High-density fermentation method and application of lactobacillus reuteri

Abstract

The invention provides a lactobacillus reuteri high-density fermentation method and application thereof, and belongs to the technical field of microbial fermentation. According to the fermentation method provided by the invention, the heme, the vitamin K compound and the inducer are exogenously added, so that the problem of active oxygen toxicity associated with aerobic respiration is effectively reduced, carbon metabolic repression generated by glucose is avoided, and the utilization efficiency of sucrose in the fermentation process is remarkably improved; and the high-speed proliferation of the thalli is ensured, and meanwhile, the extremely high cell activity is kept. Experimental data show that the viable count of the fermentation liquor can break through 1 * 10 < 11 > CFU / mL only in 8-9 hours, and the viable count of a final product can be larger than or equal to 1 * 10 < 13 > CFU / mL within 10 hours. According to the technology, the seed propagation cost and the time cost are greatly reduced, the obtained thalli are high in mechanical strength and stress resistance, and a creative solution is provided for efficient industrial production of lactobacillus reuteri.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a high-density fermentation method and application of Lactobacillus reuteri. Background Technology

[0002] Probiotics, as live microorganisms, have significant effects in regulating intestinal flora balance, enhancing the body's immune function, and preventing and adjuvantly treating various diseases. They are widely used in functional foods, health products, feed additives, and clinical microecological preparations. With the rapid development of the global probiotic industry, the market demand for highly active, high-survival-rate probiotic preparations continues to grow. The average annual growth rate of the probiotic industry has been rising year by year, with high-density, high-stability probiotic preparations becoming a key direction for industry upgrading.

[0003] Lactobacillus reuteri ( Lactobacillus reuteri Lactobacillus reuteri is a beneficial bacterium in the gut microbiota, exerting its protective effect on the gut through the production of organic acids, inhibition of pro-inflammatory cytokine release, and enhancement of intestinal barrier function. Studies have shown that Lactobacillus reuteri has unique advantages in alleviating enteritis, promoting the development of the digestive system in infants and young children, and reducing symptoms of lactose intolerance, thus it is widely used in infant formula, functional foods, and veterinary probiotics. With increasing health demands and the popularization of precision nutrition concepts, the market demand for Lactobacillus reuteri in the food and pharmaceutical fields is growing daily.

[0004] Current research focuses on high-density cultivation of *Lactobacillus reuteri*. For example, Chinese patent CN201811398938.8 proposes a culture medium primarily composed of maltose, soybean peptone, and malt extract powder. While this can increase viable cell count by approximately 43 times and improve flavor, it still suffers from high cost and insufficient industrial-scale stability. Patent CN202510259813.0 emphasizes the toxin degradation function of the strain but does not optimize the high-density, rapid fermentation process. Other technologies, such as CN108048363A and CN107739747A, utilize *Lactobacillus reuteri* to produce reuterin or selenium-enriched products. Although functional, these technologies generally suffer from long fermentation cycles, numerous conditions requiring controlled cell activity, and reliance on freeze-drying to maintain activity, resulting in high energy consumption and costs.

[0005] To meet the rapidly growing market demand of the probiotic industry, there is an urgent need to develop a fermentation process for Lactobacillus reuteri that can achieve ultra-high-density proliferation in a short period of time. High-density, rapid fermentation can not only significantly improve output and economic benefits per unit time, but also provide a higher viable count base for downstream probiotic powder preparations, functional foods, and feed products, thereby improving product stability and functional effects. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by providing a high-density fermentation method and application for *Lactobacillus reuteri*. Using the high-density rapid fermentation method for *Lactobacillus reuteri* provided by this invention, a viable cell count of ≥1×10⁻⁶ can be achieved within ≤10 hours. 13 CFU / mL, cell dry weight ≥15 g / L. Compared with the existing technologies, this invention represents a breakthrough in both fermentation cycle and yield, significantly improving the feasibility of industrial application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a high-density fermentation method for Lactobacillus reuteri, comprising culturing Lactobacillus reuteri in a culture medium containing a respiratory activator under aerobic conditions; wherein the respiratory activator is composed of a heme source and a vitamin K compound, and the carbon source system of the culture medium is composed of a main carbon source and an inducer, wherein the main carbon source is sucrose and the inducer is fucose or rhamnose.

[0008] Furthermore, the heme source is selected from heme chloride, heme, and hemoglobin, and its concentration in the culture medium is 0.01~0.05g / L.

[0009] Furthermore, the vitamin K compounds are selected from vitamin K2 and vitamin K3, and their concentration in the culture medium is 0.01~0.05 g / L.

[0010] Furthermore, the concentration in the sucrose medium is 30-50 g / L.

[0011] Furthermore, the concentration of the inducing agent in the culture medium is maintained at 5-20 mM.

[0012] Furthermore, the culture medium also contains 10-15 g / L casein digest, 84-124 g / L yeast extract, 1-5 g / L triammonium citrate, 0.05-0.2 g / L cysteine, and Mn. 2+ 0.01~0.1 g / L, Tween 80 0.2~2 g / L.

[0013] Furthermore, during the fermentation process, the aeration rate is 0.1~0.3 vvm, the pH is controlled at 4.5~6.5, and a fed-batch feeding strategy is adopted to control the residual sugar concentration in the fermentation broth to be no less than 5 g / L.

[0014] Furthermore, the fermentation process progresses until the viable cell count is not less than 1×10⁻⁶ within 8-9 hours. 11 CFU / mL.

[0015] Furthermore, the viable cell count is not less than 1×10⁻⁶ within 9-10 hours of the fermentation process. 13CFU / mL, cell dry weight not less than 15 g / L.

[0016] The second objective of this invention is to provide the application of the above-described high-density fermentation method for Lactobacillus reuteri in the preparation of probiotic formulations, feed, functional foods, and pharmaceuticals.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) The high-density fermentation method of *Lactobacillus reuteri* provided by this invention effectively reduces the reactive oxygen species toxicity associated with aerobic respiration by exogenously adding heme, vitamin K compounds, and inducers, avoids the carbon metabolism inhibition caused by glucose production, and significantly improves its utilization efficiency of sucrose during fermentation, ensuring that the bacteria maintain extremely high cell viability while proliferating rapidly. Experimental data show that, within the same fermentation time, the OD value of *Lactobacillus reuteri* cultured by the fermentation method provided by this invention reaches 45, which is significantly higher than that of traditional anaerobic fermentation, and the biomass is increased by more than 3 times.

[0018] (2) The process provided by this invention successfully eliminates the growth lag phase under the condition that the inoculum amount is only 3%~8%, enabling the cells to proliferate rapidly at the maximum specific growth rate. Experimental data confirms that this process can achieve a viable cell count of over 1×10⁻⁶ in the fermentation broth in just 8~9 hours. 11 The efficiency is 3-4 times higher than that of traditional anaerobic fermentation, with a CFU / mL concentration; the final product can achieve a viable count of ≥1×10⁻⁶ within 10 hours. 13 CFU / mL. This technology not only significantly reduces the cost and time of seed culture, but also produces bacterial cells with high mechanical strength and strong resistance, providing a pioneering solution for the efficient industrial production of Lactobacillus reuteri. Attached Figure Description

[0019] Figure 1 The growth curve of Lactobacillus reuteri in Example 1 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0021] The terminology defined and explained in this invention.

[0022] The term "respiratory chain activator" refers to a group of exogenously added compounds that can compensate for the natural deficiencies in the heme and menadione biosynthetic pathways of *Lactobacillus reuteri*, enabling it to assemble functional cytochrome oxidases and electron transport chains. In this invention, it specifically refers to a combination of heme sources and vitamin K compounds.

[0023] The term "heme source" refers to any substance with heme groups provided by any microorganism, including but not limited to heme chloride, heme, hemoglobin, myoglobin, or enzymatically hydrolyzed animal blood.

[0024] The term "aerobic conditions" refers to the presence of oxygen as the final electron acceptor in the fermentation broth through the introduction of air or oxygen. In this invention, it is preferable to maintain a "microaerobic" state that supports respiratory metabolism while avoiding excessive oxidative damage by controlling the redox potential (ORP), which can be controlled between -250mV and -100mV.

[0025] The term "OD" 600 "600nm" refers to the absorbance value at 600nm, which is usually used to reflect the relative magnitude of cell concentration. In this invention, the cell density in the fermentation broth is expressed as OD. 600 Characterization: Due to the high turbidity of the fermentation broth under high-density culture conditions, which exceeded the linear measurement range of the spectrophotometer, the fermentation broth sample was appropriately diluted, and the optical density was measured at a wavelength of 600 nm. The equivalent OD of the original fermentation broth was then calculated based on the dilution factor. 600 Value. Under the implementation conditions of this invention, the dilution ratio is adjusted according to the concentration of the original fermentation broth, generally diluted 50 to 100 times, and the equivalent OD is obtained after conversion. 600 .

[0026] The term "DCW" refers to dry cell weight, measured in g / L, and is an indicator of the actual weight of the bacterial cells. In this invention, a drying method is used for measurement. A certain volume of fermentation broth sample is taken, and after centrifugation to collect the cells, they are washed with deionized water to remove culture medium residues. The resulting cells are then dried to constant weight under constant temperature conditions. Based on the mass difference before and after drying, combined with the sample volume, the dry cell weight (g / L) per unit volume of culture medium is calculated.

[0027] The term "viable cell count" refers to the number of microorganisms capable of proliferation under given culture conditions, usually expressed as CFU / mL. In this invention, the viable cell count is measured using the plate count method. Specifically, the fermentation broth sample is serially diluted, and an appropriate amount of the diluted solution is spread on the surface of a solid culture medium. The medium is then incubated at a suitable temperature until colonies form, and the viable cell count of the original fermentation broth is calculated based on the dilution factor.

[0028] Strain source: Lactobacillus reuteri PM25, classified and named Lactobacillus reuteriThe following was deposited on June 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35014; Lactobacillus reuteri CICC 6119 was purchased from the China Industrial Microbiological Culture Collection Center.

[0029] The MRS solid culture medium consists of the following components: 10.0 g protein powder, 5.0 g beef extract powder, 4.0 g yeast extract powder, 20.0 g glucose, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15.0 g agar powder, and 1000 mL distilled water. The final pH is 6.2 ± 0.2.

[0030] The culture medium for high-density fermentation of Lactobacillus reuteri provided by this invention has the following composition: casein digest 10-15 g / L, heme source 0.01-0.05 g / L, vitamin K compounds 0.01-0.05 g / L, yeast extract 84-124 g / L, sucrose medium concentration of 30-50 g / L, triammonium citrate 1-5 g / L, cysteine ​​0.05-0.2 g / L, Mn 2+ 0.01~0.1 g / L, Tween 80 0.2~2 g / L, and 1000 mL of distilled water. Final pH 5.2~6.4.

[0031] Strain activation: Lactobacillus reuteri PM25, frozen at -80℃, was streaked onto MRS solid medium and anaerobically cultured at 37℃ for 24-48 hours.

[0032] Primary seed (shake flask) preparation: Colonies are picked from the activated plates and inoculated into seed culture medium (the medium for high-density fermentation of Lactobacillus reuteri provided in this invention), and anaerobically cultured at 37°C for 12-18 hours until OD500 is reached. 600 The concentration is approximately 5.0~7.0, resulting in a first-grade seed solution.

[0033] Secondary seed culture (1L glass jar): Inoculate the above primary seed culture into a 1L seed jar containing sterile seed culture medium at an inoculation rate of 7%, at a temperature of 37°C, stirring at 100 rpm, without aeration, and incubate for 6-8 hours until OD reaches the target level. 600 The solution was approximately 18-24, yielding a secondary seed solution.

[0034] It should be noted that both the MRS medium and the seed medium mentioned above need to be aliquoted and autoclaved at 121℃ for 20 to 25 minutes, and then cooled for later use.

[0035] The embodiments provided by this invention use a 10 L stainless steel fermenter, with a maximum actual assembly capacity of approximately 7 L, equipped with a mechanical stirring paddle (up to 1000 rpm), and online pH, DO, and ORP electrodes.

[0036] The process of preparing the heme source stock solution is as follows: weigh the heme source and dissolve it in 0.2M NaOH solution to prepare a 10 g / L stock solution, and then filter it for sterilization.

[0037] The preparation process for vitamin K compounds involves weighing vitamin K2 or K3 and dissolving it in anhydrous ethanol to prepare a 10 g / L stock solution, which is then filtered and sterilized.

[0038] Sucrose, nitrogen source, and inorganic salts in the fermenter were sterilized at 121℃ for 20 min; the feed solution was sterilized at 115℃ for 20 min; heme, vitamin K, and inducers (such as fucose and rhamnose) were added aseptically after being sterilized by a 0.22μM filter membrane.

[0039] The present invention has now been generally described, and it will be more readily understood by referring to the following examples, which are provided by way of illustration and not by way of limitation. Examples 1-4 and Comparative Examples 1-5 used *Lactobacillus reuteri* PM25, and Example 5 used *Lactobacillus reuteri* CICC 6119.

[0040] Example 1 This embodiment provides a high-density fermentation method for Lactobacillus reuteri, as detailed below: Prepare the basal culture medium: casein digest 10 g / L, yeast extract 100 g / L, sucrose 30 g / L, triammonium citrate 2 g / L, cysteine ​​0.1 g / L, Mn 2+ After sterilization, heme, vitamin K2, and L-fucose were aseptically added to 0.05 g / L, 0.5 g / L, and 1000 mL of distilled water to achieve final concentrations of 0.005 g / L, 0.005 g / L, and 2 g / L, respectively.

[0041] Feed solution: contains 500 g / L sucrose and 4.0 g / L L-fucose.

[0042] Pre-fill the fermenter with 5 L of basal culture medium. After sterilization, aseptically add heme and vitamin K2 to a final concentration of 0.005 g / L.

[0043] Inoculation (0 h): Inoculate with 300 mL of secondary seed culture.

[0044] Fermentation 0-12 h: Temperature controlled at 37℃, pH adjusted to 5.5 with ammonia, air introduced during fermentation, stirring speed adjusted to 200-400 rpm, aeration rate of 0.2-0.3 vvm, ORP controlled at -150mV±10 mV, pulse feeding used to maintain residual sugar >5 g / L. At 8 h, OD 600 The value reached 37; at 9 h, OD 600 The value reached 44; at 10 h, OD 600 The value reached 45; at 11h, OD 600 The value reached 43; at 12 h, OD 600 The value reached 42.

[0045] Example 2 The procedure is basically the same as in Example 1, except that 400 mL of secondary seed solution is inoculated.

[0046] Example 3 The procedure is basically the same as in Example 1, except that 150 mL of secondary seed solution is inoculated.

[0047] Example 4 The method is basically the same as in Example 1, except that L-fucose is replaced with rhamnose.

[0048] Comparative Example 5 It is basically the same as Example 1, except that the strain used is Lactobacillus reuteri CICC 6119.

[0049] Comparative Example 1 It is basically the same as Example 1, except that pure nitrogen gas is introduced.

[0050] Comparative Example 2 It is basically the same as Example 1, except that sucrose is replaced with glucose.

[0051] Comparative Example 3 It is basically the same as Example 1, except that sucrose is replaced with 50% sucrose + 50% glucose.

[0052] Comparative Example 4 It is basically the same as Example 1, except that L-fucose is not added.

[0053] Comparative Example 5 It is basically the same as Example 1, except that no heme is added.

[0054] Monitor OD during fermentation for 8-12 hours 600 Cell count, cell dry weight, and viable cell count were determined by plate counting. The results are shown in Table 1.

[0055] Table 1. Fermentation statistics for each example and comparative example.

[0056] ; As shown in Table 1, comparing Example 1 with Comparative Examples 1 and 5, heme significantly increased biomass by up to 3 times and had the fastest start-up, reaching its peak at 9 hours, making it suitable for short-cycle production. Comparative Example 5, due to aeration but inability to perform respiratory metabolism, resulted in severe accumulation of reactive oxygen species, leading to the worst cell growth. Comparing Example 1 with Comparative Examples 2 and 4, the OD value of Example 1 reached its peak at 10 hours and remained relatively stable, with a slow decline in viable cell count. This indicates that the fermentation process of Example 1 effectively protects the cells, better utilizes sucrose, and promotes cell growth. Although Comparative Example 2 showed rapid growth in the first 8-9 hours, its OD value and viable cell count experienced a sharp decline after 10 hours. This was because glucose produced a carbon metabolism repression effect, causing severe oxidative lysis of the cells in an aerobic environment. Comparative Example 4, lacking fucose induction, still suffered oxidative damage despite the use of sucrose to relieve the repression, resulting in rapid cell death in the later stages. Example 4 used rhamnose instead of fucose, and the OD peak reached 43.0, close to that of Example 1, proving that rhamnose is also an effective inducer of oxidative damage. Comparing Examples 1 with Examples 2 and 3, after fermentation in the original MRS medium for 14-18 h in a fermenter, the maximum OD... 600 The value was 8.96, and the viable count was 10. 9 The CFU / mL concentration is far lower than that of the fermentation method provided by this invention. Comparing Examples 1 and 5, while *Lactobacillus reuteri* CICC 6119 can grow well using the fermentation system provided by this invention, the start-up is slow; it takes until 12 hours for its viable count to reach 2.3 × 10⁻⁶. 11 CFU / mL. The high-density fermentation method of Lactobacillus reuteri provided by this invention has universality and can increase the density of Lactobacillus reuteri from different sources. However, Lactobacillus reuteri PM25 can shorten the fermentation cycle by 2-3 hours and has a superior high-density fermentation capacity, which can significantly reduce energy consumption in actual industrial production.

[0057] Depend on Figure 1 It can be seen that when Lactobacillus reuteri PM25 was fermented using the fermentation process described in Example 1, the cells were adapting to the fermentation environment from 0 to 2 hours, and entered the logarithmic growth phase from 2 to 9 hours. OD 600 It rose rapidly, from about 5 to 44, and then entered a stable period in 9-12 hours. 600 Stable at around 42-45; compared with OD 600 The trend was consistent: as time progressed, the cell dry weight reached its peak at 9 hours, with a maximum of 15 g / L. During the rapid growth phase of Lactobacillus reuteri PM25, the pH value dropped significantly. Too low a pH value (down to around 4.3) would inhibit the growth of the bacteria.

[0058] For any points not covered above, existing technologies shall apply.

[0059] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A high density fermentation process of Lactobacillus reuteri, characterized in that, The method comprises culturing Lactobacillus reuteri under aerobic conditions using a culture medium containing a respiration activator consisting of a heme source and a vitamin K compound, and a carbon source system consisting of a main carbon source and an inducer, wherein the main carbon source is sucrose and the inducer is fucose or rhamnose.

2. The high density fermentation process of Lactobacillus reuteri according to claim 1, characterized in that, The heme source is selected from the group consisting of hemin chloride, hemin and hemoglobin, and the concentration thereof in the culture medium is 0.005-0.01 g / L.

3. The high density fermentation process of Lactobacillus reuteri according to claim 1, characterized in that, The vitamin K compound is selected from the group consisting of vitamin K2 and vitamin K3, and the concentration thereof in the culture medium is 0.005-0.01 g / L.

4. The high density fermentation process of Lactobacillus reuteri according to claim 1, characterized in that, The concentration of sucrose in the culture medium is 30-50 g / L.

5. The high density fermentation process of Lactobacillus reuteri according to claim 1, characterized in that, The concentration of the inducer in the culture medium is maintained at 5-20 mM.

6. The high density fermentation process of Lactobacillus reuteri according to any one of claims 2-4, characterized in that, In the medium, casein zymohydrolysate 10 ~ 15 g / L, yeast extract 84 ~ 124 g / L, triammonium citrate 1 ~ 5 g / L, cysteine 0.05 ~ 0.2 g / L, Mn 2+ 0.01 ~ 0.1 g / L, Tween 80 0.2 ~ 2 g / L are further contained.

7. The high density fermentation process of Lactobacillus reuteri according to claim 6, characterized in that, During the fermentation process, the aeration rate is 0.1-0.3 vvm, the pH is controlled at 4.5-6.5, and the residual sugar concentration in the fermentation broth is controlled at not less than 5 g / L by using a flow feeding strategy.

8. The high density fermentation process of Lactobacillus reuteri according to claim 7, characterized in that, The fermentation process proceeds to a viable cell count of not less than 1 x 10 11 CFU / mL in 8-9 hours.

9. The high density fermentation process of Lactobacillus reuteri according to claim 7, characterized in that, The fermentation process progresses to 9~10 hours, the viable cell count is not less than 1×10 13 CFU / mL, and the cell dry weight is not less than 15 g / L.

10. Use of the high-density fermentation method of Lactobacillus reuteri according to any one of claims 7-8 in the preparation of probiotic preparations, feed, functional food and medicine.

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