A high-density selenium-rich fermentation method of lactobacillus plantarum HEW-A490

Abstract

The application relates to the technical field of microbial fermentation and functional probiotic preparation, and discloses a high-density selenium-rich fermentation method of lactobacillus plantarum HEW-A490, which comprises the following steps: after lactobacillus plantarum HEW-A490 is activated and seed cultured, the lactobacillus plantarum HEW-A490 is inoculated into a fermentation medium to carry out fermentation culture; the fermentation medium comprises double carbon sources composed of glucose and fructo-oligosaccharide and a composite nitrogen source composed of yeast extract powder and soybean peptone; during the fermentation process, the pH of the fermentation system is kept constant, glucose supplement liquid is added according to the residual sugar content in the fermentation liquor, and sodium selenite supplement liquid is supplemented to the fermentation system, and selenium-rich lactobacillus plantarum fermentation liquor is obtained after the fermentation is completed. The application aims to solve the problem that in the existing selenium-rich fermentation process of lactobacillus plantarum, high-density proliferation of the bacterial body and inorganic selenium conversion are difficult to be coordinated.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and functional probiotic preparation technology, specifically a high-density selenium-enriched fermentation method for Lactobacillus plantarum HEW-A490. Background Technology

[0002] Selenium is one of the trace elements required for the body to maintain normal physiological metabolism, and it can usually be supplemented through inorganic selenium preparations, organic selenium preparations, or selenium-enriched foods. With the development of probiotic products and selenium-enriched functional foods, the technology of using microorganisms such as lactic acid bacteria as carriers to enrich selenium has gradually attracted attention. Lactobacillus plantarum has a good foundation for food applications, and its fermentation culture usually depends on the control of carbon source, nitrogen source, inorganic salts, and culture conditions. In the process of selenium-enriched culture, inorganic selenium sources such as sodium selenite are generally added to the fermentation system, so that the bacteria can absorb or convert selenium during growth and metabolism.

[0003] In the existing Lactobacillus plantarum selenium-enriched fermentation process, the matching between the cell proliferation process and the selenium enrichment culture process is insufficient, making it difficult for the fermentation system to simultaneously ensure the stable progress of both the cell culture state and the selenium source conversion process. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of the difficulty in coordinating high-density cell proliferation and inorganic selenium conversion in the existing Lactobacillus plantarum selenium-enriched fermentation process, and to propose a high-density selenium-enriched fermentation method for Lactobacillus plantarum HEW-A490.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A high-density selenium-enriched fermentation method for Lactobacillus plantarum HEW-A490 includes the following steps: Lactobacillus plantarum HEW-A490 was activated and seed cultured before being inoculated into a fermentation medium for fermentation culture. The fermentation medium includes a dual carbon source consisting of glucose and fructooligosaccharides, and a complex nitrogen source consisting of yeast extract and soybean peptone. During fermentation, the pH of the fermentation system was kept constant, and glucose feed solution was added according to the residual sugar content in the fermentation broth. At the same time, sodium selenite feed solution was added to the fermentation system. After fermentation, selenium-enriched Lactobacillus plantarum fermentation broth was obtained.

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, the aforementioned Lactobacillus plantarum HEW-A490 was deposited at the China General Microbiological Culture Collection Center on May 27, 2016, and its classification name is Lactobacillus plantarum, with accession number CGMCC No.12554.

[0008] Furthermore, in the fermentation medium, glucose serves as a usable carbon source in the early stage of fermentation, fructooligosaccharides serve as a synergistic carbon source during the fermentation process, yeast extract serves as an organic nitrogen source and a source of growth factors, and soybean peptone serves as a source of peptides and amino acids.

[0009] Furthermore, the fermentation medium also includes inorganic salts, buffer components, and surfactant components; The inorganic salts include sodium chloride, magnesium sulfate, and manganese sulfate; the buffer components include calcium carbonate and diammonium hydrogen citrate; and the surfactant components include Tween-80.

[0010] Furthermore, in the fermentation medium, the mass ratio of glucose to fructooligosaccharides is 1.5 to 2.5:1, and the mass ratio of yeast extract to soybean peptone is 2 to 4:1. The dual carbon source and the composite nitrogen source together constitute a carbon and nitrogen nutrition system suitable for the fermentation culture of Lactobacillus plantarum HEW-A490.

[0011] Furthermore, the fermentation medium comprises, by mass / volume: 20–40 g / L glucose, 10–20 g / L fructooligosaccharides, 10–20 g / L yeast extract, 2.0–6.0 g / L soybean peptone, 1.0–4.0 g / L sodium chloride, 0.5–2.0 g / L magnesium sulfate, 0.1–0.5 g / L manganese sulfate, 1.0–3.0 g / L calcium carbonate, 2.0–4.0 g / L diammonium hydrogen citrate, 0.5–1.5 g / L Tween-80, with the balance being water.

[0012] Furthermore, in the fermentation medium, glucose and fructooligosaccharides together constitute the carbon source system, yeast extract and soybean peptone together constitute the organic nitrogen source system, sodium chloride, magnesium sulfate and manganese sulfate together constitute the inorganic salt system, calcium carbonate and diammonium hydrogen citrate together constitute the buffer system, and Tween-80 serves as the surfactant component. The carbon source system, organic nitrogen source system, inorganic salt system, buffer system, and surfactant component are used together for the fermentation culture of Lactobacillus plantarum HEW-A490.

[0013] Furthermore, the glucose feed solution and the sodium selenite feed solution are added independently, wherein the flow rate of the glucose feed solution is adjusted according to the residual sugar content of the fermentation broth, and the sodium selenite feed solution is introduced into the fermentation system during the fermentation process according to the set feed concentration.

[0014] Furthermore, the glucose feed solution has a mass concentration of 50-70%, the sodium selenite feed solution has a concentration of 10-20 μg / mL, and the residual sugar content of the fermentation broth is periodically monitored during fermentation and controlled at 3-8 g / L.

[0015] Furthermore, the inoculum size for fermentation culture is 2-5% v / v, the fermentation temperature is 33-41℃, the stirring speed is 200-300 rpm, and the tank pressure is 0.03-0.04 MPa; the pH of the fermentation system is maintained at 5.0-7.0 by adding sodium hydroxide solution, and the fermentation broth of the selenium-enriched Lactobacillus plantarum is obtained after 24-48 hours of fermentation.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: This invention involves activating and seeding *Lactobacillus plantarum* HEW-A490, then inoculating it into a specific fermentation medium for fermentation. This ensures that the bacteria entering the fermentation stage are in a relatively consistent physiological state. The fermentation medium uses a dual carbon source system composed of glucose and fructooligosaccharides, combined with a complex nitrogen source system composed of yeast extract and soybean peptone. This ensures good continuity and compatibility of carbon and nitrogen supply in the fermentation system, providing a more coordinated nutritional basis for the proliferation and metabolism of *Lactobacillus plantarum* HEW-A490 during fermentation. This avoids fluctuations in the bacterial culture state caused by rapid consumption or a single nutrient source during fermentation.

[0017] This invention maintains a constant pH in the fermentation system during fermentation and adds glucose feed solution according to the residual sugar content in the fermentation broth, so that the carbon source replenishment corresponds to the actual consumption state of the cells, reducing the fluctuation of the culture environment caused by insufficient or accumulated carbon source during fermentation; at the same time, sodium selenite feed solution is added to the fermentation system so that the selenium source can enter the fermentation system and participate in the selenium enrichment process during cell culture.

[0018] This invention integrates the nutrient supply, pH control, carbon source feeding, and selenium source supplementation required for bacterial culture into the same fermentation process, so that the high-density culture process of Lactobacillus plantarum HEW-A490 and the selenium-enriched culture process can be carried out in the same system in a coordinated manner, which is beneficial to obtaining selenium-enriched Lactobacillus plantarum fermentation broth. Attached Figure Description

[0019] Figure 1 This is a graph showing the effect of different carbon sources on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention; Figure 2 This is a graph showing the effect of different concentrations of glucose and fructooligosaccharides on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 3This is a graph showing the effect of different mixing ratios of glucose and fructooligosaccharides as mixed carbon sources on the viable count of Lactobacillus plantarum HEW-A490. Figure 4 This is a graph showing the effect of different nitrogen sources on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention; Figure 5 This is a graph showing the effect of different concentrations of yeast extract and soybean peptone on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 6 This is a graph showing the effect of different mixing ratios of yeast extract and soybean peptone as a mixed nitrogen source on the viable count of Lactobacillus plantarum HEW-A490. Figure 7 This is a response surface plot showing the effect of glucose and fructooligosaccharides on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 8 This is a response surface plot showing the effect of glucose and yeast extract powder on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 9 This is a response surface plot showing the effect of fructooligosaccharides and yeast extract powder on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 10 This is a graph showing the effect of different fermentation temperatures on the viable count of Lactobacillus plantarum HEW-A490 in this invention; Figure 11 This is a graph showing the effect of different fermentation pH on the viable count of Lactobacillus plantarum HEW-A490 in this invention; Figure 12 This is a graph showing the effect of different inoculum amounts on the viable count of Lactobacillus plantarum HEW-A490 according to the present invention; Figure 13 This is a graph showing the effect of different sodium selenite solution concentrations on the viable count and selenium enrichment rate of Lactobacillus plantarum HEW-A490 according to the present invention. Figure 14 The figure shows the effect of different fermentation times on the viable count and selenium enrichment rate of Lactobacillus plantarum HEW-A490. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0021] The strain used in this invention is *Lactobacillus plantarum* HEW-A490, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 27, 2016. Its classification name is *Lactobacillus plantarum*, and its accession number is CGMCC No. 12554. The main reagents used include glucose, fructooligosaccharides, soybean peptone, yeast extract, and sodium selenite. The culture media used include MRS liquid medium, MRS solid medium, and the high-density fermentation medium described in this invention. The main instruments and equipment used include a 5L fermenter and a UV spectrophotometer.

[0022] Before high-density fermentation and selenium-enriched fermentation, *Lactobacillus plantarum* HEW-A490 underwent strain activation and seed culture preparation. Specifically, the *Lactobacillus plantarum* HEW-A490 glycerol suspension was inoculated onto MRS solid plates using the four-zone streak method and cultured at 37.0±0.5℃ for 48 h. After culture, a single independent colony was picked and inoculated into a shake flask containing 150 mL of *Lactobacillus plantarum* seed culture medium and cultured at 37.0±0.5℃ for 22 h to obtain the primary seed culture of *Lactobacillus plantarum* for later use. Through the above activation and seed culture, the cells entering the fermentation stage are in a relatively stable physiological state, which is beneficial for subsequent high-density culture and selenium-enriched culture.

[0023] To screen suitable carbon sources for the growth of *Lactobacillus plantarum* HEW-A490, a single-factor experiment was conducted to compare different carbon sources. MRS liquid medium was used as the control group. The variable was set as different carbon sources, and the medium was prepared while keeping other components and proportions of the medium constant. The viable cell count at the fermentation endpoint was used as the evaluation index. Figure 1 As shown, in the screening experiments of different carbon sources, when the carbon source is glucose and fructooligosaccharides, the viable count of Lactobacillus plantarum HEW-A490 is slightly higher than that of other carbon source groups. Considering the cost of large-scale industrial production, glucose and fructooligosaccharides were selected as suitable carbon sources for subsequent experiments.

[0024] Furthermore, using glucose and fructooligosaccharides as carbon sources respectively, and keeping other components of the culture medium constant, the effects of glucose and fructooligosaccharide concentrations on the viable count of *Lactobacillus plantarum* HEW-A490 were investigated. Figure 2 As shown, the viable count of *Lactobacillus plantarum* HEW-A490 was higher when the concentration of glucose and fructooligosaccharides was 45 g / L. Subsequently, using glucose and fructooligosaccharides as a mixed carbon source, with a total carbon source concentration of 45 g / L, the effect of the glucose and fructooligosaccharide mixing ratio on the viable count of *Lactobacillus plantarum* HEW-A490 was investigated, while keeping other components of the culture medium constant. Figure 3As shown, the highest viable count of Lactobacillus plantarum HEW-A490 was observed when the mass ratio of glucose to fructooligosaccharides was 2:1, i.e., when the glucose concentration was 30 g / L and the fructooligosaccharide concentration was 15 g / L.

[0025] To screen for a suitable nitrogen source for the growth of *Lactobacillus plantarum* HEW-A490, MRS liquid medium was used as the control group. Different nitrogen sources were set as variables, and the medium was prepared while keeping other components and proportions constant. The viable cell count at the fermentation endpoint was used as the evaluation index. Figure 4 As shown, in the screening experiments of different nitrogen sources, when the nitrogen source was yeast extract, soybean peptone, and beef extract, the viable count of Lactobacillus plantarum HEW-A490 was higher than that of other nitrogen source groups. Considering the cost of large-scale industrial production, yeast extract and soybean peptone were selected as suitable nitrogen sources for subsequent experiments.

[0026] Furthermore, using yeast extract and soybean peptone as nitrogen sources respectively, and keeping other components of the culture medium constant, the effects of yeast extract concentration and soybean peptone concentration on the viable count of *Lactobacillus plantarum* HEW-A490 were investigated. Figure 5 As shown, when the concentration of yeast extract and soybean peptone was 20 g / L, the viable count of *Lactobacillus plantarum* HEW-A490 was relatively high. Subsequently, using yeast extract and soybean peptone as a mixed nitrogen source, with a total nitrogen source concentration of 20 g / L, the effect of the mixing ratio of yeast extract and soybean peptone on the viable count of *Lactobacillus plantarum* HEW-A490 was investigated, while keeping other components of the culture medium constant. Figure 6 As shown, when the mass ratio of yeast extract to soybean peptone is 3:1, the number of viable Lactobacillus plantarum HEW-A490 is the highest, that is, the yeast extract concentration is 15 g / L and the soybean peptone concentration is 5.0 g / L.

[0027] Based on the screening results of the above culture medium components, glucose, fructooligosaccharides, and yeast extract were selected as the main factors to be investigated. A Box-Behnken response surface methodology was designed using Design-Expert 13.0 software, with viable cell count as the response value, to determine the appropriate proportions of the main components in the culture medium. The response surface methodology factors and levels are shown in Table 1 below.

[0028] Table 1. Response surface methodology factors and levels of major components of the culture medium -1 20 10 10 0 30 15 15 1 40 20 20 The Box-Behnken experimental design and results are shown in Table 2 below.

[0029] Table 2 Box-Behnken Experimental Design and Results 1 0.000 1.000 1.000 2.96E+10 2 0.000 1.000 -1.000 3.04E+10 3 0.000 -1.000 1.000 3.26E+10 4 -1.000 -1.000 0.000 2.61E+10 5 -1.000 0.000 -1.000 2.43E+10 6 1.000 0.000 1.000 2.12E+10 7 1.000 -1.000 0.000 2.35E+10 8 1.000 0.000 -1.000 2.39E+10 9 0.000 0.000 0.000 3.71E+10 10 0.000 -1.000 -1.000 3.44E+10 11 0.000 0.000 0.000 3.91E+10 12 -1.000 1.000 0.000 2.27E+10 13 -1.000 0.000 1.000 2.31E+10 14 1.000 1.000 0.000 2.07E+10 15 0.000 0.000 0.000 3.87E+10 16 0.000 0.000 0.000 3.66E+10 17 0.000 0.000 0.000 3.90E+10 The results of the analysis of variance for the regression model are shown in Table 3 below.

[0030] Table 3. Analysis of Variance for Regression Models Model 7.426E+20 9 8.252E+19 90.64 <0.0001 ** A 5.951E+18 1 5.951E+18 6.54 0.0377 * B 2.178E+19 1 2.178E+19 23.92 0.0018 ** C 5.281E+18 1 5.281E+18 5.80 0.0469 * AB 9.000E+16 1 9.000E+16 0.0989 0.7624 AC 5.625E+17 1 5.625E+17 0.6179 0.4576 BC 2.500E+17 1 2.500E+17 0.2746 0.6164 A² 5.801E+20 1 5.801E+20 637.20 <0.0001 ** B² 4.079E+19 1 4.079E+19 44.81 0.0003 ** C² 4.413E+19 1 4.413E+19 48.48 0.0002 residual 6.372E+18 7 9.104E+17 Missing item 9.525E+17 3 3.175E+17 0.2343 0.8685 Pure error 5.420E+18 4 1.355E+18 Total error 7.490E+20 16 The experimental data in Table 2 were fitted using Design-Expert 13.0 software through multiple regression. The resulting multiple quadratic regression equation between the response value (viable bacteria count Y) and glucose A, fructooligosaccharide B, and yeast extract C is as follows: Y=3.810E+10-8.625E+08A-1.650E+09B-8.125E+08C+1.500E+08AB-3.750E+08AC+2.500E+08BC-1.174E+10A²-3.112E+09B²-3.238E+09C².

[0031] Table 3 shows that fructooligosaccharide B, quadratic terms A², B², and C² have a highly significant effect on the viable count of *Lactobacillus plantarum* HEW-A490, while glucose A and yeast extract C have a significant effect. The interaction terms AB, AC, and BC have some but insignificant effect on the viable count of *Lactobacillus plantarum* HEW-A490. The model is highly significant overall, with no significant lack-of-fit terms. The model R² is 0.9915, and R²Adj is 0.9806, indicating that the regression equation fits the experimental results well.

[0032] like Figure 7 As shown, the amounts of glucose and fructooligosaccharides (FOS) affected the viable count of *Lactobacillus plantarum* HEW-A490. With increasing amounts of glucose and FOS, the viable count initially increased and then decreased, and the interaction between glucose and FOS was not significant. Figure 8 As shown, the amounts of both glucose and yeast extract affected the viable count of *Lactobacillus plantarum* HEW-A490. With increasing amounts of both, the viable count initially increased and then decreased, and the interaction between glucose and yeast extract was not significant. Figure 9 As shown, the amounts of both fructooligosaccharides and yeast extract affected the viable count of *Lactobacillus plantarum* HEW-A490. With increasing amounts of both, the viable count initially increased and then decreased, and the interaction between fructooligosaccharides and yeast extract was not significant. The response surface methodology predicted the main components of the fermentation medium to be glucose 29.64 g / L, fructooligosaccharides 13.65 g / L, and yeast extract 14.33 g / L, with a predicted maximum viable count of 3.84 × 10¹. 0 CFU / mL.

[0033] The high-density fermentation process of *Lactobacillus plantarum* HEW-A490 was optimized using a 5L fermenter, focusing on the effects of fermentation temperature, pH, and inoculum size on the fermentation results. The viable cell count at the fermentation endpoint was used as the indicator. During fermentation, a 25% sodium hydroxide solution was added to maintain a constant pH in the culture medium, the stirring rate was 250 r / min, and the fermenter pressure was 0.03–0.04 MPa.

[0034] like Figure 10 As shown, fermentation temperature is a crucial factor affecting the fermentation growth cycle of *Lactobacillus plantarum* HEW-A490. Within the range of 33–37℃, the number of viable fermentation cells increases with increasing temperature; above 37℃, the number of viable fermentation cells decreases. When the culture temperature is low, the strain needs time to adapt to the temperature after inoculation into the culture medium, resulting in a longer lag period and a prolonged fermentation cycle; excessively high temperatures are detrimental to cell growth. Therefore, the optimal fermentation temperature for *Lactobacillus plantarum* HEW-A490 is 37℃.

[0035] like Figure 11 As shown, different pH values ​​have varying effects on the growth of *Lactobacillus plantarum* HEW-A490. pH affects the lag phase and growth rate of the strain; both excessively high and low pH values ​​are detrimental to its growth. Single-factor experiments were conducted at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5. The results showed that the optimal pH for the growth of *Lactobacillus plantarum* HEW-A490 was 5.5.

[0036] like Figure 12 As shown, different inoculum sizes directly affect the high-density culture of *Lactobacillus plantarum* HEW-A490. Appropriately increasing the inoculum size is beneficial for establishing population dominance and shortening the lag phase, thereby shortening the fermentation cycle and reducing the possibility of contamination. However, excessive inoculum size leads to a large number of viable bacteria, and the accumulation of metabolites and nutrient deficiency in the later stages may cause premature aging and decreased viability. Single-factor experiments were conducted with inoculum sizes of 1%, 2%, 3%, 4%, and 5%. The results showed that the optimal inoculum size for *Lactobacillus plantarum* HEW-A490 was 4% (v / v).

[0037] To verify the reliability of the above-mentioned high-density fermentation method, *Lactobacillus plantarum* HEW-A490 was fermented using the optimized culture medium and fermentation process. Seed culture was inoculated into the fermentation medium at a volume fraction of 4% and cultured at a constant temperature of 37℃ and pH 5.5 for 18 hours. The culture was repeated three times, and the colony count was determined using the plate count method, yielding a result of 3.88 × 10¹. 0 CFU / mL, 3.78×10¹ 0 CFU / mL and 3.65×10¹ 0 CFU / mL, with an average value of 3.77 × 10¹ 0The CFU / mL concentration showed an error of 1.8% compared to the model's predicted optimal value, indicating that the method's results are reliable. The final optimized fermentation medium composition was: glucose 29.64 g / L, fructooligosaccharides 13.65 g / L, yeast extract 14.33 g / L, soybean peptone 5.0 g / L, sodium chloride 2.3 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.25 g / L, calcium carbonate 2.0 g / L, diammonium hydrogen citrate 3.0 g / L, Tween-80 1.0 g / L, with the remainder being water.

[0038] The selenium-enriched fermentation process of Lactobacillus plantarum HEW-A490 was optimized using a 5L fermenter. During the fermentation process, glucose solution and sodium selenite solution were added simultaneously to investigate the effects of different concentrations of sodium selenite solution and fermentation time on the selenium enrichment capacity of Lactobacillus plantarum HEW-A490. The viable cell count and selenium enrichment rate of the fermentation broth at the fermentation endpoint were used as the detection indicators.

[0039] like Figure 13 As shown, the concentration of sodium selenite solution affects the viable cell count and selenium enrichment rate of *Lactobacillus plantarum* HEW-A490; both excessively high and low concentrations are detrimental to obtaining optimal fermentation results. Single-factor experiments were conducted with sodium selenite solution concentrations of 10 μg / mL, 12 μg / mL, 14 μg / mL, 16 μg / mL, 18 μg / mL, and 20 μg / mL. The results showed that the optimal concentration of sodium selenite solution was 14 μg / mL.

[0040] like Figure 14 As shown, different fermentation times affect the selenium enrichment rate of *Lactobacillus plantarum* HEW-A490. Shorter fermentation times result in lower selenium enrichment rates, while longer fermentation times reduce fermentation efficiency. Single-factor experiments were conducted with fermentation times of 24h, 28h, 32h, 36h, 40h, 44h, and 48h. The results showed that the optimal selenium enrichment fermentation time was 36h.

[0041] The high-density fermentation process of *Lactobacillus plantarum* HEW-A490 and its selenium-enriched process were validated. The primary seed culture of *Lactobacillus plantarum* HEW-A490 was inoculated into the optimized fermentation medium at a volume fraction of 4%, with an initial pH of 7.0. Fermentation was carried out at a constant temperature of 37℃ and a rotation speed of 250 rpm. During fermentation, a 25% (v / v) sodium hydroxide solution was added to maintain the pH of the fermentation system at 5.5. Simultaneously, a 60% (w / w) glucose solution and a 14 μg / mL sodium selenite solution were added during fermentation. The residual sugar content of the fermentation broth was measured every 2 hours, and the glucose flow rate was adjusted according to the residual sugar content to maintain it at 5 g / L. Fermentation was carried out for 36 hours to obtain a selenium-enriched *Lactobacillus plantarum* fermentation broth. The viable cell count of the obtained fermentation broth was 3.03 × 10¹. 0 CFU / mL, selenium enrichment rate 85.32%.

[0042] The viable cell count of *Lactobacillus plantarum* HEW-A490 fermentation broth was determined using the plate count method. Specifically, the *Lactobacillus plantarum* HEW-A490 fermentation broth was serially diluted 10-fold with sterile physiological saline. After reaching the appropriate gradient, 1 mL of each dilution was added to a petri dish, and plate counting was performed on MRS agar medium. The petri dishes were incubated at 37℃ for 48 hours before data analysis.

[0043] The selenium enrichment rate of *Lactobacillus plantarum* HEW-A490 was calculated after detecting the selenium content using the DAB spectrophotometric method. The standard curve was plotted as follows: Accurately pipette 0 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of a 10 μg / mL selenium standard solution into 100 mL beakers, add water to a final volume of 35 mL, then add 1 mL of 5 g / 100 mL EDTA-2Na solution, shake well, and adjust the pH to 2–3 with 1:1 hydrochloric acid. Add 4 mL of 0.5% DAB solution to each beaker, shake well, and place in the dark for 30 min. Adjust the pH to neutral with 5% NaOH, add the mixture to a separatory funnel, add 10 mL of toluene, shake for 2 min, allow to separate into layers, remove the aqueous layer, collect the toluene layer in a cuvette, and measure the absorbance at 420 nm to plot the standard curve.

[0044] The preparation method for bacterial culture samples is as follows: Centrifuge the bacterial fermentation broth at 5000 r / min for 10 min, discard the supernatant, add 30 mL of double-distilled water, mix well, and centrifuge again at 5000 r / min for 10 min. Discard the supernatant. Repeat this washing process three times to remove selenium adhering to the cell surface. Finally, centrifuge at 5000 r / min for 20 min to obtain the final bacterial culture, dry at 65℃, and store in a sealed container.

[0045] The method for determining the selenium content in bacterial cultures is as follows: Weigh approximately 0.1 g of dried bacterial sample into a 10 mL test tube with a graduated glass stopper, add 0.6 mL of a mixed acid digestion solution (nitric acid-perchloric acid mixture, with a volume ratio of 5:1), and let stand overnight. Then, place the test tube in a 100°C water bath and heat for 4 hours. After the yellowish-brown gas in the test tube has dissipated, remove the test tube and cool it. Add 2 mL of 30% hydrogen peroxide solution and continue digesting in a boiling water bath until the digestion solution becomes colorless or pale yellow, indicating the endpoint has been reached. After cooling, dilute the test tube to 10 mL with ultrapure water and prepare for instrumental analysis. A blank control should be performed simultaneously. The selenium enrichment rate is calculated using the following formula: Selenium enrichment rate (%) = Intracellular selenium content (μg) / Total amount of selenium added (μg) × 100%.

[0046] In Example 1, a suspension of *Lactobacillus plantarum* HEW-A490 glycerol was inoculated onto an MRS solid plate using the four-zone streak method and cultured at 37.0±0.5℃ for 48 h. A single independent colony was then picked and inoculated into a shake flask containing 150 mL of *Lactobacillus plantarum* seed culture medium and cultured at 37.0±0.5℃ for 22 h to obtain primary seed culture of *Lactobacillus plantarum*. The obtained primary seed culture was used for subsequent high-density fermentation and selenium-enriched fermentation.

[0047] In Example 2, a high-density fermentation medium for *Lactobacillus plantarum* HEW-A490 was prepared. This fermentation medium included a dual carbon source composed of glucose and fructooligosaccharides, and a complex nitrogen source composed of yeast extract and soybean peptone, as well as inorganic salts, buffer components, and surfactants. The specific formulation was: glucose 29.64 g / L, fructooligosaccharides 13.65 g / L, yeast extract 14.33 g / L, soybean peptone 5.0 g / L, sodium chloride 2.3 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.25 g / L, calcium carbonate 2.0 g / L, diammonium hydrogen citrate 3.0 g / L, Tween-80 1.0 g / L, with the remainder being water. Among them, glucose serves as a usable carbon source in the early stage of fermentation, while fructooligosaccharides serve as a synergistic carbon source during the fermentation process; yeast extract serves as an organic nitrogen source and a source of growth factors, while soybean peptone serves as a source of peptides and amino acids; sodium chloride, magnesium sulfate, and manganese sulfate together constitute an inorganic salt system, calcium carbonate and diammonium hydrogen citrate together constitute a buffer system, and Tween-80 serves as a surface-active component.

[0048] In Example 3, the primary seed culture obtained in Example 1 was inoculated into the fermentation medium prepared in Example 2 at an inoculation rate of 4% (v / v), and high-density fermentation was carried out in a 5L fermenter. The initial fermentation pH was 7.0, the fermentation temperature was 37℃, the stirring rate was 250 r / min, and the tank pressure was 0.03–0.04 MPa. During fermentation, a 25% (v / v) sodium hydroxide solution was added to maintain the pH of the fermentation system at 5.5, and fermentation was carried out for 18 hours. After fermentation, the viable cell count was determined by plate counting, and the results of three replicates were 3.88 × 10¹. 0 CFU / mL, 3.78×10¹ 0 CFU / mL and 3.65×10¹ 0 CFU / mL, with an average value of 3.77 × 10¹ 0 CFU / mL.

[0049] In Example 4, the primary seed culture obtained in Example 1 was inoculated into the fermentation medium prepared in Example 2 at an inoculation rate of 4% (v / v), and selenium-enriched fermentation was carried out in a 5L fermenter. The initial fermentation pH was 7.0, the fermentation temperature was 37℃, and the stirring rate was 250 rpm. During fermentation, a 25% (v / v) sodium hydroxide solution was added to maintain the pH of the fermentation system at 5.5. Simultaneously, a 60% (w / w) glucose solution and a 14 μg / mL sodium selenite solution were added. The residual sugar content of the fermentation broth was measured every 2 hours, and the glucose flow rate was adjusted to maintain the residual sugar content at 5 g / L. Fermentation was carried out for 36 hours to obtain a selenium-enriched *Lactobacillus plantarum* fermentation broth. The test results showed that the viable count of the obtained fermentation broth was 3.03 × 10¹. 0 CFU / mL, selenium enrichment rate 85.32%.

[0050] In Example 5, selenium-enriched fermentation was carried out according to the method of Example 4, except that the concentration of sodium selenite solution was 10 μg / mL. During fermentation, a 60% glucose solution was still fed in, and the residual sugar content was measured every 2 hours and controlled at approximately 5 g / L. The fermentation time was 36 hours. This example illustrates that *Lactobacillus plantarum* HEW-A490 can be cultured in selenium-enriched form when the sodium selenite solution concentration is within the range specified in this invention.

[0051] In Example 6, selenium-enriched fermentation was carried out according to the method of Example 4, except that the concentration of sodium selenite solution was 20 μg / mL. A 60% glucose solution was still fed-batch during fermentation, and the residual sugar content was measured every 2 hours and controlled at approximately 5 g / L. The fermentation time was 36 hours. This example illustrates that *Lactobacillus plantarum* HEW-A490 can be cultured in selenium-enriched form when the sodium selenite solution concentration is within the range specified in this invention.

[0052] In Example 7, selenium-enriched fermentation was carried out according to the method of Example 4, except that the fermentation time was 24 hours. During fermentation, a 60% glucose solution and a 14 μg / mL sodium selenite solution were added. The residual sugar content was measured every 2 hours, and the glucose flow rate was adjusted to maintain the residual sugar content at approximately 5 g / L. This example corresponds to... Figure 14 This is one of the experimental points on the effect of fermentation time on the viable count and selenium enrichment rate of Lactobacillus plantarum HEW-A490.

[0053] In Example 8, selenium-enriched fermentation was carried out according to the method of Example 4, except that the fermentation time was 48 hours. During fermentation, a 60% glucose solution and a 14 μg / mL sodium selenite solution were added. The residual sugar content was measured every 2 hours, and the glucose flow rate was adjusted to maintain the residual sugar content at approximately 5 g / L. This example corresponds to... Figure 14This is one of the experimental points on the effect of fermentation time on the viable count and selenium enrichment rate of Lactobacillus plantarum HEW-A490.

[0054] In Example 9, a fermentation medium was prepared with the following composition: 20 g / L glucose, 10 g / L fructooligosaccharides, 10 g / L yeast extract, 5.0 g / L soybean peptone, 1.0 g / L sodium chloride, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1.0 g / L calcium carbonate, 2.0 g / L diammonium hydrogen citrate, 0.5 g / L Tween-80, and the remainder being water. The primary seed culture obtained in Example 1 was inoculated into this fermentation medium at a volume fraction of 2%. The fermentation temperature was 33°C, the stirring speed was 200 rpm, and the tank pressure was 0.03 MPa. The pH of the fermentation system was maintained at 5.0 by adding sodium hydroxide solution, and a 50% glucose solution was added to control the residual sugar content within the range of 3–8 g / L. Simultaneously, a 10 μg / mL sodium selenite solution was added. After 24 hours of fermentation, a selenium-enriched *Lactobacillus plantarum* fermentation broth was obtained. This embodiment is used to illustrate that the present invention is still feasible when some parameters are at low values.

[0055] In Example 10, a fermentation medium was prepared with the following composition: 40 g / L glucose, 20 g / L fructooligosaccharides, 20 g / L yeast extract, 5.0 g / L soybean peptone, 4.0 g / L sodium chloride, 2.0 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 3.0 g / L calcium carbonate, 4.0 g / L diammonium citrate, 1.5 g / L Tween-80, and the remainder being water. The primary seed culture obtained in Example 1 was inoculated into this fermentation medium at a volume fraction of 5%. The fermentation temperature was 41°C, the stirring speed was 300 rpm, and the tank pressure was 0.04 MPa. The pH of the fermentation system was maintained at 7.0 by adding sodium hydroxide solution, and a 70% glucose solution was added to control the residual sugar content within the range of 3–8 g / L. Simultaneously, a 20 μg / mL sodium selenite solution was added. After 48 hours of fermentation, a selenium-enriched *Lactobacillus plantarum* fermentation broth was obtained. This embodiment is used to illustrate that the present invention is still feasible when some parameters are at relatively high values.

[0056] In Comparative Example 1, the difference from the present invention is that the high-density fermentation medium was replaced with ordinary MRS broth medium, and 10 μg / mL sodium selenite was added to the medium. The results showed that the viable count of *Lactobacillus plantarum* HEW-A490 fermentation broth was 1.38 × 10⁻⁶. 9 The CFU / mL concentration and selenium enrichment rate were 34.35%. Compared with Example 4, the viable cell count and selenium enrichment rate of Comparative Example 1 were significantly reduced, indicating that the high-density fermentation medium and selenium-enriched fermentation process described in this invention can significantly increase the viable cell count and selenium enrichment rate of Lactobacillus plantarum HEW-A490 fermentation broth.

[0057] In Comparative Example 2, the difference from Example 4 is that the glucose flow rate was not adjusted according to the residual sugar content of the fermentation broth during fermentation. Instead, the corresponding glucose was added all at once at the initial stage of fermentation. The remaining culture medium composition, sodium selenite solution concentration, pH control method, fermentation temperature, and fermentation time were the same as in Example 4. This comparative example illustrates that when there is a lack of residual sugar feedback feeding, the carbon source supply is difficult to match with the cell consumption state, which may affect the stability of high-density culture and selenium-enriched culture of *Lactobacillus plantarum* HEW-A490.

[0058] In Comparative Example 3, the difference from Example 4 is that no 25% sodium hydroxide solution was added during fermentation to maintain a constant pH in the fermentation system. The remaining culture medium composition, inoculum size, fermentation temperature, glucose feeding method, sodium selenite feeding method, and fermentation time were the same as in Example 4. This comparative example illustrates that without constant pH control, the pH of the fermentation system may fluctuate with cell metabolism, thereby affecting the growth status of *Lactobacillus plantarum* HEW-A490 and the selenium-enriched culture process.

[0059] In Comparative Example 4, the difference from Example 4 is that the concentration of sodium selenite solution is higher than 20 μg / mL, while the other fermentation conditions are the same as in Example 4. This comparative example is used to illustrate that when the concentration of sodium selenite solution exceeds the limits defined in this invention, the excessively high concentration of selenium source may affect the viable count and selenium enrichment rate of *Lactobacillus plantarum* HEW-A490.

[0060] The examples and comparative examples are summarized in Table 4 below.

[0061] Example 1 Strain activation and preparation of primary seed culture MRS solid plates were incubated at 37.0±0.5℃ for 48 h; shake flasks were incubated at 37.0±0.5℃ for 22 h. Obtain first-grade seed liquid Example 2 Preparation of high-density fermentation medium Glucose 29.64 g / L, fructooligosaccharides 13.65 g / L, yeast extract 14.33 g / L, soybean peptone 5.0 g / L, etc. Provides a high-density fermentation nutrient base Example 3 High-density fermentation 4% inoculum, 37℃, pH 5.5, 250 rpm, 18 h <![CDATA[Average viable bacteria count is 3.77×10¹ 0 CFU / mL]]> Example 4 Selenium-enriched fermentation verification 60% glucose solution, 14 μg / mL sodium selenite, residual sugar 5 g / L, 36 h <![CDATA[The viable count is 3.03×10¹ 0 CFU / mL, and the selenium enrichment rate is 85.32%]]> Example 5 Sodium selenite 10 μg / mL The concentration of sodium selenite solution is within the range This indicates that the selenium source concentration range is feasible. Example 6 Sodium selenite 20 μg / mL The concentration of sodium selenite solution is within the range This indicates that the selenium source concentration range is feasible. Example 7 Ferment for 24 hours correspond Figure 14 Fermentation time test points This indicates that the lower limit of fermentation time is feasible. Example 8 Fermentation for 48 hours correspond Figure 14 Fermentation time test points This indicates that the upper limit of fermentation time is feasible. Example 9 Lower parameter combinations 33℃, 2% inoculum, pH 5.0, 50% glucose, 10 μg / mL sodium selenite Covering lower value parameters Example 10 Higher parameter combinations 41℃, 5% inoculum, pH 7.0, 70% glucose, 20 μg / mL sodium selenite Higher values ​​of the covered part Comparative Example 1 Selenium-enriched fermentation in ordinary MRS broth medium Replace the high-density fermentation medium and add 10 μg / mL sodium selenite. <![CDATA[The viable bacterial count is 1.38×10 9 CFU / mL, and the selenium enrichment rate is 34.35%]]> Comparative Example 2 Feedback on insufficient residual sugar Glucose flow rate not adjusted based on residual sugar content Explain the role of residual sugar feedback feeding. Comparative Example 3 Lack of constant pH control Maintaining pH constant without adding sodium hydroxide Explain the role of constant pH control Comparative Example 4 Sodium selenite concentration exceeded the range Sodium selenite solution concentration higher than 20 μg / mL This illustrates the importance of the range of selenium source concentrations. Verified by the above experiments and examples, this invention uses *Lactobacillus plantarum* HEW-A490 as the starting strain, which, after activation and seed culture, is inoculated into a specific fermentation medium for cultivation. This fermentation medium comprises a carbon source system of glucose and fructooligosaccharides, a nitrogen source system of yeast extract and soybean peptone, and is supplemented with inorganic salts such as sodium chloride, magnesium sulfate, and manganese sulfate, buffer components such as calcium carbonate and diammonium hydrogen citrate, and Tween-80. The synergistic effect of these components provides suitable nutrition and a suitable culture environment for the proliferation of *Lactobacillus plantarum* HEW-A490 and subsequent selenium-enriched culture.

[0062] In some embodiments, the mass ratio of glucose to fructooligosaccharides can be 1.5–2.5:1, and the mass ratio of yeast extract to soybean peptone can be 2–4:1. The fermentation medium, by mass / volume, may include: 20–40 g / L glucose, 10–20 g / L fructooligosaccharides, 10–20 g / L yeast extract, 2.0–6.0 g / L soybean peptone, 1.0–4.0 g / L sodium chloride, 0.5–2.0 g / L magnesium sulfate, 0.1–0.5 g / L manganese sulfate, 1.0–3.0 g / L calcium carbonate, 2.0–4.0 g / L diammonium hydrogen citrate, 0.5–1.5 g / L Tween-80, with the balance being water.

[0063] This invention maintains a constant pH in the fermentation system during fermentation and adds glucose feed solution according to the residual sugar content in the fermentation broth, while simultaneously adding sodium selenite feed solution. The glucose and sodium selenite feed solutions are added independently. The flow rate of the glucose feed solution is adjusted according to the residual sugar content of the fermentation broth, and the glucose feed solution concentration can be 50-70%. The sodium selenite feed solution is introduced into the fermentation system at a set concentration, which can be 10-20 μg / mL. The residual sugar content of the fermentation broth is periodically monitored during fermentation and can be controlled at 3-8 g / L. The inoculum size for fermentation is 2-5% v / v, the fermentation temperature is 33-41℃, the stirring speed is 200-300 rpm, and the tank pressure is 0.03-0.04 MPa. The pH of the fermentation system is maintained at 5.0-7.0 by adding sodium hydroxide solution, and after 24-48 hours of fermentation, a selenium-enriched *Lactobacillus plantarum* fermentation broth is obtained.

[0064] As shown in the comparative examples, when using ordinary MRS broth medium with added sodium selenite, the viable cell count and selenium enrichment rate of *Lactobacillus plantarum* HEW-A490 fermentation broth were significantly lower than those of the method of this invention. The lack of residual sugar feedback feeding, the lack of constant pH control, or the sodium selenite solution concentration exceeding the suitable range may all hinder the coordinated high-density culture and selenium-enriched culture of *Lactobacillus plantarum* HEW-A490. Therefore, this invention, through a combination of dual carbon sources, a complex nitrogen source, an inorganic salt system, a buffer system, surfactant components, constant pH control, residual sugar feedback glucose feeding, and sodium selenite feeding, enables *Lactobacillus plantarum* HEW-A490 to complete both high-density and selenium-enriched culture in the same fermentation system, thereby obtaining a selenium-enriched *Lactobacillus plantarum* fermentation broth.

Claims

1. A high-density selenium-enriched fermentation method for Lactobacillus plantarum HEW-A490, characterized in that, Includes the following steps: After activation and seed culture, Lactobacillus plantarum HEW-A490 was inoculated into a fermentation medium for fermentation culture. The fermentation medium includes a dual carbon source consisting of glucose and fructooligosaccharides, and a complex nitrogen source consisting of yeast extract and soybean peptone. During fermentation, the pH of the fermentation system was kept constant, and glucose feed solution was added according to the residual sugar content in the fermentation broth. At the same time, sodium selenite feed solution was added to the fermentation system. After fermentation, selenium-enriched Lactobacillus plantarum fermentation broth was obtained.

2. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 1, characterized in that, The *Lactobacillus plantarum* HEW-A490 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 27, 2016, and its classification name is *Lactobacillus plantarum*, with accession number CGMCC No. 12554.

3. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 1, characterized in that, In the fermentation medium, glucose serves as the available carbon source in the early stage of fermentation, fructooligosaccharides serve as a synergistic carbon source during the fermentation process, yeast extract serves as an organic nitrogen source and a source of growth factors, and soybean peptone serves as a source of peptides and amino acids.

4. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 3, characterized in that, The fermentation medium also includes inorganic salts, buffer components, and surfactant components; The inorganic salts include sodium chloride, magnesium sulfate, and manganese sulfate; the buffer components include calcium carbonate and diammonium hydrogen citrate; and the surfactant components include Tween-80.

5. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 4, characterized in that, In the fermentation medium, the mass ratio of glucose to fructooligosaccharides is 1.5 to 2.5:1, and the mass ratio of yeast extract to soybean peptone is 2 to 4:

1. The dual carbon source and the composite nitrogen source together constitute a carbon-nitrogen nutrition system suitable for the fermentation culture of Lactobacillus plantarum HEW-A490.

6. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 5, characterized in that, The fermentation medium comprises, by mass / volume: 20–40 g / L glucose, 10–20 g / L fructooligosaccharides, 10–20 g / L yeast extract, 2.0–6.0 g / L soybean peptone, 1.0–4.0 g / L sodium chloride, 0.5–2.0 g / L magnesium sulfate, 0.1–0.5 g / L manganese sulfate, 1.0–3.0 g / L calcium carbonate, 2.0–4.0 g / L diammonium hydrogen citrate, 0.5–1.5 g / L Tween-80, with the balance being water.

7. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 6, characterized in that, In the fermentation medium, glucose and fructooligosaccharides together constitute the carbon source system, yeast extract and soybean peptone together constitute the organic nitrogen source system, sodium chloride, magnesium sulfate and manganese sulfate together constitute the inorganic salt system, calcium carbonate and diammonium hydrogen citrate together constitute the buffer system, and Tween-80 is used as the surfactant component. The carbon source system, organic nitrogen source system, inorganic salt system, buffer system and surfactant component are used together for the fermentation culture of Lactobacillus plantarum HEW-A490.

8. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 1, characterized in that, The glucose feed solution and the sodium selenite feed solution are added independently. The flow rate of the glucose feed solution is adjusted according to the residual sugar content of the fermentation broth, and the sodium selenite feed solution is introduced into the fermentation system during the fermentation process according to the set feed concentration.

9. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 8, characterized in that, The glucose feed solution has a mass concentration of 50-70%, and the sodium selenite feed solution has a concentration of 10-20 μg / mL. The residual sugar content of the fermentation broth is periodically monitored during the fermentation process and controlled at 3-8 g / L.

10. The high-density selenium-enriched fermentation method for *Lactobacillus plantarum* HEW-A490 according to claim 1, characterized in that, The inoculum size for fermentation culture was 2-5% v / v, the fermentation temperature was 33-41℃, the stirring speed was 200-300 rpm, and the tank pressure was 0.03-0.04 MPa. The pH of the fermentation system was maintained at 5.0-7.0 by adding sodium hydroxide solution. After fermentation for 24-48 hours, the selenium-enriched Lactobacillus plantarum fermentation broth was obtained.

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