Method for preparing calcium-rich product through fermentation of enterococcus faecium and application of calcium-rich product

Abstract

The invention provides a method for preparing a calcium-rich product through fermentation of enterococcus faecium and application, calcium is enriched through fermentation of enterococcus faecium, ultrasonic treatment is carried out in the fermentation process, the calcium-rich capacity of enterococcus faecium is improved, the influence of calcium ions on the physiological activity of enterococcus faecium is researched, and the calcium-rich product is prepared by adjusting the variety and concentration of a carbon source and a nitrogen source. The formula of the fermentation culture medium is optimized, growth of the enterococcus faecium is promoted, the capability of converting inorganic calcium into organic calcium is enhanced, meanwhile, the antioxidant activity can be improved, and the enterococcus faecium fermentation culture medium has important significance on development of novel calcium supplements.

Description

[0001] This application claims priority to a prior Chinese application, application number 2025109482436, filed on July 10, 2025; all its contents are part of this invention. Technical Field

[0002] This invention belongs to the field of microbial calcium enrichment technology, specifically relating to a method and application of preparing calcium-enriched products by fermentation with Enterococcus faecalis. Background Technology

[0003] Calcium is an essential element for life, playing a vital role in promoting bone development and maintaining normal heart function. Long-term calcium deficiency can lead to poor bone development, osteoporosis, abnormal blood clotting, and hypothyroidism. Calcium deficiency can cause various diseases such as rickets, osteoporosis, and tetany, and can also lead to problems like abnormal blood clotting and hypothyroidism. Therefore, maintaining normal calcium levels in the body is a crucial foundation for ensuring human health.

[0004] Food is an important source of calcium, but the calcium content and bioavailability in our diet are far from meeting the body's needs, leading to a growing demand for calcium supplements. The market is flooded with calcium supplements, mainly divided into inorganic and organic calcium. Inorganic calcium is primarily chemically synthesized, making it poorly absorbed by the body, but it is inexpensive. Organic calcium is easily absorbed, but it is very expensive, and most existing organic calcium products are chemically synthesized or naturally extracted from seaweed or bone meal, resulting in complex preparation processes and potential for heavy metal residues or byproducts, further compromising bioavailability. Therefore, finding a green, healthy, and low-cost method for preparing organic calcium for novel calcium supplements has become an urgent problem to solve.

[0005] In recent years, the enrichment and transformation of trace elements such as calcium by microorganisms has become a research hotspot in this field. Microorganisms have a strong enrichment and transformation effect on trace elements such as calcium, iron, zinc, and selenium. Domestic and foreign scholars have conducted research on the enrichment of calcium using microorganisms such as yeast, lactic acid bacteria, and edible fungi. Utilizing probiotics to enrich inorganic calcium into more easily absorbed organic calcium, and combining calcium with natural proteins / polysaccharides, results in higher bioavailability and holds promise as a low-cost, safe, efficient, and green calcium supplement. Many scholars have studied the mechanisms of microbial enrichment of trace elements and achieved some results. However, due to the complexity and diversity of microbial structures and species, different types of microorganisms have different enrichment mechanisms for trace elements, and even the same microorganism may have different enrichment mechanisms under different environmental influences. Therefore, the enrichment mechanisms of specific microorganisms for trace elements still require detailed research.

[0006] Enterococcus faecalis ( Enterococcus faeciumEnterococcus faecalis, a lactic acid-producing facultative anaerobic bacterium, is a normal flora widely present in the animal intestines, playing an important role in regulating the intestinal flora environment. In addition, Enterococcus faecalis also possesses probiotic functions such as inhibiting the growth of pathogenic bacteria, improving intestinal mucosal barrier function, alleviating inflammatory responses, anti-cancer effects, and alleviating obesity. Enterococcus faecalis has been widely used in the feed industry, medical field, and traditional fermented products, especially in Mediterranean cheese fermentation, where it promotes the maturation of fermented products, develops specific flavors, and inhibits pathogenic bacteria. Current research on Enterococcus faecalis mainly focuses on its probiotic properties and safety evaluation, but there are no reports on its accumulation of trace elements or calcium. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method and application for preparing calcium-enriched products through fermentation with Enterococcus faecium. Calcium is enriched by fermentation with Enterococcus faecium, and ultrasonic treatment is performed during fermentation to enhance the calcium-enriching capacity of Enterococcus faecium. The effects of calcium ions on its physiological activity were investigated. By adjusting the types and concentrations of carbon and nitrogen sources and optimizing the fermentation medium formulation, not only was the growth of Enterococcus faecium promoted, but its ability to convert inorganic calcium into organic calcium was also enhanced, while its antioxidant activity was improved. This is of great significance for the development of novel calcium supplements.

[0008] Our research group previously studied Enterococcus faecalis KQ2.6 ( Enterococcus faecium The antibacterial activity and safety of the KQ2.6 fermentation broth were evaluated. The supernatant from the Enterococcus faecalis KQ2.6 fermentation was found to have a broad antibacterial spectrum, inhibiting not only Gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, Streptococcus pyogenes, and Staphylococcus epidermidis, but also Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella paratyphi B. Furthermore, Enterococcus faecalis KQ2.6 exhibited good safety profile. Although the strain was resistant to vancomycin, it was sensitive to common antibiotics such as penicillin, chloramphenicol, tetracycline, ofloxacin, erythromycin, rifampin, and ciprofloxacin, and was not a multidrug-resistant strain. Therefore, studying the calcium-rich physiological metabolic activity and antioxidant capacity of Enterococcus faecalis for the development of novel calcium supplements holds considerable promise.

[0009] On one hand, the present invention provides a method for preparing a calcium-rich product, wherein the product is obtained by fermentation with Enterococcus faecalis, and the fermentation culture medium of Enterococcus faecalis contains calcium salts.

[0010] This invention has demonstrated that Enterococcus faecalis has a certain calcium enrichment capacity. During fermentation culture, it can convert calcium ions absorbed from the fermentation medium into bio-organic calcium, thereby effectively increasing the calcium content in the fermentation product. Moreover, the calcium in the product is combined with natural proteins / polysaccharides, resulting in higher bioavailability.

[0011] The Enterococcus faecalis KQ2.6 used in this invention is from the College of Life and Environmental Sciences, Hangzhou Normal University, with the strain preservation number CCTCC NO: M2014197.

[0012] Furthermore, the fermentation medium for the fermentation culture includes a carbon source, a nitrogen source, and a calcium salt. The carbon source includes any one or more of sucrose, lactose, glucose, maltose, and fructose. The nitrogen source includes any one or more of yeast extract, peptone, and ammonium chloride. The calcium salt includes any one or more of calcium carbonate, calcium phosphate, and calcium chloride.

[0013] This invention investigates the effects of calcium enrichment on the physiological metabolism of Enterococcus faecalis and optimizes calcium-enriched culture media to obtain the optimal conditions for calcium enrichment in Enterococcus faecalis and the influence of calcium enrichment on the nutrient composition of the bacteria, thereby further improving the calcium enrichment effect of Enterococcus faecalis.

[0014] It is understandable that when the carbon source is any one or more of sucrose, lactose, glucose, maltose, and fructose, the nitrogen source is any one or more of yeast extract, peptone, and ammonium chloride, and the calcium salt is any one or more of calcium carbonate, calcium phosphate, and calcium chloride, it can be used to prepare calcium-rich products by Enterococcus faecalis fermentation. Although different components may have some differences in calcium enrichment effect, it is undeniable that all of them can be used.

[0015] Furthermore, the fermentation medium includes MRS medium, sucrose, yeast extract, lysine, and calcium chloride.

[0016] The invention initially used MRS medium as the fermentation medium. In order to improve the calcium enrichment effect of Enterococcus faecalis, the MRS medium was optimized.

[0017] Extensive research has demonstrated that the composition of fermentation media, including the types of nitrogen and carbon sources, as well as other inorganic salts and trace elements, significantly affects the growth and calcium enrichment capacity of *Enterococcus faecium*. Some components promote strain growth but reduce calcium accumulation, while others may inhibit calcium ion absorption. This invention, through optimization of the fermentation media composition, found that adding sucrose as a carbon source, yeast extract as a nitrogen source, calcium chloride as an inorganic calcium salt, and lysine to the MRS medium effectively improves the stability and efficiency of calcium ion accumulation in *Enterococcus faecium*, enhances the strain's antioxidant activity, and reduces costs.

[0018] Furthermore, the Enterococcus faecalis needs to be subjected to ultrasonic treatment during the fermentation process.

[0019] When ultrasound acts on microorganisms, it produces a series of biophysical effects, such as ultrasonic cavitation, mechanical effects, and thermal effects. Cavitation caused by ultrasound treatment of a certain intensity can induce reversible and temporary membrane rupture, allowing for successful substance transport without causing permanent cell damage. The mechanical effect of ultrasound can create acoustic perforation, thereby altering cell membrane permeability. The thermal effect is the increase in temperature of biological tissues caused by ultrasound, resulting from the heat conversion of ultrasound energy through tissue absorption. As external stressors, these effects lead to a series of responses in microorganisms, thereby affecting their growth and metabolism.

[0020] Over long periods of evolution, microorganisms have gradually developed a complex signal transduction network capable of rapidly sensing and responding to stress signals, along with corresponding physiological, metabolic, and defense systems to maintain their life and continuous growth. Under stress conditions, cellular survival depends on the recognition, generation, and transmission of biological signals, gene expression, and metabolic regulation of external stimuli. Therefore, during microbial fermentation, ultrasonic stimulation can increase the fluid pressure on the surface of the microbial cell membrane, thereby increasing cell membrane permeability, accelerating the secretion rate of metabolites, and effectively shortening fermentation time while increasing the yield of beneficial metabolites.

[0021] This invention demonstrates that ultrasonic treatment during the fermentation of Enterococcus faecium can effectively increase the calcium content in its metabolites. This indicates that under the stress of ultrasonic cavitation, mechanical, and thermal effects, Enterococcus faecium can produce a rapid response, and its cells undergo corresponding metabolic regulation due to stress, which can help improve its calcium accumulation capacity.

[0022] Furthermore, the method includes the following steps: (1) Preparation of Enterococcus faecalis seed culture; (2) Enterococcus faecalis seed liquid is inoculated into the fermentation medium for fermentation culture, and ultrasonic treatment is performed during the fermentation process; the ultrasonic frequency of the ultrasonic treatment is 20~25KHZ, and the treatment time is 30~160s.

[0023] The intensity of ultrasound treatment needs to be controlled within an appropriate range. Excessive intensity may cause physical damage or alteration to the cell membrane structure of Enterococcus faecium, and may even lead to leakage of intracellular contents.

[0024] Furthermore, the fermentation time of the fermentation culture in step (2) is 24~36h, the fermentation temperature is 30~39℃, and the intervention time of the ultrasonic treatment begins when the fermentation time is 14~20h.

[0025] The intervention time, frequency, and duration of ultrasonic treatment all affect the fermentation process and calcium accumulation of Enterococcus faecium. It is understandable that an ultrasonic frequency of 20–50 kHz, a treatment time of 30–160 seconds, and intervention starting 14–20 hours into fermentation can all improve the calcium accumulation capacity of Enterococcus faecium.

[0026] In some methods, the power of the ultrasonic treatment is 30~100W / L. Preferably, the power of the ultrasonic treatment is 75W / L.

[0027] In some embodiments, the present invention optimizes the conditions to determine the optimal conditions for ultrasonic treatment: the ultrasonic treatment frequency is 25 kHz, the treatment time is 60 s, and the intervention time begins after 16 h of fermentation.

[0028] In some methods, the shaking speed of the fermentation culture is 160 r / min, the fermentation time is 24 h, and the fermentation temperature is 37 °C.

[0029] Furthermore, the seed culture medium for Enterococcus faecalis in step (1) includes MRS medium; the inoculation amount of Enterococcus faecalis seed liquid in step (2) is 1~5%.

[0030] In some methods, the Enterococcus faecalis seed culture is a logarithmic phase (logarithmic phase 4-12h) seed culture, and the inoculation amount is 2%.

[0031] On the other hand, the present invention provides a calcium-rich product, wherein the coating product is obtained by the preparation method described above.

[0032] In another aspect, the present invention provides the use of a fermentation medium for preparing a reagent that enhances the calcium accumulation effect and / or antioxidant activity of Enterococcus faecalis, wherein the fermentation medium comprises MRS medium, sucrose, yeast extract, lysine and calcium chloride.

[0033] Furthermore, the antioxidant activity includes any one or more of total reducing power, DPPH free radical scavenging ability, and ABTS scavenging ability.

[0034] In another aspect, the present invention provides the use of a calcium-rich environment in improving the antioxidant activity of Enterococcus faecalis fermentation broth, wherein the calcium-rich environment refers to the fermentation medium of Enterococcus faecalis containing calcium salts.

[0035] In another aspect, the present invention provides the use of Enterococcus faecium in the preparation of calcium-rich products, wherein the calcium-rich products are obtained by the preparation method described above.

[0036] In another aspect, the present invention provides the use of ultrasonic treatment to improve the calcium enrichment effect during the fermentation of Enterococcus faecalis.

[0037] The beneficial effects of this invention are as follows: (1) Enterococcus faecalis was first discovered to have a good calcium accumulation capacity; (2) It was found that when fermented and metabolized in a calcium-rich environment, the soluble protein and fat content of Enterococcus faecalis decreased, but the antioxidant indicators such as total reducing power, DPPH free radical scavenging ability and ABTS scavenging rate were improved. Moreover, the addition of calcium ions did not damage the bacterial cell membrane. (3) Ultrasonic treatment during the fermentation of Enterococcus faecalis can significantly improve the calcium enrichment effect of Enterococcus faecalis; (4) The frequency, duration and intervention time of ultrasound treatment were optimized; (5) The fermentation medium formula of Enterococcus faecalis was optimized, including the types of carbon source and nitrogen source, and the addition of lysine, to further improve the calcium enrichment effect of Enterococcus faecalis. (7) It can not only significantly increase the calcium content in the metabolites of Enterococcus faecalis, but also enhance antioxidant activity, providing theoretical support for the calcium enrichment mechanism of Enterococcus faecalis and pointing out a new direction for the efficient utilization of the strain, effectively promoting the commercialization process of microbial calcium supplements. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the changes in lipid content in bacterial cells under different culture conditions in Example 2; Figure 2 The pH change curves of the strain cultured for 24 h under calcium-rich and non-calcium-rich conditions in Example 2 are shown. Figure 3 This is a schematic diagram showing the detection results of organic calcium conversion rate of the strain after calcium enrichment culture in Example 2; Figure 4 This is a schematic diagram showing the changes in the total reducing power of the strain under different culture conditions in Example 2; Figure 5 This is a schematic diagram showing the changes in DPPH scavenging rate of the strain under different culture conditions in Example 2; Figure 6 This is a schematic diagram showing the changes in ABTS clearance rate of the strain under different culture conditions in Example 2; Figure 7 The images shown are morphological photographs of the strains observed under calcium-rich and non-calcium-rich culture conditions using scanning electron microscopy in Example 2, where (A) is the uncovered culture and (B) is the (calcium-rich) culture. Figure 8 This is a schematic diagram illustrating the effect of nitrogen source type on calcium accumulation and calcium enrichment rate of the strain in Example 3. Figure 9 This is a schematic diagram illustrating the effect of nitrogen source concentration on the calcium accumulation and calcium enrichment rate of the strain in Example 3. Figure 10This is a schematic diagram illustrating the effect of carbon source type on the calcium accumulation and calcium enrichment rate of the strain in Example 4. Figure 11 This is a schematic diagram illustrating the effect of carbon source concentration on the calcium enrichment amount and calcium enrichment rate of the strain in Example 4. Detailed Implementation

[0039] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0040] Example 1: Method for preparing calcium-enriched products by fermentation of Enterococcus faecalis The *Enterococcus faecalis* KQ2.6 strain provided in this embodiment was obtained from the College of Life and Environmental Sciences, Hangzhou Normal University, and was deposited at the China Center for Type Culture Collection (CCTCC) on May 12, 2014, with the strain accession number CCTCC NO: M2014197. The method for preparing calcium-enriched products using this strain through fermentation is as follows: 1. Preparation of seed solution; The seed culture medium (MRS medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd.) was filled into 500 mL Erlenmeyer flasks, 100 mL per flask. After autoclaving, 10 mL of bacterial solution (prepared from MRS medium and incubated at 26℃ for 7 days until the bacteria covered the slant) was added to each Erlenmeyer flask. The flasks were then placed on a shaker at 28℃ and 160 r / min for 10 h.

[0041] 2. Enterococcus faecalis fermentation and ultrasonic treatment Preparation of fermentation medium: MRS was used as the basal medium, with the addition of 10g sucrose, 40g yeast extract (purchased from Beijing Aoboxing Biotechnology Co., Ltd.), 0.10g lysine, and 1.0g calcium chloride. The mixture was thoroughly mixed to prepare 1L of fermentation medium. The liquid fermentation medium was dispensed into 500mL Erlenmeyer flasks, 100mL per flask. After autoclaving, 2.4mL of seed culture was added to each flask. The flasks were then placed on a shaker at 37℃ and 160r / min for fermentation. After 16 hours of fermentation, ultrasonic cell disruptors were used for ultrasonic irradiation. The ultrasonic probe was placed 1cm below the surface of the fermentation broth to obtain a stable ultrasonic irradiation intensity. The ultrasonic frequency was 25kHz, the treatment time was 60s, and the power was 30W / L. After treatment, the flasks were incubated on a shaker for another 24 hours.

[0042] 3. Collect fermentation products After 24 hours of fermentation, the Enterococcus faecalis fermentation broth that had reached the stable fermentation period was taken out and subjected to freeze centrifugation at 3000 r / min for 10 min. The supernatant was discarded and 6.5 g of bacterial cells were collected.

[0043] Example 2: The effect of calcium-rich environment on the fermentation process of Enterococcus faecalis This embodiment follows the method provided in Example 1 to ferment and culture Enterococcus faecalis. The fermentation process did not involve ultrasonic treatment. The study investigated the soluble protein content, fat content, antioxidant capacity, and cell membrane surface structure changes of the strains obtained under calcium-rich (1.0 g / L calcium chloride in the culture medium) and non-calcium-rich (no 1.0 g / L calcium chloride added to the culture medium, other components unchanged) conditions. The organic calcium conversion rate and acid production capacity of the strains under calcium-rich culture were also analyzed. Data were analyzed using EXCEL and SPASS, and graphs and trend lines were created. Each experiment was repeated three times, and the mean and standard deviation were calculated.

[0044] 1. Changes in soluble protein content in bacterial cells before and after calcium enrichment A protein concentration standard curve was prepared using bovine serum albumin (BSA) solution. 1.0 mL and 3.0 mL of Coomassie Brilliant Blue G250 staining solution were respectively transferred to BSA solutions (0, 20, 40, 60, 80, 100 μg / mL), vortexed, and incubated at room temperature for 5 min. The absorbance value at 595 nm was then recorded. A standard curve was plotted and fitted using a linear regression equation. The standard curve equation was y = 135.42x - 4.2365, with a correlation coefficient R0. 2 =0.993, a high correlation, suitable for subsequent experiments to determine protein content. 0.10 g of bacterial cells obtained from calcium-rich and uncovered environments (using the culture medium as in Example 1) were added to 3.0 mL of distilled water and a small amount of quartz sand, and ground into a homogenate in an ice bath. The homogenate was extracted by sonication and centrifuged at 4°C for 15 min to obtain a crude extract. 50 μL of the supernatant was added to 950 μL of distilled water and 3.0 mL of Coomassie Brilliant Blue G250 staining solution, shaken well, and incubated at room temperature for 5 min. The absorbance at 595 nm was read, and the soluble protein content was calculated based on the standard curve equation. The results are shown in Table 1.

[0045] Table 1. Changes in soluble protein content in bacterial cells before and after calcium enrichment.

[0046] Table 1 shows that the protein content in bacteria cultured without calcium enrichment was 0.57 mg / mL and in calcium enrichment culture was 0.42 mg / mL, respectively. The soluble protein content of Enterococcus faecalis KQ2.6 decreased after calcium enrichment culture. These results indicate that the addition of a certain concentration of exogenous calcium leads to a decrease in the soluble protein content in calcium-enriched bacteria, and excessively high intracellular calcium content inhibits protein synthesis.

[0047] Furthermore, previous calcium ion concentration experiments demonstrated in this embodiment that the highest calcium enrichment rate was achieved in *Enterococcus faecalis* KQ2.6 culture with exogenous calcium ion concentration of 1.0 mg / mL. This calcium concentration may have inhibited the production of soluble proteins, thus leading to a decrease in protein content after calcium-enriched culture. Additionally, calcium ion enrichment may have altered the protein structure, reducing protein solubility and promoting an increase in poorly soluble proteins.

[0048] 2. Changes in bacterial cell fat content before and after calcium enrichment This example investigated two fermentation media: 1) the original MRS medium; 2) an optimized medium (Example 1) consisting of MRS medium supplemented with sucrose, yeast extract, and lysine. Both calcium-enriched and non-calcium-enriched conditions were investigated for these two media. Fermentation was carried out according to the method provided in the example. 0.10 g of cells were collected and added to 3.0 mL of solvent (solvent: anhydrous ethanol to n-hexane, volume ratio 1:2). After evaporating the solvent to dryness, 2.0 mL of 98% sulfuric acid was added, mixed, and placed in a 90 °C water bath for 20 min. The mixture was then cooled to room temperature in an ice-water bath, and 3.0 mL of 0.25 g / L vanillin-phosphate solution was added (0.25 g vanillin was dissolved in a small amount of anhydrous ethanol and then diluted to 1000 mL with 34% phosphoric acid). After shaking, the mixture was reacted at room temperature for 20 min, and the OD was read. 368 Values ​​were determined. A triglyceride standard curve was plotted, and the total lipid content of the strain was measured.

[0049] The standard curve is plotted as y = 0.9615x + 0.1033, where R0 2 >0.99. The total lipid content of each strain in the fermentation broth was determined by the vanillin phosphate colorimetric method, and the results are as follows. Figure 1 As shown, the lipid production of the cells in the optimized fermentation medium is significantly greater than that in the original MRS culture, indicating that the optimized medium is more conducive to the growth of the strain. It can also be seen that the lipid production capacity of the strain decreases after calcium addition. It is speculated that at this calcium concentration, the strain can absorb and accumulate calcium ions, but it is not conducive to lipid production; the absorbed calcium ions may inhibit related metabolic pathways.

[0050] 3. Changes in the acid-producing capacity of strains before and after calcium enrichment The initial pH values ​​of the calcium-enriched and non-calcium-enriched media for Enterococcus faecalis were 4.97 and 5.19, respectively, indicating that the addition of calcium ions had a certain impact on the initial pH of the medium. However, the pH values ​​were not significantly different, and the initial pH was not adjusted during the experiment to avoid introducing the influence of other elements. After 24 h of culture, the pH values ​​of the calcium-enriched and non-calcium-enriched media were 3.92 and 3.00, respectively. Figure 2 The pH of the non-calcium-enriched medium was lower, suggesting a higher acid production. Figure 2 It can also be seen that the pH value of the non-calcium-enriched medium decreased significantly during the culture process, reaching the same level as the calcium-enriched medium at about 4 hours of culture. Afterward, the pH value of the non-calcium-enriched medium was significantly lower than that of the calcium-enriched medium. This may indicate that the non-calcium-enriched medium is more conducive to the production of acidic substances by Enterococcus faecium, while the addition of calcium ions may have affected the metabolic processes of the strain, leading to a decrease in its ability to produce acidic products.

[0051] 4. Determination of organic calcium conversion rate of strains after calcium-enriched culture Based on the principle that organic calcium is readily soluble in water while inorganic calcium has poor solubility, the static leaching method was used to determine the organic calcium content in mycelia. Enterococci cultured to the stable fermentation phase (24 h) were removed, centrifuged at 3000 r / min for 10 min, the supernatant was discarded, and 0.10 g of mycelia were collected. 20 mL of deionized water was added, and the mycelia were soaked for 3 h, 6 h, 12 h, 18 h, and 24 h, respectively. After centrifugation at 3000 r / min for 10 min, the supernatant was filtered, and the organic calcium content in the supernatant was determined by EDTA titration. The results are as follows: Figure 3 As shown.

[0052] Depend on Figure 3 It can be seen that the organic calcium conversion rate of the strain gradually increases with the increase of soaking time. At this time, the intracellular organic calcium dissolves in water and flows out of the cell. However, when the static leaching time is 18-24 h, the organic calcium conversion rate is stable at about 75.5%, indicating that the organic calcium has been fully leached out at this time. This shows that the strain has good organic calcium conversion efficiency and stability.

[0053] This embodiment found that Enterococcus faecalis KQ2.6 exhibited good organic calcium conversion ability in a calcium-rich environment (exogenous calcium source added at 1.0 mg / mL), with a stable conversion rate at a high level. At the same time, the organic calcium in the bacterial cells of this strain could be fully leached out in a short time, showing high conversion efficiency and rapid responsiveness. It can be seen that Enterococcus faecalis KQ2.6 has the potential to become a new calcium supplement strain.

[0054] 5. Determination of the antioxidant capacity of the strain before and after calcium enrichment 5.1 Determination of Total Reducing Power Detection method: Prepare test solutions with different concentration gradients. Take 1.0 mL of each solution into a test tube, and add 2.5 mL of 0.20 mol / L pH 6.6 phosphate buffer (PBS) and 2.5 mL of 1.0% potassium ferricyanide solution sequentially. Mix well, incubate at 50℃ for 20 min, then remove and cool rapidly. Stop the reaction by adding 2.5 mL of 10% trichloroacetic acid solution, mix well, and centrifuge at 3000 r / min for 10 min. Take 2.5 mL of the supernatant, and add 2.5 mL of distilled water and 0.50 mL of 0.10% ferric chloride solution sequentially. Mix thoroughly, let stand for 10 min, and measure the OD value at 700 nm, using anhydrous ethanol as a reference and 0.1% vitamin C solution as a positive control.

[0055] This embodiment evaluated the total reducing power of *Enterococcus faecalis* KQ 2.6 supernatant fermentation broth before and after culture medium optimization, and before and after calcium enrichment. Simultaneously, to eliminate the influence of the culture medium's own components, the inherent reducing power of the corresponding culture medium was also measured in the experiment, ensuring that the observed changes in total reducing power originated from changes in culture medium conditions. Experimental results are shown in Tables 2 and 3. Figure 4 As shown in the table, Table 2 shows the reducing power of the culture medium itself (without bacteria), and Table 3 shows the total reducing power after bacteria are added.

[0056] Table 2. Total reducing power of different culture medium components (without bacteria)

[0057] Table 3. Total reducing power of strains under different culture medium components (with added bacteria)

[0058] Tables 2 and 3 show that the total reducing power of the original MRS medium before and after calcium addition was 0.028 (2.590-2.562) and 0.061 (2.487-2.426), respectively, with a significant increase after calcium addition. The total reducing power of the optimized medium was 0.115 (0.952-0.837) and 0.406 (1.012-0.606), respectively. This indicates that the supernatant exhibited a certain reducing capacity under different medium component conditions, with the total reducing power of the optimized medium being significantly higher than that of the original MRS medium. This significant improvement may be due to the optimized medium improving the content and type of carbon and nitrogen sources, providing richer and more suitable nutritional conditions for Enterococcus faecalis KQ2.6, and promoting its metabolic activities. Simultaneously, the new nitrogen source may also serve as a precursor for reducing substances, thereby increasing the yield of reducing substances. Meanwhile, the calcium... 2+ It plays a mediating role in signal transduction, and its concentration can affect cell proliferation and differentiation. Furthermore, the calcium in the bacterial cytoplasm... 2+Increased concentration can enhance bacterial resistance to metabolic toxins. Optimizing the culture medium by adding calcium ions, due to the different new carbon source metabolites, has varying effects on calcium ion channels, enhancing the key role of calcium ions in signal transduction and metabolic pathways, promoting bacterial growth and metabolism, increasing stress resistance, and increasing the production of reducing substances.

[0059] 5.2 Determination of DPPH free radical scavenging ability Detection method: Prepare a 0.08 mmol / L DPPH-ethanol solution using anhydrous ethanol and store it in the dark for later use. Take 2.0 mL of sample solution and 2.0 mL of DPPH-ethanol solution for each concentration gradient into test tubes, mix well, and let stand at room temperature in the dark for 30 min. Measure the OD value at a wavelength of 517 nm. Simultaneously measure the OD values ​​of the 2.0 mL sample solution and 2.0 mL anhydrous ethanol mixture, as well as the OD values ​​of the 2.0 mL DPPH solution and 2.0 mL anhydrous ethanol mixture. Use anhydrous ethanol as a reference and vitamin C as a positive control. Perform three parallel measurements and take the average value. Calculate the clearance rate using the following formula: DPPH removal rate (%) = [1 - (Ai - Aj) / Ao] × 100% Where: Ai—OD value of 2mL DPPH-ethanol solution + 2mL sample solution; Aj—OD value of 2mL anhydrous ethanol + 2mL sample solution; Ao—OD value of 2mL DPPH-ethanol solution + 2mL anhydrous ethanol.

[0060] The test results are shown in Tables 4 and 5. Figure 5 As shown.

[0061] Table 4. DPPH scavenging ability of different culture medium components (without bacteria)

[0062] Table 5. DPPH scavenging ability of strains under different culture medium components (with added bacteria)

[0063] As shown in Tables 4 and 5, all culture media have a certain ability to scavenge DPPH. The optimized culture medium has a higher ability than the original MRS, and the ability to scavenge DPPH increases after calcium is added. It can be seen that a calcium-rich environment helps to improve the ability to scavenge DPPH.

[0064] Comparing the original MRS fermentation broth group and the optimized fermentation broth group, except for the optimized culture medium group, the DPPH clearance rate of the fermentation broth after cultivation in each group was actually lower than that of the original culture medium. This shows that the optimized fermentation medium can improve the DPPH clearance ability of Enterococcus faecalis KQ2.6.

[0065] The reason for this might be that *Enterococcus faecalis* KQ2.6 consumes and synthesizes reducing substances during its growth and development. If the consumption exceeds the production, the clearance rate of the fermentation broth will decrease compared to the culture medium itself. The optimized culture medium, due to changes in nitrogen and carbon sources and different types of enzymes utilizing nutrients, can alter metabolic pathways, thereby reducing the consumption of reducing substances and ultimately increasing the DPPH free radical scavenging rate. Simultaneously, the carbon source fermentation products can open bacterial calcium ion channels and activate the calcium ion efflux mechanism. Meanwhile, Ca... 2+ It plays a mediating role in signal transduction, and its concentration can affect cell proliferation and differentiation. Furthermore, the calcium in the bacterial cytoplasm... 2+ Increased concentration can enhance the bacteria's resistance to metabolic toxins. Therefore, the addition of calcium ions, due to their role in bacterial signal transduction, promotes bacterial proliferation and metabolism, enhances their resistance, increases the synthesis of reducing substances, and increases the DPPH free radical scavenging rate.

[0066] 5.3 Determination of ABTS free radical scavenging ability Detection Method: Accurately weigh and prepare equal volumes of 7.4 mmol / L ABTS solution and 2.6 mmol / L K₂S₂O₈ solution. Mix the two solutions and let them stand at room temperature for 12 hours in the dark. This stock solution is used as the working solution. Dilute the stock solution 40-50 times with anhydrous ethanol until the absorbance at 734 nm is 0.7 ± 0.02. Take 0.60 mL of sample solution for each concentration gradient and 2.4 mL of the prepared working solution in a test tube. Shake thoroughly and let stand for 6 minutes. Measure the absorbance at 734 nm (Ai). The absorbance obtained by replacing the sample solution with an equal volume of anhydrous ethanol is Ao, and the absorbance obtained by replacing the working solution with an equal volume of anhydrous ethanol is Aj. Perform three parallel measurements and take the average value. Calculate the clearance rate using the following formula: ABTS clearance rate (%) = [1 - (Ai - Aj) / Ao] × 100% Where: Ai—OD value of the experimental group, Aj—background OD value of the sample solution measured when distilled water replaces hydrogen peroxide, and Ao—OD value of the blank control group.

[0067] The test results are shown in Tables 6 and 7. Figure 6 As shown.

[0068] Table 6. ABTS scavenging ability of different culture medium components (without bacterial addition)

[0069] Table 7. ABTS scavenging ability of strains under different culture medium components (with added bacteria)

[0070] Tables 6 and 7 are included. Figure 6 The unoptimized MRS medium fermentation broth showed an ABTS clearance rate of 86.98%. After medium optimization, the clearance rate increased to 90.07%. This improvement in clearance rate is likely due to changes in the types and concentrations of nitrogen and carbon sources in the medium, leading to alterations in the enzyme activity of related substrates, which in turn caused adjustments in metabolic pathways, thereby enhancing the production of reducing substances and ultimately promoting the increase in ABTS clearance.

[0071] Meanwhile, experimental results showed that the ABTS scavenging rate increased in both the MRS fermentation broth and the optimized fermentation broth under calcium-rich conditions. This is speculated to be because the carbon source fermentation products can open bacterial calcium ion channels and activate calcium ion flow mechanisms. (The last sentence appears to be incomplete and possibly refers to Ca...) 2+ It plays a mediating role in signal transduction, and its concentration can affect cell proliferation and differentiation. Furthermore, the calcium in the bacterial cytoplasm... 2+ Increased concentration can enhance the resistance of bacteria to metabolic toxins. Therefore, under calcium-rich conditions, the metabolism of Enterococcus faecalis KQ2.6 is enhanced, the synthesis of reducing substances increases, and the ABTS clearance rate increases.

[0072] Furthermore, the experiment revealed that, except for the optimized calcium-enriched group, the ABTS scavenging rate of the fermentation broths in the other groups was lower than that of the corresponding culture media. However, this reduction decreased under calcium-enriched conditions or after culture media optimization. It is speculated that this trend occurs because microorganisms simultaneously consume and produce reducing substances in the culture medium during their growth and metabolism, and under calcium-enriched or optimized conditions, the cells can produce more reducing substances.

[0073] 6. Observation of cell membrane surface structure before and after calcium enrichment The cell membrane surface structure of the strains before and after calcium enrichment was observed using scanning electron microscopy (SEM) to analyze the changes in the cell membrane surface under calcium ion stress, helping to understand the effect of calcium ions on the growth status of the strains and the mechanism of calcium accumulation in the strains. The results are as follows: Figure 7 As shown.

[0074] Depend on Figure 7 It can be seen that Enterococcus faecalis KQ2.6 appears as a spherical or nearly spherical shape under scanning electron microscopy in the absence of calcium enrichment culture, while the strain cultured in calcium enrichment culture appears as an ellipsoid under scanning electron microscopy. The morphology of the strain is longer and the protrusions are sharper than those of the strain cultured in calcium enrichment culture. This indicates that a large number of calcium ions may have accumulated on the surface of the strain after calcium enrichment culture, which led to the change in its morphology.

[0075] It can also be seen that when cultured at a calcium ion concentration of 1.0 mg / mL, the cell membrane surface of the strain did not break or shrink, indicating that calcium ions at this concentration do not damage the cell membrane. This shows that calcium ions can attach to the cell membrane surface, promote the absorption of calcium ions by the cell, and thus affect its physiological metabolic activities.

[0076] Example 3: Optimization of Nitrogen Source In this embodiment, Enterococcus faecalis was fermented and cultured according to the method provided in Example 1. The fermentation process was not subjected to ultrasonic treatment. The original MRS was used as the base medium, and 25.0 g / L of peptone, tryptone, yeast powder, yeast extract or ammonium chloride (see Table 8) were added respectively. Then, 1.0 mg / mL of anhydrous calcium chloride was added respectively, and the fermentation culture was carried out according to the method provided in Example 1. Centrifuge 20 mL of fermentation broth, collect the bacterial cells, weigh them, and determine the organic calcium content in the cells using EDTA titration. Calculate the calcium enrichment rate (the ratio of inorganic calcium to organic calcium, specifically calculated as follows: accurately weigh 0.20 g of dry bacterial cells, pour into a digestion flask, add 10 mL of mixed acid (nitric acid:perchloric acid = 4:1), place on a muffle furnace, and digest for approximately 30 min until clear. Cool. Transfer the solution to a 50 mL volumetric flask and dilute to volume with distilled water. Determine the concentration of organic calcium in the cells according to the standard curve method, convert it to calcium ion content, and divide the calcium ion content of organic calcium in the cells by the calcium ion content of inorganic calcium to determine the optimal nitrogen source. The bacterial cell mass is shown in Table 8, and the calcium enrichment amount and calcium enrichment rate are shown in [Table 8]. Figure 8 .

[0077] Table 8. Effects of nitrogen source type on bacterial growth

[0078] As shown in Table 8, different nitrogen sources have a slight impact on the growth of the strains, with yeast extract, yeast extract and ammonium chloride being the preferred sources.

[0079] Depend on Figure 8 It is evident that the type of nitrogen source significantly affects the calcium accumulation capacity of Enterococcus faecalis KQ2.6. When yeast extract, beef extract, or tryptone were used as nitrogen sources, the calcium accumulation capacity of the strain decreased significantly compared to the original MRS medium, but the cell mass did not decrease; in fact, the cell mass of strains cultured with yeast extract as the nitrogen source was significantly increased. This indicates that these three nitrogen sources are beneficial to the growth of the strain, but reduce the calcium accumulation. This may be because these three nitrogen sources are complex nitrogen sources, containing other components that inhibit the absorption of calcium ions by the strain.

[0080] When using peptone, yeast extract, and ammonium chloride as nitrogen sources, the strains all exhibited high calcium accumulation, with yeast extract and ammonium chloride showing the most significant increases. However, ammonium chloride, as an inorganic nitrogen source, is a readily available nitrogen source in fermentation production, but its composition is relatively simple, and it may produce certain toxins during fermentation. Yeast extract, as an organic nitrogen source, has a more abundant composition, containing substances beneficial to strain growth, resulting in higher utilization and more stable strain growth. Therefore, to ensure stable production processes, high calcium accumulation, and good microbial growth, yeast extract was used as the nitrogen source for optimizing the culture medium.

[0081] Nitrogen source concentrations of 20, 25, 35, 40, 45, 50, 55, and 60 g / L were set for the addition of yeast extract. Cell mass was measured using the experimental method described above to determine the optimal nitrogen source concentration. It was found that the highest cell growth was achieved when the yeast extract concentration was 40 g / L, reaching 0.1261 g (20 mL of fermentation broth).

[0082] The calcium enrichment rate was also tested when yeast extract was added at concentrations of 35, 40, 45, 50, 55, and 60 g / L. The results are as follows: Figure 9 As shown, the lowest calcium enrichment rate was observed in the cells at a nitrogen source concentration of 3.0%. The calcium enrichment rate then increased with increasing nitrogen source concentration, reaching a peak at 40 g / L, and subsequently decreased slowly with further increases in nitrogen source concentration. This indicates that at a yeast extract concentration of 40 g / L, the cells can effectively enrich calcium ions, achieving a calcium enrichment amount of 76 mg / g and a calcium enrichment rate of 49.8%.

[0083] Example 4: Optimization of Carbon Source This embodiment follows the method provided in Example 1 for the fermentation culture of Enterococcus faecalis. The fermentation process did not involve ultrasonic treatment. MRS culture medium supplemented with 40 g / L yeast extract was used as the basal medium, with 10.0 g / L of glucose, sucrose, fructose, maltose, and lactose added respectively. Anhydrous calcium chloride was then added to each medium, and fermentation was carried out according to the method provided in Example 1. 20 mL of fermentation broth was centrifuged, and the bacterial cells were collected, weighed, and the organic calcium content in the bacterial cells was determined by EDTA titration. The calcium enrichment rate (the proportion of inorganic calcium converted to organic calcium) was calculated to determine the optimal nitrogen source. The bacterial cell mass is shown in Table 9, and the calcium enrichment amount and calcium enrichment rate are shown in Table 9. Figure 10 .

[0084] Table 9. Effect of carbon source type on bacterial growth

[0085] As shown in Table 9, different carbon sources have a slight impact on the growth of the strain, with glucose, sucrose, and lactose being the preferred sources.

[0086] Depend on Figure 10 It was found that different carbon sources significantly affected the calcium enrichment capacity of *Enterococcus faecalis* KQ2.6. Strains using glucose, sucrose, and maltose as carbon sources exhibited high calcium accumulation, while strains using sucrose and lactose as carbon sources showed high calcium enrichment rates. The influence of the carbon source on fermentation mainly depends on its properties, i.e., whether it is a rapidly utilized or slowly utilized carbon source. Slower utilization by the cells is beneficial for prolonging the synthesis time of metabolites. Based on the experimental results, it is hypothesized that sucrose and lactose, as slowly utilized carbon sources, are beneficial for the accumulation of calcium ions by the cells. Due to the higher price of lactose, sucrose was chosen as the primary carbon source.

[0087] Sucrose concentrations of 5, 10, 15, 20, 25, and 30 g / L were set, and cell mass and calcium enrichment rate were measured using the above experimental method to determine the optimal carbon source concentration. It was found that the highest cell growth was achieved when 15 g / L of sucrose was added. However, the calcium enrichment and calcium enrichment rate of the cells reached their peak at a carbon source concentration of 10 g / L, indicating that at a carbon source concentration of 10 g / L, the cells could effectively enrich calcium ions and grow well, making it more suitable for cell growth and calcium enrichment. The calcium enrichment reached 102 mg / g, and the calcium enrichment rate reached 65.7%.

[0088] Example 5: Effect of ultrasonic treatment on the calcium enrichment capacity of Enterococcus faecalis 1. Effects of ultrasonic treatment This embodiment uses the method provided in Example 1 to ferment Enterococcus faecalis. MRS culture medium supplemented with 40 g / L yeast extract and 10 g / L sucrose was used as the basal medium. The following conditions were investigated: 1. No sonication treatment; 2. Sonication treatment (Example 1). The enrichment of soluble protein, fat, and calcium in the fermented cells after fermentation, as well as the total reducing power, DPPH scavenging rate, and ABTS scavenging rate of the fermentation broth, were investigated. The detection methods are as shown in Example 2, and the detection results are shown in Table 10.

[0089] Table 10. Effects of Ultrasonic Treatment

[0090] As shown in Table 10, after ultrasonic treatment, the fermentation products of *Enterococcus faecium* showed a significant increase in soluble protein and calcium accumulation. While there was no significant increase in fat content and antioxidant activity, there was also no inhibitory effect. This indicates that under the stress of ultrasonic treatment, *Enterococcus faecium* undergoes corresponding metabolic regulation, with a significant promoting effect on calcium accumulation.

[0091] 2. Optimization of ultrasound conditions This embodiment also optimized the conditions of ultrasonic treatment by a single factor. The ultrasonic treatment frequencies of 20, 25, 30, 35, 40, 45 and 50 kHz were compared, while other conditions remained unchanged as in Example 1. The calcium enrichment and calcium enrichment rate in the prepared bacterial cells were examined, and the optimal ultrasonic treatment frequency was found to be 25 kHz.

[0092] The calcium enrichment and calcium enrichment rate in the prepared bacterial cells were examined by comparing ultrasonic treatment times of 20, 30, 40, 50, 60, 90, 120, and 160 seconds, while keeping other conditions unchanged as in Example 1. The optimal ultrasonic treatment time was found to be 60 seconds.

[0093] The calcium enrichment and calcium enrichment rate in the prepared cells were examined when the ultrasonic intervention time was 5, 8, 12, 14, 16, 18, 20, and 22 hours of fermentation, while other conditions remained unchanged as in Example 1. The optimal ultrasonic intervention time was found to be 16 hours of fermentation.

[0094] Example 5: Effect of adding lysine to the fermentation medium This embodiment uses the method provided in Example 1 to ferment Enterococcus faecalis. The fermentation media were investigated as follows: 1. MRS-based medium with 40 g / L yeast extract and 10 g / L sucrose added; 2. MRS-based medium with 40 g / L yeast extract, 10 g / L sucrose, and 0.10 g nicotinamide added; 3. MRS-based medium with 40 g / L yeast extract, 10 g / L sucrose, and 0.10 g glycine added; 4. MRS-based medium with 40 g / L yeast extract, 10 g / L sucrose, and 0.10 g lysine added; 5. MRS-based medium with 40 g / L yeast extract, 10 g / L sucrose, 0.10 g nicotinamide, and 0.10 g lysine added. All other conditions were the same as in Example 1. The enrichment of soluble protein, fat, and calcium in the fermented cells under the four conditions was detected using the method described in Example 2, and the results are shown in Table 11.

[0095] Table 11. Effects of different fermentation media

[0096] Table 11 shows that, when combined with ultrasonic treatment, adding lysine to the fermentation medium for *Enterococcus faecalis* further enhances its calcium accumulation capacity. Moreover, compared to other methods such as adding nicotinamide, glycine, or a combination of nicotinamide and lysine, only adding lysine alone significantly increases calcium accumulation without noticeably affecting the soluble protein and fat content in the cells. Therefore, the preferred fermentation medium includes MRS medium, yeast extract, sucrose, and lysine.

[0097] Example 6: Screening of Calcium Salt Types This embodiment uses the method provided in Example 1 to ferment Enterococcus faecalis, combined with ultrasonic treatment. The fermentation medium includes MRS medium, yeast extract, sucrose and lysine, and calcium carbonate, calcium phosphate or calcium chloride are added as calcium salts respectively. The effects of different calcium salts on the soluble protein, fat and calcium enrichment and calcium enrichment rate of the fermented cells are investigated. The test results are shown in Table 12.

[0098] Table 12. Screening of calcium salts

[0099] Table 12 shows that the addition of different inorganic calcium salts to the fermentation medium also affects the calcium accumulation effect of Enterococcus faecium. The addition of calcium carbonate or calcium phosphate significantly reduces the soluble protein and fat content in the bacteria, and also decreases the calcium accumulation, which may have an adverse effect on the metabolism of Enterococcus faecium. The optimal inorganic calcium salt is calcium chloride, which is more conducive to the growth of Enterococcus faecium and also more conducive to calcium accumulation.

[0100] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of producing a calcium enriched product, characterized by, The fermentation is carried out by using Enterococcus faecium, and the fermentation medium of the Enterococcus faecium contains calcium salt.

2. The production method according to claim 1, wherein The fermentation medium of the fermentation culture contains carbon source, nitrogen source and calcium salt, the carbon source contains any one or more of sucrose, lactose, glucose, maltose and fructose; the nitrogen source contains any one or more of yeast extract, peptone and ammonium chloride; and the calcium salt contains any one or more of calcium carbonate, calcium phosphate and calcium chloride.

3. The production method according to claim 2, wherein The fermentation medium contains MRS medium, sucrose, yeast extract, lysine and calcium chloride.

4. The production method according to claim 1, wherein The Enterococcus faecium needs to be subjected to ultrasonic treatment during the fermentation.

5. The production method according to claim 4, wherein The method comprises the following steps: Preparation of Enterococcus faecium seed liquid; The Enterococcus faecium seed liquid is inoculated into the fermentation medium for fermentation culture, and ultrasonic treatment is carried out during the fermentation; the ultrasonic frequency of the ultrasonic treatment is 20-50 kHz, and the treatment time is 30-160 s.

6. The production method according to claim 5, wherein The fermentation time of the fermentation culture in step (2) is 24-36 h, the fermentation temperature is 30-39℃, and the intervention time of the ultrasonic treatment is 14-20 h.

7. The production method according to claim 5, wherein The seed culture medium of the Enterococcus faecium in step (1) contains MRS medium; and the inoculation amount of the Enterococcus faecium seed liquid in step (2) is 1-5%.

8. A calcium enriched product, characterized in that, The method is obtained by using any one of claims 1-7.

9. Use of a fermentation medium for the preparation of an agent that enhances the calcium enrichment effect and / or the antioxidant activity of Enterococcus faecium, characterized in that, The fermentation medium contains MRS medium, sucrose, yeast extract, lysine and calcium chloride.

10. Use of a calcium-rich environment for increasing the antioxidant activity in Enterococcus faecium fermentation broth, characterized in that, The calcium-rich environment refers to that the fermentation medium of the Enterococcus faecium contains calcium salt. The fermentation is carried out by using Enterococcus faecium, and the fermentation medium of the Enterococcus faecium contains calcium salt. The fermentation medium of the fermentation culture contains carbon source, nitrogen source and calcium salt, the carbon source contains any one or more of sucrose, lactose, glucose, maltose and fructose; the nitrogen source contains any one or more of yeast extract, peptone and ammonium chloride; and the calcium salt contains any one or more of calcium carbonate, calcium phosphate and calcium chloride. The fermentation medium contains MRS medium, sucrose, yeast extract, lysine and calcium chloride. The Enterococcus faecium needs to be subjected to ultrasonic treatment during the fermentation. The method comprises the following steps: Preparation of Enterococcus faecium seed liquid; The Enterococcus faecium seed liquid is inoculated into the fermentation medium for fermentation culture, and ultrasonic treatment is carried out during the fermentation; the ultrasonic frequency of the ultrasonic treatment is 20-50 kHz, and the treatment time is 30-160 s. The fermentation time of the fermentation culture in step (2) is 24-36 h, the fermentation temperature is 30-39℃, and the intervention time of the ultrasonic treatment is 14-20 h. The seed culture medium of the Enterococcus faecium in step (1) contains MRS medium; and the inoculation amount of the Enterococcus faecium seed liquid in step (2) is 1-5%. The method is obtained by using any one of claims 1-7. The fermentation medium contains MRS medium, sucrose, yeast extract, lysine and calcium chloride. The calcium-rich environment refers to that the fermentation medium of the Enterococcus faecium contains calcium salt.

Images