Domestication and cultivation method and high-density fermentation process of bifidobacterium longum

Abstract

The invention relates to a domestication cultivation method and a high-density fermentation process of bifidobacterium longum, and the domestication cultivation method comprises the following steps: step 1, single colony separation: bifidobacterium longum is subjected to single colony separation, is cultured under an anaerobic condition until single colonies grow out, and is placed in an aerobic environment for several days; step 2, primary culture: during placement, selecting a plurality of grown single colonies every day, inoculating the single colonies into a dominant culture solution A for anaerobic culture to obtain a bacterial solution A, observing a bacterial turbid phenomenon or a bacterial precipitate generation condition in the bacterial solution, selecting the single colonies selected in the last day and the bacterial solution with the bacterial turbid phenomenon or the bacterial precipitate generation condition as industrial primary strains, and carrying out primary culture on the industrial primary strains; and preserving the bacterial liquid of the obtained industrial primary strain. After being domesticated and cultivated, the strain can be subjected to high-density fermentation under the aerobic condition by utilizing a pure plant nitrogen source, the fermentation condition is easy to control, the operation is simple, and the strain is suitable for industrial fermentation production.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a domestication and cultivation method of Bifidobacterium longum and a high-density fermentation process. Background Art

[0002] Bifidobacterium longum is an anaerobic Gram-positive bacillus belonging to the Actinomycetales and the Actinomycetaceae. Studies have shown that it has a variety of biological functions such as maintaining intestinal ecological balance, antibacterial, antiallergic, enhancing immunity and anti-tumor, and plays an important role in preventing and treating diseases and promoting human health. However, due to its high nutritional requirements and poor resistance to the environment, the production and practical application of Bifidobacterium longum have brought great difficulties. In particular, the wild-type Bifidobacterium longum is sensitive to oxygen in the environment, has a high degree of obligate anaerobicity, is not suitable for conventional lactic acid bacteria large-scale production requirements, and needs to be filled with nitrogen for fermentation, which not only makes the fermentation control complicated and cumbersome, but also increases energy consumption and reduces production efficiency.

[0003] In addition, most of the nitrogen sources currently used in industrial fermentation are animal nitrogen sources or microbial nitrogen sources, such as beef extract powder, bovine bone peptone, fish peptone, yeast extract powder, yeast peptone, and some contain soy peptone. On the one hand, the cost is relatively high. On the other hand, probiotic products made from animal nitrogen sources do not meet the requirements of halal food, which limits their application in the halal food field. In addition, for vegetarian enthusiasts, probiotic products fermented with pure plant nitrogen sources are also more in line with their life consumption concepts.

[0004] Therefore, how to domesticate and cultivate Bifidobacterium longum so that it can utilize pure plant nitrogen sources and ferment under aerobic conditions is crucial to simplifying the fermentation control process, reducing fermentation costs, expanding the user group of halal personnel and vegetarian enthusiasts, and improving its market competitiveness. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a method for acclimating and cultivating Bifidobacterium longum and a high-density fermentation process, which can utilize pure plant nitrogen sources for high-density fermentation under aerobic conditions, and the fermentation conditions are easy to control, the operation is simple, and it is suitable for industrial fermentation production.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for acclimatizing and cultivating industrial strains of Bifidobacterium longum comprises the following steps:

[0008] Step 1, single colony isolation: isolate Bifidobacterium longum from a single colony, culture it under anaerobic conditions until a single colony grows out, and then place it in an aerobic environment for several days;

[0009] Step 2, primary cultivation: During the placement period, a number of single colonies grown are picked every day, inoculated into the dominant culture solution A for anaerobic culture, and bacterial solution A is obtained. The bacterial turbidity or bacterial precipitation in the bacterial solution is observed, and the bacterial solution with bacterial turbidity or bacterial precipitation picked on the last day is selected as the industrial primary selection strain, and the obtained bacterial solution of the industrial primary selection strain is stored;

[0010] As a further technical solution, in step 1, before isolating the Bifidobacterium longum from single colonies, it is first activated and cultured for at least one generation;

[0011] The single colony isolation was carried out by plate streaking method.

[0012] As a further technical solution, in step 2, the bacterial solution of the selected industrial primary strain is mixed with 10-60% glycerol at a ratio of 1:1-1.5 and then stored at -20--80°C.

[0013] As a further technical solution, the method further includes step 3, re-cultivation: dividing the bacterial liquid preserved in step 2 into two portions, inoculating them respectively into the next generation dominant culture liquid B, wherein one portion is cultured in an anaerobic environment and the other portion is cultured in an aerobic environment, the culture temperature of the two portions is 37° C., and the culture time is 12-24 hours. After the culture is completed, the OD values ​​of the two fermentation liquids are measured, the ratio of the OD values ​​of aerobic culture and anaerobic culture is calculated, and the strain with a high ratio of the OD values ​​of aerobic culture and anaerobic culture is selected as the industrial strain of Bifidobacterium longum.

[0014] As a further technical solution, in step 1, the dominant culture medium A is MRS liquid culture medium;

[0015] In step 3, the dominant culture medium B uses MRS liquid culture medium supplemented with L-cysteine ​​hydrochloride.

[0016] As a further technical solution, the industrial strain of Bifidobacterium longum is activated continuously for at least 3 generations, inoculated into a pure plant nitrogen source fermentation medium, and fermented at 30-40°C. During the culture process, alkali solution is added. When the alkali solution is no longer consumed, the fermentation is terminated to obtain a high-density fermentation liquid.

[0017] As a further technical solution, after inoculation, the fermentation is allowed to stand and ferment. When the pH of the fermented liquid drops to 5.0-6.1, stirring is started, and alkali solution is added to maintain the pH of the fermentation system at 5.0-6.1 for fermentation. When the alkali solution is no longer consumed, the fermentation is terminated to obtain a high-density fermentation liquid.

[0018] As a further technical solution, the pure plant nitrogen source fermentation medium comprises the following raw materials in percentage: glucose 1.5% to 3.0%, malt extract powder 0.2% to 1.0%, rice peptone 0.5% to 2.0%, wheat peptone 0.2% to 1.0%, soybean peptide powder 0.1% to 2.0%, sodium acetate 0.2%-1.0%, sodium citrate 0.2%-1.0%, disodium hydrogen phosphate 0.2%-0.8%, MgSO4 0.01% to 0.1%, MnSO4 0.01% to 0.05%, Tween-80 0.10% to 0.30%, L-cysteine ​​hydrochloride 0.01% to 0.1%, sodium ascorbate or sodium iso-D ascorbate 0.01% to 0.1%;

[0019] The preparation method is as follows: after mixing various materials, adjust the pH value to 6.0-7.2, and sterilize at 115° C. for 20 minutes.

[0020] As a further technical solution, the alkali used in the alkali solution includes one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium bicarbonate, and ammonia water.

[0021] As a further technical solution, the obtained high-density fermentation broth contains 0.8-2.0×10 10 CFU / mL of viable bacteria.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] After the original strain of Bifidobacterium longum is domesticated and cultivated by the method of the present invention, the strain can grow and reproduce well under pure plant nitrogen source and aerobic conditions, and the number of viable bacteria can be obtained in the range of 0.8-2.0×10 10 CFU / mL high-density fermentation liquid, on the one hand, can utilize plant-derived nitrogen sources without adding animal-derived nitrogen sources and microbial-derived nitrogen sources, which reduces the fermentation cost and satisfies the consumption concept of vegetarian enthusiasts for probiotic products fermented with pure plant-derived nitrogen sources. On the other hand, it can be fermented in an aerobic environment, which simplifies the fermentation conditions and makes it suitable for industrial production, which is crucial for improving its market competitiveness. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0026] In the present invention,

[0027] The Bifidobacterium longum of the present invention adopts the Bifidobacterium longum L693 strain, whose preservation number is CGMCC No.19469, which is a published strain.

[0028] Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0029] The following is further explained with specific examples.

[0030] Example 1

[0031] A method for acclimatizing and cultivating industrial strains of Bifidobacterium longum comprises the following steps:

[0032] Step 1, single colony isolation and aerobic placement: After activating the original strain of Bifidobacterium longum L693 for one generation, streak the plate, culture under anaerobic conditions until a single colony grows, and then place it in an aerobic environment for several days;

[0033] Step 2, initial incubation: During the aerobic placement period, a number of single colonies grown are picked every day, and they are respectively inoculated into the dominant culture solution A (MRS liquid culture medium) for anaerobic culture to obtain bacterial solution A, and the bacterial turbidity or bacterial precipitation in the bacterial solution is observed. The bacterial solution with single colonies picked on the last day and with bacterial turbidity or bacterial precipitation is selected as the industrial primary strain (in this embodiment, the bacterial solution A obtained by inoculation after 9 days of aerobic placement has no bacterial turbidity and bacterial precipitation, and therefore, the bacterial solution A obtained by inoculation after the 8th day of aerobic placement has bacterial turbidity and bacterial precipitation, therefore, the bacterial solution A obtained by inoculation on the 8th day of aerobic placement is used as the industrial primary strain), and the obtained industrial primary strain bacterial solution is mixed with 10% to 60% glycerol at a mass ratio of 1:1 (M / M) to obtain an industrial primary strain bacterial suspension, which is stored at -20°C to -80°C; after the initial incubation, 8 tubes of industrial primary strains are obtained in this embodiment.

[0034] Step 3, re-cultivation: the industrial primary strain suspension preserved in step 2 is divided into two parts, and each part is inoculated into the next generation dominant culture medium B (MRS liquid medium added with L-cysteine ​​hydrochloride), one part is cultured in an anaerobic environment, and the other part is cultured in an aerobic environment. The culture temperature of the two parts is 37°C, and the culture time is 12-24h. After the culture is completed, the OD values ​​of the two fermentation broths are measured, and the OD value ratio of aerobic culture and anaerobic culture is calculated. The results are shown in Table 1. The No. 7 tube strain with the highest ratio of OD values ​​of aerobic culture and anaerobic culture is selected as the industrial strain of Bifidobacterium longum for subsequent industrial fermentation.

[0035] Table 1

[0036] Colony number Aerobic OD Anaerobic OD OD value / anaerobic OD ratio ① 9.30 8.56 1.09 ② 6.81 7.30 0.93 ③ 8.30 8.04 1.03 ④ 6.62 7.34 0.90 ⑤ 6.24 6.28 0.99 ⑥ 6.80 7.64 0.89 ⑦ 7.82 6.96 1.12 ⑧ 7.76 7.44 1.04

[0037] The fermentation culture of industrial strains of Bifidobacterium longum includes the following steps:

[0038] Step 1), activation: the industrial strain of Bifidobacterium longum domesticated and cultivated in this example is inoculated into a modified MRS (MRS medium plus L-cysteine ​​hydrochloride), and anaerobically cultured at 37° C. for 12-24 hours to obtain a first generation seed solution;

[0039] Step 2), seed liquid for subculture: the first generation seeds are inoculated in modified MRS or other suitable fermentation medium, and subcultured for three generations in an aerobic environment to obtain seed liquid for subculture.

[0040] Step 3), high density fermentation:

[0041] Fermentation medium: glucose 2.5%, malt extract powder 1.0%, rice peptone 0.5%, wheat peptone 0.8%, soybean peptide powder 0.5%, sodium acetate 0.5%, sodium citrate 0.3%, disodium hydrogen phosphate 0.3%, MgSO4 0.05%, MnSO4 0.02%, Tween-80 0.10%, L-cysteine ​​hydrochloride 0.06%, sodium ascorbate or sodium iso-D ascorbate 0.05%.

[0042] After mixing the above materials, the pH value was adjusted to 6.5 and sterilized at 115°C for 20 minutes.

[0043] Fermentation method: The seed solution of the third generation of step 2) was inoculated into 5000L fermentation medium at an inoculation rate of 3.0%, and the temperature was controlled at 37°C. After inoculation, static fermentation was first performed. When the pH of the fermentation liquid dropped to 5.0, stirring was started, and sodium hydroxide alkali solution was added to keep the pH of the fermentation liquid constant at 5.0. The fermentation was terminated when the alkali solution was no longer consumed, and the number of viable bacteria was 0.8-1.2×10 10 CFU / mL of high-density fermentation broth (6 groups of parallel experiments were carried out in this example).

[0044] Example 2

[0045] A method for acclimating and cultivating an industrial strain of Bifidobacterium longum comprises the following steps: the same as Example 1, except that there are 6 tubes of industrial primary strains, the OD value ratio of aerobic culture and anaerobic culture in the re-cultivation of the industrial primary strains, and the results are shown in Table 2, wherein tube No. 3 is the obtained industrial strain of Bifidobacterium longum.

[0046] Table 2

[0047] Colony number Aerobic OD Anaerobic OD OD value / anaerobic OD ratio ① 9.23 7.96 1.16 ② 6.88 8.06 0.85 ③ 9.84 7.46 1.32 ④ 7.88 8.18 0.96 ⑤ 8.01 8.94 0.90 ⑥ 8.64 7.14 1.21

[0048] The fermentation culture of industrial strains of Bifidobacterium longum comprises the following steps: the same as in Example 1, except that: in step 1), the culture is carried out at 37°C for 18 hours; in step 3), the fermentation medium is: fermentation medium: 3.0% glucose, 0.5% malt extract powder, 1.5% rice peptone, 1.0% wheat peptone, 0.8% soybean peptide powder, 0.8% sodium acetate, 0.2% sodium citrate, 0.5% disodium hydrogen phosphate, 0.06% MgSO4, 0.06% MnSO4, 0.15% Tween-80, 0.08% L-cysteine ​​hydrochloride, and 0.08% sodium ascorbate or sodium iso-D ascorbate. After the above materials are mixed, the pH value is adjusted to 6.8, and sterilized at 115°C for 20 minutes.

[0049] In step 3, the fermentation culture conditions are as follows: the seed solution of the third generation of step 2) is inoculated into 6000L fermentation medium at an inoculation rate of 4.0%, and the temperature is controlled at 37°C. After inoculation, static fermentation is first performed. When the pH of the fermentation liquid drops to 5.5, stirring is started, and sodium bicarbonate alkali solution is added to keep the pH of the fermentation liquid constant at 5.5. The fermentation is terminated when the alkali solution is no longer consumed, and the number of viable bacteria is 0.9-1.4×10 10 CFU / mL of high-density fermentation broth (6 groups of parallel experiments were carried out in this example).

[0050] Example 3

[0051] A method for acclimating and cultivating an industrial strain of Bifidobacterium longum comprises the following steps: the same as Example 1, except that there are 6 tubes of industrial primary strains, the OD value ratio of aerobic culture and anaerobic culture in the re-cultivation of the industrial primary strains, and the results are shown in Table 3, wherein tube No. 5 is the obtained industrial strain of Bifidobacterium longum.

[0052] Table 3

[0053] Colony number Aerobic OD Anaerobic OD OD value / anaerobic OD ratio ① 7.45 7.66 0.97 ② 8.94 8.02 1.09 ③ 8.30 8.86 0.94 ④ 6.96 7.21 0.96 ⑤ 10.08 8.77 1.15 ⑥ 7.74 8.41 0.92

[0054] The fermentation culture of industrial strains of Bifidobacterium longum comprises the following steps: the same as in Example 1, except that: in step 1), the culture is carried out anaerobicly at 37° C. for 18 h; in step 3), the fermentation medium comprises: 2.0% glucose, 1.5% malt extract powder, 1.0% rice peptone, 1.0% wheat peptone, 1.0% soybean peptide powder, 1.0% sodium acetate, 0.3% sodium citrate, 0.6% disodium hydrogen phosphate, 0.07% MgSO4, 0.03% MnSO4, 0.25% Tween-80, 0.1% L-cysteine ​​hydrochloride, and 0.04% sodium ascorbate or sodium iso-D ascorbate; after the above materials are mixed, the pH value is adjusted to 7.0, and sterilized at 115° C. for 20 min. In step 3, the fermentation culture conditions are as follows: the seed solution of the third generation of step 2) is inoculated into 7500L fermentation medium at an inoculation rate of 2.0%, and the temperature is controlled at 37°C. After inoculation, static fermentation is first performed. When the pH of the fermentation liquid drops to 5.9, stirring is started, and sodium bicarbonate alkali solution is added to keep the pH of the fermentation liquid constant at 5.9. The fermentation is terminated when the alkali solution is no longer consumed, and the number of viable bacteria is 1.5-2.0×10 10 CFU / mL of high-density fermentation broth (6 groups of parallel experiments were carried out in this example).

[0055] Comparative Example 1

[0056] The fermentation culture of Bifidobacterium longum includes the following steps: the same as in Example 1, except that the strain used for fermentation is the original strain of Bifidobacterium longum L693, and the number of viable bacteria in the obtained fermentation liquid is 4.8-5.2*10 9 CFU / mL (6 groups of parallel experiments were conducted in this example).

[0057] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for acclimating and cultivating industrial strains of Bifidobacterium longum, characterized in that: The steps include: Step 1, single colony isolation: isolate Bifidobacterium longum from a single colony, culture it under anaerobic conditions until a single colony grows out, and then place it in an aerobic environment for several days; Step 2, primary incubation: During the placement period, a number of single colonies grown are picked every day, inoculated into the dominant culture solution A for anaerobic culture, and bacterial solution A is obtained. The bacterial turbidity or bacterial precipitation in the bacterial solution is observed, and the bacterial solution with single colonies picked on the last day and bacterial turbidity or bacterial precipitation is selected as the industrial primary strain, and the obtained industrial primary strain bacterial solution is preserved.

2. The method for acclimating and cultivating an industrial strain of Bifidobacterium longum according to claim 1, characterized in that: In step 1, before isolating the Bifidobacterium longum from a single colony, the Bifidobacterium longum is first activated and cultured for at least one generation; The single colony isolation was carried out by plate streaking method.

3. The method for acclimating and cultivating an industrial strain of Bifidobacterium longum according to claim 1, characterized in that: In step 2, the bacterial solution of the selected industrial primary strain is mixed with 10-60% glycerol at a ratio of 1:1-1.5 and then stored at -20--80°C.

4. The method for acclimating and cultivating an industrial strain of Bifidobacterium longum according to claim 1, characterized in that: The method further comprises step 3, re-cultivation: dividing the bacterial solution preserved in step 2 into two portions, inoculating the two portions into the next generation dominant culture solution B respectively, wherein one portion is cultured in an anaerobic environment and the other portion is cultured in an aerobic environment, the culture temperature of the two portions is 37° C., the culture time is 12-24 hours, and after the culture is completed, the OD values ​​of the two fermentation broths are measured, the OD value ratio of aerobic culture to anaerobic culture is calculated, and the strain with a high OD value ratio of aerobic culture to anaerobic culture is selected as the industrial strain of Bifidobacterium longum.

5. The method for acclimating and cultivating an industrial strain of Bifidobacterium longum according to claim 1 or 4, characterized in that: In step 1, the dominant culture medium A is MRS liquid culture medium; in step 3, the dominant culture medium B is MRS liquid culture medium supplemented with L-cysteine ​​hydrochloride.

6. A high-density industrial fermentation process of Bifidobacterium longum, characterized in that: The steps include: After the industrial strain of Bifidobacterium longum described in any one of claims 1 to 5 is activated continuously for at least 3 generations, it is inoculated into a pure plant nitrogen source fermentation medium and fermented at 30-40° C. During the culture process, alkali solution is added. When the alkali solution is no longer consumed, the fermentation is terminated to obtain a high-density fermentation liquid.

7. The high-density industrial fermentation process of Bifidobacterium longum according to claim 6, characterized in that: After inoculation, the fermentation is allowed to stand for fermentation. When the pH of the fermentation liquid drops to 5.0-6.1, stirring is started, and alkali solution is added to maintain the pH of the fermentation system at 5.0-6.1 for fermentation. When the alkali solution is no longer consumed, the fermentation is terminated to obtain a high-density fermentation liquid.

8. The high-density industrial fermentation process of Bifidobacterium longum according to claim 7, characterized in that: The pure plant nitrogen source fermentation medium comprises the following raw materials in percentage: 1.5% to 3.0% glucose, 0.2% to 1.0% malt extract powder, 0.5% to 2.0% rice peptone, 0.2% to 1.0% wheat peptone, 0.1% to 2.0% soybean peptide powder, 0.2% to 1.0% sodium acetate, 0.2% to 1.0% sodium citrate, 0.2% to 0.8% disodium hydrogen phosphate, 0.01% to 0.1% MgSO4, 0.01% to 0.05% MnSO4, 0.10% to 0.30% Tween-80, 0.01% to 0.1% L-cysteine ​​hydrochloride, and 0.01% to 0.1% sodium ascorbate or sodium iso-D ascorbate; The preparation method is as follows: after mixing various materials, adjust the pH value to 6.0-7.2, and sterilize at 115° C. for 20 minutes.

9. The high-density industrial fermentation process of Bifidobacterium longum according to claim 7, characterized in that: The alkali used in the alkali solution includes one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium bicarbonate, and ammonia water.

10. The high-density industrial fermentation process of Bifidobacterium longum according to claim 7, characterized in that: The obtained high-density fermentation broth contained 0.8-2.0×10 10 CFU / mL of viable bacteria.