CN106978371A - One plant can efficiently utilize Lactobacillus brevis and its application of citrulling - Google Patents
One plant can efficiently utilize Lactobacillus brevis and its application of citrulling Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses Lactobacillus brevis and its application that one plant can efficiently utilize citrulling, belong to technical field of biological fermentation.The Lactobacillus brevis (Lactobacillus brevis) 2 34 of the present invention, is preserved in China typical culture collection center on March 23rd, 2017, and preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2017140.The bacterium not only can produce citrulling using arginine, and can be absorbed after logarithmic phase is grown to and utilize extracellular citrulling, and the bacterium also can be individually with the citrulling in culture medium in addition.
Description
Technical field
The present invention relates to Lactobacillus brevis and its application that one plant can efficiently utilize citrulling, belong to biofermentation technique neck
Domain.
Background technology
Urethanes (Ethyl carbamate, abbreviation EC) is a kind of carcinogenic substance, is widely present in sausage, fermentation
In the fermented foods and alcoholic beverage such as sour flour dough, soy sauce, grape wine and yellow rice wine.Research finds that EC contents are of a relatively high in yellow rice wine,
Main reacted in a heated condition by carbamyl compounds such as ethanol and urea, citrulling is formed.Therefore, EC in reduction yellow rice wine
The content of precursor urea and citrulling turns into control EC effective measures.
Urea is mainly derived from the urea cycle of yeast in yellow rice wine, and citrulling is then mainly derived from the arginine of lactic acid bacteria
De- imines enzymatic pathway.It is main at present to be sent out by the yeast of seed selection high-yield urea or the bacterial strain of the high urease-producing of screening applied to yellow rice wine
Ferment, but the content of citrulling can not be reduced simultaneously.Citrulling is not produced or melon can be absorbed if can screen
The lactic acid bacteria of propylhomoserin, then the problem of can further solving reduction EC precursors.
Have also discovered in the fermented foods such as grape wine, sausage, sourdough and alcoholic beverage production process to weigh
The Bu Shi lactobacillus Lactobacillus buchneri CUC-3 separated in the lactic acid bacteria of citrulling, such as grape wine are absorbed, are used
In the Lactobacillus saki Lactobacillus sakei CTC494 of sausage fermentation, the lactobacillus fermenti separated from sourdough
Lactobacillus fermentum IMDO130101 etc., but these bacterial strains needs could absorb profit in the presence of Arginine free
With citrulling, and part citrulling can only be absorbed, at most absorb 300mg.
Meaning of the present invention be from yellow wine fermentation liquid screening obtain it is a kind of can efficient absorption utilize the short of citrulling
Lactobacillus, and be applied in brewing yellow rice wine, so as to efficient absorption and using the citrulling in zymotic fluid, reduce EC's
Formed, improve the drinking safety of yellow rice wine.
The content of the invention
The invention provides the Lactobacillus brevis (Lactobacillus brevis) that one plant of energy efficient absorption utilizes citrulling
2-34.The Lactobacillus brevis 2-34 was preserved in China typical culture collection center on March 23rd, 2017, during preservation address is
Wuhan Wuhan University of state, deposit number is CCTCC NO:M 2017140.
The Lactobacillus brevis L.brevis 2-34 are isolated from yellow wine fermentation liquid, and separation method is as follows：Yellow wine fermentation liquid is used
Sterile saline (0.85%, w/v) presses 1:2 dilution proportions, are coated with MRS flat boards, are placed in anaerobic jar 30 DEG C and cultivate 3-5 days,
Picking single bacterium colony is seeded to MRS fluid nutrient mediums, cultivates 2 days, microscopy is determined after strain morphology, extracts genome and carries out 16S
RDNA is identified.The MRS culture mediums are 10g/L peptones, 10g/L beef extracts, 5g/L dusty yeasts, 20g/L glucose, 5g/L second
Sour sodium, 2g/L ammonium citrates, 2g/L dipotassium hydrogen phosphates, 1mL/L Tween-80s, 0.58g/L bitter salts, the water of 0.25g/L mono-
Manganese sulfate is closed, solid medium then adds 20g/L agar in addition.
The Lactobacillus brevis L.brevis 2-34 are gram-positive bacteria, shaft-like, and part cell aggregation forms short chain, mistake
Hydrogen oxide enzyme is detected as catalase feminine gender, and sports type experiment proves without motion, do not reduce nitrate, do not liquefy gelatin, can profit
With 8 kinds of carbohydrates such as arabinose, fructose, galactolipin, glucose, lactose maltose, melibiose, sucrose, xylose, breast can be produced
Acid.
The method for preserving of the Lactobacillus brevis L.brevis 2-34 is：Single bacterium on picking flat board falls within MRS Liquid Cultures
1mL bacterium solutions are drawn in glycerine cryopreservation tube after base, culture 16h, and add the glycerine of 1mL 50%.
The condition of culture of the Lactobacillus brevis L.brevis 2-34 is：50 μ L bacterium solutions are drawn from glycerol tube and are inoculated in 3mL
MRS fluid nutrient mediums, 30 DEG C stand 12~16h of activation culture and reach mid-log phase, then as needed expand seed liquor and train
Support.
The Lactobacillus brevis L.brevis 2-34 are with MRS culture medium (i.e. MRS- of the arginine (5g/L) for precursor substance
Arg culture mediums) in when growing, arginine is gradually decreased, citrulling accumulation, illustrates the bacterium using arginine and to exocytosis melon
Propylhomoserin (up to 102mg/L), during to mid-log phase, extracellular citrulling disappears, and is also said afterwards no longer to exocytosis citrulling
Bright thalline can be reuptaked and utilize citrulling.The bacterium is with MRS culture medium (i.e. MRS- of the citrulling (4g/L) for precursor substance
Cit culture mediums) in grow when, absorb citrulling simultaneously utilized, when enter logarithmic phase mid-term when, citrulling utilization rate is significantly carried
Height, can all absorb citrulling to the maturity period.
The Lactobacillus brevis L.brevis 2-34 are cultivated in MRS-Arg and MRS-Cit containing 5%~15% (V/V) alcohol
When cultivating in base, it can continue to grow and using arginine and citrulling, alcohol concentration more Seedling height speed is slower, arginine,
The utilization rate of citrulling is also lower, but by fermentation after, final cell concentration and consistent in nonalcoholic culture medium, smart ammonia
Acid and citrulling can also be absorbed and used.
Yellow rice wine is improved in brewing yellow rice wine drink peace the invention provides the above-mentioned Lactobacillus brevis L.brevis 2-34 of one kind application
The method of full property, after actication of culture is spread cultivation, prepares high density bacterium solution (108~1010Cfu/L), by 0.1%~1% (mL/L)
Ratio and distiller's yeast, water, glutinous rice, wheat koji raw material fall tank after mixing, fermented.
In one embodiment of the present invention, Lactobacillus brevis L.brevis 2-34 are seeded to activation medium activation 12-
16h, is inoculated into the automatic anaerobic fermentation tanks of 5L and carries out high density fermentation, 30 DEG C of cultivation temperature, pH 5.0, incubation time 36-48h,
High density bacterium solution (10 is prepared through 15000g refrigerated centrifuges 10min8~1010cfu/L).In the case where not changing other techniques,
By high density bacterium solution (108~1010Cfu/L) be applied to brewing yellow rice wine, when falling tank with 0.1%~1% (mL/L) ratio with
After distiller's yeast, water, glutinous rice, wheat koji raw material are mixed, fermented.
Beneficial effects of the present invention
The Lactobacillus brevis L.brevis 2-34 of the present invention carry out brewing yellow rice wine, compared to without Lactobacillus brevis L.brevis
2-34 carry out brewing yellow rice wine will be appreciated that, keep rice wine flavor on the basis of, significantly reduce melon ammonia in yellow wine fermentation liquid
The content of acid and urethanes, at the end of rear ferment, compared to without the yellow rice wine hair for adding Lactobacillus brevis L.brevis 2-34
Zymotic fluid, be vaccinated with citrulling and ethyl carbamate content in Lactobacillus brevis L.brevis 2-34 mash reduces respectively
58.2% and 29.6%.In addition, Lactobacillus brevis L.brevis 2-34 are isolated from yellow wine fermentation mash, belong to the breast of food security
Sour bacterium.
Biomaterial preservation
Lactobacillus brevis 2-34Lactobacillus brevis 2-34, Chinese Typical Representative training is preserved on March 23rd, 2017
Thing collection is supported, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2017140.
Brief description of the drawings
Cell concentration and amino acid content when Fig. 1 Lactobacillus brevis L.brevis 2-34 are cultivated in MRS-Arg culture mediums
Change.
Cell concentration and amino acid content when Fig. 2 Lactobacillus brevis L.brevis 2-34 are cultivated in MRS-Cit culture mediums
Change.
Embodiment
The Lactobacillus brevis L.brevis 2-34 screening techniques of embodiment 1
Yellow wine fermentation liquid is pressed 1 with sterile saline (0.85%, w/v):2 dilution proportions, are coated with MRS flat boards, are placed in
Cultivate 3-5 days for 30 DEG C, picking single bacterium colony is seeded to MRS fluid nutrient mediums, cultivate 2 days, microscopy determines strain morphology in anaerobic jar
Afterwards, extract genome and carry out 16S rDNA identifications.The MRS culture mediums are 10g/L peptones, 10g/L beef extracts, 5g/L yeast
Powder, 20g/L glucose, 5g/L sodium acetates, 2g/L ammonium citrates, 2g/L dipotassium hydrogen phosphates, 1mL/L Tween-80s, 0.58g/L seven
Magnesium sulfate heptahydrate, 0.25g/L Manganous sulfate monohydrates, solid medium then adds 20g/L agar in addition.
The Lactobacillus brevis L.brevis 2-34 of embodiment 2 authentication method
By yellow wine fermentation liquid and sterile saline (0.85%, w/v) with 1:2 dilution proportions, are applied on MRS flat boards,
Grown 3-5 days in anaerobic jar, picking single bacterium falls on MRS fluid nutrient medium cultures, microscopy determines to use Ezup plants after strain morphology
Formula bacterial genomes extracts kit (Shanghai life work) extracts the genome of the bacterium, using genome as template, with sequence respectively such as
SEQ ID NO:2 and SEQ ID NO:3 bacterial 16 S rDNA universal primers 27F and 1492R (5 '-
AGAGTTTGATCCTGGCTCAG-3 ' ,/5 '-GGTTACCTTGTTACGACTT-3 ') enter performing PCR, amplification 16S rDNA for primer
Gene (about 1400bp), and the sequencing of Shanghai Tian Lin biotech firms is delivered to, by sequence and the existing sequence alignments of NCBI, determine that the bacterium is
Lactobacillus brevis (sequence similarity is 99%), 16S rDNA nucleotide sequences are shown in SEQ ID NO.1.The bacterium is in March, 2017
China typical culture collection center is preserved within 23rd, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC
NO:M 2017140。
The detection of the Lactobacillus brevis L.brevis 2-34 citrulling Utilization abilities of embodiment 3
The Lactobacillus brevis L.brevis 2-34 activated in MRS culture mediums are inoculated into amino acid detection training by 1% inoculum concentration
Support base (add the arginic MRS-Arg of 5g/L in MRS culture mediums and add the MRS-Cit culture mediums of 4g/L citrulling), 30
Arginine and citrulling contains in continuous sampling in DEG C quiescent culture 72h, fermentation process, high performance liquid chromatography detection zymotic fluid
Amount.As a result show, when the bacterium grows in MRS-Arg culture mediums, arginine is gradually decreased, citrulling accumulation, to mid-log phase
When (culture 18h) citrulling accumulation 102mg/L, illustrate the bacterium using arginine and to exocytosis citrulling, hereafter, arginine
Further utilized by bacterial strain, but extracellular citrulling disappears, and bacterial strain illustrates that the bacterial strain can be again also no longer to exocytosis citrulling
Absorb and utilize citrulling (Fig. 1).When the bacterium grows in MRS-Cit culture mediums, as thalli growth is absorbable citrulling
And utilized, when culture 12h enters logarithmic phase mid-term, citrulling is significantly improved using speed, when cultivating to 21h, culture medium
In citrulling can all be absorbed and used (Fig. 2).
Embodiment 4 is without Lactobacillus brevis L.brevis 2-34 brewing yellow rice wines
Process for making yellow rice wine flow：Rice dipping-steamed rice-cooling-mixed song-falls tank-preceding ferment-rear ferment-filtering clarification.
Rice dipping：Appropriate glutinous rice is taken, added water dipped rice layer 10cm, and 28 DEG C are soaked 3 days.
Steamed rice：Glutinous rice after immersion carries out atmospheric cooking, and a large amount of steam maintain 25min after emerging.
Cooling：The glutinous rice cooked is placed in ventilation spreading for cooling to room temperature.
Mixed song：Ratio according to 130g raw wheat kojis, 40g ripe wheat kojis per 1kg glutinous rice adds wheat koji, and stand mixes uniform.
Fall tank：The glutinous rice mixed and wheat koji, water (1.7L is per 1kg glutinous rice), yeast starter liquid (10%V/V) are added together
Fermentation tank, is well mixed.
Preceding ferment：30 DEG C ferment 3-4 days.
Ferment afterwards：15 DEG C ferment 15 days or so.
Filtering clarification.
Applications of the Lactobacillus brevis L.brevis 2-34 of embodiment 5 in brewing yellow rice wine
Lactobacillus brevis L.brevis2-34 is seeded to activation medium MRS (pH5.0), 30 DEG C of activation culture 12-16h,
It is inoculated into the automatic anaerobic fermentation tanks of 5L and carries out high density fermentation, 30 DEG C of cultivation temperature, pH 5.0, incubation time 36-48h, warp
15000g refrigerated centrifuges 10min prepares high density bacterium solution (1010cfu/L).When carrying out brewing yellow rice wine, do not changing other techniques
In the case of, after when falling tank, high density bacterium solution is mixed with 0.1% (mL/L) ratio and distiller's yeast, water, glutinous rice, wheat koji raw material,
Fermented.
Applications of the Lactobacillus brevis L.brevis 2-34 of embodiment 6 in brewing yellow rice wine
Lactobacillus brevis L.brevis2-34 is seeded to activation medium MRS (pH5.0), 30 DEG C of activation culture 12-16h,
It is inoculated into the automatic anaerobic fermentation tanks of 5L and carries out high density fermentation, 30 DEG C of cultivation temperature, pH 5.0, incubation time 36-48h, warp
15000g refrigerated centrifuges 10min prepares high density bacterium solution (108cfu/L).When carrying out brewing yellow rice wine, do not changing other techniques
In the case of, after when falling tank, high density bacterium solution is mixed with 0.5% (mL/L) ratio and distiller's yeast, water, glutinous rice, wheat koji raw material,
Fermented.
Applications of the Lactobacillus brevis L.brevis 2-34 of embodiment 7 in brewing yellow rice wine
Lactobacillus brevis L.brevis2-34 is seeded to activation medium MRS (pH5.0), 30 DEG C of activation culture 12-16h,
It is inoculated into the automatic anaerobic fermentation tanks of 5L and carries out high density fermentation, 30 DEG C of cultivation temperature, pH 5.0, incubation time 36-48h, warp
15000g refrigerated centrifuges 10min prepares high density bacterium solution (109cfu/L).When carrying out brewing yellow rice wine, do not changing other techniques
In the case of, after when falling tank, high density bacterium solution is mixed with 0.5% (mL/L) ratio and distiller's yeast, water, glutinous rice, wheat koji raw material,
Fermented.
Applications of the Lactobacillus brevis L.brevis 2-34 of embodiment 8 in brewing yellow rice wine
Lactobacillus brevis L.brevis2-34 is seeded to activation medium MRS (pH5.0), 30 DEG C of activation culture 12-16h,
It is inoculated into the automatic anaerobic fermentation tanks of 5L and carries out high density fermentation, 30 DEG C of cultivation temperature, pH 5.0, incubation time 36-48h, warp
15000g refrigerated centrifuges 10min prepares high density bacterium solution (109cfu/L).When carrying out brewing yellow rice wine, do not changing other techniques
In the case of, after when falling tank, high density bacterium solution is mixed with 1% (mL/L) ratio and distiller's yeast, water, glutinous rice, wheat koji raw material,
Fermented.
Applications of the Lactobacillus brevis L.brevis 2-34 of embodiment 9 in reduction ethyl carbamate in yellow wine
Using control fermentation tank yellow rice wine mash as reference sample, using the method for gas chromatography mass spectrometry, to the addition volume ratio of embodiment 8
Urethanes in the yellow rice wine mash brewageed for 1% Lactobacillus brevis L.brevis2-34 high density bacterium solutions is measured, such as
Shown in table 1, be vaccinated with citrulling and ethyl carbamate content in Lactobacillus brevis L.brevis2-34 mash reduces respectively
58.2% and 29.6%.
The content of citrulling and urethanes in the yellow rice wine mash of table 1
| Control group mash | Add Lactobacillus brevis L.brevis2-34 mash | |
| Citrulling | 38.5±1.1 | 16.1±0.8 |
| Ethyl carbamate content (ug/L) | 43.8±3.2 | 30.88±2.6 |
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be 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
Enclose being defined of being defined by claims.
Sequence table
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<120>One plant can efficiently utilize Lactobacillus brevis and its application of citrulling
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accctggtag tccatgccgt aaacgatgag tgctaagtgt tggagggttt ccgcccttca 840
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tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaag tcgtgaggct 1260
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Claims (6)
1. one plant can efficiently utilize the Lactobacillus brevis 2-34 of citrulling, Chinese Typical Representative culture is preserved on March 23rd, 2017
Collection, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2017140.
2. applications of the Lactobacillus brevis 2-34 in brewing yellow rice wine described in claim 1.
3. application according to claim 2, it is characterised in that the application is, in the brewing yellow rice wine starting stage, will to activate
Lactobacillus brevis 2-34 after spreading cultivation falls tank after being mixed with distiller's yeast, water, glutinous rice, wheat koji raw material, is fermented.
4. application according to claim 2, it is characterised in that the application is after Lactobacillus brevis 2-34 activation is spread cultivation,
Mix tank with distiller's yeast, water, glutinous rice, wheat koji raw material in 0.1%~1%mL/L ratio.
5. the application according to any claim in claim 2 to 4, it is characterised in that the application is by short newborn bar
Bacterium 2-34 is inoculated in the MRS culture mediums that pH is 5.0, and 30 DEG C stand after 12~16h of activation culture, and spread cultivation 36~48h, through 15000g
Refrigerated centrifuge 10min prepares high density bacterium solution, and bacterial concentration is 108~1010cfu/L。
6. applications of the Lactobacillus brevis 2-34 described in claim 1 in terms of citrulling of degrading.
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| CN112586599A (en) * | 2020-12-17 | 2021-04-02 | 江南大学 | Method for preparing peanut meal |
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|---|---|---|---|---|
| CN112586599A (en) * | 2020-12-17 | 2021-04-02 | 江南大学 | Method for preparing peanut meal |
| CN112586599B (en) * | 2020-12-17 | 2022-04-29 | 江南大学 | Method for preparing peanut meal |
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| CN106978371B (en) | 2019-07-02 |
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