Culture medium for efficiently promoting growth of clostridium and preparation method thereof

Abstract

The invention relates to the technical field of microbial culture, and particularly discloses a culture medium for efficiently promoting clostridium growth and a preparation method thereof. Comprising the following raw materials in percentage by mass: 13-30% of peptone, 10-25% of bovine brain extract powder, 15-26% of bovine heart extract powder, 8-18% of yeast extract powder, 1-4% of L-cysteine, 2-6% of fructose, 2-6% of glucose, 5-14% of sodium chloride, 3-7% of disodium hydrogen phosphate and the balance of thermally inactivated probiotics, the heat-inactivated probiotics comprise 0.5-4% of extracellular polysaccharide of one or more of lactobacillus, lactococcus and pediococcus or bacteria, the pH value of the culture medium is 5.0 + / -0.5, the culture medium is specifically added with yeast extract powder, L-cysteine and fructose, and under the anaerobic culture condition, the pH value of the culture medium is 5.0 + / -0.5. On the basis of conventional fermentation culture, thermal inactivation specific probiotics are added into a culture medium, so that clostridium thalli with a probiotic effect can be promoted to be greatly increased, and the logarithmic growth period of the thalli is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and specifically to a culture medium for efficiently promoting the growth of clostridium and a preparation method thereof. Background Art

[0002] The human intestinal microbiome is a complex and dynamic ecosystem that contains trillions of microorganisms that have a profound impact on the host's health and disease status. In recent years, more and more studies have shown that the intestinal microbiome plays a key role in host immune regulation, nutrient absorption, metabolic function and disease prevention.

[0003] For example, intestinal microorganisms can produce short-chain fatty acids by fermenting undigested carbohydrates. These short-chain fatty acids are not only an important source of energy for the host, but can also regulate the immune system. In addition, certain intestinal flora disorders (such as intestinal flora disorders) are closely related to a variety of diseases such as obesity, diabetes, inflammatory bowel disease, and colon cancer. Therefore, in-depth understanding and research of intestinal microecology is of great significance for developing new treatment strategies, preventing diseases, and improving human health.

[0004] Among them, Clostridium is one of the important members of the intestinal microecology. It belongs to the Gram-positive anaerobic bacteria and is widely present in nature and the human intestine. Clostridium plays an important role in maintaining the balance of intestinal flora, inhibiting the growth of pathogens and promoting host health. Clostridium is an important genus in the study of human intestinal microecology. Taking Clostridium scindens as an example, C. scindens is a clostridium with important metabolic functions in the human intestine. It can convert bile acids into secondary bile acids, such as deoxycholic acid, which play a key role in the host's lipid metabolism and the ecological balance of the intestinal microbiota.

[0005] C. scindens is considered to be beneficial to intestinal health because secondary bile acids have antibacterial properties that can inhibit the growth of certain pathogens, thereby maintaining the stability of the intestinal microecology.

[0006] Currently, there are many challenges in culturing Clostridium in in vitro research. First, Clostridium is a strict anaerobic bacterium and is extremely sensitive to oxygen, so it needs to be cultured in a strictly controlled anaerobic environment. Commonly used anaerobic techniques include gas replacement and anaerobic jar methods. Secondly, different types of Clostridium have different nutritional requirements, so specific culture media are required to meet their growth needs. Currently commonly used Clostridium culture media include modified Thioglycollate medium, Reinforced Clostridial Medium (RCM) and Brain Heart Infusion (BHI). These culture media are usually rich in protein, carbohydrates and vitamins to meet the nutritional needs of Clostridium.

[0007] However, existing culture media still have some limitations in practical applications. For example, some culture media may not be fully suitable for specific Clostridium species, resulting in poor growth. In addition, problems such as contamination and difficulty in standardizing culture conditions during the culture process affect the reliability of research results. In particular, the requirements of some Clostridium for specific nutritional factors have not been fully clarified, making the selection and optimization of culture medium components very challenging.

[0008] Recent studies have shown that the use of inactivated bacteria as culture medium components can significantly promote the growth of certain microorganisms. This method can not only provide rich nutrition, but also simulate the complex microbial interactions in the natural ecological environment, thereby improving the growth and metabolic activity of the target microorganisms. For example, some studies have found that inactivated lactic acid bacteria can be used as a culture medium for probiotics, thereby promoting the growth of target probiotics. For example, inactivated Lactobacillus rhamnosus and Lactobacillus acidophilus were used in the culture of Bifidobacterium longum. The results showed that the cell wall components and intracellular substances provided by inactivated lactic acid bacteria can significantly promote the growth of Bifidobacterium. This promotion effect may be related to the various bioactive substances provided by inactivated bacteria, such as short-chain fatty acids, amino acids and cytokines. At the same time, inactivated bacteria can simulate the microbial interactions in natural ecosystems and provide target microorganisms with growth conditions that are closer to their natural habitats. The bioactive substances in inactivated bacteria, such as short-chain fatty acids and intracellular metabolites, can promote the metabolic activities of target microorganisms and enhance their growth rate and product yield.

[0009] In summary, there is an urgent need to develop a new Clostridium culture medium to overcome the shortcomings of existing culture media and provide a more reliable and efficient culture method. Summary of the invention

[0010] The object of the present invention is to provide a culture medium for efficiently promoting the growth of Clostridium and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] The invention discloses a culture medium comprising 13-30% peptone, 10-25% ox brain extract powder, 15-26% ox heart extract powder, 8-18% yeast extract powder, 1-4% L-cysteine, 2-6% fructose, 2-6% glucose, 5-14% sodium chloride, 3-7% disodium hydrogen phosphate, and 0.5-4% heat-killed probiotics. The pH value of the culture medium is 5.0±0.5, and the heat-killed probiotics include one or more of lactobacillus, lactococcus, and pediococcus.

[0013] A method for preparing a culture medium that efficiently promotes the growth of Clostridium comprises the following steps:

[0014] S1. Weighing mixed raw materials: weighing 43.65 g of mixed raw materials including peptone, ox brain extract powder, ox heart extract powder, yeast extract powder, fructose, glucose, sodium chloride and disodium hydrogen phosphate;

[0015] Pour the weighed mixed raw materials into 1L of distilled water and stir thoroughly with a stirrer until all ingredients are dissolved to form a culture medium solution;

[0016] Dispense the culture medium: Dispense the dissolved culture medium solution into containers;

[0017] High pressure sterilization: Place the packaged culture medium into a high pressure sterilizer, set the temperature to 121°C, and perform high pressure sterilization for 15 minutes;

[0018] After sterilization, take out the culture medium and place it at room temperature to cool down. Set it aside after it drops to an appropriate temperature.

[0019] S2. Accurately weigh 0.9 g of L-cysteine ​​and 0.225-1.8 g of heat-killed probiotics, wherein the heat-killed probiotics include one or more of Lactobacillus, Lactococcus and Pediococcus;

[0020] L-cysteine ​​and heat-killed probiotics (probiotics mainly include Lactobacillus, Lactococcus and Pediococcus) are dissolved in 1 ml of distilled water respectively, stirred with a micropipette, and the dissolved liquid is filtered and sterilized using a sterile filter with a 0.22 μm filter membrane. The filtered and sterilized L-cysteine ​​solution and heat-killed probiotics (probiotics mainly include Lactobacillus, Lactococcus and Pediococcus) solution are added to the previously prepared basal culture medium respectively, and shaken to mix.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention specifically adds yeast extract powder, L-cysteine ​​and fructose to the basic culture medium. Yeast extract powder is a supplement rich in amino acids, vitamins and nucleic acids. It can provide a variety of amino acids and vitamins required for bacterial growth, especially B vitamins, which are essential factors for microbial growth. Fructose is a simple carbohydrate that can be quickly metabolized and utilized by bacteria. For some anaerobic bacteria, fructose is a high-quality carbon source that can quickly generate energy and metabolites through fermentation pathways to support the growth and reproduction of bacteria. L-cysteine ​​is a sulfur-containing amino acid that plays a key role in bacterial metabolism. It is not only a basic unit for protein synthesis, but also can be used as an antioxidant to protect bacterial cells from oxidative damage. For anaerobic bacteria, L-cysteine ​​can also reduce the redox potential of the culture medium and create an environment more suitable for anaerobic growth. In addition, experiments have shown that under anaerobic culture conditions, by adding heat-killed probiotics (probiotics mainly include lactobacillus, lactococcus and pediococcus) to the culture medium on the basis of conventional fermentation culture, it can promote the large-scale proliferation of clostridium cells and shorten the logarithmic growth period of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a growth curve diagram of the experimental group and the reference group in the embodiment of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Embodiment 1:

[0026] See also Figure 1 As shown, a culture medium that efficiently promotes the growth of Clostridium comprises the following steps:

[0027] Comparison of the culture medium of the present invention and conventional Clostridium culture medium for Clostridium culture.

[0028] The embodiment of the present invention provides a component of a culture medium for efficiently promoting the growth of Clostridium, comprising the following raw materials in percentage by mass: 23% peptone, 17% ox brain extract powder, 22% ox heart extract powder, 11% yeast extract powder, 2% L-cysteine, 4% fructose, 4% glucose, 11% sodium chloride, 5% disodium hydrogen phosphate, and 1% heat-killed Pediococcus pentosaceus. The pH value of the culture medium is 5.0±0.2.

[0029] Preparation method: Weigh the mixed raw materials: weigh 43.65g of mixed raw materials including peptone, ox brain extract powder, ox heart extract powder, yeast extract powder, fructose, glucose, sodium chloride and disodium hydrogen phosphate; pour the weighed mixed raw materials into 1L of distilled water, and use a stirrer to fully stir until all components are dissolved to form a culture medium solution; divide the dissolved culture medium solution into containers; put the divided culture medium into an autoclave, set the temperature to 121°C, and perform autoclave sterilization for 15 minutes; take out the culture medium and place it at room temperature to cool, and set it aside after it drops to an appropriate temperature. Accurately weigh 0.9 g of L-cysteine ​​and 0.45 g of heat-killed Pediococcus pentosaceus; dissolve L-cysteine ​​and Pediococcus pentosaceus in 1 ml of distilled water, respectively, stir with a micropipette, and use a sterile filter with a 0.22 μm filter membrane to filter and sterilize the dissolved liquid; add the filtered and sterilized L-cysteine ​​solution and Pediococcus pentosaceus solution to the previously prepared basal culture medium, respectively, and shake to mix.

[0030] Experimental example:

[0031] Preparation: The conventional Clostridium culture medium is selected from American BD237500 brain heart infusion broth BHI culture medium.

[0032] The specific experimental example culture method includes the following steps:

[0033] Take 50 ml of the prepared culture medium solution of the present invention (experimental group) and BHI culture medium (control group) solution, place them in a fermentation test tube, and place them in a 37° constant temperature incubator under an anaerobic environment. Add 200 ul of Clostridiumscindens frozen solution to the culture solution for culture and fermentation. Each group of solutions is subjected to 3 biological replicates. Take an appropriate amount of fermentation solution every 2 hours, measure the absorbance of the liquid at a wavelength of 600 nm in a spectrophotometer and record it, and repeat this process. Stop measuring after 40 hours, and keep the anaerobic environment throughout the experiment.

[0034] Table 1 is a record of OD600 absorbance values ​​provided by an embodiment of the present invention. Figure 1 Table 1 shows the growth curves of Clostridium scindens in the culture medium of the present invention and conventional Clostridium culture medium solution for 40 hours provided in the embodiment of the present invention:

[0035] Table 1. OD600 absorbance value records

[0036]

[0037]

[0038] As can be seen from the above, in the implementation of the present invention, the OD600 absorbance value of the experimental group of the present invention increases the fastest, which means that the growth rate of Clostridium in the experimental group is the highest.

[0039] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0040] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A culture medium that efficiently promotes the growth of Clostridium, characterized in that: The following raw materials are included in the following mass percentages: The invention discloses a culture medium comprising 13-30% peptone, 10-25% ox brain extract powder, 15-26% ox heart extract powder, 8-18% yeast extract powder, 1-4% L-cysteine, 2-6% fructose, 2-6% glucose, 5-14% sodium chloride, 3-7% disodium hydrogen phosphate, and 0.5-4% heat-killed probiotics. The pH value of the culture medium is 5.0±0.5, and the heat-killed probiotics include one or more of lactobacillus, lactococcus, and pediococcus.

2. A method for preparing a culture medium that efficiently promotes the growth of Clostridium, characterized in that: The following steps are involved: S1. Weighing mixed raw materials: weighing 43.65 g of mixed raw materials including peptone, ox brain extract powder, ox heart extract powder, yeast extract powder, fructose, glucose, sodium chloride and disodium hydrogen phosphate; Pour the weighed mixed raw materials into 1L of distilled water and stir thoroughly with a stirrer until all ingredients are dissolved to form a culture medium solution; Dispense the culture medium: Dispense the dissolved culture medium solution into containers; High pressure sterilization: Place the packaged culture medium into a high pressure sterilizer, set the temperature to 121°C, and perform high pressure sterilization for 15 minutes; After sterilization, take out the culture medium and place it at room temperature to cool down. Set it aside after it drops to an appropriate temperature. S2. Accurately weigh 0.45-1.8 g of L-cysteine ​​and 0.225-1.8 g of heat-killed probiotics including one or more of Lactobacillus, Lactococcus and Pediococcus, or lyophilized powder of extracellular polysaccharide of bacteria; Dissolve L-cysteine ​​and heat-killed lactobacillus or extracellular polysaccharide in 1 ml of distilled water respectively, stir with a micropipette and filter the dissolved liquid with a sterile filter using a 0.22 μm filter membrane, add the filtered and sterilized L-cysteine ​​solution and heat-killed probiotic or extracellular polysaccharide solution to the previously prepared basal culture medium, and shake to mix.

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