Culture method of lactic acid bacteria adapting to vaginal environment and strongly adhering to vaginal epithelial cells

Abstract

The invention relates to a culture method of lactic acid bacteria adapting to vaginal environment and strongly adhering to vaginal epithelial cells, which comprises the following steps: inoculating a lactic acid bacteria seed solution obtained by culture into a liquid culture medium, standing and culturing until the OD600 value of the culture solution is 0.3-0.6, and adding a preparation for continuous culture, and obtaining the lactic acid bacteria strongly adhering to the vaginal epithelial cells after the fermentation is finished. The preparation comprises L-arabinose. The invention further relates to application of the L-arabinose in preparation of a preparation for improving the vaginal epithelial cell adhesion capacity of the lactic acid bacteria, and by adding the related preparation in the specific growth stage of the lactic acid bacteria, rapid proliferation of thalli and stable formation of surface hydrophobic protein can be guaranteed; therefore, the adhesion and planting capabilities of the cultured lactic acid bacteria are further improved.

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 vaginal environment and strongly adhere to vaginal epithelial cells. Background Art

[0002] There is an important relationship between vaginal microecological balance and female reproductive tract diseases. Lactobacillus is the main bacteria that colonizes the vagina of most women and plays a key role in maintaining female vaginal health. As the dominant flora in the vaginal microecology, lactic acid bacteria can not only maintain the health of vaginal microecology, but also prevent vaginal infection, playing an irreplaceable role in resisting the invasion of pathogenic microorganisms. In the vagina of healthy women, the abundance of lactic acid bacteria usually occupies a dominant position to maintain the balance of vaginal microecology. Common lactic acid bacteria include Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus johnsonii; various bacteria interact and restrict each other to achieve a dynamic balance and maintain physical health.

[0003] When the microecological balance of the reproductive tract is disrupted, the dominant bacteria will decrease and the pathogens will gradually increase, which will cause vaginal inflammation. In the past, the main clinical treatment for vaginal infection was antibiotic treatment, and the antibiotic treatment method is to indiscriminately kill vaginal microorganisms, including probiotics and some non-pathogenic vaginal organisms. This treatment method will significantly change the vaginal microbial environment, causing dysbiosis, resulting in poor treatment effect and high recurrence rate. The prerequisite for probiotics to play a probiotic role in the reproductive tract is that they can adhere to and colonize on the surface of reproductive tract cells, and inhibit reproductive tract pathogens by occupying space, preventing the colonization of other harmful bacteria, regulating the balance of vaginal flora, and achieving the purpose of preventing and assisting in regulating diseases. The adhesion ability of lactic acid bacteria is related to many factors, such as adhesins, cell surface proteins, capsular polysaccharides, and the ability of bacteria 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 a problem that needs to be solved urgently. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object 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 the preparation of 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 improving the adhesion and colonization ability of lactic acid bacteria to 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 then 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 probiotic 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 solution obtained by culture was inoculated into the liquid culture medium and cultured statically until the OD of the culture medium reached 600 When the value is 0.3-0.6, the preparation is added to continue culturing, and the lactic acid bacteria that strongly adhere to vaginal epithelial cells are obtained after the end; the preparation includes L-arabinose.

[0013] In the present invention, the adhesion and colonization ability of the cultured lactic acid bacteria is improved by adding the preparation at a specific growth stage of the lactic acid bacteria. The bacterial growth rate at this growth stage is fast, the metabolism is vigorous, and the enzyme system is active. The addition of the preparation provides a nutrient basis for the growth of microorganisms during this period, which is more conducive to ensuring the rapid proliferation of the bacteria and the stable formation of surface hydrophobic proteins and other metabolites. L-arabinose, as a prebiotic, can not only promote the proliferation of lactic acid bacteria, but also unexpectedly found in the present invention that it has an excellent promoting effect in improving the adhesion and colonization ability of lactic acid bacteria to vaginal epithelial cells.

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

[0015] When the dosage 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 improved.

[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 then 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 probiotic 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, inverting and culturing at 36-38° C. until a single colony grows, picking a single colony and inoculating it into a liquid culture medium, and statically culturing 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 point values ​​within the numerical range can be selected, and they will not be described one by one here;

[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 the numerical range can be selected, and they will not be described one by one here.

[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%, 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, which will not be repeated here.

[0024] Preferably, the temperature of the continued 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., 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 described one by one 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 the numerical range can be selected, which 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 the numerical range can be selected, which will not be described here one by one.

[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 finally obtained lactic acid bacteria postbiotic preparation has better vaginal adhesion and colonization ability.

[0032] Preferably, the inactivation protective agent is mixed 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, and the following types are exemplified but not limited to: 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 added amount of the inactivation protective agent 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, which 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] In the present invention, a new application of L-arabinose in the preparation of a preparation for improving the vaginal adhesion ability of lactic acid bacteria is provided. The adhesion and colonization ability of the cultured lactic acid bacteria is improved by adding the preparation at a specific growth stage of the lactic acid bacteria. The bacterial growth rate in this growth stage is fast, the metabolism is vigorous, and the enzyme system is active. The addition of the preparation provides a nutrient basis for the growth of microorganisms during this period, which is more conducive to ensuring the rapid proliferation of the bacterial body and the stable formation of surface proteins and other metabolites. As a prebiotic, L-arabinose can not only promote the proliferation of lactic acid bacteria, but also unexpectedly found in the present invention that it has an excellent promoting effect in improving the adhesion and colonization ability of lactic acid bacteria to vaginal epithelial cells. DETAILED DESCRIPTION

[0038] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is 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 bacteria 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 the 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 the culture is statically cultured at 37°C for 24 hours, and the culture liquid is dipped into an inoculation needle to streak on an MRS solid culture medium, and then inverted and cultured at 37°C until a single colony grows, and a single colony is picked and inoculated into MRS liquid culture medium, and the culture is statically cultured at 37°C for 24 hours to obtain the seed liquid of the three lactic acid bacteria, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus salivarius.

[0043] Example 1

[0044] This embodiment provides a lactic acid bacteria 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 culture liquid OD reached 600When the value is 0.5, the preparation (L-arabinose) is added and cultured for 18 hours at 37°C. 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 min to collect the bacteria, and the bacteria are washed three times by centrifugation with PBS solution (8000 rpm, 10 min) to obtain the lactic acid bacteria that strongly adhere to vaginal epithelial cells.

[0046] Example 2

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

[0048] The Lactobacillus acidophilus seed solution was inoculated into MRS liquid medium at a 4% (v / v) inoculation rate and cultured at 35°C until the OD of the culture medium reached 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 bacteria that strongly adheres to vaginal epithelial cells, and the preparation method thereof is as follows:

[0051] The 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 of the culture medium reached 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 combined with the lactobacillus culture liquid obtained by culture is centrifuged at 6500 rpm for 3 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.

[0052] Example 4

[0053] This embodiment 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 preparation is a combination of L-arabinose and soybean oligosaccharides in a mass ratio of 1:1, and 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 is different 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, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adheres 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 is different 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, and subsequent treatment is performed to obtain the lactic acid bacteria that strongly adheres 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 is different 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 adheres 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 is different 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 adheres to vaginal epithelial cells.

[0062] Example 9

[0063] The present embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells, which uses the plant lactobacillus culture obtained in Example 1, adds 2.5% mannitol to the culture at a ratio of 1:1 (v:v), and inactivates 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, which uses the plant lactobacillus cultured in Example 1. The preparation method is different from that of Example 9 only in that the condition for inactivating the lactobacillus is inactivation at 60° C. for 40 minutes to obtain the postbiotic that strongly adheres to vaginal epithelial cells.

[0066] Embodiment 11

[0067] This embodiment provides a postbiotic that strongly adheres to vaginal epithelial cells, which uses the plant lactobacillus cultured in Example 1. The preparation method is different from that of Example 9 only in that the condition for inactivating the lactobacillus is inactivation 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, which uses the plant lactobacillus liquid cultured in Example 1. The preparation method is different from that of Example 9 only in that the condition for inactivating the lactobacillus is inactivation 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, which uses the plant lactobacillus cultured in Example 1. The preparation method is different from that of Example 9 only in that the condition for inactivating the lactobacillus is inactivation 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 bacteria that strongly adheres to vaginal epithelial cells. The preparation method thereof is different 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. The lactic acid bacteria that strongly adhere to vaginal epithelial cells are then cultured after subsequent treatment.

[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 1.5 times the concentration of the lactic acid bacterium in the culture medium. 600 The lactic acid bacteria with strong adhesion to vaginal epithelial cells are cultured and then added when the value is 0.8, and then subsequent treatment is performed.

[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 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 h, 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 the preparation method and Example 2 is that in Example 2, no preparation is added when culturing the Lactobacillus acidophilus bacterial solution, and the culture solution is cultured until OD 600 When the value is 0.4, the culture is continued at 38° C. for 15 h, 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 preparation method thereof is different from that of Example 3 in that no preparation is added when culturing the saliva combined lactobacillus bacterial solution in Example 3, and the culture solution is cultured until OD 600 When the value is 0.6, the culture is continued at 35° C. for 18 h, 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 is different from that of Example 9 only in that no preparation is added when culturing the plant lactobacillus liquid in Example 1, and the remaining steps and parameters remain unchanged from those in Example 1. 2.5% mannitol is added to the prepared plant lactobacillus liquid at a ratio of 1:1 (v:v), and the lactic acid bacteria are inactivated in a water bath at 65°C 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 carried out.

[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 PBS buffer was used as the control group to test the OD of the aqueous phase in each 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. From the data in the table, we can see 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 a 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 lactic acid bacteria cultured.

[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 is worse than that of Example 9. It can be seen that the specific inactivation treatment conditions used 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 therefrom 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, thereby further improving its hydrophobic properties.

[0099] Test Example 2

[0100] ①Hela cell adhesion performance test

[0101] (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 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 measuring the number of pieces / g, conduct subsequent tests.

[0102] (2) Take out the cryopreserved tube of Hela cells from liquid nitrogen, quickly put it into a thermos cup filled with 37°C warm water, and then 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 in the cell operation room, and culture it 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 bottle, digest them with 1mL 0.25% trypsin-EDTA solution for 2min, gently beat the cells to detach them from the culture bottle, wash the cells with 5mL DMEM cell culture medium, and blow them evenly with a pipette 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% full, switch to RPMI-1640 cell culture medium without dual antibodies and culture for 24 hours, then 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 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, and the cover glass was removed from the cell culture plate. After washing three times with pre-cooled PBS solution, it was naturally dried at room temperature (25°C), fixed with methanol for 10 minutes, and Gram staining was performed. 50 intact Hela cells were randomly selected under an oil microscope, and the number of bacteria adhering 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 performance test

[0106] (1) The lactic acid bacteria prepared in Examples 1-8, Comparative Examples 1-4, and the lactic acid bacteria postbiotics prepared in Examples 9-13 were centrifuged and 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 measuring the number of pieces / g, conduct subsequent 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 concentration 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 for 8 h, centrifuged at 1000 rpm for 5 min, washed 3 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 microscope, 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 the 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 cultured by the present invention can achieve high-efficiency adhesion and colonization in 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 types of 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 in the preparation process 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 used 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 added at a time other than 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, thereby further enhancing the adhesion and colonization ability of lactic acid bacteria on reproductive tract epithelial cells.

[0116] Test Example 3

[0117] Lactobacillus 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℃, and detected the changes in bacterial counts 1h and 8h after mixing, and calculated the detection rate according to the following formula. The results are shown in Table 3.

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

[0120] Table 3

[0121]

[0122] It can be seen from the data in the table 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, which shows that the lactic acid bacteria postbiotics prepared by the present invention can adapt well to the vaginal environment.

[0123] In summary, in the present invention, the adhesion and colonization ability of the cultured lactic acid bacteria is improved by adding the preparation at a specific growth stage of the lactic acid bacteria. The bacterial growth rate at this growth stage is fast, the metabolism is vigorous, and the enzyme system is active. The addition of the preparation provides a nutrient basis for the growth of microorganisms during this period, which is more conducive to ensuring the rapid proliferation of the bacteria and the stable formation of surface proteins and other metabolites. L-arabinose, as a prebiotic, can not only promote the proliferation of lactic acid bacteria, but also unexpectedly found in the present invention that it has an excellent promoting effect in improving the adhesion and colonization ability of lactic acid bacteria to the vagina.

[0124] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope 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 suitable 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 preparations for improving the adhesion ability of lactic acid bacteria to vaginal epithelial cells.

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 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 solution obtained by culture was inoculated into the liquid culture medium and cultured statically until the OD of the culture medium reached 600 When the value is 0.3-0.6, the preparation is added to continue culturing, and the lactic acid bacteria that strongly adhere to vaginal epithelial cells are obtained after the end; the preparation includes L-arabinose.

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 obtained by the method according to any one of claims 4 to 7 to obtain the postbiotics that strongly adhere to vaginal epithelial cells; Preferably, the inactivation treatment is carried out at a temperature of 60-70°C and for a time of 20-40 minutes.

9. The method for preparing postbiotics according to claim 8, characterized in that: Before the inactivation treatment, the inactivation protective agent is mixed; Preferably, the added amount of the inactivation protective agent is 1%-3% of the culture solution in terms of mass percentage.

10. 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 claim 8 or 9.

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