CN108752355B - Method for co-producing various products in fresh tobacco leaves - Google Patents
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Abstract
The invention provides a co-production method of various products in fresh tobacco leaves, which can simultaneously prepare six products, namely sodium copper chlorophyllin, nicotine, solanesol, spice, protein and bacterial manure from the fresh tobacco leaves, thereby improving the comprehensive utilization rate of the fresh tobacco leaves. Compared with the prior art, the chlorophyll extraction efficiency is further improved, the yield and the purity of the prepared sodium copper chlorophyllin are also improved, and the sodium copper chlorophyllin does not contain nicotine and other impurities. Except nicotine, the content of solanesol and perfume in the extract is also improved. The extraction rate of protein is also higher. The microbial fertilizer prepared by fermenting the tobacco residue has the advantages of short period, high spore production number and the like. The technical means adopted by the invention has the advantages of low cost, simple process, high efficiency, rapidness, easy industrial amplification and the like, and is a technology for comprehensively utilizing the effective components in the fresh tobacco leaves, which has good economy and is environment-friendly.
Description
Technical Field
The invention relates to the field of bioengineering, can simultaneously prepare six products of sodium copper chlorophyllin, nicotine, solanesol, perfume, protein and bacterial manure, and particularly relates to a co-production method for preparing the six products from fresh tobacco leaves.
Background
China's tobacco planting area and yield are the first in the world, and nearly millions of tons of fresh waste tobacco leaves are generated in tobacco fields every year. The fresh tobacco leaves are rich in chlorophyll, nicotine, solanesol, spices, proteins and other natural products, wherein the chlorophyll and derivatives thereof have remarkable effects in the aspects of antibiosis, anti-mutagenesis and deodorization. The nicotine has good application prospect in the aspects of preparing pesticides and preventing senile dementia. Solanesol has antibacterial, anti-inflammatory and hemostatic effects, is an important medical intermediate for drug synthesis, is commonly used for synthesizing drugs for resisting cardiovascular diseases, cancers, ulcers and the like, and is also an important raw material for synthesizing coenzyme Q10 and vitamin K2. The perfume has high variety and content, and can be widely applied in the fields of food, daily chemical industry and the like. The content and quality of protein can be compared with soybean protein, the amino acids are complete in variety and reasonable in proportion, and the soybean protein not only contains 8 amino acids necessary for human body, but also does not contain cholesterol, and has the advantages of soft texture, easiness in digestion and the like.
Researchers at home and abroad are receiving more and more attention on the research of extracting and separating effective components in tobacco leaves. Patent CN104151140B discloses a method for comprehensively extracting multiple effective components from tobacco leaves, which specifically adopts an ultrasonic circulation extraction method and combines separation and purification means such as extraction, column chromatography, recrystallization, steam distillation and precipitation to separate solanesol, nicotine, chlorogenic acid, protein and sugar one by one, and aims to realize continuous extraction and separation of multiple effective components, thereby not only improving the utilization rate of raw materials, but also increasing the number of separated products and improving the extraction efficiency. CN102429319B discloses a method for continuously extracting tobacco, which is characterized in that the tobacco is subjected to ultrasonic alkalization treatment, solid-liquid separation is carried out to collect filtrate and filter residue, nicotine and hemicellulose are extracted and separated from the filtrate, cellulose and lignin are extracted and separated from the filter residue, and the aim of comprehensively utilizing waste is fulfilled by a continuous extraction method. The patent CN1302568A discloses a process and a device for extracting and rectifying tobacco by using supercritical multi-element fluid, which is characterized in that tobacco leaves are firstly puffed by 5-20% by using the supercritical multi-element fluid extraction technology, and then aromatic absolute oil components, nicotine, solanesol, tobacco F-1-P crystal protein and tobacco SOD superoxide dismutase in the tobacco are selectively extracted and rectified. The raw materials utilized by the three patents are all from waste roasted tobacco leaves, and an ultrasonic extraction method and a supercritical multi-element fluid extraction technology are adopted, so that the two methods not only cause higher cost, but also cause expensive equipment, and cause expensive maintenance cost in the later period of the equipment, and are not beneficial to large-scale industrial production and application in terms of economic benefit. At present, the patents for extracting effective substances from fresh tobacco leaves are less, and the patent CN1534008A discloses a method for extracting solanesol from tobacco leaves, the patent CN102643121A,
CN102887742A and CN103274784A both use fresh tobacco leaves as raw materials to produce organic fertilizer by fermentation. The research on extracting and separating effective substances from fresh tobacco leaves is less, the published patent mainly focuses on the extraction, separation and preparation of single products such as solanesol, organic fertilizer and the like, the extracted products are single, the full utilization of other components in the fresh tobacco leaves is not considered, the utilization rate of raw materials is low, and the principle of comprehensive utilization of the fresh tobacco leaves is not reflected.
Disclosure of Invention
The invention aims to provide a high-efficiency and environment-friendly comprehensive application technology of fresh tobacco leaves. The method aims to realize the extraction and separation of various products by taking fresh tobacco leaves as raw materials and designing a reasonable and effective process so as to realize the comprehensive application targets of high utilization rate of raw materials, more extraction components, high extraction efficiency and less waste discharge.
Based on the above purpose, the invention provides a co-production method of various products in fresh tobacco leaves, which comprises the following steps: .
(1) Pretreatment of raw materials: crushing fresh tobacco leaves, and then carrying out solid-liquid separation to obtain solid tobacco leaves and a precipitation liquid; the solid-liquid separation method is a natural sedimentation method, a vacuum filtration method or a plate-and-frame filter pressing method;
(2) preparation of sodium copper chlorophyllin: adding an extraction solvent I into the solid tobacco leaves obtained in the step (1) for extraction, and performing suction filtration to obtain an extracting solution I and tobacco residues; adding an alkaline substance I into the extracting solution I for saponification; removing the organic solvent from the aqueous solution of the saponified solution, and extracting by using a weak-polarity organic solvent to obtain an extract I and a raffinate I; adjusting the pH value of the raffinate I by using an acidic substance, adding a solution containing copper ions, carrying out copper treatment to obtain copper chlorophyllin acid, adding an alkaline solution II to adjust the pH value, standing to obtain sodium copper chlorophyllin crystals, washing and drying to obtain sodium copper chlorophyllin;
(3) preparing protein: extracting protein from the precipitation liquid obtained in the step (1) by adopting an isoelectric precipitation method, a flocculant precipitation method, an ethanol precipitation method or an ammonium sulfate precipitation method;
(4) preparing nicotine, solanesol and spices from the extract I in the step (2), comprising the following steps of: distilling and drying the extract I to obtain an extract, adding water into the extract under the ultrasonic condition, adjusting the pH value, and extracting by using an isovolumetric organic solvent to obtain an extract II and a raffinate II; distilling the extract II, drying, dissolving in aqueous ethanol water, performing chromatography with macroporous resin, eluting with ethanol water solution, mixing the flow-through components, concentrating to dryness to obtain essence extract refined product, mixing the eluted components, concentrating to dryness, dissolving in ethanol, methanol, acetone or acetonitrile, cooling, and crystallizing to obtain solanesol; adjusting the pH value of the raffinate II, adding an organic solvent with the same volume to extract to obtain an extract III and a raffinate III, and distilling and drying the extract III to obtain a pure nicotine product;
(5) and (3) preparing the biological bacterial fertilizer by using the tobacco residue in the step (2), and inoculating microbial strains to the tobacco residue as a raw material for solid culture.
The invention has the advantages that the extraction, separation and preparation of different substances are integrated into one process flow to be completed, and the process flow is greatly simplified, thereby becoming a process integration technology with great prospect. The inventor of the invention also finds that compared with the extraction and separation of a single substance or two substances or three substances in the prior art, the novel process for co-production of six products can furthest improve the utilization rate of fresh tobacco leaves.
Compared with the prior art, the method of the invention has the advantages that: (1) the extraction steps of various effective components of the fresh tobacco leaves are integrated into a process, so that the method is a process integration technology with great prospect; (2) the extraction, separation and preparation of six products of sodium copper chlorophyllin, nicotine, solanesol, spice, protein and bacterial manure are realized, and the advantages of more extracted products and high utilization rate of raw materials are achieved; (3) chlorophyll and protein are two most special substances in fresh tobacco leaves, the protein extracted from the fresh tobacco leaves has higher bioactivity, the amino acid composition of the protein is rich and balanced, the variety of essential amino acid is complete and accords with the proportion of human body amino acid patterns, wherein, the content of lysine is higher, and the protein can be used as an amino acid supplement. The chlorophyll has high content, and is converted into sodium copper chlorophyllin, so that the green and efficacy of the chlorophyll are maintained, and the chlorophyll is more stable to light and heat and better in water solubility, is convenient to store and apply and does not contain nicotine compared with the chlorophyll; (4) simple operation, less pollution and high extraction efficiency.
The invention provides a co-production method for simultaneously preparing six products, namely sodium copper chlorophyllin, nicotine, solanesol, spice, protein and bacterial manure from fresh tobacco leaves, and improves the comprehensive utilization rate of the fresh tobacco leaves. Compared with the prior art, the chlorophyll extraction efficiency is further improved, the yield and the purity of the prepared sodium copper chlorophyllin are also improved, and the sodium copper chlorophyllin does not contain nicotine and other impurities. Except nicotine, the content of solanesol and perfume in the extract is also improved. The extraction rate of protein is also improved to a certain extent. The microbial fertilizer prepared by fermenting the tobacco residue has the advantages of short period, high spore production number and the like. The technical means adopted by the invention has the advantages of low cost, simple process, high efficiency, rapidness, easy industrial amplification and the like, and is a technology for comprehensively utilizing the effective components in the fresh tobacco leaves, which has good economy and is environment-friendly.
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FIG. 1 is a schematic process flow diagram of a process for co-production of multiple products from fresh tobacco leaves.
Detailed Description
The invention provides a co-production method for simultaneously preparing six products, namely sodium copper chlorophyllin, nicotine, solanesol, spice, protein and bacterial manure from fresh tobacco leaves, and the process flow schematic diagram is shown in figure 1. The method comprises the steps of raw material pretreatment, sodium copper chlorophyllin preparation, protein preparation, nicotine preparation, solanesol preparation, perfume preparation and biological bacterial fertilizer preparation.
In a specific embodiment of the present invention:
in the step (1), namely the step of pretreating the raw materials, the solid-liquid separation method is a vacuum filtration method;
in the step (2), the extraction solvent I is selected from an aqueous solution of ethanol, acetone, methanol, n-propanol, isopropanol or a mixture thereof, the concentration is 60-100%, and the mass volume ratio of the feed liquid is 1: 2.5-15; the extraction times are 1-5 times; the extraction time is 20-80 min/time; the extraction method is selected from cold soaking extraction method, ultrasonic oscillation method or hot reflux method, and the hot reflux temperature is 50-70 deg.C; the alkaline substance I is sodium hydroxide, potassium hydroxide, calcium hydroxide and sodium carbonate, and the saponification pH value is 8-14; the saponification temperature is 50-70 ℃; the weak polar organic solvent is at least one of petroleum ether, ethyl acetate and n-hexane, and the extraction times are 2-5 times; the acidic substance is at least one selected from sulfuric acid, phosphoric acid and hydrochloric acid, and the adjusted pH value is 2-5; the copper ion-containing solution is copper sulfate, copper chloride solution or a mixture of copper sulfate and copper chloride solution, and the concentration of the copper ion-containing solution is 10-30%; the alkaline solution II is NaOH or NaOH ethanol solution, the concentration is 5-20%, and the adjusted pH is 10-14; the crystallization temperature is 0-10 ℃, and the time is 30 min-2 h;
in the step (3), protein is prepared by adopting an isoelectric precipitation method, the pH of the precipitation liquid is adjusted to 3-7 at the temperature of 0-20 ℃, and the precipitation time is 0.5-4.0 h; the substance for adjusting pH is at least one selected from phosphoric acid, sulfuric acid and hydrochloric acid;
in the step (4), the organic solvent is at least one of petroleum ether, ethyl acetate, n-hexane, butyl acetate or kerosene; when extract II is obtained, the pH value is adjusted to 1-6; adjusting pH to 8-14 when obtaining extract III; before macroporous resin chromatography, the concentration of ethanol in the hydrous ethanol for dissolving the sample is 70-90 percent; the macroporous resin is nonpolar or weak polar macroporous resin, and can be selected from XDA-1,801,806, XR919C, HPD600, AB-8 or HZ802 respectively and independently; the concentration of ethanol in the aqueous ethanol eluted after macroporous resin is 80-100%, and the crystallization temperature is-20-10 ℃;
in the step (5), the microbial strain is bacillus subtilis, and the inoculation amount is 10%; the thickness of the fermentation material layer is 1-5 cm, the fermentation temperature is 8-15 ℃, and the fermentation time is 5-8 days.
In another specific embodiment of the invention, the extraction solvent I in the step (2) is an ethanol aqueous solution with a concentration of 90-100%, the mass-to-volume ratio of the feed liquid is 1: 7-10, the extraction times are 2-5 times, the extraction time is 30-60 min/time, and the extraction method is a 50 ℃ thermal reflux method.
In another embodiment of the present invention, in the step (3), the substance for adjusting pH is phosphoric acid, and the pH is adjusted to 3-3.5 by using phosphoric acid at 0-4 ℃, and the precipitation time is 0.5-2.0 h.
It is easy to understand that the above mentioned preferred technical features can be combined in technical solutions to obtain a complete technical solution with more excellent technical effects. The following non-limiting examples are presented to further illustrate the aspects and effects of the present invention and to enable those of ordinary skill in the art to more fully understand the present invention, but should not be construed as limiting in any way.
Unless otherwise indicated, the materials and methods described in this specification are as follows:
1) materials: the fresh tobacco leaves used in the invention are collected in Kunming city of Yunnan province, and the fresh tobacco leaves are crushed, subpackaged and stored in a refrigerator at the temperature of-20 ℃ for later use. The extraction ethanol, petroleum ether and ethyl acetate are analytical pure reagents, and methanol in nicotine analysis is a chromatographic pure solvent. The sodium copper chlorophyllin standard substance is purchased from Shanghai Aladdin Biotechnology GmbH, and has a purity of more than 99%. Bovine Serum Albumin (BSA) standards were purchased from solibao biotechnology limited, nicotine and neophytadiene standards were purchased from shanghai feather biotechnology limited and gangrenum biotechnology limited, respectively, at purities greater than 98% and 90%, respectively. 2) The method comprises the following steps:
quantitative analysis method of chlorophyll: measuring the content of chlorophyll at 663nm and 645nm by spectrophotometer, diluting the obtained chlorophyll extractive solution by a certain multiple, and measuring the content with ethanol as control. The indexes, the content and the purity of the sodium copper chlorophyllin are determined according to the requirements of national standards GB 26406-2011 and GB 5009.260-2016. Quantitative analysis method of protein: (1) soluble protein is analyzed by adopting a Coomassie brilliant blue method, and the conditions of the Coomassie brilliant blue method are as follows: firstly, drawing a Bovine Serum Albumin (BSA) standard curve, drawing the standard curve by taking the concentration of BSA as an ordinate Y and the absorbance as an abscissa X, and determining a linear regression equation of Y-0.1432X-0.0224 and a linear correlation coefficient R20.9994, linear range of 0.01-0.08 mg ∙ mL-1The linear relationship is good in the linear range. And analyzing the content of the sample. (2) Using Kjeldahl method to treatProtein analysis is carried out, and the conditions of the Kjeldahl method are as follows: digestion: sequentially adding 0.2-1.0 g of crushed fresh tobacco leaves, 0.5g of copper sulfate-potassium sulfate mixed catalyst and 5mL of concentrated sulfuric acid into a digestion tube, placing the mixture into a digestion furnace, converting organic nitrogen into inorganic nitrogen within 400w and 2h, and judging a digestion endpoint: the solution changes from black to colorless or light green transparent liquid, which is the digestion end point. Adding 40% sodium hydroxide solution into KDN-04A azotometer to convert inorganic nitrogen into ammonia gas, distilling out with water vapor and receiving by boric acid solution. Titration: titrating by using calibrated hydrochloric acid, and judging a titration end point: the color of the material is changed from green to purple, and the material does not change color within 30 s.
Method for quantitative analysis of nicotine: the HPLC method is adopted for analysis, and the HPLC conditions are as follows: a chromatographic column: agilent 5HC-C18(2) 150X 4.6 mm; flow rates were all 1.0mg/mL, mobile phase: methanol-phosphate buffer (1:9), phosphate buffer 0.02mol/L Na2HPO40.01mol/L triethylamine, and the pH value is adjusted to 6.5 by phosphoric acid; detection wavelength: 260 nm. And (3) diluting the nicotine standard substance to different concentrations, measuring a standard curve, and analyzing the content of the sample.
The quantitative analysis method of solanesol comprises the following steps: the HPLC method is adopted for analysis, and the HPLC conditions are as follows: a chromatographic column: agilent 5HC-C18(2) 150X 4.6 mm; flow rates were all 1.0mg/mL, mobile phase: methanol and ethanol (1:1), detection wavelength: 210 nm. Diluting the solanesol standard substance to different concentrations, measuring a standard curve, and analyzing the content of the sample.
Quantitative analysis method of neophytadiene: the GC method is adopted for analysis, and the chromatographic conditions are as follows: HP-5MS column (30m × 0.25mm, particle size 0.25 μm) with N as carrier gas2The column front pressure is 400KPa, the air column front pressure is 50KPa, the hydrogen column front pressure is 60KPa, the injection port temperature is 280 ℃, and the detector temperature is 280 ℃; the sample injection amount is 1.0 mu L; the temperature rising procedure is as follows: the initial temperature is 50 ℃, the temperature is kept for 2min, the temperature is increased to 280 ℃ at the heating rate of 6 ℃/min, and the temperature is kept for 10 min. And (3) diluting the neophytadiene standard substance to different concentrations, determining a standard curve, and performing sample content analysis.
Example 1: determination of content of each effective component in fresh tobacco leaves
The contents of chlorophyll, nicotine, solanesol, flavors, proteins and the like are respectively measured by using the crushed fresh tobacco leaves as raw materials and adopting different measuring methods, and the measuring results of the contents of the effective components in the fresh tobacco leaves are shown in table 1.
TABLE 1 determination of the content of each active ingredient in fresh tobacco leaves
EXAMPLE 2 pretreatment of tobacco leaves
Fresh tobacco leaves collected in Yunnan province are firstly crushed by a traditional Chinese medicine crusher at 25000r/min for 30s, solid-liquid separation is sequentially carried out by operations of suction filtration, centrifugation and the like to obtain solid tobacco leaves and precipitated liquid, and 336g of solid tobacco leaves and 627g of precipitated liquid are obtained after 1000g of fresh tobacco leaves are pretreated, wherein the yield of the solid tobacco leaves and the precipitated liquid is 33.6 percent and 62.7 percent respectively.
Example 3: chlorophyll extraction (1)
336g of solid tobacco leaves obtained in example 2 are used as raw materials to extract chlorophyll, 90% (v%) ethanol solution is added according to the material-to-liquid ratio of 1:15(g: g), the mixture is extracted under heat reflux at 50 ℃ for 30min for 1 time, 5031mL of chlorophyll extract I and 330g of tobacco residue are obtained after suction filtration, the extraction rate of the chlorophyll is 45.3%, and the yield of the tobacco residue is 98.21%.
Example 4: chlorophyll extraction (2)
336g of solid tobacco leaves obtained in example 2 are taken as raw materials, 95% ethanol is added, the material-liquid ratio is 1:7(g: g), the extraction frequency is 2 times, each time is 30min, chlorophyll is extracted by hot reflux at 50 ℃, 4693mL of chlorophyll extraction liquid II and 327g of tobacco residue are obtained after suction filtration, the extraction rate of the chlorophyll is 79.62%, and the yield of the tobacco residue is 97.32%.
Example 5: preparation of sodium copper chlorophyllin (1)
The chlorophyll extraction solution I obtained in example 3 was adjusted to pH 12 with 67.2mL of 6mol/L sodium hydroxide solution, saponifying at 60 ℃ for 60min, vacuum concentrating the saponified solution at 35 ℃ to remove ethanol, adding petroleum ether with the volume twice of the volume of the saponified solution, extracting for 3 times to obtain an extract I and a raffinate I, adding 40.32mL of 10% sulfuric acid solution into the raffinate I to adjust the pH to be 2-3, acidifying at 60 ℃ for 30min, adding 30.24mL of 15% copper sulfate solution into the raffinate I, performing a cuprinization reaction at 60 ℃ for 60min, filtering to obtain dark green chlorophyllin copper acid precipitate, dissolving the precipitate with acetone, adding 10% NaOH solution to adjust the pH to be about 12, standing and crystallizing at room temperature for 60min to convert the chlorophyllin copper acid into sodium chlorophyllin, removing the acetone and washing the precipitate with acetone for multiple times, drying at 50 ℃, crushing to obtain 0.146g of sodium chlorophyllin copper salt, wherein the yield and the purity of the sodium chlorophyllin copper salt are 72.65% and 81% respectively.
Example 6: preparation of sodium copper chlorophyllin (2)
Adding 67.2mL of 6mol/L sodium hydroxide solution into the chlorophyll extracting solution II obtained in the embodiment 4 dropwise, adjusting the pH value to 12, carrying out saponification reaction at 60 ℃ for 60min, carrying out vacuum concentration on the saponification solution at 35 ℃ to remove ethanol, adding petroleum ether with twice volume of the saponification solution into the rest part, extracting for 3 times to obtain an extracting solution I and a raffinate I, adding 40.32mL of 10% sulfuric acid solution into the raffinate I to adjust the pH value to 2-3, carrying out acidification reaction at 60 ℃ for 30min, adding 30.24mL of 15% copper sulfate solution dropwise, carrying out a cuprinization reaction at 60 ℃ for 60min, filtering to obtain a dark green chlorophyll copper acid precipitate, firstly washing with water to neutrality, then removing unsaponifiable substances with 30-40% ethanol, and finally washing with petroleum ether until the solution is light yellow to remove impurities such as lutein and carotene. Dissolving the precipitate with acetone, adding 10% NaOH ethanol solution dropwise to adjust pH to about 12, stirring at room temperature for 10min, standing at 4 deg.C for crystallization for 60min to convert copper chlorophyllin into sodium copper chlorophyllin, filtering, washing with acetone, drying at 50 deg.C to water content below 4%, pulverizing to obtain 0.161g sodium copper chlorophyllin with yield and purity of 82.05% and 93.96%, and detecting by HPLC that no nicotine is contained.
Example 7: preparation of Nicotine, solanesol and fragrance (1)
The extract I obtained in example 5 was distilled and dried to obtain an extract I containing nicotine, solanesol and flavor, about 3.66g, with a yield of 1.09%, and extraction rates of nicotine, solanesol and flavor of 35.2%, 65.7% and 32%, respectively. Dissolving the extract I with water under ultrasound, adjusting pH to 2, adding petroleum ether, and extracting for 3 times to obtain extract II and raffinate II; concentrating the extract II, dehydrating, distilling, and drying to obtain extract II containing solanesol and perfume; dissolving the extractum II containing solanesol and the spices in 90% ethanol, loading the macroporous resin AB-8, and performing flow-through combination and concentration to dryness to obtain an extractum III containing the spices, wherein the yield of the spices is 83.17%, and the content of the neophytadiene is 10.8%. Eluting with 11BV of 100% ethanol, concentrating and drying the eluent to obtain purified solanesol, dissolving the sample in acetonitrile at 70 ℃, crystallizing at 4 ℃ for 5h, filtering and washing to obtain solanesol crystal, recrystallizing the solanesol crystal according to the same method to obtain a pure solanesol product, wherein the yield of the solanesol is 86.1%, and the purity is 95.2%. Adjusting pH of the raffinate II to 10, adding petroleum ether with the same volume for extraction for 3 times to obtain an extract III and a raffinate III, and distilling and drying the extract III to obtain a nicotine extract IV. Dissolving the nicotine extract in water, adjusting pH to 10, adding petroleum ether with the same volume for extraction for 3 times, and concentrating the extract to obtain pure nicotine product with yield of 79.21% and purity of 95%.
Example 8: preparation of Nicotine, solanesol and fragrance (2)
The saponified solution of example 6 was concentrated under vacuum at 35 ℃ to remove ethanol, and then extracted with petroleum ether for 5 times, wherein the petroleum ether phase was colorless at the time of the 5 th extraction, and the obtained extract I was concentrated under vacuum to obtain extract I of nicotine, solanesol and perfume, which was about 4.87g, with a yield of 1.33%, and the extraction rates of nicotine, solanesol and perfume were 42.53%, 95.62% and 45%, respectively (see table 2). The subsequent purification in the same manner as in example 7 also gave nicotine, solanesol and flavor in high purity.
TABLE 2 content and extraction yield of nicotine, solanesol and perfume in extract I
| Active ingredient | Content (%) | Extraction ratio (%) |
| Nicotine | 9.4 | 42.53 |
| Solanesol | 13.5 | 95.62 |
| Perfume | 2.71 | 45.00 |
Example 9: preparation of protein (1)
627g of the eluate of example 2 was used as a raw material, and an isoelectric precipitation method was used to precipitate proteins from the eluate, the pH was adjusted to 3.5 with phosphoric acid, and the precipitate was allowed to stand at room temperature and centrifuged to obtain a clear solution and a protein precipitate. The protein precipitate was freeze-dried to give crude protein powder with protein yields and purities of about 27.6% and 45.7%. The protein precipitate was dissolved in disodium hydrogenphosphate-sodium dihydrogenphosphate loading buffer (0.02mol/L, pH 8.0), centrifuged, and purified by anion exchange chromatography, the protein peak was collected, and the protein solution was concentrated to 4mL using an ultrafiltration tube with a molecular weight cut-off of 3000D, and the protein concentration was determined to be 4.5 mg/mL.
Example 10: preparation of protein (2)
627g of the eluate obtained in example 2 was used as a raw material, and an isoelectric precipitation method was used to adjust the pH to 3.0 with phosphoric acid, the precipitation time was 30min, the precipitation temperature was 4 ℃, and after centrifugation, a clear solution and a protein precipitate were obtained. The protein precipitate was freeze-dried to give crude protein powder with protein yields and purities of about 47% and 58.5%. The protein precipitate was thoroughly solubilized using 0.1mol/L NaCl solution at pH 7.5, purified by anion exchange chromatography after centrifugation, and the protein solution was concentrated to 4mL using an ultrafiltration tube with a molecular weight cut-off of 3000D, and the protein concentration was determined to be 8.48 mg/mL.
Example 11: preparation of microbial fertilizer
And (2) taking 327g of the fresh tobacco residue obtained in the example 4, wherein the fresh tobacco residue contains about 77.5% of organic solvent, naturally airing to remove ethanol to obtain about 73.58g of aired tobacco residue, inoculating bacillus subtilis to perform solid state fermentation to prepare bacterial fertilizer, wherein the inoculation amount is 10%, the thickness of a material layer is 1-5 cm, culturing for 7 days at 8-15 ℃, and fermenting to prepare the microbial fertilizer. The spore amount of Bacillus subtilis reaches 109。
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (4)
1. The co-production method of various products in fresh tobacco leaves comprises the following steps:
(1) pretreatment of raw materials: crushing fresh tobacco leaves, and then carrying out solid-liquid separation to obtain solid tobacco leaves and a precipitation liquid; the solid-liquid separation method is a natural sedimentation method, a vacuum filtration method or a plate-and-frame filter pressing method;
(2) preparation of sodium copper chlorophyllin: adding an extraction solvent I into the solid tobacco leaves obtained in the step (1) for extraction, and performing suction filtration to obtain an extracting solution I and tobacco residues; adding an alkaline substance I into the extracting solution I for saponification; removing the organic solvent from the aqueous solution of the saponified solution, and extracting by using a weak-polarity organic solvent to obtain an extract I and a raffinate I; adjusting the pH value of the raffinate I by using an acidic substance, adding a solution containing copper ions, carrying out copper treatment to obtain copper chlorophyllin acid, adding an alkaline solution II to adjust the pH value, standing to obtain sodium copper chlorophyllin crystals, washing and drying to obtain sodium copper chlorophyllin;
(3) preparing protein: extracting protein from the precipitation liquid obtained in the step (1) by adopting an isoelectric precipitation method, a flocculant precipitation method, an ethanol precipitation method or an ammonium sulfate precipitation method;
(4) preparing nicotine, solanesol and spices from the extract I in the step (2), comprising the following steps of: distilling and drying the extract I to obtain an extract, adding water into the extract under the ultrasonic condition, adjusting the pH value, and extracting by using an isovolumetric organic solvent to obtain an extract II and a raffinate II; distilling the extract II, drying, dissolving in aqueous ethanol, performing macroporous resin chromatography, eluting with ethanol water solution, mixing the flow-through components, and concentrating to dry to obtain essence extract refined product; mixing the eluted components, concentrating to dry, dissolving in ethanol, methanol, acetone or acetonitrile, cooling and crystallizing to obtain solanesol; adjusting the pH value of the raffinate II, adding an organic solvent with the same volume to extract to obtain an extract III and a raffinate III, and distilling and drying the extract III to obtain a pure nicotine product;
(5) and (3) preparing the biological bacterial fertilizer by using the tobacco residue in the step (2), and inoculating microbial strains to the tobacco residue as a raw material for solid culture.
2. The method of claim 1,
in the step (1), the solid-liquid separation method is a vacuum filtration method;
the extraction solvent I in the step (2) is selected from an aqueous solution of ethanol, acetone, methanol, n-propanol, isopropanol or a mixture thereof, the concentration is 60-100%, and the mass volume ratio of the feed liquid is 1: 2.5-15; the extraction times are 1-5 times; the extraction time is 20-80 min/time; the extraction method is selected from cold soaking extraction method, ultrasonic oscillation method or hot reflux method, and the hot reflux temperature is 50-70 deg.C; the alkaline substance I is sodium hydroxide, potassium hydroxide, calcium hydroxide and sodium carbonate, and the saponification pH value is 8-14; the saponification temperature is 50-70 ℃; the weak polar organic solvent is at least one of petroleum ether, ethyl acetate and n-hexane, and the extraction times are 2-5 times; the acidic substance is at least one selected from sulfuric acid, phosphoric acid and hydrochloric acid, and the adjusted pH value is 2-5; the copper ion-containing solution is copper sulfate, copper chloride solution or a mixture of copper sulfate and copper chloride solution, and the concentration of the copper ion-containing solution is 10-30%; the alkaline solution II is NaOH aqueous solution or NaOH ethanol solution, the concentration of the alkaline solution II is 5-20%, and the adjusted pH value is 10-14; the crystallization temperature is 0-10 ℃, and the time is 30 min-2 h;
in the step (3), protein is prepared by adopting an isoelectric precipitation method, the pH of the precipitation liquid is adjusted to 3-7 at the temperature of 0-20 ℃, and the precipitation time is 0.5-4.0 h; the substance for adjusting pH is at least one selected from phosphoric acid, sulfuric acid and hydrochloric acid;
in the step (4), the organic solvent is at least one of petroleum ether, ethyl acetate, n-hexane, butyl acetate or kerosene; when extract II is obtained, the pH value is adjusted to 1-6; adjusting pH to 8-14 when obtaining extract III; before macroporous resin chromatography, the concentration of ethanol in the hydrous ethanol for dissolving the sample is 70-90 percent; the macroporous resin is nonpolar or weak polar macroporous resin selected from XDA-1,801,806, XR919C, HPD600, AB-8 or HZ 802; the concentration of ethanol in the aqueous ethanol eluted after macroporous resin is 80-100%, and the crystallization temperature is-20-10 ℃; in the step (5), the microbial strain is bacillus subtilis, and the inoculation amount is 10%; the thickness of the fermentation material layer is 1-5 cm, the fermentation temperature is 8-15 ℃, and the fermentation time is 5-8 days.
3. The method according to claim 2, wherein in the step (2), the extraction solvent I is an ethanol aqueous solution with a concentration of 90-100%, the mass-to-volume ratio of the feed liquid is 1: 7-10, the extraction times are 2-5 times, the extraction time is 30-60 min/time, and the extraction method is a 50 ℃ thermal reflux method.
4. The method according to claim 2, wherein in the step (3), the substance for adjusting the pH is phosphoric acid, the pH is adjusted to 3-3.5 by using phosphoric acid at 0-4 ℃, and the precipitation time is 0.5-2.0 h.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3847163A (en) * | 1972-03-06 | 1974-11-12 | Brown & Williamson Tobacco | Method of making a tobacco extract |
| CN102429319A (en) * | 2011-08-26 | 2012-05-02 | 重庆恒远晋通科技有限公司 | Continuous extracting method for tobacco |
| CN104151140A (en) * | 2014-07-11 | 2014-11-19 | 武汉工程大学 | Method for comprehensively extracting multiple effective components from tobacco leaves |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3847163A (en) * | 1972-03-06 | 1974-11-12 | Brown & Williamson Tobacco | Method of making a tobacco extract |
| CN102429319A (en) * | 2011-08-26 | 2012-05-02 | 重庆恒远晋通科技有限公司 | Continuous extracting method for tobacco |
| CN104151140A (en) * | 2014-07-11 | 2014-11-19 | 武汉工程大学 | Method for comprehensively extracting multiple effective components from tobacco leaves |
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