CN1800361A - Method of nicotine biological degradation - Google Patents

Abstract

The invention relates to a method for biologically degrading nicotine. It uses nicotine-degrading bacteria (Ochrobactrum intermedium) DN2 as the strain, and uses Ochrobactrum intermedium DN2 or the nicotine-degrading enzyme of Ochrobactrum intermedium DN2 to act on recured tobacco leaves, shredded tobacco, industrial and agricultural products. Tobacco leftovers and tobacco waste, as well as the aqueous extracts of the above-mentioned various objects, cause partial or complete degradation of nicotine. The invention can degrade 40-48% of the nicotine in re-examined tobacco leaves; reduce 35-56% of nicotine in shredded tobacco; and 75-98% of nicotine in tobacco extract. The method belongs to the biodegradation method, has strong specificity, remarkable effect and low production cost, and can be used in the fields of tobacco processing, tobacco papermaking, environmental protection and the like.

Description

A method for biodegrading nicotine

technical field

The invention relates to a method for degrading nicotine by using a nicotine-degrading bacterium (Ochrobactrum intermedium) DN2, which belongs to a production method for biodegrading nicotine, and is specially used for biodegrading nicotine.

Background technique

Nicotine is an alkaloid in common tobacco, commonly known as nicotine (nicotine), its main function lies in the physiological strength it produces, that is, the so-called strength, which is proportional to the nicotine content. Nicotine has a strong stimulating and paralyzing effect on the central nervous system of people. A small amount can excite people, and a large amount can cause dizziness, vomiting and even poisoning death. Adults can be fatal if they ingest 40mg-60mg of nicotine at a time. Therefore, if the nicotine content in tobacco is too high, it will seriously endanger the health of smokers.

The tobacco leaves produced in some tobacco areas in my country, especially the upper tobacco leaves, have high nicotine content. According to reports, the average content of nicotine in flue-cured tobacco in my country is 3% to 4%, and Burley tobacco is as high as 6%. Under normal circumstances, flue-cured tobacco should be less than 3%, and Burley tobacco should be 2% to 4%. There are many upper tobacco leaves stored in major domestic tobacco factories, which are generally not easy to use in leaf group formulas because of their high nicotine content. For the upper tobacco leaves with high nicotine content, cigarette manufacturers generally realize their application by gradually digesting them. However, due to the slow digestion rate, there is still a large amount of upper tobacco leaves that cannot be effectively utilized and cause waste. In order to recover the purchase cost of tobacco leaves, some manufacturers simply use these upper tobacco leaves to produce low-grade cigarettes, so that the industrial availability and commercial additional value of the upper tobacco leaves cannot be fully reflected. In recent years, with the continuous deepening of the worldwide anti-smoking campaign and consumers’ greater concern for their own health, almost all types of cigarette products are developing towards low-tar and low-nicotine light-flavored cigarettes, and the development speed is very fast. Swift. At present, our country compulsorily stipulates that flue-cured cigarettes with a nicotine content greater than 15 mg/stick in cigarette smoke must not be produced and distributed, and this value will be reduced in the future. In addition, in order to earn foreign exchange, my country also exports a small amount of cigarettes, which generally have low nicotine content. In order to produce low-nicotine cigarettes, manufacturers of export cigarettes usually need to carefully select "superior tobacco leaves" to meet their production needs, in addition to improving the formula, which is time-consuming, laborious and expensive, and cannot guarantee the impact of mass production of such cigarettes. raw material demand. Therefore reducing the content of nicotine in cigarettes has become an urgent task for cigarette manufacturers. Although there are some methods to reduce the nicotine content in tobacco, such as agricultural measures and extraction methods, while these technologies reduce nicotine, they will also reduce those components that contribute to the quality of cigarettes.

Nicotine is also an environmentally toxic substance, and the smoke environment contains a large amount of nicotine. During the production of tobacco products, a large amount of solid, liquid and gaseous wastes are produced, and the average content of nicotine is as high as 18g nicotine/kg dry weight. In Europe, when the nicotine content per kilogram of waste (dry weight) exceeds 500mg, it is classified as "toxic and harmful substances". Since 1994, the United States has listed nicotine in the Toxics Release Inventory (TRI) of the United States. According to reports, my country produces about 1 million tons of tobacco waste every year. If these wastes are thrown into the environment without treatment, they will seriously damage the ecological environment and endanger human health.

To sum up, effectively controlling and reducing the nicotine content in cigarette products and the environment has far-reaching significance for protecting the environment and maintaining human health. At present, foreign researchers have isolated a variety of nicotine-degrading bacteria from the environment. 【Abstract】Using the culture liquid of Deharyomyces nicotianae and Micrococcus nicotianae for industrial fermentation treatment of tobacco, experiments show that these two bacterial liquids can not only improve the flavor and aroma of tobacco, but also degrade the nicotine content of tobacco by 0.45% and 0.83% respectively. %, while the nicotine content of tobacco treated with the two mixed bacterial solutions decreased by 0.61%. Tobacco, 1947, 51:6-15. [Abstract] On the synthetic medium, after treating the nicotine solution with Pseudomonas race 41 isolated from the soil for 24 hours, the nicotine content was greatly reduced, due to the decrease of the pH value and the number of colonies. Increase, the decomposition of nicotine stops, and about 75% of the starting nicotine is degraded. Arch Biochem Biophys, 1958, 72(2): 145-162. 【Abstract】The bacteria that can oxidize nicotine to γ-aminobutyric acid were isolated from tobacco leaves. Journal of Bacterial, 1958, (75): 474-479. 【Abstract】Arthrobacter nicotianae was isolated from a tobacco leaf warehouse. The bacteria multiplied on nicotine-agar and degraded nicotine. Coresta, 1959, 3:2595. 【Abstract】Using 0.2% nicotine as the only carbon source medium, bacteria capable of degrading nicotine were isolated from tobacco growth soil and tobacco leaf surface: Alcaligenes paradoxus and Arthrobacter globiformils, If glucose exists in the culture medium, they will have high nicotine degrading activity, but when the nicotine concentration is 0.5%, the growth of bacteria is inhibited; There was no obvious degradation; however, if glucose was added to the medium at the same time, a significant effect of nornicotine would be produced within two days. Science Paper, 1976, 118: 197-201. 【Abstract】A strain capable of degrading nicotine was isolated from the surface of dried tobacco leaves and identified as Enterobacter cloacae (Enterobacter cloacae) E-150. Fermentation is carried out in a medium containing nicotine (less than 5g/L) under the conditions of 34°C and pH 7.0, and the nornicotine ability of the bacterium is induced during the fermentation process. In addition, this bacteria can also metabolize niacin, but cannot degrade nornicotine and neonicotinoid. Degrader (Spain), 1983, 22:85-98. 【Abstract】Pseudomonas putida, which can degrade nicotine, and Cellulomana ssp, which can simultaneously remove nicotine and nitrate, were isolated from Puerto Rican cigar leaves by Brownway & Williamson . The microorganism is inoculated on the leaves of Burley tobacco, and sufficient water and ammonia water are added at the same time, so that the moisture content and pH value of the tobacco leaves reach 65%-75% and 6.1 respectively. Then pile up and ferment at 30°C, and the nicotine in the Burley tobacco leaves has been basically exhausted within 16 hours. The results of chemical analysis and smoking evaluation showed that compared with the cigarettes processed from untreated tobacco leaves, the nicotine in the formula and smoke of the processed tobacco leaves decreased by 48% and 42%, respectively, and the sensory evaluation and smoking results were excellent. US Patent, No.4037609 [Abstract] Degradation of nicotine in soot waste with Pseudomonas bacteria. The experiment is to take 1 kg of smoke samples (nicotine 2.0% ~ 2.5%), add 2.0 ~ 2.5L of aqueous solution containing Pseudomonas to infiltrate, so that it is completely soaked in smoke powder, put it at room temperature for several days, and occasionally stir it, After about a week, the nicotine content is between 4.0×10 ^-4 and 7.0×10 ^-4 . Domestic research in this area is less. Information Bulletin of Coresta Congress, 1994, ST8. 【Abstract】Wang Ge isolated 3 strains with strong ability to degrade nicotine from tobacco leaves. After the test strain grew to a certain concentration on the protein-free medium, the bacterial solution was evenly sprayed on the test tobacco leaves, and the treated samples and control samples were placed in a 40°C incubator for fermentation. The test results showed that after the tobacco leaves were fermented by microorganisms, the nicotine content of all treated tobacco samples was reduced to varying degrees compared with the control sample, the highest reduction was 60.15%, the lowest reduction was 5.90%, and the average reduction was 21.35%. Tobacco Science Research, 2001, (2): 66-68.

Practice has proved that biodegradation is an effective way to remove or reduce nicotine in tobacco waste. By reducing the content of nicotine in tobacco leaves, it can solve the problem of industrial usability of upper tobacco leaves in my country and increase the added value of tobacco leaves; through biodegradation, the source of raw materials for the production of low-nicotine cigarettes can be made general, saving production costs and expanding export earnings ; Degradation of nicotine in tobacco waste can protect the environment and maintain human health. Therefore, the use of biodegradable nicotine has important economic and social significance.

Nicotine-degrading bacteria (Ochrobactrum intermedium) DN2 is a nicotine-degrading bacterium isolated from soil. We reported the results of the isolation and identification of this bacterium (Acta Microbiology, 2005, 45(2): 181-184). The biological characteristics of the bacteria are: negative Gram stain, no spores, short rod shape, 2 terminal flagella; colonies on the beef extract peptone agar plate are white, smooth, and the edges are neat; the reaction of contact enzyme and oxidase is positive , Strictly aerobic, can grow with nicotine as the only carbon source, the optimum growth temperature and pH are 30°C and 7.0 respectively, oxidize glucose to produce acid, positive for nitrate reduction, negative for V.P., M.P. reaction; in King's medium B does not produce water-soluble pigments, the bacteria does not hydrolyze starch and gelatin, can utilize glucose, maltose, etc., and is not sensitive to colistin. However, the process of Ochrobactrum intermedium DN2 to degrade nicotine has not been reported yet. Using it as a bacterial strain to degrade nicotine can be used in the fields of tobacco manufacturing, tobacco leaf papermaking, environmental protection and the like.

Contents of the invention

technical problem

The object of the present invention is to provide a method for biodegrading nicotine, using nicotine degrading enzymes of nicotine degrading bacteria (Ochrobactrum intermedium) DN2 or Ochrobactrum intermedium DN2 to degrade nicotine, so as to degrade 40% to 48% of nicotine in recured tobacco leaves; The degradation of nicotine in shredded tobacco is reduced by 35-56%, and the degradation of nicotine in tobacco extract is 75-98%. By reducing the content of nicotine in tobacco leaves or tobacco extract, it can not only solve the problem of industrial usability of upper tobacco leaves in my country, but also solve the problem of environmental pollution caused by nicotine in tobacco waste.

Technical solutions

A method for biodegrading nicotine of the present invention is characterized in that

1) Expanded culture of nicotine-degrading bacteria (Ochrobactrum intermedium) DN2

Ordinary medium g/L: tryptone 11.34, beef extract 3.00, NaCl5.00, MgSO ₄ 7H ₂ O3.71, pH 7.23;

Basal medium g/L: NH ₄ NO ₃ 1.00, MgSO ₄ 7H ₂ O 0.50, (NH ₄ ) ₂ SO ₄ 0.50, KH ₂ PO ₄ 0.50, NaCl 0.50, K ₂ HPO ₄ 1.50, pH 7.0;

Tobacco extract: Tobacco waste and water are mixed at a ratio of 1:10 to form an aqueous extract, overnight, and the pH of the aqueous extract is adjusted to 7.0 with 4N NaOH or 1N HCl;

The above medium was sterilized by high-pressure steam at 121°C for 30 minutes;

2) Preparation of bacterial suspension

Ordinary culture of bacteria: Inoculate Ochrobactrum intermedium DN2 into ordinary culture medium, culture at 32°C and 120r/min shaker for 34h to obtain ordinary culture medium, centrifuge at 10000r/min for 10min, collect bacteria, and use 50mM, pH7.0 Prepare the bacterial suspension from the above bacterial cells in PBS to obtain the common cultured bacterial suspension;

Bacterial induction culture: Put the common culture solution cultured at 32°C and 120r/min for 34h into the basal medium with a nicotine concentration of 500mg/L at a ratio of 5%, and cultivate on a shaking table at 30°C and 120r/min 36h, then centrifuge the culture solution at 10000r/min for 10min, collect the bacteria, prepare the above bacteria suspension with 50mM, pH7.0PBS, and obtain the culture suspension;

3) Preparation of crude enzyme solution

For the induced culture suspension obtained by the above method, add 60mL 50mM, pH7.0PBS buffer solution to 1000mL culture suspension and ultrasonically break it. 10min, the supernatant is the crude enzyme solution;

4) Biodegradation of nicotine

The biodegradation of nicotine in re-cured tobacco is as follows: after adjusting the moisture content of re-cured tobacco leaves to 60%-85% with sterile water, the common culture suspension obtained by the above method, the induced culture suspension or the crude The enzyme solution is inserted into the re-cured tobacco leaves with 10-20% inoculation amount, fully mixed the bacteria, fermented in solid state at 25-40 °C, and ventilated by shaking every 6 hours;

Or the biodegradation of nicotine in shredded tobacco is as follows: after adjusting the moisture of shredded tobacco to 60%-85% with sterile water, the common culture suspension, induced culture suspension or crude enzyme solution obtained by the above method are respectively mixed with Put 10-20% of the inoculum into the shredded tobacco, fully mix the bacteria, ferment in solid state at 25-40°C, and shake for ventilation every 6 hours;

Or the biodegradation of nicotine in the tobacco extract is as follows: respectively inserting the common culture suspension, the induced culture suspension or the crude enzyme solution obtained by the above method into the tobacco extract with an inoculation amount of 10-20%, Cultivate on a shaker at 30°C and 120r/min.

Beneficial effect

The invention develops and utilizes a bacterium Ochrobactrum intermedium DN2 with strong nicotine degradation ability isolated from the environment for the first time. The nicotine degrading enzyme of Ochrobactrum intermedium DN2 or Ochrobactrum intermedium DN2 is used to degrade nicotine, so that the nicotine degradation of re-cured tobacco leaves is 40-48%; the nicotine degradation in shredded tobacco is reduced by 35-56%; ~98%.

By reducing the content of nicotine in the tobacco leaves, the industrial usability of the upper tobacco leaves can be solved and the added value of the tobacco leaves can be increased; through the biodegradation method, the source of raw materials for the production of low-nicotine cigarettes can be made general, saving production costs and expanding export earnings; Degradation of nicotine in tobacco waste can protect the environment and maintain human health.

Detailed ways

【Example 1】

Inoculate Ochrobactrum intermedium DN2 into common medium (g/L): tryptone 11.34, beef extract 3.00, NaCl5.00, MgSO ₄ 7H ₂ O 3.71, pH 7.23, culture at 32°C, 120r/min shaker for 34h, Centrifuge at 10000r/min for 10min, collect the thalline, prepare the bacterium suspension with the above-mentioned thalline with PBS (50mM, pH7.0), insert the tobacco extract with the inoculation amount of 15g/L (tobacco waste and water press 1: Mix at a ratio of 10, overnight, adjust the pH value of the water extract to 7.0 with 4N NaOH or 1N HCl), and stir at pH 6.5 to 8.5 at 28 to 40°C for 2 to 4 days, and the degradation rate of nicotine is 85 to 40°C. 98%.

[Example 2]

After Ochrobactrum intermedium DN2 is expanded and cultivated through ordinary medium (the method is the same as in Example 1), centrifuge at 10000r/min for 10min, collect the thalline, prepare the bacterium suspension with the above-mentioned thalline with PBS (50mM, pH7.0), inoculate with 10% The amount is added to the re-cured tobacco leaves with a moisture content of 60-85%, solid fermentation at 28-40°C, and shaking every 6h for ventilation. The degradation rate of nicotine from 2 to 4 days was 40 to 48%.

[Example 3]

After Ochrobactrum intermedium DN2 is expanded and cultivated with ordinary medium (the method is the same as in Example 1), it is centrifuged at 10000r/min for 10min to collect the thalline, and the above-mentioned thalline is prepared into a bacterial suspension with PBS (50mM, pH7.0), and inoculated with 18% The amount is inserted into shredded tobacco with a water content of 60-85%, solid fermentation is carried out at 28-40°C, and ventilation is stirred every 6 hours. The degradation rate of nicotine in 2-4 days was 40-56%.

【Example 4】

Ochrobactrum intermedium DN2 was cultured for 34 h at 32° C. and 120 r/min in common medium (same as Example 1), and then inserted into the basal medium (g/L, NH ₄ NO ₃ 1.00, MgSO ₄ . 7H ₂ O 0.50, (NH ₄ ) ₂ SO ₄ 0.50, KH ₂ PO ₄ 0.50, NaCl 0.50, K ₂ HPO ₄ 1.50, pH 7.0, nicotine 0.5), 30°C, 120r/min shaker culture 36h, then centrifuge the culture solution at 10000r/min for 10min, collect the bacteria, use PBS (50mM, pH7.0) to prepare the above bacteria into a bacterial suspension, insert it into the basal medium containing nicotine for induction culture, and collect by centrifugation The bacterium is inserted into the tobacco extract with an inoculation amount of 15g/L, stirred and reacted at pH 6.5-8.5 at 25-40°C for 2-4 days, and the nicotine degradation rate is 85-98%.

【Example 5】

Ochrobactrum intermedium DN2 was first cultured in ordinary culture medium, then inserted into basal medium containing nicotine for induction culture, the bacteria were collected by centrifugation, and the above bacteria were prepared into bacterial suspension with PBS (50mM, pH7.0) (the method was the same as Example 4), insert 10-20% inoculum amount into re-cured tobacco leaves with a moisture content of 60-85%, ferment in solid state at 28-40° C., and shake and ventilate every 6 hours. The degradation rate of nicotine from 2 to 4 days was 40 to 48%.

[Example 6]

Ochrobactrum intermedium DN2 was first cultured in ordinary culture medium, then inserted into basal medium containing nicotine for induction culture, the bacteria were collected by centrifugation, and the above bacteria were prepared into bacterial suspension with PBS (50mM, pH7.0) (the method was the same as Example 4), insert 10-20% inoculum amount into shredded tobacco with a water content of 60-85%, ferment in solid state at 28-40° C., and shake and ventilate every 6 hours. The degradation rate of nicotine in 2-4 days was 40-56%.

[Example 7]

After Ochrobactrum intermedium DN2 was cultured by nicotine induction (same as in Example 4), 1000mL of culture liquid bacterial cells plus 60mL of PBS buffer (50mM, pH7.0) was ultrasonically broken, the ultrasonic conditions were 400w, ultrasonic 5s, intermittent 10s, 90 Second-rate. Centrifuge at 4°C and 10000r/min for 10min, the supernatant is the crude enzyme solution, add the crude enzyme solution or the crude enzyme solution with 10-20% inoculation amount into the tobacco extract, at pH6.5～8.5, 25～ The reaction was stirred at 40°C for 2-4 days, and the degradation rate of nicotine was 75-86%.

[Embodiment 8]

After the Ochrobactrum intermedium DN2 was induced and cultured by nicotine, the crude enzyme solution was obtained by breaking the wall with ultrasonic waves (the method is the same as in Example 7), and the crude enzyme solution was injected into the recured tobacco leaves with a water content of 65-85% at an inoculation amount of 10-20%. During solid fermentation at 25-40°C, shake and ventilate every 6 hours. The degradation rate of nicotine from 2 to 4 days was 40 to 48%.

[Example 9]

After Ochrobactrum intermedium DN2 was cultured by nicotine induction, the crude enzyme solution was obtained by breaking the wall with ultrasonic waves (the method is the same as in Example 7), and the crude enzyme solution was inserted into cut tobacco with a water content of 65-85% at an inoculum amount of 10-20% , solid fermentation at 25 ~ 40 ° C, shaking every 6 hours to ventilate. The degradation rate of nicotine in 2-4 days is 35-45%.

Claims (1)

1, a kind of method of biological degradation nicotine is characterized in that:

1) enlarged culturing of nicotine degradation bacterium (Ochrobactrum intermedium) DN2

Ordinary culture medium g/L: Tryptones 11.34, extractum carnis 3.00, NaCl 5.00, MgSO ₄7H ₂O 3.71, pH value 7.23;

Basic medium g/L:NH ₄NO ₃1.00, MgSO ₄7H ₂O 0.50, (NH ₄) ₂SO ₄0.50, KH ₂PO ₄0.50, NaCl0.50, K ₂HPO ₄1.50, pH value 7.0;

The tobacco extract: tobacco waste and water become the water extract by 1: 10 mixed, spend the night, and the water extract is transferred to 7.0 with 4N NaOH or 1N HCl with the pH value;

121 ℃ of high pressure steam sterilization 30min of above substratum;

2) preparation of bacteria suspension

The common cultivation of thalline: Ochrobactrum intermedium DN2 is inoculated in the ordinary culture medium, 32 ℃, 120r/min shaking table cultivation 34h, obtain common nutrient solution, the centrifugal 10min of 10000r/min, collect thalline, use 50mM, the PBS of pH7.0 obtains the bacteria suspension of common cultivation with above-mentioned thalline preparation bacteria suspension;

The inducing culture of thalline: it is in the basic medium of 500mg/L that the common nutrient solution of cultivating 34h under 32 ℃, 120r/min condition is inserted nicotinic density with 5% ratio, 30 ℃, 120r/min shaking table cultivation 36h, then with the centrifugal 10min of nutrient solution 10000r/min, collect thalline, use 50mM, pH7.0 PBS obtains the bacteria suspension of inducing culture with above-mentioned thalline preparation bacteria suspension;

3) crude enzyme liquid preparation

The inducing culture bacteria suspension that adopts above method to obtain is cultivated bacteria suspension by 1000mL and is added 60mL 50mM, pH7.0PBS damping fluid ultrasonic disruption, the ultrasonic wave condition is 400w, ultrasonic 5s, intermittently 10s, 90 times, 4 ℃, the centrifugal 10min of 10000r/min, supernatant liquor is crude enzyme liquid;

4) biological degradation of nicotine

The biological degradation of nicotine is in the redrying tobacco: after with sterilized water the moisture of redried leaf tobacco being adjusted to 60%-85%, common cultivation bacteria suspension, inducing culture bacteria suspension or crude enzyme liquid that aforesaid method is obtained insert in the redried leaf tobacco with the 10-20% inoculum size, abundant mixing thalline, in 25～40 ℃ of solid fermentations, turn over every 6h and to tremble ventilation;

Perhaps the biological degradation of nicotine is in pipe tobacco: after with sterilized water the moisture of pipe tobacco being adjusted to 60%-85%, common cultivation bacteria suspension, inducing culture bacteria suspension or crude enzyme liquid that aforesaid method is obtained insert in the pipe tobacco with the 10-20% inoculum size, abundant mixing thalline, in 25～40 ℃ of solid fermentations, turn over every 6h and to tremble ventilation;

Perhaps the biological degradation of nicotine is in the tobacco extract: common cultivation bacteria suspension, inducing culture bacteria suspension or crude enzyme liquid that aforesaid method is obtained insert in the tobacco extract with the 10-20% inoculum size, 30 ℃, the cultivation of 120r/min shaking table.