CN102634460A - Rhizopus oryzae RH1-5 and separating and culturing method thereof - Google Patents
Rhizopus oryzae RH1-5 and separating and culturing method thereof Download PDFInfo
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Abstract
The invention discloses a rhizopus oryzae RH1-5 and a separating and culturing method thereof. The rhizopus oryzae RH1-5 can be obtained by the separating and culturing method, a strain of the rhizopus oryzae RH1-5 can be separated and screened from great-aromatic medium-temperature and high-temperature yeast, and culture of the rhizopus oryzae RH1-5 is filed in China General Microbiological Culture Collection Center on Nov 29, 2011 and encoded as CGMCCNo.5513. The separating and culturing method is simple in process, saccharifying fluctuation of the rhizopus oryzae RH10-5 is small and saccharifying effect of the yeast during wine brewing can be stabilized.
Description
Technical field
The present invention relates to mould fungi mikrobe and isolation cultivation method, be specifically related to a kind of Rhizopus oryzae RH1-5 and isolation cultivation method thereof.
Background technology
Daqu is as the saccharifying ferment of fragrant type brewing; In culturing process, formed special department of microbiology,, thereby formed the variety of meta-bolites for bent wine production fermentation provides abundant and useful microbial bacteria source through certain enrichment, cultivation, screening naturally, domestication; It is the complicacy of fragrance ingredient; Daqu or compound enzymic preparation contain plurality of enzymes systems such as glycase, saccharifying enzyme, zymase, esterification enzyme, proteolytic enzyme in the Daqu simultaneously, for forming complicated fragrance ingredient catalyzer are provided.But receive the influence of environmental microorganism, Daqu mikrobe fungus strain is numerous and diverse, and its saccharogenic power fluctuation is bigger, directly influences the saccharification result of Daqu in the wine brewing process.
Summary of the invention
The objective of the invention is to: a kind of Rhizopus oryzae RH1-5 and isolation cultivation method thereof are provided; Obtain Rhizopus oryzae RH1-5 through this isolation cultivation method; Isolation cultivation method technology is simple, and the fluctuation of Rhizopus oryzae RH1-5 saccharogenic power is less, stablizes the saccharification result of Daqu in the wine brewing process.
Technical solution of the present invention is: this Rhizopus oryzae RH1-5 bacterial strain is a separation screening and getting in the high-temperature daqu from giving off a strong fragrance; Bacterial classification is on November 29th, 2011 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3; Institute of Microorganism, Academia Sinica; China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, bacterium numbering is: CGMCC No. 5513, classification name: Rhizopus oryzae Rhizopus oryzae.
The isolation cultivation method of said Rhizopus oryzae RH1-5 is: take by weighing 10 grams after an amount of pulverizing of high-temperature daqu in getting, in the triangular flask that the 90mL sterilized water is housed, concussion 30min obtains bacteria suspension; Draw bacteria suspension 1.0mL with aseptic straw, put into the test tube that fills the 9mL sterilized water, be diluted to different extent of dilution with 10 times of methods successively, get 10
-2, 10
-3, 10
-4It is in the 90mm sterile petri dish that each 1mL of dilution sample adds diameter; Adopt potato dextrose agar, rose-bengal nutrient agar, czapek's solution to carry out flat board respectively and cultivate, shake up and be placed in the constant incubator 28 ℃-32 ℃ and be inverted and cultivated 3-5 days; The bacterium colony of form rule is streak culture on wheat bran juice solid slant culture base in the picking petridish, carries out repetitious separation and purification, obtains single purebred bacterium colony, and microscopy confirms as that to be preserved in wheat bran juice solid slant culture base behind the mould subsequent use; The dibbling of gained fungal strain on the screening culture medium flat board, is carried out the saccharifying mould primary dcreening operation with the transparent circle method, sift out and produce the strong mikrobe of saccharifying enzyme ability, finishing screen is selected a strain mould, is accredited as Rhizopus oryzae through 26S rDNA sequence homology analysis, name RH1-5.
Said wheat bran juice solid slant culture base: in 1.0L wheat bran juice, add its quality 1% glucose, 0.5% yeast extract paste, 1% agar, PH nature, 121 ℃ of sterilization 15min; Wherein, wheat bran juice preparation: add 5-6 doubly to the water of wheat bran weight, stir and boil 30min, took out and filter wheat bran juice.
Said potato dextrose agar (PDA substratum): 200 gram peeling potatoes are cut into small pieces; In pot, adding water boils half a hour for 1000 milliliters; Filter with double gauze, get its filtrating, boil and melt and moisturizing to 1000 milliliter with sucrose or glucose 20 grams, agar 10 grams.
Said rose-bengal agar is supported base: purchase the traditional Chinese medicines group in Shanghai.
Said czapek's solution: add SODIUMNITRATE 3 grams, potassium hydrogenphosphate 1 gram, sal epsom (MgSO in the triangular flask
47H
2O) 0.5 gram, Repone K 0.5 gram, ferrous sulfate 0.01 gram, sucrose 30 grams, agar 10 grams, zero(ppm) water are 1000 milliliters; Heating for dissolving gets mixed solution; 121 ℃ of sterilization 20min; 0.05% sodium deoxycholate of mixed solution quality is added in the cooling back, adds the 100ul Streptomycin sulphate in every again 100ml substratum.
Said screening culture medium (g/L): by Zulkovsky starch 10g, K
2HPO
40.3 g, MgCO
3Or MgSO
41g, NaCl 0.5g, KNO
31g, agar 10g form, pH7.2-7.4,121 ℃ of sterilization 30min.
The present invention carries out separation screening to the mikrobe in the Daqu and obtains strain mould a---Rhizopus oryzae; Carry out the performance comparison test with the existing preservation strain in laboratory; The result shows this bacterial strain, and not only saccharogenic power is high, and acidproof, high temperature resistant, is suitable for making the sour environment of wine cellar fermentation; Be of value to cultivating to make and strengthen high-temperature daqu, have great importance improving the yield of liquor.
Embodiment
Further specify technical solution of the present invention below in conjunction with concrete embodiment, these embodiment can not be interpreted as it is the restriction to technical solution.
Embodiment 1: according to following steps separation and Culture Rhizopus oryzae RH1-5
Take by weighing 10 grams after getting an amount of pulverizing of high-temperature daqu, in the triangular flask that the 90mL sterilized water is housed, concussion 30min obtains bacteria suspension; Draw bacteria suspension 1.0mL with aseptic straw, put into the test tube that fills the 9mL sterilized water, be diluted to different extent of dilution with 10 times of methods successively, get 10
-2, 10
-3, 10
-4It is in the 90mm sterile petri dish that each 1mL of dilution sample adds diameter; Adopting potato dextrose agar, rose-bengal nutrient agar, czapek's solution to carry out flat board respectively cultivates; Shake up and be placed in the constant incubator 28 ℃ and be inverted and cultivated 5 days, each extent of dilution do simultaneously 3 parallel; The bacterium colony of form rule is streak culture on wheat bran juice solid slant culture base in the picking petridish, carries out repetitious separation and purification, obtains single purebred bacterium colony, and microscopy confirms as that to be preserved in wheat bran juice solid slant culture base behind the mould subsequent use; The gained fungal strain is pressed the numbering dibbling on the screening culture medium flat board; Carry out the saccharifying mould primary dcreening operation with the transparent circle method, sift out and produce the strong mikrobe of saccharifying enzyme ability, finishing screen is selected a strain mould; Be accredited as Rhizopus oryzae through 26S rDNA sequence homology analysis, name RH1-5.
Wherein, said wheat bran juice solid slant culture base: in 1.0L wheat bran juice, add its quality 1% glucose, 0.5% yeast extract paste, 1% agar, PH nature, 121 ℃ of sterilization 15min; Wherein, wheat bran juice preparation: add 5 times to the water of wheat bran weight, stir and boil 30min, took out and filter wheat bran juice.
Said potato dextrose agar (PDA substratum): 200 gram peeling potatoes are cut into small pieces; In pot, adding water boils half a hour for 1000 milliliters; Filter with double gauze, get its filtrating, boil and melt and moisturizing to 1000 milliliter with sucrose or glucose 20 grams, agar 10 grams.
Said rose-bengal agar is supported base: purchase the traditional Chinese medicines group in Shanghai.
Said czapek's solution: add SODIUMNITRATE 3 grams, potassium hydrogenphosphate 1 gram, sal epsom (MgSO in the triangular flask
47H
2O) 0.5 gram, Repone K 0.5 gram, ferrous sulfate 0.01 gram, sucrose 30 grams, agar 10 grams, zero(ppm) water are 1000 milliliters; Heating for dissolving gets mixed solution; 121 ℃ of sterilization 20min; 0.05% sodium deoxycholate of mixed solution quality is added in the cooling back, adds the 100ul Streptomycin sulphate in every again 100ml substratum.
Said screening culture medium (g/L): by Zulkovsky starch 10g, K
2HPO
40.3 g, MgCO
3Or MgSO
41g, NaCl 0.5g, KNO
31g, agar 10g form, pH7.2,121 ℃ of sterilization 30min.
Embodiment 2: according to following steps separation and Culture Rhizopus oryzae RH1-5
Take by weighing 10 grams after getting an amount of pulverizing of high-temperature daqu, in the triangular flask that the 90mL sterilized water is housed, concussion 30min obtains bacteria suspension; Draw bacteria suspension 1.0mL with aseptic straw, put into the test tube that fills the 9mL sterilized water, be diluted to different extent of dilution with 10 times of methods successively, get 10
-2, 10
-3, 10
-4It is in the 90mm sterile petri dish that each 1mL of dilution sample adds diameter; Adopting potato dextrose agar, rose-bengal nutrient agar, czapek's solution to carry out flat board respectively cultivates; Shake up and be placed in the constant incubator 30 ℃ and be inverted and cultivated 4 days, each extent of dilution do simultaneously 3 parallel; The bacterium colony of form rule is streak culture on wheat bran juice solid slant culture base in the picking petridish, carries out repetitious separation and purification, obtains single purebred bacterium colony, and microscopy confirms as that to be preserved in wheat bran juice solid slant culture base behind the mould subsequent use; The gained fungal strain is pressed the numbering dibbling on the screening culture medium flat board; Carry out the saccharifying mould primary dcreening operation with the transparent circle method, sift out and produce the strong mikrobe of saccharifying enzyme ability, finishing screen is selected a strain mould; Be accredited as Rhizopus oryzae through 26S rDNA sequence homology analysis, name RH1-5.
Said wheat bran juice solid slant culture base: in 1.0L wheat bran juice, add its quality 1% glucose, 0.5% yeast extract paste, 1% agar, PH nature, 121 ℃ of sterilization 15min; Wherein, wheat bran juice preparation: add 5.5 times to the water of wheat bran weight, stir and boil 30min, took out and filter wheat bran juice.
Said potato dextrose agar (PDA substratum): 200 gram peeling potatoes are cut into small pieces; In pot, adding water boils half a hour for 1000 milliliters; Filter with double gauze, get its filtrating, boil and melt and moisturizing to 1000 milliliter with sucrose or glucose 20 grams, agar 10 grams.
Said rose-bengal agar is supported base: purchase the traditional Chinese medicines group in Shanghai.
Said czapek's solution: add SODIUMNITRATE 3 grams, potassium hydrogenphosphate 1 gram, sal epsom (MgSO in the triangular flask
47H
2O) 0.5 gram, Repone K 0.5 gram, ferrous sulfate 0.01 gram, sucrose 30 grams, agar 10 grams, zero(ppm) water are 1000 milliliters; Heating for dissolving gets mixed solution; 121 ℃ of sterilization 20min; 0.05% sodium deoxycholate of mixed solution quality is added in the cooling back, adds the 100ul Streptomycin sulphate in every again 100ml substratum.
Said screening culture medium (g/L): by Zulkovsky starch 10g, K
2HPO
40.3 g, MgCO
3Or MgSO
41g, NaCl 0.5g, KNO
31g, agar 10g form, pH7.3,121 ℃ of sterilization 30min.
Embodiment 3: according to following steps separation and Culture Rhizopus oryzae RH1-5
Take by weighing 10 grams after getting an amount of pulverizing of high-temperature daqu, in the triangular flask that the 90mL sterilized water is housed, concussion 30min obtains bacteria suspension; Draw bacteria suspension 1.0mL with aseptic straw, put into the test tube that fills the 9mL sterilized water, be diluted to different extent of dilution with 10 times of methods successively, get 10
-2, 10
-3, 10
-4It is in the 90mm sterile petri dish that each 1mL of dilution sample adds diameter; Adopting potato dextrose agar, rose-bengal nutrient agar, czapek's solution to carry out flat board respectively cultivates; Shake up and be placed in the constant incubator 32 ℃ and be inverted and cultivated 3 days, each extent of dilution do simultaneously 3 parallel; The bacterium colony of form rule is streak culture on wheat bran juice solid slant culture base in the picking petridish, carries out repetitious separation and purification, obtains single purebred bacterium colony, and microscopy confirms as that to be preserved in wheat bran juice solid slant culture base behind the mould subsequent use; The gained fungal strain is pressed the numbering dibbling on the screening culture medium flat board; Carry out the saccharifying mould primary dcreening operation with the transparent circle method, sift out and produce the strong mikrobe of saccharifying enzyme ability, finishing screen is selected a strain mould; Be accredited as Rhizopus oryzae through 26S rDNA sequence homology analysis, name RH1-5.
Said wheat bran juice solid slant culture base: in 1.0L wheat bran juice, add its quality 1% glucose, 0.5% yeast extract paste, 1% agar, PH nature, 121 ℃ of sterilization 15min; Wherein, wheat bran juice preparation: add 6 times to the water of wheat bran weight, stir and boil 30min, took out and filter wheat bran juice.
Said potato dextrose agar (PDA substratum): 200 gram peeling potatoes are cut into small pieces; In pot, adding water boils half a hour for 1000 milliliters; Filter with double gauze, get its filtrating, boil and melt and moisturizing to 1000 milliliter with sucrose or glucose 20 grams, agar 10 grams.
Said rose-bengal agar is supported base: purchase the traditional Chinese medicines group in Shanghai.
Said czapek's solution: add SODIUMNITRATE 3 grams, potassium hydrogenphosphate 1 gram, sal epsom (MgSO in the triangular flask
47H
2O) 0.5 gram, Repone K 0.5 gram, ferrous sulfate 0.01 gram, sucrose 30 grams, agar 10 grams, zero(ppm) water are 1000 milliliters; Heating for dissolving gets mixed solution; 121 ℃ of sterilization 20min; 0.05% sodium deoxycholate of mixed solution quality is added in the cooling back, adds the 100ul Streptomycin sulphate in every again 100ml substratum.
Said screening culture medium (g/L): by Zulkovsky starch 10g, K
2HPO
40.3 g, MgCO
3Or MgSO
41g, NaCl 0.5g, KNO
31g, agar 10g form, pH7.4,121 ℃ of sterilization 30min.
Embodiment 4: bacterial classification performance comparison optimization Test
The bacterial strain of embodiment 1-3 acquisition and four strain saccharification bacterial strains of laboratory preservation are carried out the performance comparison test from examination meal saccharogenic power, saccharogenic power, liquefaction power and esterification power index.
Inoculation culture saccharification: after the rice sterilization, be cooled to 35～40 ℃, insert mould spores suspension-s, fully shake up, cultivate different time with different pH, shake bottle about interval 10h 1 time in differing temps with aseptic technique; Take out the back and survey its moisture, examination meal acidity, examination meal saccharogenic power, saccharogenic power, liquefaction power, esterification power index; Inspection information learns that examination meal sugar can't react bent quality quality really, because examination meal sugar measured value is not considered the influence of inoculum size and time, examination meal saccharogenic power can be reacted bent quality relatively accurately.
Examination meal saccharogenic power: mg/gh
Examination meal saccharogenic power=examination meal sugar ÷ (bacterial classification is to the inoculum size * examination meal time of rice) * 1000
Saccharifying enzymic activity: the result restrains the over dry song in Ph4.6 solution, 35 ℃ of glucose in milligrams numbers (mg/gh) that the saccharification Zulkovsky starch was produced in 1 hour with 1
The Ye Huamei vigor: 35 ℃, during Ph4.6 solution, 1 gram over dry song is in the quality (g/gh) of 1 hour liquefaction Zulkovsky starch, ɑ-diastatic action is in ɑ-1,4 glucoside bond.
(1), different culture saccharification temperature
The different culture saccharification temperature index of table 1 are measured
Learn by table 1 data; Under identical culture saccharification temperature and time condition, Rh1-5 all shows higher examination meal saccharification performance, and saccharogenic power, liquefaction power and esterification power are relatively stable; Each index is the highest in the time of 30 ℃, and this temperature condition growth and the metabolic enzyme system of the most suitable Rh1-5 are described.
(2), different culture saccharification pH
Learn that according to different culture saccharification temperature experiment data the performance index of each bacterial strain are higher under 30 ℃ of conditions, regulate different culture saccharification pH with this understanding, contrast the performance of each bacterial strain.
30 ℃ of following different culture saccharification pH index determinings of table 2
Learn by table 2; Under identical pH condition, Rh1-5 still shows higher examination meal saccharification performance, and Rh1-5 examination meal saccharogenic power performance is best under pH4.0 and 7.0 conditions; Along with its liquefaction power of increase of pH also increases, under the pH7.0 condition, be fit to the effect of each enzyme system.
Claims (7)
1. Rhizopus oryzae RH1-5; It is characterized in that: this Rhizopus oryzae RH1-5 bacterial strain is a separation screening and getting in the high-temperature daqu from giving off a strong fragrance; Bacterial classification on November 29th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, bacterium numbering is: CGMCC No. 5513.
2. the isolation cultivation method of Rhizopus oryzae RH1-5 is characterized in that this isolation cultivation method may further comprise the steps: take by weighing 10 grams after an amount of pulverizing of high-temperature daqu in getting, in the triangular flask that the 90mL sterilized water is housed, shake 30min and obtain bacteria suspension; Draw bacteria suspension 1.0mL with aseptic straw, put into the test tube that fills the 9mL sterilized water, be diluted to different extent of dilution with 10 times of methods successively, get 10
-2, 10
-3, 10
-4It is in the 90mm sterile petri dish that each 1mL of dilution sample adds diameter; Adopt potato dextrose agar, rose-bengal nutrient agar, czapek's solution to carry out flat board respectively and cultivate, shake up and be placed in the constant incubator 28 ℃-32 ℃ and be inverted and cultivated 3-5 days; The bacterium colony of form rule is streak culture on wheat bran juice solid slant culture base in the picking petridish, carries out repetitious separation and purification, obtains single purebred bacterium colony, and microscopy confirms as that to be preserved in wheat bran juice solid slant culture base behind the mould subsequent use; The dibbling of gained fungal strain on the screening culture medium flat board, is carried out the saccharifying mould primary dcreening operation with the transparent circle method, sift out and produce the strong mikrobe of saccharifying enzyme ability, finishing screen is selected a strain mould, is accredited as Rhizopus oryzae through 26S rDNA sequence homology analysis, name RH1-5.
3. the isolation cultivation method of Rhizopus oryzae RH1-5 according to claim 2 is characterized in that said wheat bran juice solid slant culture base: in 1.0L wheat bran juice, add its quality 1% glucose, 0.5% yeast extract paste, 1% agar, PH nature, 121 ℃ of sterilization 15min; Wherein, wheat bran juice preparation: add 5-6 doubly to the water of wheat bran weight, stir and boil 30min, took out and filter wheat bran juice.
4. the isolation cultivation method of Rhizopus oryzae RH1-5 according to claim 2; It is characterized in that said potato dextrose agar (PDA substratum): 200 gram peeling potatoes are cut into small pieces; In pot, adding water boils half a hour for 1000 milliliters; Filter with double gauze, get its filtrating, boil and melt and moisturizing to 1000 milliliter with sucrose or glucose 20 grams, agar 10 grams.
5. the isolation cultivation method of Rhizopus oryzae RH1-5 according to claim 2 is characterized in that the foster base of said rose-bengal agar: purchase the traditional Chinese medicines group in Shanghai.
6. the isolation cultivation method of Rhizopus oryzae RH1-5 according to claim 2 is characterized in that said czapek's solution: add SODIUMNITRATE 3 grams, potassium hydrogenphosphate 1 gram, sal epsom (MgSO in the triangular flask
47H
2O) 0.5 gram, Repone K 0.5 gram, ferrous sulfate 0.01 gram, sucrose 30 grams, agar 10 grams, zero(ppm) water are 1000 milliliters; Heating for dissolving gets mixed solution; 121 ℃ of sterilization 20min; 0.05% sodium deoxycholate of mixed solution quality is added in the cooling back, adds the 100ul Streptomycin sulphate in every again 100ml substratum.
7. the isolation cultivation method of Rhizopus oryzae RH1-5 according to claim 2 is characterized in that said screening culture medium (g/L): by Zulkovsky starch 10g, K
2HPO
40.3 g, MgCO
3Or MgSO
41g, NaCl 0.5g, KNO
31g, agar 10g form, pH7.2-7.4,121 ℃ of sterilization 30min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818220A (en) * | 2015-04-22 | 2015-08-05 | 稼禾生物股份有限公司 | Rhizopus oryzae strain JHSW01 obtained by screening rotten straws |
| CN105779311A (en) * | 2016-05-17 | 2016-07-20 | 加加食品集团股份有限公司 | Method for rapidly screening mould strains producing high-yield protease |
| CN106753994A (en) * | 2016-11-28 | 2017-05-31 | 山西农业大学 | A kind of utilization high ester yield original inhabitants' aroma-producing yeasts strengthening porcelain improves alcohol fermentation liquid wine degree, the method for reducing isoamyl alcohol content |
| CN107189949A (en) * | 2017-06-13 | 2017-09-22 | 浙江工业大学 | Rhizopus oryzae LJH3 and the application in bioconversion Sophoricoside prepares genistein |
| CN109971657A (en) * | 2019-05-10 | 2019-07-05 | 西藏自治区农牧科学院农产品开发与食品科学研究所 | A kind of Rhizopus oryzae of high-yield glucoamylase and its application |
| CN110144298A (en) * | 2019-06-06 | 2019-08-20 | 劲牌有限公司 | A kind of Novel Rhizopus oryzae bacterial strain G1 and its cultural method and application |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818220A (en) * | 2015-04-22 | 2015-08-05 | 稼禾生物股份有限公司 | Rhizopus oryzae strain JHSW01 obtained by screening rotten straws |
| CN104818220B (en) * | 2015-04-22 | 2018-08-17 | 稼禾生物股份有限公司 | One plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk |
| CN105779311A (en) * | 2016-05-17 | 2016-07-20 | 加加食品集团股份有限公司 | Method for rapidly screening mould strains producing high-yield protease |
| CN105779311B (en) * | 2016-05-17 | 2019-04-05 | 加加食品集团股份有限公司 | A kind of rapid screening method of high proteinase yield fungal strain |
| CN106753994A (en) * | 2016-11-28 | 2017-05-31 | 山西农业大学 | A kind of utilization high ester yield original inhabitants' aroma-producing yeasts strengthening porcelain improves alcohol fermentation liquid wine degree, the method for reducing isoamyl alcohol content |
| CN106753994B (en) * | 2016-11-28 | 2020-05-19 | 山西农业大学 | Method for improving alcohol content of alcohol fermentation liquor and reducing isoamyl alcohol content by using high-ester-yield indigenous aroma-producing yeast enhanced yeast |
| CN107189949A (en) * | 2017-06-13 | 2017-09-22 | 浙江工业大学 | Rhizopus oryzae LJH3 and the application in bioconversion Sophoricoside prepares genistein |
| CN107189949B (en) * | 2017-06-13 | 2020-05-26 | 浙江工业大学 | Rhizopus oryzae LJH3 and application thereof in preparation of genistein by biotransformation of sophoricoside |
| CN109971657A (en) * | 2019-05-10 | 2019-07-05 | 西藏自治区农牧科学院农产品开发与食品科学研究所 | A kind of Rhizopus oryzae of high-yield glucoamylase and its application |
| CN109971657B (en) * | 2019-05-10 | 2023-01-31 | 西藏自治区农牧科学院农产品开发与食品科学研究所 | Rhizopus oryzae capable of producing saccharifying enzyme at high yield and application of rhizopus oryzae |
| CN110144298A (en) * | 2019-06-06 | 2019-08-20 | 劲牌有限公司 | A kind of Novel Rhizopus oryzae bacterial strain G1 and its cultural method and application |
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| CN102634460B (en) | 2013-08-14 |
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