Extraction process of probiotic preparation for nasal cavity micro-ecological health care

Abstract

The invention relates to the related technical field of probiotic fermentation, in particular to an extraction process of a probiotic preparation for nasal cavity micro-ecological health care, and the extraction process comprises the following specific steps: step 1, raw material preparation: S1, selecting a mixture of bacillus subtilis and bacillus licheniformis as a probiotic strain, and the bacterial colony number is gt; the bacterial colony count ratio of the bacillus subtilis to the bacillus licheniformis is 1 to 1; s2, a fermentation medium is an LB broth medium and comprises 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCI, 20 g / L of glucose, 2 g / L of dipotassium phosphate, 2 g / L of diammonium hydrogen citrate, 10 g / L of sodium acetate, 0.28 g / L of magnesium sulfate, 0.58 g / L of manganese sulfate, 1 g / L of Tween 80 and 0.5 g / L of cysteine hydrochloride; according to the extraction process of the probiotic preparation for nasal cavity micro-ecological health care, by optimizing the steps of strain activation, seed solution expanding culture and fermentation culture, probiotics can be cultured more efficiently, so that the overall fermentation period is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field related to probiotic fermentation, and in particular to a probiotic preparation extraction process for nasal microecological health care. Background Art

[0002] The nasal cavity is the first line of defense of the human respiratory system. The mucus layer and mucosal epithelial cells covering its surface together constitute a unique microecological environment, which is an important basis for resisting the invasion of pathogens and maintaining the homeostasis of the respiratory system. Probiotics, as a type of active microorganisms beneficial to the host, have been widely used in the field of intestinal health and have gradually expanded to mucosal sites such as the skin and oral cavity. They show the potential of "inhibiting bacteria with bacteria" and "replacing treatment with care" by regulating the host's microecological balance, enhancing barrier function and immune regulation. The nasal cavity is an open mucosal system in direct contact with the outside world. In theory, there is space for probiotics to colonize and play a role. Therefore, a probiotic preparation extraction process for nasal microecological health care is needed.

[0003] The existing extraction process of probiotic preparations for nasal microecological health care is to inoculate directly in the fermentation tank, which has a long fermentation cycle and low precision in dynamic environment control in the fermentation tank, which can easily cause stress death of bacteria. Summary of the Invention

[0004] The object of the present invention is to provide a probiotic preparation extraction process for nasal microecological health care, so as to solve the problem that the existing probiotic preparation extraction process for nasal microecological health care proposed in the above background technology is directly inoculated in a fermentation tank, the fermentation cycle is long, and the dynamic environment control accuracy in the fermentation tank is low, which easily causes stress death of bacteria.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a probiotic preparation extraction process for nasal microecological health care, the specific steps are as follows:

[0006] Step 1: Raw material preparation:

[0007] S1: The probiotic strains used are a mixture of Bacillus subtilis and Bacillus licheniformis, with colony counts of >10 billion cfu / g and a colony count ratio of Bacillus subtilis to Bacillus licheniformis of 1:1;

[0008] S2: The fermentation medium was LB broth, consisting of: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 10 g / L, magnesium sulfate 0.28 g / L, manganese sulfate 0.58 g / L, Tween 80 1 g / L, and cysteine ​​hydrochloride 0.5 g / L.

[0009] Step 2: Bacteria activation:

[0010] S1: The probiotic bacteria were restored from the frozen state to room temperature, and then inoculated into a culture medium containing LB broth at a 1% inoculum size, and cultured in a constant temperature box at 37°C for 24 hours to activate and obtain a seed solution;

[0011] Step 3: Seed liquid expansion:

[0012] S1. Transfer the activated bacterial solution to 500 mL LB medium (shake flask volume ≤ 25%) at a 1% inoculum volume;

[0013] S2, culture at 37°C and 200 rpm for 12 h (OD600≈1.0-1.5);

[0014] S3, detect the number of viable bacteria (≥1x10 8 CFU / mL is qualified);

[0015] Step 4: Fermentation culture:

[0016] Inoculate the activated seed liquid into a fermenter containing LB broth at a rate of 1% (10 parts by weight per 1000 parts by weight of LB broth) and incubate at 37-42°C and standard atmospheric pressure for 48 hours, stirring the contents of the fermenter to form a uniform bacterial suspension.

[0017] Step 5: Post-processing:

[0018] S1. Separation and extraction: Use centrifugation, filtration and other methods to separate the bacteria from the culture medium and extract the required active ingredients.

[0019] S2: Concentration: Concentrate the extracted bacteria to obtain a high-concentration bacterial suspension;

[0020] S3. Transfer the bacterial suspension to a constant temperature water bath or heat exchanger and heat at 50°C for 5 minutes (temperature fluctuation ≤ ±1°C, time error ≤ ±10 seconds). Stir continuously at a low speed (20-30 rpm) during the heat shock process to ensure uniform temperature and avoid local overheating.

[0021] S4. After the heat shock is completed, the bacterial suspension is immediately transferred to an ice bath (04°C) and rapidly cooled for 5-10 minutes to terminate the spore formation process.

[0022] Preferably, the specific mixing steps of the fermentation medium in step 1 are as follows:

[0023] 1) Dissolve the components in S2 in 800 mL of distilled water and stir until completely dissolved;

[0024] 2) Adjust the pH to 7.0 with NaOH or HCI;

[0025] 3) Adjust the volume to 1000 mL and sterilize by autoclave at 121°C for 20 minutes.

[0026] Preferably, in step 2, the probiotics are thawed on ice for ≤30 seconds, cultured at 37° C. and 200 rpm with shaking for 24 hours, and OD600≈0.5-1.0 is considered a sign of successful activation, and the number of activations is ≤3 times.

[0027] Preferably, the specific operation steps of the fermentation tank in step 4 are as follows:

[0028] 1) After sterilization, cool to 37°C and introduce sterile air (aerobic or anaerobic bacteria);

[0029] 2) Maintain low agitation (100 rpm) for the first 2 hours after inoculation to reduce mechanical damage. Increase agitation to 200-400 rpm after entering the logarithmic growth phase and monitor DO > 30% saturation;

[0030] 3) The fermentation broth enters the stable phase (OD600 reaches a plateau, Bacillus subtilis OD600≈5.0).

[0031] Preferably, after the fermentation broth enters the stable phase, the specific steps are as follows:

[0032] 1) When the fermentation broth enters the stable phase (OD600 reaches the plateau phase, such as Bacillus subtilis OD600≈5.0), start feeding. The feeding solution formula is: yeast extract 5g / L + ammonium sulfate 2g / L (provides organic and inorganic nitrogen to promote spore formation);

[0033] 2) Feeding method: pulse addition (feeding once every 4-6 hours, each feeding volume is 1%-2% of the fermentation liquid volume to avoid sudden changes in osmotic pressure caused by one-time feeding).

[0034] Preferably, the specific process of dynamic pH control of the fermentation tank in step 4 is as follows:

[0035] 1) Install a pH sensor on the fermentation tank stirring paddle, connect the pH sensor to an external PLC controller, and connect the PLC controller to the acid and alkali addition pump;

[0036] 2) The pH sensor transmits data to the controller every 10-30 seconds. When it detects that the pH deviates from the target value (such as the pH rises to 7.1), the controller triggers the acid pump to add hydrochloric acid at a flow rate of 0.1-0.5L / h. If the pH drops to 6.9, the alkali pump is triggered to add NaOH.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. By optimizing the steps of strain activation, seed liquid expansion and fermentation culture, probiotics can be cultivated more efficiently, thereby shortening the overall fermentation cycle;

[0039] 2. Optimize the fermentation environment by controlling the stirring speed, dissolved oxygen (DO) saturation, and dynamic pH control. These measures help maintain stable fermentation conditions and reduce the stress response of the bacteria;

[0040] 3. By precisely controlling fermentation conditions such as temperature, pH value and dissolved oxygen, the stress response of bacteria during the fermentation process can be effectively reduced, thereby reducing the risk of bacterial death. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the fermentation process of the present invention. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] See also Figure 1 The present invention provides a technical solution: a probiotic preparation extraction process for nasal microecological health care, the specific steps are as follows:

[0044] Step 1: Raw material preparation:

[0045] S1: The probiotic strains used are a mixture of Bacillus subtilis and Bacillus licheniformis, with colony counts of >10 billion cfu / g and a colony count ratio of Bacillus subtilis to Bacillus licheniformis of 1:1;

[0046] S2: The fermentation medium was LB broth, consisting of: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 10 g / L, magnesium sulfate 0.28 g / L, manganese sulfate 0.58 g / L, Tween 80 1 g / L, and cysteine ​​hydrochloride 0.5 g / L.

[0047] Step 2: Bacteria activation:

[0048] S1: The probiotic bacteria were restored from the frozen state to room temperature, and then inoculated into a culture medium containing LB broth at a 1% inoculum size, and cultured in a constant temperature box at 37°C for 24 hours to activate and obtain a seed solution;

[0049] Step 3: Seed liquid expansion:

[0050] S1. Transfer the activated bacterial solution to 500 mL LB medium (shake flask volume ≤ 25%) at a 1% inoculum volume;

[0051] S2, culture at 37°C and 200 rpm for 12 h (OD600≈1.0-1.5);

[0052] S3, detect the number of viable bacteria (≥1x10 8 CFU / mL is qualified);

[0053] Step 4: Fermentation culture:

[0054] Inoculate the activated seed liquid into a fermenter containing LB broth at a rate of 1% (10 parts by weight per 1000 parts by weight of LB broth) and incubate at 37-42°C and standard atmospheric pressure for 48 hours, stirring the contents of the fermenter to form a uniform bacterial suspension.

[0055] Step 5: Post-processing:

[0056] S1. Separation and extraction: Use centrifugation, filtration and other methods to separate the bacteria from the culture medium and extract the required active ingredients.

[0057] S2: Concentration: Concentrate the extracted bacteria to obtain a high-concentration bacterial suspension;

[0058] S3. Transfer the bacterial suspension to a constant temperature water bath or heat exchanger and heat at 50°C for 5 minutes (temperature fluctuation ≤ ±1°C, time error ≤ ±10 seconds). Stir continuously at a low speed (20-30 rpm) during the heat shock process to ensure uniform temperature and avoid local overheating.

[0059] S4. After the heat shock is completed, the bacterial suspension is immediately transferred to an ice bath (04°C) and rapidly cooled for 5-10 minutes to terminate the spore formation process.

[0060] Furthermore, the specific mixing steps of the fermentation medium in step 1 are as follows:

[0061] 1) Dissolve the components in S2 in 800 mL of distilled water and stir until completely dissolved;

[0062] 2) Adjust the pH to 7.0 with NaOH or HCI;

[0063] 3) Adjust the volume to 1000 mL and sterilize by autoclave at 121°C for 20 minutes.

[0064] Furthermore, the specific mixing steps of the fermentation medium, including component dissolution, pH adjustment and high-pressure sterilization, ensure the sterility and appropriate pH value of the medium, provide a stable environment for the growth of probiotics, and help improve the culture efficiency and activity of probiotics.

[0065] Furthermore, in step 2, the probiotics are thawed on ice for ≤30 seconds, cultured at 37° C. and 200 rpm with shaking for 24 hours, and OD600≈0.5-1.0 is considered a sign of successful activation, and the number of activations is ≤3 times.

[0066] Furthermore, the time and activation conditions for probiotics to be thawed on ice are specified to ensure that the probiotics are not affected by excessive heat stress during the activation process and maintain their activity. At the same time, the number of activation times is limited to avoid bacterial degradation caused by excessive cultivation.

[0067] Furthermore, the specific operation steps of the fermentation tank in step 4 are as follows:

[0068] 1) After sterilization, cool to 37°C and introduce sterile air (aerobic or anaerobic bacteria);

[0069] 2) Maintain low agitation (100 rpm) for the first 2 hours after inoculation to reduce mechanical damage. Increase agitation to 200-400 rpm after entering the logarithmic growth phase and monitor DO > 30% saturation;

[0070] 3) The fermentation broth enters the stable phase (OD600 reaches a plateau, Bacillus subtilis OD600≈5.0).

[0071] Furthermore, the specific operating steps of the fermentation tank, including sterilization, ventilation, stirring speed control and dissolved oxygen monitoring, help maintain a stable environment during the fermentation process, promote the growth and reproduction of probiotics, and reduce the stress response of the bacteria.

[0072] Furthermore, after the fermentation liquid enters the stable period, the specific steps are as follows:

[0073] 1) When the fermentation broth enters the stable phase (OD600 reaches the plateau phase, such as Bacillus subtilis OD600≈5.0), start feeding. The feeding solution formula is: yeast extract 5g / L + ammonium sulfate 2g / L (provides organic and inorganic nitrogen to promote spore formation);

[0074] 2) Feeding method: pulse addition (feeding once every 4-6 hours, each feeding volume is 1%-2% of the fermentation liquid volume to avoid sudden changes in osmotic pressure caused by one-time feeding).

[0075] Furthermore, after the fermentation broth enters the stable period, feeding is started and a pulsed addition method is adopted, which provides additional nutrients to promote the formation of spores, while avoiding the sudden change in osmotic pressure caused by one-time feeding, ensuring the stable growth of probiotics.

[0076] Furthermore, the specific process of dynamic pH control of the fermentation tank in step 4 is as follows:

[0077] 1) Install a pH sensor on the fermentation tank stirring paddle, connect the pH sensor to an external PLC controller, and connect the PLC controller to the acid and alkali addition pump;

[0078] 2) The pH sensor transmits data to the controller every 10-30 seconds. When it detects that the pH deviates from the target value (such as the pH rises to 7.1), the controller triggers the acid pump to add hydrochloric acid at a flow rate of 0.1-0.5L / h. If the pH drops to 6.9, the alkali pump is triggered to add NaOH.

[0079] Furthermore, by installing pH sensors and PLC controllers, the pH value can be monitored and adjusted in real time. This precise pH control helps maintain the optimal environment for probiotic growth and avoids the adverse effects of pH fluctuations on bacterial growth.

[0080] Working principle: First, prepare the raw materials and select a mixture of Bacillus subtilis and Bacillus licheniformis as the probiotic strains. The colony count of both is greater than 10 billion cfu / g and the ratio is 1:1. At the same time, prepare LB broth medium, which includes 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 10g / L sodium acetate, 0.28g / L magnesium sulfate, 0.58g / L manganese sulfate, Tween 80 1g / L, cysteine ​​hydrochloride 0.5g / L, and dissolve the culture medium components in 800mL distilled water, stir until completely dissolved, adjust the pH to 7.0 with NaOH or HCI, make up to 1000mL, and sterilize at 121℃ for 20 minutes. Then activate the strain. After the probiotics are restored to room temperature from the frozen state, they are inoculated into the culture medium containing LB broth medium at a 1% inoculum volume and placed in a 37℃ incubator for 24h. Thawing on ice does not exceed 30 seconds, and the activation times do not exceed 3 times. OD600≈0.5-1.0 is a sign of successful activation. Then, the seed liquid is expanded and the activated bacterial liquid is transferred to 500mL LB medium (shake flask volume ≤25%) at a 1% inoculum volume. It is cultured at 37℃ and 200rpm for 12 hours until OD600≈1.0-1.5, and the number of viable bacteria (≥1x10 9CFU / mL is qualified); then fermentation culture is carried out, the activated seed liquid is inoculated into a fermenter containing LB broth medium at a rate of 1%, and in parts by weight, 1000 parts of LB broth medium contain 10 parts of probiotic seed liquid, and cultured at 37-42°C and standard atmospheric pressure for 48 hours, during which the components in the fermenter are stirred to form a uniform bacterial suspension. The specific operation includes cooling to 37°C after sterilization, passing sterile air (aerobic bacteria or anaerobic bacteria), and maintaining low stirring (100r) for the first 2 hours after inoculation. pm) to reduce mechanical damage. After entering the logarithmic growth phase, increase the stirring to 200-400 rpm and monitor DO>30% saturation. When the fermentation liquid enters the stable phase (OD600 reaches the plateau phase, such as Bacillus subtilis OD600≈5.0), start feeding. The feeding liquid formula is yeast extract 5g / L + ammonium sulfate 2g / L. The feeding method is pulse addition, feeding once every 4-6 hours, and the feeding volume each time is 1%-2% of the fermentation liquid volume. After that, post-processing is carried out, using centrifugation, filtration, etc. Method: The bacteria in the culture medium are separated from the culture medium, the required active ingredients are extracted, and the extracted bacteria are concentrated to obtain a high-concentration bacterial suspension. The bacterial suspension is transferred to a constant temperature water bath or heat exchanger and treated at 50°C for 5 minutes (temperature fluctuation ≤+1°C, time error ≤±10 seconds). During the heat shock process, low-speed stirring (20-30rpm) is continuously performed to ensure uniform temperature and avoid local overheating. After the heat shock is completed, the bacterial suspension is immediately transferred to an ice bath (0-4°C) and rapidly cooled for 5-10 minutes to terminate spore formation. Finally, in terms of dynamic pH control of the fermenter, a pH sensor is installed on the fermenter agitator, the pH sensor is connected to an external PLC controller, and the PLC controller is connected to the acid and alkali addition pump. The pH sensor transmits data to the controller every 10-30 seconds. When it is detected that the pH deviates from the target value (such as the pH rises to 7.1), the controller triggers the acid pump to add hydrochloric acid at a flow rate of 0.1-0.5L / h. If the pH drops to 6.9, the alkali pump is triggered to add NaOH to maintain the stability of the pH value in the fermenter.

[0081] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A probiotic preparation extraction process for nasal microecological health care, characterized in that: The specific steps are as follows: Step 1: Raw material preparation: S1: The probiotic strains used are a mixture of Bacillus subtilis and Bacillus licheniformis, with colony counts of >10 billion cfu / g and a colony count ratio of Bacillus subtilis to Bacillus licheniformis of 1:1; S2: The fermentation medium was LB broth, consisting of: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 10 g / L, magnesium sulfate 0.28 g / L, manganese sulfate 0.58 g / L, Tween 80 1 g / L, and cysteine ​​hydrochloride 0.5 g / L. Step 2: Bacteria activation: S1: The probiotic bacteria were restored from the frozen state to room temperature, and then inoculated into a culture medium containing LB broth at a 1% inoculum size, and cultured in a constant temperature box at 37°C for 24 hours to activate and obtain a seed solution; Step 3: Seed liquid expansion: S1. Transfer the activated bacterial solution to 500 mL LB medium (shake flask volume ≤ 25%) at a 1% inoculum volume; S2, culture at 37°C and 200 rpm for 12 h (OD600≈1.0-1.5); S3, detect the number of viable bacteria (≥1x10 8 CFU / mL is qualified); Step 4: Fermentation culture: Inoculate the activated seed liquid into a fermenter containing LB broth at a rate of 1% (10 parts by weight per 1000 parts by weight of LB broth) and incubate at 37-42°C and standard atmospheric pressure for 48 hours, stirring the contents of the fermenter to form a uniform bacterial suspension. Step 5: Post-processing: S1. Separation and extraction: Use centrifugation, filtration and other methods to separate the bacteria from the culture medium and extract the required active ingredients. S2: Concentration: Concentrate the extracted bacteria to obtain a high-concentration bacterial suspension; S3. Transfer the bacterial suspension to a constant temperature water bath or heat exchanger and heat at 50°C for 5 minutes (temperature fluctuation ≤ ±1°C, time error ≤ ±10 seconds). Stir continuously at a low speed (20-30 rpm) during the heat shock process to ensure uniform temperature and avoid local overheating. S4. After the heat shock is completed, the bacterial suspension is immediately transferred to an ice bath (04°C) and rapidly cooled for 5-10 minutes to terminate the spore formation process.

2. The extraction process of a probiotic preparation for nasal microecological health care according to claim 1, characterized in that: The specific mixing steps of the fermentation medium in step 1 are as follows: 1) Dissolve the components in S2 in 800 mL of distilled water and stir until completely dissolved; 2) Adjust the pH to 7.0 with NaOH or HCI; 3) Adjust the volume to 1000 mL and sterilize by autoclave at 121°C for 20 minutes.

3. The extraction process of a probiotic preparation for nasal microecological health care according to claim 1, characterized in that: In the step 2, the probiotics are thawed on ice for ≤30 seconds, cultured at 37° C. and 200 rpm with shaking for 24 hours, and OD600≈0.5-1.0 is considered a sign of successful activation, and the number of activations is ≤3 times.

4. The extraction process of a probiotic preparation for nasal microecological health care according to claim 1, characterized in that: The specific operation steps of the fermentation tank in step 4 are as follows: 1) After sterilization, cool to 37°C and introduce sterile air (aerobic or anaerobic bacteria); 2) Maintain low agitation (100 rpm) for the first 2 hours after inoculation to reduce mechanical damage. Increase agitation to 200-400 rpm after entering the logarithmic growth phase and monitor DO > 30% saturation; 3) The fermentation broth enters the stable phase (OD600 reaches a plateau, Bacillus subtilis OD600≈5.0).

5. The extraction process of a probiotic preparation for nasal microecological health care according to claim 4, characterized in that: After the fermentation liquid enters the stable phase, the specific steps are as follows: 1) When the fermentation broth enters the stable phase (OD600 reaches the plateau phase, such as Bacillus subtilis OD600≈5.0), start feeding. The feeding solution formula is: yeast extract 5g / L + ammonium sulfate 2g / L (provides organic and inorganic nitrogen to promote spore formation); 2) Feeding method: pulse addition (feeding once every 4-6 hours, each feeding volume is 1%-2% of the fermentation liquid volume to avoid sudden changes in osmotic pressure caused by one-time feeding).

6. The extraction process of a probiotic preparation for nasal microecological health care according to claim 1, characterized in that: The specific process of dynamic pH control of the fermentation tank in step 4 is as follows: 1) Install a pH sensor on the fermentation tank stirring paddle, connect the pH sensor to an external PLC controller, and connect the PLC controller to the acid and alkali addition pump; 2) The pH sensor transmits data to the controller every 10-30 seconds. When it detects that the pH deviates from the target value (such as the pH rises to 7.1), the controller triggers the acid pump to add hydrochloric acid at a flow rate of 0.1-0.5L / h. If the pH drops to 6.9, the alkali pump is triggered to add NaOH.

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