CN102080058A

CN102080058A - Lactobacillus casei and breeding method thereof

Info

Publication number: CN102080058A
Application number: CN 201010579466
Authority: CN
Inventors: 陈坚; 吴重德; 张娟; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-12-09
Filing date: 2010-12-09
Publication date: 2011-06-01
Anticipated expiration: 2030-12-09
Also published as: CN102080058B

Abstract

The invention discloses a lactobacillus casei and a breeding method thereof, belonging to the field of a food biotechnology. The method in the invention comprises the following steps: carrying out chemostatic culture on the lactobacillus casei ATCC334 until the lactobacillus casei is in a stable state, reducing the pH value of a culture system, adding a culture medium to be in the stable state according to a certain dilution ratio, reducing the pH value of the system again, and adding the culture medium to be in the stable state according to a certain dilution ratio; and repeating the process until a low pH value-resistant lactobacillus casei (CCTCC NO: M2010292) is screened. The biomass liveweight of the bred bacterial strain in a culture medium with the pH value of 4.3 is improved by 50% compared with that of the original bacterial strain, and the tolerances to lactic acid with the pH value of 3.3, hydrochloric acid with the pH value of 2.5 and 1.6% cholate are respectively improved by 103 times, 4.8 times and 2.3 times. By applying the method in the invention, the tolerance of the lactobacillus casei to environmental stress is effectively improved, and the method in the invention can be used for breeding lactobacillus with environmental stress resistance in the food industry.

Description

One strain lactobacterium casei and selection thereof

Technical field

The present invention relates to a strain lactobacterium casei and a selection thereof, belong to technical field of food biotechnology.

Background technology

Lactobacterium casei is the widely used probiotic bacterium of a class, and as industrialized cell factory, lactobacterium casei becomes the main force of production in fields such as food, fermentations, have high economic worth.Yet in the Industrial processes of reality, lactobacterium casei but inevitably is faced with the influence of the various unfavourable condition of the environment that comes from the outside.Along with constantly carrying out of fermentation, the organic acid accumulation volume constantly increases, in this process, the passive tenuigenin that diffuses into of lactic acid, acetate that fermentation generates, be dissociated into the proton that can not be oozed out by cytolemma fast and carry the acid ion of electric charge, and cause the rapid decline of pH in the born of the same parents and the scarcity of ATP synthesis capability, had a strong impact on the biomass and the physiologically active of cell, limit the normal growth and the metabolism of cell, greatly reduced production efficiency.Simultaneously, produce product, in human gastrointestinal tract, also be faced with such as environment-stress such as hydrochloric acid in gastric juice, cholate with the viable bacteria benefit that lactobacterium casei is the theme.Therefore, how to keep the thalline activity of lactobacterium casei to become academia and industrial community Focal Point of Common Attention problem.

Summary of the invention

Technical problem to be solved by this invention provides a strain lactobacterium casei, this bacterial strain was on November 4th, 2010, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M 2010292, address: China, Wuhan, Wuhan University, its taxonomy called after lactobacterium casei Lb-2 ( Lactobacillus caseiLb-2).

Described lactobacterium casei under the condition of pH 4.3, OD ₆₀₀Value reaches 3.12, with respect to having improved 56.78% before the seed selection.

Another technical problem to be solved by this invention provides the method for a kind of seed selection acid resistance milk-acid bacteria.

The technical scheme that addresses the above problem is: starting strain is cultured to stable state, reduces culture system pH value, to stable state, reduce system pH once more according to certain thinning ratio fed-batch medium, according to certain thinning ratio fed-batch medium to stable state; Repeat said process, obtain a strain acid resistance milk-acid bacteria until seed selection.

Described starting strain be lactobacterium casei ( Lactobacillus casei), available from U.S. ATCC, be numbered ATCC No:334.

Described stable state is that the phalangeal cell growth reaches steady state.

Described fed-batch medium mainly is that stream adds the cultured continuously mode that growth limiting factor comes regulating and controlling microbial growth and breeding and accretion rate.

Describedly adding according to certain thinning ratio stream, is to flow into substratum with constant speed, flows out nutrient solution with constant speed, thereby has guaranteed the stable of substratum chemical ingredients, makes the microorganism in the culture systems remain the stable speed of growth.

The lactobacterium casei Lb-2(CCTCC NO:M 2010292 that seed selection of the present invention obtains) under the condition of low pH, has better growth performance, have good lactic acid, hydrochloric acid and cholate tolerance; Selection provided by the invention, simple and effect is obvious.

Embodiment

The selection of embodiment 1 lactobacterium casei (CCTCC NO:M 2010292)

With business-like lactobacterium casei ( Lactobacillus casei) ATCC334 is starting strain, and it is cultured to logarithmic phase in seed culture medium, be inoculated in the fermention medium of pH5.3 and be cultured to OD ₆₀₀After=3.0, according to thinning ratio 0.1 h ^-1Stream rate of acceleration stream in chemostat add fermention medium to stable state, reduce culture system pH to 5.0 again, stop stream and add (D=0).This moment, cell was grown in the substratum of pH5.0, when cell concentration reaches OD ₆₀₀=3.0 o'clock, stream added stream and adds fresh fermention medium until reaching stable state.So repeatable operation reduces fermentation pH to 4.3 gradually, and growth reaches balance.

Getting the final nutrient solution of perseveranceization cultivation, through suitably diluting the flat board of coating pH4.3, select single bacterium colony, is 4.3 condition bottom fermentations with single bacterium colony in pH, filters out mutant strain according to the biomass size.Obtain the lactobacterium casei that many strains have tolerance high density tolerance to environmental stress, a strain called after lactobacterium casei Lb-2 wherein, its taxonomy called after lactobacterium casei Lb-2 ( Lactobacillus caseiLb-2), be preserved in Chinese typical culture collection center on November 4th, 2010, deposit number is CCTCC NO:M 2010292.

The device of described perseveranceization cultivation is called chemostat, and available from New Brunswick company, size is 1.3 L, and liquid amount is 0.7 L.

Growth characteristics under the embodiment 2 lactobacterium casei Lb-2 acid stress conditions

1, actication of culture

To be preserved in-70 ^oStarting strain lactobacterium casei in the C glycerine pipe ( Lactobacillus casei) the lactobacterium casei Lb-2 that arrives of ATCC 334 and seed selection inserts seed culture medium with 2% inoculum size, 37 ^oC leaves standstill and cultivates 16h.

2, seed culture

Inoculum size with 2% is got the good lactobacterium casei ATCC 334 of activation and lactobacterium casei Lb-2 respectively to seed culture medium, and 37 ^OC, leave standstill and be cultured to mid-log phase.

3, fermentor cultivation

Inoculum size with 2 % changes the cultured seed nutrient solution over to fermention medium respectively, and the pH that control fermentation is irritated is 4.3, with lactic acid and hydrochloric acid as the pH regulator agent, in 37 ^oCultivate 64 h under C, the 100 rpm conditions.Measure thalline in the fermented liquid after the fermentation ends, be respectively at pH under 4.3 the condition, final cheese milk bar Lb-2, cell concentration has improved 56.78% than lactobacterium casei ATCC 334, and the result is as shown in table 1.

Described seed culture medium is: glucose 20 g L ^-1, peptone 10 g L ^-1, extractum carnis 10 g L ^-1, Yeast diffusion juice 5 g L ^-1, anhydrous sodium acetate 5 g L ^-1, potassium primary phosphate 2 g L ^-1, Triammonium citrate 2 g L ^-1, MgSO ₄7H ₂O 0.2 g L ^-1, MnSO ₄H ₂O 0.05 g L ^-1, pH 6.5.

Described fermention medium is: glucose 8 g L ^-1, peptone 10 g L ^-1, extractum carnis 10 g L ^-1, Yeast diffusion juice 5 g L ^-1, anhydrous sodium acetate 5 g L ^-1, potassium primary phosphate 2 g L ^-1, Triammonium citrate 2 g L ^-1, MgSO ₄7H ₂O 0.2 g L ^-1, MnSO ₄H ₂O 0.05 g L ^-1

Table 1 acid stress condition hypothallus growth characteristics

Bacterial strain	L．casei?ATCC334	L．casei?Lb-2
			OD ₆₀₀	1.99	3.12

The experiment of embodiment 3 environmental resistance

1, actication of culture

To be preserved in-70 ^oStarting strain lactobacterium casei in the C glycerine pipe ( Lactobacillus casei) the lactobacterium casei Lb-2 that obtains of ATCC 334 and seed selection inserts seed culture medium with 2% inoculum size, 37 ^oC leaves standstill and cultivates 16h.

2, seed culture

3, coerce experiment

The cell of taking the logarithm and growing mid-term, through 4 ℃, the centrifugal 5 min collection of 8000 rpm thalline, after thalline washs centrifugal 2 times through phosphate buffered saline buffer (pH 7.0), be resuspended in respectively in the hydrochloric acid of lactic acid, pH 2.5 of pH 4.3 and 1.6% the cholate and coerce 3 h, again use identical damping fluid centrifuge washing cell 2 times again, and be resuspended in isopyknic damping fluid, get 1 mL thalline spread plate after appropriate dilution, in 37 ^oC cultivates 48 h, calculates survival rate size (shown in table 2) respectively.Lactobacterium casei Lb-2 has improved 103 times, 4,8 times and 2.3 times to the tolerance of lactic acid, hydrochloric acid and cholate respectively than lactobacterium casei ATCC 334.

The experiment of table 2 environmental resistance

Survival rate (%)	L．casei?ATCC334	L．casei?Lb-2
			Lactic acid	0.12	12.36
Hydrochloric acid	14.92	71.62
			Cholate	1.18	2.71

Though the present invention with preferred embodiment openly as above; but it is not in order to qualification the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.

Claims

1. a strain lactobacterium casei has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M 2010292.

2. the described lactobacterium casei of a strain claim 1 is characterized in that, places the lactic acid of pH 4.3 to coerce 3 h bacterial strain, and survival rate is 12.36%.

3. the described lactobacterium casei of a strain claim 1 is characterized in that, places the hydrochloric acid of pH 2.5 to coerce 3 h bacterial strain, and survival rate is 71.62%.

4. the described lactobacterium casei of a strain claim 1 is characterized in that, bacterial strain is placed 1.6% cholate coerce 3 h, and survival rate is 2.71%.

5. the selection of the described lactobacterium casei of claim 1 comprises the steps:

1) starting strain is cultured to stable state;

2) reduce culture system pH value;

3) stream substratum to the bacterial strain that adds certain thinning ratio reaches stable state;

4) circulation step 2) progressively be reduced to target pH to step 3), so that bacterial strain reaches stable state;

5) from final nutrient solution, separate single bacterium colony.

6. method according to claim 5 is characterized in that concrete steps are as follows:

1) lactobacterium casei ATCC334 is cultured to OD ₆₀₀=3.0;

2) reduce culture system pH;

3) by thinning ratio 0.1 h ^-1Fed-batch medium is to OD ₆₀₀=3.0;

4) circulation step 2) progressively reduce pH to 4.3 to step 3), and strain growth reaches balance;

5) from final nutrient solution, separate single bacterium colony.

7. according to claim 5 or 6 described methods, it is characterized in that substratum is: glucose 8 g L ^-1, peptone 10 g L ^-1, extractum carnis 10 g L ^-1, Yeast diffusion juice 5 g L ^-1, anhydrous sodium acetate 5 g L ^-1, potassium primary phosphate 2 g L ^-1, Triammonium citrate 2 g L ^-1, MgSO ₄7H ₂O 0.2 g L ^-1, MnSO ₄H ₂O 0.05 g L ^-1