Method for detecting protein content in tobacco

Abstract

The invention provides a method for detecting the content of protein in tobacco. The method comprises the following steps: 1) mixing a tobacco sample to be detected with a solution of an enzyme preparation, and reacting; (2) adding a dimethyl sulfoxide aqueous solution into a product obtained in the step (1), mixing, and extracting protein; the enzyme preparation comprises pectinase, cellulase and hemicellulase.The method has the advantages that soluble and insoluble proteins in tobacco leaves can be sufficiently extracted by the aid of the method, and the method is high in selectivity and sensitivity and high in accuracy of detection results.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tobacco detection, and particularly relates to a method for detecting protein content in tobacco. BACKGROUND

[0002] Protein is one of the main macromolecular substances in tobacco, and its content is an important factor affecting the quality of tobacco raw materials. The protein content in fresh tobacco leaves is relatively high, and the amino acids produced by curing and aging and the Maillard products further converted are important aroma substances in tobacco. The protein content in cigarettes is closely related to smoke density and strength, and a too high content will produce a burnt feather smell and a pungent, bitter and astringent feeling during smoking. Therefore, accurate determination of the protein content in tobacco is of great significance for scientific evaluation of tobacco leaf quality and has a great demand for detection in the industry.

[0003] Currently, the existing protein detection standards in the tobacco industry, namely, "Determination of Total Protein in Tobacco and Tobacco Products" (YC / T 166-2003) and "Determination of Protein in Tobacco and Tobacco Products - Continuous Flow Method" (YC / T 294-2008), are both based on the Kjeldahl method: organic nitrogen-containing nitrogen is removed by acetic acid, and soluble protein is precipitated at the same time, and then the protein is converted into ammonia nitrogen by solid-liquid separation, high-temperature acid digestion and filtration of the residue. The protein content is then calculated by multiplying the empirical constant 6.25 after the ammonia nitrogen is detected by titration or continuous flow analyzer colorimetry. The above method has the following disadvantages: 1. The target is not accurate (the detection object is organic nitrogen rather than protein), and the detection result cannot reflect the true protein content in the sample (part of the organic nitrogen cannot be completely removed by acetic acid); 2. The process is complicated (heating, filtration, washing, digestion, constant volume, detection, etc. are required), and the accuracy of the result is greatly affected by personnel operation; 3. Strong acid (concentrated sulfuric acid) is required for high-temperature (370℃) acidolysis for a long time (5 h), which is harmful to the environment; 4. The reagents used, such as mercuric oxide, nitroso iron and sodium cyanide, are highly toxic and regulated, and are not easy to purchase and manage.

[0004] The Coomassie brilliant blue method, also known as the Bradford method, is a method widely used for protein determination. The basic principle is that under acidic conditions, Coomassie brilliant blue G-250 selectively binds with protein to form a blue protein-dye complex, which specifically absorbs at 595 nm, and the absorbance is proportional to the concentration of protein. This method has high sensitivity (the minimum protein detection concentration can reach 1 mg / L), simple and rapid operation (only about 5 min is required for a single sample determination), and less interference (not affected by pigments, polypeptides, amino acids, etc.), and is adopted by standards such as SN / T 2497.20-2010 and SN / T 3926-2014.

[0005] The proteins in tobacco can be divided into soluble proteins and insoluble proteins. In brief, the soluble proteins refer to free proteins in cytoplasm, and the soluble proteins include water-soluble proteins, alcohol-soluble proteins and oil-soluble proteins; and the insoluble proteins refer to bound proteins in cell walls, cell membranes and organelles. In order to fully extract the proteins in tobacco for detection, the extraction method and detection means need to be improved. SUMMARY

[0006] Therefore, the present application aims at the problems of the current protein determination standard method in the industry, such as complicated process, inaccurate targeting, environmental unfriendliness, and difficulty in purchasing and managing reagents, and proposes a new method for detecting the protein content in tobacco.

[0007] The present application aims to provide a method for detecting the protein content in tobacco.

[0008] To achieve the above-mentioned object, the present application adopts the following technical solutions.

[0009] The soluble and insoluble proteins in tobacco are fully extracted by using tobacco-derived biological enzymes assisted ultrasonic cell wall breaking and dimethyl sulfoxide / water solution, and finally quantitatively detected by Coomassie brilliant blue staining method.

[0010] Specifically, the present application provides a method for detecting the protein content in tobacco, comprising:

[0011] 1) mixing the tobacco sample to be detected with a solution of enzyme preparation and reacting;

[0012] 2) adding dimethyl sulfoxide aqueous solution to the product obtained in 1), mixing, and extracting proteins;

[0013] 3) performing solid-liquid separation on the product obtained in 2);

[0014] 4) detecting the protein content in the liquid obtained in 3),

[0015] wherein the enzyme preparation comprises pectinase, cellulase and hemicellulase.

[0016] In some embodiments, the enzyme activity ratio of pectinase, cellulase and hemicellulase in the enzyme preparation is 18-22:0.8-1.2:0.8-1.2. In some embodiments, the enzyme activity ratio of pectinase, cellulase and hemicellulase in the enzyme preparation is 19-21:0.9-1.1:0.9-1.1. In some embodiments, the enzyme activity ratio of pectinase, cellulase and hemicellulase in the enzyme preparation is 20:1:1.

[0017] In certain embodiments, the enzyme preparation is made by liquid fermentation of Paenibacillus amylolyticus. In certain embodiments, the enzyme preparation is made by mixing pectinase, cellulase and hemicellulase.

[0018] In certain embodiments, the enzyme preparation is made by a method comprising the following steps:

[0019] 1) inoculating Paenibacillus amylolyticus into a liquid medium;

[0020] 2) fermentation;

[0021] 3) solid-liquid separation of fermentation products;

[0022] 4) separating and collecting proteins with a molecular weight greater than or equal to 10 kDa in the resulting liquid,

[0023] wherein the liquid medium comprises tobacco powder 0.8-1.2 g / L, soybean peptone 3-7 g / L, sucrose 0.8-4 g / L, xylan 0.3-0.7 g / L, orange peel pectin 0.3-0.7 g / L, sodium carboxymethyl cellulose 0.3-0.7 g / L, manganese sulfate monohydrate 0.08-0.4 g / L, sulfuric acid heptahydrate 0.3-0.7 g / L, potassium dihydrogen phosphate 3-7 g / L and water, and the pH value of the liquid medium is 6.8-7.2.

[0024] In certain embodiments, the liquid medium comprises tobacco powder 0.9-1.1 g / L, soybean peptone 4-6 g / L, sucrose 1-3 g / L, xylan 0.4-0.6 g / L, orange peel pectin 0.4-0.6 g / L, sodium carboxymethyl cellulose 0.4-0.6 g / L, manganese sulfate monohydrate 0.1-0.3 g / L, sulfuric acid heptahydrate 0.4-0.6 g / L, potassium dihydrogen phosphate 4-6 g / L and water, and the pH value of the liquid medium is 6.9-7.1.

[0025] In certain embodiments, the liquid medium comprises tobacco powder 1 g / L, soybean peptone 5 g / L, sucrose 2 g / L, xylan 0.5 g / L, orange peel pectin 0.5 g / L, sodium carboxymethyl cellulose 0.5 g / L, manganese sulfate monohydrate 0.2 g / L, sulfuric acid heptahydrate 0.5 g / L, potassium dihydrogen phosphate 5 g / L and water, and the pH value of the liquid medium is 7.0.

[0026] In certain embodiments, the Paenibacillus amylolyticus has a preservation number of CGMCC 24247.

[0027] In some embodiments, the Paenibacillus amylolyticus is formulated into a seed solution at a concentration of 10 8 ~10 9 CFU / mL, and then inoculated into a liquid medium, preferably at an inoculation amount of 0.8-1.2 v / v% (e.g., 1 v / v%).

[0028] In some embodiments, after the Paenibacillus amylolyticus is inoculated into a liquid medium, the fermentation is carried out at 36-38°C (e.g., 37°C) and 160-200 r / min (e.g., 180 r / min) for 16-20 h (e.g., 18 h).

[0029] In some embodiments, the solid-liquid separation of the fermentation product is achieved by centrifugation, preferably at a centrifugation speed of 6000-10000 r / min (e.g., 8000 r / min).

[0030] In some embodiments, the separation of proteins with a molecular weight greater than or equal to 10 kDa is achieved by using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and the separated protein retentate is freeze-dried to obtain an enzyme preparation.

[0031] In some embodiments, the solution of the enzyme preparation is an enzyme preparation Tris-HCl buffer solution prepared by mixing the enzyme preparation and a Tris-HCl buffer.

[0032] In some embodiments, the Tris-HCl buffer is a 0.04-0.06 M, pH 5-6 Tris-HCl buffer. In some embodiments, the Tris-HCl buffer is a 0.05 M, pH 5.5 Tris-HCl buffer.

[0033] In some embodiments, the concentration of the solution of the enzyme preparation is 0.8-1.2 mg / mL (e.g., 1 mg / mL).

[0034] In some embodiments, the tobacco sample to be tested is a tobacco leaf powder, for example, a tobacco leaf powder with a moisture content of less than or equal to 8% that can pass through a 40-mesh sieve.

[0035] In some embodiments, 0.2-0.3 g of the tobacco sample to be tested is mixed with 32-48 mL of the solution of the enzyme preparation and reacted. In some embodiments, 0.25 g of the tobacco sample to be tested is mixed with 40 mL of the solution of the enzyme preparation and reacted. In some embodiments, after the tobacco sample to be tested is mixed with the solution of the enzyme preparation, it is reacted at 48-52°C for 0.8-1.21 h. In some embodiments, after the tobacco sample to be tested is mixed with the solution of the enzyme preparation, it is reacted at 50°C for 1 h.

[0036] In certain embodiments, the volume ratio of dimethyl sulfoxide to water in the aqueous dimethyl sulfoxide solution is 1:1.5 to 1.5:1. In certain embodiments, the volume ratio of dimethyl sulfoxide to water in the aqueous dimethyl sulfoxide solution is 1:1.2 to 1.2:1, for example 1:1.

[0037] In certain embodiments, the amount of the aqueous dimethyl sulfoxide solution added in step 2) is 240 mL to 400 mL per g of the tobacco sample to be tested. In certain embodiments, the amount of the aqueous dimethyl sulfoxide solution added in step 2) is 320 mL per g of the tobacco sample to be tested.

[0038] In certain embodiments, the protein is extracted by ultrasonic extraction in step 2), preferably for 1 h to 2.5 h, further preferably for 1.5 h.

[0039] In certain embodiments, the protein content in the liquid obtained in step 3) is detected by the Coomassie brilliant blue method in step 4). In certain embodiments, step 4) comprises: taking the liquid obtained in step 3), adding 10 to 50 times (for example 25 times) the volume of a Coomassie brilliant blue G-250 solution with a concentration of 80 mg / L to 120 mg / L (for example 100 mg / L), and detecting the protein content after mixing.

[0040] In certain embodiments, the method for detecting the protein content in tobacco according to the present application further comprises detecting a blank control sample, which differs from the tobacco sample to be tested in that no tobacco sample to be tested is added in step 1) when detecting. The protein content in the tobacco sample to be tested is the protein content in the liquid obtained in step 3) minus the protein content of the blank control sample.

[0041] In certain embodiments, the method for detecting the protein content in tobacco according to the present application comprises:

[0042] Experimental group: 0.25 g of tobacco powder sample was accurately weighed in the inner sleeve of the outer sleeve glass centrifuge tube, 40 mL of enzyme solution was added, and the reaction was carried out at 50°C for 1 h. After the reaction was completed, 40 mL of dimethyl sulfoxide (the enzyme solution was an aqueous solution, and at this time the volume ratio of dimethyl sulfoxide to water was 1:1) was added, ultrasonic extraction was carried out at room temperature for 1.5 h, solid-liquid separation was carried out, 0.2 mL of the extraction liquid was removed, 5 mL of Coomassie brilliant blue G-250 solution was added, and after mixing and standing for 5 min, the light absorption value at 595 nm was measured on an ultraviolet spectrophotometer. The light absorption value was converted to the protein content by the protein standard curve.

[0043] Control group: 40 mL of enzyme solution was mixed with 40 mL of dimethyl sulfoxide and sonicated at room temperature for 1.5 h. 0.2 mL of the mixture was then added to 5 mL of Coomassie Brilliant Blue G-250 solution, mixed well, and allowed to stand for 5 min before measuring the absorbance at 595 nm using a UV spectrophotometer. The protein content was obtained by converting the absorbance value to a protein standard curve.

[0044] Protein content in tobacco leaves = total protein content in the experimental group - protein content in the enzyme solution in the control group.

[0045] The enzyme solution was prepared by liquid fermentation of *Paenibacillus amylolyticus* CGMCC 24247. The fermentation medium consisted of 1 g / L tobacco powder, 5 g / L soybean peptone, 2 g / L sucrose, 0.5 g / L xylan, 0.5 g / L orange peel pectin, 0.5 g / L sodium carboxymethyl cellulose, 0.2 g / L manganese sulfate monohydrate, 0.5 g / L sulfuric acid heptahydrate, and 5 g / L potassium dihydrogen phosphate, with an initial pH of 7.0. The solvent was distilled water. The fermentation parameters were 37℃, 180 r / min, and 18 h. The enzyme preparation process involved centrifuging the fermentation broth at 8000 r / min, then using a 10 kDa ultrafiltration membrane to retain the protein. The retained solution was freeze-dried to prepare the enzyme preparation. Finally, a 1 mg / mL enzyme solution was prepared using Tris-HCl buffer (0.05 M, pH 5.5).

[0046] The preparation method for Coomassie Brilliant Blue is as follows: Weigh 100 mg of Coomassie Brilliant Blue G-250, dissolve it in 50 mL of 95% ethanol, add 120 mL of phosphoric acid, and dilute to 1 L in a brown volumetric flask with a 1:1 dimethyl sulfoxide / water solution. Filter with filter paper and store at 4℃.

[0047] The method for constructing the protein standard curve is as follows: Accurately weigh 100 mg of bovine serum albumin (BSA), dissolve it in a 1:1 (v / v) dimethyl sulfoxide / water solution, and dilute to a final volume of 100 mL in a volumetric flask to prepare a 1.0 mg / mL protein standard solution. Accurately transfer 0, 0.02, 0.04, 0.08, 0.12, 0.16, and 0.20 mL of BSA solution into 10 mL stoppered centrifuge tubes, respectively, and replenish to 0.2 mL with a 1:1 (v / v) dimethyl sulfoxide / water solution. Add 5 mL of Coomassie Brilliant Blue G-250 solution to each tube, mix well, and let stand for 5 min. Measure the absorbance of the solution at 595 nm. Plot the standard curve with absorbance as the ordinate and protein concentration as the abscissa. The regression equation is y = 1.2292x + 0.0142, R0. 2 =0.9996.

[0048] Beneficial effects of the present invention

[0049] The method for detecting protein content in tobacco provided by this invention has one or more of the following advantages:

[0050] 1. High efficiency and environmental protection: The use of dimethyl sulfoxide aqueous solution to replace traditional water bath or digestion reduces energy consumption and chemical risks, and shortens operation time.

[0051] 2. Precise cell wall disruption: The tobacco sample is treated with an enzyme preparation solution. The pectinase, cellulase, and hemicellulase in the enzyme preparation help release soluble proteins in the cytoplasm and insoluble proteins in the cell wall, thereby improving the protein extraction efficiency.

[0052] 3. High selectivity and sensitivity: The Coomassie Brilliant Blue method specifically binds to proteins, avoiding non-protein interference. The method provided by this invention can detect proteins in tobacco samples with a detection limit as low as 1.2 mg / L and a linear range of 0~1 mg / mL.

[0053] 4. High accuracy of test results: The method provided by this invention has a spiked recovery rate of 98.48% and a repeatability RSD of <5% (e.g., 0.58%~0.72%), which is consistent with the original standard method but more accurate.

[0054] 5. High applicability: The method provided by this invention can simultaneously or separately determine water-soluble and water-insoluble proteins, and is applicable to different tobacco samples. Attached Figure Description

[0055] Figure 1 This is a standard curve of protein standard concentration versus absorbance.

[0056] Figure 2 The image shows the UV-Vis spectrum of the acid hydrolysate residue after ultrasonic extraction of the sample with dimethyl sulfoxide aqueous solution.

[0057] Figure 3 This is the UV-Vis full-wavelength spectrum of an aqueous solution of dimethyl sulfoxide. Detailed Implementation

[0058] The following specific embodiments further illustrate the substantive content of this application. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this application. In the following embodiments, unless specific conditions are specified, conventional conditions or manufacturer recommendations are followed. Raw materials whose manufacturers are not specified are all commercially available conventional products.

[0059] While many of the materials and operating methods used in the following embodiments are well known in the art, this application still describes them in as much detail as possible. It will be apparent to those skilled in the art that, unless otherwise stated, the materials and operating methods used in the following embodiments are well known in the art.

[0060] Preparation of tobacco powder: After the tobacco leaves are cut into shreds, they are dried in an oven at 40°C for 2 hours, ground and pulverized, and then sieved. The portion that passes through 40 mesh is taken for testing.

[0061] Enzyme preparation A: Prepared by liquid fermentation of *Paenibacillus amylolyticus* CGMCC 24247. The fermentation medium consisted of: 1 g / L tobacco powder, 5 g / L soybean peptone, 2 g / L sucrose, 0.5 g / L xylan, 0.5 g / L orange peel pectin, 0.5 g / L sodium carboxymethyl cellulose, 0.2 g / L manganese sulfate monohydrate, 0.5 g / L sulfuric acid heptahydrate, and 5 g / L potassium dihydrogen phosphate, with an initial pH of 7.0. The solvent was distilled water. *Paenibacillus amylolyticus* was prepared into a 10... 8 ~10 9 CFU / mL seed culture was inoculated into the above fermentation medium at an inoculation rate of 1 v / v%. The fermentation parameters were: 37℃, 180 r / min, and fermentation time of 18 h. The enzyme preparation process was as follows: after centrifugation of the fermentation broth at 8000 r / min, the protein was retained by a 10 kDa ultrafiltration membrane. The retained liquid was freeze-dried to prepare the enzyme preparation, which was named "Enzyme Preparation A". Reference [Zhang Yifan, Hu Sulin, Du Wenjie, et al. Screening of a fungal strain with polysaccharide degrading enzyme activity and its application in improving tobacco quality [J]. Journal of Tobacco Science, 2024, 30(04):84-94.] The method disclosed in the article was used to detect pectinase, cellulase and hemicellulase in "Enzyme Preparation A". The enzyme activities were measured to be 100,000 U / g, 5,000 U / g and 5,000 U / g, respectively. The ratio of the three enzyme activities was 20:1:1.

[0062] Enzyme Preparation B: Commercial pectinase (purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S10007-25g, nominal enzyme activity 50,000 U / g), commercial cellulase (purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S10041-25g, nominal enzyme activity 50,000 U / g), and commercial hemicellulase (purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S10045-25g, nominal enzyme activity 20,000 U / g) were mixed according to their nominal enzyme activities. The enzyme activity ratio after mixing was 20:1:1. This mixture was named "Enzyme Preparation B".

[0063] Preparation of enzyme solutions: Enzyme preparation A and enzyme preparation B were prepared into 1 mg / mL enzyme solutions using Tris-HCl buffer (0.05 M, pH 5.5, purchased from Shanghai Yuanye, which was prepared by diluting 1 M Tris-HCl buffer at pH 5.5 with deionized water).

[0064] Preparation of Coomassie Brilliant Blue G-250 solution: Weigh 100 mg of Coomassie Brilliant Blue G-250, dissolve in 50 mL of 95% ethanol, add 120 mL of phosphoric acid, and dilute to 1 L in a brown volumetric flask with a 1:1 (v / v) dimethyl sulfoxide / water solution. Filter with filter paper and store at 4℃.

[0065] Preparation of protein standard solution: Accurately weigh 100 mg of bovine serum albumin, dissolve it in a 1:1 (v / v) dimethyl sulfoxide / water solution, and dilute to 100 mL in a volumetric flask to obtain a 1.0 mg / mL protein standard solution.

[0066] Example 1: Extraction and Total Protein Detection from Tobacco Leaves

[0067] Extraction and Detection Methods: After shredding tobacco leaves, dry them in an oven at 40℃ for 2 hours. Grind the dried leaves into a fine powder and sieve. Take the portion passing through a 40-mesh sieve as the sample to be tested. Accurately weigh 0.25 g of the tobacco powder sample into the inner sleeve of a glass-coated centrifuge tube. Add B volumes of dimethyl sulfoxide / water solution (dimethyl sulfoxide / water volume ratio A). Extract using ultrasonication at room temperature for C hours. After solid-liquid separation, transfer 0.2 mL of the extract to D volumes of Coomassie Brilliant Blue G-250 solution. Mix well and let stand for 5 min. Measure the absorbance at 595 nm using a UV spectrophotometer.

[0068] (1) Effect of the volume ratio A of dimethyl sulfoxide to water on the retention results

[0069] Following the extraction and detection methods described above, proteins in the test samples were extracted using 100 mL of dimethyl sulfoxide / water solution at different volume ratios. The extraction was performed by ultrasonic extraction at room temperature for 2 h, and the protein content was then detected. The results are shown in Table 1.

[0070] Table 1. Protein detection results with different extractant volume ratios

[0071]

[0072] The results showed that the extraction effect was better when the volume ratio of dimethyl sulfoxide to water was between 4:6 and 6:4. The preferred extraction solvent was a dimethyl sulfoxide / water solution with a volume ratio between 4:6 and 6:4 (1:1.5 to 1.5:1), and the preferred extraction solvent was a dimethyl sulfoxide / water solution with a volume ratio of 5:5 (1:1).

[0073] (2) The effect of the mention of extraction solvent B on the detection results

[0074] Following the extraction and detection methods described above, the proteins in the test samples were extracted with 20, 40, 60, 80, 100, and 120 mL of dimethyl sulfoxide / water solution at a volume ratio of 1:1. The extraction was performed by ultrasonic extraction at room temperature for 2 h, and then the protein content was detected. The results are shown in Table 2.

[0075] Table 2. Results of total protein detection with different volumes of extractant

[0076]

[0077] The results showed that the detection results reached their peak when the volume of the extraction solvent was greater than 60 mL. Therefore, the preferred volume of the extraction solvent is 60 mL to 100 mL. Considering the sample concentration and cost, the preferred volume of the extraction solvent is 80 mL.

[0078] (3) Effect of ultrasonic extraction time C on detection results

[0079] Following the extraction and detection methods described above, the protein in the sample was extracted with 80 mL of dimethyl sulfoxide / water solution at a volume ratio of 1:1. The extraction was performed by ultrasonic extraction at room temperature for 0.5, 1, 1.5, 2, and 2.5 h, respectively. The protein content was then detected, and the results are shown in Table 3.

[0080] Table 3 Protein detection results at different times

[0081]

[0082] The results showed that the detection results reached their peak after the extraction time was greater than 1 h. The optimal extraction time was 1 h to 2.5 h. Considering the sample concentration and time cost, the optimal extraction time was 1.5 h.

[0083] (4) Effect of Coomassie Brilliant Blue G-250 solution volume D on detection results

[0084] Following the extraction method described above, the protein in the sample was extracted with 80 mL of a 1:1 (v / v) dimethyl sulfoxide / water solution. The extraction was performed by ultrasonic extraction at room temperature for 1.5 h, followed by protein content determination. 0.2 mL of the extract was transferred, and 1, 2, 3, 4, 5, and 10 mL of Coomassie Brilliant Blue G-250 solution were added respectively. After mixing and standing for 5 min, the absorbance was measured using a UV spectrophotometer, tracking the changes in light absorption near 465 nm and 595 nm. The results showed that when the volume of Coomassie Brilliant Blue G-250 solution was greater than 2 mL, the absorbance did not change, indicating that adding a volume greater than 2 mL of chromogenic reagent met the analytical requirements. Therefore, the preferred volume of Coomassie Brilliant Blue G-250 solution is 2 mL to 10 mL. Considering sample concentration and cost, a volume of 5 mL of Coomassie Brilliant Blue G-250 solution was further optimized.

[0085] In summary, the preferred method for detecting protein content in tobacco is as follows: After shredding tobacco leaves, dry them in an oven at 40℃ for 2 hours, grind them into powder, and sieve them. Take the portion passing through a 40-mesh sieve for testing. Accurately weigh 0.25 g of tobacco powder sample into the inner sleeve of a glass-coated centrifuge tube, add 80 mL of dimethyl sulfoxide / water solution (dimethyl sulfoxide / water volume ratio 1:1), and sonicate at room temperature for 1.5 hours. Separate the solid and liquid phases, transfer 0.2 mL of the extract, add 5 mL of Coomassie Brilliant Blue G-250 solution, mix well, let stand for 5 minutes, and then measure the absorbance at 595 nm using a UV spectrophotometer.

[0086] (5) Accuracy assessment

[0087] The content of water-soluble protein (extracted with water) and the content of water-insoluble protein (extracted with dimethyl sulfoxide) were detected separately, and the sum of the two was compared with the detection value of the preferred method for detecting protein content in tobacco determined in (4) above.

[0088] Detection of water-soluble proteins: Accurately weigh 0.25 g of tobacco powder sample into the inner sleeve of the outer glass centrifuge tube, add 40 mL of water and sonicate for 1.5 h. Remove the inner sleeve, transfer 0.1 mL of the extract, add 0.1 mL of dimethyl sulfoxide and 5 mL of Coomassie Brilliant Blue G-250 solution, mix well and let stand for 5 min. Measure the absorbance of the sample at 595 nm on a UV spectrophotometer to determine the content of water-soluble proteins.

[0089] Detection of water-insoluble proteins: Add 40 mL of dimethyl sulfoxide to the remaining residue and sonicate for 1.5 h. Remove the core tube, transfer 0.1 mL of the extract, add 0.1 mL of water and 5 mL of Coomassie Brilliant Blue G-250 solution, mix well and let stand for 5 min. Then, use a UV spectrophotometer to measure the absorbance of the sample at 595 nm to determine the water-insoluble proteins.

[0090] The results showed that the water-soluble protein and water-insoluble protein content in the sample were 1.16% and 3.72%, respectively, with a total protein content of 4.88%.

[0091] The same sample was tested using the preferred method for detecting protein content in tobacco determined in (4) above. The result showed that the protein content was 4.85%, which was basically consistent with the result of summing the results of detecting the water-soluble protein and water-insoluble protein content in the sample separately.

[0092] It is evident that the method for detecting protein content in tobacco provided by this invention yields accurate and reliable results, is simple to operate, and can save time and costs.

[0093] (6) Using enzyme preparations to increase the extraction yield of protein in tobacco powder

[0094] Experimental group: Accurately weigh 0.25 g of tobacco powder sample into the inner core of a glass centrifuge tube, add 40 mL of enzyme solution (enzyme solution of enzyme preparation A or enzyme preparation B), react at 50℃ for 1 h, after the reaction, add 40 mL of dimethyl sulfoxide (since the enzyme solution is an aqueous solution, the volume ratio of dimethyl sulfoxide to water is 1:1 at this time), sonicate at room temperature for 1.5 h, separate solid and liquid, transfer 0.2 mL of the extract and add 5 mL of Coomassie Brilliant Blue G-250 solution, mix well and let stand for 5 min, then measure the light absorbance at 595 nm on a UV spectrophotometer.

[0095] Blank control group: 40 mL of enzyme solution (enzyme solution of enzyme preparation A or enzyme preparation B) was mixed with 40 mL of dimethyl sulfoxide. After mixing, the mixture was sonicated at room temperature for 1.5 h. 0.2 mL of the mixture was added to 5 mL of Coomassie Brilliant Blue G-250 solution, mixed well and allowed to stand for 5 min. The absorbance at 595 nm was then measured on a UV spectrophotometer.

[0096] Protein content in tobacco leaves = total protein content in the experimental group - protein content in the blank control group.

[0097] Control group: The same samples were not treated with enzyme solution and the protein content in tobacco was directly detected by the preferred method for detecting protein content in tobacco determined in (4) above.

[0098] The protein content in the samples was compared with that of enzyme solution A, enzyme solution B, and samples without enzyme treatment, as shown in Table 4, to evaluate the extraction effect of enzymes on protein in tobacco leaves.

[0099] Table 4. Extraction effects of different enzyme solutions on proteins in tobacco powder

[0100]

[0101] The results showed that both enzyme preparation A and enzyme preparation B effectively improved the extraction efficiency of proteins from tobacco powder, and enzyme preparation A had a more significant effect on improving protein extraction efficiency compared to enzyme preparation B. Without being constrained by theory, it is speculated that the pectinase, cellulase, and hemicellulase in the enzyme preparations facilitate the release of soluble proteins in the cytoplasm and insoluble proteins in the cell wall, thereby improving protein extraction efficiency. Furthermore, because enzyme preparation A uses the tobacco-derived microorganism *Paenibacillus amylolyticus* CGMCC 24247, and its fermentation medium contains added tobacco powder, it exhibits better adaptability to tobacco leaves. Therefore, enzyme preparation A is more conducive to protein extraction than enzyme preparation B.

[0102] Example 2: Linearity and Repeatability Test of the Method

[0103] Using bovine serum albumin (BSA) as a standard, accurately transfer 0, 0.02, 0.04, 0.08, 0.12, 0.16, and 0.20 mL of protein standard solution into 10 mL stoppered centrifuge tubes. Add 1:1 (v / v) dimethyl sulfoxide / water solution to a final volume of 0.2 mL. Add 5 mL of Coomassie Brilliant Blue G-250 solution to each tube, mix well, and allow to stand for 5 min. Measure the absorbance at 595 nm. Plot a standard curve with absorbance on the ordinate and protein concentration on the abscissa. Figure 1 As shown. By Figure 1 We know that the regression equation is y = 1.2292x + 0.0142, R0 2 =0.9996, which indicates that the protein absorbance in the range of 0~1 mg / mL conforms to the Lambert-Beer law.

[0104] Referring to the preferred method for detecting protein content in tobacco determined in Example 1 (4), repeatability analysis tests were performed on A, B, and C tobacco leaf samples from different origins or different grades. The results are shown in Table 5.

[0105] Table 5 Results of repeatability experiments

[0106]

[0107] The results showed that the RSDs of the six repeatability tests for samples A, B, and C were 0.58%, 0.63%, and 0.72%, respectively, indicating good reproducibility.

[0108] Different amounts of protein standards were added to tobacco sample B, and the spiking experiment was carried out according to the above-mentioned steps. The results are shown in Table 6.

[0109] Table 6. Sample recovery test results (n=3)

[0110]

[0111] The results showed that the average recovery rate was 98.48%, which was good.

[0112] Comparative Example 1: Comparison of Extractant Types

[0113] Referring to the preferred method for detecting protein content in tobacco determined in Example 1 (4), the dimethyl sulfoxide in the extraction solvent (dimethyl sulfoxide / water solution with a volume ratio of 1:1) was replaced with equal volumes of sodium hydroxide, hydrochloric acid, and urea (all at a concentration of 1 mol / L) to extract and detect protein in tobacco. The results are shown in Table 7:

[0114] Table 7. Detection results of tobacco proteins using different extractants

[0115]

[0116] The results showed that, compared to the dimethyl sulfoxide / aqueous solution of this invention, the protein content detected in the samples was lower when using hydrochloric acid / aqueous solution, sodium hydroxide / aqueous solution, and urea / aqueous solution as extraction solvents. Without being bound by theory, it is speculated that although hydrochloric acid (strong acid), sodium hydroxide (strong alkali), and urea also have the advantage of lysing cell walls and releasing proteins from tobacco, they all cause protein denaturation, precipitation, or degradation. When proteins are denatured, these denatured proteins are removed as the solid phase during solid-liquid separation. Regardless of whether the proteins are denatured and precipitated or degraded, the protein content in the extract is low. Dimethyl sulfoxide, on the other hand, avoids protein denaturation or degradation during extraction and has good compatibility with water-soluble, alcohol-soluble, and oil-soluble proteins, meeting the detection requirements.

[0117] The residue from the enzyme-treated sample followed by ultrasonic extraction with dimethyl sulfoxide / water solution was subjected to a two-step acid hydrolysis (first step: the residue was transferred to a high-pressure vessel, 1.5 mL of 72 w / w% sulfuric acid aqueous solution was added, the mixture was stirred until the residue was completely submerged in the sulfuric acid solution, and the first hydrolysis was carried out in a 30°C water bath for 2 h. After the first hydrolysis, 42 mL of ultrapure water was added to the high-pressure vessel to dilute the sulfuric acid concentration to 4 w / w); second step: the two diluted high-pressure vessels were placed in an autoclave and subjected to a second hydrolysis at 121°C for 1 h. After cooling, the hydrolysis product was vacuum filtered using dried and weighed ashless filter paper, and the filtrate and residue were collected. The filtrate was then diluted to 250 mL with ultrapure water). The results were then detected across the entire wavelength range of 190–500 nm. Figure 2 As shown, no typical absorption peaks for proteins and amino acids were found at 250 nm (proteins may be acid-digested into small molecule proteins and amino acids), indicating that the method provided by this invention extracts soluble and insoluble proteins from the sample almost completely. The dimethyl sulfoxide / water solution has excellent compatibility with various tobacco proteins, and no protein residue was found in the residue in the spectrum.

[0118] In addition, a dimethyl sulfoxide / water solution (1:1) was used for a full wavelength scan of 400–750 nm using a spectrophotometer. Figure 3 As shown, dimethyl sulfoxide has no absorption at 595 nm and does not significantly interfere with the Coomassie brightness color development.

[0119] Comparative Example 2: Comparison of the method of the present invention with the standard method

[0120] The protein content of the same tobacco sample was determined by the original standard method YC / T 294-2008 and the method of the present invention (the preferred method for detecting protein content in tobacco determined in (4) of Example 1), and the results are shown in Table 8.

[0121] Table 8. Sample recovery rate test results (%)

[0122]

[0123] The results showed that the original standard method yielded higher values ​​than the method of this invention. It is speculated that this is because the original standard method digests proteins into nitrogen before reacting and converting them. Since tobacco also contains abundant free amino acids and nicotine, these "organic nitrogen" substances would "increase" the detection results under the original standard method.

[0124] Although specific embodiments of this application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of protection of this application. The full scope of this application is given by the appended claims and any equivalents.

Claims

1. A method for detecting the protein content in tobacco, comprising: 1) Mix the tobacco sample to be tested with the enzyme preparation solution and react; 2) Add dimethyl sulfoxide aqueous solution to the product obtained in 1), mix, and extract the protein; 3) Perform solid-liquid separation on the product obtained in 2). 4) Detect the protein content in the liquid obtained in step 3). The enzyme preparations mentioned above include pectinase, cellulase, and hemicellulase.

2. The method according to claim 1, wherein the enzyme activity ratio of pectinase, cellulase and hemicellulase in the enzyme preparation is 18~22:0.8~1.2:0.8~1.2, preferably 19~21:0.9~1.1:0.9~1.1, and more preferably 20:1:

1.

3. The method of claim 2, wherein the enzyme preparation is prepared by liquid fermentation of Bacillus amyloliquefaciens, or is a mixture of pectinase, cellulase and hemicellulase.

4. The method of claim 3, wherein the enzyme preparation is prepared by a method comprising the following steps: 1) Inoculate Paenibacillus amylolyticus into liquid culture medium; 2) Fermentation; 3) Solid-liquid separation of fermentation products; 4) Separate and collect proteins with a molecular weight greater than or equal to 10 kDa from the resulting liquid. The liquid culture medium comprises: 0.8-1.2 g / L tobacco leaf powder, 3-7 g / L soybean peptone, 0.8-4 g / L sucrose, 0.3-0.7 g / L xylan, 0.3-0.7 g / L citrus peel pectin, 0.3-0.7 g / L sodium carboxymethyl cellulose, 0.08-0.4 g / L manganese sulfate monohydrate, 0.3-0.7 g / L sulfuric acid heptahydrate, 3-7 g / L potassium dihydrogen phosphate, and water. The pH of the liquid culture medium is 6.8-7.

2. Preferably, the liquid culture medium comprises: 0.9-1.1 g / L tobacco leaf powder, 4-6 g / L soybean peptone, 1-3 g / L sucrose, 0.4-0.6 g / L xylan, 0.4-0.6 g / L citrus peel pectin, 0.4-0.6 g / L sodium carboxymethyl cellulose, 0.1-0.3 g / L manganese sulfate monohydrate, 0.4-0.6 g / L sulfuric acid heptahydrate, 4-6 g / L potassium dihydrogen phosphate, and water, wherein the pH value of the liquid culture medium is 6.9-7.1; Preferably, the liquid culture medium comprises: 1 g / L tobacco powder, 5 g / L soybean peptone, 2 g / L sucrose, 0.5 g / L xylan, 0.5 g / L orange peel pectin, 0.5 g / L sodium carboxymethyl cellulose, 0.2 g / L manganese sulfate monohydrate, 0.5 g / L sulfuric acid heptahydrate, 5 g / L potassium dihydrogen phosphate, and water, wherein the pH of the liquid culture medium is 7.

0.

5. The method of claim 4, characterized by one or more of the following features: -The preservation number of Bacillus amyloliquefaciens is CGMCC 24247; - Prepare a 10% concentration of Bacillus amyloliquefaciens. 8 ~10 9 Seed culture at CFU / mL is inoculated into liquid culture medium, preferably at an inoculation rate of 0.8~1.2 v / v% (e.g., 1 v / v%). - Ferment at 36~38℃ (e.g. 37℃) and 160~200 r / min (e.g. 180 r / min) for 16~20 h (e.g. 18h). - The fermentation products are separated into solid and liquid by centrifugation, preferably at a speed of 6000~10000 r / min (e.g. 8000 r / min). - Proteins with a molecular weight greater than or equal to 10 kDa are separated by ultrafiltration membrane with a molecular weight of 10 kDa. Preferably, the separated protein retentate is freeze-dried to obtain an enzyme preparation.

6. The method of claim 1, wherein the enzyme preparation solution is a Tris-HCl buffer solution of the enzyme preparation prepared by mixing the enzyme preparation and Tris-HCl buffer. Preferably, the Tris-HCl buffer is a 0.04-0.06 M Tris-HCl buffer with a pH of 5-6, and more preferably a 0.05 M Tris-HCl buffer with a pH of 5.

5. Preferably, the concentration of the enzyme preparation solution is 0.8~1.2 mg / mL (e.g., 1 mg / mL).

7. The method according to any one of claims 1-6, characterized by one or more of the following features: - The tobacco sample to be tested is tobacco powder, such as tobacco powder with a moisture content of less than or equal to 8% that can pass through a 40-mesh sieve; - Mix 0.2~0.3 g of the tobacco sample to be tested with 32~48 mL of enzyme preparation solution and react; preferably, mix 0.25 g of the tobacco sample to be tested with 40 mL of enzyme preparation solution and react; preferably, react at 48℃~52℃ for 0.8~1.21 h, more preferably at 50℃ for 1 h; - The volume ratio of dimethyl sulfoxide to water in the aqueous solution of dimethyl sulfoxide is 1:1.5 to 1.5:1, preferably 1:1.2 to 1.2:1, for example 1:1; The amount of dimethyl sulfoxide aqueous solution added is 240 mL to 400 mL per gram of tobacco sample to be tested, preferably 320 mL per gram of tobacco sample to be tested. - Proteins are extracted by ultrasonic extraction, with an extraction time of 1 to 2.5 hours, preferably 1.5 hours; - To determine the protein content in the liquid obtained in step 3) using the Coomassie Brilliant Blue method, preferably, take the liquid obtained in step 3) and add 10 to 50 times (e.g., 25 times) the volume of Coomassie Brilliant Blue G-250 solution with a concentration of 80 mg / L to 120 mg / L (e.g., 100 mg / L), mix, and then determine the protein content.

8. The method according to any one of claims 1-6 further includes detecting a blank control sample, which is different from the tobacco leaf sample to be tested in that the tobacco leaf sample to be tested is not added in step 1) during the detection.

9. The method of claim 8, wherein the protein content in the tobacco leaf to be tested is the protein content in the liquid obtained in step 3) minus the protein content of the blank control sample.

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