CN-122024810-A - In-vitro evaluation model for evaluating bacteriostasis effect of microbial lotion based on microcalorimetry and application of in-vitro evaluation model

CN122024810ACN 122024810 ACN122024810 ACN 122024810ACN-122024810-A

Abstract

The invention discloses an in-vitro evaluation model of a vulva mode microbiota for evaluating the antibacterial effect of a microbial lotion based on a microcalorimetry and application thereof. By co-culturing representative vulva microorganisms, a healthy external female model of the microbiota comprising lactobacillus crispatus, lactobacillus grignard and staphylococcus epidermidis is constructed, and staphylococcus aureus, escherichia coli and candida albicans are introduced on the basis of the healthy external female model of the microbiota, so that a non-healthy external female model of the microbiota is constructed. The high-sensitivity isothermal microcalorimetry is utilized to conduct real-time and nondestructive monitoring on the metabolic process of the system, a power-time curve is obtained, and thermodynamic parameters of the power-time curve are analyzed, so that quantitative and qualitative evaluation on the bacteriostasis effect, bacteriostasis dynamics and selective inhibition effect on beneficial bacteria and pathogenic bacteria of different microbial lotions is achieved. The invention overcomes the defects of long time consumption, limited information quantity, incapability of dynamically reflecting the change of flora and the like of the traditional antibacterial detection method, and provides a brand-new, efficient and reliable in-vitro evaluation platform for the research and development of vulva lotion and related products with micro-ecological regulation functions.

Inventors

ZHANG YUE
SHI QUAN
Zhu Meijin
LI YI

Assignees

浙江工商大学
上海东利大健康研究院有限公司

Dates

Publication Date: 20260512
Application Date: 20251218

Claims (10)

1. An external evaluation model for evaluating the antibacterial effect of microbial lotions based on microcalorimetry for a vulva mode of microbial population is characterized in that the establishment process of the external evaluation model comprises the following steps: (a) Selecting model strains, collecting bacterial suspension of each model strain, and stably preserving to establish a standard strain library; (b) Screening culture mediums, namely analyzing thermal parameters of different culture mediums by microcalorimetry, and selecting an optimal culture medium for establishing an external evaluation model of a vulva mode microorganism population; (c) The construction of an external model for evaluating the microbial population of the external female mold, which comprises the steps of monitoring the biological metabolic process of mixed culture of each single strain and multiple strains by a microcalorimetry, analyzing the power-time curve of the biological metabolic process, and establishing a thermodynamic model of the biological metabolic process; (d) And (3) evaluating the antibacterial effect of the microbial lotion, namely comparing the regulating effect of the microbial lotion product on the human vulva microecology by taking the external evaluation model of the vulva mode microbial population established in the step (c) as a reference control.
2. The external evaluation model for vulva pattern microorganism group based on microcalorimetry for evaluation of bacteriostasis effect of microorganism washings according to claim 1, wherein the step (a) specifically comprises the following procedures: a1, summarizing and analyzing the composition condition of the vulva microorganisms and pathogenic bacteria thereof, and selecting a model strain of the vulva microorganisms; a2, using MRS culture medium, standing and culturing in a constant temperature incubator at 37 ℃ for 24 hours, using LB culture medium, staphylococcus epidermidis, staphylococcus aureus and escherichia coli, using glucose culture medium, and shaking and culturing (150 rpm) candida albicans at 37 ℃ for 24 hours; a3, performing primary subculture on each single strain bacterial suspension obtained in the step a1 to obtain active thalli in a logarithmic growth phase; a4, washing the thalli harvested in the step a2 in normal saline (0.9% NaCl solution) and re-suspending the thalli in 15% (v/v) glycerol to ensure that the final organism density is 10 8 CFU/mL, wherein the concentration of the thalli is measured by a ten-fold serial dilution method and a plate colony counting method; a5, the bacterial suspension obtained in the step a3 is aliquoted into sterile cryopreservation tubes and stored at-80 ℃ until needed.
3. A vulva model for evaluating the antibacterial effect of a microbial lotion based on microcalorimetry as claimed in claim 2, wherein, In the step a1, 6 strains of lactobacillus crispatus, lactobacillus grignard, staphylococcus epidermidis, staphylococcus aureus, escherichia coli and candida albicans are selected as model strains of a vulva mode microbiota; In the step a2, the MRS culture medium, the LB culture medium and the Saccharum sinensis Roxb culture medium are subjected to high-temperature sterilization at 121 ℃ for 15 min.
4. The vulva model for evaluating the antibacterial effect of a microbial lotion based on microcalorimetry as claimed in claim 1, wherein, The step (b) of screening culture mediums comprises the steps of analyzing thermodynamic parameters of different culture mediums through microcalorimetry, and selecting an optimal culture medium for establishing an external evaluation model of a vulva mode microorganism population, wherein the method specifically comprises the following steps of: b1, preparing an MRS culture medium and a BHI modified culture medium which are suitable for lactobacillus culture; b2, taking out the bacterial suspension stored in the step a4, immersing the bacterial suspension in a water bath at 40 ℃ for thawing for 3 minutes, and swirling the bacterial suspension for 1 minute; b3, 30. Mu.L of thawed strain culture was diluted with physiological saline and inoculated into sterile 4mL of a calorimetric glass ampoule containing 2970. Mu. LMRS medium or BHI modified medium for pure culture experiments of the single strain. For the mixed co-culture experiment, each strain is added into the same amount of hot glass ampoule; b4, sealing the ampoule by using a crimp cover and swirling for 10 seconds, placing the sample ampoule in a constant temperature pool of an isothermal microcalorimeter with stable front baseline, pushing the sample ampoule into a measuring pool after 15 minutes of temperature stabilization, stabilizing the instrument baseline after 45 minutes, and starting to monitor and record the biological metabolism heat of the sample in real time; And b5, after the step b4 is finished, drawing power-time curves and heat maps of each single strain and mixed culture, and fitting to obtain thermodynamic parameters.
5. The external evaluation model for vulva pattern microorganisms based on microcalorimetry for evaluating bacteriostatic effect of microbial lotions according to claim 4, wherein, The BHI modified culture medium in the step b1 is optimized on the BHI culture medium, comprising adding 20g/L glucose, 1mL/L Tween 80 and 5g/L yeast powder, and adjusting the pH to 6.5; In the step b2, the MRS culture medium and the BHI modified culture medium are subjected to high-temperature sterilization treatment at 121 ℃ for 15 min; In the step b3, the bacterial suspension with the concentration of 10 6 CFU/mL of single bacterial strain is used for pure culture of the single bacterial strain, and the bacterial suspension with the concentration of 10 6 CFU/mL of each bacterial strain after mixing is used for mixed co-culture of multiple bacterial strains; in the step b4, the model of an isothermal microcalorimeter is TAM IV, and the temperature is set to be 37+/-0.1 ℃ and the sterile culture medium is used as a blank reference; in step b5, statistical and fitting analysis was performed with Origin 2024 software.
6. The vulva model for evaluating the antibacterial effect of a microbial lotion based on microcalorimetry as claimed in claim 1, wherein, The construction of the evaluation model outside the vulva mode microorganism population in the step (c) comprises the steps of monitoring the biological metabolic process of mixed culture of each single strain and multiple strains by microcalorimetry, analyzing the power-time curve of the biological metabolic process, and establishing a thermodynamic model of the biological metabolic process, wherein the thermodynamic model comprises the following steps: c1, establishing a single strain characteristic thermodynamic model according to the steps b2-b 5; c2, carrying out mixed culture on lactobacillus crispatus, lactobacillus grignard and staphylococcus epidermidis according to the steps b2-b5, and establishing a healthy external female model for evaluating the microbial population; And c3, adding staphylococcus aureus, escherichia coli and candida albicans on the basis of the in-vitro evaluation model of the healthy external female model microorganism group according to the steps b2-b5 for mixed culture, and establishing the in-vitro evaluation model of the unhealthy external female model microorganism group.
7. The external evaluation model for vulva pattern microorganisms based on microcalorimetry for evaluating bacteriostatic effect of microbial lotions according to claim 6, wherein, In the step c1, isothermal trace heat experiments are carried out by using a bacterial suspension with the concentration of 10 5 、10 6 、10 7 CFU/mL of a single bacterial strain, and a thermodynamic model with the characteristics of the single bacterial strain is built; In the step c2, firstly, isothermal trace heat experiments are carried out by using lactobacillus crispatus with the concentration of 10 6 CFU/mL, respectively adding 10 4 、10 5 、10 6 CFU/mL of lactobacillus crispatus and analyzing thermodynamic information of the isothermal trace heat experiments, then isothermal trace heat experiments are carried out by using lactobacillus crispatus with the concentration of 10 6 CFU/mL, respectively adding 10 4 、10 5 、10 6 CFU/mL of lactobacillus crispatus and analyzing thermodynamic information of the isothermal trace heat experiments, and the lactobacillus crispatus with the concentration of 10 5 CFU/mL and the concentration of 10 6 CFU/mL of lactobacillus crispatus are selected as optimal inoculation proportion; Or in the step c2, the lactobacillus crispatus with the concentration of 10 5 CFU/mL and the concentration of 10 6 CFU/mL are used as the optimal inoculation proportion, staphylococcus epidermidis with the concentration of 10 4 、10 5 、10 6 CFU/mL is added for isothermal trace thermal experiments and thermodynamic information analysis, and a co-culture system with the concentration of 10 5 CFU/mL, the concentration of 10 5 CFU/mL and the concentration of 10 5 CFU/mL of lactobacillus crispatus is selected for constructing a healthy vulva model microbial population external evaluation model. In the step c3, 10 5 、10 6 、10 7 CFU/mL of staphylococcus aureus, escherichia coli or candida albicans are respectively and independently added into a healthy vulva mode microorganism population external evaluation model to carry out isothermal trace heat experiments and analyze thermodynamic information of the staphylococcus aureus, escherichia coli or candida albicans; Selecting staphylococcus aureus and escherichia coli with a concentration ratio of 1:1 as optimal inoculation proportion to be added into an external evaluation model of a healthy external female model microbial population, further adding candida albicans with a concentration ratio of 10 5 、10 6 、10 7 CFU/mL for isothermal trace heat experiment, and analyzing thermodynamic information; A co-culture system with a Lactobacillus crispatus concentration of 10 5 CFU/mL, a Lactobacillus gasseri concentration of 10 6 CFU/mL, a Staphylococcus epidermidis concentration of 10 6 CFU/mL, a Staphylococcus aureus concentration of 10 7 CFU/mL, an Escherichia coli concentration of 10 7 CFU/mL and a Candida albicans concentration of 10 7 CFU/mL was selected for constructing an unhealthy vulvar model of microbial population external evaluation.
8. The vulva model for evaluating the antibacterial effect of a microbial lotion based on microcalorimetry as claimed in claim 1, wherein, In the step (d), inoculating the in vitro model of the healthy or unhealthy external female microbiota into a modified BHI culture medium containing test lotion or plant extract, wherein the lotion is a compound antibacterial formula containing snow lotus herb extract, chinese toon bud extract, fructus cnidii extract, witch hazel extract, amur corktree bark extract, radix stemonae extract, lightyellow sophora root extract and lactobacillus metabolites.
9. The external evaluation model for vulva model microorganisms for evaluating the antibacterial effect of a microbial lotion based on microcalorimetry according to claim 1, wherein isothermal microcalorimetry results are compared with a Minimum Inhibitory Concentration (MIC) and a Minimum Bactericidal Concentration (MBC) combined analysis to verify the accuracy and reliability of microcalorimetry in the evaluation of vulvar microorganisms.
10. An application of the external evaluation model of vulva mode microorganism population based on microcalorimetry for evaluating bacteriostasis effect of microorganism lotion according to claims 1-9 in screening medicines and nursing products of other human mucosa microecologics (including vagina, intestinal tract, oral cavity, etc.).

Description

In-vitro evaluation model for evaluating bacteriostasis effect of microbial lotion based on microcalorimetry and application of in-vitro evaluation model Technical Field The invention belongs to the technical field of analysis and detection, and particularly relates to an in-vitro evaluation model for a vulva mode microbiota based on a microcalorimetry for evaluating the antibacterial effect of a microbial lotion, and application of the model in evaluation of efficacy of a vulva lotion product. Background The vulva acts as the first physiological barrier of the female reproductive system and its state of health is largely dependent on a dynamically balanced and heterogeneous microflora. The community takes lactobacillus as a main dominant flora, and simultaneously accompanies the existence of various symbiotic bacteria and conditional pathogenic bacteria. Once the vulva is dysbiosis, pathogenic bacteria are hyperproliferative, and bacterial vaginosis, vulvovaginal candida diseases and other common infectious diseases can be induced. Therefore, the evaluation of the antibacterial effect of the lotion or the antibacterial agent and the influence of the lotion or the antibacterial agent on probiotics is a key link of the research and development of clinical and daily chemical products, and is also an important index for measuring the safety and effectiveness of the products. At present, the conventional antibacterial evaluation method, such as an agar diffusion method, a micro broth dilution method, a plate counting method and the like, has the defects of complicated operation flow, subjective end point judgment, incapability of reflecting dynamic process and the like. It is worth noting that the traditional methods can only evaluate the inhibition effect on single bacterial strains respectively, and are difficult to evaluate the differentiation effect of the product on beneficial bacteria and pathogenic bacteria simultaneously and quantitatively in one system, and the 'selective antibacterial' characteristic of the product cannot be objectively reflected, which is the core target of the development of the new-generation micro-ecological friendly lotion. Isothermal microcalorimetry is a detection technology based on high-sensitivity thermal sensors, and can realize real-time, continuous and nondestructive monitoring of the whole growth and metabolism process of microorganisms by measuring heat flow signals generated or absorbed by a biological system. The method is applied to the fields of environmental microorganism activity analysis, antibacterial drug screening, biological material evaluation and the like, but the method is not yet publicly reported to be applied to the field of vulva microecology at present, and is used for constructing a multi-strain in-vitro model for simulating the health and non-health states of the vulva, and evaluating the antibacterial effect of the vulva washing liquid based on the model system. According to the vulva mode microbial population external evaluation model for evaluating the antibacterial effect of the microbial lotion based on the microcalorimetry, provided by the invention, a novel in-vitro evaluation model which can simulate an in-vivo environment, realize real-time dynamic monitoring and quantitatively evaluate the antibacterial effect is constructed through culture medium screening, metabolic thermal power curve monitoring and thermodynamic analysis of co-culture of single strain and mixed strain, so that the selective inhibition characteristics of the lotion on different bacterial populations are revealed, and a scientific and systematic evaluation basis is provided for developing a private product with a microecological regulation function. Disclosure of Invention Aiming at the problems in the background technology, the invention provides an external evaluation model of a vulva mode microorganism population based on microcalorimetry for evaluating the antibacterial effect of a microorganism lotion, which is used for simulating an in-vivo environment, realizing real-time dynamic monitoring and quantitatively evaluating the antibacterial effect of different microorganism lotions or plant extracts. The technical scheme adopted by the invention for solving the technical problems is as follows: In a first aspect, the invention provides an external evaluation model for a vulva mode microorganism population based on microcalorimetry for evaluating the bacteriostatic effect of a microorganism lotion, wherein the establishment process of the external evaluation model comprises the following steps: (a) Selecting a vulva model strain, collecting bacterial suspension of each model strain, and stably preserving to establish a standard strain library; (b) Screening culture mediums, namely analyzing thermal parameters of different culture mediums by microcalorimetry, and selecting an optimal culture medium for establishing an external evaluation model of a vulva mode microorga