High-density culture method of lactobacillus amylophilus

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-density culture method of lactobacillus amylophilus. According to the method, fermentation raw materials and a fermentation process of lactobacillus amylophilus are improved, traditional continuous culture is changed into fed-batch culture, the amount of zymophyte is increased by about one time by feeding carbon and nitrogen sources and optimizing proper feeding time and feeding volume, in addition, components such as trehalose, skim milk powder and vitamin C are adopted as spray-drying protective agents, and the production cost is reduced. Finally, according to the lactobacillus amylophilus obtained through fermentation culture with the culture method, the zymophyte amount is increased to 3 billion cfu / mL from 5 billion cfu / mL before improvement, the spray-dried bacterium amount is 135 billion cfu / g, and the powder spraying yield is 90%.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and specifically relates to a high-density culture method for Lactobacillus amyloliquefaciens. Background Technology

[0002] Lactobacillus amyloliquefaciens is a Gram-positive, facultative anaerobic, non-spore-forming lactic acid bacterium with a unique ability to hydrolyze starch. It can directly utilize raw starch or gelatinized starch to produce acid, thus showing significant application potential in fields such as feed, food, environmental protection, and biomedicine.

[0003] Currently, liquid fermentation of Lactobacillus amyloliquefaciens mostly uses traditional MRS medium or simple optimized basal medium, which faces the following core technical bottlenecks:

[0004] Firstly, it is difficult to simultaneously achieve a high bacterial yield and viability during fermentation. For example, patent CN104212743A discloses that the viable count of *Lactobacillus amyloliquefaciens* obtained through conventional liquid fermentation is only 3-5 billion CFU / mL. Although some literature records that the bacterial count of *Lactobacillus amyloliquefaciens* can reach 8 × 10⁻⁶ CFU / mL during high-density culture. 9 The concentration of live bacteria is CFU / mL, but it is easily affected by the triple stress of lactic acid accumulation, increased metabolite concentration, and excessively high bacterial density, which leads to a decrease in the integrity of bacterial cell membranes, with the damage rate rising to 35% and the starch hydrolase activity decreasing by 50%. Although the final number of live bacteria can reach a certain level, the effective bacterial cells with starch hydrolysis function account for only 60%~70%, which cannot meet the needs of industrial high-activity products (Zhang Jun et al., Stress response of lactic acid bacteria and its effect on carbohydrate metabolism [J]. China Food Journal, 2017, 17(6):1-9.).

[0005] Secondly, the culture medium is expensive and has poor adaptability. Traditional culture relies on expensive nitrogen sources such as yeast extract and peptone, resulting in a high proportion of raw material costs. While industrial by-products (such as tofu whey and sweet potato juice) can replace some raw materials, their composition fluctuates greatly, which can easily lead to significant differences in bacterial count between batches.

[0006] This invention uses inexpensive raw materials as the main fermentation raw materials, which reduces the cost of raw materials. Furthermore, by adjusting the carbon-nitrogen ratio in the fermentation formula and adopting a batch feeding process, the fermentation bacterial count is increased to 30 billion.

[0007] In addition, the production of probiotics into dry powder provides support and guarantee for the widespread application of probiotics. However, the protective agents used in the spray drying process of probiotics in the existing technology are mostly maltodextrin, sodium microcrystalline cellulose, sorbitol and other ingredients. The total yield of spray drying is only about 20%, and the loss rate of probiotics is high.

[0008] Improving the viable cell count and spray-drying survival rate of probiotic fermentation broth has become a key research focus in the industrial production of probiotic powder. Therefore, developing a suitable culture medium for *Lactobacillus amyloliquefaciens* and establishing a specific fermentation process and spray-drying protectant are crucial for obtaining high-concentration fermentation broth and high-yield bacterial powder. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention proposes a high-density cultivation method for *Lactobacillus amyloliquefaciens*. The most significant feature of this method compared to traditional methods is the optimized formulation employed during the fermentation cultivation of *Lactobacillus amyloliquefaciens*: firstly, inexpensive corn steep liquor / soybean protein powder is used to replace peptone, beef extract, and other raw materials, significantly reducing the raw material cost of the fermentation medium; secondly, a fed-batch process is used, adding starch, glucose, ammonium sulfate, and other components in batches to provide carbon and inorganic nitrogen sources for the reproduction of *Lactobacillus amyloliquefaciens*, ultimately resulting in a significant increase in the fermentation biomass of *Lactobacillus amyloliquefaciens*. Furthermore, this invention further optimizes the formulation of the spray-drying protectant. Ultimately, the fermentation biomass of *Lactobacillus amyloliquefaciens* obtained through the fermentation cultivation process of this invention increases from 5 billion to 25-30 billion, with a spray-dried biomass of 1.3 trillion and a spray-dried biomass yield of 85%-90%.

[0010] The technical solution provided by this invention is:

[0011] A method for high-density culture of Lactobacillus amyloliquefaciens includes the following steps:

[0012] S1 strain activation culture: The preserved Lactobacillus amyloliquefaciens strain is activated and cultured on a plate medium until a suspension of third-generation Lactobacillus amyloliquefaciens is obtained;

[0013] S2 Primary Seed Tank Cultivation: Take the bacterial suspension cultured in S1 and inoculate it into a seed tank containing seed culture medium at an inoculation rate of 0.3%~1% (v / v). Control the temperature at 35~40℃, the initial rotation speed at 50~100 rpm, and the aeration ratio at 0.03~0.05vvm to obtain the primary seed solution.

[0014] The seed culture medium is formulated as follows: peptone 10-20 g / L, yeast extract 10-30 g / L, glucose 10-30 g / L, beef extract powder 10-20 g / L, potassium dihydrogen phosphate 1-8 g / L, magnesium sulfate 1-8 g / L, sodium acetate 3-5 g / L, manganese sulfate 0.5-1.5 g / L, and Tween-80 0.5-1 g / L;

[0015] S3 Fermenter Cultivation: Transfer the primary seed culture from S2 to a fermenter containing fermentation medium, with an inoculum size of 5%–10% (v / v). Introduce sterile air, turn on the agitator at 60–100 rpm, maintain an aeration ratio of 0.03–0.05 vvm, and set the temperature to 36–37°C. Begin fermentation with an initial pH of 6.3–6.5. Set the dissolved oxygen (DO) to 100%, and activate pH auto-control. Maintain the pH at 6.3–6.5 and wait for net OD to develop. 600 When the concentration reaches ≥3.0, start feeding. Feeding takes 4-6 hours, and the fermentation cycle is 16-20 hours, which yields a high-density fermentation broth of Lactobacillus amyloliquefaciens.

[0016] The fermentation medium is formulated as follows: corn steep liquor / soybean protein powder: 15-40 g / L, yeast extract: 5-30 g / L, glucose: 10-30 g / L, starch: 10-20 g / L, potassium dihydrogen phosphate: 1-8 g / L, dipotassium hydrogen phosphate: 1-5 g / L, sodium acetate: 1-5 g / L, magnesium sulfate: 1-8 g / L, urea: 1-7 g / L, ammonium sulfate: 0.5-5 g / L, manganese sulfate: 0.5-1.5 g / L;

[0017] The feed formulation is: starch 300~600 g / L, ammonium sulfate 30~60 g / L, glucose 50~150 g / L, preferably: starch 500 g / L, ammonium sulfate 50 g / L, glucose 100 g / L.

[0018] In fact, the inventors discovered during their experiments that the timing of batch feeding is crucial in the culture of Lactobacillus amyloliquefaciens. If the feeding is done too early, such as at OD... 600 When the concentration is around 2.0, the nutrients in the base material have not yet been fully consumed, especially glucose. This can easily lead to the microorganisms stopping growth in the later stages of fermentation due to the inhibitory effect of substrate concentration. If the feeding is done too late, such as when the OD value is too low... 600 ≥4.0 OD will, on the one hand, prolong the fermentation cycle by an average of about 4-7 hours, and on the other hand, inhibit the growth of the microorganisms, preventing the achievement of optimal biomass. Experiments have verified that when OD... 600 The optimal time to replenish feed is when the ratio is 3.0.

[0019] In the above-mentioned culture method provided by the present invention, preferably, the formulation of the plate culture medium in S1 is as follows: peptone 5~15 g / L, beef extract 5~15 g / L, yeast extract 5~8 g / L, glucose 15~25 g / L, Tween-80 0.5~1.5 g / L, dipotassium hydrogen phosphate 1.5~3.5 g / L, sodium acetate 2.5~5 g / L, ammonium citrate 2.5~3.5 g / L, magnesium sulfate 0.5~1 g / L, manganese sulfate 0.1~0.2 g / L, L-cysteine ​​hydrochloride 0.1~0.5 g / L, and agar powder 15~20 g / L.

[0020] Preferably, in S2, pH and OD are monitored periodically during the culture process. 600 And microscopic examination, when the pH is between 4.5 and 5.0, OD 600 When ≥100 specimens are found under 100x oil immersion microscopy at a magnification of ≥3.5, a first-grade seed solution is obtained.

[0021] As a preferred embodiment, in S3, when the pH is automatically controlled, 20%~30% sodium hydroxide that has been sterilized in advance is used to adjust the pH.

[0022] As a preferred embodiment, in S3, during fermentation, depending on the foam situation, if the foam reaches the top sight glass of the tank, 20wt%~30wt% of a polyether defoamer is added.

[0023] Preferably, the number of *Lactobacillus amyloliquefaciens* in the high-density fermentation broth is ≥3×10⁻⁶. 10 cfu / mL.

[0024] The present invention also provides a probiotic powder containing Lactobacillus amyloliquefaciens, wherein the powder comprises Lactobacillus amyloliquefaciens obtained by the above-described culture method and a protectant. Based on the mass of Lactobacillus amyloliquefaciens cells, the mass percentage of each component in the protectant is as follows: skim milk powder 1%~5%, maltodextrin 1%~3%, trehalose 1%~2%, sodium microcrystalline cellulose 2%~5%, vitamin C 0.5%~1%, and sorbitol 1%~2%.

[0025] Preferably, the probiotic powder is obtained by spray drying, wherein the spray drying conditions are: inlet air temperature 125~135℃ and outlet air temperature 65~75℃.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] (1) The present invention uses inexpensive ingredients such as corn steep liquor and soybean protein powder to replace high-cost culture medium raw materials such as peptone and beef extract, which greatly saves the cost of culture medium raw materials in the fermentation process of Lactobacillus amyloliquefaciens;

[0028] (2) This invention starts with the fermentation process of Lactobacillus amyloliquefaciens, changing the traditional continuous culture to fed-batch culture. By adding carbon and nitrogen sources, optimizing the appropriate timing and volume of feeding, the amount of fermentation bacteria is increased by about 100%.

[0029] (3) The present invention uses trehalose, skim milk powder and vitamin C as spray drying protectants, which significantly improves the stability of the bacterial powder product; finally, the amount of Lactobacillus amyloliquefaciens obtained by fermentation culture through the culture method of the present invention increased from 5 billion to 30 billion, the amount of spray drying bacteria was 1.35 trillion, and the spray powder yield was 90%. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0031] Example 1

[0032] A method for high-density culture of Lactobacillus amyloliquefaciens, comprising the following steps:

[0033] S1 strain activation culture

[0034] The *Lactobacillus amyloliquefaciens* strain preserved in glycerol tubes was activated onto a first-generation agar plate in a sterile laminar flow hood and incubated at 37°C for 2 days until colonies appeared. The colonies were milky white, with neat edges, uniform size, and free of contaminants. Then, three single colonies from the first-generation plate were picked and activated onto a second-generation agar plate in a sterile laminar flow hood and incubated at 37°C for 2 days until colonies appeared. The single colonies from the second-generation agar plate were then inoculated into a liquid shake flask and incubated at 37°C for 18 hours until the bacterial cells grew and the OD (Organic Difference) was measured. 600 A suspension of third-generation Lactobacillus amyloliquefaciens is obtained when the pH is ≥3.0, pH ≤5.0, and each field of view under a microscope (100x oil immersion) contains >100 cocci and no other bacteria.

[0035] The formulation of the plate solid culture medium is as follows: peptone 10 g / L, beef extract 8 g / L, yeast extract 5 g / L, glucose 20 g / L, Tween-80 1 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 3.5 g / L, ammonium citrate 3 g / L, magnesium sulfate 0.8 g / L, manganese sulfate 0.2 g / L, L-cysteine ​​hydrochloride 0.3 g / L, agar powder 18 g / L, pH 6.8~7.0.

[0036] S2 Primary Seed Tank Cultivation

[0037] Take the bacterial suspension cultured in S1 and inoculate it into a seed tank containing the seed culture medium formulation described in Table 1 at an inoculation rate of 0.3% (v / v). Control the temperature at 37℃, the initial rotation speed at 80 rpm, and the airflow rate at 0.03 vvm. During the culture process, regularly monitor the pH and OD. 600And microscopic examination, when the pH is 4.5~5.0, OD≥3.5, and ≥100 cells per field of view are observed under 100x oil immersion, the culture cycle is about 12 hours to obtain the first-grade seed culture;

[0038] Table 1. Culture medium formulation for primary seed tank cultivation raw material Content (g / L) peptone 10 Yeast paste 10 glucose 30 Beef Powder 10 Potassium dihydrogen phosphate 1 Magnesium sulfate 1 manganese sulfate 0.5 Sodium acetate 3.5 Defoamer 0.5

[0039] Table 2 Key process parameters for fermentation in the primary seed tank content data Tank Inspection Check the fermenter's seal, replace the air filter, replace the gasket, etc. pH before sterilization 6.5 pH after sterilization 6.0 Volume before sterilization Add water to approximately 600 L Sterilized volume 800 L sterilization conditions 121℃, 30 min Dissolved oxygen control Uncontrolled Defoamer dosage 0.05% Incubation temperature 37℃ ventilation ratio 0.03 vvm cultivation cycle 12 h

[0040] S3 fermenter cultivation

[0041] Using differential pressure, all the primary seed liquid in S2 was forced into a fermenter containing the fermentation medium formula shown in Table 3. The inoculation amount was 5% (v / v). The transfer pipeline needed to be steam sterilized for about 1 hour. The pipeline was cooled with sterile air before the transfer.

[0042] The seed tank pressure is controlled at 0.15 MPa, and the fermentation tank pressure is controlled at 0.02 MPa. After all transfer is complete, the valves are closed, sterile air is introduced, the agitator is turned on at 80 rpm, the ventilation ratio is 0.03 vvm, and the temperature is set at 37℃ to start fermentation. The initial pH is around 6.3, and the DO is calibrated to 100%. When the pH drops to around 6.3, the pH is automatically controlled and maintained at 6.3~6.5 (using pre-sterilized 20% NaOH). Once the cells have grown, the net OD... 600 When the temperature reaches ≥3.0, start feeding. Feeding should continue for 5 hours. The fermentation cycle is 18 hours. The feeding formula is: corn starch 500 g / L, ammonium sulfate 50 g / L, and glucose 100 g / L.

[0043] During fermentation, defoamer is added according to the foam situation. Polyether-based defoamer is prepared at a concentration of 20%. The defoamer is added as needed when the foam reaches the top sight glass of the tank.

[0044] Table 3. Culture medium formulations for fermenter culture raw material Content (g / L) Corn syrup 38 Yeast paste 8 glucose 20 corn starch 10 Potassium dihydrogen phosphate 2 dipotassium hydrogen phosphate 3.5 Magnesium sulfate 2.6 Sodium acetate 3.7 urea 2 ammonium sulfate 1 manganese sulfate 1.5 Defoamer 0.5

[0045] Example 2

[0046] Unlike Example 1, the fermentation cycle in S3 is 17.5 h, while the remaining steps and operating conditions are the same as in Example 1.

[0047] Example 3

[0048] A probiotic powder containing Lactobacillus amyloliquefaciens is prepared as follows: The fermentation broth obtained in Example 1 is cooled to 25°C and then centrifuged using a disc centrifuge. The centrifuged heavy phase is collected. The parameters during the centrifugation process are: slag discharge cycle: 180 s, slag discharge time: 0.4 s, feed rate: 1 m³ / h.

[0049] Based on the mass of the centrifuged heavy phase, weigh out 2% skim milk powder, 2% maltodextrin, 1.6% trehalose, 3% sodium microcrystalline cellulose, 0.8% vitamin C, and 1.5% sorbitol, and add them to the centrifuged heavy phase in sequence while stirring until the materials are completely dissolved.

[0050] Open the spray tower, adjust the feed rate, inlet air temperature: 130℃, outlet air temperature: 70℃, and you will obtain the probiotic powder containing Lactobacillus amyloliquefaciens.

[0051] Comparative Example 1

[0052] Unlike Example 1, fermentation was carried out using a conventional process, as follows:

[0053] Conventional process: No additional feeding required. The basic fermentation formula is as follows:

[0054] Table 4 Basic Fermentation Formula raw material Content (g / L) peptone 15 Yeast paste 8 glucose 25 corn starch 10 Beef Powder 8 Potassium dihydrogen phosphate 2 dipotassium hydrogen phosphate 3.5 Magnesium sulfate 2.6 Sodium acetate 3.7 manganese sulfate 1.5 Defoamer 0.5

[0055] Conventional formulas use peptone and beef extract, expensive nitrogen sources, which increases production costs.

[0056] Comparative Example 2

[0057] Unlike in Example 1, when net OD 600 Feeding was started when the biomass was ≥4.0, which extended the fermentation cycle by 4-7 hours and did not achieve the optimal biomass.

[0058] Comparative Example 3

[0059] Unlike in Example 1, when net OD 600 Feeding should begin when the substrate concentration is around 2.0. At this time, the nutrients in the substrate have not yet been fully consumed, especially glucose, which will have a substrate concentration inhibition effect and affect the growth of the microorganisms.

[0060] The fermentation effects of Examples 1-2 and Comparative Example 1 are shown in Table 5 below.

[0061] Table 5. Fermentation effects of each example and comparative example. Serial Number difference Fermentation cycle Fermentation cell count (100 million CFU / mL) Microbial count in sprayed powder (100 million CFU / g) Yield (%) Comparative Example 1 conventional fermentation 16 h 50 1950 25% Example 1 Material replenishment process 18 h 300 13500 90% Example 2 Material replenishment process 17.5 h 297 13481 90%

[0062] As can be seen from the above data, after the present invention improves the raw materials and fermentation process of Lactobacillus amyloliquefaciens fermentation, the fermentation cell count can be increased to 30 billion CFU / mL. After further adjusting the spray drying protectant, the spray drying cell count is 1.35 trillion CFU / g, and the spray powder yield reaches 90%.

[0063] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for high-density culture of Lactobacillus amyloliquefaciens, characterized in that, The steps include the following: S1 strain activation culture; S2 Primary Seed Tank Cultivation: Take the bacterial suspension cultured in S1 and inoculate it into a seed tank containing seed culture medium at an inoculation rate of 0.3%~1% (v / v). Control the temperature at 35~40℃, the initial rotation speed at 50~100 rpm, and the aeration ratio at 0.03~0.05 vvm to obtain the primary seed solution. S3 Fermenter Culture: The primary seed culture from S2 is transferred to a fermenter containing fermentation medium, and fermentation is carried out until the net OD reaches zero. 600 When the concentration is ≥3.0, feeding begins, and feeding continues for 4~6 hours. The fermentation cycle is 16~20 hours, which yields a high-density fermentation broth of Lactobacillus amyloliquefaciens. The fermentation medium is formulated as follows: corn steep liquor / soybean protein powder: 15-40 g / L, yeast extract: 5-30 g / L, glucose: 10-30 g / L, starch: 10-20 g / L, potassium dihydrogen phosphate: 1-8 g / L, dipotassium hydrogen phosphate: 1-5 g / L, sodium acetate: 1-5 g / L, magnesium sulfate: 1-8 g / L, urea: 1-7 g / L, ammonium sulfate: 0.5-5 g / L, manganese sulfate: 0.5-1.5 g / L; The feed formulation is as follows: starch 300~600 g / L, ammonium sulfate 30~60 g / L, glucose 50~150 g / L.

2. The cultivation method as described in claim 1, characterized in that, The specific process described in S1 is to activate and culture the preserved Lactobacillus amyloliquefaciens strain on a plate culture medium until a suspension of third-generation Lactobacillus amyloliquefaciens is obtained. The formulation of the plate culture medium is as follows: peptone 5-15 g / L, beef extract 5-15 g / L, yeast extract 5-8 g / L, glucose 15-25 g / L, Tween-80 0.5-1.5 g / L, dipotassium hydrogen phosphate 1.5-3.5 g / L, sodium acetate 2.5-5 g / L, ammonium citrate 2.5-3.5 g / L, magnesium sulfate 0.5-1 g / L, manganese sulfate 0.1-0.2 g / L, L-cysteine ​​hydrochloride 0.1-0.5 g / L, and agar powder 15-20 g / L.

3. The cultivation method as described in claim 1, characterized in that, In S2, the seed culture medium is formulated as follows: peptone 10-20 g / L, yeast extract 10-30 g / L, glucose 10-30 g / L, beef extract powder 10-20 g / L, potassium dihydrogen phosphate 1-8 g / L, magnesium sulfate 1-8 g / L, sodium acetate 3-5 g / L, manganese sulfate 0.5-1.5 g / L, and Tween-80 0.5-1 g / L.

4. The cultivation method as described in claim 1, characterized in that, In S2, pH and OD are monitored periodically during the culture process. 600 And microscopic examination, when the pH is between 4.5 and 5.0, OD 600 When ≥100 specimens are found under 100x oil immersion microscopy at a magnification of ≥3.5, a first-grade seed solution is obtained.

5. The cultivation method as described in claim 1, characterized in that, In S3, the fermentation conditions are as follows: inoculum size is 5%~10% (v / v), sterile air is introduced, stirring is turned on at 60~100 rpm, ventilation ratio is 0.03~0.05 vvm, the temperature is set at 36~37℃, fermentation begins, initial pH is 6.3~6.5, dissolved oxygen is calibrated to 100%, pH is automatically controlled, and the pH is controlled at 6.3~6.

5.

6. The cultivation method as described in claim 1, characterized in that, In S3, when pH is automatically controlled, 20%~30% sodium hydroxide that has been sterilized in advance is used to adjust the pH.

7. The cultivation method as described in claim 1, characterized in that, In S3, during fermentation, if foam reaches the top sight glass of the tank, 20wt%~30wt% of polyether defoamer is added.

8. The cultivation method as described in claim 1, characterized in that, The high-density fermentation broth contains ≥3×10⁻⁶ Lactobacillus amyloliquefaciens bacteria. 10 cfu / mL.

9. A probiotic powder containing Lactobacillus amyloliquefaciens, characterized in that, The product includes Lactobacillus amyloliquefaciens cultured by any one of the culture methods described in claims 1 to 8 and a protectant. The protectant contains the following components in the following proportions based on the mass of Lactobacillus amyloliquefaciens cells: 1% to 5% skim milk powder, 1% to 3% maltodextrin, 1% to 2% trehalose, 2% to 5% sodium microcrystalline cellulose, 0.5% to 1% vitamin C, and 1% to 2% sorbitol.

10. The probiotic powder as described in claim 9, characterized in that, The bacterial powder is obtained by spray drying, and the spray drying conditions are: inlet air temperature 125~135℃ and outlet air temperature 65~75℃.