CN109554306B - Compound preparation of protease and protein degrading bacteria and application of compound preparation in cigars - Google Patents
Compound preparation of protease and protein degrading bacteria and application of compound preparation in cigars Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B3/00—Preparing tobacco in the factory
- A24B3/04—Humidifying or drying tobacco bunches or cut tobacco
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24C—MACHINES FOR MAKING CIGARS OR CIGARETTES
- A24C1/00—Elements of cigar manufacture
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Abstract
The invention belongs to the technical field of cigar preparation, and particularly relates to a compound preparation prepared by using protease and protein degrading bacteria and application of the compound preparation in cigars. The preparation process of the compound preparation comprises the following steps: preparing a protein degrading bacterium solution by using the bacillus pumilus SMXP-03, preparing an alkaline protease solution for later use, preparing a compound preparation and the like. The method is designed aiming at the fermentation alcoholization of the cigar tobacco leaves, and realizes the degradation of nitrogenous substances, particularly protein components, in the cigar tobacco leaves by adopting an alkaline protease and specific protein degradation strain composite preparation. Preliminary detection and application effects show that after treatment, the quality of the cigar tobacco leaves is better improved, the purposes of quality improvement and consumption reduction are better achieved, and meanwhile, the method has a better promotion effect on the production of cigars, so that the method has better practical value and popularization and application significance.
Description
Technical Field
The invention belongs to the technical field of cigar preparation, and particularly relates to a compound preparation prepared by using protease and protein degrading bacteria and application of the compound preparation in cigars.
Background
The nitrogen-containing substances, particularly the protein, are one of the main components in the tobacco leaves, the protein with a certain content is beneficial to increasing the satisfaction of the tobacco during smoking and providing rich fragrance, but the excessively high protein content can cause the tobacco leaves to generate obvious burnt feather flavor in the combustion process, so that the tobacco leaves have obvious bitter and astringent feeling during smoking, and the release of some harmful substances can be improved. Therefore, the content of nitrogen-containing substances, especially protein, in the tobacco leaves must be properly adjusted and controlled to ensure the smoking quality of the tobacco leaves.
For the perfect quality of tobacco leaves, one of the main technical means in the prior art is to degrade proteins by utilizing the action of endogenous microorganisms in the tobacco leaves through the natural alcoholization process of the tobacco leaves, but the treatment mode usually needs a long time (the natural alcoholization period is generally 1-3 years), and the protein degradation efficiency is usually limited. Therefore, in order to adjust the protein content in the tobacco leaves better, the alcoholized tobacco leaves are often subjected to further degradation treatment, so that the quality of the tobacco leaves is further improved.
As for the cigarette type, flue-cured tobacco is the main cigarette type in China, so the existing means of protein degradation and protein control mainly research and design around flue-cured tobacco leaves. Statistics shows that the protein content in the cigar tobacco is higher than that of the common flue-cured tobacco, and the protein degradation means of the existing flue-cured tobacco cannot be directly used for the cigar tobacco due to the large difference of tobacco components, so that the quality of the cigar tobacco is slowly improved, and therefore, the feasibility of related technical means can be ensured by redesigning and verifying the quality control means of the cigar tobacco which cannot be directly used for reference of the treatment mode in the flue-cured tobacco.
In the prior art, when the quality of the cigar leaves is improved, the main technical means is still mainly natural alcoholization, and then the protein content in the cigar leaves is properly reduced by using protease treatment in part in view of the existing flue-cured tobacco treatment mode. But practical tests and application effects also show that: at the initial stage of applying the protease, the effect of reducing the protein content in the cigar tobacco leaves is obvious, but the effect of reducing the protein content is also reduced rapidly along with the prolonging of time and the reduction of enzyme activity, so that the effect of reducing the related protein content is not obvious enough, and the quality of the cigar tobacco leaves cannot be effectively improved. Therefore, it is necessary to develop a technical means for reducing the protein content of the cigar leaves aiming at the characteristics of the cigar leaves, thereby laying a foundation for the continuous improvement of the cigar quality.
Disclosure of Invention
The application mainly aims to provide a compound preparation prepared by using alkaline protease and specific protein degrading bacteria, so that a certain technical basis is laid for reducing the protein content in the cigar leaves, the aim of accelerating alcoholization of the cigar leaves is fulfilled, and the technical aims of improving the quality and reducing the consumption of the cigar leaves are fulfilled.
The technical solution adopted in the present application is detailed as follows.
A compound preparation of protease and protein degrading bacteria is prepared by the following steps:
(1) preparing a protein degrading bacterium solution, specifically:
preparation of OD from Bacillus pumilus (Bacillus pumilus) SMXP-03 strain600= 0.8-1.2 (preferably OD)600= 1.0) left and right bacterial liquid for standby; the specific preparation process can be referred to as follows:
firstly, taking a mixture of sterilized tobacco powder and sodium chloride as a culture medium for standby, wherein the specific material dosage is as follows when the culture medium is prepared: 4-6 g (preferably 5 g) of tobacco powder and 0.6-1.0 g (preferably 0.8 g) of sodium chloride are added into 100mL of water;
the tobacco powder is flue-cured tobacco powder, and can be crushed and sieved by a 200-mesh sieve for facilitating subsequent fermentation treatment;
secondly, inoculating Bacillus pumilus (Bacillus pumilus) SMXP-03 strain in a culture medium, culturing at 37 ℃ for 24 hours with shaking at 120 r/min, centrifugally collecting precipitate, re-suspending the precipitate with sterile water, and adjusting OD600About = 0.8-1.2, namely the protein degrading bacteriaBacterial liquid for later use;
the protein degrading bacteria Bacillus pumilus (Bacillus pumilus) SMXP-03 belongs to publicly available strains, is stored in China Center for Type Culture Collection (CCTCC) with the collection number of CCTCC NO: m2017291;
(2) preparing an alkaline protease solution for later use, specifically:
dissolving alkaline protease to prepare 3000-10000U/mL for later use;
(3) preparing a compound preparation, specifically:
before the use, the protein degradation bacterial liquid prepared in the step (1) and the alkaline protease solution in the step (2) are uniformly mixed, and the protein degradation bacterial liquid can be used for degrading proteins in the cigar tobacco leaves; when the protein degrading bacteria liquid is mixed specifically, the protein degrading bacteria liquid in the compound preparation has a volume ratio of 50-90%, specifically, 60%, 70% and 80%.
The protease and protein degrading bacteria compound preparation is applied to cigar leaves and is used for degrading proteins in the cigar leaves.
The degradation method for reducing the protein content in the cigar tobacco leaves by utilizing the compound preparation comprises the following steps:
(1) preparing a compound preparation for later use;
(2) cigar tobacco pretreatment
Carrying out moisture regain treatment on the cigar tobacco leaves, adjusting the moisture content of the cigar tobacco leaves to be 15-20%, and preferably balancing for 24-48 h after the moisture regain treatment;
(3) fermentation treatment
Uniformly spraying the compound preparation prepared in the step (1) onto the surface of the cigar tobacco leaves in the step (2) according to the mass proportion of 1-3% (preferably 2%) of the cigar tobacco leaves in the step (2), adjusting and controlling the moisture content of the tobacco leaves to be about 20%, and standing to balance the moisture of the tobacco leaves;
stacking and placing the tobacco leaves, controlling the stack temperature to be about 30-40 ℃ (preferably 37 ℃), the ambient humidity to be about 65-80% (preferably 75%), performing stacking fermentation for about 8-12 d (preferably 10 d), and turning and loosening the tobacco leaves every 1-2 d to ensure uniform fermentation;
after fermentation is finished, stacking to dissipate heat, ventilating to reduce humidity, and finally adjusting the moisture content of the cigar tobacco leaves to 15 +/-1%.
Generally speaking, when the biological enzyme is independently adopted to treat the tobacco leaves, the enzyme substrate content in the tobacco leaves can be obviously reduced at the initial stage of adopting the biological enzyme, but the action effect is continuously reduced along with the increase of the enzymolysis time; when the microbial degradation treatment is adopted, the early treatment effect is not obvious, but the microbial planting and biomass are obviously increased along with the time, so that the fermentation effect is more continuous. Aiming at the alkaline characteristic and the high-protein content characteristic of the cigar tobacco, the alkaline protease is compounded with the existing specific microbial strain culture solution, so that the aim of stably and continuously reducing the content of protein components in the cigar tobacco can be fulfilled.
In general, the present application is designed for the fermentation and alcoholization of cigar leaves, and realizes the degradation of nitrogenous substances, especially protein components, in cigar leaves by adopting a compound preparation of alkaline protease and specific protein degradation strains. Preliminary detection and application effects show that after treatment, the quality of the cigar tobacco leaves is better improved, the purposes of quality improvement and consumption reduction are better achieved, and meanwhile, the method has a better promotion effect on the production of cigars, so that the method has better practical value and popularization and application significance.
Detailed Description
The technical solution adopted in the present application is described in detail below with reference to examples. Before describing the specific embodiments, the following description will be briefly made with respect to the background of some experimental materials, experimental instruments, etc. involved in the following embodiments.
Experimental materials:
protein degrading strains used in the following examples: bacillus pumilus (Bacillus pumilus) SMXP-03 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2017291; the applicant is the depositor of the strain and therefore keeps a copy of the strain;
alcalase alkaline protease (2.4 AU/g), purchased from Novovicin China;
tobacco leaves: hainan cigar, the upper part of the core of the cigar is the second level;
the detection method and the detection standard of part of experiments are as follows:
the following examples are carried out to determine the content of water-soluble total sugar, total nitrogen and total plant alkaloid in tobacco leaves by adopting a tobacco industry standard method, and specifically comprise the following steps: YCT 166-2003, YC/T159-2002, YC/T161, and YC/T161-2002, respectively.
Example 1
The preparation method of the compound preparation of the protease and the protein degrading bacteria is summarized as follows.
(1) Preparing a protein degrading bacterium solution, specifically:
preparation of OD from Bacillus pumilus (Bacillus pumilus) SMXP-03 strain600Bacterial liquid about =1.0 for standby; the specific preparation process can be referred to as follows:
firstly, uniformly mixing flue-cured tobacco powder (crushed and sieved by a 200-mesh sieve) and sodium chloride, adding water (5 g of tobacco powder and 0.8g of sodium chloride are added in each 100mL of water), stirring and uniformly mixing, sterilizing at 121 ℃ for 15min, and cooling to room temperature to be used as a culture medium;
secondly, inoculating Bacillus pumilus (Bacillus pumilus) SMXP-03 strain in a culture medium, culturing at 37 ℃ for 24 hours with shaking at 120 r/min, centrifugally collecting precipitate, re-suspending the precipitate with sterile water, and adjusting OD600About =1.0, namely the protein degrading bacteria liquid for standby;
(2) preparing an alkaline protease solution for later use, specifically:
dissolving alkaline protease to prepare 5000U/mL for later use;
(3) preparing a compound preparation, specifically:
before the method is used specifically, the protein degradation bacterial liquid prepared in the step (1) and the alkaline protease solution in the step (2) are mixed uniformly, and the protein degradation bacterial liquid can be used for degrading proteins in the cigar tobacco leaves.
During specific mixing, in order to determine a better proportioning range, the inventor designs mixing and compounding modes with different proportions according to the volume ratio so as to determine the optimal proportion, and the specific proportioning combination is designed as shown in the following table:
example 2
Using the compounded preparations prepared in example 1 in different proportions, the inventors carried out experiments on cigar leaves to specifically reduce the protein content, and the specific process is briefly described as follows.
Firstly, carrying out moisture regain treatment on cigar tobacco leaves, adjusting the moisture content of the cigar tobacco leaves to be about 15%, and balancing for 24 hours after the moisture regain treatment;
secondly, according to the mass ratio of 2% of the cigar tobacco leaves, the compound preparations prepared in the embodiment 1 with different ratios are uniformly sprayed on the surfaces of the cigar tobacco leaves, the moisture content of the cigar tobacco leaves is adjusted and controlled to be about 20%, and the cigar tobacco leaves are kept stand to balance the moisture;
stacking and placing the tobacco leaves, controlling the stack temperature to be about 37 ℃ and the environment humidity to be about 75%, stacking and fermenting, and turning and loosening the tobacco leaves every 1d to ensure uniform fermentation;
after fermentation is finished, stacking and radiating, gout and dampness reduction are carried out, and finally the moisture content of the cigar tobacco leaves is adjusted to 15 +/-1%.
Cigar leaf samples without any treatment were used as blank control and are designated CK.
Data were measured for fermentations 2d and 10d, respectively.
The conventional chemical component measurement is carried out on the cigar tobacco leaves after the fermentation treatment in each example, and the results of the conventional chemical analysis after 2d and 10d fermentation are shown in tables 1 and 2.
TABLE 1 results of 2d conventional chemical analysis of formulated cigar treatments
Table 2, results of conventional chemical analysis of formulated cigar treatment in 10 days
Further, sensory evaluation was performed on the cigar tobacco samples treated with the formulation for 2d and 10d, respectively, and the results are shown in tables 3 and 4 below.
Table 3, Compound formulation treatment cigar 2d sensory evaluation results
Table 4, sensory evaluation results of formulated formulation treated cigars for 10d
Note: the evaluation criteria in tables 3 and 4 are:
quality of aroma: poor 0-12, poor 13-14, moderate 15-16, preferably 17-18, preferably 19-20;
the amount of aroma: 0-12 less, 13-14 less, 15-16 more foot, 17-18 more foot, 19-20 more foot;
miscellaneous gas: 10 is not, 9 is light, 7-8 is slightly, 5-6 is heavy, and 0-4 is heavy;
concentration: very small/big 0-4, very small/big 5-6, medium 7-8, preferably 9, preferably 10;
fineness is as follows: coarse 0-4%, coarse 5-6%, medium 7-8%, fine 9%, fine 10%;
irritation: no 10, slightly 9, 7-8, 5-6 and 0-4;
aftertaste: 0-12 parts of lingualis, 13-14 parts of lingualis slightly lingualis, 15-16 parts of lingualis comfortable, 17-18 parts of lingualis comfortable and 19-20 parts of lingualis comfortable.
Comprehensive comparative analysis of the data in the above table 1 and table 2 shows that the protein content does not change significantly in the early stage of fermentation and changes significantly only in the later stage when the microbial degradation bacteria liquid (data in example 1) is used alone; when alkaline protease degradation treatment is used alone (data in example 11), the effect of reducing the protein content is obvious in the early stage of use, but the effect is not obviously changed any more in the later stage, which indicates that the reduction effect is poor in persistence; when the alkaline protease and the protein degrading bacteria liquid are compounded for use, a certain synergistic effect is shown when the degrading bacteria liquid accounts for 50-90% of the volume, and the protein content reducing effect is better. In general, after the alkaline protease and the degrading bacteria are used together for treatment, the protein degrading effect of the cigar leaves is enhanced, the total sugar content is slightly increased, the sugar-nitrogen ratio and the nitrogen-protein ratio are also increased, which is probably related to that the bacterial strain simultaneously degrades certain polysaccharide, and the best degrading effect is achieved on the cigar proteins when the volume ratio of the bacterial liquid to the protease is 6: 4.
Comparing the results of the panel tests in tables 3 and 4, it can be seen that the overall score result is substantially consistent with the result of the content test of the material components, and after the protein degradation treatment, the indexes of the cigar tobacco, such as miscellaneous odor, fineness, irritation, aftertaste, etc., are obviously improved, the quality of the aroma is slightly improved, and a better improvement effect is shown.
Claims (9)
1. A compound preparation of protease and protein degrading bacteria is characterized in that the compound preparation is prepared by the following steps:
(1) preparing a protein degrading bacterium solution, specifically:
using Bacillus pumilus (B.), (B.), (B.pumilus, B.Bacillus pumilus ) Preparation of OD by SMXP-03 Strain600Bacterial liquid of = 0.8-1.2 for later use;
the bacillus pumilus SMXP-03 is currently preserved in China center for type culture collection with the preservation number of CCTCC NO: m2017291;
(2) preparing an alkaline protease solution for later use, specifically:
dissolving alkaline protease to prepare 3000-10000U/mL for later use;
(3) preparing a compound preparation, specifically:
before the use, the protein degradation bacterial liquid prepared in the step (1) and the alkaline protease solution in the step (2) are uniformly mixed, and the protein degradation bacterial liquid can be used for degrading proteins in the cigar tobacco leaves; when the protein degrading bacteria liquid is mixed specifically, the volume ratio of the protein degrading bacteria liquid in the compound preparation is 50-90% in terms of volume ratio.
2. The preparation of claim 1, wherein in the step (3), the volume ratio of the protease-degrading bacteria solution in the preparation is 60%, 70% or 80%.
3. The preparation method of the compound preparation of the protease and the protein degrading bacteria as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparing a protein degrading bacterium solution, specifically:
preparation of OD from Bacillus pumilus SMXP-03 strain600Bacterial liquid of = 0.8-1.2 for later use;
the bacillus pumilus SMXP-03 is currently preserved in China center for type culture collection with the preservation number of CCTCC NO: m2017291;
(2) preparing an alkaline protease solution for later use, specifically:
dissolving alkaline protease to prepare 3000-10000U/mL for later use;
(3) preparing a compound preparation, specifically:
before the use, the protein degradation bacterial liquid prepared in the step (1) and the alkaline protease solution in the step (2) are uniformly mixed, and the protein degradation bacterial liquid can be used for degrading proteins in the cigar tobacco leaves; when the protein degrading bacteria liquid is mixed specifically, the volume ratio of the protein degrading bacteria liquid in the compound preparation is 50-90% in terms of volume ratio.
4. The preparation method of the compound preparation of the protease and the protein degrading bacteria as claimed in claim 3, wherein in the step (1), the specific preparation process is as follows:
firstly, taking a mixture of sterilized tobacco powder and sodium chloride as a culture medium for standby, wherein the specific material dosage is as follows when the culture medium is prepared: adding 4-6 g of tobacco powder and 0.6-1.0 g of sodium chloride into every 100mL of water;
secondly, inoculating Bacillus pumilus SMXP-03 strain in the culture medium, shaking for culture, centrifuging to collect precipitate, re-suspending the precipitate with sterile water, and adjusting OD600And (5) obtaining the protein degrading bacteria liquid by using the = 0.8-1.2.
5. The preparation method of the compound preparation of protease and protein degrading bacteria as claimed in claim 4, wherein the tobacco powder is flue-cured tobacco powder; to 100mL of water, 5g of tobacco powder and 0.8g of sodium chloride were added.
6. The use of the protease and protein degrading bacteria combination preparation of claim 1 or 2 in cigar leaves for degrading proteins in cigar leaves.
7. The use of the protease and protein degrading bacteria complex formulation as claimed in claim 6 in cigar leaves, wherein the cigar leaves are second-grade Hainan cigars on top of cigar cores.
8. A degradation method for reducing the protein content in the cigar tobacco leaves by utilizing the compound preparation of claim 1 or 2 is characterized by comprising the following steps:
(1) preparing a compound preparation for later use;
(2) cigar tobacco pretreatment
Carrying out moisture regain treatment on the cigar tobacco leaves, and adjusting the water content of the cigar tobacco leaves to 15-20%;
(3) fermentation treatment
Uniformly spraying the compound preparation prepared in the step (1) on the surface of the cigar tobacco leaves in the step (2) according to the mass ratio of 1-3% of the cigar tobacco leaves in the step (2);
then stacking and placing the tobacco leaves, controlling the stack temperature to be 30-40 ℃ and the environment humidity to be 65-80%, and performing stacking fermentation for 8-12 d; and after the fermentation is finished, adjusting the water content of the cigar tobacco leaves to be 15 +/-1%.
9. The degradation method for reducing the protein content in cigar leaves according to claim 8, wherein in the step (3), the compounded preparation is sprayed at a ratio of 2% by mass of the cigar leaves.
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| CN110693075B (en) * | 2019-10-12 | 2021-10-08 | 中国科学院青岛生物能源与过程研究所 | Four-stage fermentation method of cigar core tobacco leaves |
| CN110786542B (en) * | 2019-11-20 | 2021-07-23 | 云南省烟草农业科学研究院 | Method for reducing TSNAs of cigar tobacco leaves by using strains |
| CN113892666B (en) * | 2021-10-26 | 2022-07-26 | 四川中烟工业有限责任公司 | Enzymolysis quality-improving treatment method for cigar core tobacco leaves |
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