A method for preparing a milk tea flavor by mixed culture fermentation

Abstract

The present application relates to a kind of milk tea flavor for tobacco prepared by mixed fermentation method, belong to fermentation engineering technical field.The present application first mixes tobacco dust and tea dust, then adds water ultrasonic extraction to obtain mixed extract, carries out fermentation using Eurotium cristatum, after fermentation, constant temperature fermentation treatment is carried out by adding fresh milk and lactic acid bacteria, the fermentation product is centrifuged and concentrated, then anhydrous cream is added, emulsified and homogenized, and enzymatic reaction is carried out by adding biological enzyme, finally milk tea flavor for tobacco is obtained.The milk tea flavor for tobacco is added to cigarette, and it is found that it can increase aroma richness, give cigarette rich milk tea aroma, reduce smoke irritation and smoke is delicate and soft, so that sensory comfort is greatly improved.The method can effectively utilize tobacco dust and tea dust, solve the problem of resource waste, improve economic benefit, and can be used as a kind of tobacco flavor in cigarette to play the role of flavoring and quality improvement, has strong practicality.

Description

Technical fields:

[0001] This invention relates to a method for preparing tobacco flavoring, specifically a method for preparing tobacco milk tea flavoring using mixed microbial fermentation, belonging to the field of fermentation engineering technology. Background technology:

[0002] Flavorings and fragrances are indispensable raw materials in cigarette production, and their formulation is one of the core technologies of tobacco industrial enterprises. The application of flavorings, fragrances, and additives is closely related to the establishment and development of cigarette brands. During the tobacco product manufacturing process, flavorings and fragrances are often added to improve the aroma in order to meet production requirements and harmonize the inherent impurities of tobacco. With the implementation of the tobacco industry's "harm reduction and tar reduction" project, the resulting insufficient tobacco flavor and weakened aroma have been addressed through flavoring technology. This can compensate for the insufficient aroma of cigarettes caused by tar reduction without increasing tar production.

[0003] There are two sources of flavorings and fragrances for tobacco: natural and synthetic. Both have their limitations. Natural flavorings are expensive and have low yields, while synthetic flavorings offer limited aromas and are constrained by reaction conditions. Neither can meet the ever-growing market demand. Therefore, researchers have begun to focus on developing bio-flavorings to address this problem. Bio-flavorings utilize biotechnology such as microbial fermentation, enzyme engineering, plant tissue cell culture, and genetic engineering to prepare flavorings and fragrances. In the research and development of microbial fermentation technology for tobacco flavorings and fragrances, the screening of aroma-producing strains and the extraction and processing of plant raw materials such as tobacco are currently the main research directions in China. Many products based on bio-fermentation have shown good effects when applied to cigarettes, enhancing aroma and quality.

[0004] As the world's largest tea producer and consumer, my country has abundant tea resources, but every year a large amount of low-quality tea or tea dust generated during the production process places a certain burden on tea companies.

[0005] Meanwhile, as a major tobacco-producing country, my country ranks first in the world in both tobacco planting area and output. During the processes of tobacco leaf curing, re-drying, and cigarette production, a large amount of tobacco dust is generated that cannot be used in production, resulting in significant resource waste for tobacco companies. Improper handling of this tobacco dust can also cause environmental harm. With the development of the tobacco industry, how to efficiently utilize the potential value of this waste tobacco dust has become a hot research topic for experts in related fields.

[0006] Therefore, this application utilizes microbial mixed fermentation technology to effectively improve the utilization rate of tea leaves and tobacco dust. It effectively transforms unfavorable macromolecular substances such as starch, polysaccharides, phenols, and proteins in tea leaves and tobacco dust into a large number of aroma compounds, including alcohols, phenols, esters, aldehydes, ketones, and pyrroles. This results in the development of a high-quality natural flavoring agent with a rich and complex milk tea-like aroma, exhibiting good harmony with tobacco smoke components and effectively enhancing the aroma quality of cigarettes. Using microbial fermentation technology to prepare tobacco flavoring agents from tobacco dust and tea leaves not only aligns with the development direction of Chinese-style cigarettes proposed by the State Tobacco Monopoly Administration and meets consumer demands, but also improves resource utilization, possessing significant economic and social value. Summary of the Invention:

[0007] To address the aforementioned technical problems, the present invention aims to provide a method for preparing a milk tea-flavored flavoring for cigarettes using mixed-culture fermentation. The method first involves mixing tobacco dust and tea leaves, then adding water for ultrasonic extraction to obtain a mixed extract. Fermentation is then carried out using *Aspergillus cristatus*. After fermentation, fresh milk and lactic acid bacteria are added for constant-temperature fermentation. The fermentation product is centrifuged and concentrated, then anhydrous butter is added, followed by emulsification and homogenization. Finally, biological enzymes are added for enzymatic hydrolysis, ultimately yielding a milk tea-flavored flavoring for cigarettes. This flavoring possesses a rich and pure milk tea aroma with a naturally harmonious fragrance. When added to cigarettes, it enhances aroma richness, imparts a strong milk tea aroma, reduces smoke irritation, and produces a smooth and delicate smoke, significantly improving sensory comfort. This method effectively utilizes tobacco dust and tea leaves, solving resource waste problems, improving economic efficiency, and serves as a flavoring agent in cigarettes, enhancing aroma and quality, demonstrating strong practicality.

[0008] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0009] One of the technical solutions provided by the present invention is a method for preparing a milk tea flavoring for tobacco. The method first involves extracting tobacco dust and tea dust with water using ultrasound, followed by fermentation with *Aspergillus cristatus*. Then, milk and lactic acid bacteria are added for further fermentation. Finally, cream is added for enzymatic hydrolysis by protease and lipase to obtain the milk tea flavoring for tobacco.

[0010] Furthermore, the specific steps of the method are as follows:

[0011] (1) Take tobacco dust and tea dust, add water and extract by ultrasonication, and filter to obtain a mixed extract of tobacco dust and tea dust;

[0012] Furthermore, the tobacco dust and tea dust are pulverized and passed through an 80-150 mesh sieve;

[0013] Furthermore, the weight ratio of the tobacco dust to the tea dust is 1-5:1-5;

[0014] Furthermore, the tobacco dust is waste material generated during the tobacco leaf processing, re-drying, and cigarette production process; the tea leaves are at least one of black tea, green tea, and oolong tea.

[0015] Furthermore, during ultrasonic extraction, water is added at a total weight of 5 to 15 times that of tobacco and tea dust; the ultrasonic extraction conditions are: temperature of 30 to 70°C, and ultrasonic extraction time of 1 to 5 hours.

[0016] (2) Add *Aspergillus cristatus* seed liquid and glucose to the mixed extract of tobacco dust and tea dust for fermentation to obtain a mixed fermentation liquid of tobacco dust and tea dust;

[0017] Furthermore, the concentration of *Coronavirus cristatum* spores reached 10 after inoculation. 4 -10 6 cells / mL;

[0018] Furthermore, the concentration of glucose after addition is 0.02-0.05 g / mL;

[0019] Furthermore, the fermentation conditions are: temperature 20-30℃, fermentation time 3-7 days, 100-200 r / min;

[0020] Furthermore, the aforementioned *Aspergillus cristatus*, also known as *Aspergillus cristatus*, has the accession number CICC 41783;

[0021] Preferably, the preparation method of the *Aurotriarcha* seed liquid is as follows: *Aurotriarcha* strain is inoculated onto a CZG plate, and after being incubated at 28°C for 7 days, the *Aurotriarcha* spores scraped from a sterile glass rod are diluted with sterile water to obtain *Aurotriarcha* seed liquid;

[0022] (3) Add milk and lactic acid bacteria to the fermentation liquid of tobacco dust and tea dust for fermentation;

[0023] Furthermore, the amount of milk added is 0.9 to 10 times the weight of the fermentation liquid containing tobacco dust and tea dust;

[0024] Furthermore, the lactic acid bacteria include, but are not limited to, Lactobacillus bulgaricus, Lactobacillus casei, and Streptococcus lactis, preferably Lactobacillus bulgaricus, and more preferably Lactobacillus delbrueckii subsp. bulgaricus, accession number CICC 22153;

[0025] Furthermore, the bacterial concentration of the lactic acid bacteria after inoculation was 10. 4 -10 6 cfu / mL;

[0026] Furthermore, the fermentation conditions are: temperature of 30-40℃, fermentation time of 12-48h, and 100-200r / min;

[0027] (4) After fermentation, sterilize and centrifuge to obtain the supernatant, and then concentrate to obtain the concentrated liquid;

[0028] Furthermore, the centrifugation conditions were 6000 r / min and the centrifugation time was 5 min;

[0029] Furthermore, the volume concentration factor of the supernatant is 1 to 5 times;

[0030] (5) Add anhydrous butter to the concentrate and emulsify and homogenize;

[0031] Furthermore, the amount of anhydrous butter added is 0.3-0.7 times the mass of the concentrate;

[0032] Furthermore, the emulsification and homogenization conditions are: emulsification homogenizer speed 5000-15000 r / min, time 1-5 min;

[0033] (6) Adjust the pH to 5-7, add protease and lipase to carry out enzymatic hydrolysis, and inactivate the enzymes after the enzymatic hydrolysis to obtain the milk tea flavoring for tobacco.

[0034] Furthermore, the amount of protease added is 0.1-2% (w / w) of the weight of the concentrated emulsion after homogenization, the amount of lipase added is 0.1-2% (w / w) of the weight of the concentrated emulsion after homogenization, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 3-48h.

[0035] Furthermore, the enzyme inactivation conditions are: temperature 100–120℃, time 15–30 min;

[0036] Furthermore, after enzyme inactivation and centrifugation, the supernatant is used as a flavoring agent for tobacco milk tea.

[0037] The second technical solution provided by the present invention is a milk tea flavoring prepared by the method described in the first technical solution.

[0038] The third technical solution provided by the present invention is the application of the milk tea flavoring described in the second technical solution, especially in the field of cigarettes, and more particularly, it is used as a tobacco flavoring to enhance the aroma and quality of cigarettes.

[0039] Furthermore, the method of using the milk tea flavoring in cigarettes is as follows: the prepared milk tea flavoring is evenly sprayed into the tobacco and rolled into finished cigarettes, with the spraying amount being 0.1-0.5‰ (w / w) of the tobacco mass.

[0040] The present invention has the following beneficial effects:

[0041] (1) This invention solves the problem of waste of a large amount of low-quality tea leaves or tea dust generated during the production process of tea enterprises and a large amount of tobacco dust generated during the production process of tobacco enterprises, improves the utilization rate of resources, and has important economic value and social significance.

[0042] (2) This invention employs mixed-culture fermentation, which fills the gaps in single-culture fermentation, resulting in a richer aroma in the flavorings produced. The various enzymes secreted by *Aspergillus cristatus* transform large, undesirable molecules such as polysaccharides and proteins in tea and tobacco dust into numerous small-molecule aroma compounds, eliminating their adverse effects. Furthermore, the linalool and its oxides, benzyl alcohol, phenethyl alcohol, geraniol, palmitic acid, and other substances produced during mixed-culture fermentation enhance the aroma and quality of cigarette smoke, imparting a sweet tea and linalool fragrance. The resulting diacetone, 3-hydroxybutanone, caprylic acid, capric acid, ketones, lactones, and sulfur-containing compounds contribute to a rich milky aroma. These substances collectively impart a strong milky tea aroma to the smoke. The flavorings prepared through mixed-culture fermentation have a richer aroma quality than those produced through single-culture fermentation.

[0043] (3) This invention combines microbial fermentation and enzymatic hydrolysis to prepare a milk tea-flavored flavoring for tobacco. The resulting flavoring has a rich, pure milk tea aroma and a naturally harmonious fragrance, thus improving the overall quality of the flavoring. While enzymatic methods for preparing milk-flavored flavorings produce a strong aroma, the overall flavor profile is relatively monotonous. Microbial fermentation produces a mild, natural aroma, but the aroma intensity is insufficient and the yield of flavor compounds is low. Therefore, this invention combines the two methods. Lactic acid bacteria are used for fermentation to produce substances that constitute the characteristic milk flavor, such as diacetone and 3-hydroxybutanone, and to degrade lactose into lactic acid and organic acids. Then, enzymatic hydrolysis is used to modify the fermentation broth. Lipases hydrolyze milk fat to release free fatty acids, which enhance the milk aroma. Therefore, compared to using microorganisms for fermentation and enzymatic hydrolysis alone, the milk tea-flavored flavoring for tobacco prepared using the combination of microbial fermentation and enzymatic hydrolysis in this invention has a rich, pure milk tea aroma and a naturally harmonious fragrance, thus improving the overall quality of the flavoring.

[0044] (4) This invention uses mixed fermentation to process tea dust and tobacco dust to prepare flavorings. Nicotine in tobacco dust can increase the aroma of tobacco. Nicotine is not decomposed during mixed fermentation. It can be seen that by using the method and fermentation strain of this invention, not only can the yield of flavoring substances be increased, but also the problem of insufficient tobacco flavor caused by tar reduction can be solved.

[0045] (5) This invention utilizes the mixed fermentation of tea dust and tobacco dust by *Aspergillus cristatus* and lactic acid bacteria to prepare flavorings. It has the advantages of readily available raw materials, low cost, simple reaction, mild conditions, and unique flavoring style.

[0046] (6) The milk tea flavoring for cigarettes prepared by the present invention can increase the richness of aroma in cigarettes, give cigarettes a strong milk tea aroma, reduce the irritation of smoke and make the smoke delicate and soft, thus greatly improving the sensory comfort. Detailed implementation method:

[0047] The technical solution of the present invention will be further illustrated below through specific embodiments. However, these embodiments are only used to help understand the present invention and should not be construed as limiting the present invention.

[0048] The *Aspergillus cristatus* used in the following embodiments of the present invention is specifically *Aspergillus cristatus*, accession number CICC 41783;

[0049] The Lactobacillus bulgaricus used in the following embodiments of the present invention is specifically Lactobacillus delbrueckii subsp. bulgaricus, with accession number CICC 22153.

[0050] The present invention will be further explained and illustrated below through specific embodiments.

[0051] Example 1: A method for preparing tobacco milk tea flavoring using mixed-culture fermentation

[0052] This embodiment provides a method for preparing a milk tea flavoring for tobacco using mixed-culture fermentation. The preparation method is as follows:

[0053] (1) In a sterile laminar flow hood, *Aspergillus cristatus* inoculum was inoculated onto CZG plates and cultured at 28°C for 7 days. *Aspergillus cristatus* spores scraped from a sterile glass rod were then diluted with sterile water, and the concentration of *Aspergillus cristatus* seed culture was obtained by hemocytosis. Specifically, the concentration of *Aspergillus cristatus* spores was 10-1. 6 per mL.

[0054] (2) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0055] (3) Add 10 mL of *Eurotium cristatum* seed liquid and 20 g of glucose to the mixed extract of tobacco dust and tea dust in step (2), and ferment for 5 days at 28°C and 150 rpm to obtain the mixed fermentation liquid of tobacco dust and tea dust.

[0056] (4) Accurately weigh 550g of fresh milk, sterilize it, and then add the mixed fermentation liquid of tobacco and tea leaves from step (3). Add Lactobacillus bulgaricus inoculum, and the lactic acid bacteria concentration after inoculation is 10. 6 CFU / mL, fermented at 37℃ and 150 rpm for 24 h.

[0057] (5) After sterilizing the fermentation product in step (4), centrifuge to obtain the supernatant, and then concentrate it 4 times to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0058] (6) Add 100g of anhydrous butter to step (5), emulsify and homogenize at 10000r / min for 5min, and then adjust the pH to 7.0.

[0059] (7) In step (6), add 1.2g of protease (the amount of protease added is 3‰ (w / w) of the weight of the concentrated emulsion after homogenization) and 1.2g of lipase (the amount of lipase added is 3‰ (w / w) of the weight of the concentrated emulsion after homogenization), and enzymatically hydrolyze at 50℃ for 10h. The prepared reactants are subjected to enzyme inactivation treatment at high temperature (120℃, 15min), and then centrifuged at 6000r / min for 5min. The supernatant is the tobacco milk-flavored flavoring, which has a rich and pure milk aroma and a natural and harmonious fragrance.

[0060] The prepared tobacco flavoring was evenly sprayed onto the tobacco using an electric sprayer at a weight of 0.1‰ (w / w) based on the tobacco shreds, and then rolled into finished cigarettes. The sensory evaluation method for the single flavoring was adopted, and the evaluation was conducted by professionals with national cigarette sensory evaluation qualifications (this patent and the tobacco industry generally refer to the following reference for evaluation: Xie Jianping, Zong Yongli, Qu Zhan, "Establishment and Application of Evaluation Method for the Role of Single Flavoring in Cigarettes", Tobacco Science and Technology, 2008(04)).

[0061] The evaluation results show that the prepared tobacco flavoring has aromas such as sweet, fruity, milky, floral and refreshing sweetness, exhibiting a rich and complex milk tea aroma. Adding it to cigarettes can significantly enhance the aroma and flavor, giving cigarettes a strong milk tea aroma, reducing the irritation of the smoke, and significantly improving the smoothness and sensory comfort of the smoke.

[0062] Comparative Example 1:

[0063] This comparative example provides a method for first fermenting with *Aspergillus cristatus* followed by lactic acid bacteria fermentation. The preparation method is as follows:

[0064] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0065] (2) Add 10 mL of *Eurotium cristatum* seed liquid (see Example 1 for seed liquid preparation) and 20 g of glucose to the mixed extract of tobacco dust and tea dust in step (2). Ferment at 28°C and 150 rpm for 5 days to obtain a mixed fermented liquid of tobacco dust and tea dust, and then sterilize it.

[0066] (3) Accurately weigh 550g of fresh milk, sterilize it, and then add the mixed fermentation liquid of tobacco and tea leaves from step (3). Add 11g of Lactobacillus bulgaricus inoculum. After inoculation, the lactic acid bacteria concentration is 10. 6 CFU / mL, fermented at 37℃ and 150 rpm for 24 h.

[0067] (4) After sterilizing the fermentation product in step (3), centrifuge to obtain the supernatant, and then concentrate it to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0068] (5) Add 100g of anhydrous butter to step (4), emulsify and homogenize at 10000r / min, and then adjust the pH to 7.0.

[0069] (6) Add 1.2g of protease and 1.2g of lipase to step (5) and enzymatically hydrolyze for 10h at 50℃. The prepared reaction mixture is subjected to enzyme inactivation treatment at high temperature and then centrifuged at 6000r / min for 5min to obtain the flavoring of comparative example (1).

[0070] (7) Using an electric sprayer, the prepared milky flavoring for tobacco is evenly sprayed onto the tobacco at a rate of 0.1‰ (w / w) of the tobacco weight, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring is conducted by professionals with national cigarette sensory evaluation qualifications.

[0071] (8) The aroma evaluation results showed that the aroma of the prepared Comparative Example 1 spice tea sample was not obvious, mainly milky, with a slight sweet and floral aroma.

[0072] Comparative Example 2

[0073] This comparative example provides a method for first performing lactic acid bacteria fermentation followed by mixed fermentation with lactic acid bacteria and *Aspergillus cristatus*, the preparation method of which is as follows:

[0074] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0075] (2) Accurately weigh 550g of fresh milk, sterilize it, add the tobacco extract from step (1), add 11g of Lactobacillus bulgaricus inoculum, and after inoculation, the lactic acid bacteria concentration is 10. 6 CFU / mL, fermented at 37℃ and 150 rpm for 24 h.

[0076] (3) Add 10 mL of *Aspergillus cristatus* seed liquid (see Example 1 for seed liquid preparation) and 20 g of glucose to step (2), and ferment at 28 °C and 150 rpm for 5 days to obtain mixed fermentation broth.

[0077] (4) After sterilizing the fermentation product in step (3), centrifuge to obtain the supernatant, and then concentrate it to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0078] (6) Add 100g of anhydrous butter to step (5), emulsify and homogenize at 10000r / min, and then adjust the pH to 7.0.

[0079] (7) Add 1.2g of protease and 1.2g of lipase to step (6) and enzymatically hydrolyze for 10h at 50℃. The prepared reaction mixture is subjected to enzyme inactivation treatment at high temperature and then centrifuged at 6000r / min for 5min to obtain the flavoring of comparative example (2).

[0080] (8) Using an electric sprayer, the flavoring agent prepared in Comparative Example 2 was evenly sprayed onto the tobacco shreds at a rate of 0.1‰ (w / w) of the tobacco shreds, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring agent was conducted by professionals with national cigarette sensory evaluation certification.

[0081] The aroma evaluation results showed that the prepared Comparative Example 2 fragrance had certain floral, fruity and woody aromas, and had moderate milk tea aroma characteristics, with a relatively mild aroma.

[0082] Comparative Example 3:

[0083] This comparative example provides a method for enzymatic hydrolysis after mixed-culture fermentation without the addition of anhydrous butter. The preparation method is as follows:

[0084] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0085] (2) Add 10 mL of *Aspergillus cristatus* seed liquid (see Example 1 for seed liquid preparation) and 20 g of glucose to the mixed extract of tobacco dust and tea dust in step (2). Ferment at 28°C and 150 rpm for 5 days to obtain the mixed fermentation liquid of tobacco dust and tea dust.

[0086] (3) Accurately weigh 550g of fresh milk, sterilize it, add the mixed fermentation liquid of tobacco and tea powder in step (2), add 11g of Lactobacillus bulgaricus, and ferment at a constant temperature of 37℃ and 150rpm for 24h.

[0087] (4) After sterilizing the fermentation product in step (4), centrifuge to obtain the supernatant, and then concentrate it to 300g to obtain the comparative example (3) fragrance, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0088] (5) Using an electric sprayer, the prepared flavoring of comparative example (3) was evenly sprayed onto the tobacco shreds at a rate of 0.1‰ (w / w) based on the tobacco shred weight, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring was conducted by professionals with national cigarette sensory evaluation certification.

[0089] (6) The aroma evaluation results showed that the prepared Comparative Example 3 fragrance had a tea-like aroma and a milky aroma, but the milky aroma was monotonous and not rich, and the aroma was not harmonious.

[0090] Comparative Example 4:

[0091] This comparative example provides a method for the direct simultaneous fermentation of *Aspergillus cristatus* and lactic acid bacteria, and the preparation method is as follows:

[0092] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0093] (2) Add 10 mL of *Aspergillus cristatus* seed solution (see Example 1 for seed solution preparation) and 20 g of glucose, 550 g of sterilized fresh milk, and *Lactobacillus bulgaricus* inoculum (the lactic acid bacteria concentration after inoculation is 10) to the tobacco and tea powder mixed extract in step (1). 6 Fermentation (cfu / mL) was carried out at 28℃ and 150 rpm for 5 days.

[0094] (3) After sterilizing the fermentation product in step (2), centrifuge to obtain the supernatant, and then concentrate it to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0095] (4) Add 100g of anhydrous butter to step (3), emulsify and homogenize at 10000r / min, and then adjust the pH to 7.0.

[0096] (5) Add 1.2g of protease and 1.2g of lipase to step (4) and enzymatically hydrolyze for 10h at 50℃. The prepared reaction mixture is subjected to enzyme inactivation treatment at high temperature and then centrifuged at 6000r / min for 5min to obtain the flavoring of comparative example (4).

[0097] (6) Using an electric sprayer, the prepared flavoring from Comparative Example 4 was evenly sprayed onto the tobacco shreds at a rate of 0.1‰ of the tobacco shreds mass, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring was conducted by professionals with national cigarette sensory evaluation certification.

[0098] The aroma evaluation results showed that the prepared Comparative Example 4 fragrance had a similar floral sweet aroma with a slightly creamy fatty aroma.

[0099] Comparative Example 5:

[0100] This comparative example provides a method for preparing tobacco flavoring by fermenting only *Aspergillus cristatus*, and the preparation method is as follows:

[0101] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0102] (2) Add 10 mL of *Aspergillus cristatus* seed liquid (see Example 1 for seed liquid preparation) and 20 g of glucose to the mixed extract of tobacco dust and tea dust in step (1), and ferment at 28°C and 150 rpm for 5 days to obtain the mixed fermentation liquid of tobacco dust and tea dust.

[0103] (3) After sterilizing the fermentation product in step (2), centrifuge to obtain the supernatant, and then concentrate it to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0104] (4) Add 100g of anhydrous butter to step (3), emulsify and homogenize at 10000r / min for 5min, and then adjust the pH to 7.0.

[0105] (5) Add 1.2g of protease and 1.2g of lipase to step (4) and enzymatically hydrolyze for 10h at 50℃. The prepared reaction mixture is subjected to enzyme inactivation treatment at high temperature and then centrifuged at 6000r / min for 5min to obtain the flavoring of comparative example (5).

[0106] (6) Using an electric sprayer, the prepared tobacco flavoring is evenly sprayed onto the tobacco shreds at a ratio of 0.1‰ of the tobacco shred weight, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring is conducted by professionals with national cigarette sensory evaluation certification.

[0107] The olfactory evaluation results showed that the prepared comparative example (5) fragrance had similar floral and fatty aromas, with a slight caramel and woody aroma.

[0108] Comparative Example 6:

[0109] This comparative example provides a method for preparing flavorings using only lactic acid bacteria fermentation, and the preparation method is as follows:

[0110] (1) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 500mL of water, and ultrasonically extract at 50℃ for 2 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0111] (2) Accurately weigh 550g of fresh milk, sterilize it, add the tobacco extract from step (1), and add 11g of Lactobacillus bulgaricus inoculum (the lactic acid bacteria concentration after inoculation is 10). 6 The mixture (cfu / mL) was fermented at 37℃ and 150 rpm for 24 hours.

[0112] (3) After sterilizing the fermentation product in step (2), centrifuge to obtain the supernatant, and then concentrate it to 300g, wherein the centrifuge speed is 6000r / min and the centrifugation time is 5min.

[0113] (4) Add 100g of anhydrous butter to step (3), emulsify and homogenize at 10000r / min for 5min, and then adjust the pH to 7.0.

[0114] (5) Add 1.2g of protease and 1.2g of lipase to step (4) and enzymatically hydrolyze at 50℃ for 10h. The prepared reaction mixture is subjected to enzyme inactivation treatment at high temperature, and then centrifuged at 6000r / min for 5min to obtain the flavoring of Comparative Example 6.

[0115] (6) Using an electric sprayer, the flavoring agent prepared in Comparative Example 2 was evenly sprayed onto the tobacco shreds at a rate of 0.1‰ of the tobacco shreds mass, and then rolled into finished cigarettes. The sensory evaluation of the single flavoring agent was conducted by professionals with national cigarette sensory evaluation certification.

[0116] The aroma evaluation results showed that the prepared comparative example (6) fragrance had floral and milky aromas, a slight sweet aroma and a caramel aroma.

[0117] Experimental Example 1: Evaluation of the Sensory Effects of Fragrances

[0118] Using unflavored reference cigarettes as a carrier, the sensory evaluation method for single-component flavorings was conducted, mainly evaluating the sensory effects of the flavorings from Example 1 and Comparative Examples 1-6 in cigarettes from three aspects: aroma characteristics, smoke characteristics, and taste characteristics. The flavorings prepared in Example 1 and Comparative Examples 1-6 were evenly sprayed onto the tobacco shreds at a dosage of 0.1‰ of the tobacco shreds using an electric sprayer, and then rolled into finished cigarettes. The sensory evaluation method for single-component flavorings was used, and the evaluation was conducted by professionals with national cigarette sensory evaluation qualifications. Higher scores were awarded for better performance in each indicator (using a 5-point scoring system; a positive score indicates a better effect compared to the reference cigarette, with higher scores for greater improvement). Specific evaluation results are as follows.

[0119] Table 1. Sensory evaluation results of cigarettes

[0120]

[0121]

[0122] As shown in Table 1, compared with Comparative Examples 1-6, Example 1 exhibits the best sensory effects. The flavoring prepared in Example 1 significantly enhances the aroma quality, aroma quantity, and permeability of cigarettes, reduces off-flavors and irritation, improves the smoothness of the smoke, and enhances the aftertaste, thus significantly improving the sensory comfort of cigarettes. It also imparts a rich milky aroma and a sweet, tea-like fragrance to the cigarettes. Compared with Example 1, the flavoring prepared in Comparative Example 1 without mixed-culture fermentation and the flavoring prepared in Comparative Example 3 without enzymatic hydrolysis are less effective in improving cigarette aroma, and their milky and sweet, tea-like aromas are not as good as those in Example 1. Comparative Examples 2 and 4 have a positive effect on cigarette aroma, but the effect is worse than that of Example 1. The intensity of the milky and sweet, tea-like aromas in Comparative Examples 2 and 4 is slightly lower than that in Example 1. Comparative Examples 5 and 6, because they use only a single strain of bacteria to prepare the flavoring, have the worst effect in enhancing the aroma quality, aroma quantity, and taste comfort of cigarettes.

[0123] In addition, the sensory effects of Examples 1, 2, and 4 were better than those of Comparative Examples 1, 5, and 6, indicating that the effect of the interaction between *Aspergillus cristatus* and lactic acid bacteria was better than that of single-strain interaction; the sensory effect of Example 1 was better than that of Comparative Examples 2 and 4, indicating that the effect of fermenting *Aspergillus cristatus* first and then fermenting with lactic acid bacteria was better than the effect of fermenting with lactic acid bacteria first and then fermenting with *Aspergillus cristatus* or the effect of simultaneous fermentation.

[0124] Experimental Example 2: GC-MS Analysis of Aroma Components

[0125] The fragrances prepared in Example 1 and Comparative Examples 1-6 were filtered through a 0.45 μm organic filter membrane and the aroma components in different fragrances were detected and analyzed by GC-MS. The analysis results are shown in Table 2.

[0126] Table 2 GC-MS detection results of different fermentation products

[0127]

[0128]

[0129] Note: "trace" indicates that it was not detected.

[0130] As shown in Table 1, a total of 21 aroma substances were detected in the flavoring prepared in Example 1. The composition and content of these substances were significantly higher than those prepared in Comparative Examples 1-6. Example 1 used mixed-culture fermentation to process tobacco dust and tea dust extracts, which can transform unfavorable macromolecular substances into aroma substances such as alcohols, phenols, esters, aldehydes, ketones, and pyrroles, thereby developing a high-quality natural flavoring with a rich and abundant milk tea-like aroma, which can effectively improve the aroma quality of cigarettes.

[0131] Substances such as 3-methyl-2-butenol, linalool oxide, linalool, benzyl alcohol, phenethyl alcohol, citronellol, hydroxycitronellol, isoeugenol, methyl salicylate, β-ionone, and methylheptenone can impart rich fruity, sweet, floral, and woody aromas to cigarette smoke, significantly improving the aroma quality and quantity of cigarettes, reducing irritation and dryness, and enhancing taste comfort.

[0132] Substances such as 2-undecyl alcohol, ethyl palmitate, butyl undecyl lactone, butyl nonyl lactone, butyl decyl lactone, (E,E)-2,4-nonadienal, E-2-decenal, dimethyl diacetate, and acetoin can impart rich aromas such as milky, fatty, sweet, floral, and fruity notes to cigarette smoke, increasing the amount of aroma and sweetness, improving the transparency and smoothness of smoke, and enhancing the aftertaste.

[0133] In addition, 2-undecyl alcohol, citronellol, hydroxycitronellol, isoeugenol, and β-ionone were only detected in the flavoring of Example 1. The above flavoring compounds can significantly improve the aroma quality and richness of cigarettes. However, tea pyrrole has a caramel aroma, which has a negative effect on the sweet aroma of cigarettes. It was only detected in the flavorings prepared in Comparative Examples 1-6 and was not detected in Example 1.

[0134] Nicotine comes from tobacco dust. During the fermentation process, it is almost unaffected by the decomposition of *Aspergillus cristatus*, lactic acid bacteria, and biological enzymes. Its content is comparable to that of nicotine in the original tobacco dust. This is closely related to the fermentation strains and enzymes selected in this application. On the one hand, it decomposes macromolecular substances to increase aroma substances, and on the other hand, it maintains the nicotine content, enhances the smoking satisfaction and physiological intensity of cigarettes, and increases the smoke concentration and aroma richness.

[0135] Example 2: A milk tea flavoring for tobacco

[0136] This embodiment provides a method for preparing a milk tea flavoring for tobacco using mixed-culture fermentation. The preparation method is as follows:

[0137] (1) In a sterile laminar flow hood, *Aspergillus cristatus* inoculum was inoculated onto CZG plates and cultured at 28°C for 7 days. *Aspergillus cristatus* spores scraped from a sterile glass rod were then diluted with sterile water, and the concentration of *Aspergillus cristatus* seed culture was obtained by hemocytosis. Specifically, the concentration of *Aspergillus cristatus* spores was 10-1. 6 per mL.

[0138] (2) After crushing the tobacco powder and tea powder, pass them through a 100-mesh sieve. Accurately weigh 25g of tobacco powder and tea powder, add 250mL of water, and ultrasonically extract at 70℃ for 5 hours. After cooling to room temperature, filter to obtain a mixed extract of tobacco powder and tea powder.

[0139] (3) Add 10 mL of *Eurotium cristatum* seed liquid and 10 g of glucose to the mixed extract of tobacco dust and tea dust in step (2), and ferment for 7 days at 28°C and 150 rpm to obtain the mixed fermentation liquid of tobacco dust and tea dust.

[0140] (4) Accurately weigh 600g of fresh milk, sterilize it, and then add the mixed fermentation liquid of tobacco and tea leaves from step (3). Add Lactobacillus bulgaricus inoculum, and the lactic acid bacteria concentration after inoculation is 10. 6 CFU / mL, fermented at 37℃ and 200 rpm for 48 h.

[0141] (5) After sterilizing the fermentation product in step (4), centrifuge to obtain the supernatant, and then concentrate it 3 times, wherein the centrifuge speed is 6000 r / min and the centrifugation time is 5 min.

[0142] (6) Add anhydrous butter at 0.5 times the mass of the concentrate in step (5), emulsify and homogenize at 10000r / min for 5min, and then adjust the pH to 7.0.

[0143] (7) In step (6), add protease (the amount of protease added is 5‰ (w / w) of the weight of the concentrated emulsion after homogenization) and lipase (the amount of lipase added is 5‰ (w / w) of the weight of the concentrated emulsion after homogenization), and enzymatically hydrolyze at 50℃ for 20h. The prepared reactants are subjected to enzyme inactivation treatment at high temperature (120℃, 15min), and then centrifuged at 6000r / min for 5min. The supernatant is the tobacco milk-flavored flavoring, which has a rich and pure milky aroma and a natural and harmonious fragrance.

[0144] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A method for preparing a milk tea-flavored flavoring for tobacco, characterized in that, The method first involves ultrasonically extracting tobacco dust and tea dust with water, followed by fermentation with *Aspergillus cristatus*, then adding milk and lactic acid bacteria for further fermentation, and finally adding cream for enzymatic hydrolysis with proteases and lipases to obtain a tobacco-grade milk tea flavoring. The aforementioned *Aspergillus cristatus* is specifically *Aspergillus cristatus* (…). Aspergillus cristatus ), accession number CICC 41783; The lactic acid bacteria mentioned are specifically *Lactobacillus delbrueckii* subsp. *bulgaricus* (…). Lactobacillus delbrueckii subsp.bulgaricus ), accession number CICC 22153; The specific steps of the preparation method are as follows: (1) Take tobacco dust and tea dust, add water and ultrasonically extract, and filter to obtain a mixed extract of tobacco dust and tea dust; (2) Add *Eurotium cristatum* seed liquid and glucose to the mixed extract of tobacco dust and tea dust for fermentation to obtain a mixed fermentation liquid of tobacco dust and tea dust, without sterilization treatment; (3) Add milk and lactic acid bacteria to the mixed fermentation liquid of tobacco dust and tea dust for fermentation; (4) After fermentation, sterilize and centrifuge to obtain the supernatant, and then concentrate to obtain the concentrated liquid; (5) Add anhydrous butter to the concentrate and emulsify and homogenize; (6) Adjust the pH to 5~7, add protease and lipase to carry out enzymatic hydrolysis, and inactivate the enzyme after the enzymatic hydrolysis is completed to obtain the milk tea flavoring for tobacco.

2. The method for preparing the tobacco milk tea flavoring as described in claim 1, characterized in that, In step (1), the weight ratio of tobacco dust to tea dust is 1-5:1-5; during ultrasonic extraction, water is added at a ratio of 5 to 15 times the total weight of tobacco dust and tea dust.

3. The method for preparing the tobacco milk tea flavoring as described in claim 1, characterized in that, In step (2), the concentration of *Aspergillus cristatus* spores reached 10 after inoculation. 4 -10 6 The concentration of added glucose is 0.02-0.05 g / mL; the fermentation conditions are: temperature 20-30℃, fermentation time 3-7 days, 100-200 r / min.

4. The method for preparing the tobacco milk tea flavoring as described in claim 1, characterized in that, In step (3), the amount of milk added is 0.9-10 times the weight of the fermentation liquid containing tobacco dust and tea dust; In step (4), the volume concentration factor of the supernatant is 1-5 times.

5. The method for preparing the milk tea flavoring for tobacco as described in claim 1, characterized in that, In step (5), the amount of anhydrous butter added is 0.3-0.7 times the mass of the concentrate.

6. The method for preparing the tobacco milk tea flavoring as described in claim 1, characterized in that, In step (6), the amount of protease added is 0.1-2% of the weight of the concentrate after emulsification and homogenization, and the amount of lipase added is 0.1-2% of the weight of the concentrate after emulsification and homogenization.

7. A milk tea flavoring for tobacco prepared by the method according to any one of claims 1-6.

8. The application of the milk tea flavoring agent for tobacco as described in claim 7 in the field of cigarettes.

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