A method for culturing lactic acid bacteria that adapts to the vaginal environment and strongly adheres to vaginal epithelial cells

Abstract

The present invention relates to a method for culturing lactic acid bacteria that adapt to the vaginal environment and strongly adhere to vaginal epithelial cells. The method comprises inoculating the cultured lactic acid bacteria seed solution into a liquid culture medium, and culturing the culture medium statically until the OD value of the culture medium reaches 0. 600 When the value is 0.3-0.6, the preparation is added and culture is continued, and after completion, the lactic acid bacteria that strongly adhere to vaginal epithelial cells are obtained; the preparation includes L-arabinose. The present invention also relates to the use of L-arabinose in preparing a preparation for improving the adhesion of lactic acid bacteria to vaginal epithelial cells. By adding the relevant preparation during a specific growth stage of lactic acid bacteria, it is beneficial to ensure rapid bacterial proliferation and stable formation of surface hydrophobic proteins, thereby further improving the adhesion and colonization ability of the cultured lactic acid bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for culturing lactic acid bacteria that adapt to the vaginal environment and strongly adhere to vaginal epithelial cells. Background Art

[0002] There is a significant relationship between vaginal microecological balance and female reproductive tract diseases. Lactobacillus is the primary bacterial colonizer of the vast majority of female vaginas and plays a key role in maintaining vaginal health. As the dominant bacterial community in the vaginal microbiome, lactobacilli not only maintain vaginal microecological health but also prevent vaginal infections, playing an irreplaceable role in resisting the invasion of pathogenic microorganisms. In healthy women, lactobacilli are typically dominant in abundance, maintaining vaginal microecological balance. Common lactobacilli include Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus johnsonii. These bacteria interact and mutually restrict each other, achieving a dynamic balance and maintaining good health.

[0003] When the balance of the reproductive tract microbiome is disrupted, the dominant bacterial population decreases and the pathogenic bacteria gradually increase, leading to vaginal inflammation. Currently, the main clinical treatment for vaginal infections is antibiotics, which indiscriminately kill vaginal microorganisms, including probiotics and some non-pathogenic vaginal organisms. This treatment significantly alters the vaginal microbial environment, causing dysbiosis, which can lead to poor treatment efficacy and high recurrence rates. For probiotics to exert their beneficial effects in the reproductive tract, they must be able to adhere to and colonize the surface of reproductive tract cells. By occupying space, they can inhibit pathogenic bacteria in the reproductive tract, prevent the colonization of other harmful bacteria, regulate the balance of the vaginal flora, and achieve the purpose of preventing and assisting in the regulation of disease. The adhesion ability of lactic acid bacteria is related to multiple factors, such as adhesins, cell surface proteins, capsular polysaccharides, and the bacteria's ability to self-aggregate and co-aggregate. However, there are relatively few reports in the prior art on how to improve the adhesion and colonization ability of inactivated lactic acid bacteria (i.e., postbiotics) in the reproductive tract. Therefore, how to develop a method that can effectively improve the adhesion and colonization ability of postbiotics in the reproductive tract has become an urgent problem to be solved. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for preparing lactic acid bacteria that adapt to the vaginal environment and strongly adhere to vaginal epithelial cells.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a use of L-arabinose in preparing a preparation for improving the adhesion ability of lactic acid bacteria to vaginal epithelial cells.

[0007] L-arabinose is a prebiotic. The present invention unexpectedly discovered that it has an excellent promoting effect in enhancing the adhesion and colonization ability of lactic acid bacteria on vaginal epithelial cells. In the present invention, a new application of L-arabinose in the preparation of a preparation for improving the adhesion ability of lactic acid bacteria to vaginal epithelial cells is provided.

[0008] Preferably, the preparation is L-arabinose or a combination of L-arabinose and soybean oligosaccharides.

[0009] The present invention also creatively discovered that L-arabinose can be compounded with soybean oligosaccharides and added to the culture of lactic acid bacteria as a preparation, which has a more significant and excellent effect than a single substance or other compounding methods. The two can synergistically enhance the adhesion ability of the cultured lactic acid bacteria to vaginal epithelial cells and enhance the prebiotic effect.

[0010] Preferably, the mass ratio of L-arabinose to soybean oligosaccharides is 1:5-5:1, for example, it can be 5:1, 5:2, 5:3, 5:4, 1:1, 1:2, 1:3, 1:4, 1:5, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0011] In a second aspect, the present invention provides a method for culturing lactic acid bacteria that adapt to the vaginal environment and strongly adhere to vaginal epithelial cells, the method comprising the following steps:

[0012] The lactic acid bacteria seed liquid obtained by culture was inoculated into the liquid culture medium and cultured statically until the OD 600 When the value is 0.3-0.6, the preparation is added and culture is continued, and the lactic acid bacteria that strongly adhere to the vaginal epithelial cells are obtained after the end; the preparation includes L-arabinose.

[0013] In the present invention, the adhesion and colonization abilities of cultured lactic acid bacteria are enhanced by adding a preparation during a specific growth phase of the bacteria. During this phase, when the bacteria experience rapid growth, high metabolism, and active enzymes, the addition of the preparation provides a nutrient foundation for microbial growth, further facilitating rapid bacterial proliferation and the stable formation of surface hydrophobic proteins and other metabolites. L-arabinose, as a prebiotic, not only promotes the proliferation of lactic acid bacteria, but the present invention also unexpectedly discovered that it has an excellent promoting effect on the adhesion and colonization abilities of lactic acid bacteria on vaginal epithelial cells.

[0014] Preferably, the concentration of the preparation in the culture medium is 0.6-1 g / L, for example, it can be 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, 1 g / L, etc. Other specific point values within this numerical range can be selected, and they will not be repeated here.

[0015] When the amount of the added preparation is within the above-mentioned specific range, the adhesion and colonization ability of the lactic acid bacteria finally cultured to the vaginal epithelial cells can be further enhanced.

[0016] Preferably, the preparation further comprises soybean oligosaccharides.

[0017] The present invention also creatively discovered that L-arabinose can be compounded with soybean oligosaccharides and added to the culture of lactic acid bacteria as a preparation, which has a more significant and excellent effect than a single substance or other compounding methods. The two can synergistically enhance the adhesion ability of the cultured lactic acid bacteria to vaginal epithelial cells and enhance the prebiotic effect.

[0018] Preferably, the mass ratio of L-arabinose to soybean oligosaccharides is 1:5-5:1, for example, it can be 5:1, 5:2, 5:3, 5:4, 1:1, 1:2, 1:3, 1:4, 1:5, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0019] Preferably, the method for culturing the lactic acid bacteria seed liquid comprises streaking the activated lactic acid bacteria liquid onto a solid culture medium, incubating it upside down at 36-38° C. until a single colony grows, picking the single colony and inoculating it into a liquid culture medium, and statically culturing it at 36-38° C. for 12-14 hours to obtain the lactic acid bacteria seed liquid;

[0020] The above-mentioned "36-38°C" can be, for example, 36°C, 36.2°C, 36.5°C, 36.6°C, 36.8°C, 37°C, 37.2°C, 37.5°C, 37.6°C, 37.8°C, 37.9°C, 38°C, etc. Other specific points within this numerical range can be selected and will not be listed here one by one;

[0021] The above-mentioned "12-14h" can be, for example, 12h, 12.2h, 12.5h, 12.6h, 12.8h, 13h, 13.2h, 13.4h, 13.5h, 13.6h, 13.8h, 14h, etc. Other specific point values within this numerical range can be selected, and they will not be listed here one by one.

[0022] Preferably, the inoculation amount of the lactic acid bacteria seed liquid is 1.5%-5% of the liquid culture medium, v / v, for example, it can be 1.5%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, or 5%.

[0023] Preferably, the temperature of the static culture is 35-38°C, for example, it can be 35°C, 35.2°C, 35.5°C, 35.8°C, 36°C, 36.2°C, 36.5°C, 36.6°C, 36.8°C, 37°C, 37.2°C, 37.5°C, 37.6°C, 37.8°C, 37.9°C, 38°C, etc. Other specific point values within this numerical range can be selected, and they will not be repeated here.

[0024] Preferably, the temperature for continued cultivation is 35-38°C, for example, it can be 35°C, 35.2°C, 35.5°C, 35.8°C, 36°C, 36.2°C, 36.5°C, 36.6°C, 36.8°C, 37°C, 37.2°C, 37.5°C, 37.6°C, 37.8°C, 37.9°C, 38°C, etc., and the time is 15-20h, for example, it can be 15h, 15.2h, 15.5h, 15.8h, 16h, 16.2h, 16.5h, 16.8h, 17h, 17.5h, 18h, 18.5h, 19h, 19.5h, 19.8h, 20h, etc. Other specific point values within this numerical range can be selected, and they will not be repeated here.

[0025] Preferably, the continued cultivation is followed by centrifugation and washing.

[0026] Preferably, the centrifugal speed is 3500-6500rpm, for example, it can be 3500rpm, 3750rpm, 4000rpm, 4250rpm, 4500rpm, 4750rpm, 5000rpm, 5250rpm, 5500rpm, 6000rpm, 6250rpm, 6500rpm, etc.; the time is 3-8min, for example, it can be 3min, 3.2min, 3.5min, 3.8min, 4min, 4.2min, 4.5min, 4.8min, 5min, 5.5min, 6min, 6.5min, 6.8min, 7min, 7.2min, 7.5min, 7.8min, 8min, etc. Other specific point values within this numerical range can be selected, and they will not be repeated here.

[0027] Preferably, the washing solvent comprises PBS and / or water.

[0028] Preferably, the lactic acid bacteria include Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus reuteri or Lactobacillus crispatus.

[0029] In a third aspect, the present invention provides a method for culturing a postbiotic that adapts to the vaginal environment and strongly adheres to vaginal epithelial cells, the method comprising inactivating the lactic acid bacteria that strongly adhere to vaginal epithelial cells obtained by the method of the second aspect to obtain the postbiotic that strongly adheres to vaginal epithelial cells;

[0030] Preferably, the temperature of the inactivation treatment is 60-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 64.5°C, 64.8°C, 65°C, 65.2°C, 65.5°C, 65.8°C, 66°C, 67°C, 68°C, 69°C, 70°C, etc.; the time is 20-40min, for example, it can be 20min, 22min, 25min, 28min, 30min, 32min, 34min, 35min, 36min, 38min, 39min, 40min, etc. Other specific point values within this numerical range can be selected, and they will not be repeated here.

[0031] The present invention also unexpectedly discovered that when the inactivation conditions of the prepared lactic acid bacteria postbiotic preparation are within the above-mentioned specific range, the ultimately obtained lactic acid bacteria postbiotic preparation has better vaginal adhesion and colonization capabilities.

[0032] Preferably, the inactivation agent is mixed with the inactivation protective agent before the inactivation treatment.

[0033] In the present invention, the inactivation protective agent is added to protect the bacterial structure in the subsequent inactivation treatment. Exemplary but not limited to the following types: mannitol, trehalose, polydextrose, sodium citrate, sucrose, skimmed milk powder, sorbitol, sodium chloride and other components with relevant functions can all be added as the inactivation protective agent described in the present invention.

[0034] Preferably, the amount of the inactivation protective agent added is 1%-3% of the culture medium in mass percentage, for example, it can be 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, etc. Other specific point values within this numerical range can be selected and will not be repeated here.

[0035] In a fourth aspect, the present invention provides a postbiotic that adapts to the vaginal environment and strongly adheres to vaginal epithelial cells, wherein the postbiotic is prepared by the preparation method described in the third aspect.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention provides a novel application of L-arabinose in the preparation of a preparation for enhancing the vaginal adhesion of lactic acid bacteria. By adding the preparation during a specific growth phase of the lactic acid bacteria, the adhesion and colonization abilities of the cultured lactic acid bacteria are enhanced. During this growth phase, the bacteria experience rapid growth, vigorous metabolism, and active enzyme systems. The addition of the preparation provides a nutrient foundation for microbial growth during this period, further facilitating rapid bacterial proliferation and the stable formation of surface proteins and other metabolites. L-arabinose, as a prebiotic, not only promotes the proliferation of lactic acid bacteria, but the present invention also unexpectedly discovered that it has an excellent promoting effect on enhancing the adhesion and colonization abilities of lactic acid bacteria on vaginal epithelial cells. DETAILED DESCRIPTION

[0038] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] The following bacterial species are involved:

[0040] Lactobacillus plantarum ATCC 8014; Lactobacillus acidophilus ATCC 4796; Lactobacillus salivarius CICC 25161;

[0041] The soybean oligosaccharides mentioned below were purchased from Shanghai Yuanye Biotechnology Co., Ltd., model number S25625; RPMI-1640 cell culture medium was purchased from Thermo Fisher Scientific; vaginal epithelial cells were human vaginal epithelial cells VK2 / E6E7 (ATCC-CRL2616); and artificial simulated synthetic vaginal fluid was purchased from Chemazone Inc., model number BZ181.

[0042] The culture method of each lactic acid bacteria seed liquid involved below is as follows: Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus salivarius preserved in 50% glycerol are inoculated into MRS liquid culture medium respectively, and cultured at 37°C for 24 hours. The culture liquid is dipped into an inoculation needle and streaked on an MRS solid culture medium, and then cultured inverted at 37°C until a single colony grows. The single colony is picked and inoculated into MRS liquid culture medium, and cultured at 37°C for 24 hours to obtain seed liquid of three lactic acid bacteria: Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus salivarius.

[0043] Example 1

[0044] This embodiment provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells, and the preparation method thereof is as follows:

[0045] The plant lactic acid bacteria seed liquid was inoculated into MRS liquid medium at an inoculum volume of 2.5% (v / v) and cultured at 37°C until the OD value of the culture medium reached 0. 600When the value is 0.5, the preparation (L-arabinose) is added and cultured at 37°C for 18 hours. The concentration of the preparation in the culture medium is 0.8 g / L. The cultured Lactobacillus plantarum culture is centrifuged at 5000 rpm for 5 minutes to collect the bacteria, and the bacteria are washed three times by centrifugation with PBS solution (8000 rpm, 10 minutes) to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0046] Example 2

[0047] This embodiment provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells, and the preparation method thereof is as follows:

[0048] The Lactobacillus acidophilus seed liquid was inoculated into MRS liquid medium at a 4% (v / v) inoculation rate and cultured at 35°C until the OD value of the culture medium reached 0. 600 When the value is 0.4, the preparation (L-arabinose) is added and cultured at 38°C for 15 hours. The concentration of the preparation in the culture medium is 0.6 g / L. The cultured Lactobacillus acidophilus culture is centrifuged at 3500 rpm for 8 minutes to collect the bacteria, and the bacteria are washed three times by centrifugation with PBS solution (8000 rpm, 10 minutes) to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0049] Example 3

[0050] This embodiment provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells, and the preparation method thereof is as follows:

[0051] Saliva combined with Lactobacillus seed solution was inoculated into MRS liquid medium at an inoculum volume of 1.5% (v / v) and cultured at 38°C until the OD value of the culture medium reached 0. 600 When the value is 0.6, the preparation (L-arabinose) is added and cultured at 35°C for 18 hours. The concentration of the preparation in the culture medium is 1 g / L. The saliva obtained by culture and the lactobacillus culture are combined and centrifuged at 6500 rpm for 3 minutes to collect the bacteria. The bacteria are washed three times by centrifugation with PBS solution (8000 rpm, 10 minutes) to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0052] Example 4

[0053] This embodiment provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 1 only in that the preparation is a combination of L-arabinose and soybean oligosaccharides in a mass ratio of 1:1. The total amount of the preparation remains the same as that of Example 1, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0054] Example 5

[0055] This embodiment provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 4 only in that the mass ratio of L-arabinose to soybean oligosaccharides is 5:1, and the total amount of the preparation remains unchanged. Subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0056] Example 6

[0057] This embodiment provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 4 only in that the mass ratio of L-arabinose to soybean oligosaccharides is 1:5, and the total amount of the preparation remains unchanged. Subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0058] Example 7

[0059] This embodiment provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 4 only in that the concentration of the preparation (L-arabinose + soybean oligosaccharides) in the culture medium is 0.2 g / L, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0060] Example 8

[0061] This embodiment provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 4 only in that the concentration of the preparation (L-arabinose + soybean oligosaccharides) in the culture medium is 2 g / L, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0062] Example 9

[0063] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells. The postbiotic is prepared by using the Lactobacillus plantarum culture obtained in Example 1, adding 2.5% mannitol to the culture at a ratio of 1:1 (v:v), and inactivating the lactic acid bacteria in a 65°C water bath for 30 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0064] Example 10

[0065] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells. The postbiotic is prepared using the Lactobacillus plantarum culture obtained in Example 1. The preparation method differs from that of Example 9 only in that the inactivation of the Lactobacillus is carried out at 60° C. for 40 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0066] Example 11

[0067] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells. The postbiotic is prepared using the Lactobacillus plantarum culture obtained in Example 1. The preparation method differs from that of Example 9 only in that the Lactobacillus is inactivated at 70°C for 20 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0068] Example 12

[0069] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells. The postbiotic is prepared using the Lactobacillus plantarum culture obtained in Example 1. The preparation method differs from that of Example 9 only in that the inactivation of the Lactobacillus is carried out at 50° C. for 30 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0070] Example 13

[0071] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells. The postbiotic is prepared using the Lactobacillus plantarum culture obtained in Example 1. The preparation method differs from that of Example 9 only in that the inactivation of the Lactobacillus is carried out at 80° C. for 30 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0072] Comparative Example 1

[0073] This comparative example provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 4 only in that the lactic acid bacteria seed liquid is directly added to a liquid culture medium mixed with a preparation (L-arabinose + soybean oligosaccharides), and the concentration of the preparation in the culture medium is 0.8 g / L. Subsequently, subsequent treatment is performed to culture the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0074] Comparative Example 2

[0075] This comparative example provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells. The difference between its preparation method and that of Example 4 is that the preparation (L-arabinose + soybean oligosaccharide) is 0.0447 W / v in the culture medium. 600 The lactic acid bacteria having strong adhesion to vaginal epithelial cells are cultured and added when the value is 0.8, and then subsequent treatment is performed to obtain the lactic acid bacteria having strong adhesion to vaginal epithelial cells.

[0076] Comparative Example 3

[0077] This comparative example provides a lactic acid bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof is different from that of Example 1 only in that the L-arabinose in Example 1 is replaced with an equal amount of soybean oligosaccharides, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adheres to vaginal epithelial cells.

[0078] Comparative Example 4

[0079] This comparative example provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells. The preparation method thereof is different from that of Example 4 in that no preparation is added when culturing the plant lactobacillus liquid in Example 1, and the culture liquid is cultured until OD 600 When the value is 0.5, the culture is continued at 37° C. for 18 hours, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0080] Comparative Example 5

[0081] This comparative example provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells. The difference between its preparation method and that of Example 2 is that in Example 2, no preparation is added when culturing the Lactobacillus acidophilus liquid. The culture liquid is cultured until the OD value is 0. 600 When the value is 0.4, the culture is continued at 38° C. for 15 hours, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0082] Comparative Example 6

[0083] This comparative example provides a lactic acid bacterium that strongly adheres to vaginal epithelial cells. The difference between its preparation method and that of Example 3 is that in Example 3, no preparation is added when culturing the saliva combined with Lactobacillus bacterial liquid. The culture liquid is cultured until OD 600 When the value is 0.6, the culture is continued at 35° C. for 18 hours, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0084] Comparative Example 7

[0085] This comparative example provides a postbiotic that strongly adheres to vaginal epithelial cells. The preparation method thereof differs from that of Example 9 only in that no preparation is added when culturing the Lactobacillus plantarum liquid in Example 1. The remaining steps and parameters remain unchanged from Example 1. 2.5% mannitol is added to the prepared Lactobacillus plantarum liquid at a ratio of 1:1 (v:v), and the lactic acid bacteria are inactivated in a 65°C water bath for 30 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0086] Test Example 1

[0087] Hydrophobicity test

[0088] (1) The lactic acid bacteria prepared in Examples 1-8, Comparative Examples 1-7, and the lactic acid bacteria postbiotics prepared in Examples 9-13 were centrifuged and the bacterial solution concentration was adjusted to OD 600 =0.8 (denoted as A0), and subsequent tests were performed.

[0089] (2) After 1 mL of hydrophobic agent (dodecane, xylene, chloroform) was fully mixed with 3 mL of the above bacterial solution, the mixture was allowed to stand at room temperature (26°C) for 20 min to allow the organic phase and aqueous phase to be fully separated. The organic phase was removed and the OD of the aqueous phase of each group was tested using PBS buffer as the control group. 600Value, denoted as A t The test was repeated three times, and the hydrophobicity of each group of lactic acid bacteria (postbiotics) was calculated according to the following formula. The results are shown in Table 1.

[0090] Hydrophobicity (%) = [(A0-A t / A0]×100%

[0091] Table 1

[0092]

[0093]

[0094] Cell hydrophobicity is an important prerequisite for probiotics to adhere to and colonize in reproductive tract epithelial cells to play a probiotic role. The data in the table show that

[0095] (1) The lactic acid bacteria and lactic acid bacteria postbiotic preparations prepared by the present invention are both highly hydrophobic. L-arabinose and soybean oligosaccharides can both improve the hydrophobicity of the bacteria to varying degrees, among which L-arabinose has a better improvement effect. It is also found that when the two are used in combination, they can synergistically enhance the hydrophobicity of the bacteria.

[0096] (2) By comparing Example 4 with Examples 7-8, it can be seen that when the dosage of the preparation is not within the specific range, the hydrophobicity of the lactic acid bacteria finally cultured is worse than that of Example 4. It can be seen that the use of a specific dosage of the preparation in the present invention can further improve the hydrophobicity of the cultured lactic acid bacteria.

[0097] (4) By comparing Example 9 with Examples 12-13, it can be seen that when the inactivation temperature in the preparation process of the lactic acid bacteria postbiotic preparation is not within a specific range, the hydrophobicity of the lactic acid bacteria postbiotics prepared therefrom is worse than that of Example 9. It can be seen that the specific inactivation treatment conditions adopted in the present invention can further improve the hydrophobicity of the postbiotic preparation.

[0098] (5) By comparing Example 4 with Comparative Examples 1-2, it can be seen that when the preparation is not added at a specific growth stage, the hydrophobic properties of the lactic acid bacteria cultured are not as good as those of Example 4. It can be seen that the addition of the preparation at a specific growth stage of the bacteria in the present invention is beneficial to ensure the stable formation of hydrophobic proteins on the surface of the bacteria and further improve their hydrophobic properties.

[0099] Test Example 2

[0100] ①Hela cell adhesion performance test

[0101] (1) The lactic acid bacteria prepared in Examples 1-8 and Comparative Examples 1-7 and the lactic acid bacteria postbiotics prepared in Examples 9-13 were centrifuged and the cells were suspended in RPMI-1640 cell culture medium without double antibodies and calf serum and the bacterial concentration was adjusted to 5×10 8 CFU / g or 5×10 8 After that, conduct follow-up tests.

[0102] (2) Take out the cryopreservation tube of frozen Hela cells from liquid nitrogen and quickly place it in a thermos cup filled with 37°C warm water. Then, in the cell operation room, inoculate the frozen cells into DMEM cell culture medium containing 10% bovine serum and 0.5% double antibody (penicillin-streptomycin) pre-warmed at 37°C, and culture them in a 37°C incubator containing 5% CO2. Change the cell culture medium every 24 hours. After the Hela cells have filled the cell culture flask, digest them with 1mL of 0.25% trypsin-EDTA solution for 2 minutes, gently beat the cells to detach them from the culture flask, wash the cells with 5mL of DMEM cell culture medium, and pipette to evenly distribute them to obtain a cell suspension.

[0103] (3) Take a 6-well cell culture plate, add a sterile coverslip and 1 mL of DMEM culture medium to each well, and then add 1 mL of the above cell suspension to each well. Place the cell culture plate in a 37°C, 5% CO2 incubator for culture. When the Hela cells on the coverslip in the cell culture plate are 80% confluent, switch to RPMI-1640 cell culture medium without dual antibody and culture for 24 hours. Wash the cells three times with PBS.

[0104] (4) Add 1 mL of RPMI-1640 cell culture medium without double antibody and calf serum and 1 mL of the lactic acid bacteria suspension or lactic acid bacteria postbiotic suspension diluted in step (1) to each well of the cell culture plate, and gently shake the cell culture plate to evenly disperse the bacteria. The cell culture plate was placed in a 37°C, 5% CO2 incubator for 2 hours. The coverslip was removed from the cell culture plate, washed three times with pre-cooled PBS solution, dried naturally at room temperature (25°C), fixed with methanol for 10 minutes, and Gram-stained. 50 intact Hela cells were randomly selected under an oil immersion lens, and the number of bacteria attached to the cells was counted. The Hela cell adhesion coefficient was calculated. The results are shown in Table 1.

[0105] ② Vaginal epithelial cell adhesion test

[0106] (1) The lactic acid bacteria prepared in Examples 1-8 and Comparative Examples 1-4 and the lactic acid bacteria postbiotics prepared in Examples 9-13 were centrifuged and the cells were suspended in RPMI-1640 cell culture medium without double antibodies and calf serum and the bacterial concentration was adjusted to 2×10 7 CFU / g or 2×10 7After that, conduct follow-up tests.

[0107] (2) Vaginal epithelial cells were suspended in 10 mL of RPMI-1640 cell culture medium without double antibody and calf serum, and cultured in a cell culture incubator at 37°C and 5% CO2 until the cell density reached 1×10 5 1 mL of vaginal epithelial cell suspension was mixed with 1 mL of the lactic acid bacteria suspension or lactic acid bacteria postbiotic suspension diluted in step (1), cultured at 37°C with shaking for 8 h, centrifuged at 1000 rpm for 5 min, washed three times with PBS buffer, and lactic acid bacteria that did not adhere to or guide the epithelial cells were discarded. The precipitate was dried and smeared for Gram staining. Fifty vaginal epithelial cells were randomly counted under an oil immersion lens, and the number of bacteria adhering to the epithelial cells was counted. The results are shown in Table 2.

[0108] Table 2

[0109]

[0110] According to the data in the table,

[0111] (1) It can be seen from Examples 1-3 and Examples 9-11 that the lactic acid bacteria and lactic acid bacteria postbiotic preparations prepared by the present invention have excellent adhesion ability to Hela cells and vaginal epithelial cells, wherein Hela cells are a cell line of cervical cancer cells. The excellent Hela cell adhesion ability can also prove to a certain extent that the lactic acid bacteria and lactic acid bacteria postbiotic preparations obtained by culture of the present invention can achieve high-efficiency adhesion and colonization of the reproductive tract.

[0112] (2) By comparing Examples 1-3, Examples 4-6, Example 9 and Comparative Examples 3-7, it can be seen that both L-arabinose and soybean oligosaccharides can enhance the adhesion ability of bacteria to the two epithelial cells to varying degrees, among which L-arabinose has a better enhancement effect. In addition, it was found that when the two are used in combination, they can synergistically enhance the adhesion performance of lactic acid bacteria to reproductive tract epithelial cells.

[0113] (3) By comparing Example 4 with Examples 7-8, it can be seen that when the dosage of the preparation is not within a specific range, the adhesion ability of the lactic acid bacteria finally cultured to the two epithelial cells is worse than that of Example 4. It can be seen that the use of a specific dosage of the preparation in the present invention can further enhance the adhesion ability of the cultured lactic acid bacteria to the reproductive tract epithelial cells.

[0114] (4) By comparing Example 9 with Examples 12-13, it can be seen that when the inactivation temperature during the preparation of the lactic acid bacteria postbiotic preparation is not within a specific range, the adhesion ability of the lactic acid bacteria postbiotics prepared therefrom is worse than that of Example 9. It can be seen that the specific inactivation treatment conditions adopted in the present invention can further improve the adhesion performance of the postbiotic preparation.

[0115] (5) By comparing Example 4 with Comparative Examples 1-2, it can be seen that when the preparation is not added at a specific growth stage, the adhesion performance of the lactic acid bacteria cultured therefrom is not as good as that of Example 4. It can be seen that the addition of the preparation at a specific growth stage of the bacteria in the present invention is beneficial to ensure the rapid proliferation of the bacteria and the stable formation of surface proteins and other metabolites, further enhancing the adhesion and colonization ability of lactic acid bacteria on reproductive tract epithelial cells.

[0116] Test Example 3

[0117] Lactic acid bacteria postbiotics simulated reproductive tract adaptability test

[0118] The lactic acid bacteria postbiotics prepared in Examples 9-13 were resuspended in PBS buffer, mixed with artificial simulated synthetic vaginal fluid, and the bacterial concentration was adjusted to 1×10 9 (individuals / g), incubated at 37°C, and the changes in bacterial counts were detected 1 hour and 8 hours after mixing, and the detection rate was calculated according to the following formula. The results are shown in Table 3.

[0119] Bacteria detection rate (%) = number of detected bacteria / initial number of bacteria × 100%

[0120] Table 3

[0121]

[0122] The data in the table show that after the lactic acid bacteria cultured in the present invention are prepared into a postbiotic preparation, the bacterial count can be directly placed in a simulated vaginal environment for 8 hours without a significant decrease. This shows that the lactic acid bacteria postbiotics prepared in the present invention can adapt well to the vaginal environment.

[0123] In summary, the present invention enhances the adhesion and colonization abilities of cultured lactic acid bacteria by adding a preparation during a specific growth phase. During this phase, when the bacteria experience rapid growth, high metabolism, and active enzymes, the addition of the preparation provides a nutrient foundation for microbial growth, further facilitating rapid bacterial proliferation and the stable formation of surface proteins and other metabolites. L-arabinose, as a prebiotic, not only promotes the proliferation of lactic acid bacteria, but the present invention also unexpectedly discovered that it has an excellent promoting effect on the adhesion and colonization abilities of lactic acid bacteria in the vagina.

[0124] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0125] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0126] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Application of L-arabinose in the preparation of a preparation for improving the adhesion of lactic acid bacteria to vaginal epithelial cells; The lactic acid bacteria is selected from any one of Lactobacillus plantarum, Lactobacillus acidophilus or Lactobacillus salivarius.

2. The use according to claim 1, characterized in that The preparation is L-arabinose or a combination of L-arabinose and soybean oligosaccharides.

3. The use according to claim 2, characterized in that The mass ratio of the L-arabinose to soybean oligosaccharides is 1:5-5:

1.

4. A method for culturing lactic acid bacteria that adapt to the vaginal environment and strongly adhere to vaginal epithelial cells, characterized in that: The method comprises the following steps: The lactic acid bacteria seed liquid obtained by culture was inoculated into the liquid culture medium and cultured statically until the OD 600 When the value is 0.3-0.6, the preparation is added and culture is continued, and the lactic acid bacteria that strongly adhere to the vaginal epithelial cells are obtained after the end; the preparation includes L-arabinose; The lactic acid bacteria is selected from any one of Lactobacillus plantarum, Lactobacillus acidophilus or Lactobacillus salivarius.

5. The method according to claim 4, characterized in that The concentration of the preparation in the culture medium is 0.6-1 g / L.

6. The method according to claim 4 or 5, characterized in that Soy oligosaccharides are also included in the formulation.

7. The method according to claim 6, wherein the mass ratio of L-arabinose to soybean oligosaccharides is 1:5-5:

1.

8. A method for preparing a postbiotic that adapts to the vaginal environment and strongly adheres to vaginal epithelial cells, characterized in that: The method comprises inactivating the lactic acid bacteria that strongly adhere to vaginal epithelial cells and are obtained by the method according to any one of claims 4 to 7 to obtain the postbiotics that strongly adhere to vaginal epithelial cells.

9. The preparation method according to claim 8, characterized in that The temperature of the inactivation treatment is 60-70° C., and the time is 20-40 min.

10. The method for preparing postbiotics according to claim 8, characterized in that: The inactivation treatment is also preceded by mixing with an inactivation protective agent.

11. The method for preparing postbiotics according to claim 10, characterized in that: The added amount of the inactivation protective agent is 1%-3% of the culture solution in terms of mass percentage.

12. A postbiotic that adapts to the vaginal environment and strongly adheres to vaginal epithelial cells, characterized in that: The postbiotics are prepared by the preparation method according to any one of claims 8 to 11.

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