A freeze-drying protection system for Enterococcus faecalis, its preparation method and application

Abstract

This invention relates to the field of biotechnology, specifically disclosing a freeze-drying protection system for Enterococcus faecalis, its preparation method, and its applications. The freeze-drying protection system comprises arginine, skim milk, and glycerol, combined with low-temperature stress pretreatment. The freeze-drying protection system provided by this invention significantly increases the freeze-drying survival rate of Enterococcus faecalis L118 to 87.90±1.28%, and the survival rate remains above 90% after 28 days of storage at -20℃. The core probiotic characteristics of the freeze-dried Enterococcus faecalis L118 strain, such as acid production capacity, simulated gastric juice, and NaCl tolerance, are well preserved, providing an effective solution for preparing highly active and stable Enterococcus faecalis preparations.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a freeze-drying protection system for Enterococcus faecalis, its preparation method, and its application. Background Technology

[0002] Enterococcus faecalis ( Enterococcus faecalis Enterococcus faecalis is a Gram-positive bacterium widely recognized as a potential probiotic and is extensively used in animal feed additives. Multiple studies have confirmed that this bacterium can promote nutrient absorption, maintain intestinal epithelial barrier function, regulate intestinal flora balance, and enhance innate immunity. Furthermore, Enterococcus faecalis can produce bacteriocins, effectively inhibiting the growth of pathogenic bacteria. Its co-aggregation properties can also inhibit pathogen colonization in the intestine, maintaining intestinal flora homeostasis.

[0003] Enterococcus faecalis, a representative lactic acid bacteria, is typically preserved using vacuum freeze-drying. This technology is widely used not only in the preparation of food additives but is also a common method for long-term preservation of bacterial strains. However, during vacuum freeze-drying, ice crystals forming on the cell membrane can damage the bacterial structure. Furthermore, rapid cooling can cause cell membrane deformation, leading to decreased bacterial activity and even death. Currently, freeze-drying preservation methods for Enterococcus faecalis mainly involve combinations of various freeze-drying protectants, such as glycerol and skim milk. While these methods can maintain cell membrane integrity, prevent ice crystal damage, and increase the glass transition temperature, they still result in extremely high cell damage rates when subjected to sudden, rapid cooling and dehydration during freeze-drying, thus hindering the further widespread use of Enterococcus faecalis. Therefore, developing a novel freeze-drying protection product to maintain a high number of active cells is of significant practical importance. Summary of the Invention

[0004] To develop a novel freeze-drying protection process, this invention provides a freeze-drying protection system for Enterococcus faecalis, its preparation method, and its applications. The freeze-drying protection system provided by this invention significantly increases the freeze-drying survival rate of Enterococcus faecalis L118 to 87.90±1.28%, and the survival rate remains above 90% after 28 days of storage at -20℃. The core probiotic characteristics of the freeze-dried Enterococcus faecalis L118 strain, such as its acid-producing capacity, simulated gastric juice, and NaCl tolerance, are well preserved, providing an effective solution for preparing highly active and stable Enterococcus faecalis preparations.

[0005] This invention provides a freeze-drying protection system for Enterococcus faecalis, which, by mass-volume ratio, consists of the following raw materials: 0.5%~2.5% arginine, 5%~30% skim milk, 5%~30% glycerol, and the balance being water.

[0006] The freeze-drying protection system provided by this invention can significantly increase the freeze-drying survival rate of Enterococcus faecalis L118 to 87.90±1.28%, and the survival rate is still higher than 90% after 28 days of storage at -20℃. The core probiotic characteristics of Enterococcus faecalis L118 strain, such as acid production capacity, simulated gastric juice and NaCl tolerance, are well preserved after freeze-drying.

[0007] The present invention also provides a method for preparing a freeze-drying protection system for Enterococcus faecalis, comprising mixing arginine, skim milk and glycerol with water to obtain arginine cryoprotectant, skim milk cryoprotectant and glycerol cryoprotectant respectively; adjusting the pH of the arginine cryoprotectant to neutral, sterilizing the skim milk cryoprotectant and the arginine and glycerol cryoprotectants respectively and then mixing them to obtain the freeze-drying protection system.

[0008] This invention also provides the application of a freeze-drying protection system for Enterococcus faecalis in the preparation of freeze-dried Enterococcus faecalis powder, the preparation of which includes the following steps: Enterococcus faecalis bacterial suspension was centrifuged, washed with PBS, centrifuged again, and the bacterial cells were collected. The bacterial cells and the freeze-drying protection system were mixed in equal mass ratios to obtain a bacterial suspension; The bacterial suspension was subjected to cold stress treatment; The bacterial suspension after cold stress treatment was freeze-dried under vacuum to obtain freeze-dried bacterial powder.

[0009] Furthermore, the cold stress treatment is performed at 6℃~10℃ for 4 h~12 h.

[0010] Furthermore, the conditions for vacuum freeze drying are as follows: pre-freezing temperature -65℃ to -55℃, pre-freezing time 2 h to 6 h; vacuum degree 12 Pa to 18 Pa, drying time 46 h to 50 h.

[0011] Furthermore, the freeze-drying protection system is used to improve the freeze-dried survival rate of Enterococcus faecalis.

[0012] Furthermore, the freeze-dried survival rate of Enterococcus faecalis in the freeze-dried bacterial powder is ≥87%.

[0013] Furthermore, the freeze-drying protection system is used to maintain the acid-producing capacity of Enterococcus faecalis after freeze-drying, simulate gastric juice tolerance, and / or NaCl tolerance.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through single-factor experiments and response surface methodology, systematically optimized an optimal freeze-drying protection system consisting of arginine, skim milk, glycerol, and cold stress treatment. This system can increase the freeze-dried survival rate of Enterococcus faecalis L118 to 87.90±1.28%, significantly higher than single or traditional protectant treatment groups (e.g., a single cold stress treatment survival rate of only about 26%). The model predictions and measured values ​​show a high degree of agreement, demonstrating the scientific validity and reliability of this protection system formulation.

[0015] The freeze-drying protection system provided by this invention does not exhibit a simple additive effect of the individual components, but rather a significant synergistic interaction. Response surface methodology analysis of variance indicates that multiple interactions, including those between arginine and skim milk (AB), arginine and cold stress time (AE), skim milk and glycerol (BC), and cold stress temperature and time (DE), all have highly significant effects on survival rate (P<0.01). This synergistic effect provides comprehensive protection against various damaging factors during freeze-drying, such as ice crystal damage, membrane phase transition, and dehydration stress.

[0016] The freeze-drying protection system of this invention can significantly upregulate the expression of several key protective genes during freeze-drying, including energy metabolism and stress response genes. atpA , SOD (Enhancing cellular energy supply and antioxidant capacity), cold shock protein gene cspC (Helping cells adapt to low-temperature environments and maintain protein function), genes involved in cell membrane synthesis and modification. FabH , FabF , plsX and mprF2 (Promotes membrane lipid synthesis and modification, maintains membrane integrity and fluidity), metabolic and quorum sensing genes LDH1 , luxS (Regulating acid metabolism and signal communication between bacteria). This pattern of multi-gene synergistic upregulation indicates that the freeze-drying protection system addresses freeze-drying stress by systematically enhancing the intrinsic tolerance of cells, rather than through simple physical protection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1The effects of different freeze-drying protective factors on the survival rate of Enterococcus faecalis L118 are shown in the figure. In the figure, A represents the effect of different arginine concentrations; B represents the effect of different skim milk concentrations; C represents the effect of different glycerol concentrations; D represents the effect of different cold stress temperatures; and E represents the effect of different cold stress times.

[0019] Figure 2 This represents the relative gene expression levels of Enterococcus faecalis strain L118; in the figure, A represents... atpA Relative gene expression level; B is cspC Relative gene expression level; C is FabF Relative gene expression level; D is FabH Relative gene expression level; E is LDH1 Relative gene expression level; F is luxS Relative gene expression level; G is mprF2 Relative gene expression level; H is plsX Relative gene expression level; I is SOD Relative gene expression levels.

[0020] Figure 3 The figure shows the changes in the acid production capacity of Enterococcus faecalis L118 strain. In the figure, A is a comparison of the acid production capacity of Enterococcus faecalis L118 in the optimal freeze-drying protection system group and the fresh culture control group; B is the acid production curve and growth curve of Enterococcus faecalis L118 in the optimal freeze-drying protection system group; C is the acid production curve and growth curve of Enterococcus faecalis L118 in the fresh culture control group.

[0021] Figure 4 To assess the storage stability of Enterococcus faecalis strain L118 after treatment with the optimal freeze-drying protection system. Detailed Implementation

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0023] Example 1: A freeze-drying protection system for Enterococcus faecalis, its preparation method and application.

[0024] I. Experimental Materials Enterococcus faecalis strain L118 was purchased from the China Center for Type Culture Collection (CCTCC), accession number M2024443. This invention does not involve strain preservation.

[0025] II. Test Methods 1. Culture of Enterococcus faecalis L118 Enterococcus faecalis strain L118, cryopreserved at -80℃, was added to Brain Heart and Body Broth (BHI, Qingdao Haibo Biotechnology Co., Ltd., HB8297-1) and incubated at 37℃ for 24 h. Then, using a sterile inoculation loop, the bacterial suspension was streaked onto Enterococcus faecalis selective solid medium plates (Qingdao Haibo Biotechnology Co., Ltd., HB0268-2) and incubated at 37℃ for 24 h. Black colonies were picked and cultured separately in BHI at 37℃ for 24 h, followed by two consecutive subcultures at a 1% (v / v) inoculation rate in BHI to obtain Enterococcus faecalis strain L118 for subsequent experiments.

[0026] 2. Single-factor analysis of different factors in the freeze-drying protection system A completely randomized design was used to investigate the effects of different concentrations of arginine (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, w / v), skim milk (5%, 10%, 15%, 20%, 25%, 30%, w / v), and glycerol (5%, 10%, 15%, 20%, 25%, 30%, w / v), as well as different cold stress temperatures (8 h, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃) and cold stress durations (8℃, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) on the survival rate of Enterococcus faecalis L118. Arginine, skim milk, and glycerol were dissolved in deionized water to obtain three cryoprotectants: arginine, skim milk, and glycerol. Hydrochloric acid was added to the arginine cryoprotectant to adjust the pH to 7. Skim milk cryoprotectant was sterilized at 105°C, while two other cryoprotectants were sterilized at 115°C for 10 min each. Resuscitated Enterococcus faecalis L118 was inoculated into BHI at a 1% (v / v) inoculum and cultured for 6 h until the early stationary phase. The culture was then centrifuged at 3000 g for 10 min to collect the bacterial pellet, washed twice with 0.1 M phosphate-buffered saline (PBS), and centrifuged again at 3000 g for 10 min to collect the bacterial pellet. Different concentrations of cryoprotectant were added to the bacterial pellet at a 1:1 (w / w) ratio to resuspend the cells, obtaining bacterial suspensions. For the cold stress group, Enterococcus faecalis L118 underwent different cold stress temperatures and durations before the addition of cryoprotectants. Subsequently, the bacterial suspension was pre-frozen at -60°C for 4 h in a vacuum freeze dryer, followed by vacuum freeze-drying at 15 Pa for 48 h to obtain the freeze-dried product.

[0027] The viable cell count was determined using the standard plate count method before and after lyophilization. 100 µL of the sample was diluted to 10⁻¹⁰ with PBS. 3The lyophilized product was evenly spread on BHI agar medium (Qingdao Haibo Biotechnology Co., Ltd., HB8478) and incubated at 37℃ for 18 h. According to GB4789.2-2022, plates with a colony-forming unit (CFU) count between 30 and 300 and with uniform colony distribution were selected for counting. The survival rate was calculated using the following formula:

[0028] Survival rate (%) = viable bacteria count after freeze-drying (CFU / mL) / viable bacteria count before freeze-drying (CFU / mL) × 100%.

[0029] 3. Optimization of the freeze-drying protection system using response surface methodology Based on the optimal concentrations of arginine, skim milk, and glycerol obtained from single-factor screening, as well as the optimal temperature and time of cold stress, a three-level, five-factor Box-Behnken design (BBD) was used to further optimize the formulation and obtain the optimal freeze-drying protection system after mixing all factors. The high (1), medium (0), and low (-1) levels of each factor and their corresponding parameter settings are detailed in Table 1. The experimental factors included: A-arginine, B-skim milk, C-glycerol, D-cold stress temperature, and E-cold stress time. Forty-six experiments were designed and implemented using Expert 13.0 software. Using the survival rate of Enterococcus faecalis L118 as the response index, a quadratic polynomial regression model was established to predict the optimal freeze-drying protection system and survival rate.

[0030] Table 1. Range and level of experimental parameters for the protective factor against lyophilized Enterococcus faecalis L118 4. RT-qPCR analysis The protective mechanisms of arginine and cold stress on Enterococcus faecalis L118 strain and the synergistic effect of the optimal freeze-drying protection system were analyzed by RT-qPCR. Transcriptome analysis was performed on the arginine group, the cold stress group, and the optimal freeze-drying protection system group, with the control group being untreated Enterococcus faecalis L118 strain. Total RNA was extracted from the four groups of freeze-dried Enterococcus faecalis L118 strains using the TRIZOL method (Thermo Fisher Scientific, 15596018CN). cDNA was generated using a reverse transcription kit (Thermo Fisher Scientific, K1691) and then amplified by RT-qPCR using a SYBR Green kit (Thermo Fisher Scientific, A46110). Primers for the target gene were designed using Primer3 software (Table 2), with 16S rRNA as an internal control. Results were presented as follows: -ΔΔCt The study analyzed the effects of cold stress, arginine, and optimal freeze-drying process on the expression of the L118 gene in Enterococcus faecalis during freeze-drying.

[0031] Table 2 Primers used for RT-qPCR 5. Acid-producing capacity of Enterococcus faecalis L118 2 mL of PBS was added to the optimal freeze-drying protection system for Enterococcus faecalis L118 for rehydration, followed by centrifugation at 3000 g for 10 min. Freshly cultured Enterococcus faecalis L118 (incubated at 37℃ for 24 h) was also centrifuged at 3000 g for 10 min. After collecting the bacterial pellets, 0.1 M PBS was added to adjust the OD600 to 0.4, resulting in two bacterial suspensions. The bacterial suspensions were inoculated into BHI at a 5% (v / v) inoculum and incubated at 37℃ for 24 h. After incubation, the pH value was measured using a pH / conductivity meter to assess the acid-producing capacity of Enterococcus faecalis L118.

[0032] 6. Survival rate and NaCl tolerance of Enterococcus faecalis L118 in simulated gastric juice The optimal freeze-drying protection system for Enterococcus faecalis L118 was rehydrated in 2 mL PBS and centrifuged (3000 g, 10 min). Freshly cultured Enterococcus faecalis L118 (cultured at 37℃ for 24 h) was also centrifuged at 3000 g for 10 min. Bacterial pellets were collected and resuspended in PBS to an OD600 of 0.4, obtaining two bacterial suspensions. 1% (v / v) of the bacterial suspension was added to different concentrations of NaCl solution (3%, 4%, 5%, 6%, 7%, w / v) and simulated gastric juice (3 g / L pepsin added to 0.1 M PBS, pH adjusted to 3.0). Viability was determined after incubation at 37℃ for 3 h. The survival rate was calculated using the following formula:

[0033] Bacterial survival rate (%) = (number of viable bacteria (CFU / mL) / initial number of viable bacteria (CFU / mL)) × 100%.

[0034] 7. Storage stability test Enterococcus faecalis L118 powder treated with the optimal freeze-drying protection system was stored at -20℃, 4℃, and 25℃ for 28 days, and its survival rate at different temperatures was evaluated. The survival rate was calculated using the following formula:

[0035] Bacterial survival rate (%) = (number of viable bacteria (CFU / mL) / initial number of viable bacteria (CFU / mL)) × 100%.

[0036] III. Test Results 1. Single-factor analysis to determine the optimal values ​​of different factors in the freeze-drying protection system. Univariate analysis showed that the survival rate of Enterococcus faecalis L118 was highest at an arginine concentration of 1.5%, reaching 43.79 ± 4.21%. Figure 1(A); In 10% skim milk and 10% glycerol environments, the survival rates of Enterococcus faecalis L118 were 54.93±9.57% and 73.68±9.85%, respectively. Figure 1 (B and C); When the cold stress condition was 8℃ for 8 h, the survival rate of Enterococcus faecalis L118 reached its peak, at 26.71±0.54% and 25.98±0.85%, respectively. Figure 1 (D, E).

[0037] 2. Optimization of the freeze-drying protection system using response surface methodology Based on Table 1, an experimental design was performed using Expert 13.0. After 46 runs, the regression equation was derived as follows: Y = 88.24 - 1.37A + 1.48B - 0.0338C - 1.64D - 0.2138E - 2.89AB - 1.91AC - 3.43AD - 3.73AE - 10.09BC + 6.55BD - 5.27BE + 4.64CD + 6.32CE - 14.71DE - 8.20A 2 -12.59B 2 -11.25C 2 -16.23D 2 -8.62E 2 .

[0038] According to the ANOVA results in Table 3, the model was highly significant (P<0.0001); the Lack of Fit (P=0.9082) was not significant, and the R² value of the model was 0.9734, which is greater than 0.9. The difference between Adj R² (=0.9521) and Pred R² (=0.9178) was less than 0.2, indicating that the model is feasible. Except for the principal factors C and E, which were not significant (P>0.05), the other principal factors A, B, and D were all significant (P<0.05). The interaction of factors AB was significant (P<0.05), while the interactions of factors AD, AE, BC, BD, BE, CD, CE, and DE were all highly significant (P<0.01). The above interactions had a significant impact on the survival rate of Enterococcus faecalis L118, while the interaction of factors AC had no significant impact on the survival rate (P>0.05). Furthermore, all quadratic terms (A², B², C², D², E²) had extremely significant effects on the survival rate of Enterococcus faecalis L118 (P<0.01).

[0039] Multiple regression analysis predicted the optimal freeze-drying protection system to be 1.457% arginine, 10.392% skim milk, 9.810% glycerol, a cold stress temperature of 7.945℃, and a cold stress time of 7.966 h, with a predicted survival rate of 88.383%. Based on actual conditions, the optimal freeze-drying protection system was adjusted to 1.5% arginine, 10.4% skim milk, 9.8% glycerol, a cold stress temperature of 8℃, and a cold stress time of 8 h. Under this freeze-drying protection system, the survival rate of Enterococcus faecalis L118 was 87.90 ± 1.28%.

[0040] Table 2 Response Surface Model Analysis Note: - indicates that this item is not available.

[0041] 3. RT-qPCR analysis Figure 2 The results showed that, among the detected genes, cold stress significantly upregulated the expression of genes in strain L118. atpA , plsX , luxS and SOD Gene expression (P<0.05), while LDH1 Gene expression was significantly downregulated (P<0.05), while other genes showed no significant changes (P>0.05). Except for... luxS Gene expression was significantly downregulated (P<0.05). SOD Except for no significant changes in gene expression (P>0.05), arginine significantly upregulated the expression of other genes (P<0.05). The optimal freeze-drying protection system significantly upregulated the expression of all detected genes (P<0.05), and compared with the arginine group and the cold stress group, the optimal freeze-drying protection system significantly improved... atpA , LDH1 , FabH , FabF and plsX Gene expression (P<0.05), while in the cold stress group... luxS Gene expression was significantly higher than that of the optimal freeze-drying protection system group (P<0.05).

[0042] 4. Acid-producing capacity of Enterococcus faecalis L118 like Figure 3 As shown in A, there was no significant difference in acid production capacity between the optimal freeze-drying protection system group (pH=5.27±0.12) and the fresh culture control group (pH=5.54±0.32) (P>0.05). Figure 3Figures B and C indicate that the growth and acid production kinetics of the two groups of strains were highly consistent, with the rapid decrease in pH occurring mainly between the early logarithmic growth phase and the early stationary phase (2 h to 6 h). The results suggest that strain L118, treated with the optimal freeze-drying protection system, not only retained its acid production capacity but also maintained its acid production kinetics.

[0043] 5. Survival rate and NaCl tolerance of Enterococcus faecalis L118 in simulated gastric juice As shown in Table 4, after lyophilization using the optimal freeze-drying protection system, the survival rate and NaCl tolerance of Enterococcus faecalis L118 in simulated gastric fluid were not significantly different from those of the fresh culture control group (P>0.05), indicating that the freeze-drying protection system can stabilize the probiotic characteristics of Enterococcus faecalis L118.

[0044] Table 4. Survival rate and sodium chloride tolerance of Enterococcus faecalis L118 in simulated gastric juice. 6. Storage stability like Figure 4 As shown, the survival rate of *Enterococcus faecalis* L118 treated with the optimal freeze-drying protection system decreased with prolonged storage time, exhibiting a decreasing trend under all three temperature conditions. After storage at -20℃ for 28 days, the survival rate of this strain remained at 90.09±2.91%; at 4℃ and 25℃, the survival rate decreased to 74.16±1.79% and 35.00±3.44%, respectively. These results indicate that *Enterococcus faecalis* L118 treated with the optimal freeze-drying protection system exhibits high storage stability at -20℃.

[0045] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0046] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A freeze-drying protection system for Enterococcus faecalis, characterized in that, Based on mass-volume ratio, it consists of the following raw materials: 0.5%~2.5% arginine, 5%~30% skim milk, 5%~30% glycerol, and the balance is water.

2. A method for preparing the freeze-drying protection system for Enterococcus faecalis according to claim 1, characterized in that, Arginine, skim milk, and glycerol were mixed with water to obtain arginine cryoprotectant, skim milk cryoprotectant, and glycerol cryoprotectant, respectively. The pH of the arginine cryoprotectant was adjusted to neutral. The skim milk cryoprotectant, arginine cryoprotectant, and glycerol cryoprotectant were sterilized separately and then mixed to obtain the freeze-drying protection system.

3. The application of the freeze-drying protection system for Enterococcus faecalis as described in claim 1 in the preparation of freeze-dried Enterococcus faecalis powder, characterized in that, The preparation of freeze-dried Enterococcus faecalis powder includes the following steps: Enterococcus faecalis bacterial suspension was centrifuged, washed with PBS, centrifuged again, and the bacterial cells were collected. The bacterial cells and the freeze-drying protection system were mixed in equal mass ratios to obtain a bacterial suspension; The bacterial suspension was subjected to cold stress treatment; The bacterial suspension after cold stress treatment was freeze-dried under vacuum to obtain freeze-dried bacterial powder.

4. The application according to claim 3, characterized in that, The cold stress treatment is performed at 6℃~10℃ for 4h~12h.

5. The application according to claim 3, characterized in that, The conditions for vacuum freeze drying are as follows: pre-freezing temperature -65℃ to -55℃, pre-freezing time 2 h to 6 h; vacuum degree 12 Pa to 18 Pa, drying time 46 h to 50 h.

6. The application according to claim 3, characterized in that, The freeze-drying protection system is used to improve the survival rate of freeze-dried Enterococcus faecalis.

7. The application according to claim 3, characterized in that, The freeze-dried survival rate of Enterococcus faecalis in the freeze-dried bacterial powder is ≥87%.

8. The application according to claim 3, characterized in that, The freeze-drying protection system is used to maintain the acid-producing capacity of freeze-dried Enterococcus faecalis, simulate gastric juice tolerance, and / or NaCl tolerance.

Images