CN102816710B - 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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- CN102816710B CN102816710B CN201110435521.6A CN201110435521A CN102816710B CN 102816710 B CN102816710 B CN 102816710B CN 201110435521 A CN201110435521 A CN 201110435521A CN 102816710 B CN102816710 B CN 102816710B
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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, prepare liquid of protease method and use its liquid of protease to improve low inferior quality of tobacco, specifically utilize the method for improving low inferior quality of tobacco from the dominant strain fermentation protein enzyme liquid 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, still has larger gap, also far can not meet the needs that improve domestic cigarette quality and expand export.Raising quality of tobacco and operability are directly connected to China's tobacco industry and continue, stablize, develop in a healthy way, and are also the only ways that strengthens China's tobacco leaf competitiveness in the international market.
Due to the reasons such as kind, plantation and baking of cigarette, the 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 content and quality of tobacco, protein content is too high, not only can reduce the incendivity of tobacco product, and has the stink that seemingly burns feather, simultaneously with pungent, pained sensation.In addition can make harmful substances in flue gas as HCN equal size increases, 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 is conducive to quality of tobacco on the other hand.Therefore, reduce in tobacco leaf protein content to improving quality of tobacco, improve tobacco leaf usability and suck security very important.
Protein under proteolytic enzyme katalysis on tobacco leaf can be hydrolyzed to the small-molecule substances such as amino acid, the protein of not only having degraded, and the product of hydrolysate and further conversion can produce tobacco flavor matter.Proteolytic enzyme source can be bought commercial enzyme and also utilize microorganism fermentation preparation, but commercial enzyme price is higher, and this has increased cigarette cost virtually, and utilize, fermentation using bacteria liquid of protease is easy to operate and technique is simple.
Summary of the invention
The object of this invention is to provide a strain and produce the bacterial isolates of proteolytic enzyme and utilize fermentation using bacteria liquid of protease, use degerming after filtration and ultrafiltration and concentration liquid as the method for improving low inferior quality of tobacco.
Technical scheme of the present invention is as follows: a bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, bacterial strain preserving number is (CGMCC No. 5310), depositary institution: China Committee for Culture Collection of Microorganisms's 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 aging tobacco leaves, and product proteolytic enzyme ability is stronger.
This 0855-9 bacterial strain screening substratum adopts casein agar substratum: casein 10g, extractum carnis 3g, Na
2hPO
42g, NaCl 5g, agar 15g, distilled water 1000mL, 0.4% bromothymol blue 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 is prepared liquid of protease concrete steps:
1. the 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, access the seed culture medium of sterilizing, in 37 ℃, 150rpm constant-temperature table, cultivate 12h, in the transfer 60h that ferments of the ratio of 1:100, make fermentation protein enzyme liquid in 1L shaking flask;
2. the processing of fermentation protein enzyme liquid: the liquid of protease fermenting is through 0.22 μ m hollow fiber conduit filtration sterilization, then make crude enzyme liquid through 10000 MWCO ultrafiltration and concentration;
Above-described seed culture medium and fermention medium are all LB substratum, and its main ingredient is: NaCl 10g, and yeast powder 5g, Tryptones 10g, adding distil water, to 1000ml, regulates pH value to 7.0,121 ℃ of autoclaving 20min with NaOH solution.
Enzyme assay: 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 per minute caseinhydrolysates, be defined as 1 enzyme activity unit.
Use the liquid of protease of preparation as improving low inferior tobacco leaf step to be:
To after upper tobacco leaf moisture regain, pump offal, chopping, takes a certain amount of pipe tobacco, is divided into two parts; A copy of it pipe tobacco at room temperature (24 ℃ of left and right) ratio in 2% sprays worth crude enzyme liquid and distilled water equably, is placed in valve bag and at room temperature makes enzyme effect 12h; Then under 90 ℃ of conditions, dry 30mins and make enzyme deactivation, be placed in 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; Take this pipe tobacco as the evaluation of smokeing panel test of the laggard row of single raw material cigarette, and another part of pipe tobacco used as this group comparison object of reference.
The liquid of protease of strain fermentation of the present invention can improve tobacco aroma quality, and assorted gas alleviates, and texture, mouthfeel are better, and quality of tobacco obviously improves.Tobacco scientific worker research shows that on tobacco leaf, Institute of Micro-biology produces the conversion that enzyme can promote macromolecular substance on tobacco leaf, be conducive to improve quality of tobacco, so utilize the separated bacterium producing multi enzyme preparation fermentation protein enzyme liquid from tobacco leaf to improve low inferior quality of tobacco, there is important researching value, for utilizing fermentation using bacteria enzyme liquid to improve quality of tobacco, improve tobacco leaf usability a kind of approach is provided.
Accompanying drawing explanation
Fig. 1: proteinase activity bioassay standard 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 bacterial strain preserving number is (CGMCC No. 5310).This strains separation is from aging tobacco leaves, and product proteolytic enzyme ability is stronger, can effectively improve quality of tobacco.
1, the preparation of fermentation using bacteria liquid of protease:
1. the 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, access the seed culture medium of sterilizing, in 37 ℃, 150rpm constant-temperature table, cultivate 12h, in the transfer 60h that ferments of the ratio of 1:100, make fermentation protein enzyme liquid in 1L shaking flask.
Above-described seed culture medium and fermention medium are all LB substratum, and its main ingredient is: NaCl 10g, and yeast powder 5g, Tryptones 10g, adding distil water, to 1000ml, regulates pH value to 7.0,121 ℃ of autoclaving 20min with NaOH solution.
2. the processing of fermentation protein enzyme liquid: the liquid of protease fermenting is through 0.22 μ m hollow fiber conduit filtration sterilization, then make crude enzyme liquid through 10000 MWCO ultrafiltration and concentration.
2, enzyme assay:
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 per minute caseinhydrolysates, be defined as 1 enzyme activity unit.
3, improve the method for low inferior quality of tobacco
To after upper tobacco leaf moisture regain, pump offal, chopping, takes a certain amount of pipe tobacco, is divided into two parts.At room temperature (24 ℃ of left and right) ratio in 2% sprays worth crude enzyme liquid and distilled water equably, is placed in valve bag and at room temperature makes enzyme effect 12h; Then under 90 ℃ of conditions, dry 30mins and make enzyme deactivation, be placed in 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; Take this pipe tobacco as 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, bacterial strain preserving number is (CGMCC No. 5310), separated on the red the Nature aging tobacco leaves of fine quality.Bacterial strain screening substratum adopts casein agar substratum (casein 10g, extractum carnis 3g, Na
2hPO
42g, NaCl 5g, agar 15g, distilled water 1000mL, 0.4% bromothymol blue solution 12.5mL, pH7.4), observe hydrolysis circle production, 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, result is submitted NCBI to, by BLAST, carry out sequence homology retrieval analysis, then with CLUSTAL X software, carry out Multiple Sequence Alignment and calculate strains tested and reference bacterial strain between sequence similarity, adopt ortho position phase connection, application MEGA 4 software building systematic evolution trees.This bacterial strain is carried out to sequencing analysis, be initially identified as bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, bacterial strain preserving number is (CGMCC No. 5310).
Embodiment 2: the preparation of proteolytic enzyme crude enzyme liquid
Fermentation protein enzyme source is that bacterial strain preserving number is (CGMCC No. 5310) bacillus pumilus (Bacillus pumilus) 0855-9 bacterial strain, after the activation of LB substratum, access the seed culture medium of sterilizing, in 37 ℃ of 150rpm constant-temperature tables, cultivate 12h, in the transfer 60h that ferments of the ratio of 1:100, make fermentation protein enzyme liquid in 1L shaking flask.Above-described seed culture medium and fermention medium are all LB substratum, and its main ingredient is: NaCl 10g, and yeast powder 5g, Tryptones 10g, adding distil water, to 1000ml, regulates pH value to 7.0,121 ℃ of autoclaving 20min with NaOH solution.The liquid of protease fermenting is through 0.22 μ m hollow fiber conduit filtration sterilization, then makes crude enzyme liquid through 10000 MWCO ultrafiltration and concentration.
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 per minute caseinhydrolysates, be defined as 1 enzyme activity unit.First prepare tyrosine typical curve (Fig. 1), then by the fermented liquid pH7.2 that contains proteolytic enzyme, the phosphate buffered saline buffer of 50mmol/L is after suitable dilution, the enzyme liquid 100ul that gets dilution is incubated 2min at 37 ℃, add 2% casein food grade 100ul, at 37 ℃, be incubated after 10min, the trichoroacetic acid(TCA) termination reaction that adds 0.4mol/L, continue insulation 15min and make residual protein precipitation completely, the centrifugal 1min of 13000rpm, get supernatant liquor 100ul, then the sodium carbonate 500ul that adds 0.4mol/L, after forint phenol reagent 100ul mixes, in 37 ℃ of color development 10min, light absorption value with spectrophotometric determination 660nm place.Do a blank simultaneously, only before adding 2% casein food grade, first add trichoroacetic acid(TCA) and make enzyme deactivation, measure the light absorption value at 660nm place.
Embodiment 3: proteolytic enzyme crude enzyme liquid improves the method for low inferior quality of tobacco
To after upper tobacco leaf moisture regain, pump offal, chopping, takes the pipe tobacco of 50 grams, is divided into two parts.At room temperature (24 ℃ of left and right) ratio in 2% sprays worth crude enzyme liquid and distilled water equably, the enzyme amount of executing of 0855-9 fermenting enzyme liquid is 48 U/g tobacco leaves, be placed in valve bag and at room temperature make enzyme effect 12h, then under 90 ℃ of conditions, dry 30min and make enzyme deactivation, be placed in 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%.
The good pipe tobacco of the balance of take is the evaluation of smokeing panel test of the laggard row of single raw material cigarette, by the technique center member of smoking of Hongta Group, complete and smoke panel test, adopt < < Yuxi high-quality tobacco single-tobacco-typed cigarette aesthetic quality method > > (Yunnan Province's provincial standard, standard number DB53/T 182.3-2006) (table 1) that smokes panel test.
From evaluation and analysis result, can find out (table 2): contrast flue gas concentration is higher, assorted gas is aobvious, and aftertaste is slightly poor, sour, puckery, slightly jagged sense, and matter is coarse; LB fermention medium has no adverse effects to tobacco leaf aesthetic quality, does not also improve quality of tobacco, so LB fermention medium does not affect the result that liquid of protease is processed tobacco leaf.Smoking result shows, 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 declines, 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 processed pipe tobacco smoking result
Claims (5)
- One bacillus pumilus ( bacillus pumilus) 0855-9 bacterial strain, bacterial strain preserving number is CGMCC No.5310, and this strains separation is from aging tobacco leaves, and product proteolytic enzyme ability is stronger.
- Right to use require bacillus pumilus described in 1 ( bacillus pumilus) 0855-9 bacterial strain prepares the method for liquid of protease, it is characterized in that fermentation using bacteria prepares liquid of protease concrete steps and be:1. the 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, access the seed culture medium of sterilizing, in 37 ℃, 150rpm constant-temperature table, cultivate 12h, in the transfer 60h that ferments of the ratio of 1:100, make fermentation protein enzyme liquid in 1L shaking flask;2. the processing of fermentation protein enzyme liquid: the liquid of protease fermenting is through 0.22 μ m hollow fiber conduit filtration sterilization, then make crude enzyme liquid through 10000MWCO ultrafiltration and concentration;The substratum of above-described seed culture medium and fermentation use is all LB substratum, and its main ingredient is: NaCl10g, and yeast powder 5g, Tryptones 10g, adding distil water, to 1000ml, regulates pH value to 7.0,121 ℃ of autoclaving 20min with NaOH solution.
- Bacillus pumilus according to claim 2 ( bacillus pumilus) 0855-9 bacterial strain prepares the method for liquid of protease, it is characterized in that this bacillus pumilus 0855-9 bacterial strain screening substratum adopts casein agar substratum: casein 10g, extractum carnis 3g, Na 2hPO 42g, NaCl5g, agar 15g, distilled water 1000mL, 0.4% bromothymol blue 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.
- Bacillus pumilus according to claim 2 ( bacillus pumilus) 0855-9 bacterial strain prepares the method for liquid of protease, it is characterized in that enzyme assay: 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 per minute caseinhydrolysates, be defined as 1 enzyme activity unit.
- Bacillus pumilus according to claim 2 ( bacillus pumilus) 0855-9 bacterial strain prepares the liquid of protease purposes that the method for liquid of protease makes, it is characterized in that using the liquid of protease of preparation to improve low inferior tobacco leaf step to be:To after upper tobacco leaf moisture regain, pump offal, chopping, takes a certain amount of pipe tobacco, is divided into two parts; A copy of it pipe tobacco is under 24 ℃ of room temperatures, and crude enzyme liquid sprays in certain 2% ratio crude enzyme liquid and the distilled water making equably, is placed in valve bag and at room temperature makes enzyme effect 12h; Then under 90 ℃ of conditions, dry 30min and make enzyme deactivation, be placed in 22 ℃, the climatic chamber balance moisture in cut tobacco 24h of relative humidity 55%; Take this pipe tobacco as the evaluation of smokeing panel test of the laggard row of single raw material cigarette, and another part of pipe tobacco used as this group comparison object of reference.
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