CN102660461A - Microbial preparation for shortening tobacco fermentation period and application of microbial preparation - Google Patents
Microbial preparation for shortening tobacco fermentation period and application of microbial preparation Download PDFInfo
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Abstract
The invention relates to a microbial preparation for shortening a tobacco fermentation period and application of the microbial preparation, and relates to the technical field of microorganism and tobacco processing. The microbial preparation comprises bacillus cereus and bacillus amyloliquefaciens from tobaccos, and the ratio of the live bacillus cereus to the live bacillus amyloliquefaciens is 10-200:1-50. The powdery microbial preparation is obtained by means of separation, culture and preparation and is dissolved into sterile water according to the proportion of 0.5-5%, formed water aqua is uniformly sprayed on tobaccos according to the proportion of 3-10%, and the tobaccos are artificially fermented for 10-40 days at the temperature ranging from 25 DEG C to 50 DEG C and the humidity ranging from 65%RH to 80%RH. Tests show that cellulose, protein, pectin and the like in the tobaccos can be degraded effectively, and the tobacco fermenting period is shortened to 10-40 days. Artificially fermented tobaccos are not obviously different from naturally alcoholized tobaccos in terms of quality, the tobacco fermenting period is shortened, and the quality of the tobaccos can be improved.
Description
Technical field
The present invention relates to mikrobe and technical field of tobacco processing, separation, cultivation, preparation and the application thereof of contained mikrobe in specifically a kind of microbial preparation that shortens the tobacco fermentation cycle.
Background technology
Tobacco fermentation is the important technique measure that high quality raw material are provided for production of cigarettes.The tobacco fermentation method can be divided into aging process and artificial fermentation.
Aging process is leaving in the warehouse after the tobacco leaf process packing of the suitable moisture of modulation (comprising first, redrying) acquisition; Utilize natural environmental condition, in certain temperature, humidity range, utilize the activity of tobacco leaf endoenzyme; Ferment lentamente, to reach the purpose of improving quality of tobacco.This method improves comprehensive, thorough to quality of tobacco, be the fermentation process that China generally adopts at present.But the used time of this method is long, needs 1-2 usually, not only takies a large amount of warehouses and fund, and fermentation costs is high.
The artificial fermentation be with tobacco leaf be placed on can controlled temperature and the proving room of humidity in; The condition that adopts artificial suitable tobacco leaf interior quality to change; Impel tobacco leaf chemical composition and physical property accelerate to change, thus reach make quality of tobacco improve at short notice purpose.This method can shorten fermentation time greatly, can practice thrift mass energy, and minimizing fund and warehouse take, and be both economical.Because the condition of microbial fermentation is simply gentle; Be easy to control, production cost is low, and some mikrobes have the activity of enzyme; The mikrobe that has can also produce some enzymes; And output is very high, so biotechnology especially microbial fermentation technology becomes the problem that the tobacco scientific worker pays close attention to gradually in Application in Tobacco research.
Britain scientist Koller studied tobacco fermentation mechanism the earliest in 1858; Point out that the fermentation of cigar is similar with ethanol fermentation in some aspects; And attempt to improve the tobacco fermentation effect with mikrobe; The 1950's, the scientist of the FSU also did the test of a large amount of mikrobe tobacco fermentations, in the exploratory development of tobacco fermentation mechanism with improve aspect such as tobacco fermentation quality and obtain certain effect, but did not all see the report that successful Application is arranged.Subsequently, the scientist of FSU change method is with providing the temperature and humidity conditions of enzyme effect to create artificial fermentation's method.China tobacco scientific worker such as Han Jinfeng, Qiu Liyou etc. also attempt the dominant bacteria that is separated to is mixed and made into microbial preparation, are sprayed on the cured tobacco leaf surface, can quicken the fermentation of preparing for the postgraduate qualifying examination, and improve the tobacco fermentation quality.
Research shows that the influence that microbial preparation is formed improving the tobacco leaf overall quality is bigger.And the action effect of existing microbial preparation is mostly more single, improving quality of tobacco, certain breakthrough arranged aspect shortening the tobacco fermentation cycle, but rare successful industrial application is reported.In the fermenting process of tobacco leaf, how to effectively utilize that suitable mikrobe and zymin are regulated and control and the inherent composition that quickens tobacco leaf transforms to the aspect that helps improving cigarette quality, become difficult point and focus in the biological tobacco scientific and technical research.
At present, domesticly still seldom adopting this method, mainly is in default of suitable artificial fermentation's agent, and fermentation back quality of tobacco improves effect has gap greatly with spontaneous fermentation.With the comparatively approaching documents of the present invention be the patented claim of application number 201010565687.5 " the microbial preparation production technique is used in a kind of tobacco flue-curing ", the purpose of this application provides a kind of tobacco flue-curing with the microbial preparation production technique of improving tobacco leaf industrial applicability quality; The technical scheme foot uses " pure tobacco leaf extract solid plate high temperature separates the tobacco leaf surface method of microorganism " isolating high temperature bacterium bacterial classification, cultivates the acquisition fermented liquid through certain condition and is the microbial preparation that provides tobacco flue-curing to use.It is different that the object of the invention is compared with documents with technical scheme.
Summary of the invention
The purpose of this invention is to provide a kind of can be used in and improve quality of tobacco and the microbial preparation that shortens the tobacco fermentation cycle, another object of the present invention is this microbial preparation to be applied to make fermentation back quality of tobacco and natural alcoholization quality of tobacco no significant difference in the tobacco fermentation process and shorten traditional tobacco fermentation cycle.
The objective of the invention is to realize through following technical solution: a kind of microbial preparation that shortens the tobacco fermentation cycle mainly is made up of bacillus cereus Bacillus cereus and bacillus amyloliquefaciens Bacillus amyloliqyefaciens; Its viable count ratio is 10～200: 1～50; The mikrobe that this microbial preparation comprised all separates from tobacco leaf surface; Identify through Chinese common micro-organisms preservation administrative center; Belong to bacillus cereus Bacillus cereus respectively; Preserving number is CGMCC No.3864 and bacillus amyloliquefaciens Bacillus amyloliqyefaciens, and preserving number is CGMCC No.3866.
Said bacillus cereus Bacillus cereus and bacillus amyloliquefaciens Bacillus amyloliqyefaciens are through following method preparation: the tobacco leaf surface that shreds is added in the sterilized LB nutrient solution; After 37 ℃ of shaking tables are cultivated 24 hours; Draw nutrient solution with aseptic straw; Adopt the dilution coating method on sterilization solid LB substratum, to carry out the flat board coating; After treating that bacterium grows, on another sterilization solid LB substratum, carry out strok method with inoculating needle and separate, must list bacterium colony pure culture bacterium after the purified cultivation.
After above-mentioned single bacterium colony pure culture bacterium process test tube substratum, enlarged culturing base, liquid fermentation medium cultivation, obtain bacillus cereus and bacillus amyloliquefaciens bacterium liquid respectively.
Above-mentioned bacillus cereus and bacillus amyloliquefaciens bacterium liquid are obtained bacterium mud after directly centrifugal respectively; Mix according to viable count ratio 10～200: 1～50, add trehalose 1～2%, glycerine 1～2.8%, Sodium Glutamate 0.5～1.5% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
Further; Above-mentioned Powdered microbial preparation is dissolved in the sterilized water according to 0.5～5% ratio; Process the microbial preparation aqua, this aqua evenly sprayed application on the tobacco leaf with 3～10% ratio, with tobacco leaf in 25 ℃～50 ℃; The artificial fermentation is 10～40 days under 65%RH～80%RH, can obtain tobacco leaf behind the artificial fermentation with natural alcoholization quality of tobacco no significant difference.
Further, this microbial preparation is used for the tobacco fermentation process, has the effect that improves quality of tobacco, shortens the tobacco fermentation cycle.
Microbial preparation of the present invention has following character: the contained mikrobe of this microbial preparation is: (1) bacillus cereus Bacillus cereus, its preservation registration number is CGMCC No.3864.Cell is shaft-like, and Gram-positive forms gemma, and sporangiocyst does not expand.Bacterium colony on the LB substratum is circle, neat in edge, white, and is glossy.The VP reacting positive, VP liquid pH 6, battalion's amphimicrobian life.Catalase is positive.Methyl red test is positive.Produce acid from glucose.In pH5.7 and 7%NaCl, grow.Amylase and starch.Reduction nitrate salt.(2) bacillus amyloliquefaciens Bacillus cereus, its preservation registration number are CGMCC No.3866.Cell is shaft-like, and Gram-positive forms gemma, and sporangiocyst does not expand.Bacterium colony on the LB substratum is circle, neat in edge, white, translucent, and is smooth moistening, slightly protuberance.The VP reacting positive, VP liquid pH 6, battalion's amphimicrobian life.Catalase is positive.Methyl red test is negative.Utilize multiple kinds of carbohydrate to produce acid.In pH5.7 and 7%NaCl, grow.Amylase and starch.Reduction nitrate salt.
Test shows that microbial preparation of the present invention is used for the tobacco fermentation process, effectively Mierocrystalline cellulose, protein and the pectin etc. in the degrading tobacco; Wherein the pectin degrading rate can reach about 40%; Protein degradation rate can reach about 23%, and cellulose degradation rate can reach about 12% respectively, and the reduction of tobacco leaf pungency, blue foreign smell are reduced; Inhaling flavor improves; Fermentation back quality of tobacco and natural alcoholization quality of tobacco no significant difference can foreshorten to 10～40 days with the tobacco fermentation cycle, have outstanding advantage and significant beneficial effect.
Embodiment
The microbial preparation that the present invention adopts separation to obtain from the beneficial microorganism method of tobacco leaf own.Said preparation mainly is made up of bacillus cereus Bacillus cereus and two kinds of bacterial strains of bacillus amyloliquefaciens Bacillus amyloliqyefaciens; Its viable count ratio is 10～200: 1～50; The mikrobe that this microbial preparation comprised all separates from tobacco leaf surface; Identify through Chinese common micro-organisms preservation administrative center; Belong to bacillus cereus Bacillus cereus respectively; Preserving number is CGMCC No.3864 and bacillus amyloliquefaciens Bacillus amyloliqyefaciens, and preserving number is CGMCC No.3866.
The separation of the contained mikrobe of said microbial preparation; Be the tobacco leaf surface that shreds is added in the sterilized LB nutrient solution, after 37 ℃ of shaking tables are cultivated 24 hours, draw nutrient solution with aseptic straw; Adopt the dilution coating method on sterilization solid LB substratum, to carry out the flat board coating; After treating that bacterium grows, on another same sterilization solid LB substratum, carry out strok method with inoculating needle and separate, must list bacterium colony pure culture bacterium after the purified cultivation.After above-mentioned single bacterium colony pure culture bacterium process test tube substratum, enlarged culturing base, liquid fermentation medium cultivation, obtain bacillus cereus and bacillus amyloliquefaciens bacterium liquid respectively, specifically carry out according to following steps:
(1) test tube substratum: adopt sterilization solid LB substratum, bacillus cereus and bacillus amyloliquefaciens are inoculated into respectively on the test tube substratum, 35-38 ℃ of following activation culture in inoculation back 24 hours;
(2) enlarged culturing base: adopt sterilising liq LB substratum, bacillus cereus and bacillus amyloliquefaciens in the test tube substratum are seeded in respectively in the Erlenmeyer flask, place on the shaking table 37 ℃, shaking culture 24～48 hours is as seed liquor;
(3) liquid fermentation medium: in fermentor tank, adopt liquid LB nutrient solution; 121 ℃ of autoclavings are about 30 minutes; When treating that sterile medium is cooled to 36 ℃; With the ratio inoculation fermentation liquid of the culture in the Erlenmeyer flask, cultivated 48 hours, and can obtain bacillus cereus and bacillus amyloliquefaciens bacterium liquid respectively for 36～38 ℃ in 5-10%.
Above-mentioned bacillus cereus and bacillus amyloliquefaciens bacterium liquid are obtained bacterium mud after directly centrifugal respectively; Mix according to viable count ratio 10～200: 1～50, add trehalose 1～2%, glycerine 1～2.8%, Sodium Glutamate 0.5～1.5% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
It is the ratio according to 0.5～5% that this microbial preparation is applied to the tobacco fermentation process, and Powdered microbial preparation is dissolved in the sterilized water, processes the microbial preparation aqua.This aqua is evenly sprayed application on the tobacco leaf with 3～10% ratio, and in 25 ℃～50 ℃, the artificial fermentation is 10 days～40 days under 65%RH～80%RH, has the effect that improves quality of tobacco, shortens the tobacco fermentation cycle.
Following embodiment will specify the present invention.Yet for a person skilled in the art, these embodiment just are used for describing the present invention rather than limiting category of the present invention.
Embodiment 1:
With bacillus cereus and bacillus amyloliquefaciens bacterium mud; Mix at 4: 1 according to the viable count ratio, add trehalose 1.2%, glycerine 1.8%, Sodium Glutamate 1.0% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
Ratio according to 1% should be dissolved in the sterilized water by Powdered microbial preparation, after fully stirring, gets microbial inoculum 10Kg even spraying and adds on the 100kg tobacco leaf, and the artificial fermentation is 22 days under 45 ℃, 80%RH condition, can reach the described effect of summary of the invention.
Add the tobacco leaf of microbial preparation artificial fermentation according to the invention after 22 days (adding appearance) and compare with fermenting tobacco leaf (original kind) not, its protein contnt obviously reduces; Add the reduction of appearance pungency, blue foreign smell minimizing, characteristic perfume is obvious, inhales flavor and improves, and suitable with 60 days same kind tobacco leaf (control samples) quality of spontaneous fermentation, its chemical ingredients and aesthetic quality's detected result are following:
Table one chemical ingredients detected result
Table two aesthetic quality detected result
Embodiment 2:
With bacillus cereus and bacillus amyloliquefaciens bacterium mud; Mix at 10: 1 according to the viable count ratio, add trehalose 2%, glycerine 2%, Sodium Glutamate 1.5% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
Ratio according to 5% should be dissolved in the sterilized water by Powdered microbial preparation, after fully stirring, gets microbial inoculum 8Kg even spraying and adds on the 100kg tobacco leaf, and the artificial fermentation is 30 days under 35 ℃, 70%RH condition, can reach the described effect of summary of the invention.
Add the tobacco leaf of microbial preparation artificial fermentation according to the invention after 30 days (adding appearance) and compare with fermenting tobacco leaf (original kind) not, its protein contnt obviously reduces; Add the reduction of appearance pungency, blue foreign smell minimizing, characteristic perfume is obvious, inhales flavor and improves, and is suitable with 60 days same kind tobacco leaf (control samples) quality of spontaneous fermentation.Chemical ingredients and aesthetic quality's detected result are following:
Table three chemical ingredients detected result
Table four aesthetic quality detected result
Embodiment 3:
With bacillus cereus and bacillus amyloliquefaciens bacterium mud; Mix at 1: 5 according to the viable count ratio, add trehalose 1%, glycerine 2.8%, Sodium Glutamate 1% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
Ratio according to 3% is dissolved in this microbial preparation in the sterilized water, after fully stirring, gets microbial inoculum 5Kg even spraying and adds on the 100kg tobacco leaf, and the artificial fermentation is 52 days under 25 ℃, 70%RH condition, can reach the described effect of summary of the invention.
Add the tobacco leaf of microbial preparation artificial fermentation according to the invention after 52 days (adding appearance) and compare with fermenting tobacco leaf (original kind) not, its protein contnt obviously reduces; Add the reduction of appearance pungency, blue foreign smell minimizing, characteristic perfume is obvious, inhales flavor and improves, and is suitable with 60 days same kind tobacco leaf (control samples) quality of spontaneous fermentation.Chemical ingredients and aesthetic quality's detected result are following:
Table five chemical ingredients detected result
Table six aesthetic quality detected result
Embodiment 4:
With bacillus cereus and bacillus amyloliquefaciens bacterium mud; Mix at 1: 1 according to the viable count ratio, add trehalose 1.5%, glycerine 2.2%, Sodium Glutamate 0.8% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
Ratio according to 3% is dissolved in this microbial preparation in the sterilized water, after fully stirring, gets microbial inoculum 3Kg even spraying and adds on the 100kg tobacco leaf, and the artificial fermentation is 42 days under 40 ℃, 68%RH condition, can reach the described effect of summary of the invention.
Add the tobacco leaf of microbial preparation artificial fermentation according to the invention after 42 days (adding appearance) and compare with fermenting tobacco leaf (original kind) not, its protein contnt obviously reduces; Add the reduction of appearance pungency, blue foreign smell minimizing, characteristic perfume is obvious, inhales flavor and improves, and is suitable with 60 days same kind tobacco leaf (control samples) quality of spontaneous fermentation.Chemical ingredients and aesthetic quality's detected result are following:
Table seven chemical ingredients detected result
Table eight aesthetic quality detected result
Although the present invention has been carried out detailed explanation with above-mentioned embodiment, other embodiment also is possible, therefore, and the description that should not be limited to embodiment in this specification sheets to the essence and the scope of claim.
Claims (6)
1. a microbial preparation that shortens the tobacco fermentation cycle is characterized in that, mainly by bacillus cereus
Bacillus cereusWith bacillus amyloliquefaciens
Bacillus amyloliqyefaciensConstitute, its viable count ratio is 10～200:1～50; The mikrobe that this microbial preparation comprised all separates from tobacco leaf surface, identifies through Chinese common micro-organisms preservation administrative center, belongs to bacillus cereus respectively
Bacillus cereus,Preserving number is CGMCC No.3864 and bacillus amyloliquefaciens
Bacillus amyloliqyefaciens,Preserving number is CGMCC No.3866.
2. a kind of microbial preparation that shortens the tobacco fermentation cycle according to claim 1 is characterised in that described bacillus cereus
Bacillus cereusWith bacillus amyloliquefaciens
Bacillus amyloliqyefaciensBe through following method preparation: the tobacco leaf surface that shreds is added in the sterilized LB nutrient solution; After 37 ℃ of shaking tables are cultivated 24 hours; Draw nutrient solution with aseptic straw, adopt the dilution coating method on sterilization solid LB substratum, to carry out flat board and be coated with, treat that bacterium grows after; On another sterilization solid LB substratum, carry out strok method with inoculating needle and separate, must list bacterium colony pure culture bacterium after the purified cultivation.
3. a kind of preparation of shortening the microbial preparation in tobacco fermentation cycle according to claim 2; It is characterized in that; After above-mentioned single bacterium colony pure culture bacterium process test tube substratum, enlarged culturing base, liquid fermentation medium cultivation, obtain bacillus cereus and bacillus amyloliquefaciens bacterium liquid respectively.
4. a kind of preparation of shortening the microbial preparation in tobacco fermentation cycle according to claim 3; It is characterized in that; Above-mentioned bacillus cereus and bacillus amyloliquefaciens bacterium liquid are obtained bacterium mud after directly centrifugal respectively; Mix according to viable count ratio 10～200:1～50, add trehalose 1～2%, glycerine 1～2.8%, Sodium Glutamate 0.5～1.5% by weight percentage and carry out lyophilize as the compound lyophilized vaccine of this bacterium mud and obtain Powdered microbial preparation.
5. a kind of application of shortening the microbial preparation in tobacco fermentation cycle according to claim 4; It is characterized in that, above-mentioned Powdered microbial preparation is dissolved in the sterilized water according to 0.5～5% ratio, process mikrobe system aqua; Evenly spray application to tobacco leaf on 3～10% ratio this aqua; In 25 ℃～50 ℃, the artificial fermentation is 10～40 days under 65% RH～80% RH with tobacco leaf, can obtain tobacco leaf behind the artificial fermentation with natural alcoholization quality of tobacco no significant difference.
6. a kind of application of shortening the microbial preparation in tobacco fermentation cycle according to claim 4 is characterized in that, this microbial preparation is used for the tobacco fermentation process, has the effect that improves quality of tobacco, shortens the tobacco fermentation cycle.
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