Biofilm state lactobacillus culture method for improving survival rate of lactobacillus

Abstract

The invention discloses a biofilm state lactobacillus culture method for improving the survival rate of lactobacillus, and belongs to the technical field of strain culture. The culture method comprises the following steps: preparing a liquid culture medium; the liquid culture medium contains peptone, beef extract powder, yeast extract powder, glucose, dipotassium phosphate, sodium acetate, magnesium sulfate and manganese sulfate; inoculating lactobacillus into the liquid culture medium, and activating bacterial strains at constant temperature under an anaerobic condition to obtain bacterial suspension; inoculating the bacterial suspension into a culture plate containing the liquid culture medium, and carrying out gradient heating incubation in an anaerobic environment; after incubation is finished, floating bacteria are removed, and biofilm state lactobacillus is obtained through collection. The low-cost raw materials and simple and convenient preparation steps are adopted, the remarkable film forming effect is achieved, the biofilm yield is high, the survival rate of the finally obtained biofilm state lactobacillus in the gastric acid intestinal juice environment can be remarkably increased, and the biofilm state lactobacillus has high practical value.

Description

Technical Field

[0001] The invention relates to the technical field of strain cultivation, in particular to a method for cultivating biofilm lactobacilli for improving the survival rate of lactobacilli. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In today's probiotics industry, Lactobacillus ( Lactobacillus ) has become a key research and application target in the food and pharmaceutical fields due to its significant efficacy in regulating the intestinal microbiome, enhancing immunity, and improving host health. However, during production, processing, storage, and oral delivery, Lactobacillus is susceptible to adverse external factors (such as high temperature, low pH, high bile salts, and oxidative environments), which can significantly reduce its activity and directly restrict the stability and efficacy of Lactobacillus-based products.

[0004] In recent years, biofilm technology has provided new insights into addressing these challenges. Biofilms are structured microbial communities formed by microorganisms secreting extracellular polymers (such as exopolysaccharides, proteins, and nucleic acids), possessing unique physical barriers and biofunctional properties. Studies have found that Lactobacillus in a biofilm state can significantly enhance its tolerance to environmental stresses, such as gastrointestinal fluid tolerance and antioxidant capacity. Furthermore, the biofilm state of Lactobacillus enhances its adhesion to intestinal cells and promotes colonization in the host intestine, thereby maximizing its probiotic effects. However, in actual production and application, Lactobacillus biofilm formation is plagued by issues such as long growth cycles, low efficiency, thin biofilms, limited coverage, and low yields. Therefore, improving the stability and growth efficiency of Lactobacillus biofilms, inducing their growth on surfaces, and forming more biofilms to expand their application are urgent challenges. Summary of the Invention

[0005] In view of this, the present invention provides a method for culturing biofilm-state lactobacilli to improve the survival rate of lactobacilli. The formation process of the lactobacilli biofilm of the present invention is simple, the raw materials are easily available and low in price, the biofilm yield is increased, and the survival rate of lactobacilli in simulated gastric fluid and intestinal fluid environments can be effectively greatly improved.

[0006] The present invention provides a method for culturing biofilm lactobacilli for improving the survival rate of lactobacilli, comprising the following steps: preparing a liquid culture medium containing peptone, beef extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, sodium acetate, magnesium sulfate, and manganese sulfate; inoculating Lactobacillus into the liquid culture medium, and activating the strain at a constant temperature under anaerobic conditions to obtain a bacterial suspension; The bacterial suspension is inoculated into a culture plate containing the liquid culture medium, and incubated in an anaerobic environment with a gradient temperature increase; after the incubation is completed, the planktonic bacteria are removed and the biofilm lactobacilli are collected.

[0007] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention provides a method for culturing biofilm lactobacilli that improves the survival rate of lactobacilli. Through a specific culture medium and culture method, with low-cost raw materials and simple preparation steps, a significant film-forming effect is achieved. The biofilm lactobacilli finally obtained can significantly improve the survival rate of lactobacilli in a gastric acid and intestinal fluid environment, and has a high biofilm yield. The culture method has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0009] Figure 1 These are scanning electron microscope images of the lactobacilli of Example 1 and Comparative Example 4 of the present invention, wherein a is Example 1 and b is Comparative Example 4. DETAILED DESCRIPTION

[0010] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0011] The present invention provides a method for culturing biofilm lactobacilli for improving the survival rate of lactobacilli, comprising the following steps: preparing a liquid culture medium containing peptone, beef extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, sodium acetate, magnesium sulfate, and manganese sulfate; inoculating Lactobacillus into the liquid culture medium, and activating the strain at a constant temperature under anaerobic conditions to obtain a bacterial suspension; The bacterial suspension is inoculated into a culture plate containing the liquid culture medium, and incubated in an anaerobic environment with a gradient temperature increase; after the incubation is completed, the planktonic bacteria are removed and the biofilm lactobacilli are collected.

[0012] The present invention employs a gradient temperature incubation method under an anaerobic environment. The initial low temperature induces lactobacilli into a stress state that allows them to form biofilms. The temperature is then adjusted to the optimal temperature to promote cell metabolism and reproduction, as well as the production of stress-induced extracellular polymers (ECPs). This promotes the growth and maturation of biofilms and increases biofilm production. The biofilm-forming lactobacilli produced using the present cultivation method can achieve a survival rate that is more than 20% higher than that of planktonic lactobacilli in a simulated gastric acid and bile environment, demonstrating a significant improvement in survival.

[0013] In some embodiments of the present invention, the liquid culture medium comprises: 9-12 g / L peptone, 7-9 g / L beef extract powder, 3.5-4.5 g / L yeast extract powder, 20-25 g / L glucose, 1.5-2.5 g / L dipotassium hydrogen phosphate, 5-6 g / L sodium acetate, 0.1-0.3 g / L magnesium sulfate, 0.04-0.06 g / L manganese sulfate, and the remainder water. The sodium acetate is preferably sodium acetate trihydrate, the magnesium sulfate is preferably magnesium sulfate heptahydrate, and the manganese sulfate is preferably manganese sulfate tetrahydrate.

[0014] In some embodiments of the present invention, the pH of the liquid culture medium is 6.0-7.5, for example, 6.5, 7.0 or 7.5. The above pH conditions can promote the growth and metabolism of Lactobacillus.

[0015] In some embodiments of the present invention, the lactobacillus comprises Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus or Lactobacillus salivarius. In some specific embodiments of the present invention, the lactobacillus is selected from Lactobacillus salivarius ATCC 11741 ( Ligilactobacillus salivarius ATCC 11741)、Lactobacillus casei ATCC 15820( Lactobacillus Casei ATCC 15820) or Lactobacillus plantarum subsp. plantarum ATCC 14917 ( Lactobacillus plantarum subsp. plantarum ATCC 14917).

[0016] In some embodiments of the present invention, in the step of inoculating Lactobacillus into the liquid culture medium, the inoculation amount of Lactobacillus is 1-3% (v / v), more preferably 2% (v / v).

[0017] In some embodiments of the present invention, the rotation speed used in the step of activating the strain at constant temperature under anaerobic conditions is 100-200 rpm, preferably 150 rpm; the temperature is 36-38°C, more preferably 36.5-37.5°C, and most preferably 37°C.

[0018] In some embodiments of the present invention, in the step of activating the strain at a constant temperature under anaerobic conditions, the activated Lactobacillus is passaged twice.

[0019] In some embodiments of the present invention, the inoculation amount of the bacterial suspension is 1-3% (v / v), more preferably 2% (v / v).

[0020] In some embodiments of the present invention, the specific steps of the gradient temperature incubation under an anaerobic environment are: first, static culture at 28-31°C for 3-5 hours; then, heating to 36-38°C and static culture for 20-32 hours; more preferably, first, static culture at 30°C for 4 hours; then, heating to 37°C and static culture for 24 hours.

[0021] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no particular limitation on the sources of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0022] Example 1 This embodiment provides a method for culturing Lactobacillus salivarius in a biofilm state, comprising the following steps: (1) Prepare liquid culture medium: 10 g of peptone, 8 g of beef extract powder, 4.0 g of yeast extract powder, 20 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 5 g of sodium acetate trihydrate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate tetrahydrate, and the remainder of water. Mix the above ingredients and dissolve them evenly. Adjust the pH of the liquid culture medium to 6.5 and set aside.

[0023] (2) Activated strain: Lactobacillus salivarius ATCC 11741 ( Ligilactobacillus salivarius ATCC11741) was inoculated into the liquid culture medium provided above at an inoculum rate of 2% (v / v) and cultured in an anaerobic environment at a constant temperature (37°C, 150 rpm). The strain was subcultured twice to fully activate the strain and obtain a bacterial suspension.

[0024] (3) 5 mL of fresh liquid culture medium was added to a polystyrene six-well culture plate in advance. The bacterial suspension obtained in step (2) was inoculated into the polystyrene six-well culture plate at a 2% (v / v) inoculation rate. The culture plate was placed in an anaerobic environment and incubated at 30°C for 4 hours, and then heated to 37°C and incubated for 24 hours.

[0025] (4) After the incubation, the supernatant in the culture plate was aspirated and washed three times with PBS to remove the floating bacteria. The biofilm was gently scraped with a cell scraper and placed in an appropriate amount of physiological saline to obtain Lactobacillus salivarius in a biofilm state.

[0026] Example 2 This embodiment provides a method for culturing Lactobacillus casei in a biofilm state, comprising the following steps: (1) Prepare liquid culture medium: 9.5 g peptone, 9 g beef extract powder, 3.5 g yeast extract powder, 20 g glucose, 2.0 g dipotassium hydrogen phosphate, 6 g sodium acetate trihydrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate tetrahydrate, and the remainder water. Mix the above ingredients evenly, adjust the pH of the liquid culture medium to 8.0, and set aside.

[0027] (2) Activated strain: Lactobacillus casei ATCC 15820 (Lactobacillus Casei ATCC 15820) was inoculated into the liquid culture medium provided above at a 2% (v / v) inoculation rate and cultured in an anaerobic environment at a constant temperature (37°C, 150 rpm). The strain was subcultured twice to fully activate the strain and obtain a bacterial suspension.

[0028] (3) 4 mL of fresh liquid culture medium was added to a polystyrene six-well culture plate in advance. The bacterial suspension obtained in step (2) was inoculated into the polystyrene six-well culture plate at a 2% (v / v) inoculation rate. The culture plate was placed in an anaerobic environment and incubated at 30°C for 4 hours, and then heated to 37°C and incubated for 24 hours.

[0029] (4) After the incubation, the supernatant in the culture plate was aspirated and washed three times with PBS to remove the floating bacteria. The biofilm was gently scraped with a cell scraper and placed in an appropriate amount of physiological saline to obtain Lactobacillus casei in a biofilm state.

[0030] Example 3 This embodiment provides a method for culturing Lactobacillus plantarum subspecies plantarum in a biofilm state, comprising the following steps: (1) Prepare liquid culture medium: 11 g peptone, 7.5 g beef extract powder, 4 g yeast extract powder, 22 g glucose, 2.0 g dipotassium hydrogen phosphate, 5 g sodium acetate trihydrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate tetrahydrate, and the remainder water. Mix the above ingredients evenly, adjust the pH of the liquid culture medium to 7.5, and set aside.

[0031] (2) Activated strain: Lactobacillus plantarum subspecies ATCC 14917 (Lactobacillus plantarum subsp. plantarum ATCC 14917) was inoculated into the liquid culture medium provided above at a 2% (v / v) inoculation rate and cultured in an anaerobic environment at a constant temperature (37°C, 150 rpm). The strain was subcultured twice to fully activate the strain and obtain a bacterial suspension.

[0032] (3) Add 5 mL of fresh liquid culture medium into a polystyrene six-well culture plate in advance, inoculate the bacterial suspension obtained in step (2) into the polystyrene six-well culture plate at a 2% (v / v) inoculation rate, place the culture plate in an anaerobic environment, incubate at 30°C for 4 hours, and then heat to 37°C and incubate for 24 hours.

[0033] (4) After the incubation, the supernatant in the culture plate was aspirated and washed three times with PBS to remove the floating bacteria. The biofilm was gently scraped with a cell scraper and placed in an appropriate amount of physiological saline to obtain the biofilm state of Lactobacillus plantarum subspecies.

[0034] Comparative Example 1 The difference between this comparative example and Example 1 is that in step (3), the comparative example is subjected to constant temperature static culture at 37°C for 28 hours.

[0035] Comparative Example 2 The difference between this comparative example and Example 2 is that in step (3), the comparative example is subjected to constant temperature static culture at 37°C for 28 hours.

[0036] Comparative Example 3 The difference between this comparative example and Example 3 is that in step (3), the comparative example is subjected to constant temperature static culture at 37°C for 28 hours.

[0037] Comparative Example 4 The difference between this comparative example and Example 1 is that step (3) is not performed in this comparative example.

[0038] Comparative Example 5 The difference between this comparative example and Example 2 is that step (3) is not performed in this comparative example.

[0039] Comparative Example 6 The difference between this comparative example and Example 3 is that step (3) is not performed in this comparative example.

[0040] Test Example 1 The biofilm yields of the lactobacilli in the biofilm state of Examples 1 to 3 and Comparative Examples 1 to 3 were determined by the following steps: 200 μL of MRS culture medium was added to a polystyrene 96-well plate, and the bacterial suspension of Example (2) was inoculated into the plate at a 2% inoculation rate. The culture time and culture temperature were set according to the above examples and comparative examples. After the culture was completed, the culture medium was discarded and the biofilm formed on the bottom of the plate was gently washed twice with sterile saline. After drying for a while at room temperature, it was stained with 0.1% (w / v) crystal violet for 20 minutes; 300 μL of 95% alcohol was added and allowed to stand for 30 minutes. After the crystal violet was completely dissolved, 200 μL of crystal violet eluate was aspirated into another plate to measure its OD value.570nm The blank control was performed using MRS medium without bacterial inoculation, and the same procedure was used as above. Each group had three parallel groups, and the experiment was repeated three times. The average value was taken and the results are summarized in Table 1.

[0041] Table 1 Semi-quantitative determination data of biofilm of Lactobacillus in Examples and Comparative Examples

[0042] As can be seen from Table 1, the biofilm yields of Examples 1 to 3 were increased by about 30% to 50% compared with the corresponding Comparative Examples 1 to 3, indicating that the gradient heating method of low temperature + suitable temperature is conducive to the rapid growth of Lactobacillus biofilm.

[0043] Test Example 2 The survival rates of the biofilm-forming Lactobacilli of Examples 1 to 3 and the Lactobacilli of Comparative Examples 4 to 6 during simulated gastric digestion and intestinal digestion were determined as follows: Put 10 9 The CFU of Lactobacillus was added to 5 mL of simulated gastric fluid (pH = 2.0) and incubated anaerobically at 37°C with shaking at 100 rpm for 1 hour. The cells were then centrifuged and counted by serial dilution using PBS.

[0044] Survival rate (%) =

[0045] Put 10 9 The CFU of Lactobacillus was added to 5 mL of simulated intestinal fluid (containing 0.15 wt% bile salts) and incubated anaerobically at 37°C with shaking at 100 rpm for 1 hour. The cells were then centrifuged and serially diluted with PBS for plate count.

[0046] Survival rate (%) =

[0047] The results are summarized in Table 2.

[0048] Table 2 Survival rate test data of lactobacillus in Examples and Comparative Examples

[0049] As can be seen from Table 1, the tolerance of biofilm probiotics to gastrointestinal fluid is significantly stronger than that of planktonic probiotics, and the culture method of gradient temperature increase can also improve the tolerance of biofilm to gastrointestinal fluid to a certain extent. Figure 1 a in the figure is a scanning electron microscope image of Lactobacillus salivarius in the biofilm state of Example 1. Figure 1Figure b is a scanning electron micrograph of the planktonic Lactobacillus salivarius from Comparative Example 4. It shows that the probiotic biofilm is characterized by dense bacterial packing and a three-dimensional multilayered structure. In contrast, the electron micrograph of the planktonic probiotic bacteria shows clear bacterial morphology and little bacterial aggregation.

[0050] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for culturing biofilm lactobacilli to improve the survival rate of lactobacilli, characterized in that: The steps include: preparing a liquid culture medium containing peptone, beef extract powder, yeast extract powder, glucose, dipotassium hydrogen phosphate, sodium acetate, magnesium sulfate, and manganese sulfate; inoculating Lactobacillus into the liquid culture medium, and activating the strain at a constant temperature under anaerobic conditions to obtain a bacterial suspension; inoculating the bacterial suspension into a culture plate containing the liquid culture medium, and incubating the culture plate in an anaerobic environment with a gradient temperature increase; After the incubation, the floating bacteria were removed and the biofilm lactobacilli were collected.

2. The culture method according to claim 1, wherein The liquid culture medium comprises: 9-12 g / L of peptone, 7-9 g / L of beef extract powder, 3.5-4.5 g / L of yeast extract powder, 20-25 g / L of glucose, 1.5-2.5 g / L of dipotassium hydrogen phosphate, 5-6 g / L of sodium acetate, 0.1-0.3 g / L of magnesium sulfate, 0.04-0.06 g / L of manganese sulfate, and the remainder is water.

3. The culture method according to claim 2, wherein The pH of the liquid culture medium is 6.0-8.

5.

4. The culture method according to claim 1, wherein The lactobacillus includes Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus or Lactobacillus salivarius.

5. The culture method according to claim 1, wherein In the step of inoculating lactobacillus into the liquid culture medium, the inoculation amount of lactobacillus is 1-3% (v / v).

6. The culture method according to claim 1, wherein The step of activating the strain under anaerobic conditions adopts a rotation speed of 100-200 rpm and a temperature of 36-38°C.

7. The culture method according to claim 1, wherein In the step of activating the strain at a constant temperature under anaerobic conditions, the activated lactobacillus is passaged twice.

8. The culture method according to claim 1, wherein The inoculation amount of the bacterial suspension is 1-3% (v / v).

9. The culture method according to claim 1, wherein The specific steps of the gradient temperature incubation under the anaerobic environment are: first, static culture at 28-31° C. for 3-5 hours; then, heating to 36-38° C. and static culture for 20-32 hours.

10. The culture method according to claim 9, wherein The specific steps of the gradient temperature incubation under the anaerobic environment are: first, static culture at 30° C. for 4 hours; then, heating to 37° C. and static culture for 24 hours.

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