CN106867938A - One plant of bacillus subtilis for reducing biogenic amine and its application - Google Patents
One plant of bacillus subtilis for reducing biogenic amine and its application Download PDFInfo
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- CN106867938A CN106867938A CN201710149106.1A CN201710149106A CN106867938A CN 106867938 A CN106867938 A CN 106867938A CN 201710149106 A CN201710149106 A CN 201710149106A CN 106867938 A CN106867938 A CN 106867938A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses one plant of bacillus subtilis for reducing biogenic amine and its application, belong to fermentation technical field.Bacillus subtilis of the invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2016, deposit number is CGMCC No.13214, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.The bacterial strain has the performance of stronger 4 kinds of biogenic amines of degraded (putrescine, cadaverine, histamine, tyrasamine), and degradation efficiency reaches as high as 59.30%~68.57%.
Description
Technical field
The present invention relates to one plant of bacillus subtilis for reducing biogenic amine and its application, belong to fermentation technical field.
Background technology
Broad bean paste is brewed with fermenting raw materials such as broad beans and formed, and keeps the half flowing viscous body or half of broad bean paste original shape admittedly
State flavouring.Broad bean paste has long history, nutritious, delicious flavour, therefore depth by the joyous of consumers in general in China
Meet.But, on the one hand, current broad bean paste is still prepared using traditional fermentation technique, open fermentation, weather exposure, fermentation
Strain comes from nature, and itself is uncontrollable, may bring unknown unsafe factor；On the other hand, broad bean is rich in what is enriched
Protein, a large amount of free amino acids can generate harmful biogenic amine once by the microbial metabolism with decarboxylation ability.
Excess free enthalpy biogenic amine, may trigger the various poisoning symptoms such as headache, dizziness, nausea, respiratory distress, palpitaition.
Part population is because of disease, genetic defect or takes DAO inhibitor medicaments, and detoxification ability is weakened severely, to biogenic amine extremely
It is sensitive.Additionally, the food that healthy population eats various Content of Biogenic Amines high is likely to that enzyme system supersaturation can be made simultaneously.For to food
The consideration of product safety, people should as far as possible reduce the content of biogenic amine in food.
For biogenic amine in food, mainly there are additive, enzyme preparation, preservative, gamma-radiation irradiation, freezing, height at present
The control devices such as temperature, ultra high pressure treatment.But, some additives are relatively costly and may influence flavour of food products.Enzyme preparation DAO etc.
Animal's liver mainly is extracted from, it is with high costs to be not suitable for commercial Application.Radiation has deactivation to microorganism, it is impossible to be applied to life
The fermentation phase that thing amine is largely produced.Freezing and high temperature can then destroy flavour of food products and texture.From unlike above-mentioned control technology,
Reasonable selection leavening, especially can effectively eliminate the biology generated in food with the microorganism of degradation biological amine
Amine.And degradable fermented dose of the biogenic amine reported at present is mainly isolated from aquatic products and cheese, the production work of thick broad-bean sauce is not applied to
Skill, thereby increases and it is possible to influence local flavor.One plant of screening had both been suitable for the production environment of sauce products, and the shadow to local flavor is reduced to the full extent
Sound, and the bacterial strain of energy efficient degradation biogenic amine, the bean cotyledon jam products for preparing safety and Health have important application value.
The content of the invention
First purpose of the invention is to provide a bacillus subtilis (Bacillus subtilis), in 2016
On October 31, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC
No.13214, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Second object of the present invention is to provide the cultural method of the bacillus subtilis, be by bacillus subtilis with
Fluid nutrient medium is seeded to by the inoculum concentration of volume 1~5%, in 30~37 DEG C, 200~250rpm cultures 12h~24h.
In one embodiment of the invention, the fluid nutrient medium is LB culture mediums.
Third object of the present invention is to provide a kind of method for reducing biogenic amine, and methods described is by the withered grass gemma
Bacillus is seeded in the liquid containing biogenic amine, semisolid or solid with 1~5% inoculum concentration.
Fourth object of the present invention be to provide it is a kind of reduce broad bean paste in Content of Biogenic Amines method, methods described be
The bacillus subtilis is inoculated with broad bean paste production process.
In one embodiment of the invention, the inoculation is that bacterium solution is seeded into broad bean paste by 1~5% inoculum concentration
In.
5th purpose of the invention is to provide flavouring prepared by the bacillus subtilis.
In one embodiment of the invention, the flavouring is broad bean paste.
The present invention also provides application of the bacillus subtilis in terms of field of food reduction Content of Biogenic Amines.
Beneficial effect：The bacillus subtilis that the present invention is provided is harmless bacterium in broad bean paste fermentation process, for putrescine,
Common biogenic amine has obvious degradation in cadaverine, histamine, tyrasamine this 4 kinds of fermented foods, to putrescine, cadaverine, group
Amine, the degradation rate of tyrasamine still are able to reduce biogenic amine 35.29% up to 59.30%~68.57% in sauce unstrained spirits environment, can be effective
Content of Biogenic Amines in the fermented food production processes such as control thick broad-bean sauce, and do not change its main flavor.
Brief description of the drawings
Fig. 1 be bacterial strain in different salinity culture medium to the degradation rate figure of biogenic amine.
Biomaterial preservation
Bacillus subtilis (Bacillus subtilis), is preserved in China Microbiological bacterium on October 31st, 2016
Preservation administration committee common micro-organisms center is planted, deposit number is CGMCC No.13214, and preservation address is that Beijing is exposed to the sun
The institute 3 of area North Star West Road 1.
Specific embodiment
The screening of the bacillus subtilis of embodiment 1
1g samples are taken from the traditional zymotic bean cotyledon sauce sauce unstrained spirits of 30 days or so to be added in 9mL sterilized waters, in 30 DEG C shake
200rpm vibrations 30min is made suspension on bed, by suspension gradient dilution, coats LBGL flat boards, 37 DEG C of aerobic cultures
24h.Picking is repeated plate streaking and separates without purple halation or the less single bacterium colony of halation, the list for being purified in triplicate
Bacterium colony, for next step identification.
- 80 DEG C of glycerine storage liquid are measured with 1~2% inoculation to be inoculated in LB culture mediums, 37 DEG C of vibration quiescent culture 24h,
Genomic DNA is extracted with CTAB methods.16s rDNA are expanded to 27F/1401R with bacterial universal primers, reaction condition is：94 DEG C pre-
Enter following circulation after denaturation 3min：94 DEG C of denaturation 40s, 56 DEG C of annealing 35s, 72 DEG C of extension 80s, 30 circulations；72 DEG C of extensions
10min.It is good through 1% agarose gel electrophoresis qualification result.Amplified production is sent into Shanghai life work gene sequencing, such as SEQ ID
Shown in NO.1, BLAST sequence alignments are carried out on NCBI websites, be defined as bacillus subtilis.
Degradeds of the bacillus subtilis CGMCC No.13214 of embodiment 2 to biogenic amine
Prepare LBBA culture mediums：Peptone 10g/L；Dusty yeast 5g/L；NaCl 10g/L；Putrescine 1g/L；Cadaverine 1g/L；Group
Amine 1g/L；Tyrasamine 1g/L, pH nature.50mL triangular flasks dispense 10mL, 121 DEG C of sterilizing 20min.
- 80 DEG C of glycerine storage liquid are taken with 1% inoculum concentration (by volume) and is inoculated in fresh LB fluid nutrient mediums, 37 DEG C of shaking tables
Culture 12 hours, rotating speed 200rpm.LBBA fluid nutrient medium cultures are inoculated in 1% inoculum concentration (by volume) again, are cultivated 4 days
Afterwards, HPLC methods detect the concentration of remaining biogenic amine in culture medium, compared with blank, are inoculated with the culture medium of bacillus subtilis
In, the degradation rate of biogenic amine is respectively：Putrescine 59.30 ± 0.59%, cadaverine 68.57 ± 0.48%, histamine 61.92 ±
1.14%th, tyrasamine 63.95 ± 0.51%.
Meanwhile, with 3% inoculum concentration (by volume), bacillus amyloliquefacienses are inoculated in the LBBA trainings containing different NaCl concentrations
Support in base, the concentration of remaining biogenic amine, degradation effect are cultivated and detected under similarity condition as shown in figure 1, bacillus subtilis
CGMCC No.13214 are to the degradation rate of total biogenic amine up to 64%.In the environment of NaCl concentration is 13% (130g/L), still
With total degradation effect of the biogenic amine degradation rate more than 5%, the fermentation under compared with hyperosmosis environment can be met.
Degradeds of the bacillus subtilis CGMCC No.13214 of embodiment 3 to biogenic amine in sauce unstrained spirits
Prepare sauce unstrained spirits culture：The sauce unstrained spirits that factory ferments one month；10mL 1g/L biogenic amines mixed liquor (putrescine, cadaverine,
Histamine, tyrasamine), the packing of 250mL triangular flasks 30g, 121 DEG C of sterilizing 20min.
Take the bacterium solution that 3mL activates as stated above to be inoculated in sauce unstrained spirits culture, 37 DEG C of quiescent cultures are after 7 days, with
Bacillus subtilis CGMCC NO.12090 bacterial strains (are disclosed in the patent application document of Publication No. 105647836A
In) it is control, cultivated according to above-mentioned identical method, fermented.In HPLC methods detection culture remaining biogenic amine concentration and
Main flavor.Compared with blank, it is inoculated with the sauce unstrained spirits of bacillus subtilis, the degradation rate of biogenic amine is respectively：It is rotten
Amine 33.68 ± 0.35%, cadaverine 47.38 ± 0.28%, histamine 1 5.57 ± 0.22%, tyrasamine 31.61 ± 0.29%, total degradation
Rate 35.29 ± 0.16%, and the local flavor of sauce unstrained spirits is not influenceed；And control strain is only to the degradation rate of this 4 class alkamines material
2.31%.
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 with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>One plant of bacillus subtilis for reducing biogenic amine and its application
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1498
<212> DNA
<213>Artificial sequence
<400> 1
aatcccctta tgctccttgt tacgacttcc cccaatcatc tgtcccacct tcggcggctg 60
gctcctaaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg 120
gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat tactagcgat 180
tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga tttgtgggat 240
tggcttaacc tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg tgtgtagccc 300
aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt tgtcaccggc 360
agtcacctta gagtgcccar ctgrryrmws kmaaswawkm wmrrggktks ssyysktkss 420
gggacttaac ccaaacatct cacgacacga ggctgacgac aaycatgcas cacctgtcac 480
tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa gacctggtaa 540
ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtggcg ggcccccgtc 600
aaattccttt gagtttcagt cttgcgaccg tactccccag gcggagtgct taatgcgtta 660
gctgcagcac taaggggcgg aaacccccta acacttagca ctcatcgttt acggcgtgga 720
ctaccagggt atctaatcct gttcgctccc ccacgctttc gctcctcagc gtcagttaca 780
gaccagagag tcgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac 840
acgtggaatt ccactctcct cttctgcact caagttcccc agtttccaat gaccctcccc 900
ggttgagccg ggggctttca catcagactt aagaamccgc ctgcgagccc tttacgccca 960
ataattccgg rmmacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 1020
gtggctttct ggttaggtac cgtcaaggtg ccgccctatt tgamcggcac ttgtyyttcc 1080
ctaacammrr rsctttacga tcsgaaaacc ttcatcacyy mcrcggcgky gytscstcmg 1140
rytttcgtcc attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc 1200
tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtgagc 1260
cgttacctca ccaactagct aatgcgccgc gggtccatct gtaagtggta gccgaagcca 1320
ccttttatgt ctgaaccatg cggttcaaac aaccatccgg tattagcccc ggtttcccgg 1380
agttatccca gtcttacagg caggttaccc acgtgttact cacccgtccg ccgctaacat 1440
cagggagcaa gctcccatct gtccgctcga cttgcatgta tagcaccccg cccttgca 1498
Claims (9)
1. a bacillus subtilis (Bacillus subtilis), China Microbiological bacterium is preserved on October 31st, 2016
Preservation administration committee common micro-organisms center is planted, deposit number is CGMCC No.13214, and preservation address is that Beijing is exposed to the sun
The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica.
2. culture claim 1 described in bacillus subtilis method, it is characterised in that by bacillus subtilis by volume with
1~3% inoculum concentration is seeded to fluid nutrient medium, in 30~37 DEG C, 200~250rpm cultures 12h~24h.
3. method according to claim 2, it is characterised in that the culture medium is LB culture mediums.
4. it is a kind of reduce biogenic amine method, it is characterised in that methods described is by the bacillus subtilis described in claim 1
It is seeded in the liquid containing biogenic amine, semisolid or solid with 1~5% inoculum concentration.
5. it is a kind of reduce broad bean paste in Content of Biogenic Amines method, it is characterised in that methods described is in broad bean paste production process
Bacillus subtilis described in middle inoculation claim 1.
6. method according to claim 5, it is characterised in that the inoculation is by volume, with 1~5% inoculum concentration
It is seeded in broad bean paste.
7. the flavouring that prepared by bacillus subtilis described in application claim 1.
8. flavouring according to claim 7, it is characterised in that the flavouring is broad bean paste.
9. application of the bacillus subtilis described in claim 1 in terms of field of food reduction Content of Biogenic Amines.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456922A (en) * | 2018-11-30 | 2019-03-12 | 江苏大学 | The beach the Halophilic Bacterium bacterial strain Hua Jin bacillus of one plant of raising alec fermentation quality |
| CN109593669A (en) * | 2018-11-30 | 2019-04-09 | 江苏大学 | The heavy mud of the Halophilic Bacterium bacterial strain of one plant of raising alec fermentation quality likes salt bacillus |
| CN111248409A (en) * | 2020-02-27 | 2020-06-09 | 江南大学 | Low-salt thick broad-bean sauce fermentation method |
| CN112674258A (en) * | 2020-12-03 | 2021-04-20 | 大连工业大学 | Biological amine degrading bacterium for improving bad flavor of shrimp sauce fermentation and application thereof |
| CN115161222A (en) * | 2022-06-02 | 2022-10-11 | 渤海大学 | Application of bacillus subtilis JZXJ-7 strain in degradation of biogenic amine |
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| KR20160144614A (en) * | 2015-06-09 | 2016-12-19 | 대한민국(농촌진흥청장) | Bacillus subtilis strain JP-5 and uses thereof |
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| KR20150019435A (en) * | 2013-08-14 | 2015-02-25 | 재단법인 발효미생물산업진흥원 | Bacillus strains having degradation activity of biogenic amine and uses thereof |
| KR20160144614A (en) * | 2015-06-09 | 2016-12-19 | 대한민국(농촌진흥청장) | Bacillus subtilis strain JP-5 and uses thereof |
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| 김용상等: "Isolation of Biogenic Amines-Degrading Strains of Bacillus subtilis and Bacillus amyloliquefaciens from Traditionally Fermented Soybean Products", 《KOREAN JOURNAL OF MICROBIOLOGY》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456922A (en) * | 2018-11-30 | 2019-03-12 | 江苏大学 | The beach the Halophilic Bacterium bacterial strain Hua Jin bacillus of one plant of raising alec fermentation quality |
| CN109593669A (en) * | 2018-11-30 | 2019-04-09 | 江苏大学 | The heavy mud of the Halophilic Bacterium bacterial strain of one plant of raising alec fermentation quality likes salt bacillus |
| CN109593669B (en) * | 2018-11-30 | 2022-04-26 | 江苏大学 | Moderately halophilic bacteria strain bacillus clarkii for improving fermentation quality of fish paste |
| CN111248409A (en) * | 2020-02-27 | 2020-06-09 | 江南大学 | Low-salt thick broad-bean sauce fermentation method |
| CN112674258A (en) * | 2020-12-03 | 2021-04-20 | 大连工业大学 | Biological amine degrading bacterium for improving bad flavor of shrimp sauce fermentation and application thereof |
| CN112674258B (en) * | 2020-12-03 | 2023-08-01 | 大连工业大学 | Biological amine degrading bacteria for improving bad flavor of shrimp paste fermentation and application thereof |
| CN115161222A (en) * | 2022-06-02 | 2022-10-11 | 渤海大学 | Application of bacillus subtilis JZXJ-7 strain in degradation of biogenic amine |
| CN115161222B (en) * | 2022-06-02 | 2024-05-07 | 渤海大学 | Application of bacillus subtilis JZXJ-7 strain in degradation of biogenic amine |
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| CN106867938B (en) | 2019-09-17 |
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