CN104818221A - Fermentation cultivation method of rhizopus oryzae - Google Patents
Fermentation cultivation method of rhizopus oryzae Download PDFInfo
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Abstract
The invention discloses a fermentation cultivation method of rhizopus oryzae. The fermentation cultivation method is applicable to the rhizopus oryzae ((i) Rhizopus oryzae (/i)) strain JHSW01 with collection number of CCTCC M 2015189. After experimental verification, the fermentation cultivation of the rhizopus oryzae strain has high saccharifying enzyme activity, cellulose enzyme activity and organic acid production capacity, and can also effectively promote cellulose and hemicellulose decomposition conversion; high temperature can be resisted in the solid-state method production of the strain and a wide pH growth range is realized, and long propagation fermentation time is realized in fermentation process of forestry and agricultural residues; the culture medium formula adopted by the rhizopus oryzae strain JHSW01 is simple, is easy to prepare and contributes to cost saving of industrial production; the strain culture condition is simple, is easy to control, is convenient for industrial application and thus has good market application prospect.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of fermentation culture method of Rhizopus oryzae particularly.
Background technology
Agricultural crop straw is first renewable resources on the earth, and China's owning amount occupies first place in the world.In annual about more than the 600000000 tons of agricultural crop straws produced in the whole nation, by mechanize, also field and stack retting decomposition into field utilize agricultural crop straw about 200,000,000 tons, adopt ensiling, ammonification waited abdomen also field mode utilize about 200,000,000 tons, a small amount of agricultural crop straw is also had to be used for gasification and processing etc. in addition, 2/3 of stalk total amount achieves comprehensive utilization substantially, also have an appointment 1/3, namely about 200,000,000 tons residue stalks are not yet fully utilized, some peasants take the simplest processing mode-burning, cause topsoil, soil mineralization, the social economy that fire failure etc. are a large amount of and ecological problem, cause the extensive concern of the whole society.
Stalk itself mainly contains Mierocrystalline cellulose, hemicellulose and xylogen three kinds of material compositions, also containing a small amount of protein and mineral element, is a kind of good microorganism growth substrate.And Rhizopus oryzae is one of important mould in middle traditional Chinese medicines and distiller's yeast, a large amount of amylase, saccharifying enzyme, proteolytic enzyme can be produced, Mierocrystalline cellulose in decomposing straw and hemicellulose, be translated into the organic acids such as lactic acid, but this bacterial classification is less for the application of the fermentation sofening treatment of agriculture and forestry organic waste material at present.
Summary of the invention
For the situation that current agriculture and forestry organic waste material spontaneous fermentation efficiency is slow, the object of this invention is to provide a kind of fermentation culture method of Rhizopus oryzae, specifically go for Rhizopus oryzae to cultivate, the present invention verifies that this fermentation culture method of Rhizopus oryzae bacterial strain JHSW01 screened can obtain good technique effect by experiment, fermented bacterium does not have toxic side effect, and Rhizopus oryzae fermentation is fast, and thalli growth nutritional requirement is simple, and fermentation process is simple and easy, there is good market application foreground.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A fermentation culture method for Rhizopus oryzae, it comprises the following steps:
(1) Rhizopus oryzae is inoculated in seed culture medium carry out seed culture obtain seed culture fluid;
(2) described seed culture fluid is inoculated in solid medium carries out fermentation culture and obtain Rhizopus oryzae bacterial strain fermenting agent.
Described Rhizopus oryzae is Rhizopus oryzae bacterial strain JHSW01, and its Classification And Nomenclature is
rhizopus oryzae, its deposit number is CCTCC M 2015189.
Further: in described step (1), the condition of seed culture is: 20 ~ 40 DEG C, pH6 ~ 10, rotating speed 100 ~ 200rpm, time 24 ~ 72h.
Further: in described step (1), seed culture medium consists of: add in agriculture and forestry organic waste material extracting solution by the maltose of 5 ~ 30g/L and 2 ~ 30g/L yeast powder, adjustment pH7.0 ~ 8.0.
Further: the preparation process of described agriculture and forestry organic waste material extracting solution is: to get agriculture and forestry organic waste material powder and be added to the water according to the ratio of mass volume ratio 1:5 ~ 1:10, regulate pH to 9, boiling, filter and obtain.
Further: in described step (2), the condition of fermentation culture is: humidity is greater than 90%, temperature 20 ~ 35 DEG C, pH7 ~ 10.
Further: the solid medium in described step (2) consists of: according to quality than agricultural crop straw powder: forestry waste powder: three kinds of components mix by nitrogenous source=5 ~ 8:2 ~ 3:1 ~ 2.
Further: fermentation culture 48 ~ 72 hours in described step (2).
Further: described fermenting agent is that solid-liquid mixed fermentation material bent, dry fermentation material song, fermented supernatant fluid or wet fermentation material are bent.
Compared with prior art, advantage of the present invention and positively effect are: the present invention screens a Rhizopus oryzae bacterial strain JHSW01 from rotten stalk, through experimental verification its fermentation culture had produce that high saccharifying enzyme is lived, cellulase activity and product organic acid ability, effectively can also promote Mierocrystalline cellulose and hemicellulose decomposition and inversion; Bacterial classification solid state process of the present invention can withstand high temperatures and have pH growth scope widely in producing, in the rotten fermenting process of the heap of agriculture and forestry organic waste material, have longer breeding fermentation time; The culture medium prescription that Rhizopus oryzae bacterial strain JHSW01 of the present invention adopts is simple and easy is easy to preparation, is conducive to industrial cost savings; Spawn culture condition of the present invention is simply easy to control, and is convenient to industrial applications, so have good market application foreground.
After the specific embodiment of the present invention, the other features and advantages of the invention will become clearly.
Accompanying drawing explanation
Fig. 1 is the slat chain conveyor figure of Rhizopus oryzae JHSW01 in the present invention.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
One, the separation screening of Rhizopus oryzae bacterial strain JHSW01
On July 4th, 2010 samples more than 50 parts from Yinan, Shandong great Zhuan wheat rots stalk, gets 6g sample powder respectively and is immersed in 50 mL physiological saline, and under 20 DEG C of conditions, every 2h concussion once.Get 100 μ L sample suspension respectively at nutrient agar plate even spread, 3 ~ 7d is cultivated in 28 DEG C of constant incubators, the typical single bacterium colony method of scoring of picking form is inoculated on nutrient agar plate, obtains pure bacterium, filter out 8 strain moulds altogether by repeated multiple times line.
8 obtained strain moulds are inoculated in liquid PDA substratum with transfering loop and cultivate.The wherein strain bacterium called after Rhizopus oryzae JHSW01 obtained, it meets the biological characteristics of Rhizopus oryzae, is specially: Gram-positive; Bacterium colony is dense, and mycelia crawls to creep, initial in white, after become chocolate; Rhizoid is flourishing, and branch is root shape, in chocolate; Fibrillae of spores has Chlamydospores, sporangiophore 2 ~ 4 bunchy, sporangiophore uprightly has branch, afterwards in black, see accompanying drawing 1.
Two, the bacteria molecule genetic classification of Rhizopus oryzae bacterial strain JHSW01 is identified
First extract the DNA of bacterial strain JHSW01, with this DNA for template, 18S rDNA universal primer is primer, increases to 18S rDNA fragment.The fragment of amplification is directly carried out sequencing.Compared by blast program on the result of order-checking input NCBI website, the 18s rDNA sequence homology degree that result shows the Rhizopus oryzae in this bacterial strain 18S rDNA sequence and GenBank gene pool in Rhizopus is the highest, and homology reaches 100%.Phylogenetic analysis is carried out by the 18S rDNA sequence of DNAMAN6.0 to bacillus existing in Genbank, result shows, 18S rDNA and the Rhizopus oryzae homology of bacterial strain JHSW01 are the highest, therefore can judge the Rhizopus oryzae of this bacterial strain as Rhizopus.
Through described Identification of Biological Characteristics and the qualification of bacteria molecule genetic classification authentication method, can determine that screened bacterial strain JHSW01 is Rhizopus, Rhizopus oryzae.The Rhizopus oryzae bacterial strain JHSW01 screened is carried out culture presevation, depositary institution: China typical culture collection center (CCTCC); Address: Luojiashan, Wuchang, Wuhan City, Hubei Province, Wuhan University's preservation center; Preservation date: on April 2nd, 2015, Rhizopus oryzae (
rhizopus oryzae) deposit number be CCTCC M 2015189.
Three, the cultural method of Rhizopus oryzae bacterial strain JHSW01
The cultural method of Rhizopus oryzae bacterial strain JHSW01 specifically comprises the following steps:
(1) Rhizopus oryzae is inoculated in seed culture medium carry out cultivation obtain seed culture fluid; Seed culture medium consists of: agriculture and forestry organic waste material extracting solution 1L; Maltose 5 ~ 30g/L; Yeast powder 2 ~ 30g/L, adjustment pH7.0 ~ 8.0, the actual conditions of seed culture is: temperature 0 ~ 40 DEG C, pH6 ~ 10, rotating speed 100 ~ 200rpm, time 24 ~ 72h.
The preparation process of described agriculture and forestry organic waste material extracting solution is: get 20g stalk powder and add 200mL water, and regulate pH to 9, boiling 30min, eight layers of filtered through gauze, add water to 1L.
(2) be inoculated in solid medium by described seed culture fluid and carry out fermentation culture, solid fermentation condition is: humidity is greater than 90%, temperature 20 ~ 35 DEG C, pH7 ~ 10, ferments and within 48 ~ 72 hours, obtains Rhizopus oryzae bacterial strain fermenting agent.Solid medium consists of: according to quality than agricultural crop straw powder: forestry waste powder: three kinds of components mix by nitrogenous source=5 ~ 8:2 ~ 3:1 ~ 2, in the present embodiment nitrogenous source can select soy peptone, urea, phosphorous acid ammonia one or more.
Described fermenting agent is that solid-liquid mixed fermentation material bent, dry fermentation material song, fermented supernatant fluid or wet fermentation material are bent.
The powder of agricultural crop straw described in the present invention comprises stalk, stubble, weeds, animal excrement, processing of farm products waste etc., described forestry waste powder comprises bark, branch, timber fragment, sawdust etc., (concrete scope see " biomass energy and waste material utilize " Cui Zongjun, China Agricultyre University Press).
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (9)
1. a fermentation culture method for Rhizopus oryzae, is characterized in that it comprises the following steps:
(1) Rhizopus oryzae is inoculated in seed culture medium carry out seed culture obtain seed culture fluid;
(2) described seed culture fluid is inoculated in solid medium carries out fermentation culture and obtain Rhizopus oryzae bacterial strain fermenting agent.
2. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: described Rhizopus oryzae is Rhizopus oryzae bacterial strain JHSW01, and its Classification And Nomenclature is
rhizopus oryzae, its deposit number is CCTCC M 2015189.
3. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: in described step (1), the condition of seed culture is: 20 ~ 40 DEG C, pH6 ~ 10, rotating speed 100 ~ 200rpm, time 24 ~ 72h.
4. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: in described step (1), seed culture medium consists of: add in agriculture and forestry organic waste material extracting solution by the maltose of 5 ~ 30g/L and 2 ~ 30g/L yeast powder, adjustment pH7.0 ~ 8.0.
5. the fermentation culture method of Rhizopus oryzae according to claim 4, it is characterized in that: the preparation process of described agriculture and forestry organic waste material extracting solution is: get agriculture and forestry organic waste material powder and be added to the water according to the ratio of mass volume ratio 1:5 ~ 1:10, regulate pH to 9, boiling, filter and obtain.
6. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: in described step (2), the condition of fermentation culture is: humidity is greater than 90%, temperature 20 ~ 35 DEG C, pH7 ~ 10.
7. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: the solid medium in described step (2) consists of: according to quality than agricultural crop straw powder: forestry waste powder: three kinds of components mix by nitrogenous source=5 ~ 8:2 ~ 3:1 ~ 2.
8. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: fermentation culture 48 ~ 72 hours in described step (2).
9. the fermentation culture method of Rhizopus oryzae according to claim 1, is characterized in that: described fermenting agent is that solid-liquid mixed fermentation material bent, dry fermentation material song, fermented supernatant fluid or wet fermentation material are bent.
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Cited By (3)
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| CN106591152A (en) * | 2016-12-31 | 2017-04-26 | 沂源康源生物科技有限公司 | Rhizopus oryzae strain and method for preparing high-activity solid inoculant through rhizopus oryzae strain |
| CN108315275A (en) * | 2017-12-31 | 2018-07-24 | 三门峡龙飞生物工程有限公司 | A kind of high yield " three plain enzymes " straw decomposing inoculant and preparation method thereof |
| CN116622526A (en) * | 2023-07-20 | 2023-08-22 | 苏陀科技(北京)有限公司 | Rhizopus oryzae strain and application thereof in mycelium protein production |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591152A (en) * | 2016-12-31 | 2017-04-26 | 沂源康源生物科技有限公司 | Rhizopus oryzae strain and method for preparing high-activity solid inoculant through rhizopus oryzae strain |
| CN106591152B (en) * | 2016-12-31 | 2019-07-05 | 沂源康源生物科技有限公司 | Rhizopus oryzae bacterial strain and the method for preparing high vigor solid fungicide with it |
| CN108315275A (en) * | 2017-12-31 | 2018-07-24 | 三门峡龙飞生物工程有限公司 | A kind of high yield " three plain enzymes " straw decomposing inoculant and preparation method thereof |
| CN108315275B (en) * | 2017-12-31 | 2020-12-22 | 三门峡龙飞生物工程有限公司 | High-yield 'three-enzyme' straw decomposition agent and preparation method thereof |
| CN116622526A (en) * | 2023-07-20 | 2023-08-22 | 苏陀科技(北京)有限公司 | Rhizopus oryzae strain and application thereof in mycelium protein production |
| CN116622526B (en) * | 2023-07-20 | 2023-10-10 | 苏陀科技(北京)有限公司 | Rhizopus oryzae strain and application thereof in mycelium protein production |
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