CN1884476A - Aspergillus oryzae bacteria and its application - Google Patents
Aspergillus oryzae bacteria and its application Download PDFInfo
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Abstract
The invention relates the Aspergillus oryzae, LiT05 CGMCC No.1712. The product made with the strain has the advantages of bright-colored, light hand touch, and good agglomeration. The soy sauce has the advantages of high productivity and low energy consumption. The bacterial strain has important action in producing soy sauce.
Description
Technical field
The present invention relates to an Aspergillus oryzae bacteria and the application in production makes soy sauce thereof.
Background technology
Aspergillus oryzae (Aspergillus oryzae) is a requisite microorganism in making soy sauce, and also is the core technology in the production process of making soy sauce.The contained proteolytic enzyme of aspergillus oryzae can be decomposed into amino acid with the protein in the raw material (soya bean), and contained amylase can be sugar with the amylolysis in the raw material (flour), thereby has constituted the main component that makes soy sauce.
The aspergillus oryzae bacterial classification of good quality and high output can not only improve raw-material utilization ratio, product yield rate, can also shorten the production cycle, reduce production costs, and improves the local flavor and the quality that make soy sauce, therefore presses for a kind of aspergillus oryzae bacterial classification of good quality and high output.
Summary of the invention
The aspergillus oryzae that the objective of the invention is a strain good quality and high output.
Aspergillus oryzae bacterial strain provided by the present invention is (Aspergillus oryzae) LiT05, and this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 05 15th, 2006, and deposit number is CGMCC No.1712.
The bacterium colony of aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712 is rounded, and quality is loose, initial stage bacterium colony middle white, and the edge is transparent aplysia punctata.When bacterium colony had just formed, only the centre a bit was white in color, and grew up with bacterium colony later on, and white range also enlarges gradually, and transparent scope dwindles into edge one narrow ring-type from a slice.Grow up with bacterium colony, the central point of bacterium colony is yellow-green colour earlier, and yellow-green colour is to expansion all around then.Ripe bacterium colony is the yellow look that kills, and kills look for pale green sometimes.Can reach in 7-10 days about 6cm, conidial head is a radiation, and capsule myopia in top is spherical, and diameter is generally 40-50 μ m, and the stalk stem is arranged.The sporulation ability of this bacterial strain is 5.55 * 10
9/ g, percentage of germination are 32.5 1%.Have higher proteinase activity, aspartic protease is lived and is that it is 2227.67u/g (fermented product) that 142.5u/g (fermented product), neutral protease live, and Sumizyme MP is lived and is 62.09u/g (fermented product).
The aspergillus oryzae substratum of available routine and cultural method are cultivated aspergillus oryzae of the present invention (Asperggillusoryzae) LiT05 CGMCC No.1712.
The invention provides an Aspergillus oryzae bacteria (Aspergillus oryzae) LiT05 CGMCC No.1712.This bacterial strain is the high yield and high quality aspergillus oryzae bacterial strain through space breeding.Test manufacture experimental result proof is bright-colored by the one-tenth song of this bacterial strain preparation, and feel is light, and clumping of over grown hyphae is good, and spore and given birth to, and fermentative preparation soy sauce output height, energy consumption are low.Bacterial strain of the present invention will play a significant role in the production that makes soy sauce, and have a extensive future.
The present invention will be further described below in conjunction with specific embodiment.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and all percentage concentrations are mass percent concentration, and the solvent in all substratum is water.
Acquisition and the preservation of embodiment 1, aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712
Entrust National Space Administration, Inst. of Genetics and Development Biology, CAS carries space with aspergillus oryzae (Aspergillusoryzae) LiY05 CGMCC No.1711 (being the Li Jin of Shenzhen note Food Co., Ltd to be made soy sauce to produce at present screen the superior strain of acquisition with bacterial classification) by China's the 21st st recoverable science and experimental satellite, the space breeding that has carried out in space 27 days is cultivated experiment, this bacterial classification has satisfactorily been finished space breeding on August 29th, 2005, screen through Institute of Microorganism, Academia Sinica then, separate, the final superior strain that obtains, called after LiT05, this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 05 15th, 2006, and deposit number is CGMCC No.1712.
The comparison test of embodiment 2, aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712 and original production bacterial classification
Carry out aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712 and the above-mentioned comparison test of production that make soy sauce as follows with bacterial classification LiY05-aspergillus oryzae (abbreviation LiY05):
1, the multiple connection of aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712
Detect aspergillus oryzae LiT05 can be on the aspergillus oryzae test tube slant of routine substratum growth and breeding.Respectively at the aspergillus oryzae test tube slant of routine substratum (acid anhydride casein 4g, KH
2PO
40.36g, Na
2HPO
47H
2O 1.1g, agar 15g, water is settled to 1000mL) on inoculated 10 aspergillus oryzae LiT05 (called after T00-T10), cultivated 3 days down at 30 ℃, the whole multiple connections successes of result can be used as and produce the bacterial classification that starts that makes soy sauce.
2, the separation rejuvenation of multiple connection bacterial classification
From the slant strains of multiple connection success, filter out the aspergillus oryzae single strain that mode of appearance is good, enzyme activity is high, with strong, the high yield and high quality bacterial strain of adaptability that obtains to be fit to make soy sauce and to produce needs.Concrete grammar is: select the good test tube slant bacterial classification of mode of appearance, with its streak inoculation in separating rejuvenation plate (culture medium prescription is identical with slant medium), cultivated 5-6 days down at 30 ℃, and the aspergillus oryzae single strain that the choosing colony diameter is big, mode of appearance is good from plate is chosen and is received in the slant tube, obtain 12 strain slant tube bacterial strains altogether, difference code T 001-T012 is as first-generation test tube strains.
The preliminary screening of 3, separating the rejuvenation bacterial classification
Step 2 is inoculated in the slant tube once more through the t bacteria 001-T012 that rejuvenation obtains, cultivated 3 days down at 30 ℃, the result all inoculates success.Then these 12 slant strains are inoculated in respectively aspergillus oryzae (Aspergillus oryzae) dedicated liquid substratum (acid anhydride casein 4g, KH are housed
2PO
40.36g, Na
2HPO
47H
2O 1.1g, water is settled to 1000mL) triangular flask in carry out enlarged culturing, detect its enzyme activity with ordinary method, with LiY05 is contrast, detected result sees Table 1, select the higher T002 of enzyme activity, T005, T007, T012 four inclined-plane rejuvenation bacterial classification and former usefulness and make soy sauce production with totally 5 of bacterial strain LiY05, as soy sauce test manufacture experiment bacterial classification.
The enzyme activity detected result (units/gram) of table 1 T001-T012
| Numbering | T001 | T002 | T003 | T004 | T005 | T006 |
| Enzyme activity | 15978.96 | 17568 | 15999 | 11196 | 15472.3 | 19073.57 |
| Numbering | T007 | T009 | T010 | T012 | LiY05 | |
| Enzyme activity | 15178.8 | 18617.02 | 16720.25 | 19887.13 | 17422 |
4, the enlarged culturing of triangular flask bacterial classification
Step 3 is picked out the higher T002 of enzyme activity, T005, T007, T012 four inclined-plane rejuvenation bacterial classification and former usefulness to make soy sauce to produce to be inoculated in the triangular flask that aspergillus oryzae (Aspergillus oryzae) dedicated liquid substratum is housed with bacterial strain LiY05 and cultivates, cultivated 3 days down at 30 ℃, the result all cultivates success.And each bacterial classification carried out sense organ, mycelia microscopy, enzyme activity determination, and bacterium colony is rounded as a result, and quality is loose, initial stage bacterium colony middle white, the edge is transparent aplysia punctata.When bacterium colony had just formed, only the centre a bit was white in color, and grew up with bacterium colony later on, and white range also enlarges gradually, and transparent scope dwindles into edge one narrow ring-type from a slice.Grow up with bacterium colony, the central point of bacterium colony is yellow-green colour earlier, and yellow-green colour is to expansion all around then.Ripe bacterium colony is the yellow look that kills, and kills look for pale green sometimes.7-1 can reach about 6cm in 0 day, and conidial head is a radiation, and capsule myopia in top is spherical, and diameter is generally 40-50 μ m, and the stalk stem is arranged.The sporulation ability of this bacterial strain is about 5.55 * 10
9/ g, percentage of germination is about 32.51%.Have higher proteinase activity, aspartic protease work is about 142.5u/g (fermented product), and neutral protease work is about 2227.67u/g (fermented product), and Sumizyme MP work is about 62.09u/g (fermented product).
5, plant bent test manufacture
Under the cultivation carrier and culture condition identical with step 4, simultaneously T002, T005, T007, T012 and LiY05 are carried out the test manufacture of kind of song, be contrast with T011, to cultivate 3 days down at 30 ℃, the kind of the above-mentioned bacterial classification of result is bent all test-manufactures successfully.Simultaneously to above-mentioned kind of Qu Jinhang enzyme activity detect and subjective appreciation, the enzyme activity detected result sees Table 2, and is wherein bent best with the kind of T012, its enzyme activity reaches 19716 units/gram, and spore count is 15,000,000,000/gram, and color and luster is emerald green, Qu Xiangnong is thick, and feel is evenly loose, is particularly suitable for producing making soy sauce.
The enzyme activity detected result (units/gram) of table 2 kind of song
| Numbering | T002 | T012 | T005 | T007 | LiY05 | T011 |
| Enzyme activity | 15941.14 | 19716.28 | 9912 | 11856 | 12084.59 | 8200 |
6, soy sauce production was test-manufactured with the bent fs
, be contrast now,, reach growth, breeding situation in raw soybeans, flour to detect its adaptation situation to the aerated koji making production technique with T011 to T002, T005, T007, T012 and LiY05 kind Qu Jinhang test manufacture.Wherein, plant bent common salt concn in the aerated koji making process and see Table 1, cultivated 3 days down at 30 ℃; Unifying the materials amount during fermentation is soybean 1650kg/ days, flour 600 kg/ days, and at 30 ℃ of bottom fermentation 80-90 days.
The salt concn of table 3 kind of song
| Seed dressing kind of song | Salt concn (g/100mL) | Pond number |
| T002 | 22.72 | B08-3 |
| T012 | 21.64 | B08-4 |
| T007 | 21.96 | B07-5 |
| T005 | 22.31 | B07-3 |
| LiY05 | 21.83 | B07-4 |
1) aerated koji making interpretation of result
Aspergillus oryzae bacterial classification through space breeding is compared with the LiY05 bacterial classification, and the difference of following several respects is arranged on the sense organ:
The first, 4 bacterial classifications through space breeding need not to do special processing in kind of bent production process, promptly on the basis of the bent production technique of original seed, and fully can ordinary production.
The second, the various songs of space breeding are compared with LiY05 kind song, and the overall difference of growing state is little, but last sophisticated kind is bent, it is bigger no matter to be that color, the mycelia of kind of song difference such as life, spore amount, analyzes reason, and the management work in kind of bent making processes also has certain influence to the result.In test manufacture aerated koji making process, find, four kind songs through space breeding, all bent early 4-5 hour spore germination phase than the LiY05 kind, mycelia animated period (referring to turn over for the first time the bent time) has shifted to an earlier date 3-5 hour, the maturation time of T kind (each of space breeding kind of song) in the aerated koji making process all shifted to an earlier date like this, find at 20-24 hour that spore and give birth to, bent material beginning flavescence, the LiY05 kind just found that bent material began to change yellow in bent late 5-6 hour, so respectively become bent spore amount bigger from sense organ T kind, particularly T012 becomes bent spore to fly upward, and color and luster is yellowish green, and is deep yellow partially.But the ultimate aim of soy sauce production koji is to make aspergillus oryzae growth and breeding in raw material (soybean, flour), be to want aspergillus to produce a large amount of proteolytic enzyme and amylase, so the Cheng Quzhong mycelia to give birth to situation also very important, if single pursuit spore is many, whether grow vigorous just not comprehensive and ignore mycelia.Qu Xiangnong, feel pine, lump, spore the Cheng Qu that gives birth to not for a long time and is only ideal Cheng Qu.
The thermophilic that above-mentioned test manufacture result shows the T012 bacterial classification is stronger, can grow normal down at 32-38 ℃, the spore germination phase shortens nearly 4 hours than LiY05, with the bent koji of this kind, to make the koji time shorten to 30-32 hour from 36 hours, can save nearly 10% energy consumption, (but this only is the sense organ and the detection of once test-manufacturing in the production that is particularly useful for making soy sauce, randomness is high, any on earth bacterial classification is good, can be used as preferably makes soy sauce produces with kind of a song, and which difference will be eliminated, and still need repeatedly to test can determine Bizet).
2) system wine with dregs fermentation interpretation of result
The soy sauce color problem of former worry into koji fermentation found during the fermentation, and the fermentation of T kind system wine with dregs has color (yellow mass-tone) in the time of 24 hours, and than LiY05 kind Qu Shen, can blackening after heat treated, prove that not being is melanochrome.Produce through 6 days fermentation haematochrome, estimate the soy sauce color influence little, but in more than 10 days initial fermentation processes, amino acid generates too fast, and total acid is higher, but salt content is normal, analysis causes the higher reason of total acid, may be bad relevant with weather conditions, and this point will be kept a close eye on.
After the fermentation termination, detect organoleptic characteristics, physical and chemical index and the microbiological indicator of the soy sauce for preparing with T kind and LiY05 kind, the soy sauce for preparing of T kind is sorrel as a result, and is glossy, clarification, and sauce perfume (or spice) is honest, the flavour deliciousness, the degree of saltiness is agreeable to the taste; Amino acid peptide nitrogen content 〉=1.8g/100mL; Total number of bacterial colony (individual/mL)≤2000, intestinal bacteria are (individual/as 100mL)≤20, to meet the national standard that makes soy sauce.The soy sauce that the LiY05 kind prepares is and also meets the national standard that makes soy sauce, but amino acid peptide nitrogen content 〉=1.4g/100mL.Above-mentioned test manufacture result shows that aspergillus oryzae (Aspergillus oryzae) the LiT05 CGMCC No.1712 of space breeding of the present invention is applicable to the production that makes soy sauce.
The subordinate phase test manufacture of embodiment 3, aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712
With the condition identical aspergillus oryzae (Aspergillus oryzae) LiT05 CGMCC No.1712 is further test-manufactured, may further comprise the steps with embodiment 2:
One, triangular flask enlarged culturing
Be inoculated in the aspergillus oryzae LiT05 of embodiment 1 rejuvenation in the slant tube once more, cultivated 3 days down at 30 ℃, then with slant strains in three batches (every batch at interval 1-3 days) be inoculated in enlarged culturing in the triangular flask that aspergillus oryzae (Aspergillus oryzae) dedicated liquid substratum is housed, the enlarged culturing time of different batches sees Table 4, triangular flask bacterial classification after the enlarged culturing, mycelia is white in color, and cover with the green-yellow spore, shake bottle gently and feel fluffy, spore is tear-away to fly upward, three batches of sensory differences are little, and the spore count detected result is 101.12 hundred million/gram.Detected enzyme activity simultaneously, the enzyme biopsy is surveyed and be the results are shown in Table 4, and the kind song of different batches all has higher enzyme lives, but has certain gap.
The result is surveyed in the enzyme biopsy of the aspergillus oryzae LiT05 of table 4 enlarged culturing
| Batch | Bacterium numbering | Enzyme activity (unit) | Remarks |
| 1 (April 15) | The T-1 triangular flask | 15978.96 | Test-manufacture 2 days kind songs |
| 2 (April 19) | The T-2 triangular flask | 16532.0 | Test-manufacture 2 days kind songs |
| 3 (April 21) | The T-3 triangular flask | 15918.0 | Test-manufacture 1 day kind song |
Two, produce with kind of a bent test manufacture
In five batches (code T-00-1～T-00-5) to the aspergillus oryzae LiT05 kind Qu Jinhang test manufacture of above-mentioned different batches, intersection fed intake 10 days continuously, was organized as follows:
Each two days kind song of kind song: T-00-1, T-00-2 test manufacture on April 15, April 18 finished;
Each two days kind song of kind song: T-00-3, T-00-4 test manufacture on April 19, April 21 finished:
Each one day kind song of kind song: T-00-5 test manufacture on April 21, April 24 finished.
Use three batches of triangular flask kind songs making under the same conditions on April 15, April 19, April 21 respectively, test-manufacture.The three batches of sophisticated kinds are bent to be analyzed from sense organ, and it is yellowish green to plant bent color and luster, and it is good that spore the situation of giving birth to.The test manufacture result feels that Qu Xiangnong is thick, and feel is light, and is fluffy, and have gentle hands is touched kind of a bent spore and come off immediately and fly upward, and three batches of kind songs all are high-quality kind songs.To three crowdes of kind Qu Jinhang after the maturation enzyme activity, spore count detect.Detection method is undertaken by People's Republic of China's specialized standard (spore count assay method ZBx66028-87, protease activity amylograph SBx.10317-1999), and it is higher that detected result sees Table the enzyme activity and the spore number average of 5, three batches of kind songs.
Three batches of ripe enzyme activity, spore count detected results of planting song of table 5
| Production Time | Plant bent numbering | Enzyme activity (unit) | Spore count (hundred million/gram) | Deadline |
| April 16 | T-00-1、2 | 16984.80 | 123.36 | April 18 |
| 111.73 | ||||
| April 19 | T-00-3.4 | 12215.67 | 115.92 | April 22 |
| 122.07 | ||||
| April 23 | T-00-5 | 15428.30 | 113.58 | April 25 |
| 105.36 |
Three, aerated koji making test manufacture
Since April 26, adopt intersect every other day the method that feeds intake to five batches (code T-00-1～T-00-5) aspergillus oryzae LiT05 kind Qu Jinhang aerated koji making test manufacture, and detect enzyme activity, the enzyme biopsy is surveyed and be the results are shown in Table 6, the enzyme of the Cheng Qu of preparation is lived all higher, but different batches there are differences.
Table 6 is by the enzyme activity of the Cheng Qu of the bent preparation of five batches of aspergillus oryzae LiT05 kinds
| Mission Number | Charging time | Go out the bent time | Enzyme activity |
| T-00-1 | 4.26 | 4.28 | 1523.38 |
| T-00-2 | 4.28 | 4.30 | 1302.33 |
| T-00-3 | 4.30 | 5.2 | 1270.60 |
| T-00-4 | 5.2 | 5.4 | 1522.30 |
| T-00-5 | 5.4 | 5.6 |
See on the sense organ, the one-tenth Qu Yanse that above-mentioned five batches of aspergillus oryzae LiT05 kind songs make is more bright-coloured, no abnormal situation becomes bent feel light, and clumping of over grown hyphae is good, spore and is given birth to, but indivedual bent material are thinless because of turning over bent fragmentation, so cause Cheng Quzhong that the hard particle of diameter 4-6cm is arranged, cause mycelia, spore can't go deep into giving birth to, though little to the quality influence that becomes song, should note.
The koji latter temperature is higher, though the surface growth of bent material is good, but bent material bottom moisture content sensation is not enough, too fast because of the performance of temperature drift moisture, cause bent material that the phenomenon of shrinkage crack is arranged, and crackle does not often compress, cause wind from crackle, to run away, the air feed that has influenced the crackle periphery with become Qu Zhiliang, can be by reducing the later stage starter-making temperature and strengthening moisturizing and carry out head it off, to guarantee bent quality.
In above-mentioned aerated koji making process, the spore germination of the aspergillus oryzae LiT05 kind song of different batches, mycelia generate, the spore regenerative process is all very fast, than LiY05 kind bent fast 4-8 hour, the koji time can shorten to 30-32 hour by original 36-40 hour, made energy consumption reduce about 20%.
Four, system wine with dregs fermentation
After the fermentation termination, after the fermentation termination, detect organoleptic characteristics, physical and chemical index and the microbiological indicator of the soy sauce of each batch LiT05 preparation, the soy sauce of different batches all is sorrel as a result, and is glossy, clarification, and sauce perfume (or spice) is honest, the flavour deliciousness, the degree of saltiness is agreeable to the taste; Amino acid peptide nitrogen content 〉=1.8g/100mL; Total number of bacterial colony (individual/mL)≤2000, intestinal bacteria are (individual/as 100mL)≤20, to meet the national standard that makes soy sauce.Aspergillus oryzae (Aspergillus oryzae) the LiT05 CGMCC No.1712 that shows space breeding of the present invention is applicable to the production that makes soy sauce.
Claims (2)
1, aspergillus oryzae (Aspergillus oryzae) LiTO5 CGMCC No.1712.
2, the application of the described aspergillus oryzae of claim 1 (Aspergillus oryzae) LiTO5 CGMCC No.1712 in production makes soy sauce.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942396A (en) * | 2010-09-06 | 2011-01-12 | 华南理工大学 | Aspergillus oryzae HG76 and application thereof |
| CN102165055A (en) * | 2009-04-17 | 2011-08-24 | 龟甲万株式会社 | Aspergillus sp. having large-scale genome duplication |
| CN102643754A (en) * | 2012-04-18 | 2012-08-22 | 中国农业大学 | Aspergillus oryzae and application thereof in aspect of improving yield of alcohol |
| CN103555594A (en) * | 2013-11-08 | 2014-02-05 | 佛山市海天调味食品股份有限公司 | Aspergillus oryzae and application thereof |
| CN107586729A (en) * | 2017-10-26 | 2018-01-16 | 佛山市海天(高明)调味食品有限公司 | One Aspergillus oryzae ZA138 and its application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH0698716A (en) * | 1992-09-18 | 1994-04-12 | Fukuoka Pref Gov Shoyu Jozo Kyodo Kumiai | Production of malted rice |
| JP2002306159A (en) * | 2001-04-10 | 2002-10-22 | Yamasa Shoyu Co Ltd | Koji mold for making soy sauce and method of making soy sauce by using the same |
| CN1631239B (en) * | 2004-11-25 | 2010-09-08 | 吉林大学 | Soya sauce containing organic selenium from aspergillus oryzae and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102165055A (en) * | 2009-04-17 | 2011-08-24 | 龟甲万株式会社 | Aspergillus sp. having large-scale genome duplication |
| US8900647B2 (en) | 2009-04-17 | 2014-12-02 | Kikkoman Corporation | Koji mold having large-scale genomic duplication |
| CN101942396A (en) * | 2010-09-06 | 2011-01-12 | 华南理工大学 | Aspergillus oryzae HG76 and application thereof |
| CN102643754A (en) * | 2012-04-18 | 2012-08-22 | 中国农业大学 | Aspergillus oryzae and application thereof in aspect of improving yield of alcohol |
| CN102643754B (en) * | 2012-04-18 | 2013-05-01 | 中国农业大学 | Aspergillus oryzae and application thereof in aspect of improving yield of alcohol |
| CN103555594A (en) * | 2013-11-08 | 2014-02-05 | 佛山市海天调味食品股份有限公司 | Aspergillus oryzae and application thereof |
| CN103555594B (en) * | 2013-11-08 | 2015-05-13 | 佛山市海天调味食品股份有限公司 | Aspergillus oryzae and application thereof |
| CN107586729A (en) * | 2017-10-26 | 2018-01-16 | 佛山市海天(高明)调味食品有限公司 | One Aspergillus oryzae ZA138 and its application |
| CN107586729B (en) * | 2017-10-26 | 2018-07-27 | 佛山市海天(高明)调味食品有限公司 | One Aspergillus oryzae ZA138 and its application |
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| CN100455653C (en) | 2009-01-28 |
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