Preservation method of infantile anti-diarrhea enterococcus faecium

Abstract

The invention provides a preservation method of anti-diarrhea enterococcus faecium which is separated from excrement of a healthy baby, and particularly provides a preservation method of the anti-diarrhea enterococcus faecium which is separated from the excrement of the healthy baby. According to the method, a three-in-one composite preservation system of stress resistance protection, old-age intestinal tract adaptation and functional activity locking is constructed aiming at the adaptive pain point of an infant source strain and old-age intestinal tract micro-ecology, and a gradient cooling-vacuum freeze drying coupling process is combined; according to the present invention, the strain has characteristics of high viable bacteria survival rate, high senile intestinal tract colonization rate and anti-diarrhea function stability during the long-term preservation, and the technical problems that the existing preservation technology neglects the cross-age strain suitability and only pays attention to the viable bacteria number so as to cause poor colonization and function attenuation when the infant-derived anti-diarrhea strain is used for the elderly are solved.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a method for preserving infant-derived antidiarrheal Enterococcus faecalis, suitable for improving the intestinal health of the elderly. Background Technology

[0002] Enterococcus faecium is a Gram-positive bacterium widely distributed in the human gut and is an important member of the lactic acid bacteria family. In recent years, this bacterium has received widespread attention due to its significant probiotic functions, especially strains of Enterococcus faecium isolated from the feces of healthy infants, which have shown good anti-diarrheal activity, gut microbiota regulation capabilities, and the potential to enhance intestinal barrier function. Related studies have shown that Enterococcus faecium can provide the host with multiple health benefits by inhibiting the growth of pathogenic bacteria, regulating the body's immune response, and promoting the production of short-chain fatty acids.

[0003] Currently, the probiotic effects of Enterococcus faecium are mainly focused on its application in infants and young children, such as alleviating neonatal diarrhea and improving intestinal function. However, with the increasing aging of society, the intestinal health of the elderly has gradually become an urgent issue to be addressed. As people age, their gut microbiota becomes imbalanced, often manifesting as indigestion, constipation, and diarrhea. Simultaneously, their intestinal barrier function and immunity decline. Therefore, developing products to improve intestinal health for the elderly has significant social value and market potential.

[0004] Although Enterococcus faecalis shows great promise in improving gut health in the elderly, research and product development targeting this population are still in their early stages. Because the gut environment of the elderly differs significantly from that of infants, traditional methods of preparing and applying infant-derived anti-diarrheal Enterococcus faecalis may not fully realize its probiotic functions in the elderly. Existing preservation methods typically focus only on strain survival rates, neglecting the colonization adaptability of infant-derived strains in the elderly gut. This deficiency results in strains that, while still active after preservation, have weaker adhesion to the elderly gut environment and cannot effectively adapt to the intestinal mucus layer, thus limiting their anti-diarrheal function.

[0005] Currently disclosed patented technologies for the preservation of Enterococcus faecalis do not involve cross-age-group adaptation designs. Furthermore, small-scale probiotic preservation technologies targeting the elderly often rely on strains derived from the elderly themselves, failing to fully utilize the high anti-diarrheal activity of infant-derived Enterococcus faecalis. Therefore, to address this technological bottleneck, there is an urgent need to develop a method for preserving Enterococcus faecalis specifically designed for the elderly, ensuring that the strains maintain their activity during storage and adapt to the intestinal microecological environment of the elderly after activation, thereby maximizing their probiotic effects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preserving infantile-derived antidiarrheal Enterococcus faecalis, solving the problems of poor colonization and functional decline when infantile strains are applied to the elderly. The development of this technology not only expands the application scope of infantile-derived Enterococcus faecalis but also provides innovative solutions for intestinal health management in the elderly population.

[0007] The technical solution to achieve the above-mentioned objective is: In a first aspect, the present invention provides a method for preserving anti-diarrheal Enterococcus faecalis isolated from the feces of healthy infants, comprising the following steps: (1) Preparation of composite preservative: composed of stress-resistant core protective agent, aging intestinal adaptability factor and functional stabilizer in a mass ratio of 4.0~7.0:2.0~5.0:0.5~2.0; (2) Pretreatment of the strain: The infant-derived Enterococcus faecalis used in this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC NO.: 33919). The Enterococcus faecalis fermentation broth was centrifuged to collect the bacterial cells, washed with sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 8 × 10⁻⁶. 10 ~1.2×10 11 CFU / mL, add compound preservative and mix well to obtain preservative bacterial solution; (3) Gradient cooling: The culture solution is first pre-frozen at -18℃ for 2~2.5h, and then transferred to -85℃ for deep freezing for 3.5~4.5h; (4) Vacuum freeze drying: The deeply frozen bacterial solution is placed in a freeze dryer and dried for 7 to 8.5 hours under vacuum conditions of 8 to 16 Pa and desorption temperature of 24 to 28 °C.

[0008] According to a specific embodiment, the stress-resistant core protective agent is a compound of trehalose and xylooligosaccharide. Trehalose can form a molecular protective film to reduce freeze-drying damage, while xylooligosaccharide is a prebiotic that is beneficial to the elderly gut, creating a suitable nutritional environment for the bacterial strain in advance. The mass ratio of trehalose to xylooligosaccharide is 3:1.

[0009] According to a specific embodiment, the aging intestinal adaptor is a compound of sulfated mucin and hyaluronic acid. Sulfated mucin can mimic the characteristics of aging intestinal mucus and enhance the affinity of bacterial strains for adhesion targets. Hyaluronic acid can repair the microenvironment of the aging intestinal mucosa. The mass ratio of sulfated mucin to hyaluronic acid is 3:1.

[0010] According to a specific embodiment, the functional stabilizer is a small molecule whey protein hydrolysate, which is easily utilized by bacterial strains to maintain the activity of antibacterial related proteins, and has a molecular weight of 1500~3500 Da.

[0011] According to specific embodiments, the degree of polymerization of xylooligosaccharides is 2 to 5.

[0012] According to specific embodiments, the degree of sulfation of sulfated mucin is ≥75%.

[0013] According to specific embodiments, the final mass concentration of the composite preservative in the bacterial culture solution is 11%~14%.

[0014] According to a specific embodiment, the moisture content of the freeze-dried bacterial powder is 4.0~4.5%.

[0015] Secondly, the present invention also provides the application of the above-prepared Enterococcus faecalis powder in the preparation of anti-diarrheal products for the elderly.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention marks the first time that infant-derived anti-diarrheal Enterococcus faecalis has been applied across age groups to the elderly population. By optimizing the preservation system, it addresses the issue of strain colonization and compatibility, overcoming the limitations of single-population strain preservation techniques. The preservation method offers triple stability advantages: after 12 months of storage at 4℃, it achieves a viability rate of ≥85%, an adhesion rate of ≥68%, and a functional activity retention rate of ≥90%, effectively solving the bottlenecks of traditional preservation techniques that "preserve viability but not efficacy, or preserve efficacy but not compatibility." The strain is derived from the feces of healthy infants and its safety has been verified through natural screening. The preservation system contains no irritating components and is suitable for the intestinal physiology of the elderly. Furthermore, the process design is mild and highly reproducible, and the bacterial powder exhibits excellent flowability, meeting the needs of large-scale production of anti-diarrheal products for the elderly and providing an innovative solution for the probiotic field. Attached Figure Description

[0017] Figure 1 This is a viability graph of the preserved strain in Experiment Example 1.

[0018] Figure 2 This is a spectrum showing the adhesion rate of the preserved strain in Experiment Example 2.

[0019] Figure 3 This is a spectrum of the antidiarrheal function of the preserved strain in Experiment Example 3. Detailed Implementation

[0020] 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. Example 1

[0021] 1. Preparation of composite preservative: Weigh 12g of trehalose, 4g of xylooligosaccharide, 9g of sulfated mucin, 3g of hyaluronic acid, and 3.5g of small molecule whey protein hydrolysate; mix them and add sterile deionized water to make up to 100mL, sterilize at 115℃ for 15min to obtain a 13.5% concentration composite preservative.

[0022] 2. Strains Pretreatment: The *Enterococcus faecalis* strain used in this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC NO.: 33919). *Enterococcus faecalis* was inoculated onto MRS medium and cultured at 37°C for 16 h. The cells were collected by centrifugation at 7500 r / min for 12 min, washed three times with physiological saline, and resuspended to a concentration of 1 × 10⁻⁶. 11 CFU / mL, take 50mL of bacterial suspension and mix with 50mL of compound preservative for 15min.

[0023] 3. Gradient cooling and freeze drying: The preserved bacterial solution was first pre-frozen at -18℃ for 2.2h, and then transferred to -85℃ for deep freezing for 4h. The deep-frozen bacterial solution was placed in a freeze dryer and dried for 8h under vacuum of 12Pa and desorption temperature of 26℃ to obtain bacterial powder with a moisture content of 4.2%. Example 2

[0024] 1. Preparation of composite preservative: Weigh 10g of trehalose, 2g of xylooligosaccharide, 7g of sulfated mucin, 1g of hyaluronic acid, and 2g of small molecule whey protein hydrolysate; mix them and add sterile deionized water to make up to 100mL, sterilize at 115℃ for 15min to obtain a 10.0% concentration composite preservative.

[0025] 2. Strain pretreatment: Same as in Example 1.

[0026] 3. Gradient cooling and freeze drying: Same as in Example 1, finally obtaining bacterial powder with a moisture content of 4.5%. Example 3

[0027] 1. Preparation of composite preservative: Weigh 15g of trehalose, 6g of xylooligosaccharide, 11g of sulfated mucin, 5g of hyaluronic acid, and 5g of small molecule whey protein hydrolysate; mix them and add sterile deionized water to make up to 100mL, sterilize at 115℃ for 15min to obtain a 16.0% concentration composite preservative.

[0028] 2. Strain pretreatment: Same as in Example 1.

[0029] 3. Gradient cooling and freeze drying: Same as in Example 1, finally obtaining bacterial powder with a moisture content of 4.0%. Example 4

[0030] 1. Preparation of composite preservative: Same as in Example 1.

[0031] 2. Strain pretreatment: Same as in Example 1.

[0032] 3. Conventional cooling and freeze drying: The bacterial powder was directly placed in an environment of -85℃ for 6.2 hours and then transferred to a freeze dryer (vacuum degree 12Pa, desorption temperature 26℃) for 8 hours to obtain a moisture content of 5.8%.

[0033] During conventional cooling, water molecules inside the bacterial solution rapidly form large ice crystals. These ice crystals pierce the cell membranes and cell walls of *Enterococcus faecalis*, causing structural damage. Simultaneously, rapid cooling inactivates metabolic enzymes within the bacteria, disrupting intracellular homeostasis. Gradient cooling, on the other hand, gradually lowers the temperature, allowing water in the bacterial solution to slowly precipitate and form tiny ice crystals. This reduces physical damage to the bacteria and enables them to gradually adapt to the low-temperature environment, maintaining relative stability of the intracellular metabolic system and thus improving the survival rate of the freeze-dried bacteria. Experimental Example 1: Viability of Preserved Strains

[0034] Experimental groups: Example 1 group, Example 2 group, Example 3 group, Example 4 group, and control group (conventional trehalose + glycerol preservative, trehalose 8g + glycerol 10g, added to sterile deionized water to make up to 100mL).

[0035] The bacterial powders prepared in each group were stored at 4℃. Samples were taken at 3, 6, 9, and 12 months of storage, and the viable cell count was determined using the plate count method. Specific procedure: 0.1 g of bacterial powder was dissolved in 9.9 mL of sterile physiological saline, serially diluted, and 100 μL of the diluted solution was plated on MRS agar medium and anaerobically incubated at 37℃ for 48 h. The colony count was then counted, and the viability was calculated (viability = viable cell count after storage / viable cell count before storage × 100%).

[0036] The results are as follows Figure 1 As shown, after 12 months of storage, the viable bacterial rate was 90.5% in Group 1, 85.7% in Group 2, 88.2% in Group 3, 62.3% in Group 4, and 54.3% in the control group. This indicates that the composite preservative exhibits good preservation effects within the specified ratio range of trehalose (10-15g), xylooligosaccharides (2-6g), sulfated mucin (7-11g), hyaluronic acid (1-5g), and small molecule whey protein hydrolysate (2-5g), and the gradient cooling method is significantly superior to the conventional cooling method. Experimental Example 2: Detection of Adhesion Rate of Preserved Strains

[0037] Experimental groups: Example 1 group, Example 2 group, Example 3 group, Example 4 group, and control group (same as Example 1).

[0038] The adhesion rate was detected using an in vitro intestinal mucosal adhesion model. Mucosal sections were prepared from intestinal mucosal tissue of aged rats and placed in 24-well plates. 1 mL of bacterial suspension (concentration 1×10⁻⁶) stored for 12 months from each group was added to each well. 8 (CFU / mL), incubate at 37℃ for 1 h, wash unattached bacterial cells with phosphate-buffered saline (PBS), grind the mucosal sections, dilute and spread on MRS agar medium, count the number of attached viable bacteria, and calculate the adhesion rate (adhesion rate = number of attached viable bacteria / number of added viable bacteria × 100%).

[0039] The results are as follows Figure 2 As shown, the adhesion rate of the intestinal mucosa of the elderly in the present invention group was 71.2%, the adhesion rate of the Example 2 group was 66.5%, the adhesion rate of the Example 3 group was 69.3%, the adhesion rate of the Example 4 group was 45.8%, and the adhesion rate of the control group was 33.6%. This indicates that the preservation method of the present invention can effectively preserve the adhesion ability of the strain, and the ratio range of the composite preservative and the gradient cooling process both have a positive impact on the adhesion of the strain. Experimental Example 3: Detection of the antidiarrheal function of preserved strains

[0040] Experimental groups: Example 1 group, Example 2 group, Example 3 group, Example 4 group, control group (same as Example 1), and an original strain group (100%) before preservation was set as the baseline group.

[0041] Detection method: 1. Rehydration of bacterial powder: Take bacterial powder from each group stored for 12 months and rehydrate it with sterile physiological saline to a concentration of 1×10⁻⁶. 10 The original strain was diluted to the same concentration (CFU / mL) before preservation.

[0042] 2. Inhibition zone detection: The Oxford cup method was used. Melted LB agar medium (for *E. coli*) and BHIA agar medium (for *Clostridium difficile*) were poured into petri dishes. After cooling, 0.1 mL of the corresponding bacterial suspension (*Clostridium difficile* ATCC BAA-1870 and *E. coli* ATCC 25922, both at a concentration of 1×10⁻⁶) was evenly spread on the surface of the medium. 6 CFU / mL). Place Oxford cups on the surface of the culture medium, add 200 μL of rehydrated bacterial suspension to each Oxford cup, and incubate at 37°C for 24 h (E. coli) / 48 h (Clostridium difficile), and measure the diameter of the inhibition zone.

[0043] 3. Calculation of antibacterial activity retention rate: Antibacterial activity retention rate = (diameter of inhibition zone after preservation / diameter of inhibition zone of original strain) × 100%.

[0044] The test results are as follows Figure 3As shown, the retention rates of antibacterial activity against Clostridium difficile and Escherichia coli in the present invention group were 94.2% and 92.6%, respectively; those in Example 2 group were 89.5% and 87.8%, respectively; those in Example 3 group were 91.3% and 90.1%, respectively; those in Example 4 group were 68.4% and 65.7%, respectively; and those in the control group were only 56.8% and 60.3%, respectively. This indicates that the preservation method of the present invention can retain the antidiarrheal-related antibacterial activity of the strain to the greatest extent, and the ratio range of the compound preservative and the gradient cooling process are the key factors in maintaining the antibacterial function of the strain.

Claims

1. A method for preserving anti-diarrheal Enterococcus faecalis isolated from the feces of healthy infants, characterized in that, The method includes the following steps: (1) Preparation of composite preservative: composed of stress-resistant core protective agent, aging intestinal adaptability factor and functional stabilizer in a mass ratio of 4.0~7.0:2.0~5.0:0.5~2.0; (2) Pretreatment of bacterial strains: The Enterococcus faecalis fermentation broth was centrifuged to collect the bacterial cells, washed with sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 8 × 10⁻⁶. 10 ~1.2×10 11 CFU / mL, add compound preservative, mix well to obtain preservative bacterial solution; (3) Gradient cooling: The culture solution is first pre-frozen at -18℃ for 2~2.5h, and then transferred to -85℃ for deep freezing for 3.5~4.5h; (4) Vacuum freeze drying: The deeply frozen bacterial solution is placed in a freeze dryer and dried for 7 to 8.5 hours under vacuum of 8 to 16 Pa and desorption temperature of 24 to 28 °C to obtain freeze-dried bacterial powder.

2. The method according to claim 1, characterized in that, The stress-resistant core protective agent is a compound of trehalose and xylooligosaccharide in a mass ratio of 3:

1.

3. The method according to claim 1, characterized in that, The elderly intestinal adaptor is a compound of sulfated mucin and hyaluronic acid in a mass ratio of 3:

1.

4. The method according to claim 1, characterized in that, The functional stabilizer is a small molecule whey protein hydrolysate with a molecular weight of 1500~3500 Da.

5. The method according to claim 2, characterized in that, The degree of polymerization of the xylooligosaccharide is 2 to 5.

6. The method according to claim 3, characterized in that, The sulfated mucin has a sulfated degree ≥75%.

7. The method according to claim 1, characterized in that, The final mass concentration of the composite preservative in the culture medium is 11%~14%.

8. The method according to claim 1, characterized in that, The freeze-dried bacterial powder has a moisture content of 4.0~4.5%.

9. The application of Enterococcus faecalis powder preserved by any one of the methods described in claims 1 to 8 in the preparation of anti-diarrheal products for the elderly.

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