Freeze-drying protection method of lactobacillus rhamnosus

Abstract

The invention belongs to the technical field of microbial freeze-drying, and particularly relates to a freeze-drying protection method of lactobacillus rhamnosus and freeze-dried bacterial powder prepared by the method. According to the method, the freeze-drying survival rate and the storage stability of the strain are remarkably improved through a composite freeze-drying protective agent of the adaptive strain and a gradient freeze-drying process, the viable count is larger than or equal to 9 * 10 < 9 > CFU / g after 12 months of sealed storage at room temperature, the survival rate is larger than or equal to 90%, the process is simple, the method is suitable for industrial production, and the obtained freeze-dried bacterial powder can serve as a core raw material to be applied to corresponding probiotic preparations and has a wide application prospect. And the method has important industrial value.

Description

Technical Field

[0001] This invention belongs to the field of microbial freeze-drying protection technology, specifically relating to a specific freeze-drying protection method for Lactobacillus rhamnosus YD-S-66 (accession number CGMCC No. 33918) and a highly active freeze-dried bacterial powder prepared by the method. Background Technology

[0002] Lactobacillus rhamnosus is a normal part of the human gut flora, with high intestinal adhesion and strong colonization ability. It is one of the most widely studied probiotics, possessing functions such as balancing and improving gastrointestinal function, enhancing the body's own immunity, promoting the growth and function of Bifidobacteria and Lactobacillus acidophilus, preventing and helping to treat diarrhea, preventing respiratory infections, eliminating toxins, preventing tooth decay, and preventing allergies. Based on the multiple functions of Lactobacillus rhamnosus for the human body, an increasing number of companies are using lyophilized Lactobacillus rhamnosus powder as a raw material to produce biological products.

[0003] Freeze-drying technology is a key link in ensuring the probiotic strain library and subsequent industrialization. It can preserve the activity of strains to the maximum extent. However, there are core pain points in the freeze-drying process of existing strains: (1) Severe freeze-drying damage: The cell membrane of Lactobacillus rhamnosus is sensitive to low temperature and dehydration. Under the traditional freeze-drying process (single protectant + rapid pre-freezing), the survival rate of strains is only about 60%; (2) Poor compatibility of protectants: Existing protectants (such as single skim milk powder) cannot specifically alleviate the freeze-drying stress of Lactobacillus rhamnosus YD-S-66, resulting in rapid decline of bacterial activity after freeze-drying; (3) Insufficient stability of freeze-dried bacterial powder: After 6 months of storage at room temperature, the number of viable bacteria drops to less than 50% of the initial value.

[0004] Improved methods for freeze-drying Lactobacillus include adding protective substances (such as sugars, polysaccharides, antioxidants, and lipid compounds) during fermentation. These substances enhance cell membrane stability, reduce moisture changes, inhibit ice crystal formation, and reduce oxidative damage, thereby increasing the survival rate of the freeze-dried cells. Simultaneously, the tolerance of lactic acid bacteria can be improved by adjusting the fermentation process (such as controlling the cell growth stage, optimizing the culture medium composition, and adjusting the fermentation pH and temperature). Patent CN106434463A optimizes the culture and fermentation process of Lactobacillus rhamnosus, using a food-grade culture medium, adding the growth factor isomaltooligosaccharide, and adding food-grade NaOH solution to maintain a constant pH. A freeze-drying protectant is also added to reduce cell membrane damage. Patent CN102978143B, combining key factors affecting bacterial fermentation, innovatively employs a two-stage pH and three-stage variable temperature fermentation method, significantly improving cell yield and fermentation activity, resulting in a freeze-dried formulation with high viable cell count and good stability, and providing an excellent freeze-drying protectant.

[0005] However, while these methods have improved the freeze-drying survival rate of Lactobacillus rhamnosus to some extent, their effects are often limited, and the responses of different strains vary significantly. As research on these traditional methods gradually becomes saturated, it is becoming increasingly difficult to find ways to significantly improve freeze-drying survival rate and stability. Therefore, developing a freeze-drying protection method for Lactobacillus rhamnosus with "high survival rate and high stability" to prepare high-quality freeze-dried bacterial powder has become crucial to overcoming the bottleneck in the probiotic industry. Summary of the Invention

[0006] To address the shortcomings of existing probiotic freeze-drying technologies, a freeze-drying protection method adapted to Lactobacillus rhamnosus YD-S-66 is provided, solving the problems of low freeze-drying survival rate and poor storage stability. At the same time, a highly active freeze-dried bacterial powder prepared by this method is provided, offering high-quality raw materials for the subsequent development of probiotic products.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for using a freeze-drying protectant for Lactobacillus rhamnosus, the method specifically comprising the following steps: (1) Activation and expansion culture of strain: The preserved Lactobacillus rhamnosus strain was streaked, anaerobic, passaged and statically cultured in modified MRS medium to obtain fermentation broth; (2) Collection and pretreatment of bacterial cells: The fermentation broth obtained in step (1) was separated by freezing and centrifugation to obtain bacterial cell precipitate. The bacterial cell precipitate was washed with sterile physiological saline and collected by centrifugation to finally obtain the washed bacterial cell precipitate. (3) Mixing of bacterial cell protectants: The bacterial cell precipitate and the composite protectant solution are mixed in a certain mass ratio to prepare a bacterial suspension. The composite protectant is composed of 7.5% skim milk powder, 10.5% trehalose, 3.8% raffinose, 1.2% L-glutamine, 0.6% betaine, and 0.4% ascorbic acid. in, Skim milk powder 7.5%: Forms a protein protective film to encapsulate bacterial cells, reducing mechanical damage to cell membranes caused by ice crystal formation, while providing nutritional support; Trehalose 10.5% + Raffinose 3.8%: The disaccharides work synergistically to replace intracellular water, stabilize biomembrane and protein structures, inhibit cell rupture during freeze-drying and rehydration, and enhance resistance to drying. L-Glutamine 1.2%: Repairs damaged bacterial cell structure during freeze-drying, maintains normal physiological and metabolic functions of cells, and enhances proliferation activity after rehydration; Betaine 0.6%: Regulates cell osmotic pressure, alleviates the impact of drastic changes in osmotic pressure on probiotics during freeze-drying, and maintains intracellular environmental stability; Ascorbic acid 0.4%: It eliminates free radicals generated during freeze-drying, reduces oxidative stress damage to probiotic cells, and delays activity decay during storage.

[0008] (4) Gradient freeze-drying treatment: The bacterial suspension is pre-frozen by first pre-freezing at -20℃ for 2 hours, then rapidly freezing at -45℃ for 2.5 hours, and then pre-freezing at -60℃ for 4 hours until the bacterial suspension is completely frozen; then it is sublimated and dried under vacuum conditions with a vacuum degree ≤10Pa and a drying time of 18 hours to obtain freeze-dried bacterial powder.

[0009] According to the specific implementation method, the Lactobacillus rhamnosus is Lactobacillus rhamnosus YD-S-66, with the accession number CGMCC NO: 33918.

[0010] According to the specific implementation method, the fermentation broth in step (1) is prepared by the following method: pick up the preserved Lactobacillus rhamnosus YD-S-662-3 tube, streak it on MRS medium, anaerobic culture at 37℃ for 24h, subculture 2-3 times, pick up 2-3 single colonies with good growth, inoculate them in modified MRS broth medium, and incubate at 37℃ for about 20h to obtain the fermentation broth.

[0011] According to the specific implementation method, the modified MRS culture medium includes: 20g glucose, 10g peptone, 10g beef extract powder, 5g yeast powder, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1.0mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 15g agar, 1000mL purified water, and 5g / L whey protein peptides.

[0012] According to the specific implementation method, the bacterial cell concentration in the fermentation broth is ≥7×10⁻⁶. 9 CFU / mL.

[0013] According to the specific implementation method, the centrifugation conditions in step (2) are 4℃, 5000rpm, centrifugation for 8min, and the number of times the bacterial cells are centrifuged is 2.

[0014] According to the specific implementation method, the sterilization temperature and time of the freeze-drying protectant in step (3) are 110°C and 8 min respectively for moist heat sterilization.

[0015] According to the specific implementation method, the mass ratio of bacterial cells to compound protective agent in step (3) is 2:1.

[0016] According to a specific embodiment, the freeze-dried bacterial powder is prepared by the above-described freeze-drying protection method, and the number of viable bacteria in the freeze-dried powder is ≥1×10⁻⁶. 10 CFU / g.

[0017] According to the specific implementation method, the live bacteria count of the freeze-dried bacterial powder after 12 months of sealed storage at room temperature is ≥9×10⁶. 9 CFU / mL.

[0018] Secondly, the present invention provides a freeze-dried Lactobacillus rhamnosus powder and its application in the preparation of adjuvant drugs or health products for the treatment of dysbiosis.

[0019] Beneficial effects

[0020] This invention significantly improves the bacterial cell recovery rate (96.3%), freeze-dried cell survival rate (≥90%), and product stability by optimizing the bacterial cell activation and pretreatment steps, including improving the MRS culture medium formulation and adopting low-temperature, low-speed centrifugation. Combined with a composite protectant and gradient freeze-drying process, this results in improved bacterial cell recovery (96.3%), freeze-dried cell survival rate (≥90%), and product stability. In the freeze-drying process, the bacterial suspension is first pre-frozen at -20℃ for 2 hours, then rapidly frozen at -45℃ for 2.5 hours (to reduce ice crystal damage), and finally transferred to a -60℃ ultra-low temperature environment for another 4 hours of pre-freezing. This ensures the bacteria are fully frozen into a solid bacterial cake, effectively reducing bacterial damage during freeze-drying and ensuring high viable cell count and powder stability. Furthermore, the entire preparation process strictly uses food-grade raw materials and meets hygiene standards. The resulting *Lactobacillus rhamnosus* freeze-dried powder exhibits high activity, strong stability, and excellent storage performance, making it suitable for long-term storage and wide application in various fields. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms, or direct or indirect applications in other related technical fields, made by those skilled in the art, shall fall within the scope defined by the appended claims.

[0022] Experimental materials and instruments

[0023] 1. Strains information: The strain used in this embodiment is Lactobacillus rhamnosus YD-S-66, with accession number CGMCC NO: 33918, which was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center, Beijing, China. Before use, it was activated twice on MRS medium (anaerobic culture at 37°C for 24 hours).

[0024] 2. Culture medium and reagents MRS medium: 20g glucose, 10g peptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1.0mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 15g agar (not added for liquids), 1000mL purified water. Sterilize at 121℃ for 15min.

[0025] Modified MRS medium: 20g glucose, 10g peptone, 10g beef extract, 5g yeast powder, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1.0mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 15g agar (not added for liquids), 1000mL purified water, 5g / L whey protein peptide (0.5% addition). Sterilize at 121℃ for 15min.

[0026] 3. Sterile saline: 9 g / L sodium chloride, sterilized at 121°C for 20 min.

[0027] 4. Compound protective agent ingredients: skim milk powder 7.5%, trehalose 10.5%, raffinose 3.8%, L-glutamine 1.2%, betaine 0.6%, ascorbic acid 0.4%.

[0028] 5. Control protectants: traditional protectant (50% skim milk powder + 10% glycerin, by mass ratio), single trehalose protectant (99% purity).

[0029] Example 1: Strain activation and fermentation broth preparation

[0030] 1. Strain activation and expansion culture: Pick 2-3 tubes of preserved glycerol bacteria, streak them onto MRS medium, and anaerobic culture at 37℃ for 24 h, subculturing 2-3 times. Pick 2-3 single colonies with good growth, inoculate them into modified MRS broth medium (with 0.5% whey protein peptides added), and incubate statically at 37℃ for about 20 h to obtain the fermentation broth. The fermentation broth should have a concentration ≥7×10⁻⁶. 9 CFU / mL.

[0031] 2. Cell pretreatment: Transfer the fermentation broth to centrifuge tubes and place them in a high-speed refrigerated centrifuge. Set the temperature to 4℃ and the speed to 5000 rpm for 8 minutes. Discard the supernatant and collect the cell precipitate at the bottom. Add sterile physiological saline to the cell precipitate, gently mix by pipetting, and centrifuge again at 4℃ and 5000 rpm for 8 minutes. Discard the supernatant. Repeat the washing process twice. Finally, collect the washed cell precipitate and weigh it (12.8 g).

[0032] 3. Recovery rate calculation: The number of viable bacteria before and after washing was determined by plate counting method. The bacterial recovery rate was calculated as (total number of viable bacteria after washing / total number of viable bacteria before washing) × 100%. The results showed that the bacterial recovery rate was 96.3%.

[0033] Example 2: Preparation of freeze-dried powder

[0034] 1. Cell collection: Transfer the above fermentation broth into centrifuge tubes, place them in a high-speed refrigerated centrifuge, set the temperature to 4℃ and the speed to 6500r / min, centrifuge for 20min, discard the supernatant, and collect the cell precipitate at the bottom.

[0035] 2. Cell washing: Add 100 mL of sterile physiological saline containing 0.2% trehalose to the cell precipitate, gently pipette to mix, and centrifuge again at 4℃ and 6500 r / min for 20 min. Discard the supernatant. Repeat the washing process twice. Finally, collect the washed cell precipitate and weigh it as 12.8 g.

[0036] 3. Recovery rate calculation: The number of viable bacteria before and after washing was determined by plate counting method. The bacterial recovery rate was calculated as (total number of viable bacteria after washing / total number of viable bacteria before washing) × 100%. The results showed that the bacterial recovery rate was 96.3%.

[0037] 4. Mixing of composite protective agents (1) Preparation of protective agent solution: Weigh 7.5% skim milk powder, 10.5% trehalose, 3.8% raffinose, 1.2% L-glutamine, 0.6% betaine and 0.4% ascorbic acid according to the mass ratio, mix them, add sterile water to make up to 100mL, stir to dissolve (magnetic stirrer, speed 150r / min) to make a composite protective agent solution, sterilize at 110℃ for 8min, and cool to room temperature for later use.

[0038] (2) Preparation of bacterial suspension: The washed bacterial precipitate (12.8g) was mixed with the composite protective agent solution at a mass ratio of 2:1 to ensure that the bacterial cells and protective agent were fully mixed to prepare a uniform bacterial suspension.

[0039] 5. Gradient freeze-drying treatment (1) Pre-freezing: Dispense the bacterial suspension into lyophilized vials (each vial contains 0.5 mL of liquid), first place them in a -20℃ ultra-low temperature freezer for 2 hours, then quick-freeze them at -45℃ for 2.5 hours (to reduce ice crystal damage), and finally transfer them to a -60℃ ultra-low temperature freezer to continue pre-freezing for 4 hours until the bacterial suspension is completely frozen into a solid bacterial cake.

[0040] (2) Sublimation drying: Place the pre-frozen mushroom cake into a vacuum freeze dryer, turn on the vacuum pump, and maintain the vacuum level to ≤10Pa for 18 hours. During this period, check through the observation window to confirm that the free water in the mushroom cake has completely sublimated. After capping, seal the package and store it in a refrigerator at 4℃ for later use.

[0041] Example 3: Effect of different protectant formulations on freeze-dried survival rate

[0042] Three groups of experiments were set up, using the composite protectant of this invention, a traditional protectant (50% skim milk powder + 10% glycerol), and a single trehalose protectant, respectively. The remaining freeze-drying process parameters were the same as described above. After preparing the freeze-dried bacterial powder, the freeze-dried survival rate and storage stability were determined. The results are shown in Table 1 below: Table 1 Protective agent type viable cell count in bacterial suspension before freeze-drying (CFU / mL) viable cell count (CFU / g) in freeze-dried bacterial powder Freeze-dried survival rate (%) viable bacterial count (CFU / g) after 12 months of storage at room temperature Survival rate (%) after 12 months of storage at room temperature Composite Protective Agent of the Invention <![CDATA[1.05×10 10 ]]> <![CDATA[1.00×10 10 ]]> 92 <![CDATA[9×10 9 ]]> 90 Traditional protective agents <![CDATA[1.05×10 10 ]]> <![CDATA[6.6×10 9 ]]> 63 <![CDATA[5.2×10 9 ]]> 79 Single trehalose protectant <![CDATA[1.05×10 10 ]]> <![CDATA[7.6×10 9 ]]> 72 <![CDATA[5.9×10 9 ]]> 77 The composite protectant of this invention has a significantly better freeze-drying protection effect on Lactobacillus rhamnosus freeze-dried powder than traditional protectants and single trehalose protectants, increasing the freeze-drying survival rate by 20%-29% and the survival rate by 11%-13% after 12 months of storage at room temperature, proving that the protectant formulation has good compatibility with the strain.

[0043] Example 4: Effects of different freeze-drying processes on the properties of bacterial powder

[0044] Two experimental groups were set up. The experimental group used the gradient freeze-drying process of this invention, while the control group used the traditional rapid freeze-drying process (direct pre-freezing at -60℃ for 4 hours, with other parameters the same as the experimental group). After preparing the freeze-dried bacterial powder, the viable bacterial concentration and long-term storage stability were measured. The results are shown in Table 2 below: Table 2 Freeze-drying process types Initial viable cell concentration (CFU / g) of freeze-dried bacterial powder Viable bacterial concentration (CFU / g) after 12 months of storage at room temperature Survival rate (%) after 12 months of storage at room temperature Gradient freeze drying of the present invention <![CDATA[1.00×10 10 ]]> <![CDATA[9×10 9 ]]> 92 Traditional rapid freeze drying <![CDATA[7.0×10 9 ]]> <![CDATA[3×10 9 ]]> 42.3 The gradient freeze-drying process of this invention can effectively reduce ice crystal damage to bacteria during the pre-freezing stage. The initial viable bacteria concentration of the freeze-dried bacterial powder is increased by 30% compared with the traditional process, and the survival rate after 12 months of storage at room temperature is increased by 49.7%, which significantly improves the initial activity and long-term stability of the freeze-dried bacterial powder.

[0045] Example 5: Effect of different pretreatment processes on cell recovery rate

[0046] Two experimental groups were set up. The experimental group used the pretreatment process of this invention, while the control group used a rapid centrifugation method at 4℃, 8000 r / min, and 5 min. After recovering the bacterial cells, viable cell counts were performed, and the results are shown in Table 3 below: Table 3 Preprocessing methods Initial viable cell concentration of recovered bacteria (CFU / mL) Initial viable cell concentration of recovered bacteria (CFU / mL) Recovery rate (%) This invention relates to low-temperature and low-vulgarity centrifugation. <![CDATA[7.0×10 9 ]]> <![CDATA[6.72×10 9 ]]> 96 Traditional high-speed centrifuge <![CDATA[7.0×10 9 ]]> <![CDATA[5.67×10 9 ]]> 81 The pretreatment process of this invention, characterized by low temperature and low speed, can maximize the retention of bacterial cell activity, effectively reduce the loss rate during bacterial cell recovery, and significantly improve the initial activity of the recovered bacterial cells.

Claims

1. A method for using a freeze-drying protectant for Lactobacillus rhamnosus, characterized in that, The method specifically includes the following steps: (1) Activation and expansion culture of strain: The preserved Lactobacillus rhamnosus strain was streaked, anaerobic, passaged and statically cultured in modified MRS medium to obtain fermentation broth; (2) Collection and pretreatment of bacterial cells: The fermentation broth obtained in step (1) was separated by freezing and centrifugation to obtain bacterial cell precipitate. The bacterial cell precipitate was washed with sterile physiological saline and collected by centrifugation to finally obtain the washed bacterial cell precipitate. (3) Mixing of bacterial cell protectants: The bacterial cell precipitate and the composite protectant solution are mixed in a certain mass ratio to prepare a bacterial suspension. The composite protectant is composed of 7.5% skim milk powder, 10.5% trehalose, 3.8% raffinose, 1.2% L-glutamine, 0.6% betaine, and 0.4% ascorbic acid. (4) Gradient freeze-drying treatment: The bacterial suspension is pre-frozen by first pre-freezing at -20℃ for 2 hours, then rapidly freezing at -45℃ for 2.5 hours, and then pre-freezing at -60℃ for 4 hours until the bacterial suspension is completely frozen; then it is sublimated and dried under vacuum conditions with a vacuum degree ≤10Pa and a drying time of 18 hours to obtain freeze-dried bacterial powder.

2. The freeze-drying protection method according to claim 1, characterized in that, The Lactobacillus rhamnosus mentioned is Lactobacillus rhamnosus YD-S-66, with accession number CGMCC NO: 33918.

3. The freeze-drying protection method according to claim 1, characterized in that, The fermentation broth described in step (1) is prepared by the following method: a preserved Lactobacillus rhamnosus YD-S-662-3 tube is picked, streaked onto MRS medium, and anaerobically cultured at 37°C for 24 hours. After 2-3 subcultures, 2-3 single colonies with good growth are picked, inoculated into modified MRS broth medium, and statically cultured at 37°C for about 20 hours to obtain the fermentation broth.

4. The freeze-drying protection method according to claim 1, characterized in that, The modified MRS medium consists of: 20g glucose, 10g peptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1.0mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 15g agar, 1000mL purified water, and 5g / L whey protein peptides.

5. The freeze-drying protection method according to claim 3, characterized in that, The fermentation broth has a bacterial cell concentration ≥7×10⁻⁶. 9 CFU / mL.

6. The freeze-drying protection method according to claim 1, characterized in that, The centrifugation conditions described in step (2) are 4℃, 5000rpm, 8min, and the number of times the bacterial cells are centrifuged is 2.

7. The freeze-drying protection method according to claim 1, characterized in that, In step (3), the mass ratio of bacterial cells to compound protective agent is 2:

1.

8. A freeze-dried powder of Lactobacillus rhamnosus, characterized in that, The freeze-dried bacterial powder is prepared by any one of the freeze-drying protection methods described in claims 1 to 7, and the number of viable bacteria in the freeze-dried powder is ≥1×10⁻⁶. 10 CFU / g.

9. The *Lactobacillus rhamnosus* lyophilized powder according to claim 8, characterized in that, ≥9×10⁶ viable bacteria after 12 months of sealed storage at room temperature 9 CFU / mL.

10. The use of the freeze-dried Lactobacillus rhamnosus powder according to any one of claims 8 to 9 in the preparation of adjunctive treatment drugs or health products for dysbiosis.