CN101302487A - Method for cultivating salt stress-resistant lactococcus lactis and special culture medium therefor - Google Patents

Abstract

The invention discloses a method for cultivating salt stress resisting Lactococcus lactis and a special culture medium thereof. The Lactococcus lactis salt stress resisting culture medium is a culture medium which is obtained by adding 3.2 to 32.0 mmol/L glutathione to a culture medium for cultivating Lactococcus lactis, wherein the Lactococcus lactis is a Lactococcus lactis milk fat subspecies strain capable of absorbing the glutathione, and specifically can be Lactococcus lactis SK11. The method for cultivating the salt stress resisting Lactococcus lactis is to cultivate the Lactococcus lactis in the culture medium so as to produce the salt stress resisting Lactococcus lactis. The method for cultivating the salt stress resisting Lactococcus lactis can raise the survival rate of the Lactococcus lactis under adverse environmental conditions, ensure that the Lactococcus lactis can normally exert physiological functions thereof, increase the additional value of products, and create greater economic benefit.

Description

A kind of method and special culture media thereof of cultivating salt stress resisting lactococcus lactis

Technical field

The present invention relates to a kind of method and special culture media thereof of cultivating salt stress resisting lactococcus lactis.

Background technology

Milk-acid bacteria is the important industrial microorganism of a class as probiotic bacterium, and the history of using in food fermentation is very long.Milk-acid bacteria not only has good health-care effect, also can be used for improving Nutritive value of food, improves flavour of food products, improves food preservation and added value.Lactic acid is one of important physical and chemical index of soy sauce and flavour substances, thus in the soy sauce brewing process artificial inoculation milk-acid bacteria suitably, for the local flavor that improves soy sauce good effect is arranged.At present, but have the tolerance of salinity to reach milk-acid bacteria more than 18% in the domestic strain library.Japan successfully filters out and is fit to the halophilism milk-acid bacteria that the rare wine with dregs technology of high salt is used.We obtain the resistance to high salt milk-acid bacteria to improving the quality of sauce unstrained spirits product as soy sauce big country, create more economic benefit and are significant.

Recent findings plurality of reagents such as N-n acetylglucosamine n, pantothenic acid, pantetheine, peptide and lactulose can improve the milk-acid bacteria survival rate that is used for leavened food.

Summary of the invention

The purpose of this invention is to provide a kind of method and special culture media thereof of cultivating salt stress resisting lactococcus lactis.The bacterial strain vigor that the invention solves the inevitable loose contact environment of milk-acid bacteria in the production practice and cause descends, the practical problems of death, and method of the present invention is simple to operate, do not increase extra equipment and artificial, only by lower additive reagent input, significantly strengthened the survival efficient of thalline, and as the Lactococcus lactis of probiotic bacterium be eaten the back arrive enteron aisle can its physiological function of more effective performance, improved the added value of leavened food.

The salt stress-resistant substratum of Lactococcus lactis provided by the present invention, called after salt stress-resistant substratum is to add the substratum that the 3.2-32.0mmol/L gsh obtains in the substratum of cultivating Lactococcus lactis.

Wherein, described Lactococcus lactis is for absorbing the lactococcus lactis subsp bacterial strain of gsh.Described Lactococcus lactis is specially Lactococcus lactis SK11.

The substratum of described cultivation Lactococcus lactis can be any substratum that can cultivate Lactococcus lactis, as the substratum of being made up of following substances; K ₂HPO ₄3g/L, sodium acetate 1g/L, Triammonium citrate 0.6g/L, tyrosine 0.5g/L, vitamins C 0.5g/L, β-Phosphoric acid glycerol esters disodium 19g/L, L-Ala 0.24g/L, arginine 0.125g/L, l-asparagine 0.42g/L, cysteine hydrochloride 0.13g/L, L-glutamic acid 0.5g/L, Padil 0.175g/L, Histidine 0.15g/L, Isoleucine 0.21g/L, leucine 0.475g/L, Methionin 0.44g/L, methionine(Met) 0.125g/L, phenylalanine 0.275g/L, proline(Pro) 0.675g/L, Serine 0.34g/L, Threonine 0.225g/L, tryptophane 0.05g/L, Xie Ansuan 0.325g/L, MgCl ₂6H ₂O 0.2g/L, CaCl ₂2H ₂O 0.05g/L, MnCl ₂4H ₂O 0.016g/L, FeCl ₃6H ₂O 0.008g/L, FeSO ₄7H ₂O0.007g/L, ZnSO ₄7H ₂O 0.005g/L, CoSO ₄7H ₂O 0.0025g/L, CuSO ₄7H ₂O 0.0025g/L, (NH4) ₆Mo70242H ₂O 0.0025g/L, VITAMIN B4 0.01g/L, 0.01g/L guanine, uridylic 0.01g/L, xanthine 0.01g/L, p-benzaminic acid 10mg/L, inosine 5mg/L, vitamin B13 5mg/L, Pyridoxylamine hydrochloride 5mg/L, Thymine deoxyriboside 5mg/L, D-vitamin H 2.5mg/L, 6,8-Thioctic Acid 2.5mg/L, pyridoxine hydrochloride 2mg/L, folic acid 1mg/L, niacin 1mg/L, D-(+)-calcium pantothenate 1mg/L, riboflavin 1mg/L, thiamine hydrochloride 1mg/L, vitamin B12 1mg/L.

Another object of the present invention provides a kind of method of cultivating salt stress resisting lactococcus lactis, is Lactococcus lactis is cultivated in described salt stress-resistant substratum, obtains the Lactococcus lactis of salt stress-resistant.

Wherein, described Lactococcus lactis is for absorbing the lactococcus lactis subsp bacterial strain of gsh; Described Lactococcus lactis is specially Lactococcus lactis SK11.

Utilization of the present invention contains sulfydryl, and (reduced glutathion SH) can be removed microorganism cells and coerce the oxyradical of inducing generation down at high salt concentration; peroxidation slows down; highly unsaturated fatty acids on the protection cytolemma exempts from the characteristic of oxidation; the method of an amount of gsh is added in employing in substratum, the raising bacterial strain especially improves salt resistance to the adaptability of poor environment.

The present invention cultivates stationary phase with Lactococcus lactis in the substratum of the gsh that contains 3.2-32.0mmol/L after, handle with high salt concentration solution (5M NaCl), the survival rate of thalline is improved, and comparison improves 100-1000 doubly according to bacterial strain.In the milk-acid bacteria industrial production, the method of cultivation salt stress resisting lactococcus lactis of the present invention can improve the survival rate of Lactococcus lactis under unsuitable environmental condition, make Lactococcus lactis can normally bring into play its physiological function, strengthened value-added content of product, create bigger economic benefit.

Description of drawings

Fig. 1 is that the Lactococcus lactis of cultivating in the salt stress-resistant substratum of contrast and the GSH that contains 4.6mM passes through the colony number (CFU) that 5MNaCl handles different time

Embodiment

The Lactococcus lactis of embodiment 1, cultivation salt stress-resistant

A) substratum:

LM17 substratum: get and plant matter peptone 5.0g, polyprotein peptone 5.0g, beef extract 2.5g, agar 15g, β-Phosphoric acid glycerol esters disodium 19g, yeast extract 5.0g, xitix 0.5g, MgSO47H ₂O 0.25g is dissolved in the 1L distilled water, and making pH is 7.1, and the 15min that sterilizes under 121 ℃ of conditions adds the 15g lactose for every liter then.

Full-synthetic culture medium (CDM) (gL ^-1):

(1) gets K ₂HPO ₄3g, sodium acetate 1g (molten in advance in 400 μ l 10M NaOH), Triammonium citrate 0.6g, tyrosine 0.5g;

(2) it is molten in advance in 5ml distilled water to get vitamins C 0.5g;

(3) (1) and (2) is mixed, add β-Phosphoric acid glycerol esters disodium 19g again, be settled to 870ml with distilled water, 10 * amino acid the concentrated solution that adds 100ml subsequently, 10ml metal ion concentrated solution, 10ml Nucleotide concentrated solution, 10ml VITAMIN concentrated solution, with 5M NaOH pH is transferred to 6.8, last sterile filtration.

10 * amino acid concentrated solution (gL ^-1) form by following material: L-Ala 2.40g, arginine 1.25g, l-asparagine 4.20g, cysteine hydrochloride 1.30g, L-glutamic acid 5.00g, Padil 1.75g, Histidine 1.50g, Isoleucine 2.10g, leucine 4.75g, Methionin 4.40g, methionine(Met) 1.25g, phenylalanine 2.75g, proline(Pro) 6.75g, Serine 3.40g, Threonine 2.25g, tryptophane 0.50g, Xie Ansuan 3.25g.

100 * metal ion concentrated solution is formed (gL by following material ^-1): MgCl ₂6H ₂O 20g, CaCl ₂2H ₂O5.0g, MnCl ₂4H ₂O 1.6g, FeCl ₃6H ₂O 0.8g, FeSO ₄7H ₂O 0.7g, ZnSO ₄7H ₂O 0.5g, CoSO ₄7H ₂O 0.25g, CuSO ₄7H ₂O 0.25g, (NH ₄) ₆MO ₇O ₂₄2H ₂O 0.25g is transferred to 1.5 with 5M HCl with pH.

100 * nucleosides concentrated solution is made up of following material: 10mg VITAMIN B4, guanine, uridylic and xanthine dissolve in the 0.1M of 10ml NaOH.

100 * VITAMIN concentrated solution is formed (mgL by following material ^-1): p-benzaminic acid 1000, inferior Huang (purine nuclear) glycosides 500, vitamin B13 500, Pyridoxylamine hydrochloride 500, chest (gland pyrimi piperidine deoxidating nucleus) glycosides 500, D-vitamin H 250,6,8-Thioctic Acid 250, pyridoxine hydrochloride 200, folic acid 100, niacin 100, D-(+)-calcium pantothenate 100, riboflavin 100, thiamine hydrochloride 100, vitamin B12 100.

The salt stress-resistant substratum:

Add gsh GSH in the full-synthetic culture medium (CDM), make its final concentration difference 3.2,4.6,16 and 32.0mmol/L.

B) activation of bacterial classification

Get 0.4ml-70 ℃ of Lactococcus lactis (Lactococcus lactis) (from Dutch NIZO DSMZ, deposit number SK11) the glycerine stock solution is inoculated in the 15ml Lactococcus lactis LM17 substratum, behind static cultivation 12h under 30 ℃, as activated seed.

C) salt stress and survival rate are identified

Activated seed is transferred in fresh Lactococcus lactis CDM substratum (contrast) respectively with 1% inoculum size and is contained the salt stress-resistant substratum of the GSH of 4 kinds of different concns in the table 1, at 30 ℃ of static cultivation 16-20h down.

Get above-mentioned five kinds of fermented liquids respectively, after adjusting initial cell density, with twice of physiological saline (mass percent is 0.85% NaCl solution) washed cell, be resuspended in the 5mol/L NaCl solution of 1ml, coerce 0,6 and 24 hour after, centrifugal immediately (10,000g, 1min), with throw out physiological saline washed cell twice, to remove remaining NaCl.Subsequently, cell is resuspended in the physiological saline of 0.4ml.On the LM17 culture medium flat plate, measure CFU with different extent of dilution dibblings respectively.Place 30 ℃ to cultivate 48h down flat board, the bacterium colony on the flat board is counted.Experiment repeats 3 times.Contrast and the Lactococcus lactis that contains the salt stress-resistant culture medium culturing of different concns GSH are handled the colony number (CFU) of different time in 5mol/L NaCl solution as shown in table 1.

Table 1. contrasts and contains the Lactococcus lactis of the salt stress-resistant culture medium culturing of different concns GSH

In 5mol/L NaCl solution, handle the colony number (CFU) of different time

Experimental result shows that 5M NaCl coerces down, and 3.2-32.0mmol/L GSH all is significantly increased to the salt resistance of Lactococcus lactis, adds the survival rate of the thalline of GSH and compares about 100-1000 times of raising with control strain.

Wherein, the colony number (CFU) that the Lactococcus lactis that contrast and containing is cultivated in the salt stress-resistant substratum (among Fig. 1 with " CDM substratum add GSH " expression) of the GSH of 4.6mM is handled different time through 5M NaCl as shown in Figure 1, show in the substratum (representing with " CDM substratum " among Fig. 1) that does not add GSH, thalline is handled through high salt concentration solution (5M NaCl), and survival rate continues to descend with the prolongation of salt stress time fast; And the thalline process high salt concentration solution (5M NaCl) that adds 4.6mmol/L GSH culture medium culturing is handled after the 10h, survival rate descends not obvious, and the thalline survival rate that adds the culture medium culturing of 4.6mMGSH behind the same salt stress processing 36h improves 100 times compared with the control.

Above experimental result shows that Lactococcus lactis absorbs GSH from substratum, handles the different time through the salts solution (5MNaCl) of high density, and the survival rate of its bacterial strain is compared with control strain and improved 100 times.

Claims (7)

1, Lactococcus lactis salt stress-resistant substratum is to add the substratum that the 3.2-32.0mmol/L gsh obtains in the substratum of cultivating Lactococcus lactis.

2, substratum according to claim 1 is characterized in that: described Lactococcus lactis is for absorbing the lactococcus lactis subsp bacterial strain of gsh.

3, substratum according to claim 2 is characterized in that: described Lactococcus lactis is Lactococcus lactis SK11.

4, according to arbitrary described substratum in the claim 1 to 3, it is characterized in that: the substratum of described cultivation Lactococcus lactis is made up of following material;

K ₂HPO ₄3g/L, sodium acetate 1g/L, Triammonium citrate 0.6g/L, tyrosine 0.5g/L, vitamins C 0.5g/L, β-Phosphoric acid glycerol esters disodium 19g/L, L-Ala 0.24g/L, arginine 0.125g/L, l-asparagine 0.42g/L, cysteine hydrochloride 0.13g/L, L-glutamic acid 0.5g/L, Padil 0.175g/L, Histidine 0.15g/L, Isoleucine 0.21g/L, leucine 0.475g/L, Methionin 0.44g/L, methionine(Met) 0.125g/L, phenylalanine 0.275g/L, proline(Pro) 0.675g/L, Serine 0.34g/L, Threonine 0.225g/L, tryptophane 0.05g/L, Xie Ansuan 0.325g/L, MgCl ₂6H ₂O 0.2g/L, CaCl ₂2H ₂O 0.05g/L, MnCl ₂4H ₂O 0.016g/L, FeCl ₃6H ₂O 0.008g/L, FeSO ₄7H ₂O 0.007g/L, ZnSO ₄7H ₂O 0.005g/L, CoSO ₄7H ₂O0.0025g/L, CuSO ₄7H ₂O 0.0025g/L, (NH4) ₆Mo7O242H ₂O 0.0025g/L, VITAMIN B4 0.01g/L, 0.01g/L guanine, uridylic 0.01g/L, xanthine 0.01g/L, p-benzaminic acid 10mg/L, inosine 5mg/L, vitamin B13 5mg/L, Pyridoxylamine hydrochloride 5mg/L, Thymine deoxyriboside 5mg/L, D-vitamin H 2.5mg/L, 6,8-Thioctic Acid 2.5mg/L, pyridoxine hydrochloride 2mg/L, folic acid 1mg/L, niacin 1mg/L, D-(+)-calcium pantothenate 1mg/L, riboflavin 1mg/L, thiamine hydrochloride 1mg/L, vitamin B12 1mg/L.

5, a kind of method of cultivating salt stress resisting lactococcus lactis is that Lactococcus lactis is cultivated in arbitrary described substratum in claim 1 to 4, obtains the Lactococcus lactis of salt stress-resistant.

6, method according to claim 5 is characterized in that: described Lactococcus lactis is for absorbing the lactococcus lactis subsp bacterial strain of gsh.

7, method according to claim 6 is characterized in that: described Lactococcus lactis is Lactococcus lactis SK11.