CN111671136A - Method for solid state fermentation of tobacco stems and cut stems and application thereof - Google Patents
Method for solid state fermentation of tobacco stems and cut stems and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
Abstract
The invention discloses a method for solid-state fermentation of tobacco stems and shreds, belonging to the technical field of microbial fermentation. The invention relates to solid state fermentation of tobacco stem shreds by paecilomyces varioti fermentation liquor, which is mainly realized by the following steps: (1) slant strain culture; (2) liquid seed culture; (3) culturing paecilomyces fermentation liquor; (4) solid state fermentation culture, inoculating the cultured Paecilomyces varioti fermentation broth into a solid state fermentation culture medium, and culturing at 22-32 deg.C for 6-10 days. The tobacco stem shreds are subjected to fermentation and biotransformation by the paecilomyces varioti, so that cell wall substances of the tobacco stem shreds are decomposed and utilized by the paecilomyces varioti, efficient bioactive substances such as the paecilomyces varioti polysaccharide and the paecilomyces acidare generated, adverse factors influencing the internal quality and flavor of the tobacco stem shreds are reduced, the sensory quality during smoking is improved, and the utilization rate of the tobacco stems in cigarettes is improved.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and relates to a method for solid-state fermentation of tobacco stems and cut stems and application thereof.
Background
As a tobacco big country in China, the planting area and the yield of tobacco are at the top of the world, and a large amount of tobacco stems are generated from the tobacco big country. The existing method for treating the tobacco stems mainly comprises a physical blasting method, a chemical treatment method and a biological enzyme method, and has the defects of poor degradation effect, chemical reagent pollution, high cost and the like. At present, the bacillus pumilus V35 is reported to be prepared into a microbial inoculum for processing cut rolled stems, only cellulose and pectin are degraded, and the cell wall components, namely lignin, are not degraded, so that the degradation effect of the cell wall materials is not obvious. At present, the utilization rate of tobacco stems is low, and a large amount of overstocked tobacco stems can cause environmental pollution and waste of available resources.
The main components of tobacco stem are cell wall substances (pectin, cellulose, lignin and hemicellulose), which account for about 43% of tobacco stem, and in addition, water-soluble sugars, starch, protein, nicotine, solanesol and the like. Compared with tobacco leaves, the chemical components in the tobacco stems are characterized by generally low contents of total sugar (namely water-soluble sugar), total nitrogen and nicotine and high content of cell wall substances. Cell wall substances are considered to be adverse factors influencing the internal quality and flavor of tobacco, and the substances can generate more low-grade aldehydes under the pyrolysis effect, and can generate irritating cough and the like during smoking.
The food and medicinal fungus paecilomyces can efficiently utilize saprophytic organisms of cellulose, lignin and other substances, and simultaneously synthesize active substances such as polysaccharides, triterpenes, adenosine, alkaloid and the like, and the paecilomyces has high content of various enzymes secreted by the paecilomyces and has special fragrance.
Disclosure of Invention
The invention aims to solve the problem of low utilization rate of tobacco stems, and provides a method for solid-state fermentation of tobacco stem shreds and application thereof, so as to degrade adverse substances in the tobacco stem shreds which affect the flavor, increase the aroma components of the tobacco stem shreds and improve the utilization rate of the tobacco stems in cigarettes.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for solid-state fermentation of cut stems of tobacco stalks, comprising the following steps:
(1) slant culture of strain: inoculating the paecilomyces strain to a slant culture medium for culturing to obtain a slant strain;
(2) liquid seed culture: inoculating the slant strain obtained in the step (1) into a seed culture medium for culture to obtain liquid seeds;
(3) culturing a paecilomyces fermentation liquor: inoculating the liquid seeds obtained in the step (2) into a paecilomyces fermentation culture medium for fermentation to obtain paecilomyces fermentation liquor;
(4) solid state fermentation culture: and (4) inoculating the paecilomyces varioti fermentation liquor obtained in the step (3) into a solid state fermentation culture medium, and culturing for 6-10 days at the temperature of 22-32 ℃.
Preferably, the slant culture conditions are: culturing at 28-32 deg.C until the mycelia grow over the slant.
Preferably, the conditions of liquid seed culture and paecilomyces fermentation broth culture are as follows: culturing for 5-7 days at 28-32 deg.C and 120-.
Preferably, the liquid seeds are inoculated into the paecilomyces fermentation medium according to the inoculation amount of 1-5 percent by volume.
Preferably, the inoculation amount of the paecilomyces fermentation liquor is 10-40% by volume mass ratio.
Preferably, the slant culture medium is a potato culture medium, and comprises: potato 200g/L, glucose 20g/L and agar 20g/L, wherein the seed culture medium contains: 200g/L of potato and 20g/L of glucose.
Preferably, the paecilomyces fermentation medium contains: 20g/L glucose, 5g/L yeast powder, 10g/L peptone, MgSO4 & 7H2O 1g/L, KH2PO 41 g/L and Vb 0.1 g/L.
Preferably, the solid fermentation medium is 100% of cut stems of tobacco stalks.
Preferably, the paecilomyces sp is purchased from China general microbiological culture Collection center (CGMCC) with the preservation number of CCTCC M209240.
In a second aspect, the invention provides an application of a method for solid state fermentation of tobacco stem shreds in tobacco stem treatment.
The invention has the following beneficial effects:
1. the tobacco stem cut tobacco is subjected to solid state fermentation and microbial transformation of the paecilomyces varioti, wherein cell wall substances such as cellulose, hemicellulose, lignin and pectin are decomposed and utilized, the content of reducing sugar can be increased by 34.49%, the total sugar can be increased by 30.99%, and the utilization rate of the tobacco stem in the cigarette is effectively improved.
2. By the method, adverse substances influencing the flavor in the cut tobacco stems are degraded, and the aroma components of the cut tobacco stems are increased.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples, but the present invention is not limited to the contents of the examples in any way.
In all embodiments of the present application, the slant culture medium, the seed culture medium, the paecilomyces fermentation medium and the solid state fermentation medium are prepared according to the following methods:
(1) slant medium, i.e. potato medium (PDA medium): 200g/L of potato, 20g/L of glucose and 20g/L of agar, natural pH and 20 minutes of sterilization at 121 ℃.
(2) Seed culture medium: glucose 20g/L, potato 200g/L, natural pH, and sterilizing at 121 deg.C for 20 min.
(3) Paecilomyces fermentation medium: 20g/L glucose, 5g/L yeast powder, 10g/L, MgSO 4.7H peptone2O1g/L、KH2PO41g/L, Vb 0.1.1 g/L, natural pH, 121 ℃ sterilization for 20 minutes.
(4) Solid state fermentation medium: 100 percent of cut stem of tobacco stalk, natural pH and sterilization at 121 ℃ for 20 minutes.
In all the specific examples of the present application, the determination of cellulose, the determination of hemicellulose, the determination of lignin, the determination of pectin, the determination of reducing sugars and total sugars were determined according to the following determination methods:
(1) method for measuring cellulose
Drawing a standard curve:
taking 6 colorimetric tubes of 10mL, respectively putting 0, 0.40, 0.80, 1.20, 1.60 and 2.00mL of cellulose standard solution, respectively adding 2.00, 1.60, 1.20, 0.80, 0.40 and 0mL of distilled water, and shaking up to obtain tubes with cellulose contents of 0, 40, 80, 120, 160 and 200 mu g in sequence. Adding 0.5mL of 2% anthrone into each tube, and adding concentrated H along the tube wall2SO45mL, plugging, shaking up, heating in a boiling water bath for 5min, standing, cooling, and then determining the absorbance of the cellulose solution with different contents at the wavelength of 620 nm.
And (3) after the fermentation is finished, naturally drying the solid culture (containing the tobacco stalk shreds and the paecilomyces mycelium), crushing, and sieving by a 60-mesh sieve. Weighing about 0.3g of sample, adding 1mL of 80% ethanol, quickly homogenizing at room temperature, carrying out water bath at 90 ℃ for 20min, cooling to room temperature, centrifuging at 6000g of 25 ℃ for 10min, and discarding the supernatant. Washing the precipitate with 80% ethanol and acetone (vortex oscillation for about 2min, centrifuging at 6000g at 25 deg.C for 10min, and removing supernatant) to obtain coarse cell wall, drying the precipitate, weighing 0.2g sample in beaker, placing in cold water bath, adding 50% H2SO460mL, 40min later transfer to 100mL volumetric flask, and 50% H2SO4And (5) fixing the volume to the scale, shaking up and standing. Taking 10mL of supernatant, putting into a 100mL volumetric flask, adding distilled water on a cold water bath to dilute to a scale, shaking up, taking 2mL of solution, adding 0.5mL of 2% anthrone into a colorimetric tube, and adding concentrated H along the tube wall2SO45mL, plugging, shaking up, heating in a boiling water bath for 5min, standing and cooling, then obtaining the absorbance under the wavelength of 620nm, and calculating the content of cellulose in the tobacco stem sample according to a regression equation.
(2) Method for measuring hemicellulose
Making a standard curve: taking 8 colorimetric tubes of 25mL, adding glucose standard solutions of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4mL (1.0mg/mL) respectively, adding 2mL of distilled water, adding DNS of 1.5mL respectively, shaking uniformly, carrying out boiling water bath for 10min, cooling, fixing the volume to 25mL, and measuring the absorbance by a spectrophotometer at the wavelength of 540 nm.
0.20g of the sample was weighed into a 100mL beaker and 10mL of 60% Ca (NO) was added3)2Heating for 10min, diluting and filtering, washing filter residue with distilled water for 3 times, then placing in an oven, drying at 70 ℃, then transferring into a 250mL triangular flask, adding 10mL of HCl with the concentration of 2mol/L, then boiling in a water bath for 50min, cooling, then filtering, washing the filter residue with distilled water for 3 times, remaining and taking filtrate and washing liquid to mix uniformly, taking 0.5mL of filtrate, adding 1.5mL of DNS solution, boiling in a water bath for 10min, cooling, then fixing the volume to 25mL, measuring absorbance at the wavelength of 540nm, and then calculating the content of hemicellulose in the tobacco stem sample according to a regression equation.
(3) Method for measuring lignin
Accurately weighing 6.0mg of crushed sample powder, placing the crushed sample powder into a 25mL test tube, adding 5mL of 25% acetyl bromide-acetic acid solution (V/V) and 0.2mL of perchloric acid, sealing the tube opening of the test tube by using a sealing film, carrying out constant-temperature water bath at 80 ℃ for 40min, completely transferring sample liquid into a volumetric flask filled with 10mL of mixed liquid of 2mol/L NaOH and 25mL of glacial acetic acid, fully oscillating and uniformly mixing, and then carrying out constant volume to 100mL by using glacial acetic acid. Measuring the light absorption value at 280nm by using glacial acetic acid as a blank solution and using an ultraviolet spectrophotometer.
(4) Pectin determination method
And (3) preparing a standard curve: 0.0991g of galacturonic acid is accurately weighed, dissolved in distilled water and then fixed in volume in 100mL volumetric flasks, 0, 2.0, 4.0, 6.0, 8.0 and 10.0mL of the solution are respectively transferred into 6 100mL volumetric flasks, and distilled water is added to fix the volume to obtain standard diluent. Transferring 1.0mL of standard diluent into 6 tubes, and adding 6mL of concentrated H2SO4Shaking, heating in 85 deg.C water bath for 15min, taking out, cooling, adding 0.15% carbazole absolute ethanol solution 0.2mL, shaking, standing at room temperature in dark place, developing at 530nm wavelength for 2h, measuring absorbance (A) with lcm cuvette and blank reagent as reference, and drawing absorbance-concentration standard curve.
Weighing 5g (to the nearest 0.001g) of sample in a beaker, adding hot 70% ethanol, stirring thoroughly to extract saccharide, filtering, and repeating the operation until the filtrate does not react with saccharide. The residue was washed with 99% ethanol, then ether, and air-dried. And (3) transferring the residues in the funnel into a 250mL conical flask by using 150mL of 0.05mol/L HCl solution heated to boil, installing a condenser, heating and refluxing for 1h in a boiling water bath, cooling, transferring into a 250mL volumetric flask, adding 2 drops of methyl red indicator, adding 0.5mol/L NaOH for neutralization, diluting to a constant volume, shaking up, filtering, and collecting filtrate to obtain the total pectin extracting solution. Taking pectin extract, and diluting the pectin extract to a proper concentration (containing 10-70 mu g/L galacturonic acid) by using water. 1mL of the diluted solution was put in a test tube, and the absorbance was measured and the galacturonic acid concentration was determined from the standard curve by the method for preparing the standard curve.
(5) Method for measuring reducing sugar and total sugar
And (3) preparing a standard curve: drying analytically pure glucose at 80 ℃ to constant weight, accurately weighing 500mg, adding a small amount of distilled water for dissolving, transferring into a 100mL volumetric flask, and performing constant volume and shaking up to obtain the 5mg/mL glucose standard solution. Respectively measuring 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mL of glucose standard solution with the concentration of 5mg/mL, placing the glucose standard solution into a 25mL test tube with a plug, sucking distilled water to respectively supplement the solution to 2.0mL, and then respectively adding 2.0mL of DNS reagent. Covering a plug, heating in a boiling water bath for 6min, taking out, cooling with flowing cold water, adding distilled water to a constant volume of 25mL, shaking thoroughly, standing for 10min, and measuring absorbance on an ultraviolet visible spectrophotometer at 540nm wavelength with blank (0 mL standard solution). And solving a regression equation through regression statistics.
Weighing 100mg of ground sample (sieved by a 80-mesh sieve) into a 100mL large test tube, adding 10mL of 80% ethanol, soaking overnight, adding 10mL of hot distilled water the next day, heating in a water bath at 80 ℃ for 0.5h, filtering in a 100mL volumetric flask, repeatedly washing filter residue with the hot distilled water, and fixing the volume to obtain the reducing sugar solution to be tested. Sucking 25mL of the reducing sugar solution to be detected into a 250mL triangular flask, adding 7mL of hydrochloric acid solution with the volume ratio of 1:3 (concentrated hydrochloric acid to distilled water), placing the mixture in a boiling water bath for acidification for 0.5h, adding 1-2 drops of methyl red indicator, adjusting the pH value to be neutral or slightly alkaline (pH value is 7.0-7.5) by using 10% NaOH, transferring the mixture into a 100mL volumetric flask, and fixing the volume. The water-soluble total sugar solution to be tested is obtained. Taking 2.00mL of reducing sugar solution to be tested and 2.00mL of water-soluble total sugar solution to be tested, adding 2.00mL of 3, 5-dinitrosalicylic acid developer, determining absorbance according to a standard solution treatment method, checking out corresponding concentration from a standard curve, and calculating the content of reducing sugar and total sugar.
The contents of cellulose, hemicellulose and lignin are calculated according to the mass of a sample, and the unit is mg/g dry weight. The content of reducing sugar and total sugar are the amount of reducing sugar and total sugar contained in each gram of dry matter, namely the mass percentage, and the other methods are common methods in the field if no special indication is made.
Example 1
(1) Slant culture of strain: inoculating Paecilomyces varioti to slant culture medium (PDA culture medium), and culturing at 30 deg.C for about 7 days until the slant is full of mycelia;
(2) liquid seed culture: inoculating slant strains into a 250mL triangular flask filled with 80mL seed culture medium, and culturing for 7 days at the rotating speed of a shaking table of 150 rpm and the temperature of 30 ℃;
(3) culturing a paecilomyces fermentation liquor: scattering the liquid seeds by using glass beads and a magnetic rotor, inoculating the liquid seeds into a 250mL triangular flask filled with 80mL paecilomyces fermentation medium according to the inoculation amount of 4% in volume percentage, and culturing for 7 days at the conditions of the rotating speed of a shaking table of 150 rpm and the temperature of 30 ℃;
(4) solid state fermentation culture: the cultured paecilomyces varioti fermentation liquor is scattered and uniformly mixed, the cultured paecilomyces varioti fermentation liquor is inoculated into a 500mL triangular flask containing a solid fermentation culture medium of tobacco stalk shreds according to the inoculation amount of 10% (V/m), the culture is carried out for 6 days at the temperature of 30 ℃, and the cellulose degradation rate, the hemicellulose degradation rate, the lignin degradation rate, the pectin degradation rate, the reducing sugar increase rate and the total sugar content increase rate of the tobacco stalk shreds are respectively measured when the culture is carried out for 2 days, 4 days and 6 days. Before fermentation, the contents of cellulose, hemicellulose, lignin, pectin, reducing sugar and total sugar in the cut stems of the tobacco stems are 585.4mg/g, 167.18mg/g, 96mg/g, 66.40mg/g, 17.25% and 18.36% respectively.
Wherein, the paecilomyces is purchased from China general microbiological culture Collection center (CGMCC) with the preservation number of CCTCC M209240.
And (3) test results: the change of each component of the cut stems of the tobacco stalks after 2 days, 4 days and 6 days of fermentation is shown in table 1, wherein the degradation rate of cellulose is measured to be 18.44 percent, the degradation rate of hemicellulose is 41.43 percent, the degradation rate of lignin is 13.15 percent, the degradation rate of pectin is 12.14 percent, the increase rate of reducing sugar is 25.2 percent, and the increase rate of total sugar content is 27.6 percent after 6 days of fermentation.
Table 1:
example 2
The inoculation amount of the paecilomyces fermentation liquor is 20 percent (V/m), and other conditions are the same as those in example 1.
And (3) test results: the change of each component of the cut stems of the tobacco stalks after 2, 4 and 6 days of fermentation is shown in table 2, wherein the cellulose degradation rate is 13.75 percent, the hemicellulose degradation rate is 14.77 percent, the lignin degradation rate is 11.37 percent, the pectin degradation rate is 15.12 percent, the reducing sugar content increase rate is 34.49 percent and the total sugar increase rate is 30.99 percent after 6 days of fermentation.
Table 2:
example 3
The inoculation amount of the paecilomyces fermentation liquor is 30% (V/m), and other conditions are the same as those in example 1.
And (3) test results: the change of each component of the cut stems of the tobacco stalks after 2 days, 4 days and 6 days of fermentation is shown in table 3, wherein the degradation rate of cellulose is 6.62 percent, the degradation rate of hemicellulose is 18.47 percent, the degradation rate of lignin is 18.63 percent, the degradation rate of pectin is 14.87 percent, the increase rate of reducing sugar content is 13.97 percent and the increase rate of total sugar content is 10.35 percent, which are measured after 6 days of fermentation.
Table 3:
example 4
The inoculation amount of the paecilomyces fermentation liquor is 40% (V/m), and other conditions are the same as those in example 1.
And (3) test results: the change of each component of the cut stems of the tobacco stalks after 2, 4 and 6 days of fermentation is shown in table 4, wherein the degradation rate of cellulose is 3.58%, the degradation rate of hemicellulose is 29.03%, the degradation rate of lignin is 26.96%, the degradation rate of pectin is 18.58%, the increase rate of reducing sugar content is 13.80% and the increase rate of total sugar content is 9.20% after 6 days of fermentation.
Table 4:
through the embodiment, the method can effectively reduce the contents of cellulose, hemicellulose, lignin and pectin in the cut tobacco stems, reduce adverse factors influencing the intrinsic quality and flavor of the cut tobacco stems, and improve the sensory quality during smoking.
At present, the degradation effect of liquid fermentation on the cut tobacco stems is slightly better than that of solid fermentation on the cut tobacco stems, but the liquid fermentation has too much moisture content, and the sugar content and the flavor substance components are partially dissolved in liquid, so that a certain amount of loss is caused. The solid fermentation can not only degrade the cell wall components of the cut stems, but also increase the reducing sugar, total sugar and flavor components, so the total effect is better than that of the liquid fermentation.
Although the present invention has been described herein with reference to the illustrated embodiments thereof, which are intended to be preferred embodiments of the present invention, it is to be understood that the invention is not limited thereto, and that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure.
Claims (10)
1. A method for solid state fermentation of cut stems of tobacco stems is characterized by comprising the following steps:
(1) slant culture of strain: inoculating the paecilomyces strain to a slant culture medium for culturing to obtain a slant strain;
(2) liquid seed culture: inoculating the slant strain obtained in the step (1) into a seed culture medium for culture to obtain liquid seeds;
(3) culturing a paecilomyces fermentation liquor: inoculating the liquid seeds obtained in the step (2) into a paecilomyces fermentation culture medium for fermentation to obtain paecilomyces fermentation liquor;
(4) solid state fermentation culture: and (4) inoculating the paecilomyces varioti fermentation liquor obtained in the step (3) into a solid state fermentation culture medium, and culturing for 6-10 days at the temperature of 22-32 ℃.
2. The method for solid state fermentation of cut stems of tobacco stems according to claim 1, wherein the slant culture conditions are as follows: culturing at 28-32 deg.C until the mycelia grow over the slant.
3. The method for solid state fermentation of cut stems of tobacco stems according to claim 1, wherein the conditions of the liquid seed culture and the paecilomyces varioti fermentation broth culture are as follows: culturing for 5-7 days at 28-32 deg.C and 120-.
4. The method for solid state fermentation of cut stems of tobacco stalks according to claim 1, wherein the liquid seeds are inoculated into the paecilomyces varioti fermentation medium in an inoculation amount of 1 to 5% by volume.
5. The method for solid state fermentation of cut stems of tobacco stalks according to claim 1, wherein the inoculation amount of the paecilomyces fermentation broth is 10 to 40 percent by volume mass.
6. The method for solid state fermentation of cut stems of tobacco stems according to claim 1, wherein the slant culture medium is a potato culture medium comprising: potato 200g/L, glucose 20g/L and agar 20g/L, wherein the seed culture medium contains: 200g/L of potato and 20g/L of glucose.
7. The method for solid state fermentation of cut stems of tobacco stems according to claim 1, wherein the paecilomyces fermentation medium comprises: 20g/L glucose, 5g/L yeast powder, 10g/L peptone and MgSO4·7H2O 1g/L，KH2PO41 g/L，Vb0.1g/L。
8. The method for solid state fermentation of cut stems according to claim 1, wherein the solid state fermentation medium is 100% cut stems.
9. The method for solid state fermentation of cut stems of tobacco stalks according to claim 1, wherein the paecilomyces sp is purchased from China general microbiological culture Collection center (CGMCC) with a collection number of CCTCC M209240.
10. Use of a method of solid state fermentation of cut tobacco stems according to any one of claims 1 to 9 in the treatment of tobacco stems.
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