CN105925521A

CN105925521A - Method for supplementing salt in batches for promoting lactic acid bacteria salt resistance

Info

Publication number: CN105925521A
Application number: CN201610545726.2A
Authority: CN
Inventors: 吴重德; 邓靖程; 何桂强; 黄钧; 周荣清
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2016-07-13
Filing date: 2016-07-13
Publication date: 2016-09-07

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

A kind of method in batches mending salt raising lactic acid bacteria salt resistance ability

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

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).