Composite tobacco leaf fermented by ganoderma lucidum and paleontological bacteria and preparation method of composite tobacco leaf

Abstract

The invention discloses a ganoderma lucidum and paleontobutis fungus fermented composite tobacco leaf and a preparation method thereof, and belongs to the technical field of fermented tobacco leaves. Ganoderma lucidum fermentation liquor and tobacco leaves are subjected to mixed fermentation, and soluble components such as polysaccharide and protein in ganoderma lucidum are extracted and converted into substances with biological activity; a rich nutrient source is provided for subsequent fermentation, and metabolites beneficial to the tobacco leaves are formed.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented tobacco leaves, and specifically refers to a composite tobacco leaf fermented by a Typhus archaeobacterium and a preparation method thereof. Background Art

[0002] Ganoderma lucidum, also known as "meat ganoderma" or "Ganoderma meat fungus", is a unique microbial complex with complex and diverse components, including bacteria, yeast and other microorganisms, as well as various biological organic substances, such as polysaccharides, proteins, minerals, etc. The main components of Ganoderma lucidum include polysaccharides (such as glucan), amino acids, various trace elements, minerals (such as calcium, magnesium, zinc) and some biologically active substances, which may have the potential to enhance immunity, anti-oxidation, anti-inflammatory, antibacterial and anti-tumor. In particular, the polysaccharides in Ganoderma lucidum are considered to be its most core effective ingredients, with multiple biological activities such as activating immune cells and scavenging free radicals; the antioxidant effect of polysaccharides can help the human body resist the harmful effects of environmental pollution and foreign substances such as tobacco. The various trace elements in Ganoderma lucidum, such as zinc and selenium, are also very beneficial to human health and can support the normal functioning of metabolism and the immune system. Composite fermentation technology refers to the process of fermenting a certain raw material using the synergistic action of multiple microorganisms to change its flavor, texture and composition. In the field of tobacco processing, traditional fermentation methods mainly rely on natural fermentation or single microbial fermentation. In contrast, composite fermentation technology, by introducing a variety of microorganisms, especially the ancient bacterium Tai Sui Ji, can more comprehensively decompose the complex substances in tobacco leaves, produce rich aroma components, and reduce harmful components. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a composite tobacco leaf fermented with the archaeal bacteria of the Gastrodia elata and a preparation method thereof. The present invention mixes and ferments the Gastrodia elata fermentation liquid and tobacco leaves, so that the soluble components such as polysaccharides and proteins in the Gastrodia elata are extracted and converted into biologically active substances, which provides a rich nutrient source for subsequent fermentation and forms metabolites that are beneficial to the tobacco leaves.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention proposes a method for preparing composite tobacco leaves fermented by cyperus archaeobacteria, which specifically comprises the following steps:

[0005] S1. Place the Ganoderma lucidum body in a 75 vol% ethanol aqueous solution, soak for 5 min, take it out, wash it with sterile water for 3-4 times, and cut it into pieces with sterilized scissors to obtain pre-treated Ganoderma lucidum crushed material;

[0006] S2, placing the pre-treated Gastrodia elata crushed material prepared in step S1 in deionized water, adjusting the pH to 4.5-5.5, adding a composite enzyme for enzymatic hydrolysis, setting the enzymatic hydrolysis temperature to 45-55°C, after the enzymatic hydrolysis time is 2-3h, raising the temperature to 90°C, performing enzyme inactivation treatment, filtering after the reaction is cooled to room temperature, and saving the filtrate to obtain enzymatic hydrolysis Gastrodia elata liquid;

[0007] S3, activate Lactobacillus casei, inoculate it into the enzymatic hydrolysis solution prepared in step S2, place it in a constant temperature fermentation box, keep the temperature at 28-30 ° C, place it under aerobic conditions, ferment for 6-12 hours, then transfer to anaerobic conditions, ferment for 5-7 days, after fermentation, filter through a 0.22 μm filter membrane, collect the filtrate, and obtain fermented solution;

[0008] S4, dilute the fermented Tai Sui liquid with sterile water, adjust the pH to 5.5-6.0, cut the tobacco leaves into shreds, add them to the reaction system, mix them evenly, add them to the yeast composite seed liquid, place them in an environment of 28-30°C for fermentation, and perform static fermentation for 24-48 hours under an aerobic environment to obtain fermented composite tobacco leaves;

[0009] Preferably, in step S2, the mass concentration of the pre-treated Gastrodia elata crushed material in deionized water is 0.25-0.4 g / mL;

[0010] Preferably, in step S2, the complex enzyme comprises the following components by weight: 1-1.2 parts of pectinase, 1.5-2 parts of cellulase, and 1.5-2 parts of β-glucanase;

[0011] Preferably, in step S2, the added mass of the complex enzyme is 0.1%-0.2% of the mass of the pre-treated taro powder;

[0012] Preferably, in step S3, the bacterial content of the Lactobacillus casei in the enzymatic hydrolyzed Tai Sui liquid is 3×10 4 -5×10 4 CFU / mL;

[0013] Preferably, in step S3, the Lactobacillus paracasei adopts Lactobacillus paracasei subspecies cheese, and the activation method of the Lactobacillus paracasei subspecies cheese comprises the following steps: dissolving Lactobacillus paracasei subspecies cheese powder in 36° C. warm water for activation for 30 min to obtain initially activated Lactobacillus paracasei subspecies cheese, inoculating the initially activated Lactobacillus paracasei subspecies cheese into an MRS medium with a pH of 7.0, and culturing at 30-36° C. for 18-24 h to obtain activated Lactobacillus paracasei;

[0014] Preferably, the mass concentration of the Lactobacillus casei subspecies casei powder in warm water is 0.1-0.2 g / L;

[0015] Preferably, in step S4, the volume ratio between the yeast composite seed liquid and the fermented Tai Sui liquid is 1-2:1

[0016] Preferably, in step S4, the mass volume ratio between the tobacco leaves and the yeast composite seed liquid is 3-5 g / mL;

[0017] Preferably, in step S4, the yeast composite seed solution comprises the following components in parts by volume: 1-2 parts of Pichia seed solution, 0.3-0.7 parts of Candida seed solution, and 0.5-1.5 parts of Saccharomyces cerevisiae seed solution;

[0018] Preferably, in step S4, the method for preparing the yeast composite seed solution comprises the following steps: activating Pichia pastoris, Candida and Saccharomyces cerevisiae respectively, selecting a single colony and placing it in 15 mL of liquid culture medium, culturing it at 30°C and 150rpm until the bacterial concentration reaches 1.0×10 5 -3.0×10 5 CFU / mL, the culture was stopped to obtain a seed solution; the seed solutions of Pichia pastoris, Candida and Saccharomyces cerevisiae were mixed according to the volume fractions to obtain a yeast composite seed solution;

[0019] Preferably, the liquid culture medium formula comprises: glucose: 100 g / L, ammonium chloride: 5 g / L, potassium dihydrogen phosphate: 1 g / L, disodium hydrogen phosphate: 1 g / L, and magnesium sulfate: 0.5 g / L.

[0020] The present invention also provides a composite tobacco leaf fermented by the cyperus archaeobacteria prepared according to the preparation method.

[0021] The beneficial effects achieved by the present invention are as follows:

[0022] The invention provides a composite tobacco leaf fermented by cyperus archaea and a preparation method thereof. The invention comprises the following steps: the cyperus fermentation liquid and tobacco leaves are mixed and fermented, and soluble components such as polysaccharides and proteins in the cyperus are extracted and converted into substances with biological activity, which provides a rich nutrient source for subsequent fermentation and forms metabolites beneficial to the tobacco leaves. In the invention, the metabolic activities of lactobacillus casei and yeast produce compounds such as organic acids, esters, and alcohols, and these metabolites can not only improve the flavor of the tobacco leaves, but also have biological activities such as anti-oxidation and antibacterial activities, which are helpful to enhance the quality and shelf life of the tobacco leaves. The chemical composition of tobacco leaves changes, generating some aromatic substances with unique flavors, such as esters, alcohols, and organic acids, which help improve the aroma and taste of tobacco leaves. Enzymatic hydrolysis and microbial fermentation help reduce undesirable substances in tobacco leaves (such as aldehydes and ketones produced by the degradation of sugars or fermentation), which may improve the stability of tobacco leaves, slow down their deterioration rate during storage, and increase their shelf life. The bioactive substances generated during the fermentation process (such as antioxidants, antibacterial substances, etc.) can bring additional health benefits to tobacco leaves, which may help reduce harmful substances in tobacco leaves and improve the health experience of smokers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The sensory evaluation diagram of rolled leaves made from the fermented composite tobacco leaves described in Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0024] Figure 2 This is the total ion flow diagram of the aroma components of the fermented composite tobacco leaves prepared in Example 1 of the present invention;

[0025] Figure 3 This is a graph showing the chemical composition of part of the fermented composite tobacco leaves prepared in Example 1 of the present invention.

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] Tai Sui, collected from Shandong;

[0031] Lacticaseibacillus paracasei subsp.paracasei, strain number: CICC 22165, collection time: February 22, 2008, purchased from China Industrial Microbiological Culture Collection Management Center;

[0032] Pichia kluyveri, strain number: CICC 33440, collection time: November 5, 2020, purchased from China Industrial Microbiological Culture Collection Management Center;

[0033] Saccharomyces cerevisiae, strain number: CICC 32883, collection time: November 17, 2008, purchased from China Industrial Microbiological Culture Collection Management Center;

[0034] Candida krusei, strain number: CICC 31748, collection time: February 9, 2007, purchased from China Industrial Microbiological Culture Collection Center.

[0035] The activation method of Pichia kluyveri comprises the following steps: inoculating Pichia kluyveri into a YPD medium with a pH of 7.0, and culturing at 30° C. for 24 hours to obtain activated Pichia kluyveri, wherein the YPD medium comprises: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose;

[0036] The activation method of brewer's yeast comprises the following steps: mixing brewer's yeast powder at a concentration of 0.1 g / L in 30°C water for activation for 30 minutes to obtain initial activated brewer's yeast; mixing the initial activated brewer's yeast with 3.5 wt% glucose water at a volume ratio of 1:5; and activating the mixture in a constant temperature incubator at a constant temperature of 32-36°C for 20-30 minutes to obtain activated brewer's yeast;

[0037] The activation method of Candida kluyveri comprises the following steps: inoculating Candida kluyveri into a YPD medium with a pH of 5.5 and culturing at 30°C for 24 hours to obtain activated Candida kluyveri, wherein the YPD medium comprises: 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose.

[0038] Pectinase, the brand used is Aladdin, the product number is P755196, and the purity is 5U / mg;

[0039] Cellulase, the brand used is Aladdin, the product number is C757787, and the purity is 700 EGU / g;

[0040] β-glucanase, the brand used is Aladdin, the product number is G755113, and the purity is 1 U / mg.

[0041] Example 1

[0042] This embodiment provides a method for preparing composite tobacco leaves fermented by Cyperus archaeobacteria, which specifically comprises the following steps:

[0043] S1. Place the Ganoderma lucidum body in a 75 vol% ethanol aqueous solution, soak for 5 min, take it out, wash it with sterile water 4 times, and cut it into pieces with sterilized scissors to obtain pre-treated Ganoderma lucidum crushed material;

[0044] S2, take 3g of the pre-treated Gastrodia elata crushed material prepared in step S1 and place it in a sterile culture medium, add 10mL of sterile deionized water, adjust the pH to 5.5, set aside, accurately weigh 10mg of pectinase, 20mg of cellulase and 15mg of β-glucanase and dissolve them in 10mL of sterile water, mix them with a vortex instrument, take 1mL and add it to a sterile culture dish of the pre-treated Gastrodia elata crushed material, seal it with a sealing film, set the enzymatic hydrolysis temperature to 50°C, after enzymatic hydrolysis for 3h, place it at 90°C for enzyme inactivation for 10min, filter it after the reaction is cooled to room temperature, save the filtrate, and obtain enzymatic hydrolysis Gastrodia elata liquid;

[0045] S3, dissolving 40 mg of Lactobacillus paracasei powder in 200 mL of 36° C. warm water and activating it for 30 min to obtain an initially activated Lactobacillus paracasei subspecies, inoculating the initially activated Lactobacillus paracasei subspecies into an MRS medium with a pH of 7.0 at an inoculum size of 8%, and culturing it at 30° C. for 18 h to obtain an activated Lactobacillus paracasei culture solution, and diluting the activated Lactobacillus paracasei culture solution to a bacterial concentration of 3.82×10 5CFU / mL, take 1mL and inoculate it into the enzymatic hydrolyzed Tai Sui liquid prepared in step S2, place it in a constant temperature fermentation box, maintain the temperature at 28°C, place it under aerobic conditions, ferment for 12 hours, then transfer to anaerobic conditions, ferment for 7 days, after the fermentation is completed, filter it through a 0.22μm filter membrane, collect the filtrate, and obtain the fermented Tai Sui liquid;

[0046] S4. After activating Pichia pastoris, Candida and Saccharomyces cerevisiae respectively, single colonies were selected and placed in 15 mL of liquid culture medium (glucose: 100 g / L, ammonium chloride: 5 g / L, potassium dihydrogen phosphate: 1 g / L, sodium hydrogen phosphate: 1 g / L, magnesium sulfate: 0.5 g / L), and cultured at 30°C and 150 rpm to obtain a Pichia seed liquid bacterial concentration of 1.13×10 5 CFU / mL, Candida seed solution concentration was 1.89×10 5 CFU / mL, the concentration of Saccharomyces cerevisiae seed solution was 2.31×10 5 CFU / mL, take 1mL of Pichia pastoris culture fluid, 0.5mL of Candida culture fluid and 0.5mL of Saccharomyces cerevisiae culture fluid, mix them evenly with a vortex instrument, and obtain a yeast composite seed liquid to a seed liquid; take 2mL of fermented Tai Sui liquid and dilute it to 20mL with sterile water, adjust the pH to 6.0, obtain a fermented Tai Sui dilution liquid, add it to the yeast composite seed liquid, mix evenly, cut the tobacco leaves into shreds, take 10g of tobacco shreds, add them to the reaction system, mix evenly, and place them at 28°C for fermentation. After static fermentation for 36 hours under an aerobic environment, obtain a fermented composite tobacco leaf;

[0047] This embodiment also provides a composite tobacco leaf fermented with Cycas revoluta archaeobacteria prepared according to the above preparation method.

[0048] Example 2

[0049] This embodiment provides a method for preparing composite tobacco leaves fermented by Cyperus archaeobacteria, which specifically comprises the following steps:

[0050] S1. Place the Ganoderma lucidum body in a 75 vol% ethanol aqueous solution, soak for 5 min, take it out, wash it with sterile water for 3 times, and cut it into pieces with sterilized scissors to obtain the pre-treated Ganoderma lucidum crushed material;

[0051] S2, take 2.5g of the pre-treated Gastrodia elata crushed material prepared in step S1 and place it in a sterile culture medium, add 10mL of sterile deionized water, adjust the pH to 4.5, set aside, accurately weigh 12mg of pectinase, 18mg of cellulase and 20mg of β-glucanase and dissolve them in 10mL of sterile water, mix them with a vortex instrument, take 0.8mL and add it to a sterile culture dish of the pre-treated Gastrodia elata crushed material, seal it with a sealing film, set the enzymatic hydrolysis temperature to 45°C, after enzymatic hydrolysis for 3h, place it at 90°C for enzyme inactivation for 10min, filter it after the reaction is cooled to room temperature, save the filtrate, and obtain enzymatic Gastrodia elata liquid;

[0052] S3, dissolving 20 mg of Lactobacillus paracasei powder in 100 mL of 36° C. warm water and activating it for 30 min to obtain an initially activated Lactobacillus paracasei subspecies, inoculating the initially activated Lactobacillus paracasei subspecies into an MRS medium with a pH of 7.0 at an inoculum rate of 8%, and culturing it at 33° C. for 18 h to obtain an activated Lactobacillus paracasei culture solution, and diluting the activated Lactobacillus paracasei culture solution to a bacterial concentration of 4.87×10 5 CFU / mL, take 1mL and inoculate it into the enzymatic hydrolyzed Tai Sui liquid prepared in step S2, place it in a constant temperature fermentation box, maintain the temperature at 30°C, place it under aerobic conditions, ferment for 6 hours, then transfer to anaerobic conditions, ferment for 5 days, after the fermentation is completed, filter it through a 0.22μm filter membrane, collect the filtrate, and obtain the fermented Tai Sui liquid;

[0053] S4. After activating Pichia pastoris, Candida and Saccharomyces cerevisiae respectively, single colonies were selected and placed in 15 mL of liquid culture medium (glucose: 100 g / L, ammonium chloride: 5 g / L, potassium dihydrogen phosphate: 1 g / L, sodium hydrogen phosphate: 1 g / L, magnesium sulfate: 0.5 g / L), and cultured at 30°C and 150 rpm to obtain a Pichia seed liquid bacterial concentration of 2.85×10 5 CFU / mL, Candida seed solution concentration was 1.23×10 5 CFU / mL, the concentration of Saccharomyces cerevisiae seed solution was 2.11×10 5 CFU / mL, take 2.0mL of Pichia pastoris culture fluid, 0.7mL of Candida culture fluid and 1.3mL of Saccharomyces cerevisiae culture fluid, mix them evenly with a vortex instrument, and obtain a yeast composite seed liquid to a seed liquid; take 2mL of fermented Tai Sui liquid and dilute it to 20mL with sterile water, adjust the pH to 5.5, obtain a fermented Tai Sui dilution liquid, add it to the yeast composite seed liquid, mix evenly, cut the tobacco leaves into shreds, take 12g of tobacco shreds, add them to the reaction system, mix evenly, place them at 30°C for fermentation, and perform static fermentation for 24h under an aerobic environment to obtain a fermented composite tobacco leaf;

[0054] This embodiment also provides a composite tobacco leaf fermented with Cycas revoluta archaeobacteria prepared according to the above preparation method.

[0055] Example 3

[0056] This embodiment provides a method for preparing composite tobacco leaves fermented by Cyperus archaeobacteria, which specifically comprises the following steps:

[0057] S1. Place the Ganoderma lucidum body in a 75 vol% ethanol aqueous solution, soak for 5 min, take it out, wash it with sterile water for 3 times, and cut it into pieces with sterilized scissors to obtain the pre-treated Ganoderma lucidum crushed material;

[0058] S2, take 4g of the pre-treated Gastrodia elata crushed material prepared in step S1 and place it in a sterile culture medium, add 10mL of sterile deionized water, adjust the pH to 4.5, set aside, accurately weigh 10mg of pectinase, 15mg of cellulase and 15mg of β-glucanase and dissolve them in 10mL of sterile water, mix them with a vortex instrument, take 0.8mL and add it to a sterile culture dish of the pre-treated Gastrodia elata crushed material, seal it with a sealing film, set the enzymatic hydrolysis temperature to 45°C, after enzymatic hydrolysis for 3h, place it at 90°C for enzyme inactivation for 10min, filter it after the reaction is cooled to room temperature, save the filtrate, and obtain enzymatic Gastrodia elata liquid;

[0059] S3, dissolving 10 mg of Lactobacillus paracasei powder in 100 mL of 36° C. warm water and activating it for 30 min to obtain an initially activated Lactobacillus paracasei subspecies, inoculating the initially activated Lactobacillus paracasei subspecies into an MRS medium with a pH of 7.0 at an inoculum rate of 8%, and culturing it at 36° C. for 18 h to obtain an activated Lactobacillus paracasei culture solution, and diluting the activated Lactobacillus paracasei culture solution to a bacterial concentration of 3.07×10 5 CFU / mL, take 1mL and inoculate it into the enzymatic hydrolyzed Tai Sui liquid prepared in step S2, place it in a constant temperature fermentation box, maintain the temperature at 30°C, place it under aerobic conditions, ferment for 9 hours, then transfer to anaerobic conditions, ferment for 6 days, after the fermentation is completed, filter it through a 0.22μm filter membrane, collect the filtrate, and obtain the fermented Tai Sui liquid;

[0060] S4. After activating Pichia pastoris, Candida and Saccharomyces cerevisiae respectively, single colonies were selected and placed in 15 mL of liquid culture medium (glucose: 100 g / L, ammonium chloride: 5 g / L, potassium dihydrogen phosphate: 1 g / L, sodium hydrogen phosphate: 1 g / L, magnesium sulfate: 0.5 g / L), and cultured at 30°C and 150 rpm to obtain a Pichia seed liquid bacterial concentration of 2.15×10 5 CFU / mL, Candida seed solution concentration was 2.72×10 5 CFU / mL, the concentration of Saccharomyces cerevisiae seed solution was 1.06×105 CFU / mL, take 1.7mL of Pichia pastoris culture fluid, 0.3mL of Candida culture fluid and 1.5mL of Saccharomyces cerevisiae culture fluid, mix them evenly with a vortex instrument, and obtain a yeast composite seed liquid to a seed liquid; take 2mL of fermented Tai Sui liquid and dilute it to 20mL with sterile water, adjust the pH to 6, obtain a fermented Tai Sui dilution liquid, add it to the yeast composite seed liquid, mix evenly, cut the tobacco leaves into shreds, take 14g of tobacco shreds, add them to the reaction system, mix evenly, place them at 30°C for fermentation, and perform static fermentation for 48h under an aerobic environment to obtain a fermented composite tobacco leaf;

[0061] This embodiment also provides a composite tobacco leaf fermented with Cycas revoluta archaeobacteria prepared according to the above preparation method.

[0062] Comparative Example 1

[0063] This comparative example provides a fermented composite tobacco leaf and a preparation method thereof, which is different from Example 1 only in that the preparation method does not include step S2, and the remaining components, component contents and preparation method are the same as those in Example 1.

[0064] Comparative Example 2

[0065] This comparative example provides a fermented composite tobacco leaf and a preparation method thereof, which is different from Example 1 only in that the preparation method does not include step S3, and the remaining components, component contents and preparation method are the same as those in Example 1.

[0066] Comparative Example 3

[0067] This comparative example provides a fermented composite tobacco leaf and a preparation method thereof, which is different from Example 1 only in that, in the preparation method, the yeast composite seed liquid in step S4 only includes Pichia seed liquid, and the remaining components, component contents and preparation method are the same as those in Example 1.

[0068] Experimental Example 1

[0069] In this experimental example, the fermented composite tobacco leaves prepared in Example 1-3 and Comparative Example 1-3 were sterilized at 121°C and dried to a moisture content of 12%. After being rolled into cigarettes, they were placed in a constant temperature and humidity chamber for 48 hours. The parameters of the constant temperature and humidity chamber were set as follows: temperature 20°C, relative humidity 60%;

[0070] With reference to GB 5604.4-2005 "Cigarette Low Part 4: Sensory Technical Requirements", according to the following table, 20 testers were organized to conduct a smoking evaluation. The cigarettes made of fermented composite tobacco leaves prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to sensory evaluation by comparative smoking evaluation, and each index was scored according to the tobacco company's tobacco sensory evaluation table (as shown in the following table);

[0071] Tobacco sensory evaluation standard table

[0072]

[0073] Figure 1 The sensory evaluation diagram of the fermented composite tobacco leaves prepared in Examples 1-3 of the present invention and Comparative Examples 1-3 is shown in the figure; as shown in the figure, the fermented composite tobacco leaves prepared in the embodiments of the present invention are significantly better than the fermented composite tobacco leaves prepared in the comparative examples in terms of sensory standards, and the original macromolecules and harmful substances can be decomposed through enzymatic hydrolysis and fermentation of the tobacco leaves, and the fermented tobacco leaves can enhance the flavor and taste of tobacco.

[0074] Experimental Example 2

[0075] The aroma components of the fermented composite tobacco leaves prepared in Example 1 were extracted by distillation extraction. After the fermented composite tobacco leaves prepared in Example 1 were ground, 20 g were accurately weighed, placed in a round-bottom flask, and purified water was added for distillation. After the distillation was completed, acid, alkaline and neutral extractions were performed in sequence. After the extraction, anhydrous sodium sulfate was added for drying. After concentration to 1 mL, the mixture was filtered through a 0.22 μm filter membrane and analyzed by GC-MS. 2,6-dichlorotoluene was used as an internal standard to detect the aroma components of the tobacco leaves.

[0076] Figure 2 This is the total ion flow diagram of the aroma components of the fermented composite tobacco leaves prepared in Example 1 of the present invention, Figure 3 This is a partial chemical composition result diagram of the fermented composite tobacco prepared in Example 1 of the present invention. As shown in the figure, the fermented composite tobacco prepared in Example 1 has a variety of aroma components, including alcohols and esters.

[0077] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

[0078] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing composite tobacco leaves fermented by Cyperus rotundus, characterized in that: The specific steps include: S1. Place the Ganoderma lucidum body in a 75 vol% ethanol aqueous solution, soak for 5 minutes, take it out, wash it with sterile water for 3-4 times, and cut it into pieces with sterilized scissors to obtain pre-treated Ganoderma lucidum crushed material; S2, placing the pre-treated Gastrodia elata crushed material prepared in step S1 in deionized water, adjusting the pH to 4.5-5.5, adding a composite enzyme for enzymatic hydrolysis, setting the enzymatic hydrolysis temperature to 45-55°C, after the enzymatic hydrolysis time is 2-3h, raising the temperature to 90°C, performing enzyme inactivation treatment, filtering after the reaction is cooled to room temperature, and saving the filtrate to obtain enzymatic hydrolysis Gastrodia elata liquid; S3, activate Lactobacillus casei, inoculate it into the enzymatic hydrolysis solution prepared in step S2, place it in a constant temperature fermentation box, keep the temperature at 28-30 ° C, place it under aerobic conditions, ferment for 6-12 hours, then transfer to anaerobic conditions, ferment for 5-7 days, after fermentation, filter through a 0.22 μm filter membrane, collect the filtrate, and obtain fermented solution; S4. Dilute the fermented Tai Sui liquid with sterile water, adjust the pH to 5.5-6.0, cut the tobacco leaves into shreds, add them to the reaction system, mix them evenly, add them to the yeast composite seed liquid, place them in an environment of 28-30°C for fermentation, and perform static fermentation in an aerobic environment for 24-48 hours to obtain fermented composite tobacco leaves.

2. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 1, characterized in that: In step S2, the mass concentration of the pre-treated Gastrodia elata crushed material in deionized water is 0.25-0.4 g / mL.

3. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 2, characterized in that: In step S2, the complex enzyme includes the following components by weight: 1-1.2 parts of pectinase, 1.5-2 parts of cellulase, and 1.5-2 parts of β-glucanase.

4. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 3, characterized in that: In step S2, the added mass of the complex enzyme is 0.1%-0.2% of the mass of the pre-treated Gastrodia elata crushed material.

5. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 4, characterized in that: In step S3, the bacterial content of the Lactobacillus casei in the enzymatic hydrolyzed Tai Sui liquid is 3×10 4 -5×10 4 CFU / mL.

6. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 5, characterized in that: In step S3, the Lactobacillus casei adopts Lactobacillus casei subspecies cheese, and the activation method of the Lactobacillus casei subspecies cheese comprises the following steps: Lactobacillus casei subspecies cheese powder is dissolved in 36°C warm water and activated for 30 minutes to obtain initial activated Lactobacillus casei subspecies cheese, the initial activated Lactobacillus casei subspecies cheese is inoculated into an MRS culture medium with a pH of 7.0, and cultured at 30-36°C for 18-24 hours to obtain activated Lactobacillus casei; the mass concentration of the Lactobacillus casei subspecies cheese powder in the warm water is 0.1-0.2 g / L.

7. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 6, characterized in that: In step S4, the volume ratio between the yeast composite seed liquid and the fermented Tai Sui liquid is 1-2:1; in step S4, the mass volume ratio between the tobacco leaves and the yeast composite seed liquid is 3-5 g / mL.

8. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 7, characterized in that: In step S4, the yeast composite seed solution includes the following components in parts by volume: 1-2 parts of Pichia pastoris seed solution, 0.3-0.7 parts of Candida seed solution, and 0.5-1.5 parts of Saccharomyces cerevisiae seed solution.

9. The method for preparing a composite tobacco leaf fermented by Cyperus archaeobacteria according to claim 8, characterized in that: In step S4, the preparation method of the yeast composite seed liquid includes the following steps: after activating Pichia pastoris, Candida and Saccharomyces cerevisiae respectively, selecting a single colony and placing it in 15 mL of liquid culture medium, placing it under the conditions of 30°C and 150rpm, and culturing it until the bacterial concentration is 1.0×10 5 -3.0×10 5 CFU / mL, culturing is stopped to obtain seed liquid; Pichia pastoris, Candida and Saccharomyces cerevisiae seed liquids are mixed according to volume parts to obtain yeast composite seed liquid; the liquid culture medium formula includes: glucose: 100 g / L, ammonium chloride: 5 g / L, potassium dihydrogen phosphate: 1 g / L, sodium dihydrogen phosphate: 1 g / L, and magnesium sulfate: 0.5 g / L.

10. A composite tobacco leaf fermented by Trichoderma archaeobacteria, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 9.

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