CN114644984A - Method for separating microorganism producing amylase from soil - Google Patents
Method for separating microorganism producing amylase from soil Download PDFInfo
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- 235000019418 amylase Nutrition 0.000 title claims abstract description 51
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000002689 soil Substances 0.000 title claims abstract description 31
- 244000005700 microbiome Species 0.000 title claims abstract description 29
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- 230000000694 effects Effects 0.000 claims abstract description 23
- 241000187747 Streptomyces Species 0.000 claims abstract description 16
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 12
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- 238000004737 colorimetric analysis Methods 0.000 claims abstract description 7
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- 238000012360 testing method Methods 0.000 claims description 13
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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
- C12N1/02—Separating microorganisms from their culture media
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- 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
- C12N1/14—Fungi; Culture media therefor
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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
- 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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Abstract
The invention belongs to the field of microorganisms, and relates to a method for screening microorganisms producing amylase from soil, and strain identification is carried out. Hydrolysis rings are generated by an iodine staining method for preliminary screening, and the enzyme activity is measured by a3, 5-dinitrosalicylic acid (DNS) colorimetric method, so that the strain with superior enzyme production capability is provided for industrial production. The results show that: the 31 selected amylase-producing strains belong to 10 genera, including 9 strains of Bacillus (Bacillus), 1 strain of Paenibacillus (Paenibacillus), 4 strains of Streptomyces (Streptomyces), 2 strains of Enterobacter (Enterobacter), 4 strains of Aspergillus (Aspergillus), 2 strains of Paecilomyces (Simplicillium), 7 strains of Penicillium (Penicillium), 1 strain of Streptomyces (Alternaria), and 1 strain of Cladosporium (Cladosporium). Wherein, the amylase activity of the streptomycete, the aspergillus flavus and the aspergillus niger is higher.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a method for separating microorganisms producing amylase from soil.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Starch is a polysaccharide naturally stored by higher plants and is a homopolymer formed by the polymerization of glucose molecules. It has good biodegradability and renewability. Starch products not only remain an indispensable basic food source for humans today, but also provide a large number of important raw materials for modern industrial and production activities. The starch is iso-saccharified by amylase to produce glucose, maltodextrine, maltose, and the like.
Amylase is one of the main enzyme preparations which are firstly put into development to realize industrial production and application in China as a biocatalyst. Amylases can be classified into two major classes, alpha-amylase and beta-amylase, according to their hydrolysis modes. Alpha-amylase, a commonly used amylase in life, is a calcium metalloenzyme and does not function in the absence of calcium. It can catalyze the alpha-1, 4-glycosidic bond fracture in starch and its derivatives, and can also span alpha-1, 6-glycosidic bond. It has effect in promoting digestion of animals. Beta-amylases are a class of acting enzymes that can hydrolyze starch to maltose. Acts only on the non-reducing end, and catalyzes the alpha-1, 4-glycosidic bond mainly by taking maltose as a unit for each cleavage. It is common in sweet potato and absent in animal tissues. The production and preparation method of the amylase comprises microbial fermentation and animal and plant extraction. The microbial fermentation method for producing amylase has the advantages of high stability, high productivity, low production cost and the like, so the microbial fermentation method is preferred in industrial application. The amylase has wide application, relates to industries such as food industry, textile industry, paper making industry, medical health, feed industry and the like, plays an important role in aspects of environmental protection, chemical reagent production and the like, has higher economic value and social benefit, and the yield of the amylase accounts for more than 85 percent of the total yield of industrial enzymes. With the recent improvement of knowledge of the enzyme production industry and the development status of the enzyme production industry in academia in recent years, the breeding of excellent strains with high amylase yield becomes an important research and development hotspot. At present, the method mainly comprises natural separation of wild enzyme-producing strains and domestication or mutation breeding, and strains with higher performance need to be continuously screened and excavated from nature, so that a new choice is provided for industrial production of amylase, new power is added, and the cost of manpower, material resources and the like is reduced in the aspect of industrial production as much as possible.
The soil contains rich microbial resources and is rich in excellent strains which can produce various special enzyme functions. Most of amylase-producing strains isolated from soil microorganisms are Bacillus, for example, Bacillus megaterium (Bacillus megaterium) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and other microorganisms such as actinomycetes, Aspergillus and yeast are isolated. Research aiming at amylase currently focuses on breeding strains producing amylase and researches on mechanisms such as culture medium optimization and control based on enzyme production conditions, strain mutagenesis effect and the like. But the strain with excellent performance and high biological activity of enzyme is still lacked to meet the requirement of industrial production.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for isolating amylase-producing microorganisms from soil.
The invention is realized by the following technical scheme: the concentrations of the slurries referred to below are mass percentages unless otherwise specified; the dosage of the related reagents and medicines is relative to the absolute pulp unless specified otherwise.
In a first aspect of the invention, there is provided a method of isolating an amylase-producing microorganism from soil, comprising:
taking surface soil, adding sterile water, and uniformly mixing to obtain a sample diluent;
injecting the sample diluent into a solid screening culture medium for inverted culture, and then classifying, purifying and preserving single colonies with different forms;
and carrying out molecular biological identification, a lysosome test and enzyme activity determination on the purified strain to obtain the microorganism for producing the amylase.
The invention screens out the strains with excellent performance and higher biological activity of the enzyme, provides more target strains for the subsequent research of the amylase and provides strains with potential development value for industrial production.
In a second aspect of the present invention, there is provided an amylase producing microorganism selected by the above method, comprising: bacillus megaterium, Bacillus lautus Paenibacillus lautus, Streptomyces pratensis, Streptomyces microflavus, Streptomyces nigreus, Streptomyces diastaticus, Enterobacter diastaticus, Lecleeria adeca adecoxylata, Aspergillus niger, Aspergillus flavus, DT06 Simplicisseria lanosensis, Penicillium erythraeum, Penicillium viniferum, Penicillium chrysogenum, Alternaria Alternaria alternata, Cladosporium cladium clavatum.
The invention has the beneficial effects that:
(1) the present invention isolated 31 amylase-producing strains from soil using plates supplemented with soluble starch, identified 9 strains of Bacillus megaterium (Bacillus megaterium), 1 strain of Bacillus lautus (Paenibacillus lautus), 1 strain of Streptomyces grassland (Streptomyces pratensis), 1 strain of Streptomyces microflavus (Streptomyces microflavus)1 strain of Streptomyces nigrus (Streptomyces atratus), 1 strain of Streptomyces tanarius (Streptomyces tanhiansis), 1 strain of Enterobacterium huaxianus (Enterobacter huaxiaensis), 1 strain of non-decarboxylating bacterium Klebsiella (Lecliella adynariae), 2 strains of Aspergillus niger (Aspergillus niger), 2 strains of Aspergillus flavus (Aspergillus flavus), 2 strains of 06 (Bacillus DT), 1 strain of Penicillium erythrorhizogenes (Penicillium), 1 strain of Penicillium chrysogenum (Penicillium chrysogenum), and 5 strains of Penicillium (Penicillium chrysogenum), by gene sequence alignment. The strains which are successfully separated are preliminarily screened by using an iodine staining method, and it can be known that 29 strains can obviously observe that a transparent ring is generated, the H/C value directly reflects the relative strength of the strains on the level of enzyme production capability, and the enzyme production capability is stronger when the ratio is larger. Can initially and quickly screen out the bacterial strain with stronger enzyme production capability.
(2) The method is simple, low in cost, high in practicability and easy to popularize.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a phylogenetic tree of bacterial 16SrRNA in example 1 of the present invention;
FIG. 2 is a tree phylogenetically developed by the fungus ITS in example 1 of the present invention;
FIG. 3 is a graph of the starch hydrolysis circles for the eight strains in example 1 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
A method of isolating amylase-producing microorganisms from soil comprising:
taking surface soil, adding sterile water, and uniformly mixing to obtain a sample diluent;
injecting the sample diluent into a solid screening culture medium for inverted culture, and then classifying, purifying and preserving single colonies with different forms;
and carrying out molecular biological identification, a lysis ring test and enzyme activity determination on the purified strain to obtain the microorganism producing amylase.
In some embodiments, soil is taken at a depth of 5-10 cm from the surface of the earth.
In some embodiments, the concentration of each substance in the solid screening media is: 10-12 g/L of ammonium sulfate, 5-8 g/L of sodium chloride, 8-10 g/L of disodium hydrogen phosphate, 10-20 g/L of soluble starch, and 1.5-2% of agar powder.
In some embodiments, the concentration of each substance in the enrichment medium is: 10-12 g/L of peptone, 10-12 g/L of sodium chloride, 5-8 g/L of yeast powder and 10-12 g/L of soluble starch.
In some embodiments, the liquid starch medium: 1-3 g/L of peptone, 10-12 g/L of soluble starch, 0.5-0.8 g/L of yeast powder, 1-3 g/L of sodium chloride, 2-4 g/L of dipotassium hydrogen phosphate, 0.1-0.3 g/L of magnesium sulfate, 0.1-0.3 g/L of calcium chloride and 0.001-0.003 g/L of manganese sulfate.
In some embodiments, the conditions of the inverted culture are: culturing at 35-37 deg.C for 2-3 days.
In some embodiments, the genomic DNA of the strain is extracted using a rapid extraction yeast DNA method.
In some embodiments, assays for amylase activity are performed using 3, 5-dinitrosalicylic acid DNS colorimetry.
First embodiment，
1.1 materials
1.1.1 sample Collection
The soil is taken from the soil with the depth of 5-10 cm on the ground surface of a green land of a Linyi university campus.
1.1.2 Primary reagents
3, 5-dinitrosalicylic acid: shanghai Michelin Biochemical technology, Inc.;
phenol: tianjin, Daimao chemical reagent plant;
yeast powder, peptone: OXOID Inc.;
sodium sulfite, magnesium sulfate, dipotassium hydrogen phosphate: ron's reagent;
manganese sulfate, ammonium sulfate: tianjin Bodi chemical corporation;
soluble starch, sodium hydroxide, agar powder: hengxing chemical reagent manufacturing Co., Ltd., Tianjin;
potassium sodium tartrate, sodium chloride, calcium chloride, disodium hydrogen phosphate: tianjin, Guang Compound science and technology development Co., Ltd; iodine, potassium iodide: shandong Yidian chemical Co., Ltd.
1.1.3 culture Medium
Solid screening culture medium: 10g/L of ammonium sulfate, 5g/L of sodium chloride, 8g/L of disodium hydrogen phosphate, 10-20 g/L of soluble starch and 1.5-2% of agar powder.
Enrichment culture medium: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast powder and 10g/L of soluble starch.
Liquid starch culture medium: peptone 1g/L, soluble starch 10g/L, yeast powder 0.5g/L, sodium chloride 1g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.1g/L, calcium chloride 0.1g/L, manganese sulfate 0.001 g/L.
1.1.4 Main Instrument
An electronic balance: YP30002, shanghai youco instruments ltd;
vertical pressure steam sterilizer: model YM75, shanghai sanshen medical devices, ltd;
superclean bench: model SW-CJ-1FD, air technologies, Inc., Antai, Suzhou;
a biochemical incubator: SHP-080 model, Shanghai Jing hong experiment equipment Co., Ltd, Taicang Jing hong apparatus equipment Co., Ltd;
a centrifuge: model TGL-16, hunan instrument laboratory instrument development ltd;
gene amplification apparatus (PCR apparatus): model a300, manufactured by hangzhou langji scientific instruments ltd;
a spectrophotometer: WJH12201-7, Shanghai Jingke industries, Ltd;
a constant-temperature water bath kettle: model XR 53648, a seiko zone, new rui instrument factory, west city.
Culture dish, beaker, conical flask, test tube, pipette gun, etc
1.2 methods
1.2.1 isolation and characterization of the Strain
(1) Separation of
Some soil samples were taken 5cm below the surface and 1g of the sample was weighed on sterile paper. 99mL of sterile water was added to the previously autoclaved Erlenmeyer flask, the sample was added, and the flask was shaken for several minutes. A10 mL sterile test tube was taken, 9mL sterile water was added, 1mL of the sample solution was added, and the mixture was mixed well. The same process is repeated until the gradient is diluted to 10-7. 0.1mL of each gradient sample diluent is taken and injected onto a plate of a solid screening medium (2-3 plates are coated on each gradient). The cells were cultured in an inverted state at 35 ℃ for 2 d.
After 2d of incubation, differences in colony morphology were observed. Selecting single colonies with different forms, sequentially separating and purifying the single colonies by adopting a plate marking method, and then preserving the single colonies by using a glycerin tube.
(2) Molecular biological identification
A few colonies from the purified strains are picked, the genomic DNA of the strains is extracted by a rapid yeast DNA extraction method, the obtained DNA is used as a template for PCR amplification, the sequence of a sequencing primer is shown in a table 1-1, the PCR amplification reaction system is 50 mu L, the required content of each reagent is shown in a table 1-2, and the reaction procedure is shown in a table 1-3. After the amplification reaction was completed, a small amount (about 6. mu.L) of the amplification product was used to perform 0.8% agarose gel electrophoresis, and the band was analyzed to preliminarily classify the strains into two major classes, bacteria and fungi. The remaining amplification product is sent to Biotechnology engineering (Shanghai) GmbH to complete the subsequent sequencing work. And performing sequence splicing by using an NCBI (N-terminal hybridization) online tool Blast comparison, and constructing a phylogenetic tree by using an adjacency (NJ) method through MEGA11.0 to complete comparison of homologous sequences.
A method for rapidly extracting yeast DNA: 500 mul of sodium hydroxide with the concentration of 20mmol/L is firstly taken to be put in a centrifugal tube of 1.5ml, a single bacterial colony is selected, and a proper amount of thalli is selected to be added into the sodium hydroxide. Boiling water bath for 10min, cooling to room temperature, placing into high speed centrifuge, and centrifuging at 10000rpm/min for 5 min. The supernatant was taken as template DNA.
TABLE 1-1 sequencing primers
Tab.1-1Sequencing primer
TABLE 1-2PCR reaction System (50. mu.L)
Tab.1-2Components of the PCR system(50μL)
TABLE 1-3PCR reaction procedure
Tab.1-3PCR reaction procedure
1.2.2 Ring lysis test
100 μ L of the glycerol-preserved strain was injected into a solid plate of the screening medium and spread evenly. Respectively placing fungi and bacteria into 28 deg.C, 37 deg.C incubator, performing inverted culture for 48 hr, and activating. Picking a small amount of single colonies by using a sterile gun head, inoculating the single colonies to an enrichment medium in a point grafting mode, selecting 1-2 strains with the same name, performing point grafting, placing the strains in an incubator in an inverted mode after inoculation is completed, adjusting the temperature of the incubator to be 28 ℃, and culturing for 24 hours. After the bacterial colony grows out, diluted Lugol iodine solution is evenly sprayed on the flat plate, and after a plurality of minutes, the generation of a color circle around the bacterial colony can be obviously observed. The diameter (D, cm) of the transparent circle and the colony diameter (D, cm) were measured, and the diameter ratio (HC) thereof was calculated. And (4) screening out the bacterial strain with stronger enzyme production capability.
1.2.3 enzyme Activity assay
(1) Preparation of crude enzyme solution
mu.L of amylase-producing strain liquid (glycerol preservation) was taken by a pipette, inoculated into a liquid starch medium, activated, and cultured in an incubator at 28 ℃ for 48 hours with shaking at 200 r/min. Inoculating the activated seed liquid into a liquid starch culture medium with the inoculation amount of 5%, fermenting, and carrying out shake culture at 28 ℃ and 200r/min for 24 h. 1mL of fermentation liquor is taken and centrifuged for 20min at the speed of 4000r/min, and the obtained supernatant is the crude enzyme liquid required by the next experiment.
(2) Drawing of standard curve
Firstly preparing a glucose standard solution, then sequentially adding all reagents according to tables 1-4, shaking up, placing in boiling water, heating for 5min to complete a color reaction, taking out after heating to reduce the temperature to room temperature, adding distilled water to complement to 25ml of scale, finally standing at room temperature for 30min to stabilize, and preparing 3 parallel samples at each concentration. And (3) testing the OD values of the tubes 1-6 at the wavelength of 540nm by using a test tube No. 0 as a control, drawing a standard curve according to the obtained data, and solving a regression equation.
Tables 1-4 Standard Curve preparation
Tab.1-4standard curve making
(3) Determination of enzyme Activity
In general, the amylase activity is measured by 3, 5-dinitrosalicylic acid (DNS) colorimetry. The method comprises the following steps: first, 0.2ml of the crude enzyme solution was taken and added to a sterile test tube. Next, 2mL of the substrate was transferred to a test tube using 0.5% soluble starch solution as the substrate. Then, 0.5mL of phosphate buffer at pH 7 was added to the tube. Then, the mixture was placed in a 45 ℃ water bath and heated continuously for 30 min. After heating, 1.5ml of DNS was added to the tube and shaken well. And placing the test tube in boiling water, heating for 5min, rapidly cooling to room temperature, adding distilled water to 25ml, standing at room temperature for 30min, and measuring OD at 540nm with the inactivated bacteria solution as control.
The unit of enzyme activity is defined as: the amount of enzyme required to produce 1mg of glucose in 1min at 45 ℃ at pH 7 was defined as one unit of enzyme activity.
2 results and analysis
2.1 isolation and characterization of the strains
After coating and multiple purifications of the sample, 31 strains are separated out and marked as HE-1 to HE-31. The sequence results obtained by splicing the gene sequences of the 31 amylase-producing strains can be directly submitted to a GenBank database, and the BLAST gene sequences are compared, and the obtained gene sequence results are shown in Table 2-1. Based on the results obtained, the 31 strains were divided into bacteria and fungi, and phylogenetic trees were established, the results of which are shown in FIGS. 2-1 and 2-2.
TABLE 2-1 alignment of the sequences of the strains
Tab.2-1Results of strain sequence alignment
2.2 transparent Ring measurement
After the colonies spotted on the enrichment medium are cultured for 24 hours, Lugol iodine solution is uniformly sprayed on the enrichment medium, the diameter of the transparent ring and the diameter of the colonies are recorded, and the ratio of the diameters is calculated (see table 2-2). It was determined that the clearing zones were clearly observed in all strains except for the clearing zones produced by HE-16 and HE-30. Selecting the strain with the ratio more than or equal to 2.00 to carry out enzyme activity determination. Then, it can be seen from Table 2 that the strains having a high amylase-producing ability are HE-10, HE-11, HE-12, HE-13, HE-14, HE-19, HE-27 and HE-28 (see FIGS. 2 to 3).
TABLE 2-2 Loop test
Tab.2-2Surface dissolution ring test
2.3 Amylase Activity assay
The 8 strains obtained by primary screening were inoculated into a liquid starch medium, placed in a shaker at 28 ℃ for fermentation culture, and the enzyme activity was measured by a3, 5-dinitrosalicylic acid (DNS) colorimetric method, the results of which are shown in Table 3. As is clear from Table 3, HE-13, HE-14, HE-17 and HE-19 have high enzyme activities, and the enzyme activities reach 5.47, 6.40, 10.53 and 7.13U/ml, respectively.
TABLE 2-3 determination of the enzyme Activity of the Amylases
Tab.2-3Determination of amylase activity
3 discussion and conclusions
The soil is rich in minerals, organic matters and the like and microorganisms capable of degrading the substances, so that the soil becomes a common sample for screening bacteria. In this study, 31 amylase-producing strains were isolated from soil using plates containing soluble starch, and through gene sequence alignment, 9 strains of Bacillus megaterium (Bacillus megaterium), 1 strain of Bacillus lautus (Paenibacillus lautus), 1 strain of Streptomyces grassland (Streptomyces pratensis), 1 strain of Streptomyces microflavus (Streptomyces microflavus)1 strain of Streptomyces nigricans (Streptomyces atratus), 1 strain of Streptomyces tanarius (Streptomyces tanarius), 1 strain of Enterobacterium huaxianus (Enterobacter huaxiaensis), 1 strain of non-decarboxylating bacterium (Leclicia adynazyl), 2 strains of Aspergillus niger (Aspergillus niger), 2 strains of Aspergillus flavus (Aspergillus flavus), 2 strains of 06 (Bacillus DT), 1 strain of Penicillium rubrum (Penicillium), 1 strain of Penicillium chrysogenum (Penicillium chrysogenum), and 5 strains of Penicillium (Penicillium strain Penicillium) were identified. The strains which are successfully separated are preliminarily screened by using an iodine staining method, and it can be known that 29 strains can obviously observe that a transparent ring is generated, the H/C value directly reflects the relative strength of the strains on the level of enzyme production capability, and the enzyme production capability is stronger when the ratio is larger. Can initially and quickly screen out the bacterial strain with stronger enzyme production capability.
The ratio between the two is easily interfered by external factors, such as: the amount of the inoculation amount, the length of the culture time, the growth speed, the thickness of the solid flat plate and the like can not be accurately judged, and a3, 5-dinitrosalicylic acid (DNS) colorimetric method is adopted to further determine the enzyme activity so as to more accurately obtain the strain with stronger amylase production capacity. As the enzyme activity of the amylase-producing strain screened from the soil is lower, the result shows that the enzyme-producing capability of HE-13, HE-14 and HE-19 is stronger by using the optimized method of the paper '3, 5-dinitrosalicylic acid colorimetric method for determining the content of reducing sugar' J 'food science 2008(08): 534-536'). After being identified by a molecular biological method, HE-13, HE-14, HE-17 and HE-19 are respectively known as Streptomyces nigricans (Streptomyces atratus), Streptomyces diastaticus (Streptomyces tanashiensis), Aspergillus niger (Aspergillus niger) and Aspergillus flavus (Aspergillus flavus). Aspergillus flavus is commonly present in food and feed industries, and plays an extremely important role in industrial microorganisms for brewing wines. However, aflatoxin is easily produced by aspergillus flavus, has extremely strong biological activity, has certain damage to internal tissues and organs of human and animals, particularly liver, and can cause liver cancer and even death when the liver cancer is serious. Aspergillus niger is the most common filamentous fungus in nature in China at present, and is often used for brewing fermentation and making various syrups, beverages, sauces and the like. Aspergillus niger is in a state of diffusive growth and grows vigorously. It is basically free of toxin substances and is the earliest one of the high-quality safe pollution-free food bacterial banks which is introduced into the national FDA laboratory and certified. Aspergillus niger not only can produce amylase with high yield, but also can produce various enzymes such as cellulase, xylanase, pectinase, protease and the like, and has wide application prospect in the aspect of industrial production. The streptomyces is the highest actinomycete in nature, has various actinomycete strains, has high-efficiency bacteriostasis, anti-infection and other biological effects, can produce various beneficial biochemical metabolites quickly, and has great potential clinical application and development value and social and economic benefits. Therefore, the actinomycetes are another special microorganism resource which is to be scientifically developed and applied in the industrial food production link and has huge potential development value.
Finally, it should be noted that, although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method of isolating amylase producing microorganisms from soil comprising:
taking surface soil, adding sterile water, and uniformly mixing to obtain a sample diluent;
injecting the sample diluent into a solid screening culture medium for inverted culture, and then classifying, purifying and preserving single colonies with different forms;
and carrying out molecular biological identification, a lysis ring test and enzyme activity determination on the purified strain to obtain the microorganism producing amylase.
2. The method for isolating an amylase-producing microorganism from soil according to claim 1, wherein the soil is taken 5 to 10cm deep from the surface of the soil.
3. The method of isolating an amylase producing microorganism from soil of claim 1, wherein the concentration of each substance in the solid selection medium is: 10-12 g/L of ammonium sulfate, 5-8 g/L of sodium chloride, 8-10 g/L of disodium hydrogen phosphate, 10-20 g/L of soluble starch, and 1.5-2% of agar powder.
4. The method of isolating an amylase producing microorganism from soil of claim 1, wherein the concentration of each substance in the enrichment medium is: 10-12 g/L of peptone, 10-12 g/L of sodium chloride, 5-8 g/L of yeast powder and 10-12 g/L of soluble starch.
5. The method of isolating an amylase producing microorganism from soil of claim 1, wherein the liquid starch medium: 1-3 g/L of peptone, 10-12 g/L of soluble starch, 0.5-0.8 g/L of yeast powder, 1-3 g/L of sodium chloride, 2-4 g/L of dipotassium hydrogen phosphate, 0.1-0.3 g/L of magnesium sulfate, 0.1-0.3 g/L of calcium chloride and 0.001-0.003 g/L of manganese sulfate.
6. The method for isolating an amylase-producing microorganism from soil as claimed in claim 1, wherein the conditions of the inverted culture are: culturing at 35-37 deg.C for 2-3 days.
7. The method for isolating an amylase-producing microorganism from soil as claimed in claim 1, wherein the genomic DNA of the strain is extracted by a rapid extraction method of yeast DNA.
8. The method for isolating an amylase-producing microorganism from soil of claim 1, wherein the assay for amylase activity is performed using 3, 5-dinitrosalicylic acid DNS colorimetry.
9. An amylase-producing microorganism selected by the method according to any one of claims 1 to 8.
10. An amylase producing microorganism as claimed in claim 9, wherein said microorganism is at least one of Bacillus megaterium, Bacillus lautus Paenibacillus lautus, Streptomyces pratensis, Streptomyces microflavus, Streptomyces nigrescens, Streptomyces atrophaeus, Streptomyces diastaticus, Enterobacter albugineus, Enterobacter huacae, Escherichia decarboxyfolia, Aspergillus niger, Aspergillus flavus, DT06 Simplicium lanosporanium, Penicillium purpurogenum, Penicillium vinaceum, Penicillium chrysogenum Penicillium, Penicillium chrysogenum, Streptomyces alternifolia, Clariana, and Clariaceae.
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