder with 10% ethanol in proportion, performing reflux extraction on filtrate, performing rotary evaporation on the filtrate to remove ethanol, and sterilizing to obtain the tobacco extract; (3) preparing a fermented tobacco extract liquid: mixing the seed liquid in the step (1) and the tobacco extract liquid in the step (2) in proportion, and culturing and fermenting at 25-40 DEG C to obtain the fermented tobacco extract liquid; and (4) preparing a 2-phenethyl alcohol crude extract: carrying out solid-liquid separation on the fermented tobacco leach liquor obtained in the step (3), and extracting supernate with dichloromethane to obtain the 2-phenethyl alcohol crude extract. The method is simple in process, low in cost and high in efficiency, has remarkable economic and environmental benefits, provides a new way for high-value utilization of the waste tobacco, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial synthesis, and particularly relates to a method for preparing 2-phenylethanol using waste tobacco. Background Art

[0002] 2-Phenylethanol is an aromatic alcohol compound with a persistent and delicate floral fragrance, which is widely used in industries such as food, tobacco, and cosmetics, and is widely used and in large quantities worldwide. Natural 2-phenylethanol is mainly extracted from the flowers of plants such as roses and Chinese roses, with low extraction efficiency and high price. Most of the 2-phenylethanol in use is synthesized by chemical methods. Although the yield is high and the cost is low, there are problems such as serious environmental pollution and high process requirements, and most of the 2-phenylethanol produced has disadvantages such as many by-products and low quality. The fragrances prepared by microbial transformation belong to the category of natural fragrance raw materials, with advantages such as high efficiency, green and natural, and are the main research direction for the preparation of fragrances in recent years. The microbial synthesis of 2-phenylethanol mainly includes de novo synthesis and phenylalanine pathway, etc., which are relatively mature. Most are fermented and transformed by aroma-producing yeasts and Escherichia coli, etc., and the raw materials are generally substances such as glucose and phenylalanine. There is no report on the preparation of 2-phenylethanol using Gluconacetobacter.

[0003] Every year, a large amount of unusable tobacco such as low-grade tobacco leaves, tobacco stems, and tobacco dust forms waste, causing serious waste of resources and forming tobacco waste. Preparing tobacco flavor is an effective way to transform tobacco waste. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing 2-phenylethanol using waste tobacco, using Gluconacetobacter to ferment a tobacco matrix to prepare 2-phenylethanol, so as to be applied to tobacco flavoring, which is of great significance for the effective utilization of tobacco waste.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A method for preparing 2-phenylethanol using waste tobacco, comprising the following steps:

[0007] (1) Prepare a seed solution of Gluconacetobacter ZT-01 with the preservation number of CGMCC NO.21544;

[0008] (2) Prepare a tobacco extract: Mix waste tobacco dust with 10% ethanol at a mass ratio of 1:20 to 1:2, reflux and extract at 30°C to 120°C for 0.5 h to 5 h, sieve to obtain the filtrate, rotary evaporate, and sterilize to obtain the tobacco extract;

[0009] (3) Preparation of fermented tobacco extract: Mix the seed liquid from step (1) and the tobacco extract from step (2) at a volume ratio of 1:1000 - 1:10, and cultivate and ferment at 25 - 40 °C to obtain the fermented tobacco extract;

[0010] (4) Preparation of crude 2-phenylethanol: Perform solid-liquid separation on the fermented tobacco extract from step (3), and extract the supernatant with dichloromethane to obtain the crude 2-phenylethanol.

[0011] Preferably, in the above technical solution, in step (1), the preparation method of the seed liquid includes the following steps:

[0012] Prepare an aqueous solution containing glucose, peptone, yeast powder, and citric acid monohydrate, with a pH of 4.8 - 5, sterilize at 121 °C for 15 min, inoculate Gluconacetobacter sp. ZT-01, and then cultivate at a constant temperature of 25 - 40 °C and 80 - 200 rmp for 12 - 48 h to obtain the seed liquid.

[0013] Preferably, in the above technical solution, in step (1), in every 1 L of the aqueous solution, the mass ratio of glucose, peptone, yeast powder, and citric acid monohydrate is 20:5:5:1.

[0014] Preferably, in the above technical solution, in step (2), the waste tobacco powder is obtained by mixing waste low-grade tobacco leaves, tobacco stems, and tobacco dust at a mass ratio of 4:1:3, grinding them, and passing through a 50-mesh sieve.

[0015] Preferably, in the above technical solution, in step (2), mix the waste tobacco powder with 10% ethanol at a mass ratio of 1:10, reflux and extract at 90 °C for 2 h, filter through a 200-mesh sieve and take the filtrate, rotary evaporate the filtrate at a vacuum of 120 mbar and a temperature of 55 °C for 2 h, and then sterilize at 121 °C for 10 min to obtain the tobacco extract.

[0016] Preferably, in the above technical solution, in step (3), the volume ratio of the Gluconacetobacter sp. ZT-01 seed liquid to the tobacco extract is 1:100, and the fermentation conditions are: temperature 25 - 40 °C, rotation speed 50 - 300 r / min, and stirring fermentation time 12 - 96 h.

[0017] Preferably, in the above technical solution, in step (3), the fermentation conditions are: temperature 30 °C, rotation speed 150 r / min, and stirring fermentation time 72 h.

[0018] Preferably, in the above technical solution, step (4) specifically includes the following steps:

[0019] (41) Pass the fermented tobacco extract in step (3) through a 200-mesh sieve to fully remove the solid bacterial cellulose produced by the strain during fermentation. Then, centrifuge at 8000 r / min and 4 °C for 30 min to obtain the supernatant.

[0020] (42) Add an equal volume of dichloromethane to the supernatant and mix well. Extract at a constant temperature of 25 °C with rotary oscillation for 2 h at an oscillation frequency of 120 rpm. After extraction, let it stand still completely and separate the aqueous phase and the organic phase using a separating funnel. Continue to extract the aqueous phase with dichloromethane. After extracting the aqueous phase three times in total, mix the organic phases.

[0021] (43) In a fume hood, place the organic phase in a 70 °C water bath and stir and heat for 6 h to fully remove dichloromethane and obtain the crude extract of 2-phenylethanol.

[0022] Application of the crude extract of 2-phenylethanol prepared according to the above method in tobacco flavoring.

[0023] The above technical solution of the present invention has the following beneficial effects:

[0024] The present invention uses waste tobacco to prepare 2-phenylethanol, realizing the efficient conversion and resource utilization of waste. Through the fermentation of Gluconacetobacter sp. ZT-01, the components in waste tobacco are converted into high-value-added 2-phenylethanol, avoiding the environmental pollution and by-product problems of traditional chemical synthesis, and conforming to the concept of green chemistry. The 2-phenylethanol extracted by this method has high purity and strong aroma, can be directly applied to tobacco flavoring, and significantly improves the aroma and sensory quality of cigarettes. In addition, the process of the present invention is simple, low-cost, and high-efficiency, with significant economic and environmental benefits, providing a new way for the high-value utilization of waste tobacco and having broad application prospects. Specific embodiments

[0025] Now, various exemplary embodiments of the present invention will be described in detail. It should be noted that: unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps described in these embodiments do not limit the scope of the present invention.

[0026] Gluconacetobacter sp. ZT-01 is deposited in the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), deposit number: CGMCC No. 21544, deposit date: December 23, 2020.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials and reagents used, unless otherwise specified, can all be obtained through commercial channels. The equipment used in the experiments, unless otherwise specified, is well-known to those skilled in the art.

[0028] Example 1

[0029] (1). Preparation of Gluconacetobacter ZT-01 seed solution:

[0030] (1) Prepare an aqueous solution containing 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, and 1 g / L citric acid monohydrate, with a pH of 4.8 - 5, and sterilize it at 121 °C for 15 min.

[0031] (2) Inoculate Gluconacetobacter at 25 - 40 °C (preferably 30 °C), and culture it at a constant temperature with shaking at 80 - 200 rmp (optimally 150 rpm) for 12 - 48 h (generally optimally 24 h) to form a seed solution.

[0032] (2). Preparation of tobacco extract:

[0033] (1) Mix waste low-grade tobacco leaves, tobacco stems, and tobacco dust in a mass ratio of 4:1:3, grind them, and pass through a 50-mesh sieve to obtain waste tobacco powder.

[0034] (2) Mix the waste tobacco powder with 10% ethanol (the mass ratio of ethanol to water is 1:9) in a ratio of 1:20 - 1:2 (optimally 1:10), reflux and extract at 30 °C - 120 °C (optimally 90 °C) for 0.5 h - 5 h (optimally 2 h), filter through a 200-mesh sieve, take the filtrate, rotary evaporate the filtrate at a vacuum of 120 mbar and a temperature of 55 °C for 2 h to fully remove the ethanol in the system, and then sterilize it at 121 °C for 10 min to obtain the tobacco extract.

[0035] The above method uses a mixed solvent of ethanol and water to extract tobacco powder, which can effectively extract the water-soluble and alcohol-soluble substances in the tobacco powder. Removing ethanol can prevent the influence of ethanol on the microbial activity.

[0036] (3). Preparation of fermented tobacco extract:

[0037] Mix the Gluconacetobacter ZT-01 seed in step (1) with the tobacco extract in step (2) in a volume ratio of 1:1000 - 1:10 (optimally 1:100) (i.e., the inoculation amount is 0.1% - 10%, optimally 1%), set the culture temperature at 25 - 40 °C (optimally 30 °C), the rotation speed at 50 - 300 r / min (optimally 150 r / min), stir and ferment for 12 - 96 h (optimally 72 h), and obtain the fermented tobacco extract after fermentation.

[0038] (4) Preparation of crude extract of 2-phenylethanol:

[0039] (1) Pass the fermented tobacco extract in step (3) through a 200-mesh sieve to fully remove the solid bacterial cellulose produced by the strain during fermentation. Then, centrifuge at 8000 r / min and 4 °C for 30 min to obtain the supernatant.

[0040] (2) Add an equal volume of dichloromethane to the supernatant and mix well. Extract at a constant temperature of 25 °C with rotary oscillation for 2 h at an oscillation frequency of 120 rpm. After extraction, let it stand still fully and separate the aqueous phase and the organic phase using a separatory funnel. Continue to extract the aqueous phase with dichloromethane. After extracting the aqueous phase three times in total, mix the organic phases.

[0041] (3) In the fume hood, place the organic phase in a 70 °C water bath and stir and heat for 6 h to fully remove dichloromethane, obtaining a crude extract of 2-phenylethanol with a strong floral fragrance.

[0042] In the above method, dichloromethane is first extracted and then volatilized, which actually has the effect of extracting and concentrating 2-phenylethanol. The obtained crude extract contains the product 2-phenylethanol and also contains other tobacco components, and has high value as a flavor in cigarettes.

[0043] Application Example 1

[0044] (1) Prepare the crude extract of 2-phenylethanol according to the optimal parameters in Example 1 above, denoted as T.

[0045] (2) Use the tobacco extract without fermentation by Acetobacter gluconicum ZT-01, and the remaining steps and parameters are the same as those of T, denoted as CK.

[0046] (3) Detect the content of the obtained crude extract of 2-phenylethanol by HPLC. The yield is calculated according to the mass ratio of the newly produced 2-phenylethanol (deducting the content of 2-phenylethanol in CK) to the used waste tobacco powder. Add the two crude extracts of 2-phenylethanol to the Golden Leaf brand cigarettes at a dosage of 50 ppm, and organize professional smoking assessors for sensory evaluation.

[0047] The results are as follows:

[0048] (1) The analysis of the content of 2-phenylethanol is shown in Table 1. It can be seen that after fermentation, the total content of 2-phenylethanol in the crude extract is relatively high, which is 12.851 mg / mL, and the yield is 0.63%.

[0049] Table 1. Analysis of the content of 2-phenylethanol

[0050]

[0051] (2) The crude extract of 2-phenylethanol was added to cigarettes for sensory evaluation, and the results are shown in Table 2. After adding the unfermented crude extract CK, the aroma of the cigarettes was slightly improved, but the off-flavors increased, the aftertaste became worse, and the overall score decreased by 0.3 points. After adding the 2-phenylethanol crude extract T, the aroma of the cigarettes was significantly improved, the irritation was improved, and other indicators did not decrease. The total score increased by 1.3 points, and the sensory quality was significantly improved.

[0052] Table 2 Results of cigarette sensory evaluation

[0053] Sample Luster Aroma Harmony Off-odor Irritation Aftertaste Total score 0 5.0 27.5 5.0 10.0 17.5 22.0 87.0 CK 5.0 27.7 5.0 9.8 17.5 21.7 86.7 T 5.0 28.3 5.0 10.0 18.0 22.0 88.3

[0054] Note: "0" represents the cigarette sample without adding any substances.

[0055] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

1. A method for preparing 2-phenylethanol using waste tobacco, characterized in that: The following steps are involved: (1) preparing a seed solution of Gluconacetobacter ZT-01 with a deposit number of CGMCC NO.21544; (2) preparing tobacco extract: mixing discarded waste tobacco dust with 10% ethanol at a mass ratio of 1:20 to 1:2, reflux extraction at 30° C. to 120° C. for 0.5 h to 5 h, sieving the filtrate, rotary evaporating, and sterilizing to obtain tobacco extract; (3) preparing a fermented tobacco extract: mixing the seed solution of step (1) with the tobacco extract of step (2) at a volume ratio of 1:1000 to 1:10, culturing and fermenting at 25-40° C. to obtain a fermented tobacco extract; (4) Preparation of crude 2-phenylethanol extract: The fermented tobacco extract of step (3) is subjected to solid-liquid separation, and the supernatant is extracted with dichloromethane to obtain a crude 2-phenylethanol extract.

2. The method for preparing 2-phenylethanol from waste tobacco according to claim 1, characterized in that: In step (1), the method for preparing the seed solution comprises the following steps: An aqueous solution containing glucose, peptone, yeast powder and monohydrated citric acid was prepared with a pH of 4.8-5, sterilized at 121° C. for 15 min, inoculated with Gluconacetobacter sp. ZT-01, and cultured at 25-40° C. and 80-200 rpm for 12-48 h to obtain a seed solution.

3. The method for preparing 2-phenylethanol from waste tobacco according to claim 2, characterized in that: In step (1), in every 1L of aqueous solution, the mass ratio of glucose, peptone, yeast powder and citric acid monohydrate is 20:5:5:

1.

4. The method for preparing 2-phenylethanol from waste tobacco according to claim 1, characterized in that: In step (2), the waste tobacco dust is obtained by mixing and grinding waste low-grade tobacco leaves, tobacco stems and tobacco dust in a mass ratio of 4:1:3, and passing through a 50-mesh sieve.

5. The method for preparing 2-phenylethanol using waste tobacco according to claim 1, characterized in that: In step (2), the waste tobacco powder is mixed with 10% ethanol in a mass ratio of 1:10, refluxed at 90° C. for 2 h, filtered through a 200-mesh sieve, and the filtrate is rotary evaporated at a vacuum degree of 120 mbar and a temperature of 55° C. for 2 h, and then sterilized at 121° C. for 10 min to obtain a tobacco extract.

6. The method for preparing 2-phenylethanol from waste tobacco according to claim 1, characterized in that: In step (3), the volume ratio of Gluconacetobacter ZT-01 seed liquid to tobacco extract is 1:100, and the fermentation conditions are: temperature 25-40°C, rotation speed 50-300r / min, and stirring fermentation time 12-96h.

7. The method for preparing 2-phenylethanol from waste tobacco according to claim 1, characterized in that: In step (3), the fermentation conditions are: temperature 30°C, rotation speed 150r / min, and stirring fermentation time 72h.

8. The method for preparing 2-phenylethanol from waste tobacco according to claim 1, characterized in that: Step (4) specifically includes the following steps: (41) passing the fermented tobacco extract in step (3) through a 200-mesh sieve to remove the solid bacterial cellulose produced by the strain during the fermentation process, and then centrifuging at a speed of 8000 r / min and a temperature of 4° C. for 30 min to obtain a supernatant; (42) The supernatant was added with an equal volume of dichloromethane and mixed thoroughly. The mixture was extracted by rotary shaking at 25 °C for 2 h with an oscillation frequency of 120 rpm. After the extraction was completed, the mixture was allowed to stand for a long time and then the aqueous phase and the organic phase were separated using a separatory funnel. The aqueous phase was further extracted with dichloromethane. The aqueous phase was extracted three times and then the organic phase was mixed. (43) In a fume hood, the organic phase was placed in a 70 °C water bath and stirred for 6 h to completely remove dichloromethane and obtain a crude 2-phenylethanol extract.

9. Use of the crude 2-phenylethanol extract prepared according to any one of claims 1 to 8 in tobacco flavoring.

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