Fermentation culture process of lactobacillus buchneri

Abstract

The invention discloses a lactobacillus buchneri fermentation culture process which comprises the following steps: activating a lactobacillus buchneri strain, preparing a seed solution, inoculating 1%-5% of the seed solution into a fermentation formula after the seed solution is cultured, culturing for 20-30 hours at 36 + / -1 DEG C to obtain a fermentation solution, then sucking 1-10 mL of the fermentation solution into 99-90 mL of 0.3% of sterile tween-80 water, diluting to a proper gradient, and culturing for 2-3 hours at the temperature of 36 + / -1 DEG C to obtain the lactobacillus buchneri fermentation culture process. The lactobacillus buchneri fermentation liquor has the advantages that the lactobacillus buchneri fermentation liquor is prepared by the aid of the lactobacillus buchneri fermentation liquor fermentation process, 1mL of lactobacillus buchneri fermentation liquor is sucked into a culture dish, 15mL of MRS detection culture media is added into the culture dish and uniformly mixed, the lactobacillus buchneri fermentation liquor is cultured in an incubator at 36 + / -1 DEG C for 48h, and the viable count of lactobacillus buchneri in the lactobacillus buchneri fermentation liquor obtained by the lactobacillus buchneri fermentation process is larger than or equal to 100 * 10 < 8

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a fermentation culture process for Lactobacillus buchneri. Background Technology

[0002] Agriculture, as my country's primary industry, plays a crucial guiding role in promoting its sustainable development for other industries. However, the use of chemical fertilizers in agricultural production causes a series of problems such as soil degradation and environmental pollution, which contradicts the concept of sustainable development. Improving current agricultural production techniques that involve excessive application of chemical fertilizers and pesticides, improving soil structure, increasing the quantity and activity of beneficial microorganisms in the soil, maintaining the balance of rhizosphere microbiota, promoting plant growth, improving fertilizer utilization, and enhancing soil fertility capacity have become increasingly important research topics. These efforts are essential for restoring a healthy farmland ecological environment, achieving harmonious coexistence and common development between agricultural production and nature, and promoting the sustainable development of agriculture's ecology, economy, and society.

[0003] In recent years, driven by research in microbiology and fertilizer science, the research and application technologies and achievements of microbial fertilizers have made a qualitative leap in China, and represent a major direction for future fertilizer development. Microbial fertilizers are products containing specific living microorganisms. Through the activity of these microorganisms, they increase the supply of nutrients to plants or promote plant growth, thereby increasing crop yields and improving product quality and the agricultural ecological environment. Furthermore, they also enhance soil fertility, strengthen plant resistance to stress, assist crops in nutrient absorption, and reduce the use of chemical fertilizers, making them increasingly important in the development of sustainable agricultural strategies.

[0004] In livestock farming, a large portion of the feed consumed by poultry and animals is not digested and utilized, which not only produces odors but also pollutes the soil, water sources, and other environments. Adding microorganisms to the feed can not only effectively prevent intestinal diseases in animals but also improve the animals' utilization of nitrogen sources, reduce environmental pollution, and lower the costs for farmers.

[0005] Lactobacillus blight is a Gram-positive, short rod-shaped, non-spore-forming lactic acid bacterium that undergoes heterofermentation. It can improve intestinal health, promote nutrient digestion and absorption, enhance immunity and disease resistance, reduce feed conversion ratio and improve production performance, improve the farming environment and reduce odor in farms, and improve the aerobic stability and quality of silage. It has broad application prospects in food manufacturing (pickles, fruit and vegetable products), agriculture (production of organic compound microbial fertilizer), silage, microbial feed additives, and wastewater treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a fermentation culture process for Lactobacillus buchneri, which is simple and easy to operate and conducive to industrial promotion.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a fermentation culture process for Lactobacillus brunelli, comprising the following steps: 1) Activation of bacterial strain: Inoculate the Lactobacillus buchneri strain onto the slant of an MRS test tube and incubate it in a 36±1℃ incubator for 20-30 hours to obtain activated seed slant; 2) Seed culture preparation: Transfer the activated seeds to a 150mL Erlenmeyer flask containing 50-120mL of sterilized fermentation medium and culture for 20-30h. Then, add 1%-5% to a 5000mL Erlenmeyer flask containing 1000-3000mL of sterilized fermentation medium and culture for 20-30h to obtain the seed culture. 3) Fermentation culture: The cultured seed liquid is transferred to a 1-ton fermenter containing 600L of sterilized fermentation medium and cultured for 20-30 hours to obtain the fermentation broth; 4) Viable cell count: Dilute the fermented broth with sterile 0.3‰ Tween-80 water to a suitable gradient, take 1 mL and mix it with 15 mL of LMR S test medium in a petri dish, and incubate at 36±1℃ for 48 h to obtain the viable cell count of the fermentation broth.

[0008] Furthermore, the fermentation culture medium formulation in steps 2) and 3) is as follows: fish peptone 1-100 g / L, glucose 1-100 g / L, yeast extract 1-100 g / L, sodium acetate 1-10 g / L, dipotassium hydrogen phosphate 1-10 g / L, manganese sulfate 0.001-0.05 g / L, and L-cysteine ​​hydrochloride 1-10 g / L.

[0009] Furthermore, the culture medium formula in step 4) is as follows: 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2g ammonium citrate, 2g dipotassium hydrogen phosphate, 1.5g L-cysteine ​​hydrochloride, 1g Tween-80, 18g agar powder, and distilled water to 1L, with a pH of 6.2-6.4.

[0010] The fermentation process of this invention is simple. After activating the *Lactobacillus bruneri* strain, a seed culture is prepared. After the seed culture is cultured, 1%-5% is added to the fermentation formula, and then the mixture is incubated at 36±1℃ for 20-30 hours to obtain the fermentation broth. The fermentation broth obtained using the fermentation process of this invention has a viable *Lactobacillus bruneri* count ≥100×10⁻⁶. 8 CUF / g. Detailed Implementation

[0011] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0012] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels. Example

[0013] This embodiment provides a fermentation culture process for Lactobacillus brunelli, which includes the following steps: 1) Activation of bacterial strain: Lactobacillus buchneri strain was inoculated onto MRS test tube slants and incubated in a 35℃ incubator for 30 hours to obtain activated seed slants; 2) Seed culture preparation: Add sterile water to the cultured test tube slant to make a bacterial suspension. Take 1 mL and transfer it to a 150 mL Erlenmeyer flask containing 30 mL of sterilized fermentation medium. Incubate at 34℃ and 100 r / min for 30 h. Then transfer it to a 5000 mL Erlenmeyer flask containing 1000 mL of sterilized fermentation medium and incubate at 34℃ and 100 r / min for 30 h. This is the seed culture. 3) Fermentation culture: The seed culture is inoculated into a 1-ton tank containing 600L of sterilized fermentation medium. The tank temperature is 35℃, the tank pressure is 0.05MPa, the aeration rate is 1:0.5, the stirring speed is 100r / min, the pH is controlled at 5.0 with NaOH solution, and the culture is carried out for 30h. When the OD is about 1.0×10 and no long chains are found under a microscope, the fermentation is over and the fermentation broth is obtained.

[0014] In this embodiment, the fermentation medium formulation for steps 2) and 3) is: fish peptone 1g / L, glucose 1g / L, yeast extract 1g / L, sodium acetate 1g / L, dipotassium hydrogen phosphate 1g / L, manganese sulfate 0.001g / L, and L-cysteine ​​hydrochloride 1g / L. Example

[0015] This embodiment provides a fermentation culture process for Lactobacillus brunelli, which includes the following steps: 1) Activation of bacterial strain: Lactobacillus buchneri strain was inoculated onto MRS test tube slants and incubated in a 36℃ incubator for 24 hours to obtain activated seed slants; 2) Seed culture preparation: Add sterile water to the cultured test tube slant to make a bacterial suspension. Take 5 mL and transfer it to a 150 mL Erlenmeyer flask containing 50 mL of sterile fermentation medium. Incubate at 36℃ and 100 r / min for 24 h. Then transfer it to a 5000 mL Erlenmeyer flask containing 2000 mL of sterile fermentation medium and incubate at 36℃ and 100 r / min for 24 h. This is the seed culture. 3) Fermentation culture: The seed culture was inoculated into a 1-ton tank containing 600L of sterilized fermentation medium. The tank temperature was 36℃, the tank pressure was 0.05MPa, the aeration rate was 1:0.5, the stirring speed was 100r / min, the pH was controlled with NaOH solution to 5.0, and the culture was carried out for 24h. When the OD was about 1.0×10 and no long chains were found under a microscope, the fermentation was completed and the fermentation broth was obtained.

[0016] In this embodiment, the fermentation medium formulation for steps 2) and 3) is as follows: fish peptone 50g / L, glucose 50g / L, yeast extract 1-100g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 5g / L, manganese sulfate 0.003g / L, and L-cysteine ​​hydrochloride 5g / L. Example

[0017] This embodiment provides a fermentation culture process for Lactobacillus brunelli, which includes the following steps: 1) Activation of bacterial strain: Lactobacillus bryonicus strain was inoculated onto MRS test tube slants and incubated in a 37℃ incubator for 20 hours to obtain activated seed slants; 2) Seed culture preparation: Add sterile water to the cultured test tube slant to make a bacterial suspension. Take 10 mL and transfer it to a 150 mL Erlenmeyer flask containing 100 mL of sterilized fermentation medium. Incubate at 38℃ and 100 r / min for 20 h. Then transfer it to a 5000 mL Erlenmeyer flask containing 3000 mL of sterilized fermentation medium and incubate at 38℃ and 100 r / min for 20 h. This is the seed culture. 3) Fermentation culture: The seed culture was inoculated into a 1-ton tank containing 600L of sterilized fermentation medium. The tank temperature was 37℃, the tank pressure was 0.05MPa, the aeration rate was 1:0.5, the stirring speed was 100r / min, the pH was controlled with NaOH solution to 5.0, and the culture was carried out for 20h. When the OD was about 1.0×10 and no long chains were found under a microscope, the fermentation was completed and the fermentation broth was obtained.

[0018] In this embodiment, the fermentation medium formulation for steps 2) and 3) is: 100 g / L fish peptone, 100 g / L glucose, 100 g / L yeast extract, 10 g / L sodium acetate, 10 g / L dipotassium hydrogen phosphate, 0.05 g / L manganese sulfate, and 10 g / L L-cysteine ​​hydrochloride. Example

[0019] This embodiment is used to detect the viable count in the Lactobacillus bromide fermentation broth obtained in Examples 1-3 above, and specifically includes the following steps: Using a sterile pipette, transfer 1-10 mL of fermentation broth into a 500 mL sterile glass Erlenmeyer flask containing 99-95 mL of 0.3‰ Tween-80 sterile water. Shake on a shaker at 20℃ and 200 rpm for 1 hour to obtain the mother liquor. Transfer 1 mL of the mother liquor to a sterile test tube containing 9 mL of 0.3‰ Tween-80 sterile water and vortex until homogeneous. Repeat this dilution process until a final dilution of 10 is achieved. -7 10 -8 10 -9 Perform three gradient tests, taking 1 mL of each gradient and transferring it to three sterile culture dishes. Then, pour in 15 mL of LMR detection medium, mix thoroughly, and incubate at 36±1℃ for 48 hours. The viable count of Lactobacillus bryonicus fermentation broth can then be obtained.

[0020] The culture medium for testing was formulated as follows: 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2g ammonium citrate, 2g dipotassium hydrogen phosphate, 1.5g L-cysteine ​​hydrochloride, 1g Tween-80, and 18g agar powder, with distilled water added to a final volume of 1L. The pH was 6.2-6.4.

[0021] Testing showed that the viable cell count in the Lactobacillus bromide fermentation broth obtained in Examples 1-3 was ≥100×10⁻⁶. 8 CUF / g.

Claims

1. A fermentation culture process for Lactobacillus brunelli, characterized in that: Includes the following steps: 1) Activation of bacterial strain: Inoculate the Lactobacillus buchneri strain onto the slant of an MRS test tube and incubate it in a 36±1℃ incubator for 20-30 hours to obtain activated seed slant; 2) Seed culture preparation: Transfer the activated seeds to a 150mL Erlenmeyer flask containing 50-120mL of sterilized fermentation medium and culture for 20-30h. Then, add 1%-5% to a 5000mL Erlenmeyer flask containing 1000-3000mL of sterilized fermentation medium and culture for 20-30h to obtain the seed culture. 3) Fermentation culture: The cultured seed liquid is transferred to a 1-ton fermenter containing 600L of sterilized fermentation medium and cultured for 20-30 hours to obtain the fermentation broth; 4) Viable cell count: Dilute the fermented broth with sterile 0.3‰ Tween-80 water to a suitable gradient, take 1 mL and mix it with 15 mL of LMR S test medium in a petri dish, and incubate at 36±1℃ for 48 h to obtain the viable cell count of the fermentation broth.

2. The fermentation culture process of Lactobacillus buchneri according to claim 1, characterized in that: The fermentation medium formulations in steps 2) and 3) are as follows: fish peptone 1-100 g / L, glucose 1-100 g / L, yeast extract 1-100 g / L, sodium acetate 1-10 g / L, dipotassium hydrogen phosphate 1-10 g / L, manganese sulfate 0.001-0.05 g / L, and L-cysteine ​​hydrochloride 1-10 g / L.

3. The fermentation culture process of Lactobacillus buchneri according to claim 1, characterized in that: The culture medium formula in step 4) is as follows: 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2g ammonium citrate, 2g dipotassium hydrogen phosphate, 1.5g L-cysteine ​​hydrochloride, 1g Tween-80, 18g agar powder, and distilled water to 1L, with a pH of 6.2-6.4.