A high-density culture method of lactobacillus rhamnosus

Abstract

The application discloses a high-density culture method of lactobacillus rhamnosus, and belongs to the technical field of microbial fermentation. The high-density culture method comprises the step of adding lactobacillus buchneri to assist the fermentation culture of the lactobacillus rhamnosus. It is first proved that the lactobacillus buchneri is selected as an auxiliary strain to significantly promote the high-density fermentation of the lactobacillus rhamnosus. Compared with the prior art, the mixed fermentation of the lactobacillus buchneri and the lactobacillus rhamnosus can significantly improve the fermentation bacteria number of the lactobacillus rhamnosus by gradually optimizing the fermentation conditions. The product obtained by using the fermentation method has the advantages of high active bacteria number, natural edibility and low cost. The product has a good industrialization prospect.

Description

Technical Field

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

[0002] Lactic acid bacteria, as a class of microorganisms with various probiotic functions, have demonstrated significant application value in multiple industrial sectors. In the food industry, lactic acid bacteria are not only widely used in the development of fermented dairy products, but also optimize food quality through their metabolites. In the health industry, dietary supplements and biopharmaceutical products developed based on strain-specific functions are gradually forming a diversified product system covering areas such as intestinal microecological regulation, immune enhancement, and metabolic syndrome intervention.

[0003] Lactobacillus rhamnosus and Lactobacillus bruneri are both commonly used lactic acid bacteria in the food industry. However, Lactobacillus rhamnosus is generally difficult to grow and reproduce in solid systems. Although solid-state starter cultures for Lactobacillus rhamnosus have been developed from different technical perspectives both domestically and internationally, some of these products have complex compositions and high costs; some are made from non-natural, edible materials with poor taste; and some have poor viability, short shelf life, and are difficult to market. Moreover, at present, most solid-state starter cultures for Lactobacillus rhamnosus have a viable cell count level of only 10. 9 ~10 10 At the CFU / g level, the effective concentration of *Lactobacillus rhamnosus* is difficult to guarantee. Compared with other lactic acid bacteria, *Lactobacillus bruschetta* has the ability to metabolize lactic acid and has shown good auxiliary ability in co-fermentation with *Lactobacillus bulgaricus*. However, due to differences in the metabolic characteristics of strains, complex interactions between strains, and limitations in adaptability to the fermentation environment, it remains unknown whether it has a positive impact on the high-density fermentation of *Lactobacillus rhamnosus*.

[0004] Therefore, it is particularly important to develop a solid-state starter culture that can effectively promote high-density fermentation of Lactobacillus rhamnosus in a solid system. Summary of the Invention

[0005] The purpose of this invention is to provide a high-density culture method for *Lactobacillus rhamnosus* to solve the problems existing in the prior art. This invention involves mixed fermentation of *Lactobacillus bruneri* and *Lactobacillus rhamnosus*, which can increase the fermentation count of *Lactobacillus rhamnosus* to 1.56 × 10⁻⁶. 11 The product obtained using the fermentation method of this invention has advantages such as high viable cell count (CFU / g), natural usability, and low cost. It has good prospects for industrialization.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for high-density culture of Lactobacillus rhamnosus, including the step of adding Lactobacillus buchneri to the fermentation culture of Lactobacillus rhamnosus to assist the fermentation culture of Lactobacillus rhamnosus.

[0008] Furthermore, the fermentation culture medium components include 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.28g yeast extract, 0.22g soybean peptone, 130U / g high-temperature α-amylase, 326U / g saccharifying enzyme, and VB. 12 0.012g and tyrosine 0.008g.

[0009] Furthermore, the method for preparing the fermentation medium includes the following steps:

[0010] The wheat bran, rice flour, calcium carbonate, yeast extract, and soybean peptone are mixed to obtain a mixture. Water is added to the mixture at a ratio of 1g:1.4mL to obtain a basal culture medium.

[0011] The high-temperature α-amylase was added to the basal culture medium, and after pretreatment and sterilization, the saccharifying enzyme and VB were added. 12 Add tyrosine to obtain the fermentation medium.

[0012] Furthermore, the pretreatment conditions are: pretreatment at 100°C for 30 minutes.

[0013] Furthermore, the total inoculation amount of *Lactobacillus rhamnosus* and *Lactobacillus brunelli* during the fermentation culture was 9.29 × 10⁻⁶. 10 CFU / g.

[0014] Furthermore, the inoculation ratio of Lactobacillus rhamnosus to Lactobacillus brunelli is (1-2):1.

[0015] Furthermore, the fermentation culture temperature is 33-37℃, and the fermentation time is 48-84h.

[0016] Furthermore, the Lactobacillus buchneri is Lactobacillus buchneri B022, which was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO: M 20251048.

[0017] Furthermore, the Lactobacillus rhamnosus is Lactobacillus rhamnosus G011, which was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO: M 20251047.

[0018] The present invention discloses the following technical effects:

[0019] This invention demonstrates for the first time that selecting *Lactobacillus bruneri* as a helper strain can significantly promote high-density fermentation of *Lactobacillus rhamnosus*. Compared with existing technologies, mixed fermentation of *Lactobacillus bruneri* and *Lactobacillus rhamnosus*, through gradual optimization of fermentation conditions, can increase the total bacterial count to 1.64 × 10⁻⁶. 11 CFU / g, of which the fermentation count of Lactobacillus rhamnosus can be increased to 1.56 × 10⁻⁶. 11 CFU / g. The product obtained using the fermentation method of this invention has advantages such as high viable cell count, natural edibility, and low cost. It has good prospects for industrialization. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The effect of different culture conditions on the bacterial count of the culture;

[0022] Figure 2 The effect of different pretreatment conditions on bacterial count in cultures;

[0023] Figure 3 The effect of different nitrogen sources on bacterial counts in cultures;

[0024] Figure 4 The effects of different combinations of inorganic salt ions and growth-promoting factors on bacterial counts in cultures;

[0025] Figure 5 The effect of different lactic acid bacteria combined with Lactobacillus rhamnosus on bacterial count in cultures;

[0026] Figure 6 The effect of different fermentation media on the bacterial count of mixed cultures. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1: Isolation, Screening and Identification of Strains

[0033] 1. Preparation of culture medium

[0034] MRS liquid culture medium: glucose 20.0 g / L; peptone 10.0 g / L; yeast extract 5 g / L; beef extract powder 5.0 g / L; dipotassium hydrogen phosphate 2.0 g / L; sodium acetate 5.0 g / L; triammonium citrate 2.0 g / L; Tween-80 1.0 mL.

[0035] Modified MRS liquid culture medium: glucose 20.0 g / L; peptone 10.0 g / L; yeast extract 5 g / L; beef extract powder 5.0 g / L; dipotassium hydrogen phosphate 2.0 g / L; sodium acetate 5.0 g / L; triammonium citrate 2.0 g / L; Tween-80 1.0 mL; bromocresol purple (acid-base indicator) 0.01 g / L.

[0036] The modified MRS solid medium was prepared by adding 2% agar powder to the liquid medium, and all media were sterilized at 115°C for 25 min.

[0037] 2. Strains Isolation and Screening

[0038] Two g each of fresh feces from healthy infants and baijiu mash were placed in 100 mL of pre-sterilized modified MRS liquid medium and incubated statically for 12 h. Then, 1 mL of the culture was serially diluted and plated onto modified MRS plates. After incubation at 37°C for 48 h, yellow colonies (bromocresol purple turns yellow under acidic conditions) were selected for streak plating isolation, resulting in 15 colonies. Each single colony was transferred to 100 mL of modified MRS liquid medium and incubated at 37°C for 24 h. The pH of the culture medium was measured. After screening, the two strains with the fastest acid-producing rate were obtained:

[0039] Strain G011 produced acid at the fastest rate, with the pH of the culture medium dropping to 4.15 after 24 hours of incubation at 37°C.

[0040] Strain B022 produced acid rapidly; after 24 hours of incubation at 37°C, the pH of the culture medium dropped to 4.20.

[0041] Biochemical identification of strain G011:

[0042] This strain is rod-shaped and non-spore-forming. Colonies on modified MRS solid medium are pale yellow, round, and have regular edges. Physiological and biochemical identification shows that strain G011 is facultative anaerobic, does not reduce nitrates, does not liquefy gelatin, and is negative for both catalase and oxidase.

[0043] Biochemical identification of strain B022:

[0044] This strain is rod-shaped and non-spore-forming. Colonies on modified MRS solid medium are pale yellow, round, and have regular edges. Physiological and biochemical identification shows that strain B022 is facultative anaerobic, does not reduce nitrates, does not liquefy gelatin, and is negative for both catalase and oxidase.

[0045] 16S rDNA identification of strains G011 and B022: Single colonies of G011 and B022 were picked and amplified using universal primers. The amplified products were then subjected to 16S rDNA sequencing analysis. The sequencing results are as follows:

[0046] Strain G01116S rDNA (SEQ ID No. 1):

[0047]

[0048] Strain B02216S rDNA (SEQ ID No. 2):

[0049]

[0050] Sequencing results of the amplified products showed that the sequence of SEQ ID No. 1 had the highest similarity (95.21%) to the 16S rDNA sequence of *Lactobacillus rhamnosus*, confirming that this strain is *Lactobacillus rhamnosus*, and named *Lactobacillus rhamnosus* G011. The sequence of SEQ ID No. 2 had the highest similarity (97.51%) to the 16S rDNA sequence of *Lactobacillus buchneri*, confirming that this strain is *Lactobacillus buchneri*, and named *Lactobacillus buchneri* B022.

[0051] Strain G011 was deposited at the China Center for Type Culture Collection (CCTCC) on May 14, 2025, with the following classification and name: Lactobacillus rhamnosus G011, accession number: CCTCC NO: M 20251047, address: Wuhan University, Wuhan, China.

[0052] Strain B022 was deposited at the China Center for Type Culture Collection (CCTCC) on May 14, 2025, with the following classification and name: Lactobacillus buchneri B022, accession number: CCTCC NO: M 20251048, address: Wuhan University, Wuhan, China.

[0053] Example 2: Screening of culture conditions for Lactobacillus rhamnosus

[0054] 1. Preparation of seed liquid

[0055] Lactobacillus rhamnosus G011, preserved in glycerol tubes at -80℃, was inoculated into Erlenmeyer flasks (MRS medium) and incubated at 37℃ in a bacterial incubator for 15 hours for later use.

[0056] 2. Screening of cultivation conditions

[0057] Lactobacillus rhamnosus was cultured in a culture medium containing: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.34g yeast extract, 0.14g soybean peptone, and 44mL water. The culture conditions were set as follows: Conditions 1-9.

[0058] Condition 1: Temperature 35℃, material-to-water ratio 1:1.2, filling height 1.8cm, inoculum size 0.48×10⁻⁶ 8 CFU / g;

[0059] Condition 2: Temperature 37℃, material-to-water ratio 1:1.2, filling height 1.8cm, inoculum size 0.48×10⁻⁶ 8 CFU / g;

[0060] Condition 3: Temperature 39℃, material-to-water ratio 1:1.2, filling height 1.8cm, inoculum size 0.48×10⁻⁶ 8 CFU / g;

[0061] Condition 4: Temperature 37℃, material-to-water ratio 1:1.4, filling height 1.8cm, inoculum size 0.96×10⁻⁶ 8 CFU / g;

[0062] Condition 5: Temperature 37℃, material-to-water ratio 1:1.6, filling height 1.8cm, inoculum size 0.48×10⁻⁶ 8 CFU / g;

[0063] Condition 6: Temperature 37℃, material-to-water ratio 1:1.4, filling height 3.6cm, inoculum size 1.92×10⁻⁶ 8 CFU / g;

[0064] Condition 7: Temperature 37℃, material-to-water ratio 1:1.4, filling height 5.4cm, inoculum size 0.48×10⁻⁶ 8 CFU / g;

[0065] Condition 8: Temperature 37℃, material-to-water ratio 1:1.4, filling height 5.4cm, inoculum size 0.96×10⁻⁶ 8 CFU / g;

[0066] Condition 9: Temperature 37℃, material-to-water ratio 1:1.4, filling height 5.4cm, inoculum size 1.92×10⁻⁶ 8 CFU / g.

[0067] In conditions 1-9, the material-to-water ratio is a mass ratio, and the mass of the materials in the culture medium remains constant. The material-to-water ratio is controlled by adjusting the amount of water added.

[0068] Fermentation was carried out according to conditions 1-9. The resulting fermentation product was diluted with water, and viable cell counts were performed using the dilution plating method. The fermentation cell count is as follows: Figure 1 As shown. By Figure 1 It can be seen that the number of bacteria obtained from fermentation varies under different culture conditions. Among them, the Lactobacillus rhamnosus screened in this invention, cultured under condition 4, has the highest content of viable bacteria in its fermentation product, which is 5.61 × 10⁻⁶. 10 CFU / g, this condition was subsequently selected as the culture condition for Lactobacillus rhamnosus.

[0069] 3. Screening of culture medium pretreatment conditions

[0070] Preparation and culture conditions of fermentation medium: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.34g yeast extract, 0.14g soybean peptone, natural pH; add water at a material-to-water ratio of 1:1.4, and fill to a height of 1.8cm. Set the pretreatment conditions of the fermentation medium as Condition 1-Condition 7 respectively.

[0071] Condition 1: The amount of α-amylase added at high temperature was 65 U / g, the amount of saccharifying enzyme added was 326 U / g, and the culture medium pretreatment conditions were 121℃ for 20 min;

[0072] Condition 2: The amount of α-amylase added at high temperature was 65 U / g, the amount of saccharifying enzyme added was 326 U / g, and the culture medium pretreatment conditions were 115℃ for 30 min;

[0073] Condition 3: The amount of α-amylase added at high temperature was 65 U / g, the amount of saccharifying enzyme added was 326 U / g, and the culture medium pretreatment conditions were 100℃ for 30 min;

[0074] Condition 4: The addition amount of high-temperature α-amylase was 130 U / g, and the addition amount of saccharifying enzyme was 326 U / g. The culture medium pretreatment conditions were 100℃ for 30 min.

[0075] Condition 5: The amount of α-amylase added at high temperature was 260 U / g, the amount of saccharifying enzyme added was 326 U / g, and the culture medium pretreatment conditions were 100℃ for 30 min;

[0076] Condition 6: The amount of α-amylase added at high temperature was 260 U / g, the amount of saccharifying enzyme added was 407 U / g, and the culture medium pretreatment conditions were 100℃ for 30 min;

[0077] Condition 7: The amount of α-amylase added at high temperature was 260 U / g, the amount of saccharifying enzyme added was 489 U / g, and the culture medium pretreatment conditions were 100℃ for 30 min; the culture medium pretreatment was carried out according to the above conditions.

[0078] In conditions 1-7, high-temperature α-amylase was added to the culture medium before pretreatment, and then saccharifying enzyme was added after pretreatment, with an inoculum size of 9.6 × 10⁻⁶. 7 Inoculate with CFU / g of Lactobacillus sacchariformis seed culture and incubate at 37℃ for 48 hours in a solid state.

[0079] The fermentation product prepared according to the pretreatment conditions 1-7 was diluted with water, and viable cell counts were performed using the dilution plating method. The fermentation cell count was as follows: Figure 2 As shown in the figure, different pretreatment conditions in the culture medium resulted in different numbers of fermenting bacteria. The strain cultured under condition 4 had the highest viable bacteria content in its fermentation product, at 7.45 × 10⁻⁶. 10CFU / g, and this culture medium pretreatment condition was selected for subsequent culturing.

[0080] 4. Nitrogen source concentration screening

[0081] Preparation and culture conditions of fermentation medium: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.34g yeast extract, and 0.14g soybean peptone were added with water at a ratio of 1:1.4. The filling height was 1.8cm. The culture medium was kept at its natural pH. 130U / g of high-temperature α-amylase was added. The culture medium was pretreated at 100℃ for 30min. After pretreatment, 326U / g of saccharifying enzyme was added. The inoculum size was 9.6×10⁶. 7 CFU / g inoculated with Lactobacillus rhamnosus seed culture, and cultured in a solid state at 37℃ for 48 h; the nitrogen source concentration in the fermentation medium was set as follows: Medium 1-Medium 4:

[0082] Culture medium 1: 0.14 g yeast extract, 0.07 g soybean peptone;

[0083] Culture medium 2: 0.28g yeast extract, 0.22g soybean peptone;

[0084] Culture medium 3: 0.34g yeast extract, 0.14g soybean peptone;

[0085] Culture medium 4: 0.68g yeast extract and 0.28g soybean peptone.

[0086] The fermentation product obtained under the conditions of culture medium 1-4 was diluted with water, and viable cell count was performed according to the dilution plating method. The fermentation cell count was as follows: Figure 3 As shown in the figure, different nitrogen source concentrations result in different fermentation bacterial counts. The highest viable bacteria content in the fermentation product (9.29 × 10⁻⁶) was observed when the nitrogen source concentration was 0.28 g yeast extract and 0.22 g soybean peptone. 10 CFU / g, and this nitrogen source concentration was selected for subsequent culture conditions.

[0087] 5. Screening of different inorganic salt ions and growth factors

[0088] Preparation of fermentation medium: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.28g yeast extract, and 0.22g soybean peptone. Water was added at a ratio of 1:1.4, and the medium was filled to a height of 1.8cm. The culture medium was kept at its natural pH. 130U / g of high-temperature α-amylase was added. The pretreatment conditions were 100℃ for 30min. After pretreatment, 326U / g of saccharifying enzyme was added, along with an inoculum size of 9.6 × 10⁶ g / g. 7CFU / g of Lactobacillus rhamnosus seed culture was inoculated, and inorganic salt ions and growth factors were added to the fermentation medium as in fermentation medium A-fermentation medium L; solid-state fermentation was carried out at 37℃ for 48 h.

[0089] Fermentation medium A: 0.03 g ferrous sulfate;

[0090] Fermentation medium B: 0.06 g ferrous sulfate;

[0091] Fermentation medium C: VB 12 0.006g;

[0092] Fermentation medium D: VB 12 0.012g;

[0093] Fermentation medium E: 0.004 g tyrosine;

[0094] Fermentation medium F: 0.008 g tyrosine;

[0095] Fermentation medium G: ferrous sulfate 0.06g, VB 12 0.012g;

[0096] Fermentation medium H: ferrous sulfate 0.06g, tyrosine 0.008g;

[0097] Fermentation medium I: VB 12 0.012g, tyrosine 0.008g;

[0098] Fermentation medium J: ferrous sulfate 0.06g, tyrosine 0.008g, VB 12 0.012g.

[0099] The fermentation product obtained after adding inorganic salt ions and growth factors according to the conditions of fermentation medium AJ was diluted with water, and viable cell count was performed according to the dilution plating method. The fermentation cell count was as follows: Figure 4 As shown. Fermentation of *Lactobacillus rhamnosus* was carried out using fermentation medium I. The optimal combination of inorganic salt ions and growth-promoting factors in the medium resulted in significant fermentation effects, with a viable cell count reaching 1.37 × 10⁻⁶. 11 CFU / g.

[0100] Example 3: Screening by mixed-culture fermentation with Lactobacillus rhamnosus as the main strain

[0101] 1. Screening of auxiliary strains

[0102] Fermentation medium preparation: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.28g yeast extract, and 0.22g soybean peptone were added to a water-to-material ratio of 1:1.4. The medium was filled to a height of 1.8cm, with the pH set to natural. 130U / g of high-temperature α-amylase was added. The medium was pretreated at 100℃ for 30min. After pretreatment, 326U / g of saccharifying enzyme was added. *Lactobacillus rhamnosus* and helper strains were inoculated at a 1:1 ratio, with a total inoculum of 9.6 × 10⁻⁶. 7 CFU / g, add VB to fermentation medium 12 0.012g of tyrosine and 0.008g of other compounds were added; the mixture was fermented at 37℃ in a solid-state environment for 48 hours; the auxiliary strains in the fermentation system were designated as auxiliary strain A to auxiliary strain G.

[0103] Helper strain A: Lactobacillus paracasei;

[0104] Helper strain B: Lactobacillus reuteri;

[0105] Helper strain C: Lactobacillus plantarum;

[0106] Helper strain D: Lactobacillus brunelli B022;

[0107] Helper strain E: Lactobacillus helveticus;

[0108] Helper strain F: Pediococcus lactis;

[0109] Helper strain G: Clostridium butyricum.

[0110] The fermentation product obtained by fermentation under the conditions of the auxiliary strain AG in the fermentation system was diluted with water, and viable cell counts were performed according to the dilution plating method. The fermentation cell count was as follows: Figure 5 As shown, using helper strain D for the auxiliary fermentation of Lactobacillus rhamnosus yielded the best results, with significant fermentation effects and a viable count of up to 1.43 × 10⁻⁶. 11 CFU / g.

[0111] 2. Screening of mixed culture systems under different culture conditions

[0112] Fermentation medium preparation: 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.28g yeast extract, and 0.22g soybean peptone were added to a water-to-material ratio of 1:1.4. The medium was filled to a height of 1.8cm, with the pH set to natural. 130U / g of high-temperature α-amylase was added. The medium was pretreated at 100℃ for 30min. After pretreatment, 326U / g of saccharifying enzyme was added. *Lactobacillus rhamnosus* and *Lactobacillus brunelli* were inoculated at a 1:1 ratio, with a total inoculum of 9.6 × 10⁻⁶. 7 CFU / g, add VB to fermentation medium 120.012g of tyrosine and 0.008g of tyrosine were added; the mixture was incubated at 37℃ in a solid-state environment for 48 hours. The fermentation systems were set up as fermentation system A and fermentation system G.

[0113] Fermentation system A: Inoculum ratio 2:1;

[0114] Fermentation system B: Inoculation ratio 3:1;

[0115] Fermentation system C: Fermentation temperature 33℃;

[0116] Fermentation system D: Fermentation temperature 37℃;

[0117] Fermentation system E: Fermentation time 48h;

[0118] Fermentation system F: Fermentation time 84h;

[0119] Fermentation system G: inoculum ratio 3:1, fermentation temperature 37℃, fermentation time 84h;

[0120] Fermentation system H: inoculum ratio 3:1, fermentation temperature 37℃, fermentation time 48h;

[0121] Fermentation system I: inoculum ratio 2:1, fermentation temperature 33℃, fermentation time 84h;

[0122] Fermentation system J: inoculum ratio 2:1, fermentation temperature 33℃, fermentation time 48h.

[0123] The fermentation product obtained from fermentation under the conditions of fermentation system AJ was diluted with water, and viable cell counts were performed using the dilution plating method. The fermentation cell count was as follows: Figure 6 As shown. Fermentation system I was used to ferment the mixed culture system. The optimal culture conditions—inoculum ratio of 2:1, fermentation temperature of 33℃, and fermentation time of 84 h—showed the best auxiliary effect on *Lactobacillus rhamnosus*, with significant fermentation results. The viable count of *Lactobacillus rhamnosus* reached 1.56 × 10⁻⁶. 11 CFU / g, total fermentation count can reach 1.64×10⁻⁶. 11 CFU / g.

[0124] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for high-density culture of Lactobacillus rhamnosus, characterized in that, The step includes adding Lactobacillus buchneri during the fermentation culture of Lactobacillus rhamnosus to assist the fermentation culture of Lactobacillus rhamnosus. The fermentation culture medium components include 20g wheat bran, 5g rice flour, 6g calcium carbonate, 0.28g yeast extract, 0.22g soybean peptone, 130U / g high-temperature α-amylase, 326U / g saccharifying enzyme, and VB. 12 0.012g and 0.008g of tyrosine; The method for preparing the fermentation medium includes the following steps: The wheat bran, rice flour, calcium carbonate, yeast extract, and soybean peptone are mixed to obtain a mixture. Water is added to the mixture at a ratio of 1g:1.4mL to obtain a basal culture medium. The high-temperature α-amylase was added to the basal culture medium, and after pretreatment and sterilization, the saccharifying enzyme and VB were added. 12 Add tyrosine to obtain the fermentation medium.

2. The high-density culture method according to claim 1, characterized in that, The pretreatment conditions are 100℃ for 30 minutes.

3. The high-density culture method according to claim 1, characterized in that, The fermentation culture temperature is 33-37℃, and the fermentation time is 48-84h.

4. The high-density culture method according to claim 1, characterized in that, The Lactobacillus bruneri is Lactobacillus bruneri ( Lactobacillus buchneri B022 was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO: M 20251048.

5. The high-density culture method according to claim 1, characterized in that, The Lactobacillus rhamnosus is Lactobacillus rhamnosus ( Lactobacillus rhamnosus G011 was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO: M 20251047.

Images