Method for determining biological oxygen abstracting effect of probiotics in vitro

Abstract

The method for determining the biological oxygen abstracting effect of the probiotics in vitro comprises the following steps: (1) culturing a strain; (2) performing non-contact co-culture on aerobic probiotics and anaerobic bacteria; 3) agar sealing; and 4) biological oxygen abstracting effect determination. In the determination process, aerobic probiotics and anaerobic bacteria are not in contact with each other, so that the interference of factors such as nutrient substance competition, metabolite inhibition and dominant bacteria overpressing growth between the two microorganisms can be eliminated, and the accuracy of the result is improved; the operation is simple, and complex instruments and equipment are not needed; the agar sealed culture dish can reduce the local oxygen concentration when the culture dish is exposed in a common atmospheric environment, slow down the poisoning speed of oxygen to anaerobic bacteria, and avoid the situation that the anaerobic bacteria die before aerobic probiotics play an oxygen abstracting effect and result judgment is affected; the freezing point of the agar is low, so that the sealing operation of the culture dish can be completed without repeatedly heating the agar solution in a large-batch and long-time operation process; the result is visually observed, and the number of bacterial colonies at the bottom of the culture dish can be directly counted qualitatively and quantitatively.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a method for determining the oxygen-depriving effect of probiotics in vitro. Background Art

[0002] There are trillions of microorganisms colonizing the human and animal bodies, which far exceed the number of host cells. Most of these microorganisms live in the gastrointestinal tract. Beneficial bacteria and harmful bacteria are interdependent and mutually restricted, and are in a dynamic equilibrium state, thus maintaining normal intestinal function. Existing studies have shown that there are more than 500 species of bacteria from 30 genera inhabiting the gastrointestinal tract, mainly composed of anaerobic bacteria, facultative anaerobic bacteria and aerobic bacteria, among which obligate anaerobic bacteria are the dominant population, accounting for more than 99%. The anaerobic beneficial bacteria in the intestinal microbiome colonize on the surface of the intestinal mucosa to antagonize and inhibit the attachment of pathogens. However, when the intestinal microecological system is out of balance, the local oxygen concentration molecules will increase, the redox potential in the intestine will decrease, and the growth and reproduction of anaerobic bacteria will be inhibited.

[0003] With the rapid development of biotechnology, the definition of probiotics has become more and more perfect. At present, a relatively consensus definition has been formed, which refers to microorganisms with physiological activity. When the body ingests an appropriate amount through oral or other administration methods, it can colonize in the host and improve the host's microecological balance, thereby playing a beneficial role. The Food and Agriculture Organization of the United Nations and the World Health Organization define probiotics as "active microorganisms that are beneficial to the health of the host when ingested in sufficient quantities." Probiotics are well known for their beneficial effects such as maintaining the balance of intestinal flora, activating the immune system and regulating immune responses. In recent years, various probiotics have been widely used to promote the health of animals and humans, but the mechanism of action of probiotics is still under further research and has not been fully elucidated. The more recognized theories and doctrines in the industry include "dominant population theory", "biological barrier theory", "biological oxygen deprivation theory", "three-stream circulation theory", and "microbiome and nutrition relationship". Among them, the biological oxygen deprivation theory refers to the fact that aerobic probiotics need to consume oxygen to reproduce in the intestinal system, which can quickly create a low-oxygen environment in the intestine, support the growth of dominant anaerobic flora in the intestine (such as bifidobacteria, lactobacilli, etc.), and restore the dynamic balance of intestinal microecology.

[0004] However, the interaction between various microorganisms in the gastrointestinal tract is complex, and it is difficult to directly evaluate in vivo whether aerobic probiotics can promote the growth of anaerobic bacteria through the biological oxygen deprivation effect. In the in vitro atmospheric environment, anaerobic bacteria are continuously exposed to the oxygen environment and are easily poisoned by oxygen and die. Anaerobic workstations are often used to solve the problem of in vitro culture of anaerobic bacteria, but the anaerobic workstation equipment is expensive, and the consumption of inert gas is large and the cost is high, making it difficult to popularize in probiotic research. Some researchers have determined the biological oxygen deprivation ability of aerobic probiotics by inoculating aerobic probiotics and anaerobic bacteria in liquid culture medium for co-culture. However, the requirements of different microorganisms for culture medium are different. When studying some difficult-to-culture anaerobic intestinal microorganisms, it may not be possible to find a culture medium formula that can simultaneously meet the growth and reproduction of both microorganisms. In addition, during the culture process of this method, aerobic probiotics will compete with anaerobic bacteria for nutrients in the culture medium at the same time. The metabolites produced by the two microorganisms may also affect the growth of both, resulting in an increase in the variables affecting the growth of anaerobic bacteria, making it difficult to accurately evaluate whether aerobic probiotics can promote the growth of probiotics through the biological oxygen deprivation effect. Therefore, inventing a low-cost, simple and accurate in vitro probiotic biological oxygen-deprivation effect determination method is of great significance for clarifying the mechanism of action of probiotics and developing more new probiotics. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the oxygen-depriving effect of probiotics in vitro.

[0006] The present invention achieves the above-mentioned object through the following technical scheme: A method for determining the oxygen-depriving effect of probiotics in vitro, comprising the following steps:

[0007] 1) Culture of bacterial strains: using the Bacillus licheniformis ATCC 14580 as an aerobic probiotic, and the Bacteroides fragilis ATCC 25285 and the Bifidobacterium adolescentis ATCC 15703 as anaerobic indicator bacteria for determination; the Bacillus licheniformis is cultured in brain heart infusion broth to obtain a Bacillus licheniformis seed solution; the Bacteroides fragilis is cultured in a Bacteroides fragilis enrichment broth to obtain a Bacteroides fragilis seed solution; the Bifidobacterium adolescentis is cultured in a bifidobacterium culture medium to obtain a Bifidobacterium adolescentis seed solution;

[0008] 2) Non-contact co-culture of aerobic probiotics and anaerobic bacteria: Columbia blood agar medium is poured into the bottom of a disposable plastic culture dish with a diameter of 90 mm, and nutrient agar medium is poured onto the lid. After the medium solidifies, a sterile cotton swab is used to dip Bacillus licheniformis seed solution and evenly spread on the lid. After diluting the seed solution of Bacteroides fragilis or Bifidobacterium adolescentis, bacterial solutions of different dilution gradients are dripped onto the bottom of the dish. After the bacterial solution is completely absorbed by the culture medium, the bottom and lid of the dish are quickly turned upside down together;

[0009] 3) Agar sealing: Weigh 1-2g agar powder, add distilled water to 100mL, prepare a 1.5%-2% agar solution, sterilize at 121℃ for about 15min, use a pipette to draw the agar solution into the gap between the bottom and the lid of the dish, and seal the plastic dish;

[0010] 4) Biological oxygen deprivation effect determination: The sealed culture dish in step 3) is placed in a constant temperature incubator under normal atmospheric environment, and the growth of anaerobic bacteria is observed and the colony count is performed; a culture dish without inoculation of Bacillus licheniformis on the lid is used as a control group.

[0011] Furthermore, the Bacillus licheniformis is cultured in brain heart infusion broth at 37° C. for 24 hours.

[0012] Furthermore, the Bacteroides fragilis is anaerobically cultured in Bacteroides fragilis enrichment broth at 37° C. for 24 hours.

[0013] Furthermore, Bifidobacterium adolescentis was anaerobically cultured in a bifidobacterium culture medium at 37°C for 24 hours.

[0014] Furthermore, the sealed culture dish in step 3) is placed in a constant temperature incubator under normal atmospheric environment and cultured at 37° C. for 48 hours.

[0015] Compared with the prior art, the method of the present invention for determining the oxygen-depriving effect of probiotics in vitro has the following beneficial effects:

[0016] 1. During the determination process, aerobic probiotics and anaerobic bacteria do not contact each other, which can eliminate the interference of factors such as nutrient competition between the two microorganisms, metabolic product inhibition, and overwhelming growth of dominant bacteria, thereby improving the accuracy of the results.

[0017] 2. Simple operation, no need for complicated instruments and equipment.

[0018] 3. Agar-sealed culture dishes can reduce the local oxygen concentration when the culture dishes are exposed to the normal atmospheric environment, slow down the rate of oxygen toxicity to anaerobic bacteria, and avoid the death of anaerobic bacteria before aerobic probiotics can exert their oxygen-depriving effect, thereby affecting the results.

[0019] 4. Agar has a low freezing point. During large-scale and long-term operations, the sealing operation of the culture dish can be completed without repeatedly heating the agar solution.

[0020] 5. The results are intuitive to observe, and the number of colonies at the bottom of the culture dish can be directly counted, both qualitatively and quantitatively. DETAILED DESCRIPTION

[0021] A method for in vitro determination of the oxygen-depriving effect of probiotics, comprising the following steps:

[0022] 1) Culture of bacterial strains: Bacillus licheniformis ATCC 14580 was used as an aerobic probiotic, and Bacteroides fragilis ATCC25285 and Bifidobacterium adolescentis ATCC 15703 were used as anaerobic indicator bacteria for determination. Bacillus licheniformis was cultured in brain heart infusion broth at 37°C for 24 hours to obtain Bacillus licheniformis seed solution; Bacteroides fragilis was cultured in Bacteroides fragilis enrichment broth at 37°C for 24 hours to obtain Bacteroides fragilis seed solution; Bifidobacterium adolescentis was cultured in bifidobacterium culture medium at 37°C for 24 hours to obtain Bifidobacterium adolescentis seed solution.

[0023] 2) Non-contact co-culture of aerobic probiotics and anaerobic bacteria: Pour Columbia blood agar medium into the bottom of a disposable plastic culture dish with a diameter of 90 mm, and pour nutrient agar medium on the lid. After the medium solidifies, use a sterile cotton swab to dip Bacillus licheniformis seed liquid and evenly apply it to the lid. After a 10× series gradient dilution of Bacteroides fragilis or Bifidobacterium adolescentis seed liquid, drip the bacterial liquid of different dilution gradients onto the bottom of the dish. After the bacterial liquid is completely absorbed by the culture medium, quickly turn the bottom and lid of the dish upside down.

[0024] 3) Agar sealing: Weigh 1-2g agar powder, add distilled water to make up to 100mL, prepare an agar solution with a concentration of 1.5%-2%, sterilize at 121℃ for about 15min, use a pipette to draw the agar solution into the gap between the bottom and the cover of the dish, and seal the plastic dish.

[0025] 4) Biological oxygen deprivation effect determination: Place the sealed culture dish in step 3) in a constant temperature incubator under normal atmospheric environment, culture at 37°C for 48 hours, observe the growth of anaerobic bacteria and count the colonies. A culture dish without inoculation of Bacillus licheniformis on the lid is used as the control group.

[0026] By using the method of the present invention, the oxygen-depriving ability of aerobic probiotics can be determined in a normal atmospheric environment in vitro. The present invention has the following advantages:

[0027] 1. During the determination process, aerobic probiotics and anaerobic bacteria do not contact each other, which can eliminate the interference of factors such as nutrient competition between the two microorganisms, metabolic product inhibition, and overwhelming growth of dominant bacteria, thereby improving the accuracy of the results.

[0028] 2. Simple operation, no need for complicated instruments and equipment.

[0029] 3. Agar has a low freezing point. During large-scale and long-term operations, the sealing operation of the culture dish can be completed without repeatedly heating the agar solution.

[0030] 4. Agar-sealed culture dishes can reduce the local oxygen concentration when the culture dishes are exposed to the normal atmospheric environment, slow down the rate of oxygen toxicity to anaerobic bacteria, and avoid the death of anaerobic bacteria before aerobic probiotics can exert their oxygen-depriving effect, thereby affecting the results.

[0031] 5. The results are intuitive to observe, and the number of colonies at the bottom of the culture dish can be directly counted, both qualitatively and quantitatively.

[0032] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0033] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining the oxygen-depriving effect of probiotics in vitro, characterized in that: The following steps are involved: 1) Bacterial culture: using the Bacillus licheniformis ATCC 14580 as an aerobic probiotic, and the Bacteroides fragilis ATCC 25285 and the Bifidobacterium adolescentis ATCC 15703 as anaerobic indicator bacteria for determination; the Bacillus licheniformis is cultured in brain heart infusion broth to obtain a Bacillus licheniformis seed solution; Bacteroides fragilis is cultured in Bacteroides fragilis enrichment broth to obtain Bacteroides fragilis seed solution; Bifidobacterium adolescentis is cultured in Bifidobacterium culture medium to obtain Bifidobacterium adolescentis seed solution; 2) Non-contact co-culture of aerobic probiotics and anaerobic bacteria: Columbia blood agar medium is poured into the bottom of a disposable plastic culture dish with a diameter of 90 mm, and nutrient agar medium is poured onto the lid. After the medium solidifies, a sterile cotton swab is used to dip Bacillus licheniformis seed solution and evenly spread on the lid. After diluting the seed solution of Bacteroides fragilis or Bifidobacterium adolescentis, bacterial solutions of different dilution gradients are dripped onto the bottom of the dish. After the bacterial solution is completely absorbed by the culture medium, the bottom and lid of the dish are quickly turned upside down together; 3) Agar sealing: Weigh 1-2g agar powder, add distilled water to 100mL, prepare a 1.5%-2% agar solution, sterilize at 121℃ for about 15min, use a pipette to draw the agar solution into the gap between the bottom and the lid of the dish, and seal the plastic dish; 4) Biological oxygen deprivation effect determination: The sealed culture dish in step 3) is placed in a constant temperature incubator under normal atmospheric environment, and the growth of anaerobic bacteria is observed and the colony count is performed; a culture dish without inoculation of Bacillus licheniformis on the lid is used as a control group.

2. The method for in vitro determination of the oxygen-depriving effect of probiotics according to claim 1, characterized in that: The Bacillus licheniformis was cultured in brain heart infusion broth at 37° C. for 24 hours.

3. The method for in vitro determination of the oxygen-depriving effect of probiotics according to claim 1, characterized in that: The Bacteroides fragilis was anaerobically cultured in the Bacteroides fragilis enrichment broth at 37° C. for 24 hours.

4. The method for in vitro determination of the oxygen-depriving effect of probiotics according to claim 1, characterized in that: Bifidobacterium adolescentis was cultured anaerobically at 37°C in bifidobacterium culture medium for 24 h.

5. The method for in vitro determination of the oxygen-depriving effect of probiotics according to claim 1, characterized in that: The sealed culture dish in step 3) was placed in a constant temperature incubator under normal atmospheric environment and cultured at 37° C. for 48 h.