CN101942396A - Aspergillus oryzae HG76 and application thereof - Google Patents

Abstract

The invention discloses aspergillus oryzae HG76 and an application thereof. The aspergillus oryzae HG76 is preserved in China General Microbiological Culture Collection Center on June 25, 2010, and the preservation number is CGMCC No. 3916. Trial-production experimental results show that the strain grows fast and has good genetic stability, neutral protease and acid protease in the prepared finished koji have high vigor, and soy sauce which is prepared by fermentation has high yield. The strain can play an important role in making soy sauce and has wide application prospect.

Description

Aspergillus oryzae HG76 and application thereof

Technical field

The present invention relates to aspergillus oryzae, be specifically related to aspergillus oryzae (Aspergillus oryzae) HG76 and the application in production makes soy sauce thereof.

Background technology

Soy sauce belongs to the traditional zymotic seasoning food, and bacterial classification is the basis of fermentation, and its performance is very big to soy sauce output and quality influence.At present, China soy sauce brewing enterprise uses more than 90% is that aspergillus oryzae (Aspergillus oryzae) Shanghai makes 3.042, this bacterial classification have growth fast, produce that spore is many, enzyme system enriches, neutral protein enzymic activity advantages of higher, be the main bacteria seed of soy sauce brewing.Yet in the high-salt dilute soy fermenting process, the pH value of fermented liquid is along with the prolongation of sauce fermentation time reduces gradually, and neutral and basic protein enzyme activity descends rapidly, and the expression amount of aspartic protease in the whole enzyme of aspergillus oryzae system is on the low side relatively.This phenomenon causes the material protein utilization ratio of soy sauce and amino acid transformation efficiency on the low side, product special flavour a little less than.Therefore, the aspergillus oryzae acid protease activity is on the low side is the key factor that improves China's high-salt dilute fermented sauce industry raw material availability and quality product.

Because the saturation effect and the worry of genetically engineered selection markers aspect food safety of traditional selection by mutation, the improvement of aspergillus oryzae bacterial classification must consider to adopt other approach.The Protoplast Fusion Technique that grows up on the cell hybridization basis has easy, efficient and safe characteristics, parent's good character can be made up by many wheel reorganization, and auxiliary rational screening procedure can obtain aimed strain fast.At aspergillus oryzae acid protease activity defective on the low side, the approach that the acid protease gene of aspergillus niger is recombinated by genome is transferred to the aspergillus oryzae genome, and efficiently expresses, and can obtain high vigor aspartic protease aspergillus fusant bacterial strain.

Summary of the invention

The objective of the invention is to overcome the above-mentioned defective that prior art exists, aspergillus oryzae HG76 and application thereof are provided.

Aspergillus oryzae bacterial strain provided by the present invention is (Aspergillus oryzae) HG76, and it is CGMCC No.3916 that this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center deposit number on 06 25th, 2010.

The bacterium colony of aspergillus oryzae (Aspergillus oryzae) HG76 CGMCC No.3916 is rounded, and colonial morphology and color are partially like A.oryzae HN3042, and be 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.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 a yellow-green colour, is pistac sometimes, and HG76 mycelia branch is thicker, and branch is less, and sporocyst is loose.This bacterial strain has the proteinase activity of higher level, acid protease activity particularly, be 1605u/g (bacterium A.oryzae HN3042 has improved 85.12% than setting out), and its neutral protease activity still has 3303u/g (bacterium A.oryzae HN3042 has reduced by 0.09% than setting out).

The present invention carries out the good quality rice aspergillus tubigensis that protoplastis merges acquisition with aspergillus oryzae and aspergillus niger.Compared with prior art have following advantage and effect: the invention provides an Aspergillus oryzae bacteria (Aspergillus oryzae) HG76CGMCC No.3916.The test manufacture experimental result proves that this bacterial strain genetic stability is good, prepared one-tenth kojic acid proteolytic enzyme and neutral protease vigor height, 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 mensuration that the proteolytic enzyme enzyme is lived

Proteinase activity is measured and is pressed Folin-phenol method, and concrete operations are:

(1) making of typical curve: take by weighing an amount of L-tyrosine, dry to constant weight for 105 ℃, be dissolved in the 0.1mol/L NaCl aqueous solution, be made into the standard tyrosine mother liquor of 1mg/mL.The preparation serial dilutions, its concentration is respectively 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL.Add solution amount in will showing and dwindle 10 times simultaneously, operate by standard program.

(2) enzyme activity determination of sample: get the enzyme liquid 100 μ L after kind of song suitably dilutes, preheating 2min in 40 ℃ of water-baths, the casein solution 100 μ L that add same preheating again, accurately be incubated 10min, add 200 μ L 0.4mol/L trichoroacetic acid(TCA) termination reactions then immediately, continue to place water-bath to be incubated 20min, the centrifugal 10min of residual protein 12000rpm, get 100 μ L supernatants by the bent schedule of operation of mark, measure wherein tyrosine content, produce 1 μ g tyrosine with 40 ℃ of following per minute caseinhydrolysates and be defined as 1 protease activity unit of force.Acetic acid-the sodium-acetate buffer that is determined as pH 3.6 of aspartic protease, the phosphoric acid buffer that is determined as pH 7.0 of neutral protease.Formula is calculated in enzyme work:

$U=\frac{A\&times;4\&times;\mathrm{<figure-callout\; id="10"\; label="N"\; filenames="HSA00000262097700011.png,HSA00000262097700012.png"\; state="\{\{state\}\}">N</figure-callout>}}{10\&times;G\&times;(1-W)}---\left(1\right)$

In the formula, A is for to record the OD value by sample, looks into typical curve and gets suitable tyrosine micrograms; 4 is that 400 μ L reaction solutions take out 100 μ L mensuration (promptly 4 times); N is the multiple of enzyme liquid dilution; 10 is reaction times 10min; G is example weight (g); W is a sample moisture percentage composition.

Description of drawings

Fig. 1 be in aspergillus oryzae HG76 and 3042 pairs of sauce fermentation liquid of aspergillus oryzae total nitrogen influence figure

Fig. 2 be in aspergillus oryzae HG76 and 3042 pairs of sauce fermentation liquid of aspergillus oryzae ammonia-state nitrogen influence figure.

Embodiment

Below in conjunction with accompanying drawing concrete enforcement of the present invention is described further, but enforcement of the present invention and protection domain are not limited thereto.

Merge the preparation of strain HG76 CGMCC No.3916

Available aspergillus oryzae substratum and following cultural method are cultivated aspergillus oryzae of the present invention (Aspergillusoryzae) HG76C GMCC No.3916.

Is 10 with aseptic technique with 500 μ L concentration ⁷The aspergillus of individual/mL (aspergillus oryzae 3042 and aspergillus niger CICC2377) spore suspension is inoculated in the 50mL MPY liquid nutrient medium, and 30 ℃/100rpm shaking culture 12-24h gets mycelium pellet.With mycelium pellet through four layers of aseptic lens wiping paper suction filtration, twice of rinsed with sterile water, use twice of 10mL high osmotic buffer centrifuge washing (5000rpm * 5min) again, after suspending, the high sepage of 10mL adds 100 μ L beta-mercaptoethanols, mix back incubation 30min under 30 ℃/100rpm condition, must handle back aspergillus oryzae 3042 mycelium and aspergillus niger CICC2377 mycelium.

The mycelium of getting after the processing adds in the prozyme liquid of lywallzyme and helicase by the amount of 1.5g/10mL, carries out enzymolysis 3h under 30 ℃/80rpm condition.By two-layer 300 order nylon net filters, filtrate is in the centrifugal 10min of 2000rpm with enzymolysis solution, and high sepage damping fluid centrifuge washing twice gets aspergillus oryzae 3042 protoplastis suspension and aspergillus niger CICC2377 protoplastis suspension.

Get A.oryzae HN3042 protoplastis suspension (5.0 * 10 ⁶Individual/mL) 4mL moves in the culture dish of diameter 5.0cm.Liquid level is apart from ultraviolet light vertical range 13cm, and ultraviolet light power 15W opens ultraviolet light.Get the postradiation protoplastis suspension of 100 μ L behind the 15min in the high sepage of 900 μ L, get 100 μ L behind the mixing respectively and blend in the common soft agar (0.5%, 45 ℃) in the 5mL height, palm bounces and makes it to fall behind the mixing height and blend common regenerated plate.30 ℃ leave standstill cultivation 48h.

Get A.niger CICC2377 protoplastis suspension (3.0 * 10 ⁶Individual/mL) 1.0mL is in the 2mLEP pipe, and 65 ℃ of waters bath with thermostatic control are put into cooled on ice behind the insulation 15min immediately.Get protoplastis suspension after the 100 μ L thermal treatments in the high sepage of 900 μ L, get 100 μ L behind the mixing respectively and blend (0.5% agar, 45 ℃) in the common soft agar medium in the 5mL height, palm bounces and makes it to fall behind the mixing height and blend common regenerated plate.30 ℃ leave standstill cultivation 48h.

Equivalent parents' protoplastis after the asymmetric deactivation is mixed, be adjusted to 10 with electric shock liquid ⁶The concentration of individual/mL is drawn 500 μ L protoplastis suspension with micropipet then and is placed electric fusion instrument groove (3mm spacing), and integration slot is positioned on ice, adjusts pre-fusion strength of electric field and time to protoplastis item chain and arrange under inverted microscope.Different fusion parameters is set then carries out electricity and merge, draw 100 μ L and merge liquid in 5mL is cooled to 45 ℃ soft agar, be applied to height after palm bounces and makes it to mix and ooze on the MPY solid medium, 30 ℃ are cultured to bacterium colony and occur.With acid protease activity and middle letter proteinase activity is index, and the gained bacterium colony is screened, and must merge strain HG76 CGMCC No.3916.

Merge strain HG76 CGMCC No.3916 and parent in the bacterium colony and the mycelia poor morphology opposite sex

Merge strain HG76 and parent's aspergillus oryzae 3042 in mycelia thickness, branch degree, slipperiness, and sporocyst form aspect all exists bigger difference.Parent's aspergillus oryzae 3042 mycelia branches are thin and close, and sporocyst is loose; The aspergillus niger mycelia is thick and smooth, and branch is few, the smooth compactness of sporocyst; And merge the aspergillus oryzae that tends to that strain HG76 has on mycelia branch degree, but branch is than aspergillus oryzae more slightly, and the sporocyst form is all tended to aspergillus oryzae.

Merge strain HG76 CGMCC No.3916 and the parent otherness on acid and neutral protease vigor

To merge strain HG76 CGMCC No.3916 and parent's aspergillus oryzae 3042 respectively by 10 ⁵The concentration of individual spore/g kind song is inoculated in respectively in the good bent substratum of kind of sterilization, cultivates 72h for 30 ℃, shakes bottle once every 12～13h.After finishing, cultivation will plant bent crushing, with the phosphate buffer solution of 0.2mol/L NaCl solid-liquid ratio by 1: 5,100rpm extracts 1h under 40 ℃ of water-baths, measure aspartic protease and neutral protease vigor, the results are shown in Table 1 (genetic stability of the aspartic protease of aspergillus oryzae HG76) and table 2 (genetic stability of the neutral protease of aspergillus oryzae HG76).

HG76 CGMCC No.3916 is to the influence of sauce fermentation system total nitrogen and amino nitrogen

Soaked soybean is put into high-pressure sterilizing pot carry out boiling, exhaust 3min treats to pick up counting when temperature rises to 120 ℃, puts the steam step-down behind the boiling 20min at once, takes out soybean.Mix with flour when soybean is cooled to 50 ℃ of left and right sides, inoculate HG76 and aspergillus oryzae respectively.

The Daqu culture condition is as follows: humidity: 95%, and go out bent preceding 4h and fall humidity to 70%; Temperature: 28 ℃; Incubation time is 44h; Bent material turns over song when being cultured to 16h and 24h, gets Daqu.Daqu with after 18% salt solution mixed with 1: 2.2, was fermented 60 days.As seen from Figure 1, the total nitrogen of HG76 fermented liquid is greater than aspergillus oryzae 3042 in the whole fermentation process, and during to 30 days, total nitrogen content can reach 1.07g/100mL in the HG76 fermented liquid in fermentation, exceeds about 8.1% than the 0.99g/100mL of aspergillus oryzae 3042.As seen from Figure 2, the ammonia-state nitrogen of HG76 fermented liquid is greater than aspergillus oryzae 3042 in the whole fermentation process, and during to 30 days, total nitrogen content can reach 0.62g/100mL in the HG76 fermented liquid in fermentation, exceeds 6.9% than the 0.58g/100mL of A.oryzae HN3042.

Table 1

Table 2

Claims (2)

1. aspergillus oryzae HG76, this aspergillus oryzae (Aspergillus oryzae) was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 06 25th, 2010, and deposit number is CGMCC No.3916.

2. the application of the described aspergillus oryzae of claim 1 (Aspergillus oryzae) HG76 CGMCC No.3916 in production makes soy sauce.

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