CN115851545A - Korean bacillus and culture medium for improving activity of enzyme produced by same - Google Patents
Korean bacillus and culture medium for improving activity of enzyme produced by same Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The application relates to the technical field of microorganisms, in particular to a bacterial culture medium and application thereof, and especially relates to application of the bacterial culture medium in improving activity of enzymes produced by bacteria, or delaying the speed of activity reduction of the enzymes, or prolonging the time of activity reduction of the enzymes. The application also relates to a method which can increase the activity of the enzyme produced by the bacteria, or delay the speed of the decrease of the activity of the enzyme, or prolong the time of the decrease of the activity of the enzyme.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a bacterial culture medium and application thereof, and especially relates to application of the bacterial culture medium in improving activity of enzymes produced by bacteria, or delaying the speed of activity reduction of the enzymes, or prolonging the time of activity reduction of the enzymes. The application also relates to a method which can increase the activity of the enzyme produced by the bacteria, or delay the speed of the decrease of the activity of the enzyme, or prolong the time of the decrease of the activity of the enzyme.
Background
Korean Bacillus is a rod-shaped bacterium with yellow and endospore bacterial colony, is separated from willow root tissue by a Korean scholars Lim Jee-Min and the like for the first time in 2006 and is identified as a new species in the Bacillus, and is named as Korean Bacillus (Bacillus koreensis).
With the development of genomics, recent researchers in Canada reclassified the genus Bacillus based on CSIs after a thorough comparison of 300 Bacillus genomics systems. Accordingly, the genetic evolution branch of korean bacillus is independent of bacillus, and currently, in taxonomy, korean bacillus is formally named as korean listeria (priesta koreensis) and belongs to the genus Priestia (priesta sp.), and new classification methods have been adopted by authorities such as NCBI and DSMZ.
The Korean bacillus has abundant secondary metabolites and great development and application potential. However, there are currently few studies on the development and application of korean bacillus. In the domestic patent application, only the institute of ecological environmental science of Hubei province (patent No. CN 202111068096.1) discloses the preparation of an immobilized microbial agent using Korean Bacillus LQB126 and its use for the treatment of heavy metal pollution.
Therefore, the skilled person in the art needs to perform related research on the korean bacillus to provide technical support for the subsequent application of the korean bacillus.
Disclosure of Invention
In order to solve the above problems, in a first aspect, the present application provides a korean Bacillus (Bacillus koreensis) deposited in china general microbiological culture collection center under the collection number of CGMCC No.24131 or CGMCC No.24132.
In a second aspect, the present application provides a bacterial culture medium comprising: zymolyte of Korean Bacillus, and tobacco powder.
In some embodiments, the zymolyte of Korean Bacillus is obtained by enzymatically degrading the cells of Korean Bacillus.
In certain embodiments, the enzyme is selected from lysozyme, a protease (e.g., proteinase K), or any combination thereof.
In some embodiments, the zymolyte of the korean bacillus is obtained by degrading a bacterial solution of the korean bacillus with lysozyme and proteinase K.
In certain embodiments, the Korean bacillus is selected from Korean bacillus YL-2 with preservation number CGMCC No.24131, korean bacillus YLA-2 with preservation number CGMCC No.24132, or any combination thereof, as described in the first aspect.
In certain embodiments, the tobacco powder is tobacco leaf powder.
In certain embodiments, the tobacco powder is tobacco stem powder.
In certain embodiments, the culture medium further comprises one or more selected from the group consisting of: nutrient supplying substances, inorganic salt, growth factors and water.
In certain embodiments, the nutrient providing substance is selected from peptone, beef extract, sugars, starches (e.g., soluble starches), or any combination thereof.
In certain embodiments, the inorganic salt is selected from sodium chloride, anhydrous calcium chloride, or any combination thereof.
In certain embodiments, the culture medium comprises: zymolyte of Korean bacillus, tobacco stalk powder, peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch and water.
In certain embodiments, the culture medium is a liquid medium, a solid medium, or a semi-solid medium.
In certain embodiments, the medium is a liquid medium and comprises 0.1-10g/L of zymolyte of Korean Bacillus, 0.1-20g/L of tobacco stalk powder, 1-50g/L of peptone, 1-30g/L of beef extract, 1-50g/L of sodium chloride, 0.1-20g/L of anhydrous calcium chloride, 1-30g/L of soluble starch, and water.
In certain embodiments, the medium comprises 0.5-1g/L of zymolyte of Korean Bacillus, 0.5-1g/L of tobacco stalk powder, 1-20g/L of peptone, 1-10g/L of beef extract, 1-15g/L of sodium chloride, 0.1-5g/L of anhydrous calcium chloride, 1-10g/L of soluble starch, and water.
In certain embodiments, the medium comprises 0.5g/L of zymolyte of Korean Bacillus, 1g/L of tobacco stalk powder, 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch, and water; or,
the culture medium comprises 1g/L of zymolyte of Korean bacillus, 0.5g/L of tobacco stalk powder, 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and water.
In some embodiments, the zymolyte of the Korean bacillus is prepared by adding lysozyme and proteinase K to a bacterial solution of the Korean bacillus.
In some embodiments, the zymolyte of the korean bacillus is prepared by the following method: adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K to the bacterial liquid of Korean bacillus and performing enzymolysis in water bath to obtain an enzymolysis liquid; optionally, the enzymatic hydrolysate is inactivated and freeze dried.
In some embodiments, the zymolyte of the korean bacillus is prepared by the following method: culturing Korean Bacillus, collecting thallus in the bacterial liquid, resuspending with water to solid content of 4-15% (e.g., 4%,6%,8%,10%,12%, 14%), adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K and performing enzymolysis in water bath to obtain an enzymolysis liquid, inactivating the enzymolysis liquid and freeze-drying to water content of 2% -5% (e.g., 2%,3%,3.2%,4%, 5%).
In certain embodiments, the stem powder is obtained by sequentially soaking tobacco stems with sodium hydroxide and hydrochloric acid, drying, and making into powder.
In certain embodiments, the tobacco stem powder is prepared by the following method: 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) sodium hydroxide and 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) hydrochloric acid sequentially soak the stems of tobacco, dry to a moisture content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and then make into powder.
In certain embodiments, the cabo powder is prepared by the following method: soaking tobacco stem with 1% sodium hydroxide, washing with water, soaking tobacco stem with 1% hydrochloric acid, washing with water, and adjusting pH to 6.5-7.5; drying to water content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder.
In a third aspect, the present application provides use of the korean bacillus strain of the first aspect or the culture medium of the second aspect for culturing or fermenting bacteria.
In certain embodiments, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, listeria, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof.
In certain embodiments, the bacterium is bacillus korea.
In certain embodiments, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof.
In a fourth aspect, the present application provides the use of the korean bacillus of the first aspect or the culture medium of the second aspect for increasing the activity of an enzyme produced by a bacterium, or for delaying the rate of decrease in the activity of the enzyme, or for prolonging the time period over which the activity of the enzyme is decreased.
In certain embodiments, the medium is used to increase the viability of an enzyme in a broth or fermentation of a bacterium, or to retard the rate of, or prolong the time over which, the viability of an enzyme decreases.
In certain embodiments, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, listeria, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof.
In certain embodiments, the bacterium is bacillus korea.
In certain embodiments, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof.
In certain embodiments, the enzyme is selected from amylase, cellulase, pectinase, or any combination thereof.
In certain embodiments, the enzymes are amylases, cellulases and pectinases.
In a fifth aspect, the present application provides a method of preparing the medium of the second aspect, the method comprising: zymolyte of Korean Bacillus, tobacco stalk powder, nutrient providing material, inorganic salt, and water are mixed.
In some embodiments, zymolyte of korean bacillus, tobacco stem powder, peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch, and water are mixed.
In some embodiments, the zymolyte of the Korean bacillus is prepared by adding lysozyme and proteinase K to a bacterial solution of the Korean bacillus.
In some embodiments, the zymolyte of the korean bacillus is prepared by the following method: adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K to a bacterial solution of Korean Bacillus and performing enzymolysis in a water bath to obtain an enzymolysis solution; optionally, the enzymatic hydrolysate is inactivated and freeze dried.
In some embodiments, the zymolyte of the korean bacillus is prepared by the following method: culturing Korean Bacillus, collecting thallus in the strain solution, resuspending with water to solid content of 4-15% (e.g., 4%,6%,8%,10%,12%, 14%), adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K and performing enzymolysis in water bath to obtain an enzymolysis solution, inactivating the enzymolysis solution and freeze-drying to water content of 2% -5% (e.g., 2%,3%,3.2%,4%, 5%).
In certain embodiments, the stem powder is obtained by sequentially soaking tobacco stems with sodium hydroxide and hydrochloric acid, drying, and making into powder.
In certain embodiments, the tobacco stem powder is prepared by the following method: 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) sodium hydroxide and 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) hydrochloric acid sequentially soak the stems of tobacco, dry to a moisture content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and then make into powder.
In certain embodiments, the cabo powder is prepared by the following method: soaking tobacco stem with 1% sodium hydroxide, washing with water, soaking tobacco stem with 1% hydrochloric acid, washing with water, and adjusting pH to 6.5-7.5; drying to water content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder.
In a sixth aspect, the present application provides a method of culturing or fermenting bacteria, the method comprising: culturing or fermenting the bacteria using the medium of the second aspect under conditions suitable for growth or fermentation of the bacteria.
In certain embodiments, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof.
In certain embodiments, the bacterium is bacillus korea.
In certain embodiments, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof.
In certain embodiments, the conditions suitable for bacterial growth or fermentation are 28-38 ℃ and 120-220rpm.
In a seventh aspect, the present application provides a method of increasing the viability of an enzyme produced by a bacterium, or delaying the rate of, or extending the time over which the viability of the enzyme is reduced, the method comprising: culturing or fermenting the bacteria using the medium of the second aspect under conditions suitable for growth or fermentation of the bacteria.
In certain embodiments, the medium is used to increase the viability of an enzyme in a broth or fermentation of a bacterium, or to retard the rate of, or prolong the time over which, the viability of an enzyme decreases.
In certain embodiments, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof.
In certain embodiments, the bacterium is bacillus korea.
In certain embodiments, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof.
In certain embodiments, the enzyme is selected from amylase, cellulase, pectinase, or any combination thereof.
In certain embodiments, the enzymes are amylases, cellulases, and pectinases.
In certain embodiments, the conditions suitable for bacterial growth or fermentation are 28-38 ℃ (e.g., 28 ℃,30 ℃,32 ℃,34 ℃,36 ℃,38 ℃), 120-220rpm (e.g., 120rpm,140rpm,160rpm,180rpm,200rpm, 220rpm).
Definition of terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of biochemistry, microbiology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
As used herein, the term "korean Bacillus (Bacillus koreensis)" refers to a type of rod-shaped bacteria whose colonies are yellow, endospores. With the development of genomics, recent researchers in Canada reclassified the genus Bacillus based on CSIs after a thorough comparison of 300 Bacillus genomics systems. Accordingly, korean bacillus is independent of bacillus in genetic evolutionary branches and formally renamed to korean listeria (priesta koreensis) and also belongs to the genus Priestia (priesta sp.). The new classification method has been adopted by the authorities such as NCBI and DSMZ, but some published documents still describe the formerly known Korean Bacillus of "Korean Prisella". Therefore, in the text, both Korean Bacillus (Bacillus koreensis) and Korean Prisella (Priesia koreensis) represent the same species of bacteria and may be used interchangeably.
As used herein, the term "enzyme activity" refers to the activity of an enzyme, which may also be referred to as "enzyme activity" or "enzyme activity", all of which represent the same meaning and may be interchanged. Generally, the higher the enzyme activity, the greater the ability of the corresponding enzyme to catalyze a chemical reaction. Methods for determining the enzyme activity are known to those skilled in the art and can be determined by physical methods (e.g., optical rotation, refraction, pycnometry) or chemical methods (e.g., fiyline's method, potassium permanganate method, DNS colorimetry).
In certain embodiments, the amount of enzyme required to hydrolyze 1 micromole of substrate in 1 minute is defined as 1 enzyme activity unit U. In certain embodiments, the amount of enzyme required to hydrolyze 1.5% soluble starch, 1% sodium carboxymethylcellulose and 1% pectin to produce 1 μ g of glucose or galacturonic acid in 1 minute is defined as 1 enzyme activity unit U.
As used herein, the term "zymolyte of korean bacillus" refers to a product obtained after enzymatic degradation of korean bacillus. In some embodiments, the zymolyte of korean bacillus is a product obtained by enzyme-treating a bacterial solution of korean bacillus.
As used herein, the term "tobacco (Nicotiana tabacum l.)" is a plant of the genus Nicotiana of the family solanaceae, which is the raw material of industrial product tobacco. Tobacco is widely cultivated in various provinces in south and north China, is a temperature-loving crop, is sensitive to temperature response, and has large influence on the quality and yield of tobacco under different temperature conditions.
As used herein, the term "tobacco leaf" refers to the lamina of a plant tobacco. As used herein, the term "tobacco stem" refers to the coarse, hard veins on tobacco leaves.
As used herein, the term "fermentation" refers to a process of preparing a microbial cell itself, or a direct metabolite or a secondary metabolite, by virtue of the vital activity of the microorganism under aerobic or anaerobic conditions. As used herein, the term "fermentation broth" refers to a culture broth in which a microorganism is cultured during fermentation.
Advantageous effects of the invention
Compared with the prior art, the culture medium and the method can remarkably improve the activity of enzymes (such as amylase, cellulase and pectinase) in the fermentation liquor of the Korean bacillus, particularly can remarkably improve the highest enzyme activity of the amylase, the cellulase and the pectinase in the fermentation liquor of the Korean bacillus, can keep the enzyme activity for a longer time, and can delay the reduction speed of the enzyme activity. Therefore, the culture medium and the method of the present application enable the Korean bacillus to produce enzymes at a high yield and improve the enzyme activity of the enzymes, and have a wide prospect in subsequent applications (e.g., fermentation, degradation of substances using enzymes) of the Korean bacillus.
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.
Description of biological Material preservation
Korean Bacillus YL-2 (Bacillus koreensis YL-2) has been deposited at China General Microbiological Culture Collection Center (CGMCC) at institute No. 3 of Xilu No. 1, north Cheng, the south facing area, beijing, with a Collection number of CGMCC No.24131, and a preservation time of 2021 year, 12 months and 20 days.
Korean Bacillus YLA-2 (Bacillus koreensis YLA-2) is preserved in China General Microbiological Culture Collection Center (CGMCC) of No. 3 of Xilu No. 1 of the North Chen, the south facing the Yangtze area, beijing, and has a preservation number of CGMCC No.24132 and a preservation time of 2021 year, 12 months and 20 days.
Detailed Description
The invention will now be described with reference to the following examples which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques such as biochemistry and microbiology used IN the present invention can be found IN the METHODS of ENZYMOLOGY (METHODS IN Enzymology) series (academic Press).
In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1.
The method comprises the steps of taking Korean bacillus YL-2 (CGMCC No. 24131) after passage rejuvenation, plating in a basic fermentation medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and the balance of water, wherein the pH value is 7.0), and bottling the amount of 100/250mL. The culture was carried out at 37 ℃ and 180rpm for 40h.
Centrifuging the cultured bacteria liquid at 10000rpm, pouring out supernatant to obtain wet bacteria, adding pure water to the wet bacteria for heavy suspension, adjusting the solid content of the heavy suspension to be about 5%, adding lysozyme according to 30mg/L, adding proteinase K according to 100mg/L, performing enzymolysis in water bath at 40 ℃ for 6h, then inactivating at 121 ℃ for 20min, pre-freezing the inactivated enzymolysis liquid at-80 ℃, and performing freeze drying to obtain YL-2 bacteria zymolyte (with the water content of 3.2%) for later use.
Soaking 500g of tobacco stems (Fujian C-2021) in 1% sodium hydroxide for 18h, cleaning with tap water for 5 times, adding 1% hydrochloric acid, soaking for 2h, cleaning with tap water to pH6.5, oven drying the pretreated tobacco stems in an oven at 80 deg.C for 36h (with water content of 5.4%), pulverizing with an ultra-micro pulverizer, and sieving with a 100-mesh screen for use.
Taking prepared YL-2 thallus zymolyte and tobacco stalk powder, and preparing according to the following formula: YL-2 thallus zymolyte 0.5g/L, cabo powder 1g/L, peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, anhydrous calcium chloride 0.5g/L, soluble starch 2g/L, the balance water, and pH7.0. The prepared fermentation medium is subpackaged into 3 bottles according to the bottling amount of 100/250mL, and sterilized for 20 minutes at 121 ℃ for later use.
Taking Korean Bacillus YL-2 (CGMCC No. 24131) after passage rejuvenation, picking 3 colonies on a plate, inoculating to a fermentation medium, and fermenting at 37 ℃ and 180rpm for 48h. After 5mL of fermentation broth is respectively taken at 24, 36 and 48 hours and centrifuged at 10000rpm at 4 ℃ to remove thalli, the enzyme activity of amylase, cellulase and pectinase is respectively measured by using a 3, 5-dinitrosalicylic acid (DNS) method and taking 1.5 percent of soluble starch (pH 5.0), 1 percent of sodium carboxymethylcellulose (pH 5.0) and 1 percent of pectin (pH 9.4) as reaction substrates under the condition of 50 ℃, and the enzyme amount required for hydrolyzing the substrates within 1 minute to generate 1 mu g of glucose or galacturonic acid is defined as 1 enzyme activity unit U.
Comparative example 1.
Taking Korean Bacillus YL-2 (CGMCC No. 24131) after passage rejuvenation, selecting 3 colonies on a plate, inoculating the colonies into a basic fermentation culture medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and the balance of water, and fermenting at 37 ℃ and 180rpm for 48 hours. After centrifugation at 10000rpm at 4 ℃ for 24, 36, and 48 hours to remove cells, 5mL of fermentation broth was used to determine the enzyme activities of amylase, cellulase, and pectinase using DNS (see example 1 for details).
Example 2.
The method comprises the steps of taking Korean bacillus YAL-2 (CGMCC No. 24132) after passage rejuvenation, plating the Korean bacillus YAL-2 in a basic fermentation medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and the balance of water, wherein the pH value is 7.0), bottling the Korean bacillus YAL-2 in 100/250mL, and culturing the Korean bacillus YAL-2 at 37 ℃ and 180rpm for 60 hours.
Centrifuging the cultured bacteria liquid at 10000rpm, pouring out supernatant to obtain wet bacteria, adding pure water to the wet bacteria, re-suspending and adjusting the solid content of the re-suspended bacteria liquid to be about 8%, adding lysozyme according to 80mg/L, adding proteinase K according to 80mg/L, carrying out water bath enzymolysis at 40 ℃ for 4h, then inactivating at 121 ℃ for 20min, pre-freezing the inactivated enzymolysis liquid at-80 ℃, and carrying out freeze drying to obtain YLA-2 bacteria zymolyte (with the water content of 3.5%) for later use.
Soaking 500g of tobacco stems (Fujian C-2021) in 1% sodium hydroxide for 24h, cleaning with tap water for 5 times, adding 1% hydrochloric acid, soaking for 5h, cleaning with tap water to pH7.0, oven drying the pretreated tobacco stems in an oven at 80 deg.C for 48h (water content of 4.8%), pulverizing with an ultra-micro pulverizer, and sieving with an 80-mesh sieve.
Taking prepared YLA-2 thallus zymolyte and tobacco stalk powder, and preparing according to the following formula: 1g/L YLA-2 thallus zymolyte, 0.5g/L cabo powder, 10g/L peptone, 3g/L beef extract, 5g/L sodium chloride, 0.5g/L anhydrous calcium chloride, 2g/L soluble starch, the balance of water and pH7.0. The prepared fermentation medium is subpackaged into 3 bottles according to the bottling amount of 100/250mL, and sterilized for 20 minutes at 121 ℃ for later use.
The Korean bacillus YLA-2 (CGMCC No. 24132) after passage rejuvenation is taken, 3 colonies are picked from a plate and inoculated into a fermentation medium, and the fermentation is carried out for 48h at 37 ℃ and 200 rpm. After centrifugation at 10000rpm at 4 ℃ for 24, 36, and 48 hours to remove cells, 5mL of fermentation broth was used to determine the enzyme activities of amylase, cellulase, and pectinase using DNS (see example 1 for details).
Comparative example 2.
Taking Korean bacillus YLA-2 (CGMCC No. 24132) after passage rejuvenation, selecting 3 colonies on a plate, inoculating the colonies into a basic fermentation culture medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and the balance of water, wherein the pH value is 7.0), and fermenting at 37 ℃ and 200rpm for 48 hours. After centrifugation at 10000rpm at 4 ℃ for 24, 36, and 48 hours to remove cells, 5mL of fermentation broth was used to determine the enzyme activities of amylase, cellulase, and pectinase using DNS (see example 1 for details).
The experimental results of example 1, comparative example 1, example 2 and comparative example 2 are shown in table 1. For two Korean bacillus YL-2 and YLA-2 strains, after the fermentation medium is used, the highest enzyme activity of amylase is respectively improved by 58.9 percent and 24.1 percent, the highest enzyme activity of cellulase is respectively improved by 52.1 percent and 59.1 percent, and the highest enzyme activity of pectinase is respectively improved by 9.8 percent and 12.6 percent. In addition, examples 1 and 2 using the fermentation medium of the present application continued to increase enzyme activity at 48h (compared to 36 h) or still maintained a higher level of enzyme activity (compared to the highest enzyme activity per se), while comparative examples 1 and 2, which did not use the fermentation medium of the present application, had significantly decreased enzyme activity at 48h (compared to the highest enzyme activity per se).
Therefore, the fermentation medium of the present application can significantly improve the enzymatic activities of amylase, cellulase and pectinase of a Korean bacillus fermentation broth, maintain the enzymatic activities for a longer time, and delay the speed and time of the decrease of the enzymatic activities.
TABLE 1 results of enzyme activity
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Claims (11)
1. A Korean Bacillus (Bacillus koreensis) is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.24131 or CGMCC No.24132.
2. A bacterial culture medium comprising: zymolyte of korean bacillus, and tobacco powder;
preferably, the zymolyte of the korean bacillus is obtained by enzymatically degrading the thallus of the korean bacillus;
preferably, the enzyme is selected from lysozyme, a protease (e.g., proteinase K), or any combination thereof;
preferably, the zymolyte of the Korean bacillus is obtained by degrading a bacterial solution of the Korean bacillus by lysozyme and proteinase K;
preferably, the Korean bacillus is selected from Korean bacillus YL-2 with preservation number CGMCC No.24131, korean bacillus YLA-2 with preservation number CGMCC No.24132, or any combination thereof as claimed in claim 1;
preferably, the tobacco powder is tobacco leaf powder;
preferably, the tobacco powder is tobacco stem powder.
3. The culture medium of claim 2, wherein the culture medium further comprises one or more selected from the group consisting of: nutrient supplying substances, inorganic salt, growth factors and water;
preferably, the nutrient providing substance is selected from peptone, beef extract, sugars, starch (e.g., soluble starch), or any combination thereof;
preferably, the inorganic salt is selected from sodium chloride, anhydrous calcium chloride, or any combination thereof;
preferably, the culture medium comprises: zymolyte of Korean bacillus, tobacco stalk powder, peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch and water.
4. The culture medium of claim 3, wherein the culture medium is a liquid medium, a solid medium, or a semi-solid medium;
preferably, the culture medium is a liquid culture medium and comprises 0.1-10g/L of zymolyte of Korean bacillus, 0.1-20g/L of tobacco stem powder, 1-50g/L of peptone, 1-30g/L of beef extract, 1-50g/L of sodium chloride, 0.1-20g/L of anhydrous calcium chloride, 1-30g/L of soluble starch and water;
preferably, the culture medium comprises 0.5-1g/L of zymolyte of Korean bacillus, 0.5-1g/L of tobacco stalk powder, 1-20g/L of peptone, 1-10g/L of beef extract, 1-15g/L of sodium chloride, 0.1-5g/L of anhydrous calcium chloride, 1-10g/L of soluble starch and water;
preferably, the medium comprises 0.5g/L of zymolyte of Korean bacillus, 1g/L of tobacco stem powder, 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and water; or,
the culture medium comprises 1g/L of zymolyte of Korean bacillus, 0.5g/L of tobacco stalk powder, 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 0.5g/L of anhydrous calcium chloride, 2g/L of soluble starch and water.
5. The culture medium according to any one of claims 2 to 4, wherein the zymolyte of Korean Bacillus is prepared by adding lysozyme and proteinase K to a bacterial solution of Korean Bacillus;
preferably, the zymolyte of the korean bacillus is prepared by the following method: adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K to the bacterial liquid of Korean bacillus and performing enzymolysis in water bath to obtain an enzymolysis liquid; optionally, inactivating the enzymatic hydrolysate and freeze-drying;
preferably, the zymolyte of the korean bacillus is prepared by the following method: culturing Korean Bacillus, collecting thallus in the bacterial liquid, resuspending with water to solid content of 4-15% (e.g., 4%,6%,8%,10%,12%, 14%), adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K and performing enzymolysis in water bath to obtain an enzymolysis liquid, inactivating the enzymolysis liquid and freeze-drying to water content of 2% -5% (e.g., 2%,3%,3.2%,4%, 5%).
6. The culture medium according to any one of claims 2 to 5, wherein the stem powder is obtained by sequentially soaking tobacco stems with sodium hydroxide and hydrochloric acid, drying, and preparing into powder;
preferably, the tobacco stem powder is prepared by the following method: soaking tobacco stems sequentially with 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) sodium hydroxide and 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) hydrochloric acid, drying to water content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder;
preferably, the tobacco stem powder is prepared by the following method: soaking tobacco stem with 1% sodium hydroxide, washing with water, soaking tobacco stem with 1% hydrochloric acid, washing with water, and adjusting pH to 6.5-7.5; drying to water content of 3% -7% (e.g. 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder.
7. Use of the Korean Bacillus of claim 1 or the culture medium of any one of claims 2-6 for culturing or fermenting bacteria;
preferably, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof;
preferably, the bacterium is bacillus korea;
preferably, the Korean bacillus is selected from Korean bacillus YL-2 with a preservation number of CGMCC No.24131, korean bacillus YLA-2 with a preservation number of CGMCC No.24132, or any combination thereof.
8. Use of the korean bacillus of claim 1 or the culture medium of any one of claims 2 to 6 for increasing the viability of an enzyme produced by a bacterium, or for delaying the rate of decrease in the viability of the enzyme, or for prolonging the time of decrease in the viability of the enzyme;
preferably, the culture medium is used for improving the activity of the enzyme in a culture solution or fermentation solution of the bacteria, or is used for delaying the speed of the activity reduction of the enzyme, or is used for prolonging the time of the activity reduction of the enzyme;
preferably, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof;
preferably, the bacterium is bacillus korea;
preferably, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof;
preferably, the enzyme is selected from amylase, cellulase, pectinase, or any combination thereof;
preferably, the enzymes are amylases, cellulases and pectinases.
9. The method for preparing the korean bacillus strain of claim 1 or the culture medium of any one of claims 2 to 6, the method comprising: mixing zymolyte of Korean Bacillus, tobacco stalk powder, nutrient providing substance, inorganic salt, and water;
preferably, zymolyte of Korean bacillus, tobacco stem powder, peptone, beef extract, sodium chloride, anhydrous calcium chloride, soluble starch, and water are mixed;
preferably, the zymolyte of the Korean bacillus is prepared by adding lysozyme and proteinase K into a bacterial liquid of the Korean bacillus;
preferably, the zymolyte of the korean bacillus is prepared by the following method: adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K to a bacterial solution of Korean Bacillus and performing enzymolysis in a water bath to obtain an enzymolysis solution; optionally, inactivating the enzymatic hydrolysate and freeze-drying;
preferably, the zymolyte of the korean bacillus is prepared by the following method: culturing Korean Bacillus, collecting thallus in the bacterial liquid, resuspending with water to solid content of 4-15% (e.g., 4%,6%,8%,10%,12%, 14%), adding 10-100mg/L (e.g., 30-80 mg/L) lysozyme and 50-150mg/L (e.g., 80-100 mg/L) proteinase K, performing enzymolysis in water bath to obtain an enzymolysis liquid, inactivating the enzymolysis liquid, and freeze-drying to water content of 2% -5% (e.g., 2%,3%,3.2%,4%, 5%);
preferably, the tobacco stem powder is obtained by sequentially soaking tobacco stems in sodium hydroxide and hydrochloric acid, drying and preparing into powder;
preferably, the tobacco stem powder is prepared by the following method: sequentially soaking tobacco stems with 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) sodium hydroxide and 0.1-5% (e.g., 0.5%,1%,2%,3%,4%, 5%) hydrochloric acid, drying to water content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder;
preferably, the tobacco stem powder is prepared by the following method: soaking tobacco stem with 1% sodium hydroxide, washing with water, soaking tobacco stem with 1% hydrochloric acid, washing with water, and adjusting pH to 6.5-7.5; drying to water content of 3% -7% (e.g., 3%,4%,4.8%,5%,5.4%,6%, 7%), and making into powder.
10. A method of culturing or fermenting a bacterium, the method comprising: culturing or fermenting bacteria using the medium of any one of claims 2-5 under conditions suitable for growth or fermentation of the bacteria;
preferably, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof;
preferably, the bacterium is bacillus korea;
preferably, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof;
preferably, the conditions suitable for bacterial growth or fermentation are 28-38 ℃ and 120-220rpm.
11. A method of increasing the viability of an enzyme produced by a bacterium, or delaying the rate of, or extending the time over which the viability of the enzyme is reduced, the method comprising: culturing or fermenting the bacteria using the medium of any one of claims 2-6 under conditions suitable for growth or fermentation of the bacteria;
preferably, the culture medium is used for improving the activity of the enzyme in a culture solution or fermentation solution of the bacteria, or is used for delaying the speed of the activity reduction of the enzyme, or is used for prolonging the time of the activity reduction of the enzyme;
preferably, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, pruriester, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof;
preferably, the bacterium is bacillus korea;
preferably, the Korean bacillus is selected from Korean bacillus YL-2 having a preservation number of CGMCC No.24131, korean bacillus YLA-2 having a preservation number of CGMCC No.24132, or any combination thereof;
preferably, the enzyme is selected from amylase, cellulase, pectinase, or any combination thereof;
preferably, the enzymes are amylases, cellulases and pectinases;
preferably, the conditions suitable for bacterial growth or fermentation are from 28 to 38 deg.C (e.g., 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, 36 deg.C, 38 deg.C), 120 to 220rpm (e.g., 120rpm,140rpm,160rpm,180rpm,200rpm, 220rpm).
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