CN105925521A - Method for supplementing salt in batches for promoting lactic acid bacteria salt resistance - Google Patents
Method for supplementing salt in batches for promoting lactic acid bacteria salt resistance Download PDFInfo
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- CN105925521A CN105925521A CN201610545726.2A CN201610545726A CN105925521A CN 105925521 A CN105925521 A CN 105925521A CN 201610545726 A CN201610545726 A CN 201610545726A CN 105925521 A CN105925521 A CN 105925521A
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Abstract
The invention discloses a method for supplementing salt in batches for promoting lactic acid bacteria salt resistance and belongs to the technical field of bioengineering. The method comprises the following steps of: taking Tetragenococcus halophilus CGMCC no.3792 as original strain; respectively adding 6% (w/v), 3% and 3% NaCl into a fermentation medium after fermenting for 0 hour, 24 hours and 48 hours; statically culturing for 96 hours at 30 DEG C; collecting the acquired thallus; inoculating into the fermentation medium containing 18% NaCl; and statically culturing for 120 hours at 30 DEG C. The viable count is increased by 3 times of the viable count acquired from one-time salt-adding culture. The method provided by the invention has the advantages of simplicity and convenience in operation and application to the fermentation production of lactic acid bacteria.
Description
Technical field
The present invention relates to a kind of method in batches mending salt raising lactic acid bacteria salt resistance ability, belong to technical field of bioengineering.
Background technology
Addicted to salt tetracoccus (Tetragenococcus halophilus), it is widely present in hypersaline environment, particularly
In the salting food products such as soy sauce wine with dregs, Semen Sojae Preparatum and alec.To be applied in Semen Sojae Preparatum and sauce fermentation addicted to salt tetracoccus, create more
Volatility characteristics flavor substance, and make the color and luster of product brighter, this will assist in and improves high salt fermented seasonings
Quality.But, in these traditional zymotic product commercial Application, inevitably suffer high-salt stress ring addicted to salt tetracoccus
Border, thus seriously inhibit the normal physiological metabolism of thalline so that it is the efficiency manufacturing food reduces, and makes the quality of product be subject to
To impact.Therefore, how to improve the salt resistance ability addicted to salt tetracoccus and become industrial circle and the common focus of attention of academia
Problem.
Reply salt stress, lactic acid bacteria often induces various abiotic stress response mechanism, including regulation and control intracellular carbon metablism, induction table
Reach and coerce stress protein and keep cell membrane physiological function etc..Additionally, adaptation response is also lactic acid bacteria, common one should
Swash protected mode, when cell is in stressful environmental, a kind of adaptation response protective effect induced, can protect thin
Born of the same parents resist more rugged environment and coerce.Mending salt in the way of in batches, make thalline progressively adapt to hypersaline environment, thus it is resistance to improve it
Salt ability, these reactions may be used for improving lactic acid bacteria survival under stress conditions and improving their production technology.
Summary of the invention
It is an object of the invention to provide a kind of method utilizing and mending salt raising lactic acid bacteria salt resistance ability in batches.
The described salt mode of benefit in batches is: at fermentation initial time, add 6% NaCl in fermentation medium, in fermentation
Within 24 hours and 48 hours, add 3% NaCl, fermentation medium NaCl final concentration of 12% respectively.
Bacterial strain: addicted to salt tetracoccus (Tetragenococcus halophilus) CGMCC 3792, laboratory is from soy sauce
Wine with dregs separates, identifies through form, biophysical and biochemical tests and 16S rDNA order-checking, be preserved in Chinese microorganism strain preservation pipe
Reason committee's common micro-organisms center (China General Microbiological Culture Collection
Center, CGMCC), depositary institution address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Chinese Academy of Sciences's microorganism is ground
Study carefully institute；Preservation date is on April 29th, 2010, and deposit number is CGMCC no. 3792, and Classification And Nomenclature is addicted to salt tetrad ball
Bacterium (Tetragenococcus halophilus).
A kind of method utilizing benefit salt raising lactic acid bacteria salt resistance ability in batches, comprises the following steps:
(1) seed culture method: take-80 DEG C of preservations addicted to salt tetracoccus glycerol pipe store liquid with 5%(v/v) inoculum concentration connect
Kind in seed culture medium, 30 DEG C of quiescent culture 24 hours.Seed culture medium is: 80 g/L tryptones, 64 g/L Semen sojae atricolor eggs
White peptone, 5 g/L glucoses, 0.5 g/L ascorbic acid, 0.25 g/L MgSO4 .7H2O, pH are 7.0；
(2) fermentation culture method: take above-mentioned seed liquor, with 5%(v/v) inoculum concentration be inoculated in fermentation medium, respectively 0
Hour, within 24 hours and 48 hours, add 6%, 3% and 3% NaCl, 30 DEG C of quiescent culture 96 hours.Described fermentation medium is: 80
G/L tryptone, 64 g/L soy peptones, 5 g/L glucoses, 0.5 g/L ascorbic acid, 0.25 g/L MgSO4 .7H2O,
PH is 7.0.
The mensuration of Biomass: take fermentation liquid centrifugal (10000 r/min, 5 min, 4 DEG C) and collect thalline, with sterilized water from
The heart dilutes suitable multiple after washing 3 times, shakes up, and with TU-1901 two-beam light ultraviolet-uisible spectrophotometer, compares at 600 nm
Turbid mensuration OD value.
The mensuration of salt resistance ability: collect the thalline after fermentation culture, after washing 3 times with sterilized water, is resuspended in sterilized water,
Regulation cell concentration is to OD600 =2.0, with 5%(v/v) inoculum concentration be inoculated in containing 18% NaCl(v/v) fermentation medium
In, quiescent culture 120 hours, take fermentation liquid centrifugal (10000 r/min, 5 min, 4 DEG C) and collect thalline, be centrifuged with sterilized water
After washing 3 times, flat board, in solid plate (containing the seed culture medium of 2% agar powder), is placed in 30 DEG C of constant temperature by suitable dilution spread
After incubator is cultivated 48 hours, calculate viable count.
Beneficial effects of the present invention: in high salt sweat, uses the mode in batches mending salt, significantly improves cell
Salt resistance ability, is inoculated in the culture medium containing 18% NaCl cultivation, the cell number that viable count is cultivated than disposable salt adding by thalline
Amount improves 3 times.The present invention provides a kind of salt of in batches mending to improve the method addicted to salt tetracoccus salt resistance ability, simple, for
The fermentation of lactic acid bacteria industrialization high salt provides theoretical direction.
Detailed description of the invention
Example 1(matched group): the salt resistance ability addicted to salt tetracoccus CGMCC 3792 (table 1) that disposable salt adding is cultivated
(1) seed culture method: take the glycerin storage liquid of-80 DEG C of preservations with 5%(v/v) inoculum concentration be inoculated in seed culture medium
In, 30 DEG C of quiescent culture 24 hours.Seed culture medium is: 80 g/L tryptones, 64 g/L soy peptones, 5 g/L Fructus Vitis viniferaes
Sugar, 0.5 g/L ascorbic acid, 0.25 g/L MgSO4 .7H2O, pH are 7.0；
(2) fermentation culture method: take above-mentioned seed liquor, is seeded to containing 12%(v/v by the inoculum concentration of 5%) fermentation culture of NaCl
Base, quiescent culture 96 hours.Fermentation medium is: 80 g/L tryptones, 64 g/L soy peptones, 5 g/L glucoses,
0.5 g/L ascorbic acid, 0.25 g/L MgSO4 .7H2O, pH are 7.0；
(3) mensuration of salt resistance ability: take above-mentioned fermentation liquid centrifugal (10000 r/min, 4 DEG C, 5 min) and collect thalline, be resuspended in
In sterilized water, regulation cell concentration to OD600 =2.0, with 5%(v/v) inoculum concentration be inoculated in containing 18% NaCl(v/v)
In fermentation medium, quiescent culture 120 hours.Take fermentation liquid centrifugal (10000 r/min, 5 min, 4 DEG C) and collect thalline, use
After sterilized water centrifuge washing 3 times, flat board, in solid plate (containing the seed culture medium of 2% agar powder), is put by suitable dilution spread
After cultivating 48 hours in 30 DEG C of constant incubators, calculating viable count is 3.3 × 106Individual/mL.
Example 2(experimental group): mend the salt resistance ability addicted to salt tetracoccus CGMCC 3792 (table 1) that salt is cultivated in batches
(1) seed culture method: with example 1；
(2) fermentation culture method: take above-mentioned seed liquor, by 5%(v/v) inoculum concentration be seeded in fermentation medium, respectively 0
Hour, within 24 hours and 48 hours, add 6%, 3% and 3% NaCl, 30 DEG C of quiescent culture 96 hours.Described fermentation medium is: 80
G/L tryptone, 64 g/L soy peptones, 5 g/L glucoses, 0.5 g/L ascorbic acid, 0.25 g/L MgSO4 .7H2O,
PH is 7.0；
(3) mensuration of salt resistance ability: with example 1, and finally calculating viable count is 1.0 × 107Individual/mL.
The different salt adding mode of table 1 is on the impact addicted to salt tetracoccus salt resistance ability
。
Claims (5)
1. mend the method that salt improves lactic acid bacteria salt resistance ability in batches, comprise the steps of
(1) seed culture: take-80 DEG C of preservations lactic acid bacteria glycerol pipe store liquid, with 5%(v/v) inoculum concentration be inoculated in seed training
Support in base, 30 DEG C of quiescent culture 24 hours；
(2) fermentation culture: by above-mentioned seed liquor with 5%(v/v) inoculum concentration be seeded to 30 DEG C of quiescent culture 96 in fermentation medium
Hour；
(3) during fermentation culture, add NaCl stage by stage, be 12%(w/v to the final salinity of culture medium).
A kind of method in batches mending salt raising lactic acid bacteria salt resistance ability, it is characterised in that: described breast
Acid bacterium be addicted to salt tetracoccus (Tetragenococcus halophilus), deposit number is CGMCC no. 3792.
A kind of method in batches mending salt raising lactic acid bacteria salt resistance ability, it is characterised in that: described kind
Sub-culture medium is: 80 g/L tryptones, 64 g/L soy peptones, 5 g/L glucoses, 0.5 g/L ascorbic acid, 0.25
g/L MgSO4 .7H2O, pH 7.0.
A kind of method in batches mending salt raising lactic acid bacteria salt resistance ability, it is characterised in that: described
Ferment culture medium is: 80 g/L tryptones, 64 g/L soy peptones, 5 g/L glucoses, 0.5 g/L ascorbic acid, 0.25
g/L MgSO4 .7H2O, pH 7.0.
A kind of method in batches mending salt raising lactic acid bacteria salt resistance ability, it is characterised in that: described point
Batch mending salt mode be within the 0th hour, 24 hours and 48 hours, to add 6%(w/v respectively fermenting), 3% and the NaCl of 3%, make culture medium
Final salinity is 12%(w/v).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662860A (en) * | 2020-07-22 | 2020-09-15 | 四川大学 | Method for improving survival rate of tetragenococcus halophilus under extreme conditions |
| CN114561328A (en) * | 2022-04-18 | 2022-05-31 | 四川大学 | Preparation method of high-activity lactobacillus agent |
-
2016
- 2016-07-13 CN CN201610545726.2A patent/CN105925521A/en active Pending
Non-Patent Citations (4)
| Title |
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| 何强等: "活性污泥的耐盐驯化培养", 《环境工程学报》 * |
| 何桂强等: "多重胁迫对嗜盐四联球菌CGMCC 3792存活率及细胞成分的影响", 《食品工业科技》 * |
| 冯杰等: "不同氯化钠浓度对Candida etchellsii CICIM Y0600生长和生物特性的影响研究", 《工业微生物》 * |
| 李玲玲等: "盐度对活性污泥硝化功能的影响", 《环境污染与防治》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662860A (en) * | 2020-07-22 | 2020-09-15 | 四川大学 | Method for improving survival rate of tetragenococcus halophilus under extreme conditions |
| CN114561328A (en) * | 2022-04-18 | 2022-05-31 | 四川大学 | Preparation method of high-activity lactobacillus agent |
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Application publication date: 20160907 |