CN1264972C - Biological agent prepared by Arthrobacter AS-2 strain and its application - Google Patents

Abstract

The present invention relates to a biological preparation prepared by the bacterial strain of arthrobacter sp. AS-2, and the application thereof, which belongs to the technical field of microbes. The bacterial strain for producing the biological preparation is the bacterial strain of arthrobacter sp. AS-2 and is preserved in the China General Microbiological Culture Collection Center; the preservation number is CGMCC NO. 1282. The biological preparation is prepared through the working procedures of bacterial strain culture by cuvettes, bacterial strain enlarged culture by shaking tables, and liquid fermentation of a bacterial strain tank and a fermentation tank. The inoculum dose of the bacterial strain is from 0.5% to 2%; the adopted formula of a culture medium comprises (W/V) 0.3% of beef extract, 0.1% of yeast extract, 0.5% of peptone and 1.0% of glucose, and the pH value of the culture medium is from 6.8 to 7.2. The fermentation temperature is controlled within a range of 28 to 30 DEG C, the pH value is from 7.0 to 7.2, the fermentation time in the bacterial strain tank is from 16 to 20 hours, and the fermentation time in the fermentation tank is 24 to 48 hours; when the bacteria count in the fermentation liquid reaches 7.0*10<8> /ml, the biological preparation is obtained. The present invention has the advantages of short fermentation period, energy conservation, low cost, no poison and no pollution; the biological preparation is beneficial to be processed into various dosage forms and is convenient for store and transportation; the present invention can reduce alkali, increase fragrance and enhance the quality of tobacco, etc.

Description

A kind of biotechnological formulation with the preparation of Arthrobacter AS-2 bacterial strain

Technical field:

The present invention relates to a kind of biotechnological formulation, the microorganism belonging to genus technical field with the preparation of Arthrobacter AS-2 bacterial strain.

Background technology:

A kind of preference that tobacco sucks as people, nicotine volatilize in combustion processes and make flue gas be alkalescence and generation irritating smell, and pungency has negative effect to quality of tobacco.The height of nicotine content plays considerable effect to the quality of tobacco product, and the height of nicotine content of tobacco leaves directly has influence on the quality of quality, but the nicotine content of dissimilar tobacco leaves all has a moderate index.Nicotine content increases, and quality of tobacco is had a negative impact, and Here it is, and high-quality tobacco leaf needs one of reason of chemical ingredients coordination.

Good flue-cured tobacco upper leaf plays leading role in modern blended type cigarette and low tar Virginian-type cigarette tobacco leaf formulation, cigarette fragrance and style thereof are had very big contribution, and is in very great demand in the international market.By contrast, those quality levels are not high, style is not outstanding or the upper tobacco leaf of unfavorable industrial cost efficiency, and tobacco industry and commerce is not all had magnetism forever.People see that also under the influence of WTO and WHO, the flue-cured tobacco upper leaf will have ample scope for one's abilities.The problem matter of utmost importance that upper tobacco leaf exists on operability is that nicotine content of tobacco leaves is too high.Therefore reduce nicotine content of tobacco leaves, help to improve upper tobacco leaf operability, to improve the market competitiveness of sucking security, improving domestic tobacco leaf significant.

Dissimilar tobaccos, same type tobacco different soils are different to the influence of tobacco leaf nicotine with cultivation technique levels such as fertilising, irrigation conditionss, and nicotine content also changes with the variation of blade inserted part, and progressively improve leaf position from bottom to top nicotine content.The approach that reduces nicotine at present is: 1. the envrionment conditions of adjusting tobacco growing, use effective integrated agriculture measure: as using nitrogen phosphorus fertilizer in right amount, increase the usage quantity of potash fertilizer, with lime and the suitable cake fertilizer that uses, regulate the pH value, water by cigarette strain growth-development law, transplant in good time, according to practical situation, pinch in good time, suitably stay leaf, use growth regulator, proper mature (ripe) is gathered, optimize modulation condition and utilize ageing and fermentation to wait and comprehensively reduce nicotine content, there is nornicotine effect instability between year in this method, operation link is too much, peasant household disperses shortcomings such as not easy to operate.2. pass through to improve filtration efficiency in cigarette, dilution flue gas etc. though these measures can be regulated nicotine in the flue gas arbitrarily, often do not have suitable selectivity, make the human consumer be difficult to accept.3. utilize ageing and fermentative degradation nicotine and utilize the existing report of microbiological deterioration storage phase tobacco leaf nicotine, reported that the microorganism that can reduce nicotine comprises: Alcaligenes paradoxus alcaligenes paradoxus, Arthrobacter globiformils Arthrobacter globiformis, Arthrobacter nicotianae tobacco Arthrobacter, Arthrobacter nicotinovorans bites the nicotine Arthrobacter, Arthrobacter oxydans oxidation Arthrobacter, Bacillus megaterium bacillus megaterium, Bacillus mycoides bacillus mycoides, the Bacilluspumilus bacillus pumilus, Bacillus subtilis subtilis, Cellulomonas sp. cellulomonas cartae, Corynebacterium sp. Corynebacterium, Enterobacter cloacae enterobacter cloacae, Micrococcusnicotianae tobacco micrococci, the false pseudomonas bacillus of Pseudomonas sp., Pseudomonas nicotianae tobacco pseudomonas, the false pseudomonas bacillus of Pseudomonas putida stench, Deharyomyces nicotianae, the little gram sweat of Cunninghamella echinulata bundle spore mould, Pellicularia filamentosa silk nuclear film grass bacterium, the yeast yeast.But this measure be difficult to solve bacterial strain use age, use back moisture content in leaves content and improve problems such as causing tobacco mildew easily, never adopt in the factory.Along with the growing interest of people to objectionable constituent in the flue gas, fragrance can be the technical barrier that tobacco faces for many years for the human consumer accepts still when reducing nicotine.

Summary of the invention:

The objective of the invention is mechanism based on the microbial metabolites degrading nicotine, study and found a kind of tobacco leaf nicotine of can degrading, improve the Arthrobacter AS-2 bacterial strain of quality of tobacco, after adopting this bacterial strain through the cultivation of test tube kind, bacterial classification shaking table enlarged culturing, seeding tank, fermentor tank liquid fermenting, be prepared into and a kind ofly can reduce tobacco smoke alkaloid content but do not influence tobacco leaf aesthetic quality's biotechnological formulation.

Biotechnological formulation of the present invention prepares through liquid fermentation production method by producing bacterial strain.Wherein:

1, producing bacterial strain is Arthrobacter (Arthrobacter sp.) AS-2, and this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.1282, preservation day to be on December 28th, 2004; This bacterial strain forms oyster white bacterium colony, G on beef-protein medium ⁺, aerobic, do not form gemma, the catalase positive, decomposition of cellulose does not think that this bacterial strain belongs to genus arthrobacter.

Arthrobacter (Arthrobacter sp.) AS-2 is the bacterial strain that the inventor found in 2004.This bacterial strain is gathered the vega soil sample from Yuxi, Yunnan, coat to separate on the NA substratum through conventional serial dilution to obtain, and this bacterial strain is measured through the external nicotine of routine, and discovery can significantly reduce the content of nicotine, makes nicotine-containing substratum become blueness by white.

2, liquid fermenting production prepares the process of biotechnological formulation and is:

The preparation of microbial inoculum:

(1) test tube is cultivated: adopt the NA substratum, this bacterial classification inoculation on the test tube slant substratum, was cultivated 1-2 days for 28 ℃.

(2) shake-flask culture: adopt the NA liquid nutrient medium, be seeded in the Erlenmeyer flask of filling liquid substratum cultivating bacterium in the above-mentioned test tube, in 30 ℃ of shaking culture 1-2 of shaking table days.

The liquid fermenting production process comprises that the cultivation of test tube kind, bacterial classification shaking table enlarged culturing, seeding tank, fermentor tank liquid fermenting prepare biotechnological formulation; Wherein:

A. the test tube kind is cultivated and is: the thalline of Arthrobacter sp.AS-2 is inoculated on the test tube culture medium slant, culture medium prescription is to add extractum carnis 3g in 1000 ml distilled waters, yeast extract 1g, peptone 5g, glucose 10g, control pH6.8-7.2 cultivated 1-2 days down at 28-30 ℃, obtained the test tube kind;

B. bacterial classification shaking table enlarged culturing is: the inclined-plane seed is inoculated in the triangular flask liquid nutrient medium, culture medium prescription is to add extractum carnis 3g in 1000 ml distilled waters, yeast extract 1g, peptone 5g, glucose 10g, control pH6.8-7.2 cultivated 16-20 hour at 28-30 ℃, the shaking table of rotating speed 200-300rpm;

C. liquid seeds jar fermentation is: the bacterial classification that shaking table is enlarged is inoculated in the fermentor tank of differing capacities in the ratio of 0.5-2%, and culture medium prescription is the culture medium prescription of shaking table enlarged culturing, and culture temperature is 28-30 ℃, and incubation time is 16-20 hour;

D. liquid fermentation tank prepares biotechnological formulation and is: the bacterial classification that will enlarge step by step through the seeding tank fermentation is inoculated in the fermentor tank of differing capacities in the ratio of 1-3%, culture medium prescription is the culture medium prescription of bacterial classification shaking table enlarged culturing, culture temperature is 28-30 ℃, pH7.0-7.2, incubation time is 24-48 hour, and bacterial number reaches 7.0 * 10 in fermented liquid ⁸Individual/as during ml, to obtain biotechnological formulation.

The invention has the advantages that: 1. fermentation period is short, and save energy, production cost are low; 2. microorganism is from vega soil, and to plant and person poultry harmless, easily production, anti-storage are easy to industrialization development, and be workable; 3. can fall the alkali flavouring, improve the quality of tobacco, and the nornicotine effect not influence the tobacco leaf aesthetic quality.

Embodiment:

Embodiment 1:(is used for cured tobacco leaf)

Biotechnological formulation of the present invention by producing bacterial strain (Arthrobacter sp.) AS-2 bacterial strain, is pressed the liquid fermentation production method preparation.It is identical that concrete preparation process and condition and summary of the invention are described part.

The application of biotechnological formulation of the present invention: the tobacco leaf that maturation is good is gathered by position (in, top), enter before the barn with biotechnological formulation of the present invention by 5% amount (content of biotechnological formulation in diluent) evenly spray in tobacco leaf, top, compile bar then and go into stove, according to a conventional method baking.Tobacco leaf after the baking shows after measured, and biological system of the present invention reaches 31.68% to the nicotine degradation rate of upper tobacco leaf, and the nicotine degradation rate of middle part tobacco leaf is reached 33.86%.

Cured tobacco leaf aesthetic quality smoking result shows, adopt biotechnological formulation of the present invention to handle after, tobacco leaf strength obviously descends, perfume quantity obviously improves, and fragrance matter makes moderate progress, and pungency obviously weakens, assorted gas obviously alleviates (withered and burnt smell, the assorted gas of powder), and mouthfeel is better than contrast.

Embodiment 2:(is used for the burley tobaccos tobacco leaf)

Biotechnological formulation of the present invention by producing bacterial strain (Arthrobacter sp.) AS-2 bacterial strain, is pressed the liquid fermentation production method preparation.It is identical that concrete preparation process and condition and summary of the invention are described part.

The application of biotechnological formulation of the present invention: will cut the cigarette strain of keeping well and enter the room that dries in the air, and after one week of system of drying in the air, evenly spray at position, burley tobaccos upper, middle and lower the system of drying in the air according to a conventional method by 5% amount (content of biotechnological formulation in diluent) with biotechnological formulation of the present invention.Burley tobaccos after the system of drying in the air show after measured, and biotechnological formulation of the present invention reaches 43.56%, the nicotine degradation rate of middle part burley tobacco leaf is reached 50.22% the nicotine degradation rate of top burley tobacco leaf, and the nicotine degradation rate of bottom burley tobacco leaf is reached 53.02%.

Burley tobaccos tobacco leaf aesthetic quality smoking result shows, adopt biotechnological formulation of the present invention to handle after, tobacco leaf strength obviously descends, perfume quantity obviously improves, fragrance matter makes moderate progress, pungency obviously weakens, sensory evaluating smoking's quality is better than the CK contrast.

Claims (1)

1. biotechnological formulation with Arthrobacter AS-2 bacterial strain preparation, press the liquid fermentation production method preparation by producing bacterial strain, the production bacterial strain that it is characterized in that this biotechnological formulation is Arthrobacter (Arthrobacter sp.) AS-2 bacterial strain, this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.1282; The process that liquid fermenting production prepares biotechnological formulation is:

(1). the liquid fermenting production process comprises that the cultivation of test tube kind, bacterial classification shaking table enlarged culturing, seeding tank fermentation, fermentor tank liquid fermenting prepare biotechnological formulation; Wherein:

A. the test tube kind is cultivated and is: the thalline of Arthrobacter sp.AS-2 is inoculated on the test tube culture medium slant, culture medium prescription is to add extractum carnis 3g in 1000 ml distilled waters, yeast extract 1g, peptone 5g, glucose l0g, control pH6.8-7.2 cultivated 1-2 days down at 28-30 ℃, obtained the test tube kind;

B. bacterial classification shaking table enlarged culturing is: slant strains is inoculated in the triangular flask liquid nutrient medium, culture medium prescription is to add extractum carnis 3g in 1000 ml distilled waters, yeast extract 1g, peptone 5g, glucose l0g, control pH6.8-7.2 cultivated 16-20 hour at 28-30 ℃, the shaking table of rotating speed 200-300rpm;

C. liquid seeds jar fermentation is: the bacterial classification that shaking table is enlarged is inoculated in the fermentor tank of differing capacities in the ratio of 0.5-2%, and culture medium prescription is the culture medium prescription of shaking table enlarged culturing, and culture temperature is 28-30 ℃, and incubation time is 16-20 hour;

D. liquid fermentation tank prepares biotechnological formulation and is: the bacterial classification that will enlarge step by step through the seeding tank fermentation is inoculated in the fermentor tank of differing capacities in the ratio of 1-3%, culture medium prescription is the culture medium prescription of bacterial classification shaking table enlarged culturing, culture temperature is 28-30 ℃, pH7.0-7.2, incubation time is 24-48 hour, and bacterial number reaches 7.0 * 10 in fermented liquid ⁸Individual/as during ml, to obtain biotechnological formulation.