CN102816710A - Strain capable of generating protease, method for preparing protease liquid and method for improving quality of low-grade tobaccos by using protease liquid - Google Patents
Strain capable of generating protease, method for preparing protease liquid and method for improving quality of low-grade tobaccos by using protease liquid Download PDFInfo
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- CN102816710A CN102816710A CN2011104355216A CN201110435521A CN102816710A CN 102816710 A CN102816710 A CN 102816710A CN 2011104355216 A CN2011104355216 A CN 2011104355216A CN 201110435521 A CN201110435521 A CN 201110435521A CN 102816710 A CN102816710 A CN 102816710A
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Abstract
The invention discloses a method for improving quality of low-grade tobaccos by using bacterial fermentation protease liquid. The method includes the following steps: 1) preparation of the bacterial fermentation protease liquid: (1) bacillus strains capable of generating protease are activated by a luria-bertani (LB) medium, planted in a sterilization seed medium, cultured for 12 hours at the temperature of 37 DEG C in a 150rpm shaker with constant temperature, transplanted in a 1L shake flask according to the proportion of 1:100, and fermented for 60 hours to obtain the fermentation protease liquid; and (2) processing of the fermentation protease liquid: the fermented protease liquid is filtered and sterilized through a 0.22mum hollow fiber pipe, 10000 molecular weight cut off (MWCO) ultrafiltration and concentration are performed to obtain crude enzyme; and 2) improvement of the quality of low-grade tobaccos: the crude enzyme is sprayed on tobacco shred evenly according to the certain proportion of 2%, the tobacco shred is arranged in a ziplock bag at the room temperature to be acted for 12 hours, the enzyme is inactivated at the temperature of 90 DEG C and in 30 minutes, and the tobacco shred is arranged in a constant temperature and humidity box at the temperature of 22 DEG C and with 55% of humidity to balance moisture of the tobacco shred for 24 hours. Sensory evaluation results show that the fermented protease liquid of the strain can improve aroma quality of the tobaccos, reduces miscellaneous gases, and is good in texture and taste, and the quality of the tobaccos is improved remarkably.
Description
Technical field
The present invention relates to a strain and produce the bacterial isolates of proteolytic enzyme, preparation liquid of protease method and use its liquid of protease to improve low inferior quality of tobacco, specifically be used to the method for the low inferior quality of tobacco of dominant strain fermentation protein enzyme liquid improvement on tobacco leaf.
Background technology
China is cured tobacco production and consumption big country, and cured tobacco production occupies critical role in the development of tobacco.China's quality of tobacco is compared with international most advanced level with production level at present, and big gap is still arranged, and also far can not satisfy and improve domestic cigarette quality and the needs of expanding export.Raising quality of tobacco and operability are directly connected to China's tobacco industry and continue, stablize, develop in a healthy way, and also are the only ways that strengthens China's tobacco leaf competitiveness in the international market.
Owing to the reasons such as kind, plantation and baking of cigarette, macromolecular substance content such as China's flue-cured tobacco protein, starch are often higher.Good and bad the being closely related property that exists of protein contnt and quality of tobacco, protein contnt is too high, not only can reduce the incendivity of tobacco product, and the stink that seemingly burns feather is arranged, simultaneously with pungent, pained sensation.Can make objectionable constituent such as the increase of HCN equal size in the flue gas in addition, have a strong impact on the fragrance quality of tobacco leaf and suck security.Tobacco leaf protein matter hydrolysate can further transform and generate the flavor matter that helps quality of tobacco on the other hand.Therefore, it is very important to improving quality of tobacco, improve tobacco leaf usability and sucking security to reduce in the tobacco leaf protein contnt.
Protein on tobacco leaf under the proteolytic enzyme katalysis can be hydrolyzed to small-molecule substances such as amino acid, the protein of not only having degraded, and hydrolysate can produce the tobacco flavor matter with the product that further transforms.Proteolytic enzyme source can be bought commercial enzyme and also utilize the microbial fermentation preparation, but the commercial enzyme price is higher, and this has increased the cigarette cost virtually, and utilizes the fermentation using bacteria liquid of protease easy to operate and technology is simple.
Summary of the invention
The purpose of this invention is to provide a strain and produce the bacterial isolates of proteolytic enzyme and utilize the fermentation using bacteria liquid of protease, use process filtering bacterium and ultrafiltration and concentration liquid as the method for improving low inferior quality of tobacco.
Technical scheme of the present invention is following: a bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain; The bacterial strain preserving number is (CGMCC No. 5310); Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on September 29th, 2011; This strains separation is from refining tobacco leaf, and product proteolytic enzyme ability is stronger.
This 0855-9 bacterial strain screening substratum adopts the casein agar substratum: casein 10g, Carnis Bovis seu Bubali cream 3g, Na
2HP0
42g, NaCl 5g, agar 15g, zero(ppm) water 1000mL, 0.4% NSC 7819 solution 12.5mL, pH7.4 observes hydrolysis circle production, and the enzyme that multiple sieve adopts forint-phenol law to measure proteolytic enzyme is lived.
Fermentation using bacteria prepares the liquid of protease concrete steps:
1. activation of genus bacillus and cultivation: the bacterial strain preserving number of this genus bacillus is (CGMCC No.5310); After the activation of LB substratum, insert the seed culture medium of sterilization; Cultivate 12h in 37 ℃, 150rpm constant temperature shaking table, transfer by 1: 100 ratio and shake the 60h that ferments in the bottle in 1L and make fermentation protein enzyme liquid;
2. the processing of fermentation protein enzyme liquid: the liquid of protease that ferments makes crude enzyme liquid through the 10000MWCO ultrafiltration and concentration again through 0.22 μ m hollow fiber conduit filtration sterilization;
Above-described seed culture medium and fermention medium all are LB substratum, and its main ingredient is: NaCl 10g, yeast powder 5g, Tryptones 10g, adding distil water regulate pH value to 7.0,121 ℃ of autoclaving 20min to 1000ml with NaOH solution.
Enzyme assay: the fermentation using bacteria liquid of protease adopts forint-phenol law to measure enzymic activity, its unit of activity definition (U): produce 1 μ g tyrosine at 37 ℃ of PM caseinhydrolysates, be defined as 1 enzyme activity unit.
Use the liquid of protease of preparation to be as improving low inferior tobacco leaf step:
With taking out offal after the upper tobacco leaf moisture regain, chopping takes by weighing a certain amount of pipe tobacco, is divided into two parts; A copy of it pipe tobacco at room temperature (about 24 ℃) ratio in 2% sprays worth crude enzyme liquid and zero(ppm) water equably, places valve bag at room temperature to make enzyme effect 12h; Oven dry 30mins makes enzyme deactivation under 90 ℃ of conditions then, places 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; With this pipe tobacco is the evaluation of smokeing panel test of the laggard row of single raw material cigarette, and another part pipe tobacco uses as this group comparison object of reference.
The liquid of protease of strain fermentation of the present invention can improve tobacco incense makings, and assorted gas alleviates, and texture, mouthfeel are better, and quality of tobacco obviously improves.Tobacco scientific worker research shows that tobacco leaf attendes Institute of Micro-biology and produce the conversion that enzyme can promote macromolecular substance on the tobacco leaf; Help improving quality of tobacco; Improve low inferior quality of tobacco important researching value is arranged so utilize to separate bacterium producing multi enzyme preparation fermentation protein enzyme liquid from tobacco leaf, for utilizing fermentation using bacteria enzyme liquid to improve quality of tobacco, improving tobacco leaf usability a kind of approach is provided.
Description of drawings
Fig. 1: proteolytic enzyme enzyme activity determination typical curve.
Embodiment
Fermentation protein enzyme liquid of the present invention source: bacterium producing multi enzyme preparation is bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, and the bacterial strain preserving number is (CGMCC No.5310).This strains separation is from refining tobacco leaf, and product proteolytic enzyme ability is stronger, can improve quality of tobacco effectively.
1, the preparation of fermentation using bacteria liquid of protease:
1. activation of genus bacillus and cultivation: the bacterial strain preserving number of this genus bacillus is (CGMCC No.5310); After the activation of LB substratum, insert the seed culture medium of sterilization; Cultivate 12h in 37 ℃, 150rpm constant temperature shaking table, transfer by 1: 100 ratio and shake the 60h that ferments in the bottle in 1L and make fermentation protein enzyme liquid.
Above-described seed culture medium and fermention medium all are LB substratum, and its main ingredient is: NaCl 10g, yeast powder 5g, Tryptones 10g, adding distil water regulate pH value to 7.0,121 ℃ of autoclaving 20min to 1000ml with NaOH solution.
2. the processing of fermentation protein enzyme liquid: the liquid of protease that ferments makes crude enzyme liquid through the 10000MWCO ultrafiltration and concentration again through 0.22 μ m hollow fiber conduit filtration sterilization.
2, enzyme assay:
The fermentation using bacteria liquid of protease adopts forint-phenol law to measure enzymic activity, its unit of activity definition (U): produce 1 μ g tyrosine at 37 ℃ of PM caseinhydrolysates, be defined as 1 enzyme activity unit.
3, improve the method for low inferior quality of tobacco
With taking out offal after the upper tobacco leaf moisture regain, chopping takes by weighing a certain amount of pipe tobacco, is divided into two parts.At room temperature (about 24 ℃) ratio in 2% sprays worth crude enzyme liquid and zero(ppm) water equably, places valve bag at room temperature to make enzyme effect 12h; Oven dry 30mins makes enzyme deactivation under 90 ℃ of conditions then, places 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; With this pipe tobacco is the evaluation of smokeing panel test of the laggard row of single raw material cigarette.
Embodiment 1: the screening of bacteria produced proteinase strain and evaluation
Bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, the bacterial strain preserving number is (CGMCC No.5310), separates on the red the Nature alcoholization of fine quality tobacco leaf.The bacterial strain screening substratum adopts casein agar substratum (casein 10g, Carnis Bovis seu Bubali cream 3g, Na
2HPO
42g, NaCl 5g, agar 15g, zero(ppm) water 1000mL, 0.4% NSC 7819 solution 12.5mL pH7.4), observes hydrolysis circle production, and the enzyme that multiple sieve adopts forint-phenol law to measure proteolytic enzyme is lived.
Utilize a pair of universal primer of 16S rRNA gene to carry out pcr amplification; Connection carrier transforms and checks order; The result submits NCBI to, carries out the sequence homology retrieval analysis through BLAST, carries out the multisequencing comparison and calculates the sequence similarity between strains tested and the reference bacterial strain with CLUSTAL X software then; Adopt the ortho position method of joining, use MEGA4 software building systematic evolution tree.This bacterial strain is carried out sequencing analysis, is bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain with its preliminary evaluation, and the bacterial strain preserving number is (CGMCC No.5310).
Embodiment 2: the preparation of proteolytic enzyme crude enzyme liquid
The fermentation protein enzyme source is (CGMCC No.5310) bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain for the bacterial strain preserving number; After the activation of LB substratum, insert the seed culture medium of sterilization; Cultivate 12h in 37 ℃ of 150rpm constant temperature shaking tables, transfer by 1: 100 ratio and shake the 60h that ferments in the bottle in 1L and make fermentation protein enzyme liquid.Above-described seed culture medium and fermention medium all are LB substratum, and its main ingredient is: NaCl 10g, yeast powder 5g, Tryptones 10g, adding distil water regulate pH value to 7.0,121 ℃ of autoclaving 20min to 1000ml with NaOH solution.The liquid of protease that ferments makes crude enzyme liquid through the 10000MWCO ultrafiltration and concentration again through 0.22 μ m hollow fiber conduit filtration sterilization.
The fermentation using bacteria liquid of protease adopts forint-phenol law to measure enzymic activity, its unit of activity definition (U): produce 1 μ g tyrosine at 37 ℃ of PM caseinhydrolysates, be defined as 1 enzyme activity unit.At first prepare tyrosine typical curve (Fig. 1), the fermented liquid that will contain proteolytic enzyme is then used pH7.2, and the phosphate buffered saline buffer of 50mmol/L is after suitable dilution; The enzyme liquid 100ul that gets dilution adds 2% casein food grade 100ul at 37 ℃ of following insulation 2min, behind 37 ℃ of following insulation 10min; The trichoroacetic acid(TCA) termination reaction that adds 0.4mol/L continues insulation 15min and makes the residual protein deposition fully, the centrifugal 1min of 13000rpm; Get supernatant 100ul, add the yellow soda ash 500ul of 0.4mol/L then, behind the forint phenol reagent 100ul mixing; In 37 ℃ of color development 10min, with the light absorption value at spectrophotometric determination 660nm place.Do a blank simultaneously, only before adding 2% casein food grade, add trichoroacetic acid(TCA) earlier and make enzyme deactivation, measure the light absorption value at 660nm place.
Embodiment 3: the proteolytic enzyme crude enzyme liquid improves the method for low inferior quality of tobacco
Take out offal with after the upper tobacco leaf moisture regain, chopping takes by weighing the pipe tobacco of 50 grams, is divided into two parts.At room temperature (about 24 ℃) ratio in 2% sprays worth crude enzyme liquid and zero(ppm) water equably; The enzyme amount of executing of 0855-9 fermenting enzyme liquid is the 48U/g tobacco leaf; Place valve bag at room temperature to make enzyme effect 12h; Oven dry 30min makes enzyme deactivation under 90 ℃ of conditions then, places 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%.
With the good pipe tobacco of balance is the evaluation of smokeing panel test of the laggard row of single raw material cigarette; Smoke panel test by the completion of Hongta Group's technique center person of smokeing panel test; Adopt " Yuxi high-quality tobacco single-tobacco-typed cigarette aesthetic quality smoke panel test method " (Yunnan Province's provincial standard, standard numbering DB53/T182.3-2006) (table 1).
The result can find out (table 2) from evaluation and analysis: the contrast flue gas concentration is higher, and assorted gas shows, and aftertaste is poor slightly, and is sour, puckery, slightly jagged sense, and matter is coarse; The LB fermention medium has no adverse effects to the tobacco leaf aesthetic quality, does not also improve quality of tobacco, so the LB fermention medium does not influence the result that liquid of protease is handled tobacco leaf.Smoking result shows that the liquid of protease of this strain fermentation is comparatively obvious to the improvement of quality of tobacco, shows as fragrance matter and improves, and assorted gas alleviates, and texture, mouthfeel are all better, and strength slightly descends, and pungency makes moderate progress.
Table 1: Hongta Group's monomer tobacco leaf organoleptic quality evaluation method and index
Table 2:0855-9 bacterial strain is handled the pipe tobacco smoking result
Claims (5)
1. a bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, the bacterial strain preserving number is (CGMCC No.5310), and this strains separation is from refining tobacco leaf, and it is stronger to produce the proteolytic enzyme ability.
2. bacillus pumilus according to claim 1 (Bacillus pumilus) is characterized in that this 0855-9 bacterial strain screening substratum adopts the casein agar substratum: casein 10g, Carnis Bovis seu Bubali cream 3g, Na
2HPO
42g, NaCl 5g, agar 15g, zero(ppm) water 1000mL, 0.4% NSC 7819 solution 12.5mL, pH7.4 observes hydrolysis circle production, and the enzyme that multiple sieve adopts forint-phenol law to measure proteolytic enzyme is lived.
3. use the described bacillus pumilus of claim 1 (Bacillus pumilus) 0855-9 bacterial strain to prepare the method for liquid of protease, it is characterized in that fermentation using bacteria prepares the liquid of protease concrete steps and is:
1. activation of genus bacillus and cultivation: the bacterial strain preserving number of this genus bacillus is (CGMCC No.5310); After the activation of LB substratum, insert the seed culture medium of sterilization; Cultivate 12h in 37 ℃, 150rpm constant temperature shaking table, transfer by 1: 100 ratio and shake the 60h that ferments in the bottle in 1L and make fermentation protein enzyme liquid;
2. the processing of fermentation protein enzyme liquid: the liquid of protease that ferments makes crude enzyme liquid through the 10000MWCO ultrafiltration and concentration again through 0.22 μ m hollow fiber conduit filtration sterilization;
Above-described seed culture medium and fermention medium all are LB substratum, and its main ingredient is: NaCl 10g, yeast powder 5g, Tryptones 10g, adding distil water regulate pH value to 7.0,121 ℃ of autoclaving 20min to 1000ml with NaOH solution.
4. bacillus pumilus according to claim 3 (Bacillus pumilus) 0855-9 bacterial strain prepares liquid of protease; It is characterized in that enzyme assay: the fermentation using bacteria liquid of protease adopts forint-phenol law to measure enzymic activity; Its unit of activity definition (U): produce 1 μ g tyrosine at 37 ℃ of PM caseinhydrolysates, be defined as 1 enzyme activity unit.
5. bacillus pumilus according to claim 3 (Bacillus pumilus) 0855-9 bacterial strain prepares liquid of protease, it is characterized in that using the liquid of protease of preparation as improving low inferior tobacco leaf step to be:
With taking out offal after the upper tobacco leaf moisture regain, chopping takes by weighing a certain amount of pipe tobacco, is divided into two parts; A copy of it pipe tobacco at room temperature (about 24 ℃) ratio in 2% sprays worth crude enzyme liquid and zero(ppm) water equably, places valve bag at room temperature to make enzyme effect 12h; Oven dry 30mins makes enzyme deactivation under 90 ℃ of conditions then, places 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; With this pipe tobacco is the evaluation of smokeing panel test of the laggard row of single raw material cigarette, and another part pipe tobacco uses as this group comparison object of reference.
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