CN106399190A - Bacillus amyloliquefaciens capable of efficiently utilizing arginine and citrulline and application of bacillus amyloliquefaciens - Google Patents

Bacillus amyloliquefaciens capable of efficiently utilizing arginine and citrulline and application of bacillus amyloliquefaciens Download PDF

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CN106399190A
CN106399190A CN201610926520.4A CN201610926520A CN106399190A CN 106399190 A CN106399190 A CN 106399190A CN 201610926520 A CN201610926520 A CN 201610926520A CN 106399190 A CN106399190 A CN 106399190A
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bacillus amyloliquefaciens
citrulline
cfu
arginine
fermentation
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CN106399190B (en
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方芳
张梦寒
王博
陈坚
堵国成
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Jiangnan University
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Abstract

The invention discloses bacillus amyloliquefaciens capable of efficiently utilizing arginine and citrulline and application of the bacillus amyloliquefaciens, and belongs to the technical field of food microorganisms. The bacillus amyloliquefaciens JY06-C12 capable of efficiently utilizing the arginine and the citrulline is obtained by means of plasma mutation breeding. The bacillus amyloliquefaciens and the application have the advantages that the bacillus amyloliquefaciens which is a strain can efficiently utilize the arginine and the citrulline under high-salinity conditions and is excellent in passage stability; the content of citrulline which is a precursor of ethyl carbamate can be reduced by 15.7% if the strain is added into soy sauce in soy sauce brewing procedures, and accordingly the bacillus amyloliquefaciens is beneficial to controlling and reducing the content of the ethyl carbamate in the soy sauce.

Description

The bacillus amyloliquefaciens of a kind of efficient utilization arginine and citrulline and its application
Technical field
The present invention relates to the bacillus amyloliquefaciens of a kind of efficient utilization arginine and citrulline and its application, belong to food Microbial technology field.
Background technology
Urethanes (Ethyl carbamate, EC) are a kind of carcinogen, and being continued Excess free enthalpy by human body may The serous tumors diseases such as the lung cancer caused, hepatocarcinoma of meeting, and the immune system of human body is damaged.
Soy sauce is a kind of widely used fermented seasonings of food industry, is have unique color nutritious Functional food, in China's production history of existing 2500.In recent years result of study shows, potential carcinogen amino first Acetoacetic ester (EC), is widely present in the soy sauce that especially Japanese technique is produced in soy sauce, what this was serious have impact on soy sauce Foodsafety.
The main producers material of middle urethanes of making soy sauce is citrulline and ethanol.Therefore reduce melon ammonia in soy sauce The content of acid is that we reduce one of urethanes effective measures in soy sauce.Produce early stage in soy sauce, raw material hydrolysis is big Amount arginine, these arginine, in the presence of the lactic acid bacteria of a big class anaerobism or amphimicrobian, are converted into citrulline.If The bacterial strain that these arginine are had the de- imino group approach of complete arginine by some utilizes, and conversion of Arginine is become ornithine, just The citrulline accumulation in soy sauce can be reduced.Therefore, one plant of screening is resistant to high salt, and being capable of Efficient Conversion arginine and not The bacterial strain of accumulation citrulline, has significant application value for the content reducing urethanes in soy sauce.
Content of the invention
First purpose of the present invention is to provide a bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) JY06-C12, is preserved in China typical culture collection center, preservation on 19th in September in 2016 Numbering is CCTCC NO:M 2016498, preservation address is Wuhan, China Wuhan University.
Second object of the present invention is to provide the cultural method of described bacillus amyloliquefaciens, is by described solution starch bud Spore bacillus BBE-JY06-C12 is seeded to LB fluid medium, 30 DEG C of quiescent culture 24～48h.
In one embodiment of the invention, described LB culture medium also contains 80～150g/LNaCl.
In one embodiment of the invention, described bacillus amyloliquefaciens JY06-C12 condition of culture is：By preservation Mutagenic strain rule on the LB solid medium containing 100g/L NaCl, in 37 DEG C of quiescent culture 2d.
Third object of the present invention is to provide a kind of method reducing citrulline content in soy sauce, and methods described is by institute State bacillus amyloliquefaciens JY06-C12 to be seeded in moromi.
In one embodiment of the invention, methods described is the 0～3d inoculation solution starch spore in sauce fermentation Bacillus reaches 10 to bacillus amyloliquefaciens in moromi6CFU/mL to 108CFU/mL.
In one embodiment of the invention, methods described, specifically：(1) yeast production：Defatted soybean and cooked wheat are through leaching Bubble, sterilizing, after cooling, add wheat bran, flour, and inoculate aspergillus oryzae spore, culture, treat Qu Biancheng peak green, yeast production completes； (2) ferment：Cheng Qu mixes saline, start ferment, fermentation initiate to the 0th～3 day in inoculate bacillus amyloliquefaciens, temperature control 10 ～20 DEG C, fermentation 7d inoculation 107The Lu Shi yeast of CFU/mL (the finally concentration in moromi), fermentation 14d inoculates Shandong Family name's yeast is to 107CFU/mL (the finally concentration in moromi), 30 DEG C of ferment at constant temperature, 90d terminates to ferment.
Beneficial effect：Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the JY06-C12 energy of the present invention Enough efficient utilization arginine and citrulline under high salt conditions, it has reached 96.3% to arginic utilization rate, does not accumulate melon Propylhomoserin, and possess good mitotic stability.This bacterial strain is added to soy sauce brewing, urethanes can be made Precursor citrulline content reduces by 15.7%, contributes to controlling and reduce the content of soy sauce urethanes.
Brief description
Fig. 1 is the hereditary stability of mutant JY06-C12；JH06, bacillus amyloliquefaciens BBE-JY06；C12, Xie Dian Afnyloliquefaciens JY06-C12；* represent the bacterial strain after passing on for 200 generations.
Biomaterial preservation
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JY06-C12, in September in 2016 19 days It is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2016498, preservation address is Wuhan, China, Wuhan University.
Specific embodiment
Aminoacid detects culture medium：Yeast extract 5g/L, Carnis Bovis seu Bubali cream 5g/L, tryptone 5g/L, NaCl 180g/L, Fructus Vitis viniferae Sugared 0.5g/L, Tween-80 1g/L, MgSO4·7H2O 0.2g/L, MnSO4·H2O 0.05g/L, FeSO40.4g/L, citric acid Triamine 2g/L, CaCO30.1g/L, pyridoxal 5-phosphate 0.05g/L, K2HPO42g/L, pH 6.0.For detecting specific amino acids When, add aminoacid 6g/L to be measured.
Bacillus amyloliquefaciens JY06-C12 cultural method：By the bacterial strain of preservation in the LB solid culture containing 100g/LNaCl Rule on base, in 37 DEG C of quiescent culture 2d, picking single bacterium colony accesses the LB fluid medium containing 100g/L NaCl, 37 DEG C of shaking tables Supernatant, the phosphate buffer resuspension of use pH 7.0 of obtained thalline is removed after culture 36h, 8000rpm centrifugation 1min.
Aminoacid detection method：It is measured using high performance liquid chromatography (HPLC method).Before sample being carried out before measuring Process：Take 1mL fermentation liquid, remove thalline after 12000rpm/min centrifugation 10min and take supernatant.Trichloroacetic acid with 5% is by supernatant After liquid dilutes 5 times, via hole diameter is 0.22 μm of membrane filtration, and the sample after process is placed in -20 DEG C of preservations.
Chromatographic condition is：Agilent 1200 chromatograph, chromatographic column C18, aperture 250 × 4.6mm.Detector is that VWD is purple External detector, Detection wavelength 338nm, 40 DEG C of column temperature.
Mobile phase A phase (1L)：Anhydrous sodium acetate 5g, oxolane 5mL, triethylamine 200 μ L, pH are 7.2.
Mobile phase B phase (1L)：Anhydrous sodium acetate 5g, ultra-pure water 200mL, methanol 400mL, acetonitrile 400mL, pH are 7.2.
Embodiment 1：The screening of bacillus amyloliquefaciens
(1) with deposit number for CCTCC NO:Bacillus amyloliquefaciens BBE-JY06 (the letter in embodiment of M 2015423 Writing bacillus amyloliquefaciens JY06) as starting strain, bacillus amyloliquefaciens JY06 is seeded to LB fluid medium, 37 DEG C, 220rpm/min shaking table culture 4～6h is to exponential phase.
(2) the bacteria suspension Deca taking 10 μ L, on the microscope slide after sterilizing cooling, carries out plasma mutation, mutation with ARTP Time is respectively 0s, 5s, 10s, 15s, 20s, 40s, 60s.
(3) bacteria suspension after mutation is transferred in the sterile tube equipped with 1mL normal saline, then by diluted sample extremely 10-1～10-3Three concentration, take diluent to coat in screening culture medium, are placed in culture 24～36h in 37 DEG C of constant incubators, Each is processed in triplicate.
(4) picking colony carries out arginine colour developing secondary screening.
(5) pipette 50 μ L and meet supernatant corresponding to the bacterial strain of primary dcreening operation condition in the centrifuge tube of 1.5mL, then divide again To in centrifuge tube, do not add 40g/L NaOH solution, 80g/L alpha naphthol normal propyl alcohol solution and 0.5mL/L biacetyl normal propyl alcohol solution Each 1mL, after concussion shakes up, puts into chromogenic reaction 15min in 30 DEG C of water-baths.After taking out cooling a period of time, in spectrophotometric Light absorption value OD is surveyed on meter540, using nonvaccinated culture medium as comparison.Light absorption value OD540Less bacterial strain is efficient utilization essence The bacterial strain of propylhomoserin.
Embodiment 2：Bacillus amyloliquefaciens JY06-C12 utilizes arginine capability analysis
The original strain bacillus amyloliquefaciens JY06 activating on picking flat board respectively and mutant strain solution starch spore The single bacterium colony of bacillus JY06-C12 is inoculated in the LB fluid medium of 100g/LNaCl, 37 DEG C of shaking table cultures 36 hours, takes 5mL Bacterium solution centrifugation 1min (8000rpm, 4 DEG C) abandons supernatant, and obtained thalline adds the phosphate buffer resuspension of 1mLpH7.0.
The arginic aminoacid detection culture medium that 4mL contains 10g/L, the suspension of inoculation 500 μ L is added in 5mL centrifuge tube Bacterium solution, OD after inoculation600For 7.8.30 DEG C of quiescent culture use the method for HPLC to detect arginine and citrulline in culture medium after 3 days And ornithine content.
Table 1 arginine metabolism related amino acid content
Note：ΔArg(g/L)：Arginic consumption；ΔCit(g/L)：The growing amount of citrulline；ΔOrn(g/L)：Bird The growing amount of propylhomoserin
As shown in Table 1, the arginic ability of bacillus amyloliquefaciens JY06-C12 metabolism with compare bacillus amyloliquefaciens JY06 compares and improves 18%, and arginine also improves 64% to the conversion ratio of ornithine.
Embodiment 3：Bacillus amyloliquefaciens JY06-C12 mitotic stability
Respectively bacillus amyloliquefaciens JY06, mutant JY06-C12 are seeded to LB fluid medium to cultivate, 37 DEG C, 220rmp/min cultivates, and is forwarded in new LB fluid medium every 24h, after culture 20d, takes the centrifugation of 4mL bacterium solution to remove supernatant, With twice of sterile water wash thalline, isopyknic arginine is added to utilize culture medium, 30 DEG C of quiescent culture 3～5d.
Take 1mL sample, after centrifugation, take supernatant.After supernatant is diluted 20 times by the trichloroacetic acid with 5%, via hole diameter is 0.22 μm membrane filtration.With arginine content in high effective liquid chromatography for measuring sample.
As shown in figure 1, mutant JY06-C12 is good to the hereditary stability of arginine utilization power.After passing on for 200 generations, The arginic ability of mutant JY06-C12 metabolism is 1.97 times of original strain JY06.
Embodiment 4：Application bacillus amyloliquefaciens JY06-C12 produces soy sauce
(1) yeast production：Defatted soybean and cooked wheat soak 8h, 121 DEG C of sterilizing 5min, are cooled to 80 DEG C, be proportionally added into wheat bran, Flour, defatted soybean, cooked wheat, wheat bran, the mass ratio of flour are 20:15:1:1, it is concurrently accessed aspergillus oryzae spore, inoculum concentration is 8 ×109Individual/g initial feed, cultivates in 30 DEG C of calorstats, and 6-8h turns over Qu Yici, cultivates 46-48h, Qu Biancheng peak green, yeast production Complete；
(2) ferment：Saline (the w of Cheng Quyu 20%:V) with volume ratio for 1:1.7 ratio mixed fermentation, 0-3d is respectively Inoculation bacillus amyloliquefaciens JY06 and mutant JY06-C12,10-20 DEG C of temperature control, fermentation 7d inoculation is final concentration of 107The Lu Shi yeast of CFU/mL, fermentation 14d inoculates Lu Shi yeast to final concentration of 10 in moromi7CFU/mL, 30 DEG C Ferment at constant temperature, 90d terminates to ferment.
In sweat, moromi sample is sampled, measures wherein amino acid content, result is as shown in table 2.Table 2 data Show, 0～3d with the addition of 10 respectively7CFU/mL～108CFU/mL bacillus amyloliquefaciens JY06's and mutant JY06-C12 In soy sample, can be transferred through arginine desimidase metabolic pathway and arginine is converted in a large number ornithine.And 0～3d With the addition of 107CFU/mL～108In the soy sample of CFU/mL mutant JY06-C12, the content of citrulline is minimum, and with the addition of The soy sample of initial strains JY06 is compared, and citrulline content reduces 15.7%, EC content and reduces 19.3%.
Arginine metabolism related amino acid and EC content analysis in crude oil after table 2 squeezing
Note：1 is without JY06；2 is that 0～3d adds 107CFU/mL～108The JY06 of CFU/mL；3 add for 0～3d Plus 107CFU/mL～108The JY06-C12 of CFU/mL.
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

Claims (8)

1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JY06-C12, in September 19 in 2016 Day is preserved in China typical culture collection center, and deposit number is CCTCC NO:M 2016498, preservation address is that China is military Chinese Wuhan University.
2. the cultural method of bacillus amyloliquefaciens described in claim 1 is it is characterised in that be to be seeded to LB culture medium at 37 DEG C Lower culture 24～48h.
3. method according to claim 2 is it is characterised in that described LB culture medium also contains 80～150g/LNaCl.
4. a kind of method reducing ethyl carbamate content in soy sauce is it is characterised in that methods described is by claim 1 institute The bacillus amyloliquefaciens JY06-C12 stating is seeded in moromi.
5. method according to claim 4 is it is characterised in that the 0～3d inoculation in sauce fermentation solves starch spore bar Bacterium reaches 10 to bacillus amyloliquefaciens in moromi6CFU/mL to 108CFU/mL.
6. method according to claim 4 is it is characterised in that methods described, specifically：(1) yeast production：Defatted soybean and stir-fry Wheat is soaking, sterilizing, after cooling, add wheat bran, flour, and inoculate aspergillus oryzae spore, culture, treats Qu Biancheng peak green, yeast production Complete；(2) ferment：Cheng Qu mixes saline, start ferment, fermentation initiate to the 0th～3 day in inoculate bacillus amyloliquefaciens, 10～20 DEG C of temperature control, fermentation 7d inoculation 107The Lu Shi yeast of CFU/mL (the finally concentration in moromi), fermentation 14d is again Inoculation Lu Shi yeast is to 107CFU/mL (the finally concentration in moromi), 30 DEG C of ferment at constant temperature, 90d terminates to ferment.
7. the application in terms of the urethanes in reducing food of bacterial strain described in claim 1.
8. bacterial strain described in claim 1 is preparing food, the application in health product.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108902748A (en) * 2018-08-24 2018-11-30 江南大学 A kind of method of urethanes in reduction thick broad-bean sauce

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018381A (en) * 2015-07-23 2015-11-04 江南大学 Bacillus amyloliquefaciens utilizing arginine without accumulation of citrulline
CN105639590A (en) * 2015-12-29 2016-06-08 江南大学 Method for applying bacillus amyloliquefaciens to soy sauce brewage
CN106282071A (en) * 2016-10-26 2017-01-04 江南大学 One strain degraded urethanes and the bacillus amyloliquefaciens of carbamide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018381A (en) * 2015-07-23 2015-11-04 江南大学 Bacillus amyloliquefaciens utilizing arginine without accumulation of citrulline
CN105639590A (en) * 2015-12-29 2016-06-08 江南大学 Method for applying bacillus amyloliquefaciens to soy sauce brewage
CN106282071A (en) * 2016-10-26 2017-01-04 江南大学 One strain degraded urethanes and the bacillus amyloliquefaciens of carbamide

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108902748A (en) * 2018-08-24 2018-11-30 江南大学 A kind of method of urethanes in reduction thick broad-bean sauce

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