CN104818220B - One plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk - Google Patents
One plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk Download PDFInfo
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Abstract
The present invention provides one plant to screen the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk, and the deposit number of this bacterial strain is CCTCC M 2015189.Through the present invention experiments have shown that this bacterial strain has an ability for generating high saccharification enzyme activity, can also cellulase-producing, while the synthesis secretion of flora cellulase in agriculture and forestry organic waste material putrefaction process can also be effectively facilitated；Utilize screened Rhizopus oryzae bacterial strain JHSW01 can effectively decomposition of cellulose characteristic, the agriculture and forestry organic waste materials such as fermented stalk can be used it for, take full advantage of this renewable resource of stalk, and solve stalk and directly burn the pollution problem brought to environment；The culture medium prescription that Rhizopus oryzae bacterial strain JHSW01 of the present invention is used simply is easy to prepare, and is conducive to industrial cost savings；Spawn incubation condition of the present invention is simply easily controllable, is convenient for industrial applications, so having good market application prospect.
Description
Technical field
The invention belongs to biotechnologies, and in particular to one plant is screened the Rhizopus oryzae bacterial strain obtained from rotten stalk
JHSW01。
Background technology
Agricultural crop straw is the first big renewable resource on the earth, and China's owning amount occupies first place in the world.National annual generation
About more than 600,000,000 in ton agricultural crop straw, 200,000,000 tons of left sides of agricultural crop straw are utilized by mechanization returning to the field and stack retting decomposition into field
The right side, using ensiling, ammonification waited abdomen returning to the field mode utilize 200,000,000 tons or so, in addition also a small amount of agricultural crop straw for gasify and
Processing etc., the 2/3 of stalk total amount realizes comprehensive utilization substantially, not yet there is also about the remaining stalk in 1/3, i.e. 200,000,000 ton or so
To comprehensive utilization, some peasants take simplest processing mode-burning, cause atmosphere pollution, soil mineralization, fire thing
Therefore a large amount of social economy and ecological problem are waited, cause the extensive concern of the whole society.
Stalk itself mainly has three kinds of cellulose, hemicellulose and lignin material compositions, also contain a small amount of protein and
Mineral element is a kind of good microorganism growth substrate.And Rhizopus oryzae is one of the important mould in middle traditional Chinese medicines and distiller's yeast,
A large amount of amylase, carbohydrase, protease, the cellulose in decomposing straw and hemicellulose can be generated, lactic acid is translated into
Equal organic acids have large quantities of scholars and start with the experiment that stalk cooking liquor carries out Rhizopus oryzae fermentation lactic acid, have article to point out this
Strain can convert 80% sugar to lactic acid under suitable aerobic conditions.But the strain is used for the hair of agriculture and forestry organic waste material at present
The application of ferment sofening treatment is less.
Invention content
The object of the present invention is to provide one plant to screen the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk, the present invention
The Rhizopus oryzae bacterial strain JHSW01 screened is as a kind of fermenting microbe produced used in food, not only without toxic side effect, but also
Rhizopus oryzae fermentation is fast, and thalli growth nutritional requirement is simple, can be used for the agriculture and forestry organic waste material that ferments, before having the application of good market
Scape.
For achieving the above object, the present invention is achieved using following technical proposals：
One plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk, and Classification And Nomenclature isRhizopus oryzae, deposit number is CCTCC M 2015189.
The Rhizopus oryzae bacterial strain JHSW01 Gram-positives；Bacterium colony is dense, and mycelia crawls to creep, initially white,
After become dark brown；Rhizoid is flourishing, and branch is in root shape, is in dark brown；There are Chlamydospores, 2 ~ 4 bunchy of sporangiophore, spore on fibrillae of spores
Upright stand of capsule has branch, is in black afterwards.
Further, the Rhizopus oryzae bacterial strain JHSW01 is inoculated into culture generation enzyme activity in fluid nutrient medium and is up to 110-
The carbohydrase of 200 U/mL.
Further, the Rhizopus oryzae bacterial strain JHSW01 is inoculated into culture in solid medium and generates enzyme activity up to 50 ~ 100U/
The cellulase of g.
Further, the Rhizopus oryzae bacterial strain JHSW01 has the characteristic of effective decomposition of cellulose and hemicellulose.
Compared with prior art, the advantages and positive effects of the present invention are：The present invention screens from rotten stalk and obtains one
Rhizopus oryzae bacterial strain JHSW01, experiments verify that its can cellulase-producing and with the ability for generating high saccharification enzyme activity, can
Effective decomposition of cellulose and hemicellulose；Utilize screened Rhizopus oryzae bacterial strain JHSW01 can effective decomposition of cellulose
Characteristic, can use it for the agriculture and forestry organic waste materials such as fermented stalk, not only take full advantage of this renewable resource of stalk, but also solve
Stalk of having determined directly burns the pollution problem brought to environment；The culture medium that Rhizopus oryzae bacterial strain JHSW01 of the present invention is used
Formula simply is easy to prepare, and is conducive to industrial cost savings；Spawn incubation condition of the present invention is simply easily controllable, is convenient for
Industrial applications, so having good market application prospect.
After specific implementation mode in conjunction with the present invention, the other features and advantages of the invention will become clearer.
Description of the drawings
Fig. 1 is the tablet culture figure of Rhizopus oryzae JHSW01 in the present invention.
Specific implementation mode
Technical scheme of the present invention is described in further detail in the following with reference to the drawings and specific embodiments.
Embodiment 1
One, the separation screening of Rhizopus oryzae bacterial strain JHSW01
On July 4th, 2010 samples more than 50 parts in rotting stalk from Shandong Yinan great Zhuan wheats, and 6g sample powders is taken to soak respectively
Bubble is in 50 mL physiological saline, and under the conditions of 20 DEG C, per 2h, concussion is primary.Take 100 μ L samples suspension flat in nutrient agar respectively
Plate even spread, 3 ~ 7d is cultivated in 28 DEG C of constant incubators, and the typical single bacterium colony of picking form is inoculated into nutrition fine jade with method of scoring
On fat tablet, pure bacterium is obtained by repeated multiple times scribing line, filters out 8 plants of moulds altogether.
Obtained 8 plants of moulds are inoculated into oese in liquid PDA culture medium and are cultivated.Wherein one plant obtained
Bacterium is named as Rhizopus oryzae JHSW01, meets the biological characteristics of Rhizopus oryzae, specially：Gram-positive；Bacterium colony is thick
Close, mycelia crawls to creep, initially it is white, after become dark brown；Rhizoid is flourishing, and branch is in root shape, is in dark brown；On fibrillae of spores
There are Chlamydospores, 2 ~ 4 bunchy of sporangiophore, sporangiophore uprightly to have branch, is in afterwards black, referring to attached drawing 1.
Two, the bacteria molecule genetic classification identification of Rhizopus oryzae bacterial strain JHSW01
The DNA of extraction bacterial strain JHSW01 first, using the DNA as template, 18S rDNA universal primers are primer, to 18S
RDNA segments are expanded.The segment of amplification is directly subjected to sequencing.It will be on the websites result input NCBI of sequencing
Blast program is compared, and as a result shows in bacterial strain 18S rDNA sequences and GenBank gene pools in Rhizopus
The 18s rDNA sequence homology degree highests of Rhizopus oryzae, homology reach 100%.By DNAMAN6.0 to Genbank
In existing bacillus 18S rDNA sequences carry out phylogenetic analysis, the results show that the 18S of bacterial strain JHSW01
RDNA and Rhizopus oryzae homology highests, therefore can be determined that the bacterial strain is the Rhizopus oryzae of Rhizopus.
It is identified through the Identification of Biological Characteristics and bacteria molecule genetic classification identification method, it may be determined that screened
Bacterial strain JHSW01 be rhizopus, Rhizopus oryzae.The Rhizopus oryzae bacterial strain JHSW01 screened is subjected to culture presevation, preservation list
Position：China typical culture collection center（CCTCC）；Address：Luojiashan, Wuchang, Wuhan City, Hubei Province, in Wuhan University's preservation
The heart；Preservation date：On April 2nd, 2015, Rhizopus oryzae（Rhizopus oryzae）Deposit number be CCTCC M 2015189.
Three, Rhizopus oryzae bacterial strain JHSW01 generates high saccharification enzyme activity during the fermentation
Weigh wheat bran 2.4g, soy peptone 1.6g, soluble starch 0.8g, K2HPO40.16g, MgSO4.7H2O
It after 0.04g, KCl 0.04g are dissolved in the distilled water of 80mL, is placed in 250mL triangular flasks, 121 DEG C of sterilizing 20min.It is inoculated with after cooling
Two ring inclined-plane Rhizopus oryzae JHSW01 mycelium, are placed in 30 DEG C, and ferment 40h under the conditions of 190rpm, and centrifuging and taking 1mL supernatants measure enzyme
It is living.The qualification result that Rhizopus oryzae carbohydrase enzyme activity is carried out using DNS methods is as follows.
The carbohydrase enzyme activity experimental result of 1 Rhizopus oryzae JHSW01 of table
| Number | 1 | 2 | 3 | Average enzyme activity |
| Rhizopus oryzae group | 112.21U/g | 112.83 U/g | 113.06 U/g | 112.7U/g |
As can be seen from Table 1, which ferments in being inoculated into culture medium can generate higher saccharification enzyme activity,
The enzyme activity that is saccharified is tests prove that can reach the saccharification enzyme activity of 110-200 U/mL.
Four, the measurement that Rhizopus oryzae bacterial strain JHSW01 fermented stalk cellulase-producings are lived
According to《People's Republic of China's light industry professional standard》Carboxymethyl cellulose in QB2583-2003（Reducing sugar method）
Enzyme activity（CMCA-DNS）Assay method measures Rhizopus oryzae enzymatic productivity.
Experimental program is：3g stalk powders and 0.1g urea is taken to be added to the 500mL triangular flasks containing 100mL distilled water
In, 121 DEG C of sterilizings, 20min.What bacterium 2 ring Rhizopus oryzae JHSW01 mycelium of cooling access, control group do not take over.Fermentation system exists
30 DEG C, 96h is cultivated under the conditions of 190rpm, and fermented supernatant fluid is taken to measure the cellulase activity of two groups of fermentation systems.Experiment setting altogether
4 groups of experiments, wherein three groups are the parallel laboratory test for being inoculated with Rhizopus oryzae, another group be the control group for not connecing bacterium, experimental result such as table 2
It is shown：
The cellulase activity experimental result of 2 Rhizopus oryzae JHSW01 of table
| Number | 1 | 2 | 3 | Average enzyme activity |
| Rhizopus oryzae group | 58.31U/g | 59.87 U/g | 59.46 U/g | 59.21U/g |
Control group is internal after sterilizing not to have bacterial growth, therefore without generating cellulose enzyme activity, but control group can
To provide the reduction sugar amount generated after straw under high temperature sterilizing, going out so having removed stalk powder itself before experiment enzyme activity determination
The reduced sugar generated during bacterium influences, and can generate cellulase in Rhizopus oryzae fermentation process as can be seen from Table 2.
Five, the facilitation that Rhizopus oryzae JHSW01 generates cellulase in mixed fermentation
Weigh two parts of the 100g stalk powders of moisture content 58.3%, add 5g urea and 200g water respectively, inoculum concentration be according to
Volume mass is than 15%（15mL bacterium solutions are such as added in 100g stalk powders by volume mass）, it is stacked in the beaker of 2L, sets
The 90h in 30 DEG C of thermostat water bath, it is primary per turning for 24 hours in fermentation process.One group of experiment and one group of control is arranged in fermentation altogether
（Control group is the distilled water that 15% Rhizopus oryzae seed liquor is replaced with to 15%）, 1g stalk powders is taken to add 9mL's after fermentation
The citric acid-sodium citrate buffer solution of pH4.8,0.05mol/L, impregnate 30min, and centrifuging and taking supernatant measures cellulase activity.
According to《People's Republic of China's light industry professional standard》Carboxymethyl cellulose in QB2583-2003（Reducing sugar method）
Enzyme activity（CMCA-DNS）Assay method measures Rhizopus oryzae enzymatic productivity.Experimental result is as shown in table 3：
Table 3：The facilitation experimental result that Rhizopus oryzae generates cellulase in mixed fermentation
| 1 | 2 | 3 | Average enzyme activity | |
| Control group | 85.62U/g | 86.01U/g | 85.98U/g | 85.87U/g |
| Rhizopus oryzae group | 142.31U/g | 140.87 U/g | 143.06 U/g | 142.08U/g |
From table 3 it is observed that the cellulase activity being added after Rhizopus oryzae in fermented stalk has reached 142.08U/g, than
The fermentation system for being not added with Rhizopus oryzae has been higher by 1.65 times, since this Rhizopus oryzae itself cellulase-producing enzyme amount is relatively low, thus can see
Go out to add Rhizopus oryzae has actively impact effect for the secretion of stalk fermentation flora cellulase-producing, this is mainly the Rhizopus oryzae
The sugar that fermentative microflora decomposition of cellulose and hemicellulose generate can be efficiently used, lactic acid is translated into, reduces substrate suppression
It makes and uses, may advantageously facilitate the secretion of fermentative microflora cellulase.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these are changed or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (2)
1. one plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk, it is characterised in that：Its Classification And Nomenclature is rice root
It is mould（Rhizopus oryzae）, deposit number is CCTCC M 2015189；
The Rhizopus oryzae bacterial strain JHSW01 Gram-positives；Bacterium colony is dense, and mycelia crawls to creep, initially white, rear to become
For dark brown；Rhizoid is flourishing, and branch is in root shape, is in dark brown；There are Chlamydospores, 2 ~ 4 bunchy of sporangiophore, sporangiophore on fibrillae of spores
Uprightly there is branch, is in black afterwards；
The Rhizopus oryzae bacterial strain JHSW01 is inoculated into culture in fluid nutrient medium and generates the saccharification that enzyme activity is up to 110-200 U/mL
Enzyme；
The Rhizopus oryzae bacterial strain JHSW01 is inoculated into the cellulase cultivated in solid medium and generate enzyme activity up to 50 ~ 100U/g.
2. Rhizopus oryzae bacterial strain JHSW01 according to claim 1, it is characterised in that：The Rhizopus oryzae bacterial strain JHSW01 has
The characteristic of effective decomposition of cellulose and hemicellulose.
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| CN111334440B (en) * | 2020-04-16 | 2022-07-08 | 浙江树人学院（浙江树人大学） | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide |
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| CN102174419A (en) * | 2011-03-24 | 2011-09-07 | 南京工业大学 | Rhizopus oryzae strain, mutagenic screening method thereof and method for producing fumaric acid through fermentation |
| CN102634460A (en) * | 2012-03-08 | 2012-08-15 | 江苏今世缘酒业股份有限公司 | Rhizopus oryzae RH1-5 and separating and culturing method thereof |
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| CN102174419A (en) * | 2011-03-24 | 2011-09-07 | 南京工业大学 | Rhizopus oryzae strain, mutagenic screening method thereof and method for producing fumaric acid through fermentation |
| CN102634460A (en) * | 2012-03-08 | 2012-08-15 | 江苏今世缘酒业股份有限公司 | Rhizopus oryzae RH1-5 and separating and culturing method thereof |
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