CN102417892B - Feeding lactobacillus johnsonii high-density fermentation medium and application thereof - Google Patents

Abstract

The invention belongs to the technical field of lactic acid bacteria cultivation, and relates to a high-density fermentation medium of Lactobacillus johnsonii for feeding and an application thereof. Lactobacillus johnsonii fermented by using the high-density fermentation medium of the present invention and its fermentation method, the thalline density is significantly improved compared with ordinary fermentation, the number of viable bacteria is not less than 10 ¹⁰ cfu/ml, and the number of viable bacteria cultured compared with ordinary MRS medium The increase is more than 80 times, and the high-density fermentation of Lactobacillus johnsonii is realized; the fermentation medium and its application of the present invention also reduce the separation cost of biomass, shorten the production cycle, thereby reducing production cost and improving production efficiency.

Description

A kind of feeding Lactobacillus johnsonii high-density fermentation medium and application thereof

technical field

The invention belongs to the technical field of lactic acid bacteria cultivation, and relates to a high-density fermentation medium of Lactobacillus johnsonii for feeding and an application thereof.

Background technique

Lactic acid bacteria are normal bacteria commonly found in the digestive tract of animals. The history of using this type of bacteria to develop probiotics is the earliest. This bacteria has been widely used in fermented food, animal feed and health food.

Lactobacillus johnsonii (Lactobacillus johnsonii) is a kind of lactic acid bacteria. Based on the current literature reports about Lactobacillus johnsonii, its beneficial function to animals is mainly reflected in the following two aspects. On the one hand, Lactobacillus johnsonii has a natural defense effect on animals. System and immune function have a regulatory effect; on the other hand, Lactobacillus johnsoni showed a specific preventive effect on animal dermatitis. Tanaka et al. found that the incidence of atopic dermatitis (AD) in mice fed with Lactobacillus johnsonii was reduced, and at the same time, it could effectively inhibit epidermal hyperplasia in mice.

The fermentation density of the bacteria in the fermentation liquid obtained by the traditional static fermentation method is low, and the number of viable Lactobacillus johnsonii is about 500-100 million. In order to obtain more viable bacteria, it is necessary to increase the volume of the bioreactor Or increase the number of fermentations, etc., thereby increasing the separation cost of biomass and prolonging the production cycle, thereby increasing production costs and reducing production efficiency.

Lactobacillus johnsoni is still difficult to achieve high-density fermentation. The main problem that is difficult to achieve is the fermentation medium and its fermentation method. The lack of nutrients and the accumulation of metabolites (mainly lactic acid) in the later stage of fermentation inhibit the growth of bacteria, making high-density fermentation difficult. accomplish.

Contents of the invention

(1) Technical problems to be solved

The object of the invention is to provide a high-density fermentation medium for feeding Lactobacillus johnsonii.

Another object of the present invention is to provide the application of the high-density fermentation medium in the fermentation of Lactobacillus johnsonii.

(2) Technical solution

The purpose of the present invention is achieved through the following technical solutions:

The invention provides a high-density fermentation medium of Lactobacillus johnsonii for feeding. The main components and contents of the medium are as follows: glucose 20-30g/L, tryptone 10-15g/L, beef extract Or beef powder 6~15g/L, yeast powder 5~10g/L, K ₂ HPO ₄ 2~3g/L, CH ₃ COONa 5~8g/L, sodium citrate 2~4g/L, Mg ²⁺ water soluble Compound 0.2-0.4g/L, Fe ²⁺ water-soluble compound 0.1-0.2g/L and Mn ²⁺ water-soluble compound 45-55mg/L.

Wherein, the Mg ²⁺ water-soluble compound is any one of MgSO ₄ and MgCl ₂ ; the Fe ²⁺ water-soluble compound is FeSO ₄ , FeCl ₂ , (NH ₄ ) ₂ Fe(SO ₄ ) ₂ Any one of them; the Mn ²⁺ water-soluble compound is MnSO ₄ .

The preparation method of fermentation medium of the present invention is as follows:

(1) Weigh each component according to the content, dissolve with distilled water, wherein glucose is dissolved separately;

(2) Glucose is sterilized separately at 115°C for 20 minutes, and other components are put into a fermenter and sterilized at 121°C for 30 minutes;

(3) Before inoculation, glucose is added into the fermenter through aseptic operation, stirred and mixed evenly, and the obtained product is obtained.

The high-density fermentation medium of Lactobacillus johnsonii for feeding of the present invention can be applied in the fermentation of Lactobacillus johnsonii, and in order to maximize the effect of the culture medium of the present invention, when using the culture medium of the present invention to ferment Lactobacillus johnsonii, its The fermentation process is as follows:

(1) Activate cryopreserved Lactobacillus johnsonii three times on the MRS agar plate, pick a single colony and inoculate it into the deep MRS liquid test tube, cultivate it at 37°C for 10 hours, transfer it to the MRS liquid medium with 1% inoculation amount, and culture After 8 to 10 hours, it is used as a seed solution;

(2) Prepare a high-density fermentation medium for Lactobacillus johnsonii. When the medium drops to the required temperature, connect a dissolved oxygen electrode, and mark the dissolved oxygen as 100%;

(3) inoculating the seed liquid obtained in step (1) in the fermentation medium of step (2) with an inoculation amount of 5% to 10%;

(4) Fermentation, maintain the pH value of the fermentation broth at 6-6.5 during the fermentation process, when the pH exceeds the set value, add feed liquid; when the pH is lower than the set value, automatically add alkaline neutralizer ammonia water, KOH , any one of NaOH; ferment for 10-14 hours at a temperature of 37-39°C and a rotational speed of 100 rpm, without aeration or N ₂ during the fermentation process, and maintain dissolved oxygen less than 1%;

(5) When the pH of the added feed liquid no longer decreases or the absorbance value of the fermented liquid no longer increases at 600nm wavelength, the fermentation is terminated, and the fermented liquid with the viable count of Lactobacillus johnsonii reaching more than 10 ¹⁰ cfu/ml is obtained.

Wherein, the main components and contents of the feed liquid are as follows: 500 g/L sucrose, 150 g/L peptone, 150 g/L beef powder or beef extract, 75 g/L yeast extract or yeast extract powder.

The high-density fermentation medium and the fermentation method thereof of the present invention can also be applied to other lactic acid bacteria, but in order to achieve the best inventive effect, it is preferably applicable to Lactobacillus johnsonii.

(3) Beneficial effects

The present invention adopts the fed-batch fermentation strategy of pH feedback control, and at the same time reduces the concentration of lactic acid in the fermentation liquid, thereby releasing the feedback inhibition effect of lactic acid, using the high-density fermentation medium and its fermentation method of the present invention to ferment Lactobacillus johnsonii, The cell density is significantly higher than that of ordinary fermentation, and the number of viable bacteria is not less than 10 ¹⁰ cfu/ml, which is a fermentation standard that is difficult to achieve with existing technologies. Compared with ordinary MRS medium, the number of viable bacteria cultured is 5.1×10 ⁸ cfu/ml, The increase is more than 80 times, and the high-density fermentation of Lactobacillus johnsonii is realized; the fermentation medium and its application of the present invention also reduce the separation cost of biomass, shorten the production cycle, thereby reducing production cost and improving production efficiency.

Detailed ways

The present invention will be described more specifically through the following examples, but it should be understood that the examples are only for illustrating the present invention, not limiting the scope of the present invention in any way.

The used instrument model, reagent and source thereof of the following examples of the present invention are as follows:

Fermentation tank: BioFlo/Celligen115 type of New Brunswick company;

Circulating water machine: DX-208 type of Beijing Changliu Scientific Instrument Company;

Air compressor: HF-6100C type of Beijing Haofu Electromechanical Technology Development and Application Co., Ltd.;

Spectrophotometer: Shanghai Precision Scientific Instrument Co., Ltd. Model 721N;

Microscope: OLYMPUS CX31;

Ultra-clean bench: Sujing Antai SW-CJ-2FD type;

Biochemical incubator: Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory SPX-250B-Z type

Yeast extract, beef powder, and tryptone were purchased from Beijing Aoboxing Biotechnology Co., Ltd., and other reagents were purchased from Beijing Chemical Reagent Company.

Lactobacillus johnsonii: It comes from the China General Microorganism Culture Collection Management Center, and the preservation number is CGMCC No.4926.

Preparation and fermentation comparison of embodiment 1 fermentation medium

1. The components and content of Lactobacillus johnsonii high-density fermentation medium are:

Glucose: 20g/L, tryptone: 10g/L, beef extract: 10g/L, yeast powder: 5g/L, K ₂ HPO ₄ : 2g/L, CH ₃ COONa: 5g/L, sodium citrate: 2g/L L. MgSO ₄ : 0.2g/L, FeSO ₄ : 0.1g/L, MnSO ₄ : 55mg/L.

2. Medium preparation method:

(1) Weigh each component according to the above-mentioned content, dissolve with distilled water, wherein glucose is dissolved separately;

(2) Glucose is sterilized separately at 115°C for 20 minutes, and other components are put into a fermenter and sterilized at 121°C for 30 minutes;

(3) Before inoculation, glucose is added into the fermenter through aseptic operation, stirred and mixed evenly, and the obtained product is obtained.

3. Comparison between high-density fermentation and existing technology fermentation

3.1 The main steps of high-density fermentation are as follows

(1) Activate cryopreserved Lactobacillus johnsonii three times on the MRS agar plate, pick a single colony and inoculate it into the deep MRS liquid test tube, cultivate it at 37°C for 10 hours, transfer it to the MRS liquid medium with 1% inoculation amount, and culture After 10 hours as a seed solution;

(2) Prepare the fermentation medium, when the medium drops to the required temperature, connect the dissolved oxygen electrode, and mark the dissolved oxygen as 100%;

(3) inoculate the seed liquid obtained in step (1) in the fermentation medium of step (2) with an inoculum size of 5%;

(4) Fermentation, maintain the pH value of the fermentation broth at 6.0 during the fermentation process. When the pH exceeds 6.0, add about 100ml of feed liquid; when the pH is lower than 6.0, automatically add alkaline neutralizer ammonia water; Ferment for 10 hours under the condition of rotating speed 100rpm, the fermentation process is not ventilated, and the dissolved oxygen is maintained at less than 1%;

(5) When the pH of the added feed liquid no longer decreases, the fermentation is terminated to obtain a fermentation liquid in which the viable count of Lactobacillus johnsonii reaches 4.3×10 ¹⁰ cfu/ml.

3.2 Control experiment

(1) The components and contents of Lactobacillus johnsonii fermentation medium (MRS medium) are:

Glucose: 20g/L, peptone: 10g/L, beef extract: 5g/L, yeast powder: 4g/L, K ₂ HPO ₄ : 2g/L, CH ₃ COONa: 5g/L, sodium citrate: 2g/L , MgSO ₄ : 0.2g/L, MnSO ₄ : 50mg/L, Tween 80: 1ml.

(2) fermentation

The cryopreserved Lactobacillus johnsonii was activated three times on the MRS agar plate, and a single colony was picked and inoculated into the deep MRS liquid test tube, and cultured at 37°C for 10 hours. Transfer to MRS liquid medium with 1% inoculum amount, cultivate for 10 hours as seed liquid, and inoculate fermentation medium with 10% inoculum amount. After static culture at 37°C for 10 hours, the fermentation was terminated, and the viable count of Lactobacillus johnsonii was 5.1×10 ⁸ cfu/ml.

3.3 Result analysis

Apply the culture medium of the present invention and its fermentation method, can obtain the fermented liquid that the Lactobacillus johnsonii viable count reaches 4.3×10 ¹⁰ cfu/ml, and apply current fermentation culture medium (MRS culture medium) and its fermentation method, obtain The viable count of Lactobacillus johnsonii was only 5.1×10 ⁸ cfu/ml. Experiments have proved that the number of viable Lactobacillus johnsonii obtained by the medium and the fermentation method of the present invention is nearly 80 times that of the prior art, and high-density fermentation of Lactobacillus johnsonii has been realized.

Preparation and fermentation comparison of embodiment 2 fermentation medium

1. The components and contents of Lactobacillus johnsonii high-density fermentation medium are:

Glucose: 25g/L, tryptone: 10g/L, beef powder: 15g/L, yeast powder: 10g/L, K ₂ HPO ₄ : 2g/L, CH ₃ COONa: 7g/L, sodium citrate: 3g/L L, MgCl ₂ : 0.2g/L, FeCl ₂ : 0.1g/L, MnSO ₄ : 50mg/L.

2. Medium preparation method:

(1) Weigh each component according to the content, dissolve with distilled water, wherein glucose is dissolved separately;

(2) Glucose is sterilized separately at 115°C for 20 minutes, and other components are put into a fermenter and sterilized at 121°C for 30 minutes;

(3) Before inoculation, glucose is added into the fermenter through aseptic operation, stirred and mixed evenly, and the obtained product is obtained. 3. Comparison between high-density fermentation and existing technology fermentation

3.1 The main steps of high-density fermentation are as follows

(1) Activate cryopreserved Lactobacillus johnsonii three times on the MRS agar plate, pick a single colony and inoculate it into the deep MRS liquid test tube, cultivate it at 37°C for 10 hours, transfer it to the MRS liquid medium with 1% inoculation amount, and culture 8 hours later as seed liquid;

(2) Prepare the fermentation medium, when the medium drops to the required temperature, connect the dissolved oxygen electrode, and mark the dissolved oxygen as 100%;

(3) inoculate the seed liquid obtained in step (1) in the fermentation medium of step (2) with an inoculum size of 5%;

(4) Fermentation. During the fermentation process, the pH value of the fermented liquid is maintained at 6.3. When the pH exceeds the set value, about 100ml of feed liquid is added; when the pH is lower than the set value, the alkaline neutralizer ammonia water is automatically added; Ferment for 12 hours at a temperature of 38°C and a rotation speed of 100 rpm, without ventilation during the fermentation process, and maintain dissolved oxygen less than 1%;

(5) When the OD ₆₀₀ of the fermented liquid of the added feed liquid does not increase any more, the fermentation is terminated, and the fermented liquid in which the viable count of Lactobacillus johnsonii reaches 5.9×10 ¹⁰ cfu/ml is obtained.

3.2 Control experiment:

(1) The components and content of Lactobacillus johnsonii fermentation medium (MRS medium) are:

Glucose: 20g/L, peptone: 10g/L, beef powder: 5g/L, yeast powder: 4g/L, K ₂ HPO ₄ : 2g/L, CH ₃ COONa: 5g/L, sodium citrate: 2g/L , MgSO ₄ : 0.2g/L, MnSO ₄ : 50mg/L, Tween 80: 1ml.

(2) Fermentation:

The cryopreserved Lactobacillus johnsonii was activated three times on the MRS agar plate, and a single colony was picked and inoculated into the deep MRS liquid test tube, and cultured at 37°C for 10 hours. Transfer to MRS liquid medium with 1% inoculum amount, cultivate for 10 hours as seed liquid, and inoculate fermentation medium with 10% inoculum amount. Static culture was carried out at 38°C for 12 hours, and the fermentation was terminated, and the number of viable Lactobacillus johnsonii was 6.0×10 ⁸ cfu/ml.

3.3 Result analysis

Applying the culture medium of the present invention and its fermentation method, can obtain the fermented liquid that the Lactobacillus johnsonii viable count reaches 5.9×10 ¹⁰ cfu/ml, and apply the current fermentation culture medium (MRS culture medium) and its fermentation method, obtain The viable count of Lactobacillus johnsonii is only 6.0×10 ⁸ cfu/ml. Experiments have proved that the number of viable Lactobacillus johnsonii obtained by the culture medium and the fermentation method of the present invention is 98 times that of the prior art, and high-density fermentation of Lactobacillus johnsonii has been realized.

Preparation and fermentation comparison of embodiment 3 fermentation medium

1. The components and contents of Lactobacillus johnsonii high-density fermentation medium are:

Glucose: 30g/L, tryptone: 15g/L, beef extract: 6g/L, yeast powder: 6g/L, K ₂ HPO ₄ : 3g/L, CH ₃ COONa: 8g/L, sodium citrate: 4g/L L, (NH ₄ ) ₂ Fe(SO ₄ ) ₂ : 0.2 g/L, MgSO ₄ : 0.4 g/L, MnSO ₄ : 45 mg/L.

2. Medium preparation method:

(1) Weigh each component according to the content, dissolve with distilled water, wherein glucose is dissolved separately;

(2) Glucose is sterilized separately at 115°C for 20 minutes, and other components are put into a fermenter and sterilized at 121°C for 30 minutes;

(3) Before inoculation, glucose is added into the fermenter through aseptic operation, stirred and mixed evenly, and the obtained product is obtained.

3. Comparison between high-density fermentation and existing technology fermentation

3.1 The main steps of high-density fermentation are as follows

(1) Activate cryopreserved Lactobacillus johnsonii three times on the MRS agar plate, pick a single colony and inoculate it into the deep MRS liquid test tube, cultivate it at 37°C for 10 hours, transfer it to the MRS liquid medium with 1% inoculation amount, and culture After 10 hours as a seed solution;

(2) Prepare the fermentation medium, when the medium drops to the required temperature, connect the dissolved oxygen electrode, and mark the dissolved oxygen as 100%;

(3) inoculate the seed liquid obtained in step (1) in the fermentation medium of step (2) with an inoculum size of 10%;

(4) Fermentation, maintain the pH value of the fermented liquid during the fermentation process at 6.5, when the pH exceeds the set value, add about 100ml of feed liquid; when the pH is lower than the set value, automatically add alkaline neutralizer ammonia water; Ferment for 14 hours at a temperature of 39°C and a rotation speed of 100 rpm, nitrogen gas is introduced during the fermentation process, and dissolved oxygen is maintained at less than 1%;

(5) When the pH of the added feed liquid no longer decreases, the fermentation is terminated, and the fermentation liquid in which the viable count of Lactobacillus johnsonii reaches 4.9×10 ¹⁰ cfu/ml is obtained.

3.2 Control experiment:

(1) The components of Lactobacillus johnsonii fermentation medium (MRS medium) are:

Glucose: 20g/L, peptone: 10g/L, beef extract: 5g/L, yeast powder: 4g/L, K ₂ HPO ₄ : 2g/L, CH ₃ COONa: 5g/L, sodium citrate: 2g/L , MgSO ₄ : 0.2g/L, MnSO ₄ : 50mg/L, Tween 80: 1ml.

(2) fermentation

The cryopreserved Lactobacillus johnsonii was activated three times on the MRS agar plate, and a single colony was picked and inoculated into the deep MRS liquid test tube, and cultured at 37°C for 10 hours. Transfer to MRS liquid medium with 1% inoculum amount, cultivate for 10 hours as seed liquid, and inoculate fermentation medium with 10% inoculum amount. After static culture at 39°C for 14 hours, the fermentation was terminated, and the viable count of Lactobacillus johnsonii was 4.8×10 ⁸ cfu/ml.

3.3 Result analysis

Apply the culture medium of the present invention and its fermentation method, can obtain the fermented liquid that the number of live bacteria of Lactobacillus johnsonii reaches 4.9×10 ¹⁰ cfu/ml, and apply the current fermentation culture medium (MRS culture medium) and its fermentation method, obtain The viable count of Lactobacillus johnsonii was only 4.8×10 ⁸ cfu/ml. Experiments have proved that the number of viable Lactobacillus johnsonii obtained by the medium and the fermentation method of the present invention is nearly 102 times that of the prior art, and high-density fermentation of Lactobacillus johnsonii has been realized. In production, the realization of high-density fermentation can reduce the cost of biomass separation, shorten the production cycle, reduce production costs, improve production efficiency, etc., which is of great significance in the contemporary society of high efficiency and energy saving.

Claims (2)

1. Lactobacillus johnsonii (Lactobacillus johnsonii) CGMCC No.4926 high-density fermentation method for feeding is characterized in that the fermentation process is as follows: (1) Activate cryopreserved Lactobacillus johnsonii three times on the MRS agar plate, pick a single colony and inoculate it into the deep MRS liquid test tube, cultivate it at 37°C for 10 hours, transfer it to the MRS liquid medium with 1% inoculation amount, and culture After 8 to 10 hours, it is used as a seed solution; (2) Prepare a high-density fermentation medium, when the medium drops to the required temperature, connect the dissolved oxygen electrode, and mark the dissolved oxygen as 100%; (3) inoculating the seed liquid obtained in step (1) in the fermentation medium of step (2) with an inoculation amount of 5% to 10%; (4) Fermentation, maintain the pH value of the fermentation broth at 6-6.5 during the fermentation process, when the pH exceeds the set value, add feed liquid; when the pH is lower than the set value, automatically add alkaline neutralizer ammonia water, KOH , any one of NaOH; ferment for 10-14 hours at a temperature of 37-39°C and a rotational speed of 100 rpm, without aeration or N ₂ during the fermentation process, and maintain dissolved oxygen less than 1%; (5) When the pH of the added feed liquid no longer decreases or the absorbance value of the fermented liquid no longer increases at 600nm wavelength, the fermentation is terminated, and the fermented liquid in which the viable count of Lactobacillus johnsonii reaches more than ¹⁰ cfu/ml is obtained, Wherein, the main components and contents of the high-density fermentation medium are as follows: glucose 20-30g/L, tryptone 10-15g/L, beef extract or beef powder 6-15g/L, yeast powder 5-10g /L, K ₂ HPO ₄ 2～3g/L, CH ₃ COONa 5～8g/L, Sodium citrate 2～4g/L, Mg ²⁺ water soluble compound 0.2～0.4g/L, Fe ²⁺ water soluble compound 0.1～0.2g/L and Mn ²⁺ water-soluble compound 45～55mg/L, Wherein, the main components and contents of the feed liquid are as follows: 500 g/L sucrose, 150 g/L peptone, 150 g/L beef powder or beef extract, 75 g/L yeast extract or yeast extract powder. 2. The high-density fermentation method according to claim 1, characterized in that, the Mg ²⁺ water-soluble compound is any one of MgSO ₄ , MgCl ₂ ; the Fe ²⁺ water-soluble compound is FeSO _4. Any one of FeCl ₂ , (NH ₄ ) ₂ Fe(SO ₄ ) ₂ ; the Mn ²⁺ water-soluble compound is MnSO ₄ . 3. The high-density fermentation method as claimed in claim 1 or 2, wherein the preparation method of the fermentation medium is as follows: (1) Weigh each component according to the content, dissolve with distilled water, wherein glucose is dissolved separately; (2) Glucose is sterilized separately at 115°C for 20 minutes, and other components are put into a fermenter and sterilized at 121°C for 30 minutes; (3) Before inoculation, glucose is added into the fermenter through aseptic operation, stirred and mixed evenly, and the obtained product is obtained.